Journal: RSC Chemical Biology

Article Title: Covalent targeting of PSMD14 by Eupalinolide B induces oncoprotein degradation and apoptosis in acute promyelocytic leukemia cells

doi: 10.1039/d5cb00197h

Figure Lengend Snippet: (A) Co-localization of PSMD14 (green) and EB-P (red) in HL-60 cells confirmed by immunofluorescence staining (scale bar = 10 μm). (B) Pull-down assay using EB-P, followed by Western blotting, confirming that EB binds to PSMD14 in situ . (C and D) Cellular thermal shift assay (CETSA)-WB indicating the direct interaction between EB and PSMD14. (E, Left) Structural model of the predicted PSMD14–EB complex. The protein is shown as a gray cartoon, and EB is depicted in orange sticks. Residues forming the predicted binding pocket are highlighted. (E, Right) Detailed view of the predicted binding interface. The key residue His183 of PSMD14 is represented as sticks and is labeled. (F) Mapping of the EB binding site on recombinant human PSMD14 by liquid chromatography-tandem mass spectrometry (LC-MS/MS). (G) Western blot showing PSMD14 protein levels in HL-60 cells incubated with or without EB (15 μM). (H) In vitro activity assay showing that EB (100 μM or 200 μM) significantly inhibits PSMD14 enzymatic activity. (I) Both EB (15 μM) and CZM (40 μM, a known PSMD14 inhibitor) markedly inhibited HL-60 cell proliferation. (J) Cell viability was assessed by CCK-8 assay following PSMD14 knockdown. (K) HL-60 cells transfected with siPSMD14-2 or siNC were treated with different concentrations of EB, and cell viability was measured by CCK-8 assay. (L–O) Cell cycle distribution of HL-60 cells treated with CZM. (P–S) PSMD14 knockdown significantly altered cell cycle progression in HL-60 cells. ** P < 0.01, *** P < 0.001, ns = not significant.

Article Snippet: To identify the direct binding site of EB on PSMD14, 40 μg of recombinant human PSMD14 protein (Signalchem Lifesciences) was incubated with 40 μM EB for 2 h at 37 °C.

Techniques: Immunofluorescence, Staining, Pull Down Assay, Western Blot, In Situ, Thermal Shift Assay, Binding Assay, Residue, Labeling, Recombinant, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Incubation, In Vitro, Activity Assay, CCK-8 Assay, Knockdown, Transfection

Journal: RSC Chemical Biology

Article Title: Covalent targeting of PSMD14 by Eupalinolide B induces oncoprotein degradation and apoptosis in acute promyelocytic leukemia cells

doi: 10.1039/d5cb00197h

Figure Lengend Snippet: (A and B) WB analysis of AKT1 and CDK4 protein expression following PSMD14 knockdown in HL-60 cells. (C and D) WB analysis of PSMD14, AKT1, and CDK4 in HL-60 cells treated with the PSMD14 inhibitor CZM. (E and F) Expression of PSMD14, AKT1, and CDK4 after treatment with cycloheximide (CHX, 20 μM), analyzed by Western blot. (G and H) WB analysis of PSMD14, AKT1, and CDK4 in HL-60 cells transfected with siPSMD14-2 and subsequently treated with EB; siNC was used as the negative control. (I–K) Quantification of PSMD14 (I), AKT1 (J), and CDK4 (K) protein levels with or without EB treatment. * P < 0.05, ** P < 0.01, *** P < 0.001, ns = not significant. Identical letters indicate no statistically significant difference, while different letters indicate P < 0.05.

Article Snippet: To identify the direct binding site of EB on PSMD14, 40 μg of recombinant human PSMD14 protein (Signalchem Lifesciences) was incubated with 40 μM EB for 2 h at 37 °C.

Techniques: Expressing, Knockdown, Western Blot, Transfection, Negative Control

Journal: bioRxiv

Article Title: PSMD14 drives melanoma cell survival and MAPK inhibitor resistance through histone H2A deubiquitination

doi: 10.64898/2026.02.27.708453

Figure Lengend Snippet: (A) Schematic diagram illustrating the strategy to identify deubiquitinases (DUBs) affecting proliferation and survival in BRAF mutant melanoma cells. siRNA knockdown in 501Mel cells was used, with confluence measured by IncuCyte at 96 h and results normalized to a non-targeting control. (B) Dot plots show normalized cell confluency for each siRNA versus siCtl. Candidates within the blue square demonstrate over 50% inhibition of confluence. PSMD14, highlighted in red, appears as one of the top hits. (C) Bar graph showing the correlation between dependency scores and candidates from siRNA DUB screening in skin cancer cell lines, using DepMap CRISPR data. Significant dependencies < -1 (red panel) and > -1 (green panel) illustrate essential genes and non-essential genes, respectivey. PSMD14 shows the strongest impact on cell survival. (D) Dependency scores of PSMD14 across pan-cancer cell lines, based on DepMap CRISPR data (21Q4). Melanoma cell lines, highlighted in red, are among the most affected by PSMD14 depletion. (E) PSMD14 expression levels in primary melanoma tumors and normal skin tissues, using GEPIA interactive analysis. *, P <0.05. ( F) PSMD14 expression across melanoma progression stages (GSE3189). (G) Kaplan-Meier overall survival curves in melanoma patients with high or low PSMD14 expression from TGCA SKCM dataset were obtained through SurvExpress (p=0.008; log-rank test). (H) GSEA of the TCGA SKCM dataset shows enrichment of hallmark gene sets including E2F targets, MYC targets and mitotic spindle in PSMD14^high tumors (positive NES; significant FWER-adjusted p-values)

Article Snippet: Primary antibodies used were as follows: PSMD14 (Elabscience, Cat# E-AB-63456), p21^Waf1/Cip1 (clone 12D1; Cell Signaling Technology, Cat# 2947), HSP90 (Santa Cruz Biotechnology, Cat# sc-13119), PARP (Cell Signaling Technology, Cat# 9542S), Cleaved Caspase-3 (Asp175; Cell Signaling Technology, Cat# 9661), γH2AX (Cell Signaling Technology, Cat# 9718S), H2Aub (Lys119) (clone D27C4; Cell Signaling Technology, Cat# 8240S), ubiquitinylated proteins (Merck Millipore, Cat# 04-263), Histone H2A (Cell Signaling Technology, Cat# 12349S), HA tag (Sigma-Aldrich, Cat# H9658), PSMD7 (Santa Cruz Biotechnology, Cat# sc-390705), PSMD12 (Santa Cruz Biotechnology, Cat# sc-398279), MCL-1 (Cell Signaling Technology, Cat# 94296), BCL-2 (Cell Signaling Technology, Cat# 15071), and phospho-ERK1/2 (Thr202/Tyr204; Cell Signaling Technology, Cat# 9101).

Techniques: Mutagenesis, Knockdown, Control, Inhibition, CRISPR, Expressing

Journal: bioRxiv

Article Title: PSMD14 drives melanoma cell survival and MAPK inhibitor resistance through histone H2A deubiquitination

doi: 10.64898/2026.02.27.708453

Figure Lengend Snippet: (A) Proportion of PSMD14 alterations in 471 SKCM patient samples from the cBioPortal database. (B) Hallmark enrichment and GSEA analyses associated with PSMD14 expression. (C) TCGA skin melanoma data showing a significant correlation between PSMD14 expression and melanoma aggressiveness–related hallmarks.

