Detection of alterations of autophagy related protein LC3-II as a possible method of post mortem age estimation: A useful approach?

Detection of alterations of autophagy related protein LC3-II as a possible

method of post mortem age estimation:

A useful approach?

16.01.2017

Research Project by Lisa Jansen, Identity code: 10634584

Master of Science in Forensic Science, University of Amsterdam, Science Park 107, 1098 XG Amsterdam, Netherlands

36 European Credits

Time period: 08.07.2016 – 16.01.2017

Research Project performed at the Institute of Forensic Medicine, University Medical Center Duesseldorf, Germany

Supervisor: Prof. Dr. med. Stefanie Ritz-Timme Examiner: Prof. Dr. Roelof-Jan Oostra

Suggested journal: Journal of Forensic and Legal Medicine E-mail: lisa.jansen@student.uva.nl; lisa-jansen89@web.de Address: Pescher Str. 120, 41352 Korschenbroich, Germany

Abstract

The identification of unknown human remains is a frequent task in forensic case work. One highly important aspect is the estimation of the deceased person’s age. Bio-medical basic research revealed age related alterations in the expression of some proteins involved in cellular autophagy processes. This pilot project addresses the question, if such alterations can be used as basis for age estimation. The two aims of this study were (1) to find out if the autophagy marker microtubule-associated protein 1A/1B-light chain 3 (LC3-II) can be detected in post mortem drawn samples of human skeleton muscle and if so, (2) to clarify, if age dependent changes in protein expression can be observed. A suitable protein isolation method was developed. However, western blotting analysis revealed no age dependent alteration in the amount of LC3-II. As a side finding an age dependent decrease of the amount of β-actin was observed. Therefore, LC3-II analysis of muscle tissues seems not to be suitable for age estimation but the decrease of the amount of β-actin might be interesting for further research.

Introduction

The identification of unknown human remains is an important task in forensic routine work. This process typically involves the estimation of the deceased person’s age [1].

To date, this task is mainly accomplished by the evaluation of morphologic features of the whole body, or of bones and teeth. Only few molecular methods, like age estimation based on the racemization of aspartic acid, have been established [2]. However, especially in cases of advanced stages of decay or when only parts of a body are found, post mortem age estimation may remain difficult. Morphological methods are quite reliable in young ages, whereas in cases of deceased adults they become imprecise [3]. Molecular methods, on the other hand, depend on the presence of specific sample material, like the body’s teeth and may be compromised by putrefaction processes. Some methods are often not standardized yet, because of too less applications, especially in different forensic scenarios [4]. In order to meet the demand of the various and in part very complex cases, forensic research is working on the development of new, additional methods for age estimation. Recently, bio-medical basic research has shown that cellular autophagy processes present age related alterations during life time [5-9]. Autophagy is a protective degradation process inside the cells of a body. It permits the cells to decompose unwanted or no longer required cellular structures, such as damaged organelles or parts of the cytosol. This contributes to a balance in cell growth, development and cell death [10]. During aging, this essential process seems to change. One of the commonly used approaches to detect and investigate autophagy is the analysis of a protein called LC3. It exists in two forms, LC3-I and –II, the latter serving as an indicator for autophagic activity [11-12]. To measure this autophagic activity, usually the ratio between LC3-II and LC3-I or between LC3-II and a loading control is detected [12]. Several studies, investigating alteration of LC3-II ratios during the ageing process, have already been published [13-20]. Although the findings of these studies were not convergent and it is not yet completely clear if LC3-II expression increases or decreases during age, measuring autophagic activity might be an interesting approach for forensic age estimation.

To find out more about the possible usage of LC3-II for age estimation in forensic case work, we designed our project to address the following questions:

1. Can LC3-II be detected in post mortem drawn, skeletal tissue samples? 2. If yes: is there an age dependent increase or decrease of LC3-II?

