Journal: JID Innovations

Article Title: The Volume-Regulated Anion Channel LRRC8 is Involved in the Initiation of Epidermal Differentiation and is Deregulated in Psoriasis

doi: 10.1016/j.xjidi.2025.100357

Figure Lengend Snippet: LRRC8A protein is primarily localized in transient amplifying cells of the basal epidermal layer. ( a ) LRRC8A was stained via immunohistochemistry in punch biopsies from healthy donors. LRRC8A is localized in the basal epidermal layer, as described previously ( ; ). Bar represents 20 μm. ( b ) Native human skin was stained with a LRRC8A specific antibody (red) and an anti-CD271 antibody (green). Nuclei were stained with DAPI. Greyscale images are shown for each channel as well as an overlay image of the red and green channel. Scale bars represent 20 μm. ( c ) Keratinocyte populations were separated into KSC, TAC, and PMC based on their adhesion to collagen IV. Protein lysates from these populations were analyzed by Western blotting. LRRC8A was mainly detected in KSC and TAC, but not PMC, with the highest level in TAC. ( d ) Keratinocyte populations were differentiated by the addition of 2 mM CaCl 2 for 72 hours, analyzed by Western blotting, and quantified by densitometry (n = 16 independent experiments, mean + SEM). KSC, keratinocyte stem cell; LRRC8A shows its highest expression in early (-CaCl 2 ) TAC. LRRC8, leucine-rich repeat-containing protein 8; PMC, post-mitotic cell; TAC, transiently amplifying cell.

Article Snippet: For immunohistochemistry, the primary anti-LRRC8A antibody (NBP2-32158 1:100, Novus Biologicals) or concentration-adjusted isotype control antibody (Cell Signaling Technology) was applied overnight after antigen retrieval with EDTA solution.

Techniques: Staining, Immunohistochemistry, Western Blot, Expressing

Journal: JID Innovations

Article Title: The Volume-Regulated Anion Channel LRRC8 is Involved in the Initiation of Epidermal Differentiation and is Deregulated in Psoriasis

doi: 10.1016/j.xjidi.2025.100357

Figure Lengend Snippet: LRRC8A gene expression increases with progressing epidermal maturation. RNA was isolated from keratinocyte populations and used for RNA sequencing (n = 4 independent experiments). Read counts were normalized and expression analysis was conducted using DESeq2. ( a ) To verify proper separation of keratinocyte populations, selected marker genes of basal or differentiating keratinocytes were normalized to the highest expressing population and compared to the others. Color changes from green to red indicate an increase in gene expression. ( b ) LRRC8A was differentially expressed among different populations (p: 3.6 × 10 −8 , Likelihood Ratio Test) with its transcripts significantly increasing during maturation. ( c ) All other LRRC8 family members, whose transcripts are severely less abundant than those of LRRC8A , are also differentially expressed among different populations ( P values: B: 9.1 × 10 −9 , C: 5.8 × 10 −10 , D: 7.01 × 10 −8 , E: 1.31 × 10 −25 , Likelihood Ratio Test). LRRC8B and LRRC8E increase during differentiation, while LRRC8C and LRRC8D decrease. LRRC8, leucine-rich repeat-containing protein 8.

Article Snippet: For immunohistochemistry, the primary anti-LRRC8A antibody (NBP2-32158 1:100, Novus Biologicals) or concentration-adjusted isotype control antibody (Cell Signaling Technology) was applied overnight after antigen retrieval with EDTA solution.

Techniques: Gene Expression, Isolation, RNA Sequencing, Expressing, Marker

Journal: JID Innovations

Article Title: The Volume-Regulated Anion Channel LRRC8 is Involved in the Initiation of Epidermal Differentiation and is Deregulated in Psoriasis

doi: 10.1016/j.xjidi.2025.100357

Figure Lengend Snippet: LRRC8A gene and protein expression can be recapitulated in vitro. Through the addition of 2 mM CaCl 2 , harvested every 24 hours, and analyzed via RT-qPCR, 5.7 × 10 4 /cm 2 cells were differentiated ( a ) or Western blots ( b ). RNA is upregulated and stays increased during further differentiation (n = 11 independent experiments, mean + SEM) (Repeated measures ANOVA and Tukey post-test to correct for multiple comparisons, asterisks indicate changes compared to 0 hours [other comparisons n.s.]). In contrast, the amount of LRRC8A protein increases 24 hours after Ca 2+ addition and is downregulated afterward. Blots were quantified by densitometry (b, bottom) (n = 7 independent experiments, mean + SEM) (Repeated measures ANOVA and Tukey post-test to correct for multiple comparisons, asterisks indicate changes compared to 24 hours [other comparisons n.s.]). LRRC8, leucine-rich repeat-containing protein 8; n.s., not significant.

