Fig 2: Extended information on the role hepatocyte caspase-8-induced meteorin in HSC activation, related to Fig. 4.a, THLE2 cells were treated with scrambled RNA (Scr) or siMETRN. After 24 h, the media were changed to DMEM/0.1% FBS, and after an additional 24 h, the THLE2 cells were assayed for METRN mRNA and meteorin protein. Data are presented as mean ± SEM; ***p=0.0004, unpaired two-tailed t-test; n=3 per group. b, Upper, AML12 cells were treated 24 h with ZVAD or ITED, and Metrn mRNA was measured. Lower, AML12 cells were treated for 24 h with or without 5 mg/ml Jo2 to induce apoptosis in the presence of vehicle (DMSO) or 20 mm ZVAD or 20 mm ITED. The cleaved forms of caspase-3 (cl-Casp3) and caspase-8 (cl-Casp8), which are a measure of their activity in apoptosis, were assayed by immunoblot. Data are presented as mean ± SEM; n.s., no significant difference, unpaired two-tailed t-test; n=3 per group. c, Scr- or siCASP8-treated primary human hepatocytes were transfected with constructs expressing GFP control or DEDs. After 48 h, the cells were assayed for mRNAs encoding METRN and DED domains. Data are presented as mean ± SEM; *p=0.022, ***p=0.0007, ****p<0.0001, one-way ANOVA; n=3 per group. d, Immunoblot of caspase-10 (CASP10) in normal and MASH human livers, with β-actin as loading control. *p=0.029, unpaired two-tailed t-test; n=3 per group. e, Immunoblot and quantification of caspase-10 in primary human hepatocytes from normal and MASH livers. Data are presented as mean ± SEM; *p=0.026, unpaired two-tailed t-test; n=3. f, CASP10 and METRN mRNA in primary human hepatocytes treated with scrambled RNA (Scr) or siCASP10. Data are presented as mean ± SEM; *p=0.019, unpaired two-tailed t-test; n=4 per group. g, Primary mouse HSCs were transfected with scrambled RNA (Scr), siKit, or siStat3. After 48 h, Kit and Stat3 mRNA were measured. Data are presented as mean ± SEM; *p=0.024 (kit) *p=0.029 (Stat3), unpaired two-tailed t-test; left, n=3 per group, right, n=4 per group. h, Hepatocyte nuclear extracts from AML12 cells treated with scrambled RNA (Scr) or siCasp8 were subjected to Pol-II ChIP using anti-Pol-II or IgG control. The Metrn promoter region sequence was amplified by qPCR and normalized to the values obtained from input DNA. Data are presented as mean ± SEM; ***p=0.0003, one-way ANOVA; n=4 per group. Source data

Fig 3: Extended information on hepatocyte caspase-8-induced HSC activation, related to Fig. 3.a, Immunoblot of caspase-8 in AML12 cells treated with scrambled RNA (Scr) or siCasp8. b, CASP8 mRNA in THLE2 cells treated with scrambled RNA (Scr) or siCASP8. Data are presented as mean ± SEM; ***p=0.0002, unpaired two-tailed t-test; n=6 per group. c, THLE2 human HCs were transfected with scrambled RNA (Scr) or siCasp8. After 24 h, the media were changed to DMEM/0.1% FBS, and after an additional 24 h, the conditioned media from the cells was transferred to quiescent primary mouse HSCs. After 72 h, the HSCs were assayed for Timp1, Spp1, and Col1a1 mRNA. Data are presented as mean ± SEM; *p=0.017, **p=0.0066, two-way ANOVA; n=4 per group. d, Control or heat-inactivated (HI; 100 °C x 5 min) CM from Scr- or siCasp8-treated AML12 HCs was added to quiescent primary mouse HSCs as in panel a. After 72 h, the HSCs were assayed for the indicated mRNAs. Data are presented as mean ± SEM; ***p=0.0004, ****p<0.0001, n.s., non-significant, two-way ANOVA; n=4 per group. e, Left, CASP8 mRNA in primary human hepatocytes treated with scrambled RNA (Scr) or siCASP8. Data are presented as mean ± SEM; ****p<0.0001, unpaired two-tailed t-test; n=4 per group. Right, Primary human hepatocytes (HCs) were transfected with scrambled RNA (Scr) or siCasp8. After 24 h, the media were changed to DMEM/0.1% FBS, and after an additional 24 h, the conditioned media (CM) were transferred to human primary HSCs. After 48 h, the HSCs were assayed for TIMP1 and SPP1 mRNA. Data are presented as mean ± SEM; *p=0.024 (TIMP1), *p=0.023 (SPP1: grey vs. white), *p=0.016 (SPP1: hash vs. grey), **p = 0.0020, two-way ANOVA; n=4 per group. f, Human primary hepatic 3D spheroids treated with scrambled RNA (Scr) or siCASP8 (n=3 per group). Left, CASP8 mRNA. Data are presented as mean ± SEM; *p=0.036, unpaired two-tailed t-test. Right, The indicated mRNAs were assayed, Data are presented as mean ± SEM; *p=0.025 (SPP1), *p=0.012 (COL1A1), *p=0.049 (COL3A1), two-way ANOVA. g, AML12 mouse hepatocytes cultured in DMEM/0.1% FBS were treated ± IETD. After 24 h, the conditioned media (CM) were transferred to quiescent primary mouse HSCs, with one set of control HSCs receiving no CM (HSC only). After 72 h, the HSCs were assayed for Timp1 and Col1a1 mRNA. Data are presented as mean ± SEM; n.s., non-significant, one-way ANOVA; n=4 per group. h, Scr- or siCasp8-treated AML12 cells were transfected with GFP control, wild-type Casp8, or C360A-mutant Casp8. After 24 h, the media were changed to DMEM/0.1% FBS, and after another 24 h, the CM from the hepatocytes were transferred to quiescent primary mouse HSCs. After 72 h, the HSCs were assayed for Timp1 and Col1a1 mRNA. Data are presented as mean ± SEM; *p=0.012 (Timp1), **p=0.0055 (Timp1), *p=0.011 (Col1a1: GFP vs. CASP8), *p=0.033 (Col1a1: GFP vs. C360A), one-way ANOVA; n=4 per group. i, Scr- or siCASP8-treated THLE2 cells were assayed for CASP8 and METRN mRNA and for caspase-8 and meteorin protein. Data are presented as mean ± SEM; **p=0.0015 (CASP8 and METRN), unpaired two-tailed t-test; n=6 per group. Source data