Journal: The Journal of Biological Chemistry

Article Title: S-nitrosylation of the scaffold protein STRAP enhances oxidative stress–induced apoptosis

doi: 10.1016/j.jbc.2026.111141

Figure Lengend Snippet: S-nitrosylation of STRAP abolishes its ability to suppress ASK1 activity and disrupts their interaction. A , S-nitrosylated STRAP (SNO-STRAP) after in vitro SNO donor Cys-NO treatment. B , ASK1 kinase activity in the presence of unmodified STRAP or SNO-STRAP. n = 5. C , interaction between STRAP and ASK1 following iNOS induction. FLAG-STRAP and Myc-ASK1 were coexpressed in HEK-iNOS cells. Cell lysates were immunoprecipitated with anti-FLAG antibody and probed with anti-Myc antibody. “−” indicates the absence of doxycycline (Dox); “+” indicates the presence of Dox. n = 3. D , expression of iNOS and STRAP in HEK-iNOS cells transfected with no-targeting negative control siRNA or STRAP siRNA and induced by Dox. β-tubulin was used as a loading control. E , phosphorylation levels of MKK3 (Ser189) and p38 (Thr180/Tyr182) in HEK-iNOS cells transfected with control siRNA or STRAP siRNA. Cells were treated with H 2 O 2 , with or without Dox induction (“−” or “+”). n = 3. F and G , quantification of MKK3 phosphorylation and p38 phosphorylation levels in E . Phosphorylation levels of MKK3 and p38 (ImageJ signal intensities in E ) were first normalized to the corresponding pan-MKK3 and pan-p38 levels, respectively, and then were normalized to the si-control condition without Dox (−) and H 2 O 2 treatment (0 μM). All data in B , F , and G are presented as mean ± SD. Each data point represents one biological replicate. Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test. ∗ p < 0.05 and ∗∗ p < 0.01. ASK1, apoptosis signal–regulating kinase 1; H 2 O 2 , hydrogen peroxide; HEK, human embryonic kidney cell line; iNOS, inducible nitric oxide synthase; MKK, mitogen-activated protein kinase kinase; NO, nitric oxide; SNO, S-nitrosolthiol; STRAP, serine–threonine kinase receptor–associated protein.

Article Snippet: Phosphorylation levels were analyzed by SDS-PAGE, followed by immunoblotting using antibodies from Cell Signaling Technology, including phospho-MKK3 (9231S; 1:1000 dilution) and phospho-p38 (9215S; 1:1000 dilution), as well as a mouse anti-p97 antibody (10R-P104a; 1:2000 dilution) from Fitzgerald Industries.

Techniques: Activity Assay, In Vitro, Immunoprecipitation, Expressing, Transfection, Negative Control, Control, Phospho-proteomics

Journal: The Journal of Biological Chemistry

Article Title: S-nitrosylation of the scaffold protein STRAP enhances oxidative stress–induced apoptosis

doi: 10.1016/j.jbc.2026.111141

Figure Lengend Snippet: Mutation of SNO sites in STRAP alters ASK1 activity. A , detection of S-nitrosylated STRAP (SNO-STRAP) in HEK-iNOS cells overexpressing WT STRAP or candidate SNO-site mutants (C152S, C270S, C305S, C340S, and C152/270S). Cells were induced with doxycycline (Dox). Reaction without ascorbate (−Ascorbate) was used as a negative control. n = 3. B , quantification of SNO-STRAP levels shown in A . SNO level of all samples (ImageJ signal intensities in A ) was first normalized to the corresponding input, respectively, and then were normalized to the SNO level of STRAP-WT. C , ASK1 kinase activity in the presence of STRAP-WT or STRAP-C152/270S, with or without CysNO treatment (“−” or “+”). ASK1 activity in all samples was normalized to the average ASK1 activity of the first column (without STRAP protein). D , phosphorylation of MKK3 (Ser189) and p38 (Thr180/Tyr182) in HEK-iNOS cells expressing STRAP-WT or STRAP-C152/270S, following H 2 O 2 treatment with or without Dox induction (“−” or “+”). The p97 was used as a loading control. n = 3. E and F , quantification of MKK3 phosphorylation and p38 phosphorylation levels in D . Phosphorylation levels of MKK3 and p38 (ImageJ signal intensities in D ) were first normalized to the corresponding pan-MKK3 and pan-p38 levels, respectively, and then normalized to the STRAP-WT condition without Dox (−) and H 2 O 2 treatment (0 μM). All data in B , E , and F are presented as mean ± SD, with each data point representing one biological replicate. Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. ASK1, apoptosis signal–regulating kinase 1; H 2 O 2 , hydrogen peroxide; HEK, human embryonic kidney cell line; iNOS, inducible nitric oxide synthase; MKK3, mitogen-activated protein kinase kinase; SNO, S-nitrosolthiol; STRAP, serine–threonine kinase receptor–associated protein.

Article Snippet: Phosphorylation levels were analyzed by SDS-PAGE, followed by immunoblotting using antibodies from Cell Signaling Technology, including phospho-MKK3 (9231S; 1:1000 dilution) and phospho-p38 (9215S; 1:1000 dilution), as well as a mouse anti-p97 antibody (10R-P104a; 1:2000 dilution) from Fitzgerald Industries.

Techniques: Mutagenesis, Activity Assay, Negative Control, Phospho-proteomics, Expressing, Control

Journal: The Journal of Biological Chemistry

Article Title: S-nitrosylation of the scaffold protein STRAP enhances oxidative stress–induced apoptosis

doi: 10.1016/j.jbc.2026.111141

Figure Lengend Snippet: S-nitrosylation of STRAP abolishes its ability to suppress ASK1 activity and disrupts their interaction. A , S-nitrosylated STRAP (SNO-STRAP) after in vitro SNO donor Cys-NO treatment. B , ASK1 kinase activity in the presence of unmodified STRAP or SNO-STRAP. n = 5. C , interaction between STRAP and ASK1 following iNOS induction. FLAG-STRAP and Myc-ASK1 were coexpressed in HEK-iNOS cells. Cell lysates were immunoprecipitated with anti-FLAG antibody and probed with anti-Myc antibody. “−” indicates the absence of doxycycline (Dox); “+” indicates the presence of Dox. n = 3. D , expression of iNOS and STRAP in HEK-iNOS cells transfected with no-targeting negative control siRNA or STRAP siRNA and induced by Dox. β-tubulin was used as a loading control. E , phosphorylation levels of MKK3 (Ser189) and p38 (Thr180/Tyr182) in HEK-iNOS cells transfected with control siRNA or STRAP siRNA. Cells were treated with H 2 O 2 , with or without Dox induction (“−” or “+”). n = 3. F and G , quantification of MKK3 phosphorylation and p38 phosphorylation levels in E . Phosphorylation levels of MKK3 and p38 (ImageJ signal intensities in E ) were first normalized to the corresponding pan-MKK3 and pan-p38 levels, respectively, and then were normalized to the si-control condition without Dox (−) and H 2 O 2 treatment (0 μM). All data in B , F , and G are presented as mean ± SD. Each data point represents one biological replicate. Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test. ∗ p < 0.05 and ∗∗ p < 0.01. ASK1, apoptosis signal–regulating kinase 1; H 2 O 2 , hydrogen peroxide; HEK, human embryonic kidney cell line; iNOS, inducible nitric oxide synthase; MKK, mitogen-activated protein kinase kinase; NO, nitric oxide; SNO, S-nitrosolthiol; STRAP, serine–threonine kinase receptor–associated protein.

Article Snippet: The PVDF membrane was stripped using Restore PLUS Western Blot Stripping Buffer (Thermo Scientific) for 10 min and reblocked with antibodies rabbit anti-MKK3 (8535S; 1:1000 dilution) and anti-p38 (8690S; 1:1000 dilution) from Cell Signaling Technology.

