Journal: Science Advances

Article Title: Disrupting USP14-mediated PARP1 dynamics reinstates MIC-A/B–driven antigen-independent CD8 + T cell killing in glioma

doi: 10.1126/sciadv.aeb3800

Figure Lengend Snippet: ( A ) Schematic of the DUB-focused screening strategy. ( B ) Validation of top siRNA candidates identifies USP14 as a negative regulator of MIC-A/B. NC, negative control; JAMM, JAB1/MPN/Mov34 metalloprotease; OTU, ovarian tumor proteases; UCH, ubiquitin C-terminal hydrolase; USP, ubiquitin-specific protease. ( C ) Representative flow cytometry plots showing MIC-A/B up-regulation upon USP14 inhibition. ( D and E ) Quantification of MIC-A/B elevation after USP14 inhibition (D) or USP14 knockout (E). ( F ) Representative MRI images of orthotopic glioma-bearing mice treated with IU1. ( G ) Kaplan-Meier survival analysis (log-rank test; n = 10). ( H ) Tumor growth in BALB/c nude and C57BL/6J mice bearing subcutaneous GL261 tumors treated with IU1 or control. ( I ) Tumor inhibition rates in C57BL/6J and BALB/c nude mice after IU1 treatment. ( J ) Functional enrichment analysis showing immune-related pathways enriched in IU1-treated tumors. ( K ) CIBERSORT deconvolution indicating increased CD8 + T cell infiltration in IU1-treated tumors. ( L ) Representative immunofluorescence images showing increased CD8 + T cell infiltration and Rae1 expression following IU1 treatment. ( M ) Quantification of CD8 + T cell infiltration and Rae1 expression in (L). ( N and O ) Cytotoxicity assays showing that USP14 knockout in tumor cells increases susceptibility to CD8 + T cell–mediated killing. Data are means ± SD. Two-tailed unpaired Student’s t tests were used for (D), (F), (H), (I), and (M) and two-way ANOVA for (O). Survival in (G) was analyzed by log-rank test. ** P < 0.01, *** P < 0.001, and **** P < 0.0001. DC, dendritic cell; a.u., arbitrary unit.

Article Snippet: The USP14 inhibitor IU1 was synthesized by Selleck (Groningen, the Netherlands).

Techniques: Biomarker Discovery, Negative Control, Ubiquitin Proteomics, Flow Cytometry, Inhibition, Knock-Out, Control, Functional Assay, Immunofluorescence, Expressing, Two Tailed Test

Journal: Science Advances

Article Title: Disrupting USP14-mediated PARP1 dynamics reinstates MIC-A/B–driven antigen-independent CD8 + T cell killing in glioma

doi: 10.1126/sciadv.aeb3800

Figure Lengend Snippet: ( A ) Experimental workflow and data preprocessing. ( B and C ) UMAP plot demonstrates that all 46,056 cells are clustered into 12 major clusters according to classical markers. ( D ) Bar plot of variations in proportions of cell subtypes after USP14 inhibition. ( E and F ) UMAP plot displays 11 clusters of T cells according to specific markers. ( G ) Proportion of each Cd8 + T cell subgroup after USP14 inhibition. ( H ) Monocle 2 trajectory plot showing the pseudotime curve and the dynamics of CD8 + T subclusters. ( I ) Dynamics of Cd8 + T subclusters between Ctrl and IU1-treated groups. ( J ) Heatmap depicting genes in a branch-dependent manner for branch point 1. Data were means ± SD. Statistical significance was calculated by two-tailed unpaired Student’s t tests for (D). * P < 0.05.

Article Snippet: The USP14 inhibitor IU1 was synthesized by Selleck (Groningen, the Netherlands).

Techniques: Inhibition, Two Tailed Test

Journal: Science Advances

Article Title: Disrupting USP14-mediated PARP1 dynamics reinstates MIC-A/B–driven antigen-independent CD8 + T cell killing in glioma

