Fig. 4

The crosstalk between ER stress and autophagy in the TNF-α-exposed Caco-2 cells. Caco-2 cells were incubated with TNF-α (0–50 ng/mL) for 24 h. a The cell viability was measured by the CCK-8 assay (n = 6); b-d The mRNA abundance of inflammatory cytokines was measured by qRT-PCR analysis (n = 4). Caco-2 cells were co-incubated with TNF-α and tunicamycin (TM), 4-phenylbutyric acid (4PBA), rapamycin (RAP), or 3-methyladenine (3MA). e-g The mRNA abundance of inflammatory cytokines was measured by qRT-PCR analysis (n = 4); h Representative scatter plots and quantitative analysis of apoptotic cells in experimental groups as analyzed by the flow cytometry (n = 4); i Western blot analysis was conducted to determine the protein levels of autophagy-associated molecules (n = 4); j Autophagic flux of Caco-2 cells was analyzed through transfection with AdPlus-mCherry-GFP-LC3B. The representative immunofluorescent photographs were indicated. GFP dots are green. mCherry dots are red (n = 4); k RT-PCR assay was conducted to detect the mRNA expression of sXBP-1 in Caco-2 cells (n = 4); l, m Western blot analysis was carried out to detect the protein levels of ER stress markers and UPR sensors in Caco-2 cells (n = 4). Data from at least three independent experiments were presented as mean ± SE. ns means no significance; ^*P < 0.05 vs. CON group; ^#P < 0.05 vs. TNF-α group