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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 Tet2 WT -CCl 4 and Tet2 ΔMye -CCl 4 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 Tet2 WT -CCl 4 and Tet2 ΔMye -CCl 4 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 <t>Ybx1</t> 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 Tet2 WT -CCl 4 and Tet2 ΔMye -CCl 4 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 Tet2 WT -CCl 4 and Tet2 ΔMye -CCl 4 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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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 Tet2 WT -CCl 4 and Tet2 ΔMye -CCl 4 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 Tet2 WT -CCl 4 and Tet2 ΔMye -CCl 4 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 <t>Ybx1</t> 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 Tet2 WT -CCl 4 and Tet2 ΔMye -CCl 4 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 Tet2 WT -CCl 4 and Tet2 ΔMye -CCl 4 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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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 Tet2 WT -CCl 4 and Tet2 ΔMye -CCl 4 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 Tet2 WT -CCl 4 and Tet2 ΔMye -CCl 4 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 <t>Ybx1</t> 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 Tet2 WT -CCl 4 and Tet2 ΔMye -CCl 4 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 Tet2 WT -CCl 4 and Tet2 ΔMye -CCl 4 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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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 Tet2 WT -CCl 4 and Tet2 ΔMye -CCl 4 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 Tet2 WT -CCl 4 and Tet2 ΔMye -CCl 4 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 <t>Ybx1</t> 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 Tet2 WT -CCl 4 and Tet2 ΔMye -CCl 4 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 Tet2 WT -CCl 4 and Tet2 ΔMye -CCl 4 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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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 Tet2 WT -CCl 4 and Tet2 ΔMye -CCl 4 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 Tet2 WT -CCl 4 and Tet2 ΔMye -CCl 4 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 <t>Ybx1</t> 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 Tet2 WT -CCl 4 and Tet2 ΔMye -CCl 4 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 Tet2 WT -CCl 4 and Tet2 ΔMye -CCl 4 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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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 Tet2 WT -CCl 4 and Tet2 ΔMye -CCl 4 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 Tet2 WT -CCl 4 and Tet2 ΔMye -CCl 4 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 <t>Ybx1</t> 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 Tet2 WT -CCl 4 and Tet2 ΔMye -CCl 4 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 Tet2 WT -CCl 4 and Tet2 ΔMye -CCl 4 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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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 Tet2 WT -CCl 4 and Tet2 ΔMye -CCl 4 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 Tet2 WT -CCl 4 and Tet2 ΔMye -CCl 4 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 <t>Ybx1</t> 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 Tet2 WT -CCl 4 and Tet2 ΔMye -CCl 4 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 Tet2 WT -CCl 4 and Tet2 ΔMye -CCl 4 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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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 Tet2 WT -CCl 4 and Tet2 ΔMye -CCl 4 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 Tet2 WT -CCl 4 and Tet2 ΔMye -CCl 4 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 Tet2 WT -CCl 4 and Tet2 ΔMye -CCl 4 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 Tet2 WT -CCl 4 and Tet2 ΔMye -CCl 4 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).

Journal: The Journal of Experimental Medicine

Article Title: Tet2 deficiency–induced expansion of monocyte-derived macrophages promotes liver fibrosis

doi: 10.1084/jem.20251114

Figure Lengend Snippet: 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 Tet2 WT -CCl 4 and Tet2 ΔMye -CCl 4 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 Tet2 WT -CCl 4 and Tet2 ΔMye -CCl 4 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 Tet2 WT -CCl 4 and Tet2 ΔMye -CCl 4 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 Tet2 WT -CCl 4 and Tet2 ΔMye -CCl 4 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).

Article Snippet: Captured proteins were analyzed by western blot using Abs against Ybx1 (11910-1-AP; Proteintech), Elavl1 (20339-1-AP; Proteintech), and Zfp36 (12737-1-AP; Proteintech).