Techniques: Activity Assay, In Vitro, Immunoprecipitation, Expressing, Transfection, Negative Control, Control, Phospho-proteomics

Journal: The Journal of Biological Chemistry

Article Title: S-nitrosylation of the scaffold protein STRAP enhances oxidative stress–induced apoptosis

doi: 10.1016/j.jbc.2026.111141

Figure Lengend Snippet: Mutation of SNO sites in STRAP alters ASK1 activity. A , detection of S-nitrosylated STRAP (SNO-STRAP) in HEK-iNOS cells overexpressing WT STRAP or candidate SNO-site mutants (C152S, C270S, C305S, C340S, and C152/270S). Cells were induced with doxycycline (Dox). Reaction without ascorbate (−Ascorbate) was used as a negative control. n = 3. B , quantification of SNO-STRAP levels shown in A . SNO level of all samples (ImageJ signal intensities in A ) was first normalized to the corresponding input, respectively, and then were normalized to the SNO level of STRAP-WT. C , ASK1 kinase activity in the presence of STRAP-WT or STRAP-C152/270S, with or without CysNO treatment (“−” or “+”). ASK1 activity in all samples was normalized to the average ASK1 activity of the first column (without STRAP protein). D , phosphorylation of MKK3 (Ser189) and p38 (Thr180/Tyr182) in HEK-iNOS cells expressing STRAP-WT or STRAP-C152/270S, following H 2 O 2 treatment with or without Dox induction (“−” or “+”). The p97 was used as a loading control. n = 3. E and F , quantification of MKK3 phosphorylation and p38 phosphorylation levels in D . Phosphorylation levels of MKK3 and p38 (ImageJ signal intensities in D ) were first normalized to the corresponding pan-MKK3 and pan-p38 levels, respectively, and then normalized to the STRAP-WT condition without Dox (−) and H 2 O 2 treatment (0 μM). All data in B , E , and F are presented as mean ± SD, with each data point representing one biological replicate. Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. ASK1, apoptosis signal–regulating kinase 1; H 2 O 2 , hydrogen peroxide; HEK, human embryonic kidney cell line; iNOS, inducible nitric oxide synthase; MKK3, mitogen-activated protein kinase kinase; SNO, S-nitrosolthiol; STRAP, serine–threonine kinase receptor–associated protein.

Article Snippet: The PVDF membrane was stripped using Restore PLUS Western Blot Stripping Buffer (Thermo Scientific) for 10 min and reblocked with antibodies rabbit anti-MKK3 (8535S; 1:1000 dilution) and anti-p38 (8690S; 1:1000 dilution) from Cell Signaling Technology.

Techniques: Mutagenesis, Activity Assay, Negative Control, Phospho-proteomics, Expressing, Control

Journal: Molecular Biomedicine

Article Title: Ubiquitin-specific protease 13 promotes colorectal cancer progression by stabilizing mitogen-activated protein kinase kinase 3

doi: 10.1186/s43556-025-00375-3

Figure Lengend Snippet: USP13 interacts with MKK3. a HCT116 cells overexpressing FLAG-USP13 or FLAG-USP13-AE were harvested for Western blot with the indicated antibodies. b Lysates of HEK293T cells transfected with MYC-p38α and FLAG-USP13 as indicated were immunoprecipitated with anti-FLAG beads, followed by Western blot with the indicated antibodies. c Lysates of HEK293T cells transfected with MYC-MKK3, MYC-MKK6 and FLAG-USP13 as indicated were immunoprecipitated with anti-FLAG beads, followed by Western blot with the indicated antibodies. d Lysates of HEK293T cells transfected with MYC-MKK3, FLAG-USP13 as indicated were immunoprecipitated with anti-MYC beads, followed by Western blot with the indicated antibodies. e Immunoprecipitation were performed using MKK3 or USP13 antibodies, followed by Western blot to identify the interaction between endogenous USP13 and MKK3. f The colocalization of USP13 and MKK3 was detected with immunofluorescence. MKK3 was labeled with red fluorescence, USP13 with green fluorescence, and the nucleus was stained with DAPI. Scale bar, 5 μm. g Lysates of HEK293T cells transfected with MYC-MKK3, FLAG-USP13-WT or AE were immunoprecipitated with anti-FLAG beads, followed by Western blot with the indicated antibodies. h Lysates of HEK293T cells transfected with empty vector, MYC-MKK3, MYC-MKK3-AA or EE, together with FLAG-USP13, were immunoprecipitated with anti-MYC antibody, followed by Western blot. i Schematic diagram of MKK3 and USP13 and their truncation mutants. j MYC-MKK3 truncations and FLAG-USP13 were transfected into HEK293T cells, followed by co-immunoprecipitation with anti-MYC antibody. k FLAG-USP13 truncations and MYC-MKK3 were transfected into HEK293T cells, followed by co-immunoprecipitation with anti-FLAG antibody

Article Snippet: Anti-rabbit antibody against MKK3 (#8535, 1:1,000), phospho-MKK3 (#12,289, 1:1,000), E-Cadherin (#3195, 1:2,000), Snail (#3879, 1:1,000), p38 (#8690, 1:2,000), phospho-p38 (#4511, 1:1,000), Erk1/2 (#4695, 1:2,000), phospho-Erk1/2 (#4370, 1:1,000), JNK (#67,096, 1:2,000) and phospho-JNK (#4668, 1:1,000) were purchased from CST and anti-mouse antibody against USP13 (#66,176–1-Ig, 1:2,000) were purchase from Proteintech.

Techniques: Western Blot, Transfection, Immunoprecipitation, Immunofluorescence, Labeling, Fluorescence, Staining, Plasmid Preparation

Journal: Molecular Biomedicine

Article Title: Ubiquitin-specific protease 13 promotes colorectal cancer progression by stabilizing mitogen-activated protein kinase kinase 3

doi: 10.1186/s43556-025-00375-3

Figure Lengend Snippet: USP13 promotes the stabilization of MKK3. a Western blot was performed to detect the protein levels of MKK3 and pMKK3 in HCT116 cells overexpressing FLAG-USP13-WT or AE. b A gradient transfection of FLAG-USP13 was performed, followed by Western blot with the indicated antibodies. c Western blot was performed to detect the protein levels of MKK3, pMKK3, p38, and p-p38 in control and USP13 knockout HCT116 cells. d Control and USP13 knockout HCT116 cells untreated or treated with MG132 (10 μM,12 h) were subjected to Western blot with the indicated antibodies. e CHX (50 μg/mL) and MG132 (10 μM) was added to HCT116 cells for 12 h, followed by Western blot with the indicated antibodies. f FLAG-USP13 was transfected into HCT116 cells, and 24 h later, CHX (50 μg/mL) was added for 12 h. Western blot was performed using the indicated antibodies. g MG132 (10 μM) was added to HCT116 cells stably overexpressing MYC-MKK3-WT or AA/EE for 12 h. The expression levels of the exogenous protein were detected using the MYC antibody. h CHX (50 μg/mL) was added to HCT116 cells stably overexpressing MYC-MKK3 or AA/EE, and cells were collected at the indicated time points, followed by Western blot with the indicated antibodies. i HCT116 cells expressing MYC-MKK3 or AA/EE were treated with CHX (50 μg/mL) and MG132 (10 μM) for 12 h, and followed by Western blot with the indicated antibodies. j HCT116 cells expressing MYC-MKK3 or AA/EE were transfected with FLAG-USP13. After 24 h, CHX (50 μg/mL) was added for 12 h, and followed by Western blot with the indicated antibodies. k-l CHX (50 μg/mL) was added to HCT116 cells, and cells were collected at the indicated time points, followed by Western blot with the indicated antibodies. ImageJ was used to quantify the gray values of the proteins at each time point, and relative values were calculated by comparing with the internal control, setting the 0-h time point as the baseline. A line graph was plotted to show the protein levels over time ( h , k, l ). The date were presented as means ± SD at least three independent experiments. Two-way ANOVA test was performed. ns: no significance, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Anti-rabbit antibody against MKK3 (#8535, 1:1,000), phospho-MKK3 (#12,289, 1:1,000), E-Cadherin (#3195, 1:2,000), Snail (#3879, 1:1,000), p38 (#8690, 1:2,000), phospho-p38 (#4511, 1:1,000), Erk1/2 (#4695, 1:2,000), phospho-Erk1/2 (#4370, 1:1,000), JNK (#67,096, 1:2,000) and phospho-JNK (#4668, 1:1,000) were purchased from CST and anti-mouse antibody against USP13 (#66,176–1-Ig, 1:2,000) were purchase from Proteintech.

