Journal: Advanced Science

Article Title: PSMD14 Stabilizes SLC7A11 to Ameliorate Glucocorticoid‐Induced Osteoporosis by Suppressing Osteocyte Ferroptosis

doi: 10.1002/advs.202414902

Figure Lengend Snippet: PSMD14 acts as a major regulator for SLC7A11 stability in response to DEX stimulation. A) Schematic illustration of the preparation of SLC7A11‐binding protein sample in MLO‐Y4 cells for LC‐MS/MS analysis to identify the deubiquitinase. B) After proofreading using the UbiBrowser library, the SLC7A11‐bound deubiquitinases were ranked according to sequence coverage. C) LC‐MS/MS analysis of SLC7A11‐bound deubiquitinase eluted from MLO‐Y4 cell lysate. PSM: Peptide Spectrum Match. D) Immunoprecipitation of SLC7A11 or control IgG was performed on MLO‐Y4 cells treated with PBS or DEX (100 µM) for 8 h. E) Immunoprecipitation of PSMD14 or control IgG was performed on MLO‐Y4 cells. F) Immunoprecipitation of the interaction between SLC7A11 and PSMD14 was performed on plasmid‐transfected MLO‐Y4 cells with the indicated antibodies. After transfection was completed, MLO‐Y4 cells were treated with MG132 (10 µM) for 8 h while adding PBS or DEX (100 µM). G) Western blot and quantitative analysis of Flag‐PSMD14 and SLC7A11 protein was performed on MLO‐Y4 cells transfected with indictaed plasmid (n = 5 per group). H) Western blot and quantitative analysis of PSMD14 protein was performed on MLO‐Y4 cells transfected with si‐RNA (50 nM si‐NC or si‐PSMD14 #1‐3 for 24 h) (n = 5 per group). I) Western blot and quantitative analysis of PSMD14 and SLC7A11 proteins were performed on siRNA‐transfected MLO‐Y4 cells (50 nM si‐NC or si‐PSMD14 for 24 h). After transfection was completed, MLO‐Y4 cells were treated with PBS or DEX (100 µM) for 8 h (n = 5 per group). J) Immunoprecipitation of SLC7A11 ubiquitination and its binding to PSMD14 were performed on siRNA‐transfected MLO‐Y4 cells (50 nM si‐NC or si‐PSMD14 for 24 h). After transfection was completed, MLO‐Y4 were treated with MG132 (10 µM) for 8 h while adding PBS or DEX (100 µM). K) Predicted binding complex model of SLC7A11 and PSMD14 (left). The picture showed the hydrogen bonds in the protein interaction region and the corresponding amino acid residues (right). L) Binding energy analysis of the PSMD14‐SLC7A11 complex and the PSMD14‐SLC7A11‐DEX complex based on molecular dynamics simulations. Data are expressed as mean ± SD, with biologically individual data points shown. p values were determined by one‐way ANOVA test with Tukey's multiple comparisons (H) and two‐way ANOVA test with Tukey's multiple comparisons (G,I), * p < 0.05, ** p < 0.01.

Article Snippet: To investigate the binding affinity between PT (MedChemExpress) and recombinant mouse PSMD14 protein, SPR analysis was conducted using a BIAcore T200 instrument (GE Healthcare, USA).

Techniques: Binding Assay, Liquid Chromatography with Mass Spectroscopy, Sequencing, Immunoprecipitation, Control, Plasmid Preparation, Transfection, Western Blot, Ubiquitin Proteomics

Journal: Advanced Science

Article Title: PSMD14 Stabilizes SLC7A11 to Ameliorate Glucocorticoid‐Induced Osteoporosis by Suppressing Osteocyte Ferroptosis