doi: 10.1126/sciadv.aeb3800

Figure Lengend Snippet: ( A ) Images of GL261-Luc growth in mice after USP14 knockout and knockdown of Rae1 expression. n = 5 for each group. ( B ) Quantitative analysis of biofluorescence in different groups. ( C ) Images of GL261-Luc growth in cDC1 knockout mice after USP14 knockout. n = 5 for each group. ( D ) Quantitative analysis of biofluorescence in different groups. ( E ) Survival times of tumor-bearing mice were analyzed using the Kaplan-Meier method with the log-rank test. ( F and G ) Percent of Gzmb + and Pd1 + Cd8 + T cells in tumors from wild-type and Batf3 −/− mice. ( H ) Images of GL261-Luc growth in mice treated with IgG, anti-PD1, IU1, and anti-PD1 plus IU1. n = 8 for each group. ( I ) Quantitative analysis of biofluorescence in IgG, anti-PD1, IU1, and anti-PD1 plus IU1 groups. ( J ) Survival times of tumor-bearing mice treated with IgG, anti-PD1, IU1, and anti-PD1 plus IU1 were analyzed using the Kaplan-Meier method with the log-rank test. ( K to M ) Percent of γ-Ifn + , Gzmb + , and Pd1 + Cd8 + T cells in tumors from IgG, anti-PD1, IU1, and anti-PD1 plus IU1 groups. Data were means ± SD. Statistical significance was calculated by two-way ANOVA for (B), (F), (G), (I) and (K) to (M). Survival analysis of (E) and (J) was performed by the log-rank test and two-tailed unpaired Student’s t tests for (D). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: The USP14 inhibitor IU1 was synthesized by Selleck (Groningen, the Netherlands).

Techniques: Knock-Out, Knockdown, Expressing, Two Tailed Test

Journal: Science Advances

Article Title: Disrupting USP14-mediated PARP1 dynamics reinstates MIC-A/B–driven antigen-independent CD8 + T cell killing in glioma

doi: 10.1126/sciadv.aeb3800

Figure Lengend Snippet: ( A ) Functional enrichment analysis of differential proteins. ( B ) Heatmap of the top 20 differential proteins. ( C ) Co-IP demonstrates the exogenous interaction between USP14 and PARP1. ( D and E ) Co-IP demonstrates the endogenous interaction between USP14 and PARP1. ( F ) Immunofluorescence staining presented the co-localization of USP14 and PARP1. ( G and H ) GST-USP14 and HIS-PARP1 proteins can pull down each other from total protein extracts. ( I and J ) Western blot analyses and quantification of the indicated cells knocking out USP14. ( K and L ) Western blot analyses and quantification of the indicated cells transfected with USP14 plasmids. ( M ) PARP1 mRNA expression after USP14 inhibition in U251MG and T98G. Data were means ± SD. Statistical significance was calculated by one-way ANOVA for (J) and (L) and two-tailed unpaired Student’s t tests for (M). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: The USP14 inhibitor IU1 was synthesized by Selleck (Groningen, the Netherlands).

Techniques: Functional Assay, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Western Blot, Transfection, Expressing, Inhibition, Two Tailed Test

Journal: Science Advances

Article Title: Disrupting USP14-mediated PARP1 dynamics reinstates MIC-A/B–driven antigen-independent CD8 + T cell killing in glioma

doi: 10.1126/sciadv.aeb3800

Figure Lengend Snippet: ( A and B ) USP14 antagonized E3 ligase-mediated degradation of PARP1. ( C and D ) CHX-chase analysis of PARP1 protein half-life in USP14-kncokout tumor cells. CHX, cycloheximide. ( E to H ) Indicated cells were treated with IU1 or DMSO for the indicated time and lysates were analyzed. ( I and J ) CHX-chase analysis of PARP1 protein half-life in HEK293T cells. Data were means ± SD. Statistical significance was calculated by two-way ANOVA for (B) to (D) and (H) and (J). ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: The USP14 inhibitor IU1 was synthesized by Selleck (Groningen, the Netherlands).

Techniques:

Journal: Science Advances

Article Title: Disrupting USP14-mediated PARP1 dynamics reinstates MIC-A/B–driven antigen-independent CD8 + T cell killing in glioma

doi: 10.1126/sciadv.aeb3800

Figure Lengend Snippet: ( A ) USP14 knockout tumor cells were treated with MG132 for 6 hours, and lysates were immunoprecipitated with anti-PARP1. ( B ) Tumor cells treated with IU1 (100 μM) for 24 hours and MG132 (10 μM) for 6 hours and lysates were immunoprecipitated with anti-PARP1. ( C ) HEK293T cells were transfected with HA-ubiquitin and Flag-PARP1 with or without Myc-USP14, and lysates were immunoprecipitated with anti-Flag. ( D ) HEK293T cells were transfected with HA-ubiquitin and Flag-PARP1 with mutant Myc-USP14, and lysates were immunoprecipitated with anti-Flag. ( E ) Levels of different K-linked PARP1 ubiquitination upon Flag-USP14 transfection were assessed by anti-HA antibody. ( F ) Immunofluorescence and colocalization analysis of PARP1 and proteasome (PSMB9). ( G ) Western blot analysis of proteasome-associated PARP1 levels after knocking out USP14. ( H ) Western blot analysis of proteasome-associated PARP1 levels after inhibiting out USP14. ( I ) HEK293T cells were transfected with mutant Flag-PARP1, with or without Myc-USP14, to examine changes in ubiquitination. IB, immunoblot; WT, wild type.