Techniques: Western Blot, Transfection, Control, Knock-Out, Stable Transfection, Expressing

Journal: Molecular Biomedicine

Article Title: Ubiquitin-specific protease 13 promotes colorectal cancer progression by stabilizing mitogen-activated protein kinase kinase 3

doi: 10.1186/s43556-025-00375-3

Figure Lengend Snippet: USP13 targets MKK3 for deubiquitination. a HCT116 wild-type or USP13 knockout cells were transfected with MYC-MKK3 and HA-Ub, followed by MG132 treatment (10 μM, 12 h). Ubiquitination assay was performed to detect the ubiquitination levels of MKK3. b HCT116 cells were transfected with MYC-MKK3 and FLAG-USP13-WT or AE followed by MG132 treatment (10 μM, 12 h). Ubiquitination assay was performed to detect the ubiquitination levels of MKK3. c-d HEK293T cells were transfected with FLAG-USP13, MYC-MKK3, and HA-Ub mutants (K48, K63 or K48R, K63R) and treated with MG132 (10 μM, 12 h), followed by ubiquitination assay. e Ubiquitination assay was performed to analyze the ubiquitination of MKK3 in HEK293T cells transfected with MYC-MKK3 and HA-Ub mutants (K6, K11, K27, K29 or K33) together with or without Flag-USP13. f HEK293T cells were transfected with MYC-MKK3 or its AA/EE mutants as indicated, together with HA-Ub. Cell lysates were subjected to ubiquitination assay. g 293 T cells were transfected with MYC-MKK3-WT or AA/EE mutants, HA-Ub, and FLAG-USP13. After 24 h, cells were treated with MG132 (10 μM, 12 h). Cell lysates were subjected to ubiquitination assay. h HEK293T cells were transfected with MYC-MKK3 or its mutants (K32R, K80/93R, K200R, K243R, K332/340R) as indicated, together with HA-ubiquitin. Cell lysates were subjected to ubiquitination assay. i HEK293T cells were transfected with FLAG-USP13, HA-Ub, and MYC-MKK3 or its mutants (K32R or K332/340R), followed by ubiquitination assay

Article Snippet: Anti-rabbit antibody against MKK3 (#8535, 1:1,000), phospho-MKK3 (#12,289, 1:1,000), E-Cadherin (#3195, 1:2,000), Snail (#3879, 1:1,000), p38 (#8690, 1:2,000), phospho-p38 (#4511, 1:1,000), Erk1/2 (#4695, 1:2,000), phospho-Erk1/2 (#4370, 1:1,000), JNK (#67,096, 1:2,000) and phospho-JNK (#4668, 1:1,000) were purchased from CST and anti-mouse antibody against USP13 (#66,176–1-Ig, 1:2,000) were purchase from Proteintech.

Techniques: Knock-Out, Transfection, Ubiquitin Proteomics

Journal: Molecular Biomedicine

Article Title: Ubiquitin-specific protease 13 promotes colorectal cancer progression by stabilizing mitogen-activated protein kinase kinase 3

doi: 10.1186/s43556-025-00375-3

Figure Lengend Snippet: MKK3 promotes colorectal cancer progression. a Western blot was performed to detect the expression of MKK3 in CRC cells infected with MYC-MKK3 lentivirus. b Western blot was performed to detect the expression of MKK3 in CRC cells infected with sgMKK3 lentivirus. c Western blot was performed to detect the expression of MKK3-AA/EE mutants in CRC cells infected with MYC-MKK3-AA/EE lentivirus. d-f Colony formation ability was determined in HCT116 cells. g-h CCK-8 assay was performed to detect cell proliferation ability of HCT116 cells. i-j EdU assay was performed to determine the DNA synthesis activity in RKO cells. Representative images were presented. Scale bar, 100 μm. k-l Transwell assay was performed to determine the migration ability of RKO cells. Representative images were presented. Scale bar, 100 μm. m–o HCT116 cells were subcutaneously injected into the nude mice ( n = 5 per group). The tumor growth curve ( m ), photographs ( n ) and weight ( o ) were represented. The date were presented as means ± SD at least three independent experiments. Student’s t -tests ( d-f , i-l and o ) and two-way ANOVA test ( g , h and m ) were performed, ns: no significance, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Anti-rabbit antibody against MKK3 (#8535, 1:1,000), phospho-MKK3 (#12,289, 1:1,000), E-Cadherin (#3195, 1:2,000), Snail (#3879, 1:1,000), p38 (#8690, 1:2,000), phospho-p38 (#4511, 1:1,000), Erk1/2 (#4695, 1:2,000), phospho-Erk1/2 (#4370, 1:1,000), JNK (#67,096, 1:2,000) and phospho-JNK (#4668, 1:1,000) were purchased from CST and anti-mouse antibody against USP13 (#66,176–1-Ig, 1:2,000) were purchase from Proteintech.

Techniques: Western Blot, Expressing, Infection, CCK-8 Assay, EdU Assay, DNA Synthesis, Activity Assay, Transwell Assay, Migration, Injection

Journal: Molecular Biomedicine

Article Title: Ubiquitin-specific protease 13 promotes colorectal cancer progression by stabilizing mitogen-activated protein kinase kinase 3

doi: 10.1186/s43556-025-00375-3

Figure Lengend Snippet: Blocking K32 ubiquitination stabilizes MKK3 and promotes colorectal cancer. a-b Lysates of HEK293T cells transfected with MYC-MKK3-WT or K32R and FLAG-USP13 as indicated were immunoprecipitated with anti-FLAG/MYC antibodies, followed by immunoblot analysis with the indicated antibodies. c HCT116 cells expressing MYC-MKK3-WT or K32R were treated with CHX (50 μg/mL), and cells were collected at indicated time points. The expression levels of exogenous MYC-MKK3 proteins were detect by Western blot. The gray values of the protein at each time point were quantified using ImageJ, and relative values were calculated by comparing with the loading control. A line graph was plotted to show the changes in protein levels over time. d CCK-8 assay was performed to detect cell proliferation ability of MYC-MKK3-WT or K32R overexpressing HCT116 cells. e Colony formation ability was determined in MYC-MKK3-WT or K32R overexpressing HCT116 cells. f Transwell assay was performed to determine the migration and invasion ability of MYC-MKK3-WT or K32R overexpressing HCT116 cells. Representative images were presented. Scale bar, 100 μm. The date were presented as means ± SD at least three independent experiments. Student’s t -tests ( e–f ) and two-way ANOVA test ( c-d ) were performed, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Anti-rabbit antibody against MKK3 (#8535, 1:1,000), phospho-MKK3 (#12,289, 1:1,000), E-Cadherin (#3195, 1:2,000), Snail (#3879, 1:1,000), p38 (#8690, 1:2,000), phospho-p38 (#4511, 1:1,000), Erk1/2 (#4695, 1:2,000), phospho-Erk1/2 (#4370, 1:1,000), JNK (#67,096, 1:2,000) and phospho-JNK (#4668, 1:1,000) were purchased from CST and anti-mouse antibody against USP13 (#66,176–1-Ig, 1:2,000) were purchase from Proteintech.

