Fig 2: TRPV6 is necessary for KLB regulating TJ proteins. (a) The protein levels of TRPV6 in the EtOH-treated cells, KLBOE group and KLBi group (n=3). * P<0.05 vs. (-)EtOH; # P<0.05 vs. KLB NC; ? P<0.05 vs. KLB NCi. (b) Representative images of immunofluorescence of TRPV6 (red) in EtOH treated Caco2 cells (i-ii), KLBOE cells (iii-iv) and KLBi cells (v-vi) (n=3). (c) The protein levels of Occludin and ZO-1 in KLBOE cells with or without TRPV6i (upper panel, n=3), and Occludin/ZO-1 expression in KLBi cells with or without TRPV6OE (lower panel, n=4). (d) Representative images of immunofluorescence of Occludin and ZO-1 in KLBOE cells with or without TRPV6i (i-iv), and in KLBi cells with or without TRPV6OE (v-viii) (n=3). (e, f) TEER and Papp values in KLBOE cells with or without TRPV6i (e) and in KLBi cells with or without TRPV6OE (f), n=5. * P<0.05 vs. KLBOE(+)/TRPV6i(-); # P<0.05 vs. KLBi(+)/TRPV6OE(-). Statistical analysis was performed using t-test. Data are mean ± SEM.TEER, transepithelial electrical resistance; Papp, apparent permeability coefficient; NC, negative control; NCi, negative control for RNA interference; KLBOE, KLB overexpression; KLBi. inhibition of KLB; TRPV6OE, TRPV6 overexpression; TRPV6i, inhibition of TRPV6.

Fig 3: KLB interacts with FXR1 to regulate TRPV6 mRNA stability. (a) The mRNA levels of TRPV6 in EtOH treated Caco2 cells, KLBOE cells, and KLBi cells. n=3, # P<0.05 vs. (-)EtOH; ? P<0.05 vs. KLB NC; * P<0.05 vs. KLB NCi. (b) The activity of TRPV6 promoter in NC and KLBOE cells. (c) The TRPV6 mRNA stability in NC and KLBOE cells in the presence of ActD. n=3, * P<0.05 vs. KLB NC. (d) The Co-IP of KLB and FXR1 (n=3). (e) Relative enrichment of TRPV6 mRNA 3’UTR in FXR1 protein of NC and KLBOE cells. n=3, * P<0.05 vs. KLB NC. The KLBOE or KLBNC cells were transfected with FXR1 overexpression (FXR1OE) or control plasmid (FXR1NC), respectively. (f) The mRNA stability of TRPV6, the mRNA levels (g) and protein levels of TRPV6 (h) in these cells. n=3, # P<0.05 vs. KLBNC(FXR1NC); * P<0.05 vs. KLBOE(FXR1NC). Statistical analysis was performed using one-way/two-way ANOVA followed with Tukey post-hoc test or t-test. Data are mean ± SEM. (i) The Schematic diagram for the mechanisms of alcohol reduces intestinal KLB expression via HNF4A, resulting in intestinal barrier disruption and subsequent liver injury. The intestinal KLB could interacts with FXR1 to enhance TRPV6 mRNA stability, inhibiting the endocytosis and degradation of tight junction proteins. NC, negative control; NCi, negative control for RNA interference; KLBOE, KLB overexpression; KLBi. inhibition of KLB; ActD, actinomycin D.