Figure S4.
Effect of Tet2 deficiency on Ccl2 and Ccl8 mRNA stability and IL-6 neutralization on liver fibrosis progression. (A) The frequency of Ccr2+ and Ccr3+ pMDMs in Tet2WT-CCl4 and Tet2ΔMye-CCl4 mice (n = 4 for each group). (B) Expression of Ccr2 and Ccr3 on monocytes, Tet2+/+ pMDMs, and Tet2−/− pMDMs (n = 4 for each group). (C) The infiltration difference of other Ccr2 or Ccr3 expressing cell types in Tet2WT-CCl4 and Tet2ΔMye-CCl4 mice (n = 4 for each group). (D) GAPDH mRNA decay curve in Tet2+/+ pMDMs and Tet2−/− pMDMs (n = 6 for each group). (E) Transcriptional level of Elavl1, Znf36, and Ybx1 in Tet2+/+ pMDMs and Tet2−/− pMDMs (n = 4 for each group). (F–I) Serum levels of (F) IL-1α, (G) IL-2, (H) TNFα, and (I) IL-1β in livers of Tet2WT-CCl4 and Tet2ΔMye-CCl4 mice (n = 5 for each group). (J) Serum IL-6 levels in scramble, WT-MT, and KO-MT mice (n = 4 for each group). (K) Il-6 levels in CD45.2+ pMDMs isolated from livers of WT-MT and KO-MT mice (n = 4). (L) mRNA levels of Acta2 and Col1a1 in Tet2+/+and Tet2−/− HSCs (n = 4 for each group). (M and N) Effect of anti–IL-6 Abs treatment on mRNA levels of Col1a1 (M) and Acta2 (N) in Tet2+/+and Tet2−/− HSCs co-cultured with Tet2+/+or Tet2−/− MDMs detected by RT-PCR in vitro (n = 3 for each group). (O) Effect on recombinant Ccl2 and Ccl8 on Il-6 expression in Tet2+/+ pMDMs and Tet2−/− pMDMs (n = 3 for each group). (P) Detection of MDMs in livers by flow cytometry after Bindarit or IL-6 Abs treatment for 2 wk (n = 5 for each group). (Q) H&E staining of liver tissues treated with PBS, Bindarit, IL-6 Abs, or Bindarit plus IL-6 Abs in Tet2WT-CCl4 and Tet2ΔMye-CCl4 mice (n = 4 for each group). Data are representative of at least two independent experiments with similar results (A–P). All data are shown as mean ± SD and were analyzed by two-tailed, unpaired Student’s t test (A, C, D, E, K, L, and O) or one-way ANOVA with Tukey’s multiple comparison test (B and J) or two-way ANOVA with Sidak’s multiple comparison test (F–I, M, and N). ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; P > 0.05 not significant (ns).

Effect of Tet2 deficiency on Ccl2 and Ccl8 mRNA stability and IL-6 neutralization on liver fibrosis progression. (A) The frequency of Ccr2+ and Ccr3+ pMDMs in Tet2WT-CCl4 and Tet2ΔMye-CCl4 mice (n = 4 for each group). (B) Expression of Ccr2 and Ccr3 on monocytes, Tet2+/+ pMDMs, and Tet2−/− pMDMs (n = 4 for each group). (C) The infiltration difference of other Ccr2 or Ccr3 expressing cell types in Tet2WT-CCl4 and Tet2ΔMye-CCl4 mice (n = 4 for each group). (D) GAPDH mRNA decay curve in Tet2+/+ pMDMs and Tet2−/− pMDMs (n = 6 for each group). (E) Transcriptional level of Elavl1, Znf36, and Ybx1 in Tet2+/+ pMDMs and Tet2−/− pMDMs (n = 4 for each group). (F–I) Serum levels of (F) IL-1α, (G) IL-2, (H) TNFα, and (I) IL-1β in livers of Tet2WT-CCl4 and Tet2ΔMye-CCl4 mice (n = 5 for each group). (J) Serum IL-6 levels in scramble, WT-MT, and KO-MT mice (n = 4 for each group). (K) Il-6 levels in CD45.2+ pMDMs isolated from livers of WT-MT and KO-MT mice (n = 4). (L) mRNA levels of Acta2 and Col1a1 in Tet2+/+and Tet2−/− HSCs (n = 4 for each group). (M and N) Effect of anti–IL-6 Abs treatment on mRNA levels of Col1a1 (M) and Acta2 (N) in Tet2+/+and Tet2−/− HSCs co-cultured with Tet2+/+or Tet2−/− MDMs detected by RT-PCR in vitro (n = 3 for each group). (O) Effect on recombinant Ccl2 and Ccl8 on Il-6 expression in Tet2+/+ pMDMs and Tet2−/− pMDMs (n = 3 for each group). (P) Detection of MDMs in livers by flow cytometry after Bindarit or IL-6 Abs treatment for 2 wk (n = 5 for each group). (Q) H&E staining of liver tissues treated with PBS, Bindarit, IL-6 Abs, or Bindarit plus IL-6 Abs in Tet2WT-CCl4 and Tet2ΔMye-CCl4 mice (n = 4 for each group). Data are representative of at least two independent experiments with similar results (A–P). All data are shown as mean ± SD and were analyzed by two-tailed, unpaired Student’s t test (A, C, D, E, K, L, and O) or one-way ANOVA with Tukey’s multiple comparison test (B and J) or two-way ANOVA with Sidak’s multiple comparison test (F–I, M, and N). ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; P > 0.05 not significant (ns).

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