Techniques: Neutralization, Expressing, Isolation, Cell Culture, Reverse Transcription Polymerase Chain Reaction, In Vitro, Recombinant, Flow Cytometry, Staining, Two Tailed Test, Comparison

Tet2 deficiency enhances Ccl2 and Ccl8 mRNA stability by modifying 5hmC-dependent RNA – protein interactions. (A and B) Ccl2 (A) and Ccl8 (B) mRNA decay in Tet2 +/+ and Tet2 −/− MDMs ( n = 6 for each group). (C) Tet2 -mediated oxidation of Ccl2 and Ccl8 mRNA 5mC disrupts its binding with Ybx1, Elavl1, and Zfp36. Pull-down assay was performed by incubating C, 5mC, and 5hmC oligos of Ccl2 and Ccl8 mRNA with cell lysate from THP1-derived pMDMs ( n = 3 for each group). (D) Effect of Tet2 deficiency on the binding enrichment of Ybx1, Elavl1, and Zfp36 at 3′UTR of Ccl2 and Ccl8 mRNA. Tet2-binding sites were mapped in the mRNA of Ccl2 and Ccl8 by qPCR of Ybx1, Elavl1, and Zfp36 RIP product in THP1-derived pMDMs ( n = 3 for each group). (E and F) Effect of enzymatic inactivation of Tet2 via catalytic domain mutation on stabilization of Ccl2 (F) and Ccl8 (G) transcripts ( n = 4 for each group). Data are the accumulative results from at least two independent experiments (A, B, D, E, and F) or are representative of at least two independent experiments with similar results (C and D). All data are shown as mean ± SD and were analyzed by two-tailed, unpaired Student’s t test (A, B, and D–F). ***P < 0.001; **P < 0.01; *P < 0.05; P > 0.05 not significant (ns). Source data are available for this figure: .

Journal: The Journal of Experimental Medicine

Article Title: Tet2 deficiency–induced expansion of monocyte-derived macrophages promotes liver fibrosis

doi: 10.1084/jem.20251114

Figure Lengend Snippet: Tet2 deficiency enhances Ccl2 and Ccl8 mRNA stability by modifying 5hmC-dependent RNA – protein interactions. (A and B) Ccl2 (A) and Ccl8 (B) mRNA decay in Tet2 +/+ and Tet2 −/− MDMs ( n = 6 for each group). (C) Tet2 -mediated oxidation of Ccl2 and Ccl8 mRNA 5mC disrupts its binding with Ybx1, Elavl1, and Zfp36. Pull-down assay was performed by incubating C, 5mC, and 5hmC oligos of Ccl2 and Ccl8 mRNA with cell lysate from THP1-derived pMDMs ( n = 3 for each group). (D) Effect of Tet2 deficiency on the binding enrichment of Ybx1, Elavl1, and Zfp36 at 3′UTR of Ccl2 and Ccl8 mRNA. Tet2-binding sites were mapped in the mRNA of Ccl2 and Ccl8 by qPCR of Ybx1, Elavl1, and Zfp36 RIP product in THP1-derived pMDMs ( n = 3 for each group). (E and F) Effect of enzymatic inactivation of Tet2 via catalytic domain mutation on stabilization of Ccl2 (F) and Ccl8 (G) transcripts ( n = 4 for each group). Data are the accumulative results from at least two independent experiments (A, B, D, E, and F) or are representative of at least two independent experiments with similar results (C and D). All data are shown as mean ± SD and were analyzed by two-tailed, unpaired Student’s t test (A, B, and D–F). ***P < 0.001; **P < 0.01; *P < 0.05; P > 0.05 not significant (ns). Source data are available for this figure: .

Article Snippet: Captured proteins were analyzed by western blot using Abs against Ybx1 (11910-1-AP; Proteintech), Elavl1 (20339-1-AP; Proteintech), and Zfp36 (12737-1-AP; Proteintech).

Techniques: Binding Assay, Pull Down Assay, Derivative Assay, Mutagenesis, Two Tailed Test