Journal: Advanced Science

Article Title: TRIM40 Drives Pathological Cardiac Hypertrophy and Heart Failure via Ubiquitination of PKN2

doi: 10.1002/advs.202521337

Figure Lengend Snippet: TRIM40 interacts directly with PKN2. (A) Schematic diagram of the quantitative proteomic screening workflow for identifying TRIM40‐interacting proteins. (B) Tandem mass spectrum of representative peptide fragments from PKN2. (C) Amino acid sequence information of the identified PKN2 peptides. (D, E) Co‐IP assays using anti‐Flag antibody in NRVMs (D) and HEK‐293T cells (E) transfected with Flag‐tagged TRIM40, followed by immunoblotting to detect PKN2 association. IgG served as a NC for Co‐IP (n = 3). (F) Endogenous PKN2 binding was detected by immunoblotting after Co‐IP with anti‐TRIM40 antibody from mouse heart tissue lysates. IgG was used as a control (n = 6). (G) Schematic representation of the domain deletion mutants of PKN2. (H) HEK‐293T cells were co‐transfected with HA‐tagged full‐length PKN2 or its deletion mutants together with Flag‐TRIM40. Immunoprecipitation was performed using anti‐HA antibody, followed by immunoblotting to detect Flag‐TRIM40 binding (n = 3). (I) Schematic diagrams of TRIM40 domain deletion mutants and its catalytically inactive mutant (C29S). (J) HEK‐293T cells were co‐transfected with Flag‐tagged full‐length TRIM40 or its mutants together with HA‐PKN2. Immunoprecipitation with anti‐Flag antibody was used to assess HA‐PKN2 binding (n = 3). (K) Ubiquitination assay of PKN2 in HEK‐293T cells co‐expressing Myc‐Ub, HA‐PKN2, and the catalytically inactive mutant Flag‐TRIM40‐C29S. HA immunoprecipitates were analyzed by immunoblotting to detect PKN2 ubiquitination. (n = 3) (L) Confocal microscopy images showing the effects of different TRIM40 variants (full‐length, deletion mutants, and C29S mutant) on F‐actin cytoskeleton organization in NRVMs. Cells were stained with rhodamine‐conjugated phalloidin (red, labeling F‐actin) and anti‐TRIM40 antibody (green, indicating transfected TRIM40 variants), with nuclei counterstained by DAPI (blue). Scale bar = 50 µm (n = 3). (M) Structural basis of the TRIM40‐PKN2 interaction.

Article Snippet: Antibodies against TRIM40 (24526‐1‐AP), MYH7 (22280‐1‐AP), ANP (27426‐1‐AP), BNP (13299‐1‐AP), PKN2 (14608‐1‐AP), cTnT (68300‐1‐Ig), Rabbit IgG (B900610), His (6005‐1‐lg), Flag (20543‐1‐AP) and HA (51064‐2‐AP) were purchased from Proteintech (IL, USA).

Techniques: Sequencing, Co-Immunoprecipitation Assay, Transfection, Western Blot, Binding Assay, Control, Immunoprecipitation, Mutagenesis, Ubiquitin Proteomics, Expressing, Confocal Microscopy, Staining, Labeling

Journal: Advanced Science

Article Title: TRIM40 Drives Pathological Cardiac Hypertrophy and Heart Failure via Ubiquitination of PKN2