Material and methods

Human skeleton muscle samples (M. iliopsoas major) were obtained from 30 forensic autopsy cases (including 15 male and 15 female). Samples were categorized in 7 age groups: 20-29, 30-39, 40-49, 50-59, 60-69, 70-79 and 80-89. In addition, 3 individuals younger than 20 years were included. Only cases with a post mortem interval of less than 2 days and without signs of putrefaction were included. Exclusion criteria were diseases affecting muscle tissue and intoxications that could be detected in the prehistory. The samples were immediately frozen and stored at -80 °C. The experimental procedure (study number 5375) was approved by the ethic committee of the Heinrich-Heine-University Duesseldorf.

Used antibodies:

The following antibodies were used in this study: A rabbit polyclonal primary antibody against LC3B (1:666, Cell Signaling), a polyclonal peroxydase-labelled anti-rabbit secondary antibody (1:5000, Cell Signaling), a mouse monoclonal primary antibody against β-actin (1:5000, Sigma-Aldrich) and a peroxydase-linked anti-mouse immunoglobulin G secondary antibody (1:5000, GE Healthcare). Tissue preparation, sodium dodecyl sulfate polyamide gel electrophoresis (SDS-PAGE), and Western blotting:

0.5 g of each muscle sample were homogenized with an 19-foldhomogenization buffer (0.125 mol/L Tris/HCl pH 6.4, 4 mol/L urea, 4 % SDS, 10 % glycerol and 10% mercapto ethanol, final pH 6.8) containing regular amount of Protease Inhibitor Tablets® (cOmplete Tablets, Mini, Roche). The samples were mixed by an Ultra Turrax Homogenizer and heated for 4 minutes in boiling water. After centrifugation the protein concentrations of the supernatants were measured by Lowry protein assay. 50 µg of each muscle lysate were denaturated by heat shock at 95°C for 5 min. Each sample was filled in 3 lanes and separated on 16 % SDS-PAGE. The separated proteins were transferred to polyvinylidenedifluoride (PVDF) membranes by Western blotting. The membranes were blocked one hour at room temperature with 5% skimmed milk powder (Sucofin) soluted in phosphate-buffered saline (PBS), supplemented with 0.1% Tween (PBST). After washing, the membrane was cut horizontally in two pieces. The part containing the higher molecular weight was incubated with anti-β-actin antibodies for 1-h at room temperature. The part with a lower weight was incubated with anti-LC3B antibodies over night at 4°C. Afterwards, the blots were washed and incubated 1 -h with anti-rabbit or anti-mouse secondary antibodies. Both halves were developed simultaneously with enhanced chemiluminescence substrate (SuperSignal West Pico, Thermo Scientific). All films were digitalized by a Luminescenct Image Analyzer (Fujifilm LAS-4000) and the band intensities were measused by Multi Gauge. Corresponding box sizes were chosen for the background values and set below each band of interest. On each membrane also a positive control (Ht 1080 cells) was blotted that contains concentrated and defined amounts of LC3-I, -II and β-actin.

The measured intensitity of LC3-II (faster moving band) was set relative to the intensity of LC3-I (slower moving band), as well as the intensity of LC3-II to the intensity of the normalization protein β-actin.

Besides luminescence controls, the LC3-II intensity was compared to the intensity of the complete protein content on the blot that was stained with 0.1 % Amido black.

Calculations of different intensity ratios representing autophagic activity

In order to calculate different ratios as a measure of autophagic activity, the signal intensities of the relevant bands were measured and backgrounds were substracted. In case there were several samples of one donor on the same gel (usually 3 to 5), the means of the bands were calculated. The intensity ratio was determined by dividing the two means. In order to compare the rations of two different membranes, they had to be multiplied with the intensity of a control band of extracts from Ht 1080 cells (C), which were loaded on each gel with a standardized protein concentration. Mean LC3-II/LC3-I intensity ratio

= 𝐿𝐶3−𝐼𝐼 1 − 𝑏𝑔 1 + … + 𝐿𝐶3−𝐼𝐼 𝑥 − 𝑏𝑔 𝑥 𝑥 𝐿𝐶3−𝐼 1 − 𝑏𝑔 1 + … + 𝐿𝐶3−𝐼 𝑥 − 𝑏𝑔 𝑥 𝑥 * C