Techniques: Blocking Assay, Ubiquitin Proteomics, Transfection, Immunoprecipitation, Western Blot, Expressing, Control, CCK-8 Assay, Transwell Assay, Migration

Journal: Molecular Biomedicine

Article Title: Ubiquitin-specific protease 13 promotes colorectal cancer progression by stabilizing mitogen-activated protein kinase kinase 3

doi: 10.1186/s43556-025-00375-3

Figure Lengend Snippet: MKK3 alleviate USP13 deficient induced phenotype. a MYC-MKK3 was introduced into USP13-knockout cells. Western blot was performed to confirm USP13 knockout and exogenous MYC-MKK3 expression. b HCT116 cells were seeded into 12-well plates at a density of 250 or 500 cells per well. After 7 days, colonies were stained with crystal violet and quantified. c MYC-MKK3-AA/EE mutants were introduced into USP13-knockout cells. Western blot was performed to confirm USP13 knockout and exogenous MYC-MKK3 expression. d HCT116 cells were seeded into 12-well plates at a density of 250 or 500 cells per well. After 7 days, colonies were stained with crystal violet and quantified. e–f CCK-8 assay was performed to assess cell proliferation ability of USP13 knockout or MKK3 reconstitution HCT116 or RKO cells. g EdU assay was performed to assess the DNA synthesis activity in USP13 knockout or MKK3 reconstitution HCT116 cells. Representative images were presented. Scale bar, 100 μm. h Transwell assay was performed to assess the migration ability of USP13 knockout or MKK3 reconstitution RKO cells. Representative images were presented. Scale bar, 100 μm. i-k USP13 knockout or p38α reconstitution HCT116 cells were subcutaneously injected into the nude mice ( n = 5 per group). l-n Control or USP13 knockout HCT116 cells were subcutaneously injected into the nude mice ( n = 5 per group). Tumor-bearing mice were treated daily with either vehicle (10% DMSO, 40% PEG300, 5% Tween-80, and 45% saline, i.p.) or SB202190 (5 mg/kg in 10% DMSO, 40% PEG300, 5% Tween-80, and 45% saline, i.p.). The tumor growth curve ( i , l ), photographs ( j , m ) and weight ( k , n ) were represented. The date were presented as means ± SD at least three independent experiments. Student’s t -tests ( b , d , g , h , k and n ) and two-way ANOVA test ( e , f , i and l ) were performed, ns: no significance, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Anti-rabbit antibody against MKK3 (#8535, 1:1,000), phospho-MKK3 (#12,289, 1:1,000), E-Cadherin (#3195, 1:2,000), Snail (#3879, 1:1,000), p38 (#8690, 1:2,000), phospho-p38 (#4511, 1:1,000), Erk1/2 (#4695, 1:2,000), phospho-Erk1/2 (#4370, 1:1,000), JNK (#67,096, 1:2,000) and phospho-JNK (#4668, 1:1,000) were purchased from CST and anti-mouse antibody against USP13 (#66,176–1-Ig, 1:2,000) were purchase from Proteintech.

Techniques: Knock-Out, Western Blot, Expressing, Staining, CCK-8 Assay, EdU Assay, DNA Synthesis, Activity Assay, Transwell Assay, Migration, Injection, Control, Saline

Journal: Molecular Biomedicine

Article Title: Ubiquitin-specific protease 13 promotes colorectal cancer progression by stabilizing mitogen-activated protein kinase kinase 3

doi: 10.1186/s43556-025-00375-3

Figure Lengend Snippet: USP13-MKK3-p38 promotes tumor formation. a-c MC38 cells were subcutaneously injected into 8-week-old Usp13 + / + and Usp13 −/− C57 mice ( n = 5 per group). Tumor size was measured every other day once tumors became visible. At the end of the experiment, tumors were excised, photographed, and weighed. The tumor growth curve ( a ), photographs ( b ) and weight ( c ) were represented. d-e H&E staining and Ki-67 immunostaining were performed on tumor samples, followed by quantification of the percentage of Ki-67-positive cells. Scale bar, 100 μm. f–h Tissue microarrays were prepared from CRC patient samples and stained with USP13, MKK3, and Ki-67 antibodies. The percentage of positive cells for each antibody was quantified and calculated ( n = 30). Scale bar, 100 μm. i Western blot was performed to detect the protein levels of USP13 and MKK3 in CRC samples. The representative images were presented. j The band density of USP13 and MKK3 in was quantified using ImageJ software and normalized to the corresponding loading control. The correlation analysis of USP13 and MKK3 protein levels in 30 CRC samples. k Mechanism diagram of USP13-mediated stabilization of MKK3 through deubiquitination (Created by Biorender: https://BioRender.com/jze3xva ). The date was presented as means ± SD at least three independent experiments. Student’s t -tests ( c and e–h ) and two-way ANOVA test ( a ) were performed, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Anti-rabbit antibody against MKK3 (#8535, 1:1,000), phospho-MKK3 (#12,289, 1:1,000), E-Cadherin (#3195, 1:2,000), Snail (#3879, 1:1,000), p38 (#8690, 1:2,000), phospho-p38 (#4511, 1:1,000), Erk1/2 (#4695, 1:2,000), phospho-Erk1/2 (#4370, 1:1,000), JNK (#67,096, 1:2,000) and phospho-JNK (#4668, 1:1,000) were purchased from CST and anti-mouse antibody against USP13 (#66,176–1-Ig, 1:2,000) were purchase from Proteintech.

Techniques: Injection, Staining, Immunostaining, Western Blot, Software, Control

Journal: Molecular Biomedicine

Article Title: Ubiquitin-specific protease 13 promotes colorectal cancer progression by stabilizing mitogen-activated protein kinase kinase 3

doi: 10.1186/s43556-025-00375-3

Figure Lengend Snippet: USP13 interacts with MKK3. a HCT116 cells overexpressing FLAG-USP13 or FLAG-USP13-AE were harvested for Western blot with the indicated antibodies. b Lysates of HEK293T cells transfected with MYC-p38α and FLAG-USP13 as indicated were immunoprecipitated with anti-FLAG beads, followed by Western blot with the indicated antibodies. c Lysates of HEK293T cells transfected with MYC-MKK3, MYC-MKK6 and FLAG-USP13 as indicated were immunoprecipitated with anti-FLAG beads, followed by Western blot with the indicated antibodies. d Lysates of HEK293T cells transfected with MYC-MKK3, FLAG-USP13 as indicated were immunoprecipitated with anti-MYC beads, followed by Western blot with the indicated antibodies. e Immunoprecipitation were performed using MKK3 or USP13 antibodies, followed by Western blot to identify the interaction between endogenous USP13 and MKK3. f The colocalization of USP13 and MKK3 was detected with immunofluorescence. MKK3 was labeled with red fluorescence, USP13 with green fluorescence, and the nucleus was stained with DAPI. Scale bar, 5 μm. g Lysates of HEK293T cells transfected with MYC-MKK3, FLAG-USP13-WT or AE were immunoprecipitated with anti-FLAG beads, followed by Western blot with the indicated antibodies. h Lysates of HEK293T cells transfected with empty vector, MYC-MKK3, MYC-MKK3-AA or EE, together with FLAG-USP13, were immunoprecipitated with anti-MYC antibody, followed by Western blot. i Schematic diagram of MKK3 and USP13 and their truncation mutants. j MYC-MKK3 truncations and FLAG-USP13 were transfected into HEK293T cells, followed by co-immunoprecipitation with anti-MYC antibody. k FLAG-USP13 truncations and MYC-MKK3 were transfected into HEK293T cells, followed by co-immunoprecipitation with anti-FLAG antibody

Article Snippet: Anti-rabbit antibody against MKK3 (#8535, 1:1,000), phospho-MKK3 (#12,289, 1:1,000), E-Cadherin (#3195, 1:2,000), Snail (#3879, 1:1,000), p38 (#8690, 1:2,000), phospho-p38 (#4511, 1:1,000), Erk1/2 (#4695, 1:2,000), phospho-Erk1/2 (#4370, 1:1,000), JNK (#67,096, 1:2,000) and phospho-JNK (#4668, 1:1,000) were purchased from CST and anti-mouse antibody against USP13 (#66,176–1-Ig, 1:2,000) were purchase from Proteintech.

