RPS6 Antibody - #AF7831

Figure 4. Regorafenib induces the formation of autophagy by inhibiting the TGF-β1/mTOR signal pathway. (A) BLM-PPF cells were treated with RG (2 µM, 4 µM) for 24 h, and the expression levels of autophagy-related proteins (Atg3, Atg7, p62, and LC3-II/I) were detected. Densitometric analysis are shown beside; (B) Mlg cells were exposed to chloroquine (CQ) and/or RG (2 µM, 4 µM) for 24 h, and Western blot technology was used to detect the p62 protein expression level; (C) Mlg cells were exposed to bafilomycin A1 (Baf A1) and/or RG (2 µM, 4 µM) for 24 h. Densitometric analysis are shown below; (D,E) The plasmids of GFP-LC3B (green fluorescent protein-microtubule-associated protein 1 light chain 3B) and mCherry-GFP-LC3B (mCherry fluorescent protein-green fluorescent protein-microtubule-associated protein 1 light chain 3B) was transferred into NIH-3T3 cells, and these cells subsequently were incubated with RG (2 µM, 4 µM) and/or TGF-β1 (5 ng/mL) for 12 h. DNA was counterstained with DAPI (blue). Scale bars: 50 µm; (F) Mlg cells were incubated with RG (2 µM, 4 µM) and/or TGF-β1 (5 ng/mL) for 12 h, and the expression levels of mTOR (mechanistic target of rapamycin), ULK-1 (protein kinase ULK1/autophagy-related protein 1), p70 S6K (p70 S6 kinase), and S6RP (S6 ribosomal protein), and their phosphorylations were detected. Densitometric analysis are shown beside; (G) BLM-PPF cells were explored to RG (2 µM, 4 µM) for 12 h, and Western Blot were used to detect the expression levels of mTOR, ULK-1, and S6RP, and their phosphorylations. Densitometric analysis are shown beside. Data in (A–G) are means ± standard error of mean (SEM), and GAPDH or β-tubulin were used as a loading control. # p < 0.05, ### p < 0.001, ** p < 0.01, *** p < 0.001(one-way ANOVA). NS: not significant.

FIGURE 4 Ellagic acid promotes pulmonary fibroblast autophagy mainly via inhibiting Wnt-mTOR signaling pathway (A,B) Mlg cells were exposed to CQ (20 µM) and Baf A1 (100 nM) with or without Ellagic acid (5 µM, 10 µM) to analyze the p62 expression level by using western blot. Densitometric analyses were shown beside (C,D) The plasmids of GFP-LC3B and mCherry-GFP-LC3B were transfected to NIH3T3 cells with PEI, and these cells were subsequently exposed to Ellagic acid (5 µM, 10 µM) and/or Wnt3a (100 ng/ml) for 12 h. DNA was counterstained with DAPI (blue). Quantitative analyses are showed beside. Scale bars: 50 μm (E) BLM-PPF cells were treated with Ellagic acid (5 µM, 10 µM) for 24 h, and the Atg16L1, Beclin1 and LC3-II/I expression levels were detected by Western blot. Densitometric analyses were shown below (F) BLM-PPF cells were treated with Ellagic acid (5 µM, 10 µM) for 12 h, and protein expression levels of mTOR, S6RP and their phosphorylation were detected by Western blot. Densitometric analyses were shown below. Data are means ± Standard Error of Mean, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, and NS: nonsignificant (one-way ANOVA). β-tubulin or GAPDH were used as a loading control.

FIGURE 4 |Densitometric analyses were shown below (F) BLM-PPF cells were treated with Ellagic acid (5 µM, 10 µM) for 12 h, and protein expression levels of mTOR, S6RP and their phosphorylation were detected by Western blot. Densitometric analyses were shown below. Data are means ± Standard Error of Mean, n
3, *p < 0.05, **p < 0.01,***p < 0.001, and NS: nonsignificant (one-way ANOVA). β-tubulin or GAPDH were used as a loading control.