Mean LC3-II/β-actin intensity ratio

= 𝐿𝐶3−𝐼𝐼 1 − 𝑏𝑔 1 + … + 𝐿𝐶3−𝐼𝐼 𝑥 − 𝑏𝑔 𝑥 𝑥 ß−𝑎𝑐𝑡𝑖𝑛 1 − 𝑏𝑔 1 + … + ß−𝑎𝑐𝑡𝑖𝑛 𝑥 − 𝑏𝑔 𝑥 𝑥 * C

Mean LC3-II/total protein intensity ratio

= 𝐿𝐶3−𝐼𝐼 1 − 𝑏𝑔 1 + … + 𝐿𝐶3−𝐼𝐼 𝑥 − 𝑏𝑔 𝑥 𝑥 𝑤𝑝 1 − 𝑏𝑔 1 + … + 𝑤𝑝 𝑥 − 𝑏𝑔 𝑥 𝑥 ∗ 𝐶 bg = Background

x = Frequency of measuring one sample wp = Total protein measured by Amido black

C = Intensity of the LC3-I band of Ht 1080 control sample (applied when samples of different membranes are compared to each other)

Variability testing

The variability of the ratios of the intensities of LC3-II/LC3-I, LC3-II/β-actin and LC3-II/total protein staining was tested by blotting one sample five times on one membrane, and three times on two membranes. In addition, two samples from different parts of one muscle of one donor were drawn and blotted five times on one membrane. The variability for the first test was determined by calculating the standard deviation. The variability for the other two tests a means of the multiple intensity measurements and their percentage differences were calculated.

Normalization tests of β-actin

The normalization efficiency of β-actin was tested by measuring the signal intensities of β-actin bands of different muscle samples and relating them to the total protein amount. The results were compared to the age of the sample donor.

Histologic examination – H & E staining:

Muscle sections from the same part of the muscle as the samples for protein extraction were fixed in formalin and stained with hematoxylin and eosin.

Statistical analysis

For age relation measurements, each sample was blotted three times, and the mean was calculated. The final scatterplot was evaluated using the value of Pearson’s correlation coefficient r. Correlations below 0.5 (or above -0.5) were considered as no relationship between the LC3-II ratio and age, correlations higher than 0.5 (or lower than -0.5) were considered as a weak relationship, correlations higher than 0,8 (lower than -0,8) were considered as a high relationship, correlations = 1 (or -1) were considered as a strong relationship. In addition the p-value was determined and the relation between LC3-II ratio and age was considered as significant if p ≤ 0,05.

Results

In post mortem obtained human skeleton muscle samples, all proteins, relevant for calculating the intensity ratios representing autophagy activity, (LC3-I, LC3-II and β-actin) could be detected. The total protein staining by Amido black was also possible.

1. Calculation of autophagic activity

Figure 1 presents the results of the calculated intensity ratios of LC3-II/LC3-I, LC3-II/β-actin and LC3-II/total protein related to the donor’s age. In these graphs, both sexes are included.

The LC3-II/LC3-I intensity ratio shows a correlation coefficient r = 0,328 and a p-value p = 0.076 when all samples are regarded; only male donors: r = 0,158, p = 0,575; only female donors: r = 0,33, p = 0,230. The LC3-II/β-actin intensity ratio shows a correlation coefficient r = 0,232 and a p-value p = 0,217 when all samples are regarded; only male donors: r = 0,146, p = 0,604; only female donors: r = 0,253, p = 0,363. The LC3-II/total protein intensity ratio shows a correlation coefficient of 0,154 and a p-value of 0,416 when all samples are regarded; only male donors: r = 0,277, p = 0,317; only female donors: r = 0,030, p = 0,917. All correlation coefficients and p-values showed no significant relationship between the LC3-II amount and age.

Fig. 2 depicts that in general, the results suggest a four-fold increase of the LC3-II/LC3-I intensity ratio between the age of 20 and 80. However, there is no constant rise in the different age groups. In each age interval high differences between the minimum and maximum intensity values were detected, ranging from 8.0 million in the 50-59 year group to 22.8 million in the 80-89 year group.

