Fig 2: Panx3 regulates HaCaT cell differentiation. (A) Immunostaining of Panx3 (green), Filaggrin (red), and Notch1 (purple), Ki67 (yellow) in pCMV6-mock or pCMV6-Panx3 transfected HaCaT cells cultured in normal condition media (a). Relative fluorescence intensity comparison of images and % of Ki67 positive cells were analyzed (b). *p < 0.01. Error bars represent the mean ± SD; N = 3. The nuclei were counterstained with DAPI (blue in merged). (B) qPCR for K1, K10, K5, and K14 in pCMV6-mock or pCMV6-Panx3 transfected HaCaT cells. Results represent the mean ± SD; N = 3. *p < 0.01.

Fig 3: Epfn deficiency does not interfere with Panx3 expression. (A) qPCR for Panx3 and Epfn using total RNA prepared from skin of 6 weeks old Epfn+/+ and Epfn-/- mice. Results represent the mean ± SD; N = 3. *p < 0.01. NS, not significant. (B) Immunostaining of Panx3 (green) and Epfn (red) in si-Control or si-Epfn transfected HaCaT cells cultured in normal medium. The nuclei were counterstained with DAPI (blue in merged) (a). Relative fluorescence intensity comparison of images were analyzed (b). (C) Western blots of Epfn and Notch1 in Panx3 overexpressed HaCaT cells transfected with or without si-Epfn (a). Quantification of the ratios of Epfn/a-Tubulin (upper panel) and Notch1/a-Tubulin (lower panel). (D) Western blots of Epfn and Notch1 in shPanx3 transfected HaCaT cells with or without pcDNA3.1-Epfn (a). Quantification of the ratios of Epfn/a-Tubulin (upper panel) and Notch1/a-Tubulin (lower panel). *p < 0.01. NS, not significant.

Fig 4: Panx3 ER Ca2+ channel regulates Epfn via Akt/NFAT signaling pathway. HaCaT cells were cultured for 3 days in normal media. Western blotting was performed with antibodies to Panx3, Epfn, Notch1, phospho-Akt (P-Akt), Akt, phospho-NFATc1 (P-NFATc1), NFATc1, and a-Tubulin. The cropped blots were used in the figures. Full blot is shown in the Supplementary Information (Full Original Blots-I–IV). (B) Quantification of the ratios of Epfn/a-Tubulin (upper panel of A,b and B,b) and Notch1/a-Tubulin (lower panel of A,b and B,b), P-Akt/Akt (upper panel of A,c and B,c) and P-NFATc1/NFATc1 (lower panel of A,c and b,c). **p < 0.05, *p < 0.01. Error bars represent the mean ± SD; N = 3.