doi: 10.1002/advs.202414902

Figure Lengend Snippet: PSMD14 maintains SLC7A11 expression by cleaving K48‐linked polyubiquitin chains from SLC7A11. A) Schematic diagram of the PSMD14‐SLC7A11 protein complex and the PSMD14 mutant plasmids (residues 1–233 and 234–310) used in subsequent immunoprecipitation assays. B) Immunoprecipitation of the interaction between SLC7A11 and each PSMD14 fragment was performed on plasmid‐transfected MLO‐Y4 cells. C) Immunoprecipitation of the interaction between SLC7A11 and PSMD14 (234‐310) fragment was performed on plasmid‐transfected MLO‐Y4 cells. After transfection was completed, MLO‐Y4 cells were treated with PBS or DEX (100 µM) for 8 h. D,E) Immunoprecipitation was performed to identify the type of polyubiquitination of SLC7A11 in MLO‐Y4 cell. MLO‐Y4 cells were cotransfected with Myc‐SLC7A11 plasmid, Flag‐PSMD14 plasmid and HA‐Ub K48/K63 or HA‐Ub K48R/K63R plasmid. After transfection was completed, MLO‐Y4 cells were treated with MG132 (10 µM) for 8 h. F) Immunoprecipitation of SLC7A11 ubiquitination and its binding to PSMD14 was HA‐Ub K48/K48R plasmid‐treated MLO‐Y4 cells. After transfection was completed, MLO‐Y4 cells were treated with MG132 (10 µM) and supplemented with DEX (100 µM) and/or THL (2 µM) for 8 h. G) Stable PSMD14 knockout MLO‐Y4 cells were constructed by CRISPR‐Cas9 strategy. H) Deubiquitination of SLC7A11 requires the intact PSMD14 fragment. PSMD14 knockout MLO‐Y4 cells treated with HA‐Ub K48 plasmid, Myc‐SLC7A11 plasmid, and PSMD14 full‐length or deletion mutant plasmids upon MG132 (10 µM) treatment for 8 h. I) Deubiquitination of SLC7A11 requires the intact PSMD14 fragment. Stable PSMD14 knockout MLO‐Y4 cells were treated with HA‐Ub K48 plasmid, Myc‐SLC7A11 plasmid, and Flag‐PSMD14 or PSMD14 mutants upon MG132 (10 µM) treatment for 8 h. The experiments were repeated three times independently with similar results.

Article Snippet: To investigate the binding affinity between PT (MedChemExpress) and recombinant mouse PSMD14 protein, SPR analysis was conducted using a BIAcore T200 instrument (GE Healthcare, USA).

Techniques: Expressing, Mutagenesis, Immunoprecipitation, Plasmid Preparation, Transfection, Ubiquitin Proteomics, Binding Assay, Knock-Out, Construct, CRISPR

Journal: Advanced Science

Article Title: PSMD14 Stabilizes SLC7A11 to Ameliorate Glucocorticoid‐Induced Osteoporosis by Suppressing Osteocyte Ferroptosis

doi: 10.1002/advs.202414902

Figure Lengend Snippet: Activation of PSMD14 with Pantethine suppresses osteocyte ferroptosis and bone loss. A) Schematic diagram of screening and identification of PSMD14 agonist. B) Docking scores of the top 20 candidates based on virtual screening. C) CCK‐8 assay was performed on MLO‐Y4 cells treated with different 20 candidates (10 µM) and DEX for 48 h (n = 5 per group). D) Chemical structures of five drug candidates (CA, TA, TB, PT and PC). E) Western blot and quantitative analysis of GPX4 protein were performed on MLO‐Y4 cells treated with DEX (100 µM) supplemented with or wihtout candidates (CA, TA, TB, PT or PC 10 µM) for 48 h. F) Cystine uptake assay was performed on MLO‐Y4 cells treated with DEX (100 µM) supplemented with or wihtout candidates (CA, TA, TB, PT or PC 10 µM) for 48 h (n = 9 per group). G) Immunoprecipitation of SLC7A11 ubiquitination and its binding to PSMD14 were performed on MG132 and DEX‐exposed MLO‐Y4 cells (MG132 10µM and DEX 100 µM). MG132 and DEX‐expousred MLO‐Y4 cells were treated with PT (10 µM) and/or THL (2 µM) for 8 h. H) Binding affinity of PT with recombinant PSMD14 was determined using an SPR assay (K D = 5.14 µM). I) Recombinant PSMD14 were incubated with PT, followed by the measurement of the absorbance at OD 445 nm to detect PSMD14 activity using Ubiquitin‐AMC assay (n = 5 per group). J) CCK‐8 assay was performed on DEX‐exposed MLO‐Y4 cells treated with PT (0–100 µM) and/or THL (2 µM) for 48 h (n = 5 per group). K–N) Western blot and quantitative analysis of GPX4 protein (K), MDA concentration detection (L), C11‐BODIPY 581/591 staining (M) and quantitative analysis (N) were performed on DEX‐exposed MLO‐Y4 cells treated with PT (100 µM) and/or THL (2 µM) for 48 h (n = 5 per group). O) Schematic showing the experimental protocol for 8‐weeks of PT / PT + THL injections in GIOP mice. P) Micro‐CT 3D restruction and H&E staining of the distal femur of mice in each group. The processing details of each group are shown in (O). Q) Distal femur BV/TV, Tb.Th, Tb.Sp, Tb.N, and BMD of mice in each group were measured by micro‐CT (n = 6 per group). Quantitative analysis of the empty lacunae in cortical bone (Number of empty lacunae with respect to bone area, N. Empt. Lc./B. Ar. per mm 2 ) based on H&E staining (n = 6 per group). R) GPX4, SLC7A11 and PSMD14 IHC staining of the distal femur of mice in each group. The processing details of each group are shown in (O). S) Quantification of GPX4, SLC7A11 and PSMD14‐positive osteocytes in mouse cortical femurs based on IHC staining (n = 6 per group). T) MDA content in tibia tissue of mice in each group (n = 6 per group). U) Maximum load and Maximum deflection of femoral cortical bone evaluated by the three‐point bending test (n = 6 per group). Data are expressed as mean ± SD, with biologically individual data points shown. p values were determined by one‐way ANOVA test with Tukey's multiple comparisons (C,E,F,H,I,L,N,Q,S–U) and two‐way ANOVA test with Tukey's multiple comparisons (J,K), ns, p > 0.05, * p < 0.05, ** p < 0.01.