Differences were also observed for donors of the same age: One 72 year old woman showed an LC3-II/LC3-I intensity ratio of ~23 million whereas a man of the same age showed a ratio of 6.5 million. 2. Variability testing

2.1. Variability of intensity ratios representing autophagic activity on one membrane

Blotting of one sample five times on one membrane resulted in a variability (standard deviation) of 11% for the intensity ratio II/I, of 41% for ratio II/β-actin, and of 12% for ratio LC3-II/total protein.

2.2. Variability of calculated intensity ratios representing autophagic activity on several membranes

Exemplary blotting of one sample three times on two different membranes resulted in a variability (percentage difference of the means) of 29% for intensity ratio II/I, 34% for ratio LC3-II/β-actin, and 15% for ratio LC3-II/total protein.

2.3. Biopsy variability

Two different samples of one donor presented an exemplary variability (percentage difference of the means) of 9% for intensity ratio LC3-II/LC3-I, of 51 % for ratio LC3-II/β-actin, and of 25% for ratio LC3-II/total protein.

The variability example of the LC3-II/LC3-I intensity ratio is presented in Figure 3. 3. Normalization tests of β-actin

Normalization test for the extracted amounts of β-actin compared to the total amount of the extracted proteome showed a decrease of β-actin depending on the age of the donor (figure 4). Regarding female and male samples, r was 0,463 and p was 0,030. Male samples alone resulted in r = 0,381 and p = 0,221; analysis of female samples alone resulted in r = 0,643 and p = 0,045.

4. Histologic examination – H & E staining:

Light microscope evaluation showed no morphological signs of diseases.

Discussion

Forensic estimation of a person’s age is a highly important aspect in the identification of unknown human remains. The number of unidentified bodies and the number of missing persons has risen over the last years. Though there obviously is a high demand for methods for the identification of unknown bodies and although several approaches have already been examined, identification remains difficult in forensic practice. In order to meet the demand of the various and in part very complex cases, forensic research is working on the development of new, additional methods for age estimation [2]. Thus, the detection and evaluation of molecular aging markers could be important for forensic practice.

Recently, bio-medical basic research has shown that cellular autophagy processes present age related alterations during life time [5-9]. LC3-II is an autophagy marker that can be used to monitor such age related changings. LC3-proteins are ubiquitin-like, mammalian homologues of yeast Atg8 and expressed in nearly all tissues. In mammalian cells LC3 is present in three different isoforms, LC3A, LC3B, and LC3C, but only LC3B corresponds to the levels of autophagy. For this reason, this study focuses on this LC3B isoform. LC3B exists in two different forms: LC3-I and LC3-II. When autophagy is induced, cytosolic LC3-I is converted into the membrane-associated LC3-II form which participates in the autophagic selection of cargo components for degradation. Thus, LC3-II serves as a marker for autophagic activity [11-12]. Both forms, LC3-I and LC3-II can be visualized by SDS-PAGE and Western blot analysis, because LC3-II moves faster through the gel compared to LC3-I [15]. In vitro studies revealed that the level of autophagy is increased in aging human fibroblasts by observing their total LC3 (LC3-I and LC3-II) content by immunofluorescence microscopy. Also Western blotting of the fibroblasts showed a rise during age by measuring the intensity ratio between LC3-II/LC3-I normalized by β-actin [7]. In 2014, Tashiro, K., et al. also

applied immunofluorescence microscopy with cultured dermal fibroblasts from women of different ages [8]. The amount of LC3 dots was higher in cells of elderly women (68 and 70 years old), as compared to those of young women (23 and 27 years old). By Western blot the single LC3-II band intensities were optically observed and also showed a rise during age.

In vivo studies focusing on autophagic activity have also been conducted. Experiments investigating mice hearts or thymus and liver, respectively, have also applied immunofluorescence microscopy and Western blot. The first method showed a strongly reduction of the LC3 (LC3 -I and LC3-II) fluorescence intensity in thymus and liver during age. Also Western blot showed in all three tissues that the band intensities of LC3-II decrease with the age of the aminals [15, 18]. In contrast, another study observed an age dependent increase of the LC3-II/LC3-I intensity ratio normalized by β-actin in skeletal muscle tissue of dogs [17]. The Western blot investigation of extraocular muscles of mice revealed a first increase followed by a decrease of LC3-I as well as from LC3-II intensity in older mice [14]. And finally, another study observed by Western blotting no marked change of the LC3-II, but of the LC3-I level in rat kidneys during aging, resulting in an increase of the LC3-II/LC3-I intensity ratio [20]. Although there is no definite knowledge, in which way autophagy changes with age, the results of these studies at least show that there may be alterations that might be interesting for forensic research.

