Fig. 3: UDCA induces p62-dependent autophagic degradation of TGF-β.

a–j IB analysis of TGF-β1 (a, c, d, g, h) and FC analysis of Foxp3⁺CD4⁺ T cells (b, e, f, i, j) in naïve CD4⁺ T cells treated with 0.25 μM SBI-115 (a, b), 5 μM MDL12330A (c, e), 2 μM H89 (d, f), or 10 μM IBMX (g, i) or in naïve CD4⁺ T cells with PRKACA overexpression (h, j) and treated with anti-CD3, anti-CD28, and 50 μM UDCA for 24 h (a, c, d, g, h) or for 4 days (b, e, f, i, j). k-n IB analysis of TGF-β1 (k, m) and statistical analysis of TGF-β1 stability (l, n) in naïve CD4⁺ T cells stimulated with anti-CD3, anti-CD28, 10 nM Baf-A1 (k, l) or 20 μM MG132 (m, n) for the indicated times. o-s IB analysis of TGF-β1 (o–q) and FC analysis of Foxp3⁺CD4⁺ T cells (r, s) in WT (o), Atg3-deficient or Atg5-deficient (p, q) naïve CD4⁺ T cells stimulated with anti-CD3, anti-CD28, or 50 μM UDCA with (o) or without (p, q) MG132 treatment for 24 h (o–q) or for 4 days (r, s). t Immunofluorescence analysis of LC3B and TGF-β1 in naïve CD4⁺ T cells stimulated with anti-CD3, anti-CD28, and 50 μM UDCA for 24 h. Scale bar, 2 μm. u Immunoelectron microscopy analysis of TGF-β1 localization in autophagosomes of HEK293T cells. N, nucleus; C, cytoplasm. Scale bar, 500 nm. v Immunofluorescence analysis of p62 and TGF-β1 in naïve CD4⁺ T cells stimulated with anti-CD3, anti-CD28, and 50 μM UDCA for 24 h. Scale bar, 2 μm. w IB analysis of p62 and TGF-β1 in lysates of HEK293T cells transfected with vectors expressing Flag-TGF-β1 and Myc-p62 with or without UDCA and 1 nM Baf-A1 treatment for 24 h. IB analysis was conducted after IP with anti-Flag. Representative results from two (t, v) or three (a-s, u, w) independent experiments are shown (n = 3 in b, e, f, i, j, l, n, r, s; n = 14 in t; n = 15 in v). *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant (unpaired two-tailed Student’s t test except for one-way ANOVA followed by Newman–Keuls test in (e, f, i); mean and s.d.). See Source Data file for the exact P-values.