Article Snippet: Primary antibodies used were as follows: PSMD14 (Elabscience, Cat# E-AB-63456), p21^Waf1/Cip1 (clone 12D1; Cell Signaling Technology, Cat# 2947), HSP90 (Santa Cruz Biotechnology, Cat# sc-13119), PARP (Cell Signaling Technology, Cat# 9542S), Cleaved Caspase-3 (Asp175; Cell Signaling Technology, Cat# 9661), γH2AX (Cell Signaling Technology, Cat# 9718S), H2Aub (Lys119) (clone D27C4; Cell Signaling Technology, Cat# 8240S), ubiquitinylated proteins (Merck Millipore, Cat# 04-263), Histone H2A (Cell Signaling Technology, Cat# 12349S), HA tag (Sigma-Aldrich, Cat# H9658), PSMD7 (Santa Cruz Biotechnology, Cat# sc-390705), PSMD12 (Santa Cruz Biotechnology, Cat# sc-398279), MCL-1 (Cell Signaling Technology, Cat# 94296), BCL-2 (Cell Signaling Technology, Cat# 15071), and phospho-ERK1/2 (Thr202/Tyr204; Cell Signaling Technology, Cat# 9101).

Techniques: Expressing

Journal: bioRxiv

Article Title: PSMD14 drives melanoma cell survival and MAPK inhibitor resistance through histone H2A deubiquitination

doi: 10.64898/2026.02.27.708453

Figure Lengend Snippet: (A) Time-course heatmap showing cell confluency following PSMD14 knockdown with three independent siRNA sequences (siPSMD14#1, #2, and #3) compared to control siRNA (siCtl) in 501Mel and A375 BRAF-mutant melanoma cells. (B) Western blot analysis of PSMD14, CDK2, and p21Cip1 expression in 501Mel and A375 melanoma cells transfected with control siRNA (siCtl) or siPSMD14#1, #2, and #3. HSP90, loading control. (C) Quantification of cell survival after 72 h of PSMD14 depletion in melanoma cell lines harboring distinct oncogenic mutations. Data are mean ± SEM (n=6). **** P <0.0001, Wilcoxon signed-rank test. (D) Western blot analysis with anti-PSMD14 antibody showing expression of exogenous HA-PSMD14 and HA-PSMD14 JAMM M compared to endogenous PSMD14 in A375 cells. HSP90, loading control. (E) Bar graph showing cell confluency of HA-PSMD14, HA-PSMD14 JAMM M and HA-GFP-expressing A375 cells after 72 h. Data are presented as mean ± SEM (n = 3). **** P <0.0001, one-way ANOVA. (F) IC 50 determination after dose response of PSMD14i (8TQ) on human and murine melanoma cell lines with various oncogenic mutations. The corresponding IC 50 were determined by measuring the cell viability following AnnexinV and DAPI staining and by flow cytometry analysis at 72 h (mean ± SEM, n=3, two-way ANOVA). (G) Flow cytometry analysis of apoptosis after 72 h of PSMD14i treatment in A375 cells using Annexin V-FITC and DAPI staining. Bar graphs display the percentage of Annexin V- and DAPI-positive cells. Data are mean ± SEM (n = 6), ** P <0.01, **** P <0.0001, two-way ANOVA. (H) Flow cytometry analysis of apoptosis on A375 cells transfected for 72h with siCtl or siPSMD14. Bar graphs display the percentage of Annexin V- and DAPI-positive cells (mean ± SEM, n = 3, **** P <0.0001, two-way ANOVA. (I) Western blot analysis of apoptosis markers following PSMD14 inhibition or knockdown. HSP90, loading control. (J) Western blot analysis of gH2AX expression in a dose- and time-dependent manner following PSMD14i treatment in A375 and 501Mel cells. (K) Confocal microscopy analysis shows a marked increase in γH2AX-rich nuclear foci in PSMD14-depleted (siPSMD14, 75 ± 10 foci per nucleus) compared to control (siCtl, 5 ± 3 foci per nucleus) A375 cells with γH2AX foci in red and DAPI-stained nuclei in blue. Scale bar, 10 µm.

Article Snippet: Primary antibodies used were as follows: PSMD14 (Elabscience, Cat# E-AB-63456), p21^Waf1/Cip1 (clone 12D1; Cell Signaling Technology, Cat# 2947), HSP90 (Santa Cruz Biotechnology, Cat# sc-13119), PARP (Cell Signaling Technology, Cat# 9542S), Cleaved Caspase-3 (Asp175; Cell Signaling Technology, Cat# 9661), γH2AX (Cell Signaling Technology, Cat# 9718S), H2Aub (Lys119) (clone D27C4; Cell Signaling Technology, Cat# 8240S), ubiquitinylated proteins (Merck Millipore, Cat# 04-263), Histone H2A (Cell Signaling Technology, Cat# 12349S), HA tag (Sigma-Aldrich, Cat# H9658), PSMD7 (Santa Cruz Biotechnology, Cat# sc-390705), PSMD12 (Santa Cruz Biotechnology, Cat# sc-398279), MCL-1 (Cell Signaling Technology, Cat# 94296), BCL-2 (Cell Signaling Technology, Cat# 15071), and phospho-ERK1/2 (Thr202/Tyr204; Cell Signaling Technology, Cat# 9101).

Techniques: Knockdown, Control, Mutagenesis, Western Blot, Expressing, Transfection, Staining, Flow Cytometry, Inhibition, Confocal Microscopy

Journal: bioRxiv

Article Title: PSMD14 drives melanoma cell survival and MAPK inhibitor resistance through histone H2A deubiquitination

doi: 10.64898/2026.02.27.708453

Figure Lengend Snippet: (A) Representative crystal violet–stained images showing residual adherent cells following PSMD14 depletion (siPSMD14 #1, #2, and #3) versus control siRNA (siCtl) in UACC62, A375, and 501Mel cells, followed by crystal violet staining and quantification. (B) Quantification of long-term crystal violet survival assays shown in (A). Bar graphs show fold-change of cell survival in siPSMD14 treated cells relative to control (siCtl). Data are the mean ± SEM (n=3). **** P <0.0001, two-way ANOVA. (C) Time-lapse analysis of cell confluency in A375 cells expressing wild-type (WT) or mutant (JAMM M ) HA-PSMD14, compared with HA-GFP controls. Statistical significance for longitudinal proliferation curves was assessed using a two-way repeated-measures ANOVA . **** P <0.0001. (D) Long-term crystal violet survival assay showing the effect of HA-GFP, HA-PSMD14 WT and HA-PSMD14 JAMM M overexpression on A375 cell survival. Cells were stained with crystal violet after 2 weeks. A representative image of two independent experiments is shown . (E) Levels of poly-ubiquitinated proteins (poly-Ub) in 501Mel and A375 cells depleted of PSMD14 (siPSMD14 #1, #2, and #3), assessed by Western blot. (F) Levels of poly-ubiquitinated proteins (poly-Ub) in A375 cell lysates treated with increasing concentrations of PSMD14i 8-TQ, assessed by Western blot. HSP90, loading control. (G) Western blot analysis of γH2AX levels in PSMD14-depleted cells at 24 h, 48 h, and 72 h. HSP90, loading control.