Techniques: Western Blot, Transfection, Immunoprecipitation, Immunofluorescence, Labeling, Fluorescence, Staining, Plasmid Preparation

Journal: Molecular Biomedicine

Article Title: Ubiquitin-specific protease 13 promotes colorectal cancer progression by stabilizing mitogen-activated protein kinase kinase 3

doi: 10.1186/s43556-025-00375-3

Figure Lengend Snippet: USP13 promotes the stabilization of MKK3. a Western blot was performed to detect the protein levels of MKK3 and pMKK3 in HCT116 cells overexpressing FLAG-USP13-WT or AE. b A gradient transfection of FLAG-USP13 was performed, followed by Western blot with the indicated antibodies. c Western blot was performed to detect the protein levels of MKK3, pMKK3, p38, and p-p38 in control and USP13 knockout HCT116 cells. d Control and USP13 knockout HCT116 cells untreated or treated with MG132 (10 μM,12 h) were subjected to Western blot with the indicated antibodies. e CHX (50 μg/mL) and MG132 (10 μM) was added to HCT116 cells for 12 h, followed by Western blot with the indicated antibodies. f FLAG-USP13 was transfected into HCT116 cells, and 24 h later, CHX (50 μg/mL) was added for 12 h. Western blot was performed using the indicated antibodies. g MG132 (10 μM) was added to HCT116 cells stably overexpressing MYC-MKK3-WT or AA/EE for 12 h. The expression levels of the exogenous protein were detected using the MYC antibody. h CHX (50 μg/mL) was added to HCT116 cells stably overexpressing MYC-MKK3 or AA/EE, and cells were collected at the indicated time points, followed by Western blot with the indicated antibodies. i HCT116 cells expressing MYC-MKK3 or AA/EE were treated with CHX (50 μg/mL) and MG132 (10 μM) for 12 h, and followed by Western blot with the indicated antibodies. j HCT116 cells expressing MYC-MKK3 or AA/EE were transfected with FLAG-USP13. After 24 h, CHX (50 μg/mL) was added for 12 h, and followed by Western blot with the indicated antibodies. k-l CHX (50 μg/mL) was added to HCT116 cells, and cells were collected at the indicated time points, followed by Western blot with the indicated antibodies. ImageJ was used to quantify the gray values of the proteins at each time point, and relative values were calculated by comparing with the internal control, setting the 0-h time point as the baseline. A line graph was plotted to show the protein levels over time ( h , k, l ). The date were presented as means ± SD at least three independent experiments. Two-way ANOVA test was performed. ns: no significance, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Anti-rabbit antibody against MKK3 (#8535, 1:1,000), phospho-MKK3 (#12,289, 1:1,000), E-Cadherin (#3195, 1:2,000), Snail (#3879, 1:1,000), p38 (#8690, 1:2,000), phospho-p38 (#4511, 1:1,000), Erk1/2 (#4695, 1:2,000), phospho-Erk1/2 (#4370, 1:1,000), JNK (#67,096, 1:2,000) and phospho-JNK (#4668, 1:1,000) were purchased from CST and anti-mouse antibody against USP13 (#66,176–1-Ig, 1:2,000) were purchase from Proteintech.

Techniques: Western Blot, Transfection, Control, Knock-Out, Stable Transfection, Expressing

Journal: Molecular Biomedicine

Article Title: Ubiquitin-specific protease 13 promotes colorectal cancer progression by stabilizing mitogen-activated protein kinase kinase 3

doi: 10.1186/s43556-025-00375-3

Figure Lengend Snippet: USP13 targets MKK3 for deubiquitination. a HCT116 wild-type or USP13 knockout cells were transfected with MYC-MKK3 and HA-Ub, followed by MG132 treatment (10 μM, 12 h). Ubiquitination assay was performed to detect the ubiquitination levels of MKK3. b HCT116 cells were transfected with MYC-MKK3 and FLAG-USP13-WT or AE followed by MG132 treatment (10 μM, 12 h). Ubiquitination assay was performed to detect the ubiquitination levels of MKK3. c-d HEK293T cells were transfected with FLAG-USP13, MYC-MKK3, and HA-Ub mutants (K48, K63 or K48R, K63R) and treated with MG132 (10 μM, 12 h), followed by ubiquitination assay. e Ubiquitination assay was performed to analyze the ubiquitination of MKK3 in HEK293T cells transfected with MYC-MKK3 and HA-Ub mutants (K6, K11, K27, K29 or K33) together with or without Flag-USP13. f HEK293T cells were transfected with MYC-MKK3 or its AA/EE mutants as indicated, together with HA-Ub. Cell lysates were subjected to ubiquitination assay. g 293 T cells were transfected with MYC-MKK3-WT or AA/EE mutants, HA-Ub, and FLAG-USP13. After 24 h, cells were treated with MG132 (10 μM, 12 h). Cell lysates were subjected to ubiquitination assay. h HEK293T cells were transfected with MYC-MKK3 or its mutants (K32R, K80/93R, K200R, K243R, K332/340R) as indicated, together with HA-ubiquitin. Cell lysates were subjected to ubiquitination assay. i HEK293T cells were transfected with FLAG-USP13, HA-Ub, and MYC-MKK3 or its mutants (K32R or K332/340R), followed by ubiquitination assay

Article Snippet: Anti-rabbit antibody against MKK3 (#8535, 1:1,000), phospho-MKK3 (#12,289, 1:1,000), E-Cadherin (#3195, 1:2,000), Snail (#3879, 1:1,000), p38 (#8690, 1:2,000), phospho-p38 (#4511, 1:1,000), Erk1/2 (#4695, 1:2,000), phospho-Erk1/2 (#4370, 1:1,000), JNK (#67,096, 1:2,000) and phospho-JNK (#4668, 1:1,000) were purchased from CST and anti-mouse antibody against USP13 (#66,176–1-Ig, 1:2,000) were purchase from Proteintech.

Techniques: Knock-Out, Transfection, Ubiquitin Proteomics

Journal: Molecular Biomedicine

Article Title: Ubiquitin-specific protease 13 promotes colorectal cancer progression by stabilizing mitogen-activated protein kinase kinase 3

doi: 10.1186/s43556-025-00375-3

Figure Lengend Snippet: MKK3 promotes colorectal cancer progression. a Western blot was performed to detect the expression of MKK3 in CRC cells infected with MYC-MKK3 lentivirus. b Western blot was performed to detect the expression of MKK3 in CRC cells infected with sgMKK3 lentivirus. c Western blot was performed to detect the expression of MKK3-AA/EE mutants in CRC cells infected with MYC-MKK3-AA/EE lentivirus. d-f Colony formation ability was determined in HCT116 cells. g-h CCK-8 assay was performed to detect cell proliferation ability of HCT116 cells. i-j EdU assay was performed to determine the DNA synthesis activity in RKO cells. Representative images were presented. Scale bar, 100 μm. k-l Transwell assay was performed to determine the migration ability of RKO cells. Representative images were presented. Scale bar, 100 μm. m–o HCT116 cells were subcutaneously injected into the nude mice ( n = 5 per group). The tumor growth curve ( m ), photographs ( n ) and weight ( o ) were represented. The date were presented as means ± SD at least three independent experiments. Student’s t -tests ( d-f , i-l and o ) and two-way ANOVA test ( g , h and m ) were performed, ns: no significance, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Anti-rabbit antibody against MKK3 (#8535, 1:1,000), phospho-MKK3 (#12,289, 1:1,000), E-Cadherin (#3195, 1:2,000), Snail (#3879, 1:1,000), p38 (#8690, 1:2,000), phospho-p38 (#4511, 1:1,000), Erk1/2 (#4695, 1:2,000), phospho-Erk1/2 (#4370, 1:1,000), JNK (#67,096, 1:2,000) and phospho-JNK (#4668, 1:1,000) were purchased from CST and anti-mouse antibody against USP13 (#66,176–1-Ig, 1:2,000) were purchase from Proteintech.

