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. 2022 Apr 7;14(2):245–270. doi: 10.1016/j.jcmgh.2022.04.002

Figure 3.

Figure 3

Knockdown of TM9SF4 promoted inflammation, increased ER stress, and increased cell deaths in colon epithelial cells. (A) Expression levels of TM9SF4 in HCECs, Caco-2, and HT-29 cells were compared by Western blot (n = 3). HCECs with Scr-shRNA or TM9SF4-shRNAs were challenged with (B) 100 ng/mL LPS or (C) 1 μg/mL PGN for 24 hours. The mRNA levels of TNFα, IL1β, and IL6 were assessed by qRT-PCR (n = 3). (D) HCECs with Scr-shRNA or TM9SF4-shRNAs were challenged with LPS. The levels of TM9SF4, GRP78, ATF4, cleaved ATF6, spliced XBP1, CHOP, COX2, and cleaved caspase 3 were detected by Western blot. Representative from 3 experiments. (E) ROS measurement in HCECs by DHE fluorescence. Representative from 4 experiments. Scale bar: 100 μm. (F) Representative FACS analysis and data summary (right) of Annexin V and PI staining in LPS-challenged HCECs (n = 4). KEGG analysis of altered signaling pathways after TM9SF4 knockdown in (G) basal condition (vehicle) and (H) inflammatory condition (LPS) in HCECs. HCECs were transfected with scrambled-shRNA (SCR) or TM9SF4-shRNA 1 (KD) for RNA sequencing analysis. Shown are means ± SEM. ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. ECM, extracellular matrix; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GnRH, gonadotropin releasing hormone; KD, knockdown clone; PI, propidium iodide; SCR, scrambled control; TGF, transforming growth factor; TRP, trasient receptor potential.