Article Snippet: For immunohistochemistry, the primary anti-LRRC8A antibody (NBP2-32158 1:100, Novus Biologicals) or concentration-adjusted isotype control antibody (Cell Signaling Technology) was applied overnight after antigen retrieval with EDTA solution.

Techniques: Expressing, In Vitro, Quantitative RT-PCR, Western Blot

Journal: JID Innovations

Article Title: The Volume-Regulated Anion Channel LRRC8 is Involved in the Initiation of Epidermal Differentiation and is Deregulated in Psoriasis

doi: 10.1016/j.xjidi.2025.100357

Figure Lengend Snippet: Knockdown of LRRC8A interferes with epidermal differentiation. Keratinocytes (5.7 × 10 4 /cm 2 ) 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 CaCl 2 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.

Article Snippet: For immunohistochemistry, the primary anti-LRRC8A antibody (NBP2-32158 1:100, Novus Biologicals) or concentration-adjusted isotype control antibody (Cell Signaling Technology) was applied overnight after antigen retrieval with EDTA solution.

Techniques: Knockdown, Transfection, Quantitative RT-PCR, Western Blot, Marker, Control, Small Interfering RNA

Journal: JID Innovations

Article Title: The Volume-Regulated Anion Channel LRRC8 is Involved in the Initiation of Epidermal Differentiation and is Deregulated in Psoriasis

doi: 10.1016/j.xjidi.2025.100357

Figure Lengend Snippet: Knockdown of LRRC8A does not interfere with keratinocyte proliferation. Primary keratinocytes (2.1 × 10 4 /cm 2 ) were seeded and transfected 24 hours later with 10 pmol siRNA specific for LRRC8A (siLRRC8A) or unspecific siCtrl. ( a ) Cells were trypsinized and counted every 24 hours. Cell count was normalized to t = 0 hours (first harvest 8 hours after transfection). No differences between siCtrl and siLRRC8A were detected (n = 7 independent experiments, mean ± SEM). ( b ) After counting cell numbers for proliferation, RNA was isolated and knockdown was verified via RT-qPCR (n = 7 independent experiments, mean + SEM) (2-way ANOVA with Sidak post-test to correct for multiple comparisons). LRRC8, leucine-rich repeat-containing protein 8; siCtrl, siRNA control; siLRRC8A, LRRC8A- specific siRNA; siRNA, small interfering RNA.

Article Snippet: For immunohistochemistry, the primary anti-LRRC8A antibody (NBP2-32158 1:100, Novus Biologicals) or concentration-adjusted isotype control antibody (Cell Signaling Technology) was applied overnight after antigen retrieval with EDTA solution.

Techniques: Knockdown, Transfection, Cell Counting, Isolation, Quantitative RT-PCR, Control, Small Interfering RNA

Journal: JID Innovations

Article Title: The Volume-Regulated Anion Channel LRRC8 is Involved in the Initiation of Epidermal Differentiation and is Deregulated in Psoriasis

doi: 10.1016/j.xjidi.2025.100357

Figure Lengend Snippet: Efficient knockdown of LRRC8A with 2 specific siRNAs interferes with the expression of differentiation markers. ( a ) Keratinocytes (5.7 × 10 4 /cm 2 ) were seeded and transfected with 2 LRRC8A siRNAs (siLRRC8A 1 and 2) or an unspecific siCtrl or left untreated and harvested after 48 hours. Protein lysates were subjected to Western blotting with the indicated antibodies. ( b ) Keratinocytes (5.7 × 10 4 /cm 2 ) were seeded and transfected with 2 LRRC8A siRNAs or an unspecific siCtrl. Seven to 8 hours after transfection, cells were stimulated with 2 mM CaCl 2 to induce differentiation, harvested every 24 hours, and analyzed by Western blotting with the indicated antibodies. LRRC8, leucine-rich repeat-containing protein 8; siCtrl, siRNA control; siLRRC8A, LRRC8A- specific siRNA; siRNA, small interfering RNA.