We decided to analyze LC3-II in skeletal muscle tissue, because it is present in nearly all body parts and relatively stable to putrefaction processes. Kimura et al. have tested the effects of post mortem putrefaction on the LC3-II/LC3-I and LC3-II/control protein intensity ratio in wounds of mice and found no relevant changes for several days [21]. This finding also indicated that the detection of LC3-II as a marker of autophagic activity for post mortem age estimation might be feasible.

We were able to extract and measure LC3-II from post mortem drawn muscle samples. However, all calculated intensity ratios of LC3-II, as an indicator for autophagic activity, showed no satisfying correlation with the donors’ age. The highest correlation was found for the in the intensity ratio of LC3-II/LC3-I with a correlation coefficient of r = 0,328 and p = 0,076. The ratios LC3-II/β-actin and LC3-II/total protein resulted in an even lower correlation. With a view to the detailed results of the LC3-II/LC3-I ratio it becomes obvious, that there is a high variation of the results in the pre-determined age groups.

A reason for such differences could be that autophagic activity not only depends on the age of a person but also on other influences. One relevant influence could be the personal fitness and sport activity of a person. Elderly persons are often reduced in training leading to less strength and muscular atrophy (sarcopenia) [22]. But not only life time activity might play a role, also momentary exercise levels might have an impact. Two studies have investigated the effect of acute [23] and chronic resistance exercise on autophagy [24]. Acute exercise resulted in a decrease of the LC3-II/LC3-I intensity ratio in muscles of young and older human participants [23]. Also the tests of chronic resistance exercise of rats showed a significant lower LC3-II/LC3-I intensity ratio compared to that in untrained rat muscles [24]. This might be one explanation for the wide differences in each age interval.

Also nutrition habits or fasting could be a reason for different results. Ogata et al. showed that the Western blot intensity of LC3-II rises in rat muscles after fasting times [25]. This might also account for humans and therefore might alter autophagic activity as well. It could be one explanation for one of the upwards outliers in figure 1 a and b, belonging to a 72 year old female person who was underweighted. Perhaps that is one reason for the high LC3-II intensity ratios in both diagrams. Moreover, several studies indicated that autophagy can be affected by diseases such as

neurodegeneration, infections and cancer [26]. By immunohistochemical staining, Avalos et al. showed by immunohistochemical staining that an increase of the LC3 intensity could already be observed in human pancreas cancer [27]. In figure 1 a and c, a 43 year old woman also resulted in upward outliers. Medical prehistory of the woman reported on breast cancer in the past. Perhaps the fact itself or therapeutic therapies could have led to such high LC3-II/LC3-I and LC3-II/total protein ratios. Since there is a high chance that deceased persons have been affected by some kind of disease, whether a chronic one like atherosclerosis or an acute one like an infection, we cannot rule out that this also alters the results of the calculated, autophagic activity.

Overall, our results show clearly, that the post mortem evaluation of autophagic activity by measuring LC3-II ratios in skeleton muscle is not suitable as a method for age estimation.

Interestingly, as a side finding, the normalization tests for β-actin revealed that there is a decrease of the amount of this structural protein in muscle tissue, obviously correlating with the age of the tissue donor. Our results revealed a weak but significant relation (r = 0,463, p = 0,03) between β-actin/total protein intensity ratio and the donor’s age. A study of Vigelso et al. revealed similar results. They tested the reliability of β-actin as a normalization protein in human skeletal muscles and also observed a decrease of β-actin with aging. Here the sole β-actin amount of five young (23 ±1 years) and five older (66 ± 2 years) men were measured by Wester blotting and resulted in an intensity decrease with a p < 0,05 results [28]. Although those findings indicate a relationship between β-actin and age, it is obviously not yet close enough for age estimation in forensic case work. Further studies might be needed to verify this hypothesis.