Article Snippet: To investigate the binding affinity between PT (MedChemExpress) and recombinant mouse PSMD14 protein, SPR analysis was conducted using a BIAcore T200 instrument (GE Healthcare, USA).

Techniques: Activation Assay, CCK-8 Assay, Western Blot, Immunoprecipitation, Ubiquitin Proteomics, Binding Assay, Recombinant, SPR Assay, Incubation, Activity Assay, Ub-AMC Assay, Concentration Assay, Staining, Micro-CT, Immunohistochemistry

Journal: Advanced Science

Article Title: PSMD14 Stabilizes SLC7A11 to Ameliorate Glucocorticoid‐Induced Osteoporosis by Suppressing Osteocyte Ferroptosis

doi: 10.1002/advs.202414902

Figure Lengend Snippet: Schematic diagram illustrating the mechanism of GC‐mediated osteocyte ferroptosis. Top: Under physiological conditions, PSMD14 binds to and deubiquitinates SLC7A11 to stabilize SLC7A11 expression and the cystine uptake capacity of osteocytes, thereby ensuring the GSH content and GPX4 activity in osteocytes to maintain cellular function and vitality. Down: During DEX exposure, SLC7A11 is degraded due to limited binding with PSMD14, leading to insufficient cystine in osteocytes and triggering ferroptosis. Combined with virtual screening, we identified PT as a PSMD14 agonist that stabilizes SLC7A11 expression against DEX‐mediated ferroptosis.

Article Snippet: To investigate the binding affinity between PT (MedChemExpress) and recombinant mouse PSMD14 protein, SPR analysis was conducted using a BIAcore T200 instrument (GE Healthcare, USA).

Techniques: Expressing, Activity Assay, Cell Function Assay, Binding Assay

Journal: EBioMedicine

Article Title: The deubiquitinating enzyme PSMD14 facilitates tumor growth and chemoresistance through stabilizing the ALK2 receptor in the initiation of BMP6 signaling pathway

doi: 10.1016/j.ebiom.2019.10.039

Figure Lengend Snippet: Characterization of DUB enzymes involved in regulation of BMP signaling in human colorectal cancer cells. (a) After HCT116 and RKO cells were reverse-transfected with specific siRNA against PSMD14 and control siRNA (siCON) and treated with 100 ng/ml BMP6 for the indicated times, immunoblotting assays were performed with the indicated antibodies. (b) Expressions of ID1, ID3 , and SMAD6 mRNAs were analyzed by quantitative real-time RT-PCR (qRT-PCR) in PSMD14 -knockdown and control HCT116 or RKO cells. Expression of the mRNAs detected by qRT-RCR were normalized to Gapdh mRNA. (c) BRE-Luc luciferase reporter assays were performed in PSMD14 -knockdown and control HCT116 and RKO cells. In (b) and (c) , bar graphs show the mean ± s .d. from three independent experiments. ** P <0.01 (one-way ANOVA followed by Dunnett's test, n = 3, compared to the indicated controls). (d) After Smad4-null HT29 cells were reverse-transfected with PSDM14 -specific siRNA or siCON and treated with BMP6, immunoblots were performed with the indicated antibodies. (e) HCT116 or RKO cells overexpressing Flag-PSMD14 were immunoblotted with the indicated antibodies upon treatment of BMP6. (f) Expressions of ID1, ID3 , and SMAD6 mRNAs were analyzed by qRT-PCR in PSMD14-overexpressing HCT116 and RKO cells. In all immunoblot analyses, β-actin expression was used as a loading control and the images are representative of three independent experiments.

