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. 2021 Oct 29;12(11):1016. doi: 10.1038/s41419-021-04274-7

Fig. 2. TM sequentially induces ER stress, autophagy, fibrosis, and apoptosis in HK-2 cells.

Fig. 2

A, B HK-2 cells were exposed to TM (100 nM) for various time (0, 1, 2, 4, 8, and 24 h). DMSO was used as vehicle control. Cell lysates were analyzed for BiP, p-PERK, PERK, p-eIF2α, eIF2α, LC3, FN, Cleaved Caspase-3, and GAPDH by immunoblot analysis (A) and quantified by densitometry (B). C–E HK-2 cells were transfected with RFP-GFP-LC3 and then treated with 100 nM TM or DMSO (vehicle control) for 2 h. C Representative fluorescence microscopy images showing GFP and RFP LC3 puncta in HK-2 cells. Bar = 5 μm. D Quantitative analysis of GFP and RFP LC3 puncta. Data are expressed as mean ± SD. #P < 0.05 vs. control; ^P < 0.05 vs GFP-LC3 puncta in TM-treated group. E Analysis of autophagic flux rate. F–H Cells were incubated with 100 nM TM for 0–24 h for phase contrast and TUNEL staining and immunofluorescence analysis of Collagen I. (F) Representative images. Scale bar = 100 μm. G, H Quantification of TUNEL staining positive cells and Collagen I intensity. Data are expressed as mean ± SD. n = 4. *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant. vs. control.