Techniques: Western Blot, Expressing, Infection, CCK-8 Assay, EdU Assay, DNA Synthesis, Activity Assay, Transwell Assay, Migration, Injection

Journal: Molecular Biomedicine

Article Title: Ubiquitin-specific protease 13 promotes colorectal cancer progression by stabilizing mitogen-activated protein kinase kinase 3

doi: 10.1186/s43556-025-00375-3

Figure Lengend Snippet: Blocking K32 ubiquitination stabilizes MKK3 and promotes colorectal cancer. a-b Lysates of HEK293T cells transfected with MYC-MKK3-WT or K32R and FLAG-USP13 as indicated were immunoprecipitated with anti-FLAG/MYC antibodies, followed by immunoblot analysis with the indicated antibodies. c HCT116 cells expressing MYC-MKK3-WT or K32R were treated with CHX (50 μg/mL), and cells were collected at indicated time points. The expression levels of exogenous MYC-MKK3 proteins were detect by Western blot. The gray values of the protein at each time point were quantified using ImageJ, and relative values were calculated by comparing with the loading control. A line graph was plotted to show the changes in protein levels over time. d CCK-8 assay was performed to detect cell proliferation ability of MYC-MKK3-WT or K32R overexpressing HCT116 cells. e Colony formation ability was determined in MYC-MKK3-WT or K32R overexpressing HCT116 cells. f Transwell assay was performed to determine the migration and invasion ability of MYC-MKK3-WT or K32R overexpressing HCT116 cells. Representative images were presented. Scale bar, 100 μm. The date were presented as means ± SD at least three independent experiments. Student’s t -tests ( e–f ) and two-way ANOVA test ( c-d ) were performed, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Anti-rabbit antibody against MKK3 (#8535, 1:1,000), phospho-MKK3 (#12,289, 1:1,000), E-Cadherin (#3195, 1:2,000), Snail (#3879, 1:1,000), p38 (#8690, 1:2,000), phospho-p38 (#4511, 1:1,000), Erk1/2 (#4695, 1:2,000), phospho-Erk1/2 (#4370, 1:1,000), JNK (#67,096, 1:2,000) and phospho-JNK (#4668, 1:1,000) were purchased from CST and anti-mouse antibody against USP13 (#66,176–1-Ig, 1:2,000) were purchase from Proteintech.

Techniques: Blocking Assay, Ubiquitin Proteomics, Transfection, Immunoprecipitation, Western Blot, Expressing, Control, CCK-8 Assay, Transwell Assay, Migration

Journal: Molecular Biomedicine

Article Title: Ubiquitin-specific protease 13 promotes colorectal cancer progression by stabilizing mitogen-activated protein kinase kinase 3

doi: 10.1186/s43556-025-00375-3

Figure Lengend Snippet: MKK3 alleviate USP13 deficient induced phenotype. a MYC-MKK3 was introduced into USP13-knockout cells. Western blot was performed to confirm USP13 knockout and exogenous MYC-MKK3 expression. b HCT116 cells were seeded into 12-well plates at a density of 250 or 500 cells per well. After 7 days, colonies were stained with crystal violet and quantified. c MYC-MKK3-AA/EE mutants were introduced into USP13-knockout cells. Western blot was performed to confirm USP13 knockout and exogenous MYC-MKK3 expression. d HCT116 cells were seeded into 12-well plates at a density of 250 or 500 cells per well. After 7 days, colonies were stained with crystal violet and quantified. e–f CCK-8 assay was performed to assess cell proliferation ability of USP13 knockout or MKK3 reconstitution HCT116 or RKO cells. g EdU assay was performed to assess the DNA synthesis activity in USP13 knockout or MKK3 reconstitution HCT116 cells. Representative images were presented. Scale bar, 100 μm. h Transwell assay was performed to assess the migration ability of USP13 knockout or MKK3 reconstitution RKO cells. Representative images were presented. Scale bar, 100 μm. i-k USP13 knockout or p38α reconstitution HCT116 cells were subcutaneously injected into the nude mice ( n = 5 per group). l-n Control or USP13 knockout HCT116 cells were subcutaneously injected into the nude mice ( n = 5 per group). Tumor-bearing mice were treated daily with either vehicle (10% DMSO, 40% PEG300, 5% Tween-80, and 45% saline, i.p.) or SB202190 (5 mg/kg in 10% DMSO, 40% PEG300, 5% Tween-80, and 45% saline, i.p.). The tumor growth curve ( i , l ), photographs ( j , m ) and weight ( k , n ) were represented. The date were presented as means ± SD at least three independent experiments. Student’s t -tests ( b , d , g , h , k and n ) and two-way ANOVA test ( e , f , i and l ) were performed, ns: no significance, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Anti-rabbit antibody against MKK3 (#8535, 1:1,000), phospho-MKK3 (#12,289, 1:1,000), E-Cadherin (#3195, 1:2,000), Snail (#3879, 1:1,000), p38 (#8690, 1:2,000), phospho-p38 (#4511, 1:1,000), Erk1/2 (#4695, 1:2,000), phospho-Erk1/2 (#4370, 1:1,000), JNK (#67,096, 1:2,000) and phospho-JNK (#4668, 1:1,000) were purchased from CST and anti-mouse antibody against USP13 (#66,176–1-Ig, 1:2,000) were purchase from Proteintech.

Techniques: Knock-Out, Western Blot, Expressing, Staining, CCK-8 Assay, EdU Assay, DNA Synthesis, Activity Assay, Transwell Assay, Migration, Injection, Control, Saline

Journal: Molecular Biomedicine

Article Title: Ubiquitin-specific protease 13 promotes colorectal cancer progression by stabilizing mitogen-activated protein kinase kinase 3

doi: 10.1186/s43556-025-00375-3

Figure Lengend Snippet: USP13-MKK3-p38 promotes tumor formation. a-c MC38 cells were subcutaneously injected into 8-week-old Usp13 + / + and Usp13 −/− C57 mice ( n = 5 per group). Tumor size was measured every other day once tumors became visible. At the end of the experiment, tumors were excised, photographed, and weighed. The tumor growth curve ( a ), photographs ( b ) and weight ( c ) were represented. d-e H&E staining and Ki-67 immunostaining were performed on tumor samples, followed by quantification of the percentage of Ki-67-positive cells. Scale bar, 100 μm. f–h Tissue microarrays were prepared from CRC patient samples and stained with USP13, MKK3, and Ki-67 antibodies. The percentage of positive cells for each antibody was quantified and calculated ( n = 30). Scale bar, 100 μm. i Western blot was performed to detect the protein levels of USP13 and MKK3 in CRC samples. The representative images were presented. j The band density of USP13 and MKK3 in was quantified using ImageJ software and normalized to the corresponding loading control. The correlation analysis of USP13 and MKK3 protein levels in 30 CRC samples. k Mechanism diagram of USP13-mediated stabilization of MKK3 through deubiquitination (Created by Biorender: https://BioRender.com/jze3xva ). The date was presented as means ± SD at least three independent experiments. Student’s t -tests ( c and e–h ) and two-way ANOVA test ( a ) were performed, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Anti-rabbit antibody against MKK3 (#8535, 1:1,000), phospho-MKK3 (#12,289, 1:1,000), E-Cadherin (#3195, 1:2,000), Snail (#3879, 1:1,000), p38 (#8690, 1:2,000), phospho-p38 (#4511, 1:1,000), Erk1/2 (#4695, 1:2,000), phospho-Erk1/2 (#4370, 1:1,000), JNK (#67,096, 1:2,000) and phospho-JNK (#4668, 1:1,000) were purchased from CST and anti-mouse antibody against USP13 (#66,176–1-Ig, 1:2,000) were purchase from Proteintech.

