Journal: bioRxiv

Article Title: XPC loss-of-function triggers melanomagenesis through CDKN2A downregulation

doi: 10.1101/2025.04.03.646637

Figure Lengend Snippet: Xpc var/var mice did not express Cdkn2a in their tail skin melanocytes. ( A ) UMAP displays unsupervised clustering of all cells, identified using the shared nearest neighbor (SNN) modularity optimization-based clustering algorithm in Seurat, in the wild-type and Xpc var/var mice. The cell-type annotation of the clusters is based on established marker genes, as determined by the SCSA analysis, with clusters listed alphabetically. The number in the cluster name (e.g., T-cells 1 and T-cells 2) shows that the clusters were defined by different gene sets. The cell-type annotation of the clusters is based on established marker genes, as determined by the SCSA analysis, with clusters listed alphabetically. ( B ) UMAP displays Xpc expression levels in the different clusters of the wild-type and Xpc var/var mice. ( C ) UMAP displays Xpc expression levels in melanocytes of the wild-type and Xpc var/var mice. ( D ) Violin plot showing Xpc expression within melanocytes of wild-type and Xpc var/var mice. ( E ) Violin plot showing Cdkn2a expression within melanocytes of wild-type and Xpc var/var mice. ( F ) Circos plot showing the relationship between Xpc and Cdkn2a expression in the melanocytes of wild-type and Xpc var/var mice.

Article Snippet: Cells fixed and permeabilized using the Inside Stain Kit (Miltenyi Biotec, #130-090-477), following the manufacturer’s protocol, were incubated with DyLight 594-conjugated human XPC antibody (Boster Biological Technology, #A00473-1-Dyl594) and Alexa Fluor 488-conjugate CDKN2A/p16INK4a antibody (Bioss Antibodies, #bs-4592R-A488) for 10 min at room temperature in the dark, after which, fluorescence data from triplicate samples were acquired on a BD Fortessa (BD Biosciences) and analyzed using FlowJo v10.10.

Techniques: Marker, Expressing

Journal: bioRxiv

Article Title: XPC loss-of-function triggers melanomagenesis through CDKN2A downregulation

doi: 10.1101/2025.04.03.646637

Figure Lengend Snippet: XPC regulates CDKN2A expression. ( A-D) . qRT-PCR analysis for XPC (A, C) and CDKN2A (B, D) expression in HEK-293 (A, B) and WM-164 (C, D) parent, vector control (VC), and XPC knockdown (XPC-KD) cells. The expression levels were normalized to GAPDH and presented as mean ± SEM of the relative quantification (RQ) values from 3 independent experiments. *p<0.05, determined using one-way ANOVA with Tukey’s multiple comparisons. ( E ) Representative western blot for XPC and p16 INK4A on the parent, VC, and XPC knockdown (XPC-KD) HEK-293, WM-164, and SK-MEL-28 cells. GAPDH was used as the loading control. ( F ) Representative western blot for XPC and p16 INK4A fibroblasts derived from the patients and their parents. GAPDH was used as the loading control. ( G ) Representative western blot for XPC and p16 INK4A on the parent, VC, and XPC overexpressing (XPC) HEK-293, WM-164, and SK-MEL-28 cells. GAPDH was used as the loading control. ( H ) Representative western blot for XPC and p16 INK4A on the patient-derived fibroblasts transfected with either the VC or XPC overexpression plasmid. GAPDH was used as the loading control. ( I ) Representative dot plots of XPC and CDKN2A expression in control (HDFa) and patient-derived fibroblasts transfected with either the VC or XPC overexpression plasmid determined using flow cytometry. ( J ) Percentage cells not expressing p16 INK4A in the patient and control (HDFa) and patient-derived fibroblasts transfected with either the VC or XPC overexpression plasmid, determined by flow cytometry. Data expressed as mean ± SEM of 3 independent experiments. *p<0.05, determined using Student’s t -test.

Article Snippet: Cells fixed and permeabilized using the Inside Stain Kit (Miltenyi Biotec, #130-090-477), following the manufacturer’s protocol, were incubated with DyLight 594-conjugated human XPC antibody (Boster Biological Technology, #A00473-1-Dyl594) and Alexa Fluor 488-conjugate CDKN2A/p16INK4a antibody (Bioss Antibodies, #bs-4592R-A488) for 10 min at room temperature in the dark, after which, fluorescence data from triplicate samples were acquired on a BD Fortessa (BD Biosciences) and analyzed using FlowJo v10.10.

Techniques: Expressing, Quantitative RT-PCR, Plasmid Preparation, Control, Knockdown, Quantitative Proteomics, Western Blot, Derivative Assay, Transfection, Over Expression, Flow Cytometry

Journal: bioRxiv

Article Title: XPC loss-of-function triggers melanomagenesis through CDKN2A downregulation

doi: 10.1101/2025.04.03.646637

Figure Lengend Snippet: XPC binds to the CDKN2A promoter and is required for CDKN2A expression. ( A ) Schematic showing the gene structure of CDKN2A and the promotor regions. ( B ) ChIP fold enrichment of DNA fragments around the CDKN2A promoter regions by ChIP-qPCR. Four primer sets were used: Primer sets 1 and 2 targeted Promotor 1 and Prime sets 3 and 4 targeted Promotor 2 of the CDKN2A gene. Data expressed as mean ± SEM of 3 independent experiments. *p<0.05, determined using Student’s t -test. ( C ) Fold change in relative luminescence units (RLU) in wild-type (WT) and XPC-knock-down (XPC-KD) HEK-293 and WM-164 cells transiently transfected with a luciferase construct with the Promotor 2 region of CDKN2A or a random negative control (Rand) 48 h after transfection. Data from 3 independent experiments are shown. Error bar indicates SEM. *p<0.05, determined using two-way ANOVA with Tukey’s multiple comparisons. ( D ) Fold change in RLU in control fibroblasts and fibroblasts derived from the patients and their parents transiently transfected with a luciferase construct with the Promotor 2 region of CDKN2A or a random negative control (Rand) 48 h after transfection. Data from 3 independent experiments are shown. Error bar indicates SEM. *p<0.05, determined using two-way ANOVA with Tukey’s multiple comparisons. ( E ) Representative western blot for XPC and p16 INK4A in control fibroblasts (HDFa) and patient (Son)-derived fibroblasts treated with or without different concentrations of gentamicin. GAPDH was used as the loading control. ( F ) Fold change in RLU in control fibroblasts and patient (Son)-derived fibroblasts treated with different concentrations of gentamicin and transiently transfected with a luciferase construct with the Promotor 2 region of CDKN2A or a random negative control (Rand) 48 h after transfection. Data from 3 independent experiments are shown. Error bar indicates SEM. *p<0.05, determined using two-way ANOVA with Tukey’s multiple comparisons.

Article Snippet: Cells fixed and permeabilized using the Inside Stain Kit (Miltenyi Biotec, #130-090-477), following the manufacturer’s protocol, were incubated with DyLight 594-conjugated human XPC antibody (Boster Biological Technology, #A00473-1-Dyl594) and Alexa Fluor 488-conjugate CDKN2A/p16INK4a antibody (Bioss Antibodies, #bs-4592R-A488) for 10 min at room temperature in the dark, after which, fluorescence data from triplicate samples were acquired on a BD Fortessa (BD Biosciences) and analyzed using FlowJo v10.10.

Techniques: Expressing, ChIP-qPCR, Knockdown, Transfection, Luciferase, Construct, Negative Control, Control, Derivative Assay, Western Blot