Fig 1: Inhibition of mTORC1 signaling promotes differentiation.(a) Increasing cell numbers of HaCaT cells were seeded and after 24h 100 nM Rapamycin, 250 nM Torin or solvent control (DMSO) were added and differentiation was allowed to proceed for 72h. Protein lysates were subjected to Western blotting and proteins were detected with the indicated antibodies. Quantification of 3–6 similar blots is depicted below. Statistical significance was calculated with two-way ANOVA and Bonferroni multiple comparison, comparing different treatments with the corresponding cell density in the control group (*p ≤0.05, ***p ≤0,001, ns: non-significant). (b) In NHK cells differentiation was induced with 2mM CaCl2 in the presence of 100nM Rapamycin, 250 nM Torin or solvent control for 48h. Protein lysates were subjected to Western blotting and proteins were detected with the indicated antibodies. Below a quantification of six similar blots is shown. Statistical significance was calculated with one-way ANOVA and Bonferroni multiple comparison (*p ≤0.05). (c) NHK cells were treated with 2 mM CaCl2 and 100nM Rapamycin or DMSO control. RNA was isolated and quantitative RT-PCR was performed to measure expression of the indicated differentiation markers. Graph presents mean ± SEM (n = 5–8). Statistical significance was calculated with two-way ANOVA and Bonferroni multiple comparison. (*p ≤0.05, ****p ≤0.0001, ns: non-significant). (d+e) HaCaT (d) or NHK (e) cells were transfected with siRNA specific for Raptor (Rap), mTOR or an siRNA control (si contr) and differentiation was induced either by post-confluent growth (d) or with 2mM CaCl2 (e) for 48h. Protein lysates were analyzed by Western blotting with the indicated antibodies. Below each blot a quantification of IVL and P-S6 band relative to actin bands of 4–6 similar blots is shown. Statistical significance was calculated with 2-way ANOVA and Bonferroni multiple comparison, comparing Raptor or mTOR knockdown with the corresponding differentiation state in the control group (*p ≤0.05, ** p ≤0,01, ****p ≤0,0001).
Fig 2: LRRC8A regulation during epidermal differentiation is disturbed in an inflammatory environment. (a) LRRC8A was stained in punch biopsies from NN and PP or PN skin from psoriasis vulgaris patients using anti-LRRC8A antibody. Representative staining was selected (left). Scale bars represent 50 μm. Staining intensities were estimated semi-quantitatively on a scale from 0 to 3 (right) (n = 5 to 10 independent experiments, mean + SEM) (1-way ANOVA with Tukey post-test to correct for multiple comparisons). (b, c) In KGM-2, 5.7 × 104/cm2 cells were differentiated by Ca2+ without supplements in the presence or absence of Th1 cytokines (Mix 1 and 2) for the indicated time points. Mix 1 consisted of IL-1β, IL-17A, and TNF-α, and Mix 2 of IL-22 and TNF-α (20 ng/μl each). (b) Protein lysates were subjected to Western blotting with antibodies for K10 and IVL, to show impaired differentiation and AKT activation under inflammatory conditions. Densitometric quantification of blots indicates that LRRC8A upregulation was inhibited by cytokines (n = 6 to 8 independent experiments, mean + SEM) (2-way ANOVA with Sidak post-test to correct for multiple comparisons). (c) RT-qPCR in contrast showed that LRRC8A mRNA levels are not dysregulated by Th1 inflammation (n = 4 to 6 independent experiments, mean ± SEM). AKT, serine/threonine proteine kinase homolog to v-akt1 oncogene; LRRC8, leucine-rich repeat-containing protein 8; NN, healthy donors; PN, non-lesional skin; PP, lesional skin.
Fig 3: Knockdown of LRRC8A interferes with epidermal differentiation. Keratinocytes (5.7 × 104/cm2) were seeded and transfected with LRRC8A-specific siRNA (siLRRC8A) or an unspecific siCtrl. Seven to 8 hours after transfection, cells were stimulated with 2 mM CaCl2 to induce differentiation, harvested every 24 hours, and analyzed by RT-qPCR (a) or Western blot (b). (a) Knockdown of LRRC8A reduces mRNA levels of MKI67, IVL, and FLG, with the strongest effect after 72 hours (n = 7–9 independent experiments, mean + SEM) (2-way ANOVA with Sidak post-test to correct for multiple comparisons). (b) Western blot analysis shows a slight reduction of IVL after 72 hours in knockdown cells. Blots were quantified by densitometry (n = 5–6 independent experiments, mean + SEM) (2-way ANOVA with Sidak post-test to correct for multiple comparisons). FLG, filaggrin; IVL, involucrin; LRRC8, leucine-rich repeat-containing protein 8; MKI67, marker of proliferation Ki-67; siCtrl, siRNA control; siLRRC8A, LRRC8A-specific siRNA; siRNA, small interfering RNA.
Fig 4: LRRC8A modulates the transition from KSC to TAC. Keratinocyte populations were separated based on their adhesion to collagen IV, transfected with 2 different LRRC8A specific siRNAs (siLRRC8A 1 and 2) or an unspecific siCtrl, and differentiated with 2 mM CaCl2. RNA was isolated after 72 hours and analyzed by RT-qPCR for the indicated genes. Knockdown of LRRC8A (a) decreases expression of KRT1 (c) and IVL (d) in all populations, although it was not significant in all cases. Markers of basal cell types such as MKI67 (b), NGFR (e), and FOXM1 (f) were reduced in LRRC8A knockdown with both siRNAs in cells transitioning from KSC to TAC (KSC/TAC) but not in cells transitioning from TAC to PMC (TAC/PMC) indicative of a reduced entry into the differentiation process (n = 7 to 17 independent experiments, mean + SEM) (2-way ANOVA with Tukey post-test to correct for multiple comparisons). FOXM1, forkhead box protein M1; IVL, involucrin; KRT1, cytokeratin 1; KSC, keratinocyte stem cell; LRRC8, leucine-rich repeat-containing protein 8; MKI67, marker of proliferation Ki-67; NGFR, neuronal growth factor receptor; PMC, post-mitotic cell; siCtrl, siRNA control; siLRRC8A, LRRC8A-specific siRNA; siRNA, small interfering RNA; TAC, transiently amplifying cell.
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