Conclusion:

Collectively, our study revealed that LC3-II can be extracted from post-mortem drawn human muscle tissue and protein intensity ratios representing autophagic activities can be calculated. However we detected no clear increase or decrease with the age of the tissue donors. Thus, the determination of LC3-II and autophagic activities are not suitable for forensic age-estimation. As a side finding, by testing the normalization ability of β-actin, a decrease of the measured amount showing a weak correlation with the age of the donor was observed. Further studies should be carried out to find more about a possible use of this phenomenon as a method for age estimation.

r = 0,154 p = 0,416 -1 2 3 4 5 6 7 0 50 100 LC3 -II /wh o le p ro te in in te n si ty ratio (*1 0^ 6) Age (years)

LC3-II/total protein ratio related

to age

Figures:

Fig. 1: Calculated intensity ratios representing autophagic activity. The graph shows the measured

intensity ratios of LC3-II to LC3-I (a), LC3-II to β-actin (b) and LC3-II to the total protein amount (c) plotted against the age of the donors. Both sexes are included. r = 0,328 p = 0,076 -5,00 10,00 15,00 20,00 25,00 30,00 35,00 40,00 0 50 100 LC3 -II /LC3 -I in te n si ty ratio (*1 0^ 6) Age (years)

LC3-II/LC3-I intensity ratio related

to age

LC3-II/β-actin intensity ratio

related to age

b

a

Fig. 2: Means, minimum and maximum LC3-II/LC3-I intensity ratios per age interval. Each bar represents

the mean of one age interval including 10 years. The stripes show the lowest and highest LC3-II/LC3-I intensity ratio of each age interval. Both sexes are taken into account. The intervals are expressed in years.

-5,00 10,00 15,00 20,00 25,00 30,00 35,00 40,00 Age intervals M e an LC3 -II /LC3 -I in te n si ty ratios (*1 0^ 6)

LC3-II/LC3-I intensity ratios in pre-determined age intervals

20-29 30-39 40-49 50-59 60-69 70-79 80-89

Fig. 3: Variability of LC3II/LC3-I-ratio. a Lane 1-5 were filled with the same amount of one sample. The

picture illustrates a chemiluminescence blot for β-actin, LC3-I and LC3-II on the left side and total protein staining by Amido black on the right side. b shows the calculated ratios of LC3-II and LC3-I; The standard deviation was 11%. c shows the variability of the LC3II/LC3-I intensity ratio of one sample analyzed on two membranes and of two samples from one muscle. y. = years.

0 5 10 15 20 25 30 35 40 LC3 -II /LC3 -I in te n si ty ratios (*1 0^ 6) Lane 1 2 3 4 5

LC3-II/-I Ratios on one membrane b -5 10 15 20 25 30 35 40

Variability 2 gels, 55 y. Biopsy variability, 81 y.

R at io m e ans LC3 -I I/LC 3 -I inten sit y ra ti o s ( *1 0 ^6 )

Variability on two membranes and two autopsy samples

29 %

9 % c

Fig.4: β-actin/total protein intensity ratio. The graphs shows the calculated intensity ratios of β-actin to total

protein compared to the age of the donors. Both sexes are taken into account.

r = 0,463, p = 0,03 0,00 0,05 0,10 0,15 0,20 0,25 0 20 40 60 80 100 β -ac tin /wh o le p ro te in r atio Age (years)

Appendix:

Development of suitable methods

The first aim of this project was to detect both forms of LC3 (LC3-I and LC3-II), in post mortem drawn muscle samples by establishing a suitable method to a) extract proteins out of post-mortem drawn human muscle samples and b) to detect LC3-I and -II by Western blotting.

Development of the right protein isolation method

The aim of the proteome isolation was to extract the total proteins out of post-mortem drawn, human skeleton muscle tissue. Therefore, two different isolation protocols were tested. The trichloroacetic acid (TCA)/acetone isolation is a standard proceeding in protein extraction. This protocol consisted of homogenization by ultrasonic treatment, a precipitation of the proteome by the addition of TCA and acetone and centrifugation. The second protocol was based on a methodology of Anderson and Davison [29]. In contrast to the TCA/acetone protocol it did not include a precipitation step. Instead, the muscle tissue was homogenized by a dispersing machine, exposed to heat and centrifuged.