Article Snippet: For immunohistochemistry, the primary anti-LRRC8A antibody (NBP2-32158 1:100, Novus Biologicals) or concentration-adjusted isotype control antibody (Cell Signaling Technology) was applied overnight after antigen retrieval with EDTA solution.

Techniques: Knockdown, Expressing, Transfection, Western Blot, Control, Small Interfering RNA

Journal: JID Innovations

Article Title: The Volume-Regulated Anion Channel LRRC8 is Involved in the Initiation of Epidermal Differentiation and is Deregulated in Psoriasis

doi: 10.1016/j.xjidi.2025.100357

Figure Lengend Snippet: 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 CaCl 2 . 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.

Article Snippet: For immunohistochemistry, the primary anti-LRRC8A antibody (NBP2-32158 1:100, Novus Biologicals) or concentration-adjusted isotype control antibody (Cell Signaling Technology) was applied overnight after antigen retrieval with EDTA solution.

Techniques: Transfection, Isolation, Quantitative RT-PCR, Knockdown, Expressing, Marker, Control, Small Interfering RNA

Journal: JID Innovations

Article Title: The Volume-Regulated Anion Channel LRRC8 is Involved in the Initiation of Epidermal Differentiation and is Deregulated in Psoriasis

doi: 10.1016/j.xjidi.2025.100357

Figure Lengend Snippet: 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 × 10 4 /cm 2 cells were differentiated by Ca 2+ 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.

Article Snippet: For immunohistochemistry, the primary anti-LRRC8A antibody (NBP2-32158 1:100, Novus Biologicals) or concentration-adjusted isotype control antibody (Cell Signaling Technology) was applied overnight after antigen retrieval with EDTA solution.

Techniques: Staining, Western Blot, Activation Assay, Quantitative RT-PCR

Journal: JID Innovations

Article Title: The Volume-Regulated Anion Channel LRRC8 is Involved in the Initiation of Epidermal Differentiation and is Deregulated in Psoriasis

doi: 10.1016/j.xjidi.2025.100357

Figure Lengend Snippet: The membrane potential does not change during keratinocyte differentiation. ( a ) A sample of 1.5 × 10 4 was differentiated with 2 mM CaCl 2 24 hours after seeding in 96-well plates. At the indicated time points cells were stained for 60 minutes with 1 μM DiBAC 4 (3), and fluorescence was recorded as a measure for the MP. Afterwards, 20 μg/ml gramicidin was added for 30 minutes and the measurement was repeated. Fluorescence values were normalized to untreated cells (0 hours) (n = 7 independent experiments, mean ± SEM). ( b ) A sample of 1.5 × 10 4 keratinocytes was seeded in 96-well plates and transfected with siRNA after attachment overnight and treated with 2 mM CaCl 2 . Cells were stained for 60 minutes with 1 μM DiBAC 4 (3), fluorescence measured, and normalized to siCtrl cells without Ca 2+ (0 hours) (n = 5 independent experiments, mean ± SEM). MP, membrane potential; siCtrl, siRNA control; siLRRC8A, LRRC8A- specific siRNA; siRNA, small interfering RNA.

Article Snippet: For immunohistochemistry, the primary anti-LRRC8A antibody (NBP2-32158 1:100, Novus Biologicals) or concentration-adjusted isotype control antibody (Cell Signaling Technology) was applied overnight after antigen retrieval with EDTA solution.

Techniques: Membrane, Staining, Fluorescence, Transfection, Control, Small Interfering RNA

Journal: JID Innovations

Article Title: The Volume-Regulated Anion Channel LRRC8 is Involved in the Initiation of Epidermal Differentiation and is Deregulated in Psoriasis

doi: 10.1016/j.xjidi.2025.100357

Figure Lengend Snippet: LRRC8A is involved in early differentiation. LRRC8A is upregulated when KSCs divide and develop into TACs, where LRRC8A interacts with essential factors of early differentiation such as FOXM1 and NGFR. We hypothesize that this is mediated by the interaction of downstream signaling molecules and not by the ion channel function of LRRC8A itself, because the MP is not altered by the absence of LRRC8A. In psoriatic skin, LRRC8A is less abundantly expressed, which potentially contributes to the differentiation defects seen in these skin lesions. FOXM1, forkhead box protein M1; KSC, keratinocyte stem cell; LRRC8, leucine-rich repeat-containing protein 8; MP, membrane potential; NGFR, neuronal growth factor receptor; PMC, post-mitotic cell; TAC, transient amplifying cell.

