Journal: Journal of Biological Chemistry

Article Title: Catalytically Active Proteasomes Function Predominantly in the Cytosol

doi: 10.1074/jbc.m115.712406

Figure Lengend Snippet: FIGURE 3. Proteasome localization in cultured human cells. A, mammalian HEK293T cells were fractionated, and proteasome peptidase activity was determined in the nuclear and cytosolic fractions. Error bars represent S.E. from four independent experiments. Peptidase activity was detected solely in the cytoplasmic lysate, using either equal amount of protein (conc.) or proportional volumes of lysates (vol.) following fractionation. B, total, cytoplasmic, and nuclear fractions were prepared from HEK293T cells and separated in a native polyacrylamide gel. LLVY-AMC hydrolysis was examined in situ. A 5-fold higher loading of the nuclear extract (5 Nuc) was also examined. The positions of the single-capped and double-capped proteasomes are shown. C, protein extracts fromHEK293TcellswereseparatedbySDS-PAGEandtransferredtonitrocellulose,andthefiltersstainedwithPonceau-S.Adjustingthelysatestoequalprotein concentrations resulted in significant over-representation of nuclear proteins (lanes 1–3), as noted above (Fig. 2). Antibody reactions against tubulin (cytosolic) andhistoneH3(nuclear)showedthatthefractionationyieldednucleithatwerefreeofcytoplasmicproteins.Anasteriskinlane3identifiesanover-represented nuclear protein that cross-reacted with anti-Rpn12 antibody (seeD, below). D, immunoblot in C was reacted with antibodies indicated on theright. Proteasome subunits (19S and 20S subunits) and tubulin were detected only in the cytosol, and nuclear proteins (XPC, p53, and H3) were detected only in the nuclear fraction.

Article Snippet: Tubulin antibody was purchased from Life Technologies, Inc.; XPC antibody was purchased from Novus; proteasome and immunoproteasome antibodies were purchased from Enzo Biochem.

Techniques: Cell Culture, Activity Assay, Fractionation, In Situ, Western Blot

Journal: Nucleic Acids Research

Article Title: Single-molecule analysis of DNA-binding proteins from nuclear extracts (SMADNE)

doi: 10.1093/nar/gkad095

Figure Lengend Snippet: DNA-binding interactions of other DNA repair proteins. ( A ) The structure of eGFP-XPC (PDB codes 6CFI of Rad4 the yeast homolog to XPC and 4EUL). ( B ) A cartoon depiction of the DNA substrate used for XPC binding characterization, with UV damage sites shown in yellow and XPC binding shown in blue. Also shown is an example kymograph of eGFP-XPC binding and diffusing along the DNA in yellow. ( C ) CRTD analysis of XPC binding DNA with UV damage. ( D ) Distribution of motile and nonmotile XPC events. ( E ) An example MSD plot for analyzing XPC diffusion on DNA (D, in μm 2 /s). ( F ) Diffusion and α values for the diffusion of XPC on DNA. Event marked with asterisk was too short to determine an α value so it was defined as 1.0. ( G ) A structural model of APE1-tGFP from PDB code (5WNO and 4EUL). ( H ) Schematic and example kymograph of APE1 binding to DNA with nicks. ( I ) CRTD analysis of APE1 binding nicked DNA, with fit shown in blue. ( J ) A structural model of polβ-tGFP, taken from PDB codes (4KLO and 4EUL) and the tGFP modeled in. ( K ) Example schematics of polβ binding DNA containing nicks as well as a corresponding kymograph of an observation of polβ binding. ( L ) CRTD analysis of polβ binding nicked DNA, with the fit shown in blue. See .

Article Snippet: Primary antibodies used: PARP1 (1:100; abcam #ab227244), DDB2 (1:1000; abcam #ab181136), DDB1 (1:1000; Invitrogen #37-6200), XPC (1:1000; Novus #NB100-477) Polβ (1:1000; proteintech #18003-1-AP), OGG1 (1:1000; abcam #ab124741), and APE1 (1:100; Abcam #ab194).

Techniques: Binding Assay, Diffusion-based Assay

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