The best results were obtained with the Anderson and Davison method. In contrast to the other method, clear bands of LC3-I and LC3-II could be seen in the consecutive Western blotting (figure 5 b). Proteome isolation with the TCA/acetone protocol, on the other hand, only presented a single LC3-I band in Western blots (figure 5 a). Due to these results the methodology of Anderson and Davison was chosen for the final method of the research project.

a b

Fig. 5: Western blotting analysis of LC3-I and LC3-II in human muscle tissues. The proteomes were

isolated by TCA/acetone precipitation (a) or the method of Anderson and Davison (b) before.

Western blot and detection of LC3-II

To optimize the Western blotting procedure, two different blotting types were tested: Semi-Dry and Tank Transfer Blotting. After Semi-Dry blotting the protein patterns were very blurred and showed vague edges. The results of Tank Transfer Blotting presented much clearer band patterns. For this reason, the latter blotting type was selected for further tests.

Linearity testing:

To ensure Western blot quantification is not over- or underloaded, serial amounts of one muscle sample (female, 68 years old) were blotted (Fig.6). For whole protein staining, two different box sizes were tested: one thin strip in the middle of the lane and one covering its whole broadness (figure 6 A). These two sizes were chosen in previous tests. The bands of β-actin, LC3-I and LC3-II are shown in Fig. B, C and D. Each protein’s band size raze until an amount of 60 µg, or of 70 µg for LC3-II, respectively. At 80 µg all band sizes dropped down again, which could be due to pipetting mistakes or blotting inaccuracy. These optical results also resemble the intensity measurements in figure 6 E. The percentage values of band intensities for each amount of protein are demonstrated here. LC3-I and –II showed a nearly consistent graph-course as well as both Amido black intensities. The intensities of those four measurements rose until they reached the 100% at a volume of 70 µg. In contrast, the 100% maximum of β-actin was already reached at 60 µg followed by a slightly lower value at 70 µg (>95%).

Because no other protein showed its maximum already at 60 µg, β-actin could be saturated from this point on. For the experiments a volume containing 50 µg protein was used for each sample. So it could be prevented that LC3-II intensities are underloaded, and those of β-actin exceed the point of saturation.

Fig. 6: Linearity of the different Western measurements used in this study. Different amounts of one protein

sample were blotted, labeled with specific antibodies, and afterwards stained with Amido black. The last lane represents the Ht cells. A: Amido black was measured in two boxes with different broadness. The blot was labelled with anti-β-actin antibodies and anti-LC3B antibodies. B represents β-actin bands, C LC3-I bands, and

D LC3-II bands. Their boxes for intensity measurements had the same size as the corresponding background

boxes which were placed on empty areas below or next to the bands/lanes. E: The graphs demonstrate the measured intensities as a percentage of their maximum (%) per protein amount (µg).

0 20 40 60 80 100 30 40 50 60 70 80 Pr o ce n tu al v al u e s o f b an d in te n si ties Amount of protein (µg) LC3-I LC3-II

ß-Actin Amido black thin

Amido black thick E

Literature

1. Schmeling, A., et al., Age estimation. Forensic Sci Int, 2007. 165(2-3): p. 178-81.

2. Ritz-Timme, S., et al., Age estimation: the state of the art in relation to the specific demands

of forensic practise. Int J Legal Med, 2000. 113(3): p. 129-36.

3. Meissner, C. and S. Ritz-Timme, Molecular pathology and age estimation. Forensic Sci Int, 2010. 203(1-3): p. 34-43.

4. Cunha, E., et al., The problem of aging human remains and living individuals: a review. Forensic Sci Int, 2009. 193(1-3): p. 1-13.

5. Cuervo, A.M., et al., Autophagy and aging: the importance of maintaining "clean" cells. Autophagy, 2005. 1(3): p. 131-40.

6. Martinez-Lopez, N., D. Athonvarangkul, and R. Singh, Autophagy and aging. Adv Exp Med Biol, 2015. 847: p. 73-87.

7. Demirovic, D., C. Nizard, and S.I. Rattan, Basal level of autophagy is increased in aging human

skin fibroblasts in vitro, but not in old skin. PLoS One, 2015. 10(5): p. e0126546.