Article Snippet: Primary antibodies used were as follows: PSMD14 (Elabscience, Cat# E-AB-63456), p21^Waf1/Cip1 (clone 12D1; Cell Signaling Technology, Cat# 2947), HSP90 (Santa Cruz Biotechnology, Cat# sc-13119), PARP (Cell Signaling Technology, Cat# 9542S), Cleaved Caspase-3 (Asp175; Cell Signaling Technology, Cat# 9661), γH2AX (Cell Signaling Technology, Cat# 9718S), H2Aub (Lys119) (clone D27C4; Cell Signaling Technology, Cat# 8240S), ubiquitinylated proteins (Merck Millipore, Cat# 04-263), Histone H2A (Cell Signaling Technology, Cat# 12349S), HA tag (Sigma-Aldrich, Cat# H9658), PSMD7 (Santa Cruz Biotechnology, Cat# sc-390705), PSMD12 (Santa Cruz Biotechnology, Cat# sc-398279), MCL-1 (Cell Signaling Technology, Cat# 94296), BCL-2 (Cell Signaling Technology, Cat# 15071), and phospho-ERK1/2 (Thr202/Tyr204; Cell Signaling Technology, Cat# 9101).

Techniques: Staining, Control, Expressing, Mutagenesis, Clonogenic Cell Survival Assay, Over Expression, Western Blot

Journal: bioRxiv

Article Title: PSMD14 drives melanoma cell survival and MAPK inhibitor resistance through histone H2A deubiquitination

doi: 10.64898/2026.02.27.708453

Figure Lengend Snippet: (A, B) Effect of PSMD14 inhibitor (8TQ; 15 mg/kg) on tumor growth in (A) YUMM1.7 murine melanoma (Braf V600E /Pten null /Cdkn2a null ) and (B) MaNRAS melanoma (Nras Q61K ) models. Tumor volumes were measured over time in mice treated with vehicle (n = 10) or PSMD14i (n = 8). Statistical significance was determined using two-way repeated-measures ANOVA followed by Bonferroni’s multiple comparisons test (**P < 0.01; ***P < 0.001). (C) Representative immunohistochemical staining of Ki67, γH2AX, and cleaved caspase-3 in YUMM1.7 tumors following 2 weeks of treatment with vehicle or PSMD14i. Scale bar, 100 µm.

Article Snippet: Primary antibodies used were as follows: PSMD14 (Elabscience, Cat# E-AB-63456), p21^Waf1/Cip1 (clone 12D1; Cell Signaling Technology, Cat# 2947), HSP90 (Santa Cruz Biotechnology, Cat# sc-13119), PARP (Cell Signaling Technology, Cat# 9542S), Cleaved Caspase-3 (Asp175; Cell Signaling Technology, Cat# 9661), γH2AX (Cell Signaling Technology, Cat# 9718S), H2Aub (Lys119) (clone D27C4; Cell Signaling Technology, Cat# 8240S), ubiquitinylated proteins (Merck Millipore, Cat# 04-263), Histone H2A (Cell Signaling Technology, Cat# 12349S), HA tag (Sigma-Aldrich, Cat# H9658), PSMD7 (Santa Cruz Biotechnology, Cat# sc-390705), PSMD12 (Santa Cruz Biotechnology, Cat# sc-398279), MCL-1 (Cell Signaling Technology, Cat# 94296), BCL-2 (Cell Signaling Technology, Cat# 15071), and phospho-ERK1/2 (Thr202/Tyr204; Cell Signaling Technology, Cat# 9101).

Techniques: Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: PSMD14 drives melanoma cell survival and MAPK inhibitor resistance through histone H2A deubiquitination

doi: 10.64898/2026.02.27.708453

Figure Lengend Snippet: (A) Schematic illustration of the strategy to study the PSMD14 interactome in melanoma cells. Lysates from A375 expressing HA-GFP or HA-PSMD14 cells are immunoprecipitated using anti-HA coated agarose beads and analyzed by quantitative mass spectrometry. (B) Volcano plot showing significantly differential enrichment of PSMD14 interactors over GFP. Relative intensity-based label-free quantification (LFQ) was processed using the MaxLFQ algorithm. A two-sample t -test was performed using permutation-based FDR-controlled at 0.5 %. The p- value was adjusted using a scaling factor S0 with a value of 1. The difference LFQ intensity was plotted against the −log10 of the p- value. The bold line indicated the applied threshold values ( p- value < 0.05; fold change ≥ 1.5). Among PSMD14 (blue dot), components of the proteasome (green dots) and histones (red dots) are highlighted. (C) Bar graph showing gene ontology (GO) analysis of the identified PSMD14-interacting proteins grouped into functional categories. (D) Schematic representation of the proposed function of PSMD14 in regulating H2A ubiquitination (H2Aub) and relieving H2Aub-dependent transcriptional repression. (E) Co-immunoprecipitation analysis of HA-PSMD14 confirming the binding of PSMD14 to proteasome-associated proteins PSMD7 and PSMD12, to histone H2A in A375 cells. (F) Co-immunoprecipitation of HA–PSMD14 and FLAG–H2A overexpressed in HEK293T cells. (G) Western blot analysis of H2Aub and total H2A levels following overexpression of HA–PSMD14 alone or in combination with PSMD14 inhibitor (PSMD14i) or following overexpression of HA-PSMD14 JAMM M mutant in HEK293T cells. (H) Western blot analysis of H2Aub levels in A375 melanoma cells treated with increasing concentrations of PSMD14i for 24h or over time with 1µM of PSMD14i. (I) Western blot detection of elevated H2Aub levels in A375 cells after 72 h of PSMD14 depletion (siPSMD14) compared to control (siCtl). HSP90, loading control. (J) Western blot analysis of A375 cells transfected with or without PSMD14 siRNA and subsequently treated with MG132 (20 μg/mL for 4 h), using the indicated antibodies. HSP90, loading control.

Article Snippet: Primary antibodies used were as follows: PSMD14 (Elabscience, Cat# E-AB-63456), p21^Waf1/Cip1 (clone 12D1; Cell Signaling Technology, Cat# 2947), HSP90 (Santa Cruz Biotechnology, Cat# sc-13119), PARP (Cell Signaling Technology, Cat# 9542S), Cleaved Caspase-3 (Asp175; Cell Signaling Technology, Cat# 9661), γH2AX (Cell Signaling Technology, Cat# 9718S), H2Aub (Lys119) (clone D27C4; Cell Signaling Technology, Cat# 8240S), ubiquitinylated proteins (Merck Millipore, Cat# 04-263), Histone H2A (Cell Signaling Technology, Cat# 12349S), HA tag (Sigma-Aldrich, Cat# H9658), PSMD7 (Santa Cruz Biotechnology, Cat# sc-390705), PSMD12 (Santa Cruz Biotechnology, Cat# sc-398279), MCL-1 (Cell Signaling Technology, Cat# 94296), BCL-2 (Cell Signaling Technology, Cat# 15071), and phospho-ERK1/2 (Thr202/Tyr204; Cell Signaling Technology, Cat# 9101).

