Figure 1. TXNIP levels were lower in lung tissues of bleomycin-challenged mice. (A) Male C57/BL6J mice were intratracheally challenged with PBS or bleomycin (0.045 U/mouse). After 3 weeks, lung tissue lysates were analyzed by immunoblotting with HO-1, TXNIP, and β-actin antibodies. (B) Intensities of TXNIP and HO-1 were quantified by ImageJ software. n = 3–5. **p < 0.01 compared to PBS-treated mice. (C) Re-anaysis of TXNIP gene transcript in GDS4902 and GDS5078 databases. (D) Single cell RNAseq analysis of gene expression of TXNIP in lung cells from control (cont) and IPF patients (Kaminski/Rosas data base from ipfcellatlas.com). Red circle: alveolar epithelial cells; black circle: fibroblasts; blue circle: endothelial cells.

Figure 2. Overexpression of TXNIP attenuates TGF-β1-induced phosphorylation of Smad2/3 and FN expression in lung fibroblasts. (A) IMR90 cells were transfected with plasmid encoding HA-tagged TXNIP (TXNIP-HA) for 48 h, then treated with TGF-β1 (10 ng/mL) for 0.5 h. Cell lysates were analyzed by immunoblotting with phopho (p)-Smad2/3, Smad3, HA tag, and β-actin antibodies. (B) Intensities of p-Smad2/3 were quantified by ImageJ software and normalized to Smad2/3. n = 3. **p < 0.01 compared to TGF-β1 alone. (C) IMR90 cells were transfected with plasmid encoding HA-tagged TXNIP (TXNIP-HA) for 48 h, then treated with TGF-β1 (10 ng/mL) for 24 h. Cell lysates were analyzed by immunoblotting with FN and β-actin antibodies. (D) Intensities of FN were quantified by ImageJ software and normalized to β-actin. n = 3. *p < 0.05 compared to TGF-β1 alone. Representative blots from three independent experiments are shown.

Figure 3. TGF-β1 treatment lowers TXNIP protein levels, not mRNA levels. (A) IMR90 cells were treated with TGF-β1 (10 ng/mL) for 0–48 h. Cell lysates were analyzed by immunoblotting with FN, TXNIP, and β-actin antibodies. (B) Intensities of TXNIP were quantified by ImageJ software and normalized to β-actin. n = 3. ** p < 0.01 compared to 0 h. (C) IMR90 cells were treated with BMP4 (0–10 ng/mL) for 24 h. Cell lysates were analyzed by immunoblotting with TXNIP and β-actin antibodies. (D) Intensities of TXNIP were quantified by ImageJ software and normalized to β-actin. n = 3. n.s. p > 0.05 compared to 0 ng/mL. (E) IMR90 cells were treated with TGF-β1 (10 ng/mL) for 24 h, and total RNA was extracted. FN and TXNIP mRNA levels were measured by realtime PCR and normalized to GAPDH mRNA levels. n = 4. *p < 0.05, n.s. p > 0.05 compared to vehicle (Veh). (F) IMR90 cells were treated with CRM1 inhibitor III (CRM1i, 10 M) for 1 h prior to TGF-β1 (10 ng/mL) treatment for 6 h. Cell lysates were analyzed by immunoblotting with TXNIP and β-actin antibodies. G. Intensities of TXNIP were quantified by ImageJ software and normalized to β-actin. n = 3. n.s. p > 0.05 compared to TGF-β1 alone. Representative blots from three independent experiments are shown.