8. Tashiro, K., et al., Age-related disruption of autophagy in dermal fibroblasts modulates

extracellular matrix components. Biochem Biophys Res Commun, 2014. 443(1): p. 167-72.

9. Wohlgemuth, S.E., et al., Autophagy in the heart and liver during normal aging and calorie

restriction. Rejuvenation Res, 2007. 10(3): p. 281-92.

10. Reggiori, F. and D.J. Klionsky, Autophagy in the eukaryotic cell. Eukaryot Cell, 2002. 1(1): p. 11-21.

11. Barth, S., D. Glick, and K.F. Macleod, Autophagy: assays and artifacts. J Pathol, 2010. 221(2): p. 117-24.

12. Wang, W., et al., The carboxyl-terminal amino acids render pro-human LC3B migration similar

to lipidated LC3B in SDS-PAGE. PLoS One, 2013. 8(9): p. e74222.

13. Sakuma, K., et al., p62/SQSTM1 but not LC3 is accumulated in sarcopenic muscle of mice. J Cachexia Sarcopenia Muscle, 2016. 7(2): p. 204-12.

14. McMullen, C.A., et al., Age-related changes of cell death pathways in rat extraocular muscle. Exp Gerontol, 2009. 44(6-7): p. 420-5.

15. Uddin, M.N., et al., Autophagic activity in thymus and liver during aging. Age (Dordr), 2012. 34(1): p. 75-85.

16. Wenz, T., et al., Increased muscle PGC-1alpha expression protects from sarcopenia and

metabolic disease during aging. Proc Natl Acad Sci U S A, 2009. 106(48): p. 20405-10.

17. Pagano, T.B., et al., Age related skeletal muscle atrophy and upregulation of autophagy in

dogs. Vet J, 2015. 206(1): p. 54-60.

18. Taneike, M., et al., Inhibition of autophagy in the heart induces age-related cardiomyopathy. Autophagy, 2010. 6(5): p. 600-6.

19. Liu, Y., et al., Impaired autophagic function in rat islets with aging. Age (Dordr), 2013. 35(5): p. 1531-44.

20. Cui, J., et al., Age-related changes in the function of autophagy in rat kidneys. Age (Dordr), 2012. 34(2): p. 329-39.

21. Kimura, A., et al., Autophagy in skin wounds: a novel marker for vital reactions. Int J Legal Med, 2015. 129(3): p. 537-41.

22. Ogborn, D.I., et al., Effects of age and unaccustomed resistance exercise on mitochondrial

transcript and protein abundance in skeletal muscle of men. Am J Physiol Regul Integr Comp

Physiol, 2015. 308(8): p. R734-41.

23. Fry, C.S., et al., Skeletal muscle autophagy and protein breakdown following resistance

exercise are similar in younger and older adults. J Gerontol A Biol Sci Med Sci, 2013. 68(5): p.

24. Luo, L., et al., Chronic resistance training activates autophagy and reduces apoptosis of

muscle cells by modulating IGF-1 and its receptors, Akt/mTOR and Akt/FOXO3a signaling in aged rats. Exp Gerontol, 2013. 48(4): p. 427-36.

25. Ogata, T., et al., Fasting-related autophagic response in slow- and fast-twitch skeletal muscle. Biochem Biophys Res Commun, 2010. 394(1): p. 136-40.

26. Arroyo, D.S., et al., Autophagy in inflammation, infection, neurodegeneration and cancer. Int Immunopharmacol, 2014. 18(1): p. 55-65.

27. Fujii, S., et al., Autophagy is activated in pancreatic cancer cells and correlates with poor

patient outcome. Cancer Sci, 2008. 99(9): p. 1813-9.

28. Vigelso, A., et al., GAPDH and beta-actin protein decreases with aging, making Stain-Free

technology a superior loading control in Western blotting of human skeletal muscle. J Appl

Physiol (1985), 2015. 118(3): p. 386-94.

29. Anderson, L.V. and K. Davison, Multiplex Western blotting system for the analysis of muscular