Techniques: Expressing, Immunoprecipitation, Mass Spectrometry, Quantitative Proteomics, Functional Assay, Ubiquitin Proteomics, Binding Assay, Western Blot, Over Expression, Mutagenesis, Control, Transfection

Journal: bioRxiv

Article Title: PSMD14 drives melanoma cell survival and MAPK inhibitor resistance through histone H2A deubiquitination

doi: 10.64898/2026.02.27.708453

Figure Lengend Snippet: (A, B) Correlation between the H2Aub gene signature (Supplementary Table X) and the expression of (A) PSMD14 or (B) RING1B in the TCGA SKCM dataset (n = 421; UCSC Xena Browser). Correlations are assessed using Spearman’s rank-order test. (C) Heatmap showing mRNA expression of selected H2Aub target genes involved in apoptosis and metabolism in A375 melanoma cells stably expressing HA-PSMD14 or HA-PSMD14 JAMM M compared to control cells (HA-GFP). (D) Western blot analysis showing the effects of increasing expression of HA-PSMD14 on H2Aub, MCL1 and BLC2 levels. (E) Western blot analysis of H2Aub and MCL1 levels in A375 cells expressing HA-PSMD14 WT or HA-PSMD14 JAMM M compared to control cells (HA-GFP). (F) Heatmap showing expression changes in selected H2Aub target genes upon PSMD14 depletion in A375 cells. (G) Effect of PSMD14 knockdown (siPSMD14#1 and #2) in A375 cells on H2Aub, MCL1 and BLC2 levels compared to control (siCtl). HSP90, loading control. (H) Crystal violet staining (left) and quantification (right) of cell survival one week after knockdown of PSMD14 (siPSMD14) or RING1B (siRING1B) or the combined knockdown (siPSMD14/siRING1B). Bar graph shows cell survival relative to the control condition (siCtl). Data are mean ± SEM. Statistical significance was assessed using two-way ANOVA followed by Bonferroni’s multiple comparisons test. ****P ≤ 0.0001. (I) Western blot analysis of apoptotic markers (PARP, MCL1 and BCL2) and H2Aub levels following PSMD14 knockdown alone or in combination with RING1B depletion. HSP90, loading control.

Article Snippet: Primary antibodies used were as follows: PSMD14 (Elabscience, Cat# E-AB-63456), p21^Waf1/Cip1 (clone 12D1; Cell Signaling Technology, Cat# 2947), HSP90 (Santa Cruz Biotechnology, Cat# sc-13119), PARP (Cell Signaling Technology, Cat# 9542S), Cleaved Caspase-3 (Asp175; Cell Signaling Technology, Cat# 9661), γH2AX (Cell Signaling Technology, Cat# 9718S), H2Aub (Lys119) (clone D27C4; Cell Signaling Technology, Cat# 8240S), ubiquitinylated proteins (Merck Millipore, Cat# 04-263), Histone H2A (Cell Signaling Technology, Cat# 12349S), HA tag (Sigma-Aldrich, Cat# H9658), PSMD7 (Santa Cruz Biotechnology, Cat# sc-390705), PSMD12 (Santa Cruz Biotechnology, Cat# sc-398279), MCL-1 (Cell Signaling Technology, Cat# 94296), BCL-2 (Cell Signaling Technology, Cat# 15071), and phospho-ERK1/2 (Thr202/Tyr204; Cell Signaling Technology, Cat# 9101).

Techniques: Expressing, Stable Transfection, Control, Western Blot, Knockdown, Staining

Journal: bioRxiv

Article Title: PSMD14 drives melanoma cell survival and MAPK inhibitor resistance through histone H2A deubiquitination

doi: 10.64898/2026.02.27.708453

Figure Lengend Snippet: (A) Scatter plot with linear regression shows significantly correlated PSMD14 and MCL-1 proteins levels using DepMap-proteomic from a collection of 30 metastatic melanoma cell lines. (B) Spearman correlation matrix showing the association between PSMD14 and MCL1 mRNA expression in the TCGA SKCM dataset. (C) Representative genomic tracks of H2AK119ub enrichment at the MCL1 (chr1) and BCL2 (chr18) loci. ChIP-seq signals for H2AK119ub are shown for cells transfected with a control siRNA (siCtl, blue) or a siRNA targeting PSMD14 (siPSMD14, red). The displayed genomic regions are aligned with the corresponding gene annotations. Shaded green areas highlight regions with notable modulation of H2AK119ub levels. (D) Time lapse monitoring of cell death after transfection of A375 cells with control siRNA (siCtl), siPSMD14, siRING1B or the combination of siPSMD14 and siRING1B. Quantification of dead cells is determined using Incucyte® Cytotox red dye relative to cell confluency. Data are the mean ± SEM; ****P < 0.0001, two-way ANOVA test.

Article Snippet: Primary antibodies used were as follows: PSMD14 (Elabscience, Cat# E-AB-63456), p21^Waf1/Cip1 (clone 12D1; Cell Signaling Technology, Cat# 2947), HSP90 (Santa Cruz Biotechnology, Cat# sc-13119), PARP (Cell Signaling Technology, Cat# 9542S), Cleaved Caspase-3 (Asp175; Cell Signaling Technology, Cat# 9661), γH2AX (Cell Signaling Technology, Cat# 9718S), H2Aub (Lys119) (clone D27C4; Cell Signaling Technology, Cat# 8240S), ubiquitinylated proteins (Merck Millipore, Cat# 04-263), Histone H2A (Cell Signaling Technology, Cat# 12349S), HA tag (Sigma-Aldrich, Cat# H9658), PSMD7 (Santa Cruz Biotechnology, Cat# sc-390705), PSMD12 (Santa Cruz Biotechnology, Cat# sc-398279), MCL-1 (Cell Signaling Technology, Cat# 94296), BCL-2 (Cell Signaling Technology, Cat# 15071), and phospho-ERK1/2 (Thr202/Tyr204; Cell Signaling Technology, Cat# 9101).

Techniques: Expressing, ChIP-sequencing, Transfection, Control

Journal: bioRxiv

Article Title: PSMD14 drives melanoma cell survival and MAPK inhibitor resistance through histone H2A deubiquitination

doi: 10.64898/2026.02.27.708453

Figure Lengend Snippet: (A) Bubble plot showing Gene Ontology (GO) normalized enrichment scores (NES) in BRAFV600E melanoma cells treated with BRAFi versus DMSO (GSE98314). GO terms related to protein deubiquitination and H2Aub target genes are highlighted. FDR-adjusted p -values are represented as –log 10 color scale. (B) PSMD14 expression in A375 cells after BRAF inhibitor (GSK2118436) or MEK inhibitor (GSK1120212) administration alone or in combination (GSE35230). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Two-way ANOVA. (C) PSMD14 expression in A375 and 501MEL melanoma cell lines exposed to DMSO or the combination of vemurafenib (10µM) and cobimetinib (1µM) for 24h. Data are presented as mean ± SEM (n= 4). *P < 0.05, **P < 0.01, one way ANOVA. (D) Western blot analysis of pERK1/2 and H2Aub levels on NRAS mutant melanoma cells SBcl2 exposed to increasing dose of trametinib (MEKi) for 24h.

Article Snippet: Primary antibodies used were as follows: PSMD14 (Elabscience, Cat# E-AB-63456), p21^Waf1/Cip1 (clone 12D1; Cell Signaling Technology, Cat# 2947), HSP90 (Santa Cruz Biotechnology, Cat# sc-13119), PARP (Cell Signaling Technology, Cat# 9542S), Cleaved Caspase-3 (Asp175; Cell Signaling Technology, Cat# 9661), γH2AX (Cell Signaling Technology, Cat# 9718S), H2Aub (Lys119) (clone D27C4; Cell Signaling Technology, Cat# 8240S), ubiquitinylated proteins (Merck Millipore, Cat# 04-263), Histone H2A (Cell Signaling Technology, Cat# 12349S), HA tag (Sigma-Aldrich, Cat# H9658), PSMD7 (Santa Cruz Biotechnology, Cat# sc-390705), PSMD12 (Santa Cruz Biotechnology, Cat# sc-398279), MCL-1 (Cell Signaling Technology, Cat# 94296), BCL-2 (Cell Signaling Technology, Cat# 15071), and phospho-ERK1/2 (Thr202/Tyr204; Cell Signaling Technology, Cat# 9101).

