Fig 2: Effects of TMEM16A on LPS induced IEC-6 cell apoptosis measured by flow cytometry.Cells were cultured for 24 h with media contained siRNA targeting TMEM16A (TMEM16A-) or adenovirus stimulating TMEM16A (TMEM16A+) in the presence and absence of high dose LPS (10 µg/mL). (n = 6/group). Representative (a) and statistical results (b) were shown. Data are expressed as the mean ± SD. Values in the normal control (NC) group are set to 100% and other values are given relative to those in the NC group, **p < 0.01 compared with NC group. ##p < 0.01 compared with corresponding TMEM16A- or TMEM16A+ group.

Fig 3: The effects of ANO1 inhibition on HaCaT cells. (a) Proliferation of cells exposed to 30 µM T16Ainh-A01 for 3 days. (b) Proliferation of cells exposed to 10 µM Ani9 for 3 days. (c) Proliferation of cells following ANO1 knock-down for 3 days. After knock-down of ANO1 using siRNA targeting ANO1 and in negative controls (mock and si-NC), proliferation rates of HaCaT cells were measured for 3 days. (d) Immunoblots of ANO1 and ERK and AKT signaling pathway-related proteins after ANO1 knock-down for 2 days. (e) Densitometric analysis for the relative intensity of ANO1 and ERK and AKT signaling pathway-related proteins. Treatments with the two blockers (T16Ainh-A01 and Ani9) and ANO1 knock-down were performed as four independent replicates to guarantee reliable results. To compare the differences between control (vehicle or Mock [si-NC]) and blocker-treated or ANO1 knock-down cells, two-way ANOVA and Tukey’s post-hoc tests were used. *: significantly different from vehicle in (a,b), from Mock and si-NC (c), and from Mock (e) (* p < 0.05, ** p < 0.01, and *** p < 0.001). #: significantly different from si-NC (e) (# p < 0.05 and ## p < 0.01).

Fig 4: Changes in histopathological structure and ANO1-related proteins in mouse ears showing psoriasis-like symptoms caused by inhibiting ANO1. The right ears of the mice in all four groups (control, IMQ, IMQ + 30 µM A01, and IMQ + 100 µM A01 groups) were obtained on the eighth day after psoriasis-like skin injury and inhibition of ANO1. (a) H&E staining and ANO1 immunoreaction in the right ears of control, IMQ, and IMQ + 100 µM A01 groups. Scale bar = 500 µm. (b) The quantification of acanthosis in the right ears of control (n = 3), IMQ (n = 3), and IMQ + 100 µM A01 (n = 3) groups. (c) The signal intensity of ANO1 per ten cells in the epidermis of right ears of control (n = 3), IMQ (n = 3), and IMQ + 100 µM A01 (n = 3) groups. Significant differences of acanthosis (b) or ANO1 intensity (c) among control, IMQ, and IMQ + 100 µM A01 groups were analyzed using one-way ANOVA followed by Tukey’s multiple comparison test. *: significantly different from IMQ group (** p < 0.01 and *** p < 0.001). (d) The expression levels of ERK and AKT proteins. The phosphorylation of ERK was increased by IMQ cream but was decreased by A01 in a dose-dependent manner. Imiquimod, IMQ; T16Ainh-A01, A01.

Fig 5: Alleviation of psoriasis-like symptoms in mouse ears by inhibiting ANO1. To induce psoriasis-like skin injury, three mouse groups (IMQ [n = 7], IMQ + 30 µM A01 [n = 7], and IMQ + 100 µM A01 [n = 7]) received a daily dose of 25 mg of 5% IMQ cream on their right ears for 7 consecutive days; IMQ + 30 µM A01 and IMQ + 100 µM A01 groups additionally received 15 µL of 30 µM and 100 µM A01, respectively, at 30 min before and after IMQ treatment. The control group received a similar dose of Vaseline cream on their right ears. (a) Changes in right ear thickness caused by IMQ and A01. (b–d) Thickness, erythema, and scaling of right ears were scored daily. (e) The cumulative score (thickening plus erythema plus scaling) was described. Symbols per day depict mean ± SEM of seven mice ears per group. The significant differences among groups obtained from statistical analysis are presented in Figure S2. (f) Phenotypic representation of the right ears in mice treated with IMQ or IMQ + 100 µM A01 for 7 days. Imiquimod, IMQ; T16Ainh-A01, A01.