Article Snippet: The USP14 inhibitor IU1 was synthesized by Selleck (Groningen, the Netherlands).

Techniques: Knock-Out, Immunoprecipitation, Transfection, Ubiquitin Proteomics, Mutagenesis, Immunofluorescence, Western Blot

Journal: Science Advances

Article Title: Disrupting USP14-mediated PARP1 dynamics reinstates MIC-A/B–driven antigen-independent CD8 + T cell killing in glioma

doi: 10.1126/sciadv.aeb3800

Figure Lengend Snippet: ( A ) Images of GL261-Luc growth in mice after USP14 knockout and rescue of Parp1 expression. n = 5 for each group. ( B ) Quantitative analysis of biofluorescence in (A). ( C ) Survival times of tumor-bearing mice were analyzed using the Kaplan-Meier method with the log-rank test. ( D to G ) Percent of Gzmb + , γ-Ifn + , Tnf-α + , and Pd1 + Cd8 + T cells in tumors. ( H to M ) Quantitative analysis of the indicated markers shown in fig. S11C. Data were means ± SD. Statistical significance was calculated by two-way ANOVA for (B) and (D) to (M). Survival analysis of (C) was performed by the log-rank test. * P < 0.1, ** P < 0.01, and *** P < 0.001.

Article Snippet: The USP14 inhibitor IU1 was synthesized by Selleck (Groningen, the Netherlands).

Techniques: Knock-Out, Expressing

Journal: Science Advances

Article Title: Disrupting USP14-mediated PARP1 dynamics reinstates MIC-A/B–driven antigen-independent CD8 + T cell killing in glioma

doi: 10.1126/sciadv.aeb3800

Figure Lengend Snippet: ( A and B ) Western blotting images of USP14 expression in pri-tumor tissues and tumor tissues. ( C ) TCGA database shows the expression levels of USP14 in normal brain tissue and glioma. ( D ) Representative images of USP14 and PARP1 IHC staining in human glioma specimens. ( E and F ) Expression and correlation of USP14 and PARP1 in glioma. ( G ) TCGA database shows the expression levels of USP14 in glioma of different grades. ( H ) Immunofluorescence staining of GFAP and USP14 in glioma tissue. ( I ) Single-cell sequencing reveals the cell subtype expression of USP14 and PARP1. ( J ) USP14 IHC of glioma tissue microarray. ( K ) Survival curves of patients with high versus low USP14 expression. ( L ) Immunofluorescence staining of GZMB (a marker of CD8 + T cell activation) and TOX (a marker of T cell exhaustion) in glioma from the high and low USP14 expression groups. ( M ) Mechanism diagram. Data were means ± SD. Statistical significance was calculated by two-tailed unpaired Student’s t tests for (B) and (C); expression correlations were calculated using the Pearson’s correlation algorithm for (E) and one-way ANOVA for (F) and (G). Survival analysis of (K) was performed by the log-rank test. **** P < 0.0001.

Article Snippet: The USP14 inhibitor IU1 was synthesized by Selleck (Groningen, the Netherlands).

Techniques: Western Blot, Expressing, Immunohistochemistry, Immunofluorescence, Staining, Single Cell, Sequencing, Microarray, Marker, Activation Assay, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Andrographolide targets syndecan4 to impair its interaction with syntenin and inhibits the biogenesis of small extracellular vesicles

doi: 10.1016/j.jbc.2026.111182

Figure Lengend Snippet: MG132 inhibits the degradation of SDC4-CTF. A and B , Western blot of HCT116 cells treated with 0.5, 1, 2.5, or 5 μM MG132 for 12 h, or with 2.5 μM MG132 for 0 to 12 h. C , Western blot of full-length SDC1 and SDC4 in HCT116 cells treated with MG132 (0.5–5 μM, 12 h). D and E , immunofluorescence detection and quantification of HCT116 cells transfected with SDC1-GFP or SDC4-GFP, with/without 5 μM MG132 (4 h; n = 6). F , Western blot of multiple colorectal cancer cell lines treated with 10 μM MG132 (12 h). G , Western blot of SDC1-CTF and SDC4-CTF in HCT116 cells treated with 0.1, 0.5, or 1 μM proteasome inhibitors (Carfilzomib, Ixazomib, Bortezomib) for 24 h. H , Western blot of SDC1-CTF and SDC4-CTF in HCT116 cells treated with PD150606 (2, 5, 10 μM) for 12 h. I , quantification of DQ-BSA fluorescence with MG132 or Earle’s balanced salt solution (EBSS) treatment (n = 4). J , quantification of lysosomal activity using LysoTracker with MG132 or EBSS treatment (n = 4). K , Western blot of SDC1-CTF and SDC4-CTF in HCT116 cells with siRNA knockdown of proteasome subunits PSMD14, PSMD2, USP14, PSMB5, PSMA6. Data: mean ± SD (≥3 experiments). Statistics: unpaired two-tailed t test (∗∗∗ p < 0.0005, ∗∗∗∗ p < 0.0001). SDC4, syndecan4; CTF, C-terminal transmembrane (TM) fragment; EBSS, Earle’s balanced salt solution.