Techniques: Expressing, Western Blot, Mutagenesis

Journal: bioRxiv

Article Title: PSMD14 drives melanoma cell survival and MAPK inhibitor resistance through histone H2A deubiquitination

doi: 10.64898/2026.02.27.708453

Figure Lengend Snippet: (A) Heatmap showing transcript levels of genes involved in protein deubiquitination biological process (GO:0016579) in BRAF-mutant melanoma cell lines treated with BRAF inhibitor dabrafenib with or without MEK inhibitor trametinib compared to DMSO (GSE98314). Each column represents one cell line. Red: high expression, blue: low expression. PSMD14 is marked by a red arrow. HSP90, loading control. (B) Western blot analysis of PSMD14 and H2Aub levels in A375 cells treated with increasing doses and combinations of BRAFi and MEKi. HSP90, loading control. (C) Enrichment analysis of the H2Aub gene signature in BRAF-mutant melanoma cells exposed to BRAFi and/or MEKi (GSE98314). (D) Long-term crystal violet survival assay showing the effect of PSMD14 and RING1B depletion on residual cell survival after 21 days of BRAFi/MEKi treatment. Left, representative images of crystal violet cell staining. Right, bar graph showing the quantification of 2 independent experiments. Data are presented as mean ± SEM. Significance was determined with two-way ANOVA followed by Bonferroni’s multiple comparisons test. ****P ≤ 0.0001. (E) Long-term crystal violet survival assay showing the effect of HA-PSMD14 WT or HA-PSMD14 JAMM M overexpression, in the presence or absence of PSMD14i (1 µM), on cell survival after 21 days of BRAFi/MEKi treatment. Cells were stained with crystal violet. A representative image of two independent experiments is shown. (F) Western blot analysis of PSMD14, H2Aub and P-ERK1/2 levels in A375 cells across stages of MAPKi treatment. P (untreated parental cells), TX (BRAFi/MEKi 24h), DTP (drug tolerant persister cells BRAFi/MEKi 21 days), DR (BRAFi/MEKi drug resistant cells). (G) PSMD14 expression (left panel) and H2Aub gene signature (right panel) scores were extracted from single cell RNA sequencing of the MEL006 patient-derived xenograft model at drug-response phases (GSE116237) : pre-treatment (T0), 4 days of BRAFi/MEKi treatment (Phase 1), minimal residual disease (28 days on BRAFi/MEKi, Phase 2), and resistance phase (Phase 3). *P < 0.05. ****P < 0.0001. Two-way ANOVA. (H) Cell death analysis on A375 cells transfected with control siRNA (siCtl) or siPSMD14 and exposed or not to BRAFi/MEKi for 24 h. Cell death was monitored in real time using Incucyte® with Cytotox Red labelling. Data are mean ± SEM and statistical significance was assessed with two-way repeated-measures ANOVA. ****P ≤ 0.0001. (I) Synergy score determined according to the Loewe additivity model on A375 cells treated with the indicated combination of PSMD14i and BRAFi/MEKi. Scores >1 are characteristic of synergy above additivity and also visible in 501MEL and UACC62 BRAF-mutant melanoma cells (inset). (J) Schematic illustration of the A375 xenograft murine model of melanoma response to targeted therapies (left panel) and tumor volume changes in xenografts treated with BRAFi+MEKi (vemurafenib 30 mg/kg + cobimetinib 7 mg/kg, orally every 2 days) alone or combined with PSMD14i (8TQ, 15 mg/kg) (right panel). Vehicle-treated mice served as control. Group sizes were: Vehicle n = 10, BRAFi+MEKi n = 11, and BRAFi+MEKi+PSMD14i n = 8. Data represent mean ± SEM. **** P <0.0001, two way repeated measures ANOVA followed by Bonferroni correction.

Article Snippet: Primary antibodies used were as follows: PSMD14 (Elabscience, Cat# E-AB-63456), p21^Waf1/Cip1 (clone 12D1; Cell Signaling Technology, Cat# 2947), HSP90 (Santa Cruz Biotechnology, Cat# sc-13119), PARP (Cell Signaling Technology, Cat# 9542S), Cleaved Caspase-3 (Asp175; Cell Signaling Technology, Cat# 9661), γH2AX (Cell Signaling Technology, Cat# 9718S), H2Aub (Lys119) (clone D27C4; Cell Signaling Technology, Cat# 8240S), ubiquitinylated proteins (Merck Millipore, Cat# 04-263), Histone H2A (Cell Signaling Technology, Cat# 12349S), HA tag (Sigma-Aldrich, Cat# H9658), PSMD7 (Santa Cruz Biotechnology, Cat# sc-390705), PSMD12 (Santa Cruz Biotechnology, Cat# sc-398279), MCL-1 (Cell Signaling Technology, Cat# 94296), BCL-2 (Cell Signaling Technology, Cat# 15071), and phospho-ERK1/2 (Thr202/Tyr204; Cell Signaling Technology, Cat# 9101).

Techniques: Mutagenesis, Expressing, Control, Western Blot, Clonogenic Cell Survival Assay, Staining, Over Expression, Single Cell, RNA Sequencing, Derivative Assay, Transfection

Journal: bioRxiv

Article Title: PSMD14 drives melanoma cell survival and MAPK inhibitor resistance through histone H2A deubiquitination

doi: 10.64898/2026.02.27.708453

Figure Lengend Snippet: (A) Schematic diagram illustrating the strategy to identify deubiquitinases (DUBs) affecting proliferation and survival in BRAF mutant melanoma cells. siRNA knockdown in 501Mel cells was used, with confluence measured by IncuCyte at 96 h and results normalized to a non-targeting control. (B) Dot plots show normalized cell confluency for each siRNA versus siCtl. Candidates within the blue square demonstrate over 50% inhibition of confluence. PSMD14, highlighted in red, appears as one of the top hits. (C) Bar graph showing the correlation between dependency scores and candidates from siRNA DUB screening in skin cancer cell lines, using DepMap CRISPR data. Significant dependencies < -1 (red panel) and > -1 (green panel) illustrate essential genes and non-essential genes, respectivey. PSMD14 shows the strongest impact on cell survival. (D) Dependency scores of PSMD14 across pan-cancer cell lines, based on DepMap CRISPR data (21Q4). Melanoma cell lines, highlighted in red, are among the most affected by PSMD14 depletion. (E) PSMD14 expression levels in primary melanoma tumors and normal skin tissues, using GEPIA interactive analysis. *, P <0.05. ( F) PSMD14 expression across melanoma progression stages (GSE3189). (G) Kaplan-Meier overall survival curves in melanoma patients with high or low PSMD14 expression from TGCA SKCM dataset were obtained through SurvExpress (p=0.008; log-rank test). (H) GSEA of the TCGA SKCM dataset shows enrichment of hallmark gene sets including E2F targets, MYC targets and mitotic spindle in PSMD14^high tumors (positive NES; significant FWER-adjusted p-values)

Article Snippet: The PSMD14 inhibitor 8-(tosylamino)quinoline (8-TQ) was purchased from Merck.