Techniques: Injection, Staining, Immunostaining, Western Blot, Software, Control

Journal: Molecular Biomedicine

Article Title: Ubiquitin-specific protease 13 promotes colorectal cancer progression by stabilizing mitogen-activated protein kinase kinase 3

doi: 10.1186/s43556-025-00375-3

Figure Lengend Snippet: USP13 interacts with MKK3. a HCT116 cells overexpressing FLAG-USP13 or FLAG-USP13-AE were harvested for Western blot with the indicated antibodies. b Lysates of HEK293T cells transfected with MYC-p38α and FLAG-USP13 as indicated were immunoprecipitated with anti-FLAG beads, followed by Western blot with the indicated antibodies. c Lysates of HEK293T cells transfected with MYC-MKK3, MYC-MKK6 and FLAG-USP13 as indicated were immunoprecipitated with anti-FLAG beads, followed by Western blot with the indicated antibodies. d Lysates of HEK293T cells transfected with MYC-MKK3, FLAG-USP13 as indicated were immunoprecipitated with anti-MYC beads, followed by Western blot with the indicated antibodies. e Immunoprecipitation were performed using MKK3 or USP13 antibodies, followed by Western blot to identify the interaction between endogenous USP13 and MKK3. f The colocalization of USP13 and MKK3 was detected with immunofluorescence. MKK3 was labeled with red fluorescence, USP13 with green fluorescence, and the nucleus was stained with DAPI. Scale bar, 5 μm. g Lysates of HEK293T cells transfected with MYC-MKK3, FLAG-USP13-WT or AE were immunoprecipitated with anti-FLAG beads, followed by Western blot with the indicated antibodies. h Lysates of HEK293T cells transfected with empty vector, MYC-MKK3, MYC-MKK3-AA or EE, together with FLAG-USP13, were immunoprecipitated with anti-MYC antibody, followed by Western blot. i Schematic diagram of MKK3 and USP13 and their truncation mutants. j MYC-MKK3 truncations and FLAG-USP13 were transfected into HEK293T cells, followed by co-immunoprecipitation with anti-MYC antibody. k FLAG-USP13 truncations and MYC-MKK3 were transfected into HEK293T cells, followed by co-immunoprecipitation with anti-FLAG antibody

Article Snippet: The slides were then incubated overnight at 4 °C with primary antibodies targeting USP13 (Proteintech, 1:200), MKK3 (CST, 1:200), and Ki-67 (CST, 1:200), after a 1-h blocking step using 5% BSA at room temperature.

Techniques: Western Blot, Transfection, Immunoprecipitation, Immunofluorescence, Labeling, Fluorescence, Staining, Plasmid Preparation

Journal: Molecular Biomedicine

Article Title: Ubiquitin-specific protease 13 promotes colorectal cancer progression by stabilizing mitogen-activated protein kinase kinase 3

doi: 10.1186/s43556-025-00375-3

Figure Lengend Snippet: USP13 promotes the stabilization of MKK3. a Western blot was performed to detect the protein levels of MKK3 and pMKK3 in HCT116 cells overexpressing FLAG-USP13-WT or AE. b A gradient transfection of FLAG-USP13 was performed, followed by Western blot with the indicated antibodies. c Western blot was performed to detect the protein levels of MKK3, pMKK3, p38, and p-p38 in control and USP13 knockout HCT116 cells. d Control and USP13 knockout HCT116 cells untreated or treated with MG132 (10 μM,12 h) were subjected to Western blot with the indicated antibodies. e CHX (50 μg/mL) and MG132 (10 μM) was added to HCT116 cells for 12 h, followed by Western blot with the indicated antibodies. f FLAG-USP13 was transfected into HCT116 cells, and 24 h later, CHX (50 μg/mL) was added for 12 h. Western blot was performed using the indicated antibodies. g MG132 (10 μM) was added to HCT116 cells stably overexpressing MYC-MKK3-WT or AA/EE for 12 h. The expression levels of the exogenous protein were detected using the MYC antibody. h CHX (50 μg/mL) was added to HCT116 cells stably overexpressing MYC-MKK3 or AA/EE, and cells were collected at the indicated time points, followed by Western blot with the indicated antibodies. i HCT116 cells expressing MYC-MKK3 or AA/EE were treated with CHX (50 μg/mL) and MG132 (10 μM) for 12 h, and followed by Western blot with the indicated antibodies. j HCT116 cells expressing MYC-MKK3 or AA/EE were transfected with FLAG-USP13. After 24 h, CHX (50 μg/mL) was added for 12 h, and followed by Western blot with the indicated antibodies. k-l CHX (50 μg/mL) was added to HCT116 cells, and cells were collected at the indicated time points, followed by Western blot with the indicated antibodies. ImageJ was used to quantify the gray values of the proteins at each time point, and relative values were calculated by comparing with the internal control, setting the 0-h time point as the baseline. A line graph was plotted to show the protein levels over time ( h , k, l ). The date were presented as means ± SD at least three independent experiments. Two-way ANOVA test was performed. ns: no significance, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: The slides were then incubated overnight at 4 °C with primary antibodies targeting USP13 (Proteintech, 1:200), MKK3 (CST, 1:200), and Ki-67 (CST, 1:200), after a 1-h blocking step using 5% BSA at room temperature.

Techniques: Western Blot, Transfection, Control, Knock-Out, Stable Transfection, Expressing

Journal: Molecular Biomedicine

Article Title: Ubiquitin-specific protease 13 promotes colorectal cancer progression by stabilizing mitogen-activated protein kinase kinase 3

doi: 10.1186/s43556-025-00375-3

Figure Lengend Snippet: USP13 targets MKK3 for deubiquitination. a HCT116 wild-type or USP13 knockout cells were transfected with MYC-MKK3 and HA-Ub, followed by MG132 treatment (10 μM, 12 h). Ubiquitination assay was performed to detect the ubiquitination levels of MKK3. b HCT116 cells were transfected with MYC-MKK3 and FLAG-USP13-WT or AE followed by MG132 treatment (10 μM, 12 h). Ubiquitination assay was performed to detect the ubiquitination levels of MKK3. c-d HEK293T cells were transfected with FLAG-USP13, MYC-MKK3, and HA-Ub mutants (K48, K63 or K48R, K63R) and treated with MG132 (10 μM, 12 h), followed by ubiquitination assay. e Ubiquitination assay was performed to analyze the ubiquitination of MKK3 in HEK293T cells transfected with MYC-MKK3 and HA-Ub mutants (K6, K11, K27, K29 or K33) together with or without Flag-USP13. f HEK293T cells were transfected with MYC-MKK3 or its AA/EE mutants as indicated, together with HA-Ub. Cell lysates were subjected to ubiquitination assay. g 293 T cells were transfected with MYC-MKK3-WT or AA/EE mutants, HA-Ub, and FLAG-USP13. After 24 h, cells were treated with MG132 (10 μM, 12 h). Cell lysates were subjected to ubiquitination assay. h HEK293T cells were transfected with MYC-MKK3 or its mutants (K32R, K80/93R, K200R, K243R, K332/340R) as indicated, together with HA-ubiquitin. Cell lysates were subjected to ubiquitination assay. i HEK293T cells were transfected with FLAG-USP13, HA-Ub, and MYC-MKK3 or its mutants (K32R or K332/340R), followed by ubiquitination assay

Article Snippet: The slides were then incubated overnight at 4 °C with primary antibodies targeting USP13 (Proteintech, 1:200), MKK3 (CST, 1:200), and Ki-67 (CST, 1:200), after a 1-h blocking step using 5% BSA at room temperature.