Article Snippet: Antibodies against LC3 (14600-1-AP), LAMP2 (66301-1-Ig), p62 (18420-1-AP), Syntenin (22399-1-AP), CD63 (25682-1-AP), TSG101 (28283-1-AP), CD9 (20597-1-AP), α-tubulin (11,224–1-AP), PSMA6 (67695-1-Ig), PSMED2 (14748-1-AP), USP14 (67746-1-Ig), and SDC1 (10593-1-AP) for SDC1-FL detection were obtained from Proteintech. β-catenin (ab16051) and PSMD14 (ab109123) antibodies were obtained from Abcam.

Techniques: Western Blot, Immunofluorescence, Transfection, Fluorescence, Activity Assay, Knockdown, Two Tailed Test

Journal: Cell reports

Article Title: Proteasome activity maintains cell-type-specific gene expression

doi: 10.1016/j.celrep.2026.116973

Figure Lengend Snippet: (A) Lysates from HCT116 WT, ΔRAZUL, or hRpn10 VWA cells were resolved and analyzed by immunoprobing for hRpn10, E6AP, or β-actin. (B) Lysates from WT, ΔhRpn13, trRpn13, ΔRAZUL, hRpn10 VWA , or ΔUCHL5 cells were resolved and immunoprobed for K48-linked (left) or bulk (right) ubiquitin chains or β-actin. (C) Immunoblots probing for ubiquitin or hRpn2 of proteasomes immunoprecipitated by anti-hRpt3 antibodies from WT, ΔhRpn13, trRpn13, ΔRAZUL, hRpn10 VWA , or ΔUCHL5 without (left) or with (right) DSP crosslinker. IgG antibodies were included as a control. (D) Boxplot representation plotting the range and mean value of protein abundance for hRpn1, hRpn10, hRpn8, hRpn11, hRpn13, hRpn2, hRpt3, β5, E6AP, UCHL5, and USP14 across three samples as measured by TMT-MS. (E) Lysates from HCT116 WT, ΔhRpn13, trRpn13, ΔRAZUL, hRpn10 VWA , or ΔUCHL5 cells resolved and analyzed by immunoprobing for hRpn1, hRpn2, hRpn10, hRpn13, hRpn8, hRpn11, USP14, UCHL5, β5, or β-actin. (F) Graphical plot of protein and mRNA abundance in hRpn10 VWA compared to WT by TMT-MS ( x axis) and qPCR ( y axis), respectively, for RP subunits hRpn1, hRpn10, hRpn11, and hRpn13, CP β5, or deubiquitinases UCHL5 and USP14. A dotted line indicates no change. (G) Volcano plot of fold-change in protein abundance ( x axis, log 2 ) and adjusted p value ( y axis, −log 10 ) in hRpn10 VWA cells relative to WT. Vertical and horizontal lines are included at ±1 and 1.3, respectively. Proteins with expression values statistically greater or less than 2-fold difference are colored and labeled according to their molecular function as indicated in the legend. (H) Plot of the change in protein abundance (log 2 ) relative to WT by TMT-MS for ΔRAZUL ( x axis) and hRpn10 VWA ( y axis). The Spearman rank correlation value is included. Data with adjusted p value ≥ 0.05 are excluded from this analysis. Color coding follows (G). FC, fold change. (I) Illustration of the aftereffects that arise following deletion of hRpn10 UIMs and RAZUL domain. Weak binding of ubiquitinated substrates to proteasomes causes dysregulated protein levels in multiple functional pathways and induces upregulated gene expression of proteasome subunits. hRpn10 VWA , ubiquitin, substrate, peptide, RP, and CP α or β subunits are colored purple, yellow, brown, dark gray, light yellow, gray, or light gray, respectively. See also , , and .

Article Snippet: TaqMan probe for USP14 , Applied Biosystem , Hs00193036_m1.