Techniques: Mutagenesis, Knockdown, Control, Inhibition, CRISPR, Expressing

Journal: bioRxiv

Article Title: PSMD14 drives melanoma cell survival and MAPK inhibitor resistance through histone H2A deubiquitination

doi: 10.64898/2026.02.27.708453

Figure Lengend Snippet: (A) Proportion of PSMD14 alterations in 471 SKCM patient samples from the cBioPortal database. (B) Hallmark enrichment and GSEA analyses associated with PSMD14 expression. (C) TCGA skin melanoma data showing a significant correlation between PSMD14 expression and melanoma aggressiveness–related hallmarks.

Article Snippet: The PSMD14 inhibitor 8-(tosylamino)quinoline (8-TQ) was purchased from Merck.

Techniques: Expressing

Journal: bioRxiv

Article Title: PSMD14 drives melanoma cell survival and MAPK inhibitor resistance through histone H2A deubiquitination

doi: 10.64898/2026.02.27.708453

Figure Lengend Snippet: (A) Time-course heatmap showing cell confluency following PSMD14 knockdown with three independent siRNA sequences (siPSMD14#1, #2, and #3) compared to control siRNA (siCtl) in 501Mel and A375 BRAF-mutant melanoma cells. (B) Western blot analysis of PSMD14, CDK2, and p21Cip1 expression in 501Mel and A375 melanoma cells transfected with control siRNA (siCtl) or siPSMD14#1, #2, and #3. HSP90, loading control. (C) Quantification of cell survival after 72 h of PSMD14 depletion in melanoma cell lines harboring distinct oncogenic mutations. Data are mean ± SEM (n=6). **** P <0.0001, Wilcoxon signed-rank test. (D) Western blot analysis with anti-PSMD14 antibody showing expression of exogenous HA-PSMD14 and HA-PSMD14 JAMM M compared to endogenous PSMD14 in A375 cells. HSP90, loading control. (E) Bar graph showing cell confluency of HA-PSMD14, HA-PSMD14 JAMM M and HA-GFP-expressing A375 cells after 72 h. Data are presented as mean ± SEM (n = 3). **** P <0.0001, one-way ANOVA. (F) IC 50 determination after dose response of PSMD14i (8TQ) on human and murine melanoma cell lines with various oncogenic mutations. The corresponding IC 50 were determined by measuring the cell viability following AnnexinV and DAPI staining and by flow cytometry analysis at 72 h (mean ± SEM, n=3, two-way ANOVA). (G) Flow cytometry analysis of apoptosis after 72 h of PSMD14i treatment in A375 cells using Annexin V-FITC and DAPI staining. Bar graphs display the percentage of Annexin V- and DAPI-positive cells. Data are mean ± SEM (n = 6), ** P <0.01, **** P <0.0001, two-way ANOVA. (H) Flow cytometry analysis of apoptosis on A375 cells transfected for 72h with siCtl or siPSMD14. Bar graphs display the percentage of Annexin V- and DAPI-positive cells (mean ± SEM, n = 3, **** P <0.0001, two-way ANOVA. (I) Western blot analysis of apoptosis markers following PSMD14 inhibition or knockdown. HSP90, loading control. (J) Western blot analysis of gH2AX expression in a dose- and time-dependent manner following PSMD14i treatment in A375 and 501Mel cells. (K) Confocal microscopy analysis shows a marked increase in γH2AX-rich nuclear foci in PSMD14-depleted (siPSMD14, 75 ± 10 foci per nucleus) compared to control (siCtl, 5 ± 3 foci per nucleus) A375 cells with γH2AX foci in red and DAPI-stained nuclei in blue. Scale bar, 10 µm.

Article Snippet: The PSMD14 inhibitor 8-(tosylamino)quinoline (8-TQ) was purchased from Merck.

Techniques: Knockdown, Control, Mutagenesis, Western Blot, Expressing, Transfection, Staining, Flow Cytometry, Inhibition, Confocal Microscopy

Journal: bioRxiv

Article Title: PSMD14 drives melanoma cell survival and MAPK inhibitor resistance through histone H2A deubiquitination

doi: 10.64898/2026.02.27.708453

Figure Lengend Snippet: (A) Representative crystal violet–stained images showing residual adherent cells following PSMD14 depletion (siPSMD14 #1, #2, and #3) versus control siRNA (siCtl) in UACC62, A375, and 501Mel cells, followed by crystal violet staining and quantification. (B) Quantification of long-term crystal violet survival assays shown in (A). Bar graphs show fold-change of cell survival in siPSMD14 treated cells relative to control (siCtl). Data are the mean ± SEM (n=3). **** P <0.0001, two-way ANOVA. (C) Time-lapse analysis of cell confluency in A375 cells expressing wild-type (WT) or mutant (JAMM M ) HA-PSMD14, compared with HA-GFP controls. Statistical significance for longitudinal proliferation curves was assessed using a two-way repeated-measures ANOVA . **** P <0.0001. (D) Long-term crystal violet survival assay showing the effect of HA-GFP, HA-PSMD14 WT and HA-PSMD14 JAMM M overexpression on A375 cell survival. Cells were stained with crystal violet after 2 weeks. A representative image of two independent experiments is shown . (E) Levels of poly-ubiquitinated proteins (poly-Ub) in 501Mel and A375 cells depleted of PSMD14 (siPSMD14 #1, #2, and #3), assessed by Western blot. (F) Levels of poly-ubiquitinated proteins (poly-Ub) in A375 cell lysates treated with increasing concentrations of PSMD14i 8-TQ, assessed by Western blot. HSP90, loading control. (G) Western blot analysis of γH2AX levels in PSMD14-depleted cells at 24 h, 48 h, and 72 h. HSP90, loading control.

Article Snippet: The PSMD14 inhibitor 8-(tosylamino)quinoline (8-TQ) was purchased from Merck.

Techniques: Staining, Control, Expressing, Mutagenesis, Clonogenic Cell Survival Assay, Over Expression, Western Blot

Journal: bioRxiv

Article Title: PSMD14 drives melanoma cell survival and MAPK inhibitor resistance through histone H2A deubiquitination

doi: 10.64898/2026.02.27.708453

Figure Lengend Snippet: (A, B) Effect of PSMD14 inhibitor (8TQ; 15 mg/kg) on tumor growth in (A) YUMM1.7 murine melanoma (Braf V600E /Pten null /Cdkn2a null ) and (B) MaNRAS melanoma (Nras Q61K ) models. Tumor volumes were measured over time in mice treated with vehicle (n = 10) or PSMD14i (n = 8). Statistical significance was determined using two-way repeated-measures ANOVA followed by Bonferroni’s multiple comparisons test (**P < 0.01; ***P < 0.001). (C) Representative immunohistochemical staining of Ki67, γH2AX, and cleaved caspase-3 in YUMM1.7 tumors following 2 weeks of treatment with vehicle or PSMD14i. Scale bar, 100 µm.

Article Snippet: The PSMD14 inhibitor 8-(tosylamino)quinoline (8-TQ) was purchased from Merck.