Article Snippet: For immunofluorescence assays, PKH26 (PKH26GL, 1:1000; Sigma-Aldrich) was administered to cells for 10 min and cold methanol at −20 °C was used to fix cells for 10 min, and subsequently, 5% BSA in PBS solution was used for blocking at room temperature for 30 min. Next, samples were incubated with primary antibodies at 4 °C for 15 h. Mouse polyclonal anti-PSMD14 antibody (SAB1408741, 1:100; Sigma-Aldrich), rabbit polyclonal ALK2 antibody (orb314588, 1:100; Biorbyt, San Francisco, USA) were used to detect endogenous PSMD14 and ALK2, respectively.

Techniques: Transfection, Control, Western Blot, Quantitative RT-PCR, Knockdown, Expressing, Luciferase

Journal: EBioMedicine

Article Title: The deubiquitinating enzyme PSMD14 facilitates tumor growth and chemoresistance through stabilizing the ALK2 receptor in the initiation of BMP6 signaling pathway

doi: 10.1016/j.ebiom.2019.10.039

Figure Lengend Snippet: The role of PSMD14 in the BMP6 signaling pathway is independent of the 26S proteasome system. (a) After PSMD14, PSMB5 or PSMB4 expression was depleted by specific siRNA against each mRNA in HCT116 cells and cells were subsequently treated with 100 ng/ml BMP6, cells were immunoblotted with the indicated antibodies. (b) Expression of ID3 mRNA was analyzed by qRT-PCR in PSMD14, PSMB4 or PSMB5-knockdown HCT116 cells, which were treated with BMP6. Expressions of ID3 mRNA were normalized to Gapdh mRNA. Bar graphs show the mean ± s .d. from three independent experiments. * P <0.05, ** P <0.01, *** P < 0.001, ns; not significant (one-way ANOVA followed by Dunnett's test, n = 3, compared to the indicated controls). (c) After PSMD14 -knockdown HCT116 cells were pre-treated with protease inhibitor MG132 or bortezomib, cells were treated with BMP6 for the indicated times and subsequently immunoblotted with the indicated antibodies. Expression of β-actin was used as a loading control. The images in all immunoblot analyses are representative of three independent experiments.

Article Snippet: For immunofluorescence assays, PKH26 (PKH26GL, 1:1000; Sigma-Aldrich) was administered to cells for 10 min and cold methanol at −20 °C was used to fix cells for 10 min, and subsequently, 5% BSA in PBS solution was used for blocking at room temperature for 30 min. Next, samples were incubated with primary antibodies at 4 °C for 15 h. Mouse polyclonal anti-PSMD14 antibody (SAB1408741, 1:100; Sigma-Aldrich), rabbit polyclonal ALK2 antibody (orb314588, 1:100; Biorbyt, San Francisco, USA) were used to detect endogenous PSMD14 and ALK2, respectively.

Techniques: Expressing, Quantitative RT-PCR, Knockdown, Protease Inhibitor, Control, Western Blot

Journal: EBioMedicine

Article Title: The deubiquitinating enzyme PSMD14 facilitates tumor growth and chemoresistance through stabilizing the ALK2 receptor in the initiation of BMP6 signaling pathway