Techniques: Ubiquitin Proteomics, Western Blot, Immunoprecipitation, Control, Quantitative Proteomics, Expressing, Labeling, Binding Assay, Functional Assay, Gene Expression

Journal: Discover Oncology

Article Title: USP14 promotes epithelial-mesenchymal transition and functions as a multifaceted biomarker in lung adenocarcinoma

doi: 10.1007/s12672-026-04682-1

Figure Lengend Snippet: USP14 expression was upregulated in LUAD tissues and associated with poorer survival. A A pan-cancer analysis of USP14 expression in TIMER database. B Survival Forest plots of pan-cancer database by SangerBox online tools. C Survival curve of overall survival (OS) stratified by optimal cut-off value for LUAD in TCGA+GTEx database (HR = 1.70, p = 7.6e-4). D , E USP14 mRNA expression in LUSC and LUAD in TCGA database. F , G USP14 mRNA expression in paired tumor and adjacent normal tissues in LUSC and LUAD in TCGA database. H , I Survival curves of OS stratified by the median expression level of USP14 in TCGA database. J , K Survival curves of OS stratified by optimal cut-off value in CPTAC database. L Forest plot of OS analysis stratified by optimal cut-off value in GSE30219 , GSE11969 , GSE101929 , and GSE41271 database. M Survival curve of disease specific survival (DSS) stratified by the median expression level of USP14 D in TCGA-LUAD database. N USP14 protein expression in LUAD in Academia Sinica LUAD-100 Proteome

Article Snippet: Proteins were then separated by SDS-PAGE and transferred to PVDF membranes, and blocked with a protein-free solution for 30 min or 3% BSA for 2 h. The membranes were incubated at 4 degrees Celsius overnight with primary antibodies against human USP14 (14517-1-AP, Proteintech), N-Cadherin (bsm-52389R, Bioss), E-Cadherin (#3195, Cell Signaling Technology), Vimentin (10366-1-AP, Proteintech), PI3 Kinase p85 (#4257, Cell Signaling Technology), Phospho-PI3K p85 (Tyr458)[Tyr467]/p55 (Tyr199) (#AF3242, Affinity Biosciences), Akt (#9272, Cell Signaling Technology), Phospho-Akt (Ser473) (#4060, Cell Signaling Technology), mTOR (#2983, Cell Signaling Technology), Phospho-mTOR (Ser2448) (#2971, Cell Signaling Technology), p70(S6K) (14485-1-AP, Proteintech), and Phospho-p70(S6K) (Thr389)/p85(S6K) (Thr412) (28735-1-AP, Proteintech).

Techniques: Expressing

Journal: Discover Oncology

Article Title: USP14 promotes epithelial-mesenchymal transition and functions as a multifaceted biomarker in lung adenocarcinoma

doi: 10.1007/s12672-026-04682-1

Figure Lengend Snippet: Prognostic value and prognostic modeling of USP14 based on various clinicopathologic features. A – F The mRNA expression levels of USP14 in LUAD in TCGA database based on T stages ( A ), N stages ( B ), M stages ( C ), TNM stages ( D ), OS event ( E ) and primary therapy outcome ( F ). G Sankey diagram showing the association of USP14 expression with multiple clinical features. H , I The protein expression level of USP14 in LUAD in Academia Sinica LUAD-100 Proteome based on N stages ( H ), clinical stages ( I ). J , K The univariate ( J ) and multivariate ( K ) Cox regression analysis of USP14 expression with clinical features. L Construction of the nomogram model for LUAD patients based on Cox regression analysis. M , N Diagnostic ROC analysis of USP14 for the binary outcome (Tumor vs. Normal) in LUAD ( M ) and NSCLC ( N ). The “Normal” tissue group was designated as the reference category. X-axis indicates false-positive rates, and Y-axis indicates true-positive rates

Article Snippet: Proteins were then separated by SDS-PAGE and transferred to PVDF membranes, and blocked with a protein-free solution for 30 min or 3% BSA for 2 h. The membranes were incubated at 4 degrees Celsius overnight with primary antibodies against human USP14 (14517-1-AP, Proteintech), N-Cadherin (bsm-52389R, Bioss), E-Cadherin (#3195, Cell Signaling Technology), Vimentin (10366-1-AP, Proteintech), PI3 Kinase p85 (#4257, Cell Signaling Technology), Phospho-PI3K p85 (Tyr458)[Tyr467]/p55 (Tyr199) (#AF3242, Affinity Biosciences), Akt (#9272, Cell Signaling Technology), Phospho-Akt (Ser473) (#4060, Cell Signaling Technology), mTOR (#2983, Cell Signaling Technology), Phospho-mTOR (Ser2448) (#2971, Cell Signaling Technology), p70(S6K) (14485-1-AP, Proteintech), and Phospho-p70(S6K) (Thr389)/p85(S6K) (Thr412) (28735-1-AP, Proteintech).