Techniques: Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: PSMD14 drives melanoma cell survival and MAPK inhibitor resistance through histone H2A deubiquitination

doi: 10.64898/2026.02.27.708453

Figure Lengend Snippet: (A) Schematic illustration of the strategy to study the PSMD14 interactome in melanoma cells. Lysates from A375 expressing HA-GFP or HA-PSMD14 cells are immunoprecipitated using anti-HA coated agarose beads and analyzed by quantitative mass spectrometry. (B) Volcano plot showing significantly differential enrichment of PSMD14 interactors over GFP. Relative intensity-based label-free quantification (LFQ) was processed using the MaxLFQ algorithm. A two-sample t -test was performed using permutation-based FDR-controlled at 0.5 %. The p- value was adjusted using a scaling factor S0 with a value of 1. The difference LFQ intensity was plotted against the −log10 of the p- value. The bold line indicated the applied threshold values ( p- value < 0.05; fold change ≥ 1.5). Among PSMD14 (blue dot), components of the proteasome (green dots) and histones (red dots) are highlighted. (C) Bar graph showing gene ontology (GO) analysis of the identified PSMD14-interacting proteins grouped into functional categories. (D) Schematic representation of the proposed function of PSMD14 in regulating H2A ubiquitination (H2Aub) and relieving H2Aub-dependent transcriptional repression. (E) Co-immunoprecipitation analysis of HA-PSMD14 confirming the binding of PSMD14 to proteasome-associated proteins PSMD7 and PSMD12, to histone H2A in A375 cells. (F) Co-immunoprecipitation of HA–PSMD14 and FLAG–H2A overexpressed in HEK293T cells. (G) Western blot analysis of H2Aub and total H2A levels following overexpression of HA–PSMD14 alone or in combination with PSMD14 inhibitor (PSMD14i) or following overexpression of HA-PSMD14 JAMM M mutant in HEK293T cells. (H) Western blot analysis of H2Aub levels in A375 melanoma cells treated with increasing concentrations of PSMD14i for 24h or over time with 1µM of PSMD14i. (I) Western blot detection of elevated H2Aub levels in A375 cells after 72 h of PSMD14 depletion (siPSMD14) compared to control (siCtl). HSP90, loading control. (J) Western blot analysis of A375 cells transfected with or without PSMD14 siRNA and subsequently treated with MG132 (20 μg/mL for 4 h), using the indicated antibodies. HSP90, loading control.

Article Snippet: The PSMD14 inhibitor 8-(tosylamino)quinoline (8-TQ) was purchased from Merck.

Techniques: Expressing, Immunoprecipitation, Mass Spectrometry, Quantitative Proteomics, Functional Assay, Ubiquitin Proteomics, Binding Assay, Western Blot, Over Expression, Mutagenesis, Control, Transfection

Journal: bioRxiv

Article Title: PSMD14 drives melanoma cell survival and MAPK inhibitor resistance through histone H2A deubiquitination

doi: 10.64898/2026.02.27.708453

Figure Lengend Snippet: (A, B) Correlation between the H2Aub gene signature (Supplementary Table X) and the expression of (A) PSMD14 or (B) RING1B in the TCGA SKCM dataset (n = 421; UCSC Xena Browser). Correlations are assessed using Spearman’s rank-order test. (C) Heatmap showing mRNA expression of selected H2Aub target genes involved in apoptosis and metabolism in A375 melanoma cells stably expressing HA-PSMD14 or HA-PSMD14 JAMM M compared to control cells (HA-GFP). (D) Western blot analysis showing the effects of increasing expression of HA-PSMD14 on H2Aub, MCL1 and BLC2 levels. (E) Western blot analysis of H2Aub and MCL1 levels in A375 cells expressing HA-PSMD14 WT or HA-PSMD14 JAMM M compared to control cells (HA-GFP). (F) Heatmap showing expression changes in selected H2Aub target genes upon PSMD14 depletion in A375 cells. (G) Effect of PSMD14 knockdown (siPSMD14#1 and #2) in A375 cells on H2Aub, MCL1 and BLC2 levels compared to control (siCtl). HSP90, loading control. (H) Crystal violet staining (left) and quantification (right) of cell survival one week after knockdown of PSMD14 (siPSMD14) or RING1B (siRING1B) or the combined knockdown (siPSMD14/siRING1B). Bar graph shows cell survival relative to the control condition (siCtl). Data are mean ± SEM. Statistical significance was assessed using two-way ANOVA followed by Bonferroni’s multiple comparisons test. ****P ≤ 0.0001. (I) Western blot analysis of apoptotic markers (PARP, MCL1 and BCL2) and H2Aub levels following PSMD14 knockdown alone or in combination with RING1B depletion. HSP90, loading control.

Article Snippet: The PSMD14 inhibitor 8-(tosylamino)quinoline (8-TQ) was purchased from Merck.

Techniques: Expressing, Stable Transfection, Control, Western Blot, Knockdown, Staining

Journal: bioRxiv

Article Title: PSMD14 drives melanoma cell survival and MAPK inhibitor resistance through histone H2A deubiquitination

doi: 10.64898/2026.02.27.708453

Figure Lengend Snippet: (A) Scatter plot with linear regression shows significantly correlated PSMD14 and MCL-1 proteins levels using DepMap-proteomic from a collection of 30 metastatic melanoma cell lines. (B) Spearman correlation matrix showing the association between PSMD14 and MCL1 mRNA expression in the TCGA SKCM dataset. (C) Representative genomic tracks of H2AK119ub enrichment at the MCL1 (chr1) and BCL2 (chr18) loci. ChIP-seq signals for H2AK119ub are shown for cells transfected with a control siRNA (siCtl, blue) or a siRNA targeting PSMD14 (siPSMD14, red). The displayed genomic regions are aligned with the corresponding gene annotations. Shaded green areas highlight regions with notable modulation of H2AK119ub levels. (D) Time lapse monitoring of cell death after transfection of A375 cells with control siRNA (siCtl), siPSMD14, siRING1B or the combination of siPSMD14 and siRING1B. Quantification of dead cells is determined using Incucyte® Cytotox red dye relative to cell confluency. Data are the mean ± SEM; ****P < 0.0001, two-way ANOVA test.

Article Snippet: The PSMD14 inhibitor 8-(tosylamino)quinoline (8-TQ) was purchased from Merck.

Techniques: Expressing, ChIP-sequencing, Transfection, Control

Journal: bioRxiv

Article Title: PSMD14 drives melanoma cell survival and MAPK inhibitor resistance through histone H2A deubiquitination

doi: 10.64898/2026.02.27.708453

Figure Lengend Snippet: (A) Bubble plot showing Gene Ontology (GO) normalized enrichment scores (NES) in BRAFV600E melanoma cells treated with BRAFi versus DMSO (GSE98314). GO terms related to protein deubiquitination and H2Aub target genes are highlighted. FDR-adjusted p -values are represented as –log 10 color scale. (B) PSMD14 expression in A375 cells after BRAF inhibitor (GSK2118436) or MEK inhibitor (GSK1120212) administration alone or in combination (GSE35230). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Two-way ANOVA. (C) PSMD14 expression in A375 and 501MEL melanoma cell lines exposed to DMSO or the combination of vemurafenib (10µM) and cobimetinib (1µM) for 24h. Data are presented as mean ± SEM (n= 4). *P < 0.05, **P < 0.01, one way ANOVA. (D) Western blot analysis of pERK1/2 and H2Aub levels on NRAS mutant melanoma cells SBcl2 exposed to increasing dose of trametinib (MEKi) for 24h.

Article Snippet: The PSMD14 inhibitor 8-(tosylamino)quinoline (8-TQ) was purchased from Merck.