Techniques: Knock-Out, Transfection, Ubiquitin Proteomics

Journal: Molecular Biomedicine

Article Title: Ubiquitin-specific protease 13 promotes colorectal cancer progression by stabilizing mitogen-activated protein kinase kinase 3

doi: 10.1186/s43556-025-00375-3

Figure Lengend Snippet: MKK3 promotes colorectal cancer progression. a Western blot was performed to detect the expression of MKK3 in CRC cells infected with MYC-MKK3 lentivirus. b Western blot was performed to detect the expression of MKK3 in CRC cells infected with sgMKK3 lentivirus. c Western blot was performed to detect the expression of MKK3-AA/EE mutants in CRC cells infected with MYC-MKK3-AA/EE lentivirus. d-f Colony formation ability was determined in HCT116 cells. g-h CCK-8 assay was performed to detect cell proliferation ability of HCT116 cells. i-j EdU assay was performed to determine the DNA synthesis activity in RKO cells. Representative images were presented. Scale bar, 100 μm. k-l Transwell assay was performed to determine the migration ability of RKO cells. Representative images were presented. Scale bar, 100 μm. m–o HCT116 cells were subcutaneously injected into the nude mice ( n = 5 per group). The tumor growth curve ( m ), photographs ( n ) and weight ( o ) were represented. The date were presented as means ± SD at least three independent experiments. Student’s t -tests ( d-f , i-l and o ) and two-way ANOVA test ( g , h and m ) were performed, ns: no significance, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: The slides were then incubated overnight at 4 °C with primary antibodies targeting USP13 (Proteintech, 1:200), MKK3 (CST, 1:200), and Ki-67 (CST, 1:200), after a 1-h blocking step using 5% BSA at room temperature.

Techniques: Western Blot, Expressing, Infection, CCK-8 Assay, EdU Assay, DNA Synthesis, Activity Assay, Transwell Assay, Migration, Injection

Journal: Molecular Biomedicine

Article Title: Ubiquitin-specific protease 13 promotes colorectal cancer progression by stabilizing mitogen-activated protein kinase kinase 3

doi: 10.1186/s43556-025-00375-3

Figure Lengend Snippet: Blocking K32 ubiquitination stabilizes MKK3 and promotes colorectal cancer. a-b Lysates of HEK293T cells transfected with MYC-MKK3-WT or K32R and FLAG-USP13 as indicated were immunoprecipitated with anti-FLAG/MYC antibodies, followed by immunoblot analysis with the indicated antibodies. c HCT116 cells expressing MYC-MKK3-WT or K32R were treated with CHX (50 μg/mL), and cells were collected at indicated time points. The expression levels of exogenous MYC-MKK3 proteins were detect by Western blot. The gray values of the protein at each time point were quantified using ImageJ, and relative values were calculated by comparing with the loading control. A line graph was plotted to show the changes in protein levels over time. d CCK-8 assay was performed to detect cell proliferation ability of MYC-MKK3-WT or K32R overexpressing HCT116 cells. e Colony formation ability was determined in MYC-MKK3-WT or K32R overexpressing HCT116 cells. f Transwell assay was performed to determine the migration and invasion ability of MYC-MKK3-WT or K32R overexpressing HCT116 cells. Representative images were presented. Scale bar, 100 μm. The date were presented as means ± SD at least three independent experiments. Student’s t -tests ( e–f ) and two-way ANOVA test ( c-d ) were performed, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: The slides were then incubated overnight at 4 °C with primary antibodies targeting USP13 (Proteintech, 1:200), MKK3 (CST, 1:200), and Ki-67 (CST, 1:200), after a 1-h blocking step using 5% BSA at room temperature.

Techniques: Blocking Assay, Ubiquitin Proteomics, Transfection, Immunoprecipitation, Western Blot, Expressing, Control, CCK-8 Assay, Transwell Assay, Migration

Journal: Molecular Biomedicine

Article Title: Ubiquitin-specific protease 13 promotes colorectal cancer progression by stabilizing mitogen-activated protein kinase kinase 3

doi: 10.1186/s43556-025-00375-3

Figure Lengend Snippet: MKK3 alleviate USP13 deficient induced phenotype. a MYC-MKK3 was introduced into USP13-knockout cells. Western blot was performed to confirm USP13 knockout and exogenous MYC-MKK3 expression. b HCT116 cells were seeded into 12-well plates at a density of 250 or 500 cells per well. After 7 days, colonies were stained with crystal violet and quantified. c MYC-MKK3-AA/EE mutants were introduced into USP13-knockout cells. Western blot was performed to confirm USP13 knockout and exogenous MYC-MKK3 expression. d HCT116 cells were seeded into 12-well plates at a density of 250 or 500 cells per well. After 7 days, colonies were stained with crystal violet and quantified. e–f CCK-8 assay was performed to assess cell proliferation ability of USP13 knockout or MKK3 reconstitution HCT116 or RKO cells. g EdU assay was performed to assess the DNA synthesis activity in USP13 knockout or MKK3 reconstitution HCT116 cells. Representative images were presented. Scale bar, 100 μm. h Transwell assay was performed to assess the migration ability of USP13 knockout or MKK3 reconstitution RKO cells. Representative images were presented. Scale bar, 100 μm. i-k USP13 knockout or p38α reconstitution HCT116 cells were subcutaneously injected into the nude mice ( n = 5 per group). l-n Control or USP13 knockout HCT116 cells were subcutaneously injected into the nude mice ( n = 5 per group). Tumor-bearing mice were treated daily with either vehicle (10% DMSO, 40% PEG300, 5% Tween-80, and 45% saline, i.p.) or SB202190 (5 mg/kg in 10% DMSO, 40% PEG300, 5% Tween-80, and 45% saline, i.p.). The tumor growth curve ( i , l ), photographs ( j , m ) and weight ( k , n ) were represented. The date were presented as means ± SD at least three independent experiments. Student’s t -tests ( b , d , g , h , k and n ) and two-way ANOVA test ( e , f , i and l ) were performed, ns: no significance, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: The slides were then incubated overnight at 4 °C with primary antibodies targeting USP13 (Proteintech, 1:200), MKK3 (CST, 1:200), and Ki-67 (CST, 1:200), after a 1-h blocking step using 5% BSA at room temperature.

Techniques: Knock-Out, Western Blot, Expressing, Staining, CCK-8 Assay, EdU Assay, DNA Synthesis, Activity Assay, Transwell Assay, Migration, Injection, Control, Saline

Journal: Molecular Biomedicine

Article Title: Ubiquitin-specific protease 13 promotes colorectal cancer progression by stabilizing mitogen-activated protein kinase kinase 3

doi: 10.1186/s43556-025-00375-3

Figure Lengend Snippet: USP13-MKK3-p38 promotes tumor formation. a-c MC38 cells were subcutaneously injected into 8-week-old Usp13 + / + and Usp13 −/− C57 mice ( n = 5 per group). Tumor size was measured every other day once tumors became visible. At the end of the experiment, tumors were excised, photographed, and weighed. The tumor growth curve ( a ), photographs ( b ) and weight ( c ) were represented. d-e H&E staining and Ki-67 immunostaining were performed on tumor samples, followed by quantification of the percentage of Ki-67-positive cells. Scale bar, 100 μm. f–h Tissue microarrays were prepared from CRC patient samples and stained with USP13, MKK3, and Ki-67 antibodies. The percentage of positive cells for each antibody was quantified and calculated ( n = 30). Scale bar, 100 μm. i Western blot was performed to detect the protein levels of USP13 and MKK3 in CRC samples. The representative images were presented. j The band density of USP13 and MKK3 in was quantified using ImageJ software and normalized to the corresponding loading control. The correlation analysis of USP13 and MKK3 protein levels in 30 CRC samples. k Mechanism diagram of USP13-mediated stabilization of MKK3 through deubiquitination (Created by Biorender: https://BioRender.com/jze3xva ). The date was presented as means ± SD at least three independent experiments. Student’s t -tests ( c and e–h ) and two-way ANOVA test ( a ) were performed, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: The slides were then incubated overnight at 4 °C with primary antibodies targeting USP13 (Proteintech, 1:200), MKK3 (CST, 1:200), and Ki-67 (CST, 1:200), after a 1-h blocking step using 5% BSA at room temperature.

Techniques: Injection, Staining, Immunostaining, Western Blot, Software, Control