Techniques: Expressing, Diagnostic Assay

Journal: Discover Oncology

Article Title: USP14 promotes epithelial-mesenchymal transition and functions as a multifaceted biomarker in lung adenocarcinoma

doi: 10.1007/s12672-026-04682-1

Figure Lengend Snippet: The genetic alteration characteristics of USP14 in LUAD. A Lollipop diagram displaying the mutation sites of USP14 in pan-cancer. B The proportion of USP14 mutations in 2274 LUAD samples in cBioPortal database. C Mutation types of USP14 in 5 databases. D Waterfall plot displaying the somatic mutation landscape of USP14 in LUAD. Top 15 somatic mutation genes were exhibited in the cohort. E – G Correlation analysis between USP14 expression and Tumor Mutational Burden (TMB, Pearson r =0.26, p <4.9e-9) ( E ), Homologous Recombination Deficiency (HRD, Pearson r =0.32, p <1.1e-13) ( F ), Loss of Heterozygosity (LOH, Pearson r =0.23, p <3.8e-7) ( G )

Article Snippet: Proteins were then separated by SDS-PAGE and transferred to PVDF membranes, and blocked with a protein-free solution for 30 min or 3% BSA for 2 h. The membranes were incubated at 4 degrees Celsius overnight with primary antibodies against human USP14 (14517-1-AP, Proteintech), N-Cadherin (bsm-52389R, Bioss), E-Cadherin (#3195, Cell Signaling Technology), Vimentin (10366-1-AP, Proteintech), PI3 Kinase p85 (#4257, Cell Signaling Technology), Phospho-PI3K p85 (Tyr458)[Tyr467]/p55 (Tyr199) (#AF3242, Affinity Biosciences), Akt (#9272, Cell Signaling Technology), Phospho-Akt (Ser473) (#4060, Cell Signaling Technology), mTOR (#2983, Cell Signaling Technology), Phospho-mTOR (Ser2448) (#2971, Cell Signaling Technology), p70(S6K) (14485-1-AP, Proteintech), and Phospho-p70(S6K) (Thr389)/p85(S6K) (Thr412) (28735-1-AP, Proteintech).

Techniques: Mutagenesis, Expressing, Homologous Recombination

Journal: Discover Oncology

Article Title: USP14 promotes epithelial-mesenchymal transition and functions as a multifaceted biomarker in lung adenocarcinoma

doi: 10.1007/s12672-026-04682-1

Figure Lengend Snippet: USP14 overexpression promotes LUAD cell migration and invasion. A , B The expression levels of USP14 in bronchial epithelial cells (BEAS-2B) and NSCLC cell lines (including A549, NCI-H1975) were analyzed through qRT-PCR ( A ) and western blot ( B ). C Gene Effect scores for USP14 in lung cancer cell lines derived from CRISPR knockout screens published by Broad’s Achilles and Sanger’s SCORE projects. Data sourced from DepMap project (CCLE database). D , E EMT scores related to USP14 expression calculated by EMT_gene_list based on R package “imogimap”. F – I The over-expression of USP14 in LUAD stable cell lines was detected by qRT-PCR ( G , I ) and western blot. The expression of EMT markers was detected by western blot ( F , H ). J , K The wound-healing assays were conducted to assess the effect of USP14 overexpression on cell’s migration ability. L , M Transwell assays were used to detect the effect of USP14 overexpression on cell’s migration and invasion ability

Article Snippet: Proteins were then separated by SDS-PAGE and transferred to PVDF membranes, and blocked with a protein-free solution for 30 min or 3% BSA for 2 h. The membranes were incubated at 4 degrees Celsius overnight with primary antibodies against human USP14 (14517-1-AP, Proteintech), N-Cadherin (bsm-52389R, Bioss), E-Cadherin (#3195, Cell Signaling Technology), Vimentin (10366-1-AP, Proteintech), PI3 Kinase p85 (#4257, Cell Signaling Technology), Phospho-PI3K p85 (Tyr458)[Tyr467]/p55 (Tyr199) (#AF3242, Affinity Biosciences), Akt (#9272, Cell Signaling Technology), Phospho-Akt (Ser473) (#4060, Cell Signaling Technology), mTOR (#2983, Cell Signaling Technology), Phospho-mTOR (Ser2448) (#2971, Cell Signaling Technology), p70(S6K) (14485-1-AP, Proteintech), and Phospho-p70(S6K) (Thr389)/p85(S6K) (Thr412) (28735-1-AP, Proteintech).