Article Snippet: For immunohistochemistry, the primary anti-LRRC8A antibody (NBP2-32158 1:100, Novus Biologicals) or concentration-adjusted isotype control antibody (Cell Signaling Technology) was applied overnight after antigen retrieval with EDTA solution.

Techniques: Membrane

Journal: JID Innovations

Article Title: The Volume-Regulated Anion Channel LRRC8 is Involved in the Initiation of Epidermal Differentiation and is Deregulated in Psoriasis

doi: 10.1016/j.xjidi.2025.100357

Figure Lengend Snippet: LRRC8A protein is primarily localized in transient amplifying cells of the basal epidermal layer. ( a ) LRRC8A was stained via immunohistochemistry in punch biopsies from healthy donors. LRRC8A is localized in the basal epidermal layer, as described previously ( ; ). Bar represents 20 μm. ( b ) Native human skin was stained with a LRRC8A specific antibody (red) and an anti-CD271 antibody (green). Nuclei were stained with DAPI. Greyscale images are shown for each channel as well as an overlay image of the red and green channel. Scale bars represent 20 μm. ( c ) Keratinocyte populations were separated into KSC, TAC, and PMC based on their adhesion to collagen IV. Protein lysates from these populations were analyzed by Western blotting. LRRC8A was mainly detected in KSC and TAC, but not PMC, with the highest level in TAC. ( d ) Keratinocyte populations were differentiated by the addition of 2 mM CaCl 2 for 72 hours, analyzed by Western blotting, and quantified by densitometry (n = 16 independent experiments, mean + SEM). KSC, keratinocyte stem cell; LRRC8A shows its highest expression in early (-CaCl 2 ) TAC. LRRC8, leucine-rich repeat-containing protein 8; PMC, post-mitotic cell; TAC, transiently amplifying cell.

Article Snippet: LRRC8A antibodies were obtained from Novus Biologicals (NBP2-32158) or Sigma Aldrich (HPA016811—discontinued).

Techniques: Staining, Immunohistochemistry, Western Blot, Expressing

Journal: JID Innovations

Article Title: The Volume-Regulated Anion Channel LRRC8 is Involved in the Initiation of Epidermal Differentiation and is Deregulated in Psoriasis

doi: 10.1016/j.xjidi.2025.100357

Figure Lengend Snippet: LRRC8A gene expression increases with progressing epidermal maturation. RNA was isolated from keratinocyte populations and used for RNA sequencing (n = 4 independent experiments). Read counts were normalized and expression analysis was conducted using DESeq2. ( a ) To verify proper separation of keratinocyte populations, selected marker genes of basal or differentiating keratinocytes were normalized to the highest expressing population and compared to the others. Color changes from green to red indicate an increase in gene expression. ( b ) LRRC8A was differentially expressed among different populations (p: 3.6 × 10 −8 , Likelihood Ratio Test) with its transcripts significantly increasing during maturation. ( c ) All other LRRC8 family members, whose transcripts are severely less abundant than those of LRRC8A , are also differentially expressed among different populations ( P values: B: 9.1 × 10 −9 , C: 5.8 × 10 −10 , D: 7.01 × 10 −8 , E: 1.31 × 10 −25 , Likelihood Ratio Test). LRRC8B and LRRC8E increase during differentiation, while LRRC8C and LRRC8D decrease. LRRC8, leucine-rich repeat-containing protein 8.

Article Snippet: LRRC8A antibodies were obtained from Novus Biologicals (NBP2-32158) or Sigma Aldrich (HPA016811—discontinued).