Techniques: Expressing, Western Blot, Mutagenesis

Journal: bioRxiv

Article Title: PSMD14 drives melanoma cell survival and MAPK inhibitor resistance through histone H2A deubiquitination

doi: 10.64898/2026.02.27.708453

Figure Lengend Snippet: (A) Heatmap showing transcript levels of genes involved in protein deubiquitination biological process (GO:0016579) in BRAF-mutant melanoma cell lines treated with BRAF inhibitor dabrafenib with or without MEK inhibitor trametinib compared to DMSO (GSE98314). Each column represents one cell line. Red: high expression, blue: low expression. PSMD14 is marked by a red arrow. HSP90, loading control. (B) Western blot analysis of PSMD14 and H2Aub levels in A375 cells treated with increasing doses and combinations of BRAFi and MEKi. HSP90, loading control. (C) Enrichment analysis of the H2Aub gene signature in BRAF-mutant melanoma cells exposed to BRAFi and/or MEKi (GSE98314). (D) Long-term crystal violet survival assay showing the effect of PSMD14 and RING1B depletion on residual cell survival after 21 days of BRAFi/MEKi treatment. Left, representative images of crystal violet cell staining. Right, bar graph showing the quantification of 2 independent experiments. Data are presented as mean ± SEM. Significance was determined with two-way ANOVA followed by Bonferroni’s multiple comparisons test. ****P ≤ 0.0001. (E) Long-term crystal violet survival assay showing the effect of HA-PSMD14 WT or HA-PSMD14 JAMM M overexpression, in the presence or absence of PSMD14i (1 µM), on cell survival after 21 days of BRAFi/MEKi treatment. Cells were stained with crystal violet. A representative image of two independent experiments is shown. (F) Western blot analysis of PSMD14, H2Aub and P-ERK1/2 levels in A375 cells across stages of MAPKi treatment. P (untreated parental cells), TX (BRAFi/MEKi 24h), DTP (drug tolerant persister cells BRAFi/MEKi 21 days), DR (BRAFi/MEKi drug resistant cells). (G) PSMD14 expression (left panel) and H2Aub gene signature (right panel) scores were extracted from single cell RNA sequencing of the MEL006 patient-derived xenograft model at drug-response phases (GSE116237) : pre-treatment (T0), 4 days of BRAFi/MEKi treatment (Phase 1), minimal residual disease (28 days on BRAFi/MEKi, Phase 2), and resistance phase (Phase 3). *P < 0.05. ****P < 0.0001. Two-way ANOVA. (H) Cell death analysis on A375 cells transfected with control siRNA (siCtl) or siPSMD14 and exposed or not to BRAFi/MEKi for 24 h. Cell death was monitored in real time using Incucyte® with Cytotox Red labelling. Data are mean ± SEM and statistical significance was assessed with two-way repeated-measures ANOVA. ****P ≤ 0.0001. (I) Synergy score determined according to the Loewe additivity model on A375 cells treated with the indicated combination of PSMD14i and BRAFi/MEKi. Scores >1 are characteristic of synergy above additivity and also visible in 501MEL and UACC62 BRAF-mutant melanoma cells (inset). (J) Schematic illustration of the A375 xenograft murine model of melanoma response to targeted therapies (left panel) and tumor volume changes in xenografts treated with BRAFi+MEKi (vemurafenib 30 mg/kg + cobimetinib 7 mg/kg, orally every 2 days) alone or combined with PSMD14i (8TQ, 15 mg/kg) (right panel). Vehicle-treated mice served as control. Group sizes were: Vehicle n = 10, BRAFi+MEKi n = 11, and BRAFi+MEKi+PSMD14i n = 8. Data represent mean ± SEM. **** P <0.0001, two way repeated measures ANOVA followed by Bonferroni correction.

Article Snippet: The PSMD14 inhibitor 8-(tosylamino)quinoline (8-TQ) was purchased from Merck.

Techniques: Mutagenesis, Expressing, Control, Western Blot, Clonogenic Cell Survival Assay, Staining, Over Expression, Single Cell, RNA Sequencing, Derivative Assay, Transfection

Journal: RSC Chemical Biology

Article Title: Covalent targeting of PSMD14 by Eupalinolide B induces oncoprotein degradation and apoptosis in acute promyelocytic leukemia cells

doi: 10.1039/d5cb00197h

Figure Lengend Snippet: (A) Co-localization of PSMD14 (green) and EB-P (red) in HL-60 cells confirmed by immunofluorescence staining (scale bar = 10 μm). (B) Pull-down assay using EB-P, followed by Western blotting, confirming that EB binds to PSMD14 in situ . (C and D) Cellular thermal shift assay (CETSA)-WB indicating the direct interaction between EB and PSMD14. (E, Left) Structural model of the predicted PSMD14–EB complex. The protein is shown as a gray cartoon, and EB is depicted in orange sticks. Residues forming the predicted binding pocket are highlighted. (E, Right) Detailed view of the predicted binding interface. The key residue His183 of PSMD14 is represented as sticks and is labeled. (F) Mapping of the EB binding site on recombinant human PSMD14 by liquid chromatography-tandem mass spectrometry (LC-MS/MS). (G) Western blot showing PSMD14 protein levels in HL-60 cells incubated with or without EB (15 μM). (H) In vitro activity assay showing that EB (100 μM or 200 μM) significantly inhibits PSMD14 enzymatic activity. (I) Both EB (15 μM) and CZM (40 μM, a known PSMD14 inhibitor) markedly inhibited HL-60 cell proliferation. (J) Cell viability was assessed by CCK-8 assay following PSMD14 knockdown. (K) HL-60 cells transfected with siPSMD14-2 or siNC were treated with different concentrations of EB, and cell viability was measured by CCK-8 assay. (L–O) Cell cycle distribution of HL-60 cells treated with CZM. (P–S) PSMD14 knockdown significantly altered cell cycle progression in HL-60 cells. ** P < 0.01, *** P < 0.001, ns = not significant.

Article Snippet: To identify the direct binding site of EB on PSMD14, 40 μg of recombinant human PSMD14 protein (Signalchem Lifesciences) was incubated with 40 μM EB for 2 h at 37 °C.

Techniques: Immunofluorescence, Staining, Pull Down Assay, Western Blot, In Situ, Thermal Shift Assay, Binding Assay, Residue, Labeling, Recombinant, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Incubation, In Vitro, Activity Assay, CCK-8 Assay, Knockdown, Transfection

Journal: RSC Chemical Biology

Article Title: Covalent targeting of PSMD14 by Eupalinolide B induces oncoprotein degradation and apoptosis in acute promyelocytic leukemia cells

doi: 10.1039/d5cb00197h

Figure Lengend Snippet: (A and B) WB analysis of AKT1 and CDK4 protein expression following PSMD14 knockdown in HL-60 cells. (C and D) WB analysis of PSMD14, AKT1, and CDK4 in HL-60 cells treated with the PSMD14 inhibitor CZM. (E and F) Expression of PSMD14, AKT1, and CDK4 after treatment with cycloheximide (CHX, 20 μM), analyzed by Western blot. (G and H) WB analysis of PSMD14, AKT1, and CDK4 in HL-60 cells transfected with siPSMD14-2 and subsequently treated with EB; siNC was used as the negative control. (I–K) Quantification of PSMD14 (I), AKT1 (J), and CDK4 (K) protein levels with or without EB treatment. * P < 0.05, ** P < 0.01, *** P < 0.001, ns = not significant. Identical letters indicate no statistically significant difference, while different letters indicate P < 0.05.

Article Snippet: To identify the direct binding site of EB on PSMD14, 40 μg of recombinant human PSMD14 protein (Signalchem Lifesciences) was incubated with 40 μM EB for 2 h at 37 °C.

Techniques: Expressing, Knockdown, Western Blot, Transfection, Negative Control