Techniques: Over Expression, Migration, Expressing, Quantitative RT-PCR, Western Blot, Derivative Assay, CRISPR, Knock-Out, Stable Transfection

Journal: Discover Oncology

Article Title: USP14 promotes epithelial-mesenchymal transition and functions as a multifaceted biomarker in lung adenocarcinoma

doi: 10.1007/s12672-026-04682-1

Figure Lengend Snippet: USP14 knockdown inhibits LUAD cell migration and invasion. A – D USP14 siRNA transfection in LUAD cell lines was followed by the assessment of USP14 mRNA and protein expression via qRT-PCR ( B , D ) and western blot. The expression of EMT markers was detected by western blot ( A , C ). E , F The wound-healing assays were conducted to assess the effect of USP14 knockdown on cell’s migration ability. G , H Transwell assays were used to detect the effect of USP14 knockdown on cell’s migration and invasion ability

Article Snippet: Proteins were then separated by SDS-PAGE and transferred to PVDF membranes, and blocked with a protein-free solution for 30 min or 3% BSA for 2 h. The membranes were incubated at 4 degrees Celsius overnight with primary antibodies against human USP14 (14517-1-AP, Proteintech), N-Cadherin (bsm-52389R, Bioss), E-Cadherin (#3195, Cell Signaling Technology), Vimentin (10366-1-AP, Proteintech), PI3 Kinase p85 (#4257, Cell Signaling Technology), Phospho-PI3K p85 (Tyr458)[Tyr467]/p55 (Tyr199) (#AF3242, Affinity Biosciences), Akt (#9272, Cell Signaling Technology), Phospho-Akt (Ser473) (#4060, Cell Signaling Technology), mTOR (#2983, Cell Signaling Technology), Phospho-mTOR (Ser2448) (#2971, Cell Signaling Technology), p70(S6K) (14485-1-AP, Proteintech), and Phospho-p70(S6K) (Thr389)/p85(S6K) (Thr412) (28735-1-AP, Proteintech).

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

Journal: Discover Oncology

Article Title: USP14 promotes epithelial-mesenchymal transition and functions as a multifaceted biomarker in lung adenocarcinoma

doi: 10.1007/s12672-026-04682-1

Figure Lengend Snippet: USP14 activated the PI3K-AKT-mTOR signaling pathway. A , B The volcano map and heatmap were used to visualize the differentially expressed genes ( DEG ) between the high expression group vs. the low expression group of USP14 in the TCGA-LUAD database. The adjusted p value ≤ 0.05 and |log₂FC| ≥ 1 were considered as the significance filtering threshold. C , D GO and KEGG enrichment analysis was conducted on 796 DEGs filtered above, and terms with p and q value ≤ 0.05 were believed to be enriched significantly. E , F Stratification of samples based on the median USP14 expression level in the TCGA-LUAD database was followed by enrichment analysis employing the h.all.v2025.1.Hs.symbols.gmt gene set. GSEA demonstrates that USP14 expression is associated with the MTORC1 and the PI3K-AKT-MTOR signaling pathway. G – J Correlation analysis between USP14 expression and PIK3R1, AKT1, MTOR, and RPS6KB1 through GEPIA. K – R Western blot was used to detect protein changes of PI3K-AKT and mTOR signaling pathway markers in LUAD cell lines

Article Snippet: Proteins were then separated by SDS-PAGE and transferred to PVDF membranes, and blocked with a protein-free solution for 30 min or 3% BSA for 2 h. The membranes were incubated at 4 degrees Celsius overnight with primary antibodies against human USP14 (14517-1-AP, Proteintech), N-Cadherin (bsm-52389R, Bioss), E-Cadherin (#3195, Cell Signaling Technology), Vimentin (10366-1-AP, Proteintech), PI3 Kinase p85 (#4257, Cell Signaling Technology), Phospho-PI3K p85 (Tyr458)[Tyr467]/p55 (Tyr199) (#AF3242, Affinity Biosciences), Akt (#9272, Cell Signaling Technology), Phospho-Akt (Ser473) (#4060, Cell Signaling Technology), mTOR (#2983, Cell Signaling Technology), Phospho-mTOR (Ser2448) (#2971, Cell Signaling Technology), p70(S6K) (14485-1-AP, Proteintech), and Phospho-p70(S6K) (Thr389)/p85(S6K) (Thr412) (28735-1-AP, Proteintech).