doi: 10.1002/advs.202521337

Figure Lengend Snippet: TRIM40 Positively Regulates the Phosphorylation of PKN2 by Mediating Its K63‐Linked Ubiquitination. (A) HEK‐293T cells were transfected with HA‐PKN2, Myc‐Ub, and Flag‐TRIM40, and treated with 10 µ m MG132 for 6 h before harvesting. The ubiquitination level of PKN2 was detected by immunoblotting using an anti‐HA antibody (Control = IgG) (n = 3). (B) HEK‐293T cells were co‐transfected with HA‐PKN2, Flag‐TRIM40, and various types of Myc‐Ub (including WT, K6‐, K11‐, K27‐, K29‐, K33‐, K48‐, and K63‐linked ubiquitin chains). After immunoprecipitation with anti‐HA magnetic beads, the ubiquitination of PKN2 was analyzed by immunoblotting. Cells were pretreated with 10 µ m MG132 for 6 h before harvesting (n = 3). (C) HEK‐293T cells were transfected with HA‐PKN2, Myc‐Ub, EV, Flag‐TRIM40, and Flag‐TRIM40‐29S, treated with 10 µ m MG132 for 6 h before harvesting. The ubiquitination level of PKN2 was detected by immunoblotting using an anti‐HA antibody (n = 3). (D) NRVMs were transfected with siRNA targeting TRIM40, followed by treatment with 1 µ m Ang II for 12 h. The phosphorylation level of PKN2 was detected by immunoblotting, with GAPDH used as the loading control (n = 3). (E) Quantitative data of the blot intensity of corresponding proteins determined by Image J software in panel D (n = 3). (F) NRVMs were transfected with a TRIM40 expression vector, followed by treatment with 1 µ m Ang II for 12 h. The phosphorylation level of PKN2 was detected by immunoblotting, with GAPDH used as the loading control (n = 3). (G) Quantitative data of the blot intensity of corresponding proteins determined by Image J software in panel F (n = 3). (H) NRVMs overexpressing Flag‐TRIM40 or Flag‐TRIM40‐C29S were treated with Ang II (1 µ m ) for 24 h. Phosphorylation of PKN2 at Ser815 (p‑PKN2) was detected by Western blot. GAPDH served as a loading control (n = 3). (I) Densitometric quantification of the Western blot bands from Figure (n = 3). (J) The level of p‐PKN2 was detected by immunoblotting in whole heart tissue lysates from mice infused with Ang II for 4 weeks (n = 6). (K) Quantitative data of the blot intensity of corresponding proteins determined by Image J software in panel J (n = 6). (L) The level of p‐PKN2 was detected by immunoblotting in whole heart tissue lysates from mice subjected to TAC surgery (n = 6). (M) Quantitative data of the blot intensity of corresponding proteins determined by Image J software in panel L (n = 6). (N) NRVMs overexpressing Flag‐TRIM40 or Flag‐TRIM40‐C29S were treated with Ang II (1 µ m ) for 24 h. Protein levels of MYH7, ANP and BNP were detected by Western blot. GAPDH served as a loading control (n = 3). (O) Densitometric quantification of the Western blot bands from Figure (n = 3). All quantitative data are presented as mean ± SEM. Data between two groups were compared by independent‐sample two‐tailed Student's t‐test. Data among multiple groups were compared by one‐way ANOVA test, followed by Tukey post hoc test; ** p < 0.01.

Article Snippet: Antibodies against TRIM40 (24526‐1‐AP), MYH7 (22280‐1‐AP), ANP (27426‐1‐AP), BNP (13299‐1‐AP), PKN2 (14608‐1‐AP), cTnT (68300‐1‐Ig), Rabbit IgG (B900610), His (6005‐1‐lg), Flag (20543‐1‐AP) and HA (51064‐2‐AP) were purchased from Proteintech (IL, USA).

Techniques: Phospho-proteomics, Ubiquitin Proteomics, Transfection, Western Blot, Control, Immunoprecipitation, Magnetic Beads, Software, Expressing, Plasmid Preparation, Two Tailed Test

Journal: Advanced Science

Article Title: TRIM40 Drives Pathological Cardiac Hypertrophy and Heart Failure via Ubiquitination of PKN2

doi: 10.1002/advs.202521337

Figure Lengend Snippet: TRIM40 exacerbates Ang II‐induced cardiomyocyte hypertrophy and fibrosis by regulating PKN2. C57BL/6 mice received two injections of AAV9 encoding TRIM40 at one‐month intervals, followed by continuous infusion of saline or Ang II for two weeks. (A) Mouse systolic blood pressure was measured weekly using a non‐invasive tail‐cuff system (n = 6). (B) Serum Ang II concentration was detected in mice (n = 6). (C) Representative echocardiographic images of mice from each experimental group (n = 6). (D–F) Cardiac function parameters showing EF (D), FS (E), and IVRT (F) (n = 6). (G) Serum CK‐MB levels in mice (n = 6). (H) HW/BW of mice in each group (n = 6). (I) Representative freshly isolated heart specimens photographed against a white background (scale bar = 5 mm) (n = 6). (J) Cardiomyocyte size was assessed by fluorescein‐conjugated WGA staining (scale bar = 50 µm) (n = 6). (K) H&E staining of heart tissue sections (scale bar = 50 µm) (n = 6). (L, M) Myocardial fibrosis was evaluated by Masson's trichrome staining (L) and Picrosirius red staining (M) (scale bar = 50 µm) (n = 6). (N) Western blot analysis of MYH7, ANP, and BNP in myocardial tissues, with GAPDH as a loading control (n = 6). (O, P) mRNA expression levels of hypertrophy‐associated genes (O) and inflammation‐related genes (P) in myocardial tissues, normalized to Actb (n = 6). (Q) p‐PKN2 levels were detected by immunoblotting in whole heart lysates from mice infused with Ang II for 4 weeks (n = 6). (R) Quantitative data of the blot intensity of corresponding proteins determined by Image J software in panel Q (n = 6). All quantitative data are presented as mean ± SEM. Data between the two groups were compared by independent‐sample two‐tailed Student's t‐test. Data among multiple groups were compared by one‐way ANOVA test, followed by a Tukey post hoc test. ns indicates not statistically significant; * p < 0.05, ** p < 0.01.

Article Snippet: Antibodies against TRIM40 (24526‐1‐AP), MYH7 (22280‐1‐AP), ANP (27426‐1‐AP), BNP (13299‐1‐AP), PKN2 (14608‐1‐AP), cTnT (68300‐1‐Ig), Rabbit IgG (B900610), His (6005‐1‐lg), Flag (20543‐1‐AP) and HA (51064‐2‐AP) were purchased from Proteintech (IL, USA).