Techniques: Gene Expression, Isolation, RNA Sequencing, Expressing, Marker

Journal: JID Innovations

Article Title: The Volume-Regulated Anion Channel LRRC8 is Involved in the Initiation of Epidermal Differentiation and is Deregulated in Psoriasis

doi: 10.1016/j.xjidi.2025.100357

Figure Lengend Snippet: LRRC8A gene and protein expression can be recapitulated in vitro. Through the addition of 2 mM CaCl 2 , harvested every 24 hours, and analyzed via RT-qPCR, 5.7 × 10 4 /cm 2 cells were differentiated ( a ) or Western blots ( b ). RNA is upregulated and stays increased during further differentiation (n = 11 independent experiments, mean + SEM) (Repeated measures ANOVA and Tukey post-test to correct for multiple comparisons, asterisks indicate changes compared to 0 hours [other comparisons n.s.]). In contrast, the amount of LRRC8A protein increases 24 hours after Ca 2+ addition and is downregulated afterward. Blots were quantified by densitometry (b, bottom) (n = 7 independent experiments, mean + SEM) (Repeated measures ANOVA and Tukey post-test to correct for multiple comparisons, asterisks indicate changes compared to 24 hours [other comparisons n.s.]). LRRC8, leucine-rich repeat-containing protein 8; n.s., not significant.

Article Snippet: LRRC8A antibodies were obtained from Novus Biologicals (NBP2-32158) or Sigma Aldrich (HPA016811—discontinued).

Techniques: Expressing, In Vitro, Quantitative RT-PCR, Western Blot

Journal: JID Innovations

Article Title: The Volume-Regulated Anion Channel LRRC8 is Involved in the Initiation of Epidermal Differentiation and is Deregulated in Psoriasis

doi: 10.1016/j.xjidi.2025.100357

Figure Lengend Snippet: Knockdown of LRRC8A interferes with epidermal differentiation. Keratinocytes (5.7 × 10 4 /cm 2 ) 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 CaCl 2 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.

Article Snippet: LRRC8A antibodies were obtained from Novus Biologicals (NBP2-32158) or Sigma Aldrich (HPA016811—discontinued).

Techniques: Knockdown, Transfection, Quantitative RT-PCR, Western Blot, Marker, Control, Small Interfering RNA

Journal: JID Innovations

Article Title: The Volume-Regulated Anion Channel LRRC8 is Involved in the Initiation of Epidermal Differentiation and is Deregulated in Psoriasis

doi: 10.1016/j.xjidi.2025.100357

Figure Lengend Snippet: Knockdown of LRRC8A does not interfere with keratinocyte proliferation. Primary keratinocytes (2.1 × 10 4 /cm 2 ) were seeded and transfected 24 hours later with 10 pmol siRNA specific for LRRC8A (siLRRC8A) or unspecific siCtrl. ( a ) Cells were trypsinized and counted every 24 hours. Cell count was normalized to t = 0 hours (first harvest 8 hours after transfection). No differences between siCtrl and siLRRC8A were detected (n = 7 independent experiments, mean ± SEM). ( b ) After counting cell numbers for proliferation, RNA was isolated and knockdown was verified via RT-qPCR (n = 7 independent experiments, mean + SEM) (2-way ANOVA with Sidak post-test to correct for multiple comparisons). LRRC8, leucine-rich repeat-containing protein 8; siCtrl, siRNA control; siLRRC8A, LRRC8A- specific siRNA; siRNA, small interfering RNA.

Article Snippet: LRRC8A antibodies were obtained from Novus Biologicals (NBP2-32158) or Sigma Aldrich (HPA016811—discontinued).

Techniques: Knockdown, Transfection, Cell Counting, Isolation, Quantitative RT-PCR, Control, Small Interfering RNA

Journal: JID Innovations

Article Title: The Volume-Regulated Anion Channel LRRC8 is Involved in the Initiation of Epidermal Differentiation and is Deregulated in Psoriasis

doi: 10.1016/j.xjidi.2025.100357

Figure Lengend Snippet: Efficient knockdown of LRRC8A with 2 specific siRNAs interferes with the expression of differentiation markers. ( a ) Keratinocytes (5.7 × 10 4 /cm 2 ) were seeded and transfected with 2 LRRC8A siRNAs (siLRRC8A 1 and 2) or an unspecific siCtrl or left untreated and harvested after 48 hours. Protein lysates were subjected to Western blotting with the indicated antibodies. ( b ) Keratinocytes (5.7 × 10 4 /cm 2 ) were seeded and transfected with 2 LRRC8A siRNAs or an unspecific siCtrl. Seven to 8 hours after transfection, cells were stimulated with 2 mM CaCl 2 to induce differentiation, harvested every 24 hours, and analyzed by Western blotting with the indicated antibodies. LRRC8, leucine-rich repeat-containing protein 8; siCtrl, siRNA control; siLRRC8A, LRRC8A- specific siRNA; siRNA, small interfering RNA.