Techniques: Expressing, Western Blot

Journal: Discover Oncology

Article Title: USP14 promotes epithelial-mesenchymal transition and functions as a multifaceted biomarker in lung adenocarcinoma

doi: 10.1007/s12672-026-04682-1

Figure Lengend Snippet: Suppressive immune infiltration landscape of USP14 in LUAD. A Heatmap of the immune cell infiltration profiles of USP14 by 8 algorithms (CIBERSORT, EPIC, ESTIMATE, IPS, MCP_counter, Quanti-seq, TIMER, xCell) based on IOBR package. B Correlation analysis between USP14 expression and four ESTIMATE scores, including Tumor Purity, Immune Score, Stromal Score and Estimate Score. C Estimate score of the high expression group vs. the low expression group of USP14 by ESTIMATE algorithm in TCGA database. D USP14 mRNA expression in immune subtypes: Wound Healing (C1); IFN-γ Dominant (C2); Inflammatory (C3); Lymphocyte Depleted (C4); Immunologically Quiet (C5); TGF-β Dominant (C6). E USP14 mRNA expression in TME subtypes: Immune-Enriched, Fibrotic (IE/F); Immune-Enriched, Non-Fibrotic (IE); Fibrotic (F); Immune-Depleted (D)

Article Snippet: Proteins were then separated by SDS-PAGE and transferred to PVDF membranes, and blocked with a protein-free solution for 30 min or 3% BSA for 2 h. The membranes were incubated at 4 degrees Celsius overnight with primary antibodies against human USP14 (14517-1-AP, Proteintech), N-Cadherin (bsm-52389R, Bioss), E-Cadherin (#3195, Cell Signaling Technology), Vimentin (10366-1-AP, Proteintech), PI3 Kinase p85 (#4257, Cell Signaling Technology), Phospho-PI3K p85 (Tyr458)[Tyr467]/p55 (Tyr199) (#AF3242, Affinity Biosciences), Akt (#9272, Cell Signaling Technology), Phospho-Akt (Ser473) (#4060, Cell Signaling Technology), mTOR (#2983, Cell Signaling Technology), Phospho-mTOR (Ser2448) (#2971, Cell Signaling Technology), p70(S6K) (14485-1-AP, Proteintech), and Phospho-p70(S6K) (Thr389)/p85(S6K) (Thr412) (28735-1-AP, Proteintech).

Techniques: Expressing

Journal: Discover Oncology

Article Title: USP14 promotes epithelial-mesenchymal transition and functions as a multifaceted biomarker in lung adenocarcinoma

doi: 10.1007/s12672-026-04682-1

Figure Lengend Snippet: Immune infiltration analysis and immunotherapy response prediction of USP14 in LUAD. A Barplot shows the proportion of 22 kinds of TICs of the high expression group vs. the low expression group of USP14 in the TCGA-LUAD database by CIBERSORT algorithm. Column names of plot are sample ID which not shown in the plot. B Clustered barplot shows the ratio differentiation of 22 kinds of immune cells between the low expression and high expression group of USP14 in the TCGA-LUAD database, and Wilcoxon rank sum was used for the significance test. C Heatmap depicting the correlation between 22 immune cells and USP14 expression, where each color square represents the corresponding correlation value. D Correlation analysis between USP14 expression and 24 immune cells by ssGSEA algorithm. E – H The violin diagrams show the differences in response index between the high expression group vs. the low expression group of USP14 among four Immunophenotype Scores ( IPS ) subgroups. I – L The comparison of treatment response rates ( I ), Tumor Immune Dysfunction and Exclusion (TIDE) scores ( J ), PD-L1 expression ( K ), Myeloid-Derived Suppressor Cells (MDSC) scores ( L ) between the low expression and high expression group of USP14

Article Snippet: Proteins were then separated by SDS-PAGE and transferred to PVDF membranes, and blocked with a protein-free solution for 30 min or 3% BSA for 2 h. The membranes were incubated at 4 degrees Celsius overnight with primary antibodies against human USP14 (14517-1-AP, Proteintech), N-Cadherin (bsm-52389R, Bioss), E-Cadherin (#3195, Cell Signaling Technology), Vimentin (10366-1-AP, Proteintech), PI3 Kinase p85 (#4257, Cell Signaling Technology), Phospho-PI3K p85 (Tyr458)[Tyr467]/p55 (Tyr199) (#AF3242, Affinity Biosciences), Akt (#9272, Cell Signaling Technology), Phospho-Akt (Ser473) (#4060, Cell Signaling Technology), mTOR (#2983, Cell Signaling Technology), Phospho-mTOR (Ser2448) (#2971, Cell Signaling Technology), p70(S6K) (14485-1-AP, Proteintech), and Phospho-p70(S6K) (Thr389)/p85(S6K) (Thr412) (28735-1-AP, Proteintech).

Techniques: Expressing, Comparison, Derivative Assay