Techniques: Saline, Concentration Assay, Isolation, Staining, Western Blot, Control, Expressing, Software, Two Tailed Test

Journal: Advanced Science

Article Title: TRIM40 Drives Pathological Cardiac Hypertrophy and Heart Failure via Ubiquitination of PKN2

doi: 10.1002/advs.202521337

Figure Lengend Snippet: TRIM40 exacerbates TAC‐induced cardiomyocyte hypertrophy and fibrosis by regulating PKN2. C57BL/6 mice received two injections of AAV9 encoding TRIM40 at one‐month intervals, followed by sham surgery or TAC surgery for two weeks. (A) Representative echocardiographic images of mice from each experimental group (n = 6). (B–D) Cardiac function parameters showing EF (B), FS (C), and IVRT (D) (n = 6). (E) Serum CK‐MB levels in mice (n = 6). (F) HW/BW of mice in each group (n = 6). (G) Representative freshly isolated heart specimens photographed against a white background (scale bar = 5 mm) (n = 6). (H) Cardiomyocyte size was assessed by fluorescein‐conjugated WGA staining (scale bar = 50 µm) (n = 6). (I) Cardiomyocyte hypertrophy evaluated by fluorescein‐conjugated WGA staining (scale bar = 50 µm) (n = 6). (J) H&E staining of heart tissue sections (scale bar = 50 µm) (n = 6). (K, L) Myocardial fibrosis was evaluated by Masson's trichrome staining (K) and Picrosirius red staining (L) (scale bar = 50 µm) (n = 6). (M) Western blot analysis of MYH7, ANP, and BNP in myocardial tissues, with GAPDH as a loading control (n = 6). (N, O) mRNA expression levels of hypertrophy‐associated genes (N) and inflammation‐related genes (O) in myocardial tissues, normalized to Actb (n = 6). (P) p‐PKN2 levels were detected by immunoblotting in whole heart lysates from mice infused with Ang II for 4 weeks (n = 6). (Q) Quantitative data of the blot intensity of corresponding proteins determined by Image J software in panel P (n = 6). All quantitative data are presented as mean ± SEM. Data between the two groups were compared by independent‐sample two‐tailed Student's t‐test. Data among multiple groups were compared by one‐way ANOVA test, followed by Tukey post hoc test; ** p < 0.01.

Article Snippet: Antibodies against TRIM40 (24526‐1‐AP), MYH7 (22280‐1‐AP), ANP (27426‐1‐AP), BNP (13299‐1‐AP), PKN2 (14608‐1‐AP), cTnT (68300‐1‐Ig), Rabbit IgG (B900610), His (6005‐1‐lg), Flag (20543‐1‐AP) and HA (51064‐2‐AP) were purchased from Proteintech (IL, USA).

Techniques: Isolation, Staining, Western Blot, Control, Expressing, Software, Two Tailed Test

Journal: Advanced Science

Article Title: TRIM40 Drives Pathological Cardiac Hypertrophy and Heart Failure via Ubiquitination of PKN2

doi: 10.1002/advs.202521337

Figure Lengend Snippet: A Mechanism of TRIM40 Driving Cardiac Hypertrophy through PKN2 Ubiquitination.

Article Snippet: Antibodies against TRIM40 (24526‐1‐AP), MYH7 (22280‐1‐AP), ANP (27426‐1‐AP), BNP (13299‐1‐AP), PKN2 (14608‐1‐AP), cTnT (68300‐1‐Ig), Rabbit IgG (B900610), His (6005‐1‐lg), Flag (20543‐1‐AP) and HA (51064‐2‐AP) were purchased from Proteintech (IL, USA).

Techniques: Ubiquitin Proteomics

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: PKN2 enhances the immunosuppressive activity of polymorphonuclear myeloid-derived suppressor cells in esophageal carcinoma by mediating fatty acid oxidation.

doi: 10.1186/s10020-025-01132-6

Figure Lengend Snippet: Fig. 1 PKN2 is highly expressed in PMN-MDSCs of ESCC tumor tissues. (A) The GEO datasets (GSE20347) revealed a significant elevation in PKN2 expres sion in ESCC tumor tissues (N = 17) relative to normal tissues (N = 17). (B) Immunohistochemistry of PKN2 antibody for EC tissues and adjacent tissues. (C) The expression and distribution characteristics of PKN2 in the BioGPS database (http://biogps.org/#goto=genereport_id=5586). (D) Immunofluores cence staining results showed that PKN2 was expressed on MDSCs in esophageal cancer tissues. (E) The expression of mRNA and protein levels of PKN2 in normal mouse immune cells, including macrophages from abdominal cavity (AC), B cells and monocytes from peripheral blood (PB), and plasma cells and MDSCs from bone marrows (BM), was