Article Snippet: LRRC8A antibodies were obtained from Novus Biologicals (NBP2-32158) or Sigma Aldrich (HPA016811—discontinued).

Techniques: Knockdown, Expressing, Transfection, Western Blot, Control, Small Interfering RNA

Journal: JID Innovations

Article Title: The Volume-Regulated Anion Channel LRRC8 is Involved in the Initiation of Epidermal Differentiation and is Deregulated in Psoriasis

doi: 10.1016/j.xjidi.2025.100357

Figure Lengend Snippet: 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 CaCl 2 . 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.

Article Snippet: LRRC8A antibodies were obtained from Novus Biologicals (NBP2-32158) or Sigma Aldrich (HPA016811—discontinued).

Techniques: Transfection, Isolation, Quantitative RT-PCR, Knockdown, Expressing, Marker, Control, Small Interfering RNA

Journal: JID Innovations

Article Title: The Volume-Regulated Anion Channel LRRC8 is Involved in the Initiation of Epidermal Differentiation and is Deregulated in Psoriasis

doi: 10.1016/j.xjidi.2025.100357

Figure Lengend Snippet: 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 × 10 4 /cm 2 cells were differentiated by Ca 2+ 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.

Article Snippet: LRRC8A antibodies were obtained from Novus Biologicals (NBP2-32158) or Sigma Aldrich (HPA016811—discontinued).

Techniques: Staining, Western Blot, Activation Assay, Quantitative RT-PCR

Journal: JID Innovations

Article Title: The Volume-Regulated Anion Channel LRRC8 is Involved in the Initiation of Epidermal Differentiation and is Deregulated in Psoriasis

doi: 10.1016/j.xjidi.2025.100357

Figure Lengend Snippet: The membrane potential does not change during keratinocyte differentiation. ( a ) A sample of 1.5 × 10 4 was differentiated with 2 mM CaCl 2 24 hours after seeding in 96-well plates. At the indicated time points cells were stained for 60 minutes with 1 μM DiBAC 4 (3), and fluorescence was recorded as a measure for the MP. Afterwards, 20 μg/ml gramicidin was added for 30 minutes and the measurement was repeated. Fluorescence values were normalized to untreated cells (0 hours) (n = 7 independent experiments, mean ± SEM). ( b ) A sample of 1.5 × 10 4 keratinocytes was seeded in 96-well plates and transfected with siRNA after attachment overnight and treated with 2 mM CaCl 2 . Cells were stained for 60 minutes with 1 μM DiBAC 4 (3), fluorescence measured, and normalized to siCtrl cells without Ca 2+ (0 hours) (n = 5 independent experiments, mean ± SEM). MP, membrane potential; siCtrl, siRNA control; siLRRC8A, LRRC8A- specific siRNA; siRNA, small interfering RNA.

Article Snippet: LRRC8A antibodies were obtained from Novus Biologicals (NBP2-32158) or Sigma Aldrich (HPA016811—discontinued).

Techniques: Membrane, Staining, Fluorescence, Transfection, Control, Small Interfering RNA

Journal: JID Innovations

Article Title: The Volume-Regulated Anion Channel LRRC8 is Involved in the Initiation of Epidermal Differentiation and is Deregulated in Psoriasis

doi: 10.1016/j.xjidi.2025.100357

Figure Lengend Snippet: LRRC8A is involved in early differentiation. LRRC8A is upregulated when KSCs divide and develop into TACs, where LRRC8A interacts with essential factors of early differentiation such as FOXM1 and NGFR. We hypothesize that this is mediated by the interaction of downstream signaling molecules and not by the ion channel function of LRRC8A itself, because the MP is not altered by the absence of LRRC8A. In psoriatic skin, LRRC8A is less abundantly expressed, which potentially contributes to the differentiation defects seen in these skin lesions. FOXM1, forkhead box protein M1; KSC, keratinocyte stem cell; LRRC8, leucine-rich repeat-containing protein 8; MP, membrane potential; NGFR, neuronal growth factor receptor; PMC, post-mitotic cell; TAC, transient amplifying cell.

Article Snippet: LRRC8A antibodies were obtained from Novus Biologicals (NBP2-32158) or Sigma Aldrich (HPA016811—discontinued).

Techniques: Membrane