doi: 10.1016/j.ebiom.2019.10.039

Figure Lengend Snippet: PSMD14 stabilizes ALK2 protein through direct binding. (a) After HEK293FT cells were co-transfected with the indicated plasmids, cells were immunoprecipitated (IP) with anti-HA antibody and subsequently immunoblotted (IB) with the anti-Flag or anti-HA antibody. (b) After HCT116 cells were treated with 100 ng/ml BMP6, endogenous PSMD14 was immunoprecipitated with anti-PSMD14 antibody and subsequently immunoblotted with the indicated antibodies against endogenous ALK2 and PSMD14 proteins. (c) Expression of endogenous ALK2 and ID3 proteins in PSMD14 -depleted or control (siCON) HCT116 cells were examined by immunoblot analysis (left). Expression of ALK2 mRNA in PSMD14 -depleted or control HCT116 cells was analyzed by qRT-PCR (right). (d) After a plasmid encoding HA-ALK2 was co-transfected into 293FT cells with dose-dependent expression of Flag-PSMD14, cells were immunoblotted with the indicated antibodies. (e) PSMD14-depleted or control (siCON) HCT116 cells were treated with 20 μM cycloheximide (CHX; protein synthesis inhibitor) for the indicated times. Cells were immunoblotted with anti-ALK2 and anti-PSMD14 antibodies. (f) After plasmids encoding wild-type (WT) Flag-PSMD14 and catalytic inactive DUB mutants of PSMD14 were respectively transfected into HCT116 cells, cells were treated with 20 μM CHX for the indicated times and immunoblotted. In (e) and (f) , ALK2 levels were quantified by using ImageJ software and normalized to β-actin expression. The data were statistically analyzed and the bars represent the mean ± s .d. from three independent experiments. * P <0.05, *** P < 0.001 (one-way ANOVA followed by Dunnett's test, n = 3, compared to the indicated controls). (g) PSMD14 -depleted and control (siCON) HCT116 cells were treated with 100 ng/ml BMP6 for 10 min and immunostained with the indicated antibodies to detect endogenous PSMD14 (green) and ALK2 (red). PKH26 molecule (yellow) and DAPI (blue) were used to stain the cell membrane and nuclei. Scale bar; 10 μm. (h) After HCT116 cells were treated with BMP6 for 10 min, cells were fractionated into membrane (Mem), cytoplasmic (Cyt) and nuclear (Nuc) extracts, which were subsequently immunoblotted with the indicated antibodies. Expressions of α-Tubulin, Lamin B1 and E-cadherin were used as markers and loading controls of cytosolic, nuclear and membrane fractions, respectively. Except for ( h ), expression of β-actin was used as a loading control of immunoblot analysis. The images in this figure are representative of three independent experiments.

Article Snippet: For immunofluorescence assays, PKH26 (PKH26GL, 1:1000; Sigma-Aldrich) was administered to cells for 10 min and cold methanol at −20 °C was used to fix cells for 10 min, and subsequently, 5% BSA in PBS solution was used for blocking at room temperature for 30 min. Next, samples were incubated with primary antibodies at 4 °C for 15 h. Mouse polyclonal anti-PSMD14 antibody (SAB1408741, 1:100; Sigma-Aldrich), rabbit polyclonal ALK2 antibody (orb314588, 1:100; Biorbyt, San Francisco, USA) were used to detect endogenous PSMD14 and ALK2, respectively.

Techniques: Binding Assay, Transfection, Immunoprecipitation, Expressing, Control, Western Blot, Quantitative RT-PCR, Plasmid Preparation, Software, Staining, Membrane

Journal: EBioMedicine

Article Title: The deubiquitinating enzyme PSMD14 facilitates tumor growth and chemoresistance through stabilizing the ALK2 receptor in the initiation of BMP6 signaling pathway

doi: 10.1016/j.ebiom.2019.10.039

Figure Lengend Snippet: PSMD14 stabilizes ALK2 protein through deubiquitinating Smurf1-mediated polyubiquitination of ALK2 (a, b) Plasmids encoding Flag-ALKs, Flag-PSMD14 or HA-Ubi were co-transfected into HEK293FT cells according to the indicated combinations. Ubiquitination of Flag-ALK2, Flag-ALK3, and Flag-ALK6 were examined by immunoprecipitation (IP) and immunoblots (IB) with the indicated antibodies. (c) Flag-ALK2 and Flag-PSMD14 were co-transfected into HEK293FT cells with a plasmid encoding wild-type or lysine mutant (K48 or K63) HA-Ubi in the indicated combinations. When the K48 mutant of His-Ubi was transfected, MG132 was treated to prevent ALK2 degradation. (d) The catalytically inactive DUB mutant of Flag-PSMD14 (H113Q, C120A or C120S) and wild-type Flag-PSMD14 were co-transfected into HEK293FT cells with Flag-ALK2 and HA-Ubi in the indicated combinations. (e) After PSMD14-depleted and control HCT116 cells were treated with 100 ng/ml BMP6 for the indicated times, ubiquitination of endogenous ALK2 protein was examined by IP and IB with the indicated antibodies. IgG was used as a negative control for IP. (f) After a plasmid encoding HA-ALK2 was co-transfected into HEK293FT cells with plasmids encoding Flag-Smurf1, Flag-Smurf2, Flag-CHIP, or Flag-TRIM33, respectively, co-immunoprecipitation assays were performed with the indicated antibodies. (g) Flag-Smurf1 or Flag-Smurf2 was co-transfected into HEK293FT cells with HA-Ubi and Flag-ALK2 in the indicated combinations. (h) Flag-ALK2 was co-transfected into HEK293FT cells with wild-type His-Ubi or a lysine mutant (K48 or K63) of HA-Ubi in the absence or presence of Flag-Smurf1. (i) Flag-Smurf1 and the K48 lysine mutant of HA-Ubi were co-transfected into HEK293FT cells with wild-type Flag-PSMD14 or the catalytic inactive DUB mutant of PSMD14 (H113Q). In (a) - (d) , ubiquitination of Flag-ALK proteins were examined by immunoprecipitation (IP) and immunoblots (IB) with the indicated antibodies. In (g) - (i) , ubiquitination of the ALK2 protein was analyzed by IP and IB with the indicated antibodies. In (h) and (i) , cells were pre-treated with MG132 to prevent protein degradation. Expression of β-actin was used as a loading control. The images in immunoblot analyses are representative of three independent experiments.

