Figure 12.

TM9SF4 knockdown disrupted the intestinal epithelial barrier. (A–C) Caco-2 cells were stably transfected with Scr-shRNA or TM9SF4-shRNAs. Relative transepithelial electrical resistance was measured (A) (n = 5). The levels of claudin-1 and ZO-1 were detected by (B) qRT-PCR and (C) Western blot (n = 3). (D–F) HT-29 cells were overexpressed with TM9SF4 or vector pcDNA6. (D) Relative transepithelial electrical resistance was measured (n = 5). The levels of claudin-1 and ZO-1 were detected by (E) qRT-PCR (n = 3) and (F) Western blot (n = 3). (G–J) Phorbol 12-myristate 13-acetate (PMA)-primed THP-1 cells were treated with LPS, IL1β, and IFNγ, and co-cultured with Caco-2 cells. Shown are the schematic diagram of (G) co-culture study, (H) relative transepithelial resistance (n = 5), (I) expressional level of claudin-1 and ZO-1 by qRT-PCR (n = 3), and (J) Western blot (n = 3). (K) Data summary of serum FITC–dextran concentration in bone marrow–chimeras (n = 6). (L) Immunofluorescence staining of claudin-1 (green) in colon tissue sections from 2% DSS-treated bone marrow–chimeras (n = 6). Blue, Hoechst nuclear stain. Scale bars: 100 μm. (M and N) HCECs were transfected with Scr-shRNA or TM9SF4-shRNA1. Supernatants were collected from IL4/IL13-treated PMA-primed THP-1 cells that were stably expressed with Scr-shRNA or with TM9SF4-shRNA1. The supernatants were added into HCECs. (M) Cell migration was measured by wound healing assays (n = 8–15 per group). (N) Cell proliferation was quantified by Cell Counting Kit-8 (CCK8) from day 0 to day 4 after THP-1 conditional medium-treatment (n = 4). Means ± SEM. ∗∗P < .01, ∗∗∗P < .001. pcDNA, plasmid cloning DNA; SCR, scrambled control.