Figure 4. TGF-β1 induces TXNIP degradation in the ubiquitin-proteasome system. (A) IMR90 cells were treated with MG-132 (20 μM) or leupeptin (100 μM) for 1 h, and then challenged with TGF-β1 (10 ng/mL) for additional 6 h. Cell lysates were analyzed by immunoblotting with TXNIP and β-actin antibodies. (B) Intensities of TXNIP were quantified by ImageJ software and normalized to β-actin. n = 4. ** p < 0.01, n.s. p > 0.05 compared to TGF-β1 alone. (C) A549 cells were treated with MG-132 (20 μM) or leupeptin (100 μM) for 1 h, and then challenged with TGF-β1 (10 ng/mL) for additional 6 h. Cell lysates were analyzed by immunoblotting with TXNIP and β-actin antibodies. (D) Intensities of TXNIP were quantified by ImageJ software and normalized to β-actin. n = 3. ** p < 0.01, n.s. p > 0.05 compared to TGF-β1 alone. (E) MLE12 cells were treated with MG-132 (20 μM) for 1 h, then challenged with CHX (20 ng/mL) for additional 0–4 h. Cell lysates were analyzed by immunoblotting with TXNIP and β-actin antibodies. (F) Intensities of TXNIP were quantified by ImageJ software and normalized to β-actin. n = 3. ** p < 0.01 compared to CHX 2 and 4 h. Shown are representative blots from three to four independent experiments. (G) HEK293 cells were transfected with plasmids encoding HA-tagged ubiquitin for 48 h, and then treated with MG-132 (20 μM) for 1 h prior to TGF-β1 treatment (10 ng/mL, 3 h). Denatured cell lysates were subjected to immunoprecipitation with rabbit IgG and TXNIP antibody, followed by immunoblotting with HA tag antibody.

Figure 5. TGF-β1 induces TXNIP degradation in the ubiquitin-proteasome system. (A) IMR90 cells were treated with MG-132 (20 μM) or leupeptin (100 μM) for 1 h, and then challenged with TGF-β1 (10 ng/mL) for additional 6 h. Cell lysates were analyzed by immunoblotting with TXNIP and β-actin antibodies. (B) Intensities of TXNIP were quantified by ImageJ software and normalized to β-actin. n = 4. ** p < 0.01, n.s. p > 0.05 compared to TGF-β1 alone. (C) A549 cells were treated with MG-132 (20 μM) or leupeptin (100 μM) for 1 h, and then challenged with TGF-β1 (10 ng/mL) for additional 6 h. Cell lysates were analyzed by immunoblotting with TXNIP and β-actin antibodies. (D) Intensities of TXNIP were quantified by ImageJ software and normalized to β-actin. n = 3. ** p < 0.01, n.s. p > 0.05 compared to TGF-β1 alone. (E) MLE12 cells were treated with MG-132 (20 μM) for 1 h, then challenged with CHX (20 ng/mL) for additional 0–4 h. Cell lysates were analyzed by immunoblotting with TXNIP and β-actin antibodies. (F) Intensities of TXNIP were quantified by ImageJ software and normalized to β-actin. n = 3. ** p < 0.01 compared to CHX 2 and 4 h. Shown are representative blots from three to four independent experiments. (G) HEK293 cells were transfected with plasmids encoding HA-tagged ubiquitin for 48 h, and then treated with MG-132 (20 μM) for 1 h prior to TGF-β1 treatment (10 ng/mL, 3 h). Denatured cell lysates were subjected to immunoprecipitation with rabbit IgG and TXNIP antibody, followed by immunoblotting with HA tag antibody.

Figure 6. USP13 stabilizes TXNIP. (A) IMR90 cells were transfected with plasmids encoding V5-tagged USP13 for 48 h, and then treated with TGF-β1 (10 ng/mL) for additional 6 h. Cell lysates were analyzed by immunoblotting with TXNIP, V5 tag, and β-actin antibodies. (B) Intensities of TXNIP were quantified by ImageJ software and normalized to β-actin. n = 3. ** p < 0.01, compared to TGF-β1 alone. (C) IMR90 cells were transfected with control or USP13 siRNA for 72 h, and then treated with TGF-β1 (10 ng/mL) for additional 6 h. Cell lysates were analyzed by immunoblotting with TXNIP and β-actin antibodies. (D) Intensities of TXNIP were quantified by ImageJ software and normalized to β-actin. n = 3. ** p < 0.01, compared to control. Shown are representative blots from three independent experiments.

Figure 7. USP13 targets and deubiquitinates TXNIP. (A) IMR90 cell lysates were subjected to immunoprecipitation with rabbit IgG and TXNIP antibody, followed by immunoblotting analysis with USP13 antibody. (B) IMR90 cells were treated with MG-132 (20 M, 1 h) prior to addition of spautin-1 (5 M, 6 h). Denatured cell lysates were subjected to immunoprecipitation with IgG or Ubi antibody, followed by TXNIP immunoblotting. C. IMR90 cells were transfected with plasmids encoding USP13-V5 for 48 h, and then denatured cell lysates were subjected to immunoprecipitation with IgG or Ubi antibody, followed by TXNIP immunoblotting. Input cell lysates were analyzed by immunoblotting with V5 antibody.