Article Snippet: For immunofluorescence assays, PKH26 (PKH26GL, 1:1000; Sigma-Aldrich) was administered to cells for 10 min and cold methanol at −20 °C was used to fix cells for 10 min, and subsequently, 5% BSA in PBS solution was used for blocking at room temperature for 30 min. Next, samples were incubated with primary antibodies at 4 °C for 15 h. Mouse polyclonal anti-PSMD14 antibody (SAB1408741, 1:100; Sigma-Aldrich), rabbit polyclonal ALK2 antibody (orb314588, 1:100; Biorbyt, San Francisco, USA) were used to detect endogenous PSMD14 and ALK2, respectively.

Techniques: Transfection, Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Plasmid Preparation, Mutagenesis, Control, Negative Control, Expressing

Journal: EBioMedicine

Article Title: The deubiquitinating enzyme PSMD14 facilitates tumor growth and chemoresistance through stabilizing the ALK2 receptor in the initiation of BMP6 signaling pathway

doi: 10.1016/j.ebiom.2019.10.039

Figure Lengend Snippet: PSMD14 and ALK2 are required for BMP-mediated colon cancer tumorigenesis. (a) For the analysis of cell proliferation, 1 × 10 3 cells for each cell line were cultured in 12-well plates in the absence or presence of 100 ng/ ml BMP6 for the indicated times. Cell numbers were counted at the indicated time points. (b) BrdU incorporation assays were used to detect cell proliferation. (c) MTT assays were performed to analyze cell viability. (d) For colony forming assays, cells were seeded in 6-well plates with soft-agar media and incubated for 14 days. Colonies were counted and described as graphs demonstrating the percentage of colonies in the field. In (a) - (d) , the data were statistically analyzed and the error bras represent the mean ± s .d. * P <0.05, ** P <0.01, *** P < 0.001, ns; not significant (one-way ANOVA followed by Dunnett's test. n = 3 per group, compared to the indicated controls). (e) 1 × 10 6 PSMD14 -depleted, ALK2 -depleted or control HCT116 cells were injected in the NOD-SCID mouse group ( n = 5 per group). The tumorigenesis experiment was processed for 30 days. (f) Cell lysates isolated from each tumor xenograft were immunoblotted with the indicated antibodies. Expression of β-actin was used as a loading control. The images in immunoblot analyses are representative of three independent experiments. (g) Tumor sizes ( n = 5 per group) were calculated every 5 days. (h) After isolating each tumor from mice ( n = 5 per group), tumor weights were measured and calculated as the average per group. In (g) and (h) , the data were statistically analyzed and the error bras represent the mean ± s .d. *** P < 0.001, ns; not significant (one-way ANOVA followed by Dunnett's test. n = 5 per group, compared to the indicated controls). In (a) - (h) , PSMD14 - and ALK2 -depleted HCT116 cells were generated by the infection of recombinant lentiviruses expressing shRNAs targeting ALK2 or PSMD14 .

Article Snippet: For immunofluorescence assays, PKH26 (PKH26GL, 1:1000; Sigma-Aldrich) was administered to cells for 10 min and cold methanol at −20 °C was used to fix cells for 10 min, and subsequently, 5% BSA in PBS solution was used for blocking at room temperature for 30 min. Next, samples were incubated with primary antibodies at 4 °C for 15 h. Mouse polyclonal anti-PSMD14 antibody (SAB1408741, 1:100; Sigma-Aldrich), rabbit polyclonal ALK2 antibody (orb314588, 1:100; Biorbyt, San Francisco, USA) were used to detect endogenous PSMD14 and ALK2, respectively.

Techniques: Cell Culture, BrdU Incorporation Assay, Incubation, Control, Injection, Isolation, Expressing, Western Blot, Generated, Infection, Recombinant

Journal: EBioMedicine

Article Title: The deubiquitinating enzyme PSMD14 facilitates tumor growth and chemoresistance through stabilizing the ALK2 receptor in the initiation of BMP6 signaling pathway

doi: 10.1016/j.ebiom.2019.10.039

Figure Lengend Snippet: PSMD14 depletion reduces BMP6-mediated colorectal cancer stemness. (a, b) FACS analysis of CD133 + /CD44 + cells in PSMD14 - or ALK2 -depleted HCT116 cells, which were treated with 100 ng/ml BMP6 for 48 h. The proportion of the CD133 + /CD44 + fraction was described with the density plots (a) and a bar graph (b) . shGFP-expressing HCT116 cells were used as a control. (c) Sphere forming assay of PSMD14 - or ALK2 - depleted HCT116 cells. Spheres with a diameter above 50 μm were counted and described in a bar graph. Scale bars, 50 μm. (d) PSMD14 - or ALK2 -depleted HCT116 cells were treated with BMP6. Expression of pluripotent transcription factors were analyzed by immunoblotting with the indicated antibodies. siCON-expressing HCT116 cells were used as a control. (e, h) 2 × 10 4 cells of PSMD14 -, ALK2 -, ABCA7 - or ABCC4 -depleted HCT116 were respectively treated with 20 μM oxaliplatin and 30 μM DAPT and their viabilities were measured at 6 h. shGFP-expressing HCT116 cells were used as a control. (f, g) PSMD14 - or ALK2 -depleted HCT116 cells were treated with BMP6 for 6 h. Expressions of ABCA7 and ABCC4 were measured by quantitative RT-PCR. The data were statistically analyzed by two-way ANOVA followed by Bonferroni's multiple comparison test ( n = 3, *** P < 0.001 compared to the indicated controls. ns; not significant). The bars represent the mean ± s .d. The images in this figure are representative of three independent experiments. In (b), (c), (e) and (h) were statistically analyzed by one-way ANOVA followed by Dunnett's test ( n = 3, ** P <0.01, *** P < 0.001 compared to the indicated controls. ns; not significant).

Article Snippet: For immunofluorescence assays, PKH26 (PKH26GL, 1:1000; Sigma-Aldrich) was administered to cells for 10 min and cold methanol at −20 °C was used to fix cells for 10 min, and subsequently, 5% BSA in PBS solution was used for blocking at room temperature for 30 min. Next, samples were incubated with primary antibodies at 4 °C for 15 h. Mouse polyclonal anti-PSMD14 antibody (SAB1408741, 1:100; Sigma-Aldrich), rabbit polyclonal ALK2 antibody (orb314588, 1:100; Biorbyt, San Francisco, USA) were used to detect endogenous PSMD14 and ALK2, respectively.

Techniques: Expressing, Control, Western Blot, Quantitative RT-PCR, Comparison

Journal: EBioMedicine

Article Title: The deubiquitinating enzyme PSMD14 facilitates tumor growth and chemoresistance through stabilizing the ALK2 receptor in the initiation of BMP6 signaling pathway

doi: 10.1016/j.ebiom.2019.10.039

Figure Lengend Snippet: Higher expression of PSMD14 and ALK2 confer poor prognosis in human colorectal cancer. (a) Cell lysates isolated from the indicated colorectal cancer cell lines were immunoblotted with the ALK2 and PSMD14 antibodies. Expression of β-actin was used as a loading control. The images in immunoblot analyses are representative of three independent experiments. (b, c) Using the Kaplan-Meier (KM) plotter tool, expression of PSMD14 and ALK2 mRNA between normal and tumor tissues of colorectal cancer patients in a public GEO dataset (GSE21510; n = 148) were analyzed. (d) To verify the correlation of ALK2 mRNA expression in normal colon tissues and tumor tissues with the expression levels of PSMD14, a public GSE dataset (GSE21510) was analyzed by the KM plotter tool. In (b) - (d) , the boxes represent the interquartile range, centre is the median, and the minimum and maximum values are represented in the whiskers. *** P <0.001, ns; not significant (Student t -test, compared to normal tissues or PSMD14-low samples). (e) Correlation of ALK2 and PSMD14 mRNAs in normal colon tissues, polyps in colon, and colorectal tumor tissues of human colorectal cancer patient samples (GSE68468, n = 288). Samples were classified into three groups (double low expression of ALK2 and PSMD14 , single high expression of ALK2 or PSMD14 , and double high expression of ALK2 and PSMD14 ) in normal tissues, polyps and tumors. (f, g) Scatter dot plot analysis represents the scores of the expression of PSMD14 and ALK2 protein in the matched normal and tumor tissues of human colon cancer patients (normal tissue n = 70, cancer tissue n = 70) analyzed by immunohistochemistry. *** P <0.001 (Student t -test, compared to normal tissues). (h) Scatter dot plot shows Spearman correlations between ALK2 and PSMD14 protein expression according to scoring within all samples. *** P < 0.001 (Student t -test). The Spearman r indicates the Spearman correlation coefficients. (i, j) Correlation of PSMD14 or ALK2 mRNA expression with overall survival rates of human colorectal cancer patients were analyzed by a KM plot analysis in a public GSE dataset (GSE17538; n = 224 patients). P = 0.0372, P = 0.0021 (Log-rank test). HR = hazard ratio.

Article Snippet: For immunofluorescence assays, PKH26 (PKH26GL, 1:1000; Sigma-Aldrich) was administered to cells for 10 min and cold methanol at −20 °C was used to fix cells for 10 min, and subsequently, 5% BSA in PBS solution was used for blocking at room temperature for 30 min. Next, samples were incubated with primary antibodies at 4 °C for 15 h. Mouse polyclonal anti-PSMD14 antibody (SAB1408741, 1:100; Sigma-Aldrich), rabbit polyclonal ALK2 antibody (orb314588, 1:100; Biorbyt, San Francisco, USA) were used to detect endogenous PSMD14 and ALK2, respectively.

Techniques: Expressing, Isolation, Control, Western Blot, Immunohistochemistry

Journal: EBioMedicine

Article Title: The deubiquitinating enzyme PSMD14 facilitates tumor growth and chemoresistance through stabilizing the ALK2 receptor in the initiation of BMP6 signaling pathway

doi: 10.1016/j.ebiom.2019.10.039

Figure Lengend Snippet: Schematic representation of the proposed mechanism of BMP6 signaling pathway regulated by PSMD14-ALK2 axis in colorectal cancers. ALK2 type I receptor is polyubiquitinated by E3 ligase Smurf1 and subsequently degraded in the absence of BMP6. Upon treatment of BMP6, PSMD14 binds to ALK2 protein and deubiquitinates the K48-linked polyubiquitin chains of ALK2 protein, resulting in the increase of ALK2 stability and thus leads to the initiation of BMP6 signaling pathway. This initiation of BMP6 signaling pathway facilitates tumor growth, cancer stemness and chemoresistance in colorectal cancers.

Article Snippet: For immunofluorescence assays, PKH26 (PKH26GL, 1:1000; Sigma-Aldrich) was administered to cells for 10 min and cold methanol at −20 °C was used to fix cells for 10 min, and subsequently, 5% BSA in PBS solution was used for blocking at room temperature for 30 min. Next, samples were incubated with primary antibodies at 4 °C for 15 h. Mouse polyclonal anti-PSMD14 antibody (SAB1408741, 1:100; Sigma-Aldrich), rabbit polyclonal ALK2 antibody (orb314588, 1:100; Biorbyt, San Francisco, USA) were used to detect endogenous PSMD14 and ALK2, respectively.

Techniques:

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: Anti-Cancer Drugs

Article Title: Serum heat shock protein family A member 9 protein as a biomarker for bortezomib resistance and poor prognosis in patients with multiple myeloma

doi: 10.1097/CAD.0000000000001764

Figure Lengend Snippet: Expression levels of HSPA9, DKK1, PSMD14, and TRIM21 proteins in MM patients. Serum samples were collected from 46 MM patients and 52 healthy controls, and ELISA was performed to detect the levels of HSPA9 (a), DKK1 (b), TRIM21 (c), and PSMD14 (d) proteins. Data are expressed as mean ± SD from three independent experiments. DKK1, dickkopf Wnt signaling pathway inhibitor 1; HSPA9, heat shock protein family A member 9; MM, multiple myeloma; PSMD14, proteasome 26S subunit non-ATPase 14; TRIM21, tripartite motif containing 21.

Article Snippet: The following ELISA kits were used in the study: HSPA9 (order no. D711242; Sangon Biotech, Shanghai, China), DKK1 (order no. D711029; Sangon Biotech), PSMD14 (order no. CSB-PA018904OB01HU; CUSABIO, Wuhan, China), and TRIM21 (order no. ml106271; Enzyme-linked Biotechnology, Shanghai, China).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay