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The interplay of the Notch signaling in hepatic stellate cells and macrophages determines the fate of liver fibrogenesis

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The interplay of the Notch signaling in hepatic stellate cells and macrophages determines the fate of liver fibrogenesis

Ruchi Bansal1*; Joop van Baarlen2; Gert Storm1,3; Jai Prakash1

Supplementary Table 1: Antibodies used for the immunohistochemistry

Primary Antibody Source Dilution

Polyclonal rabbit α-SMA Abcam 1:100

Polyclonal goat anti-collagen I Southern Biotech 1:100

Polyclonal goat anti-desmin Santa Cruz 1:100

Polyclonal rat anti-F4/80 (MCA497) AbD Serotec 1:100

Polyclonal goat anti-vimentin (sc-7557) Santa Cruz 1:100 Polyclonal rabbit anti-Notch1 (NBP1-78292) Novus Biologicals 1:100 Polyclonal rabbit anti-Notch3 (M134, sc-5593) Santa Cruz 1:100 Monoclonal rat MHC-II (ER-TR3, sc-59318) Santa Cruz 1:100 Polyclonal goat anti-chitinase 3-like 3/ECF-L (YM1) R & D systems 1:100

Secondary Antibody Source Dilution

Polyclonal Goat anti-rabbit IgG DAKO 1:100

Polyclonal Rabbit anti-rat IgG DAKO 1:100

(2)

Supplementary Table 2: Sequence of the mouse primers used for quantitative real-time PCR

Supplementary Table 3: Sequence of the human primers used for quantitative real-time PCR Gene Forward primer sequence (5’-3’) Reverse primer sequence (5’-3’) Arginase I GTGAAGAACCCACGGTCTGT CTGGTTGTCAGGGGAGTGTT Collagen I TGACTGGAAGAGCGGAGAGT ATCCATCGGTCATGCTCTCT

Desmin ATGCAGCCACTCTAGCTCGT CTCATACTGAGCCCGGATGT

Dll1 TCTAGAACACTCTGGGAGCGG CTATTAGGAAGCGGCGGTCT

Dll4 TATACCTGCACCTGTCTCCCA TTACAGCTGCCACCATTTCG

GAPDH ACAGTCCATGCCATCACTGC GATCCACGACGGACACATTG

Hes1 GCGGAATCCCCTGTCTACC GTCTTAGGGCTACTTAGTGATCG

IL-1β GCCAAGACAGGTCGCTCAGGG CCCCCACACGTTGACAGCTAGG

IL-6 TGATGCTGGTGACAACCACGGC TAAGCCTCCGACTTGTGAAGTGGTA

NOS2 GGTGAAGGGACTGAGCTGTT GCTACTCCGTGGAGTGAACAA

Jag1 ACTGGTGTGTAAGGAAGCGG CGCACATTGTTGGTGGTGTT

MRC1 GGGACGTTTCGGTGGACTGTGG TTGTGGGCTCTGGTGGGCGA

Notch1 TGTGGCTTCCTTCTACTGCG CTTTGCCGTTGACAGGGTTG

Notch2 AGCTTGGGCAGGTTACATCC TGGACATGTGCTTCCCTTCC

Notch3 GACTGCTCACTGAACGTGGA CACACCGGCTGTTGTTGAAG

SOX9 GTGCAAGCTGGCAAAGTTGA TGCTCAGTTCACCGATGTCC

Vimentin TCCAGAGAGAGGAAGCCGAA AAGGTCAAGACGTGCCAGAG

α-SMA ACTACTGCCGAGCGTGAGAT CCAATGAAAGATGGCTGGAA

Gene Forward primer sequence (5’-3’) Reverse primer sequence (5’-3’) Collagen I GTACTGGATTGACCCCAACC CGCCATACTCGAACTGGAAT

Desmin GCGGGTTTCGGCTCTAAGG AGAAACTCCTGGTTCACCGC

Dll1 GAGGCACTGTGACGACAACG GCACACTCGCACACATAGCG

Dll4 AAGGCTGCGCTACTCTTACC GTCATTGCGCTTCTTGCACA

GAPDH TCCAAAATCAAGTGGGGCGA TGATGACCCTTTTGGCTCCC

Hes1 CTGAGCACAGACCCAAGTGT GAGTGCGCACCTCGGTATTA

Jag1 TGAGGCCGTTGCTGACTTAG TGAGATGCGGCACTCGATTT

Notch1 GAATGGTCAATGCGAGTGGC CCGCAGAGGGTTGTATTGGT

Notch2 AGAATCAGCCCTGCCAGAAT CAATGCCCTGGATGGAAAATGG

Notch3 CATGGTATCTGCACCAACCT TTGATGTCCTGATCGCAGGAAG

Vimentin AAATGGCTCGTCACCTTCGT CAGCTTCCTGTAGGTGGCAA

(3)

Supplementary Table 4: Antibodies used for western blot analysis

Primary Antibody Source Dilution

Polyclonal rabbit anti-Notch1 (NBP1-78292) Novus Biologicals 1:250 Polyclonal rabbit anti-Notch3 (M134, sc-5593) Santa Cruz 1:250 Monclonal mouse β-actin antibody Sigma Aldrich 1:5000

Secondary Antibody Source Dilution

Polyclonal Goat anti-rabbit IgG DAKO 1:2000

Polyclonal goat anti-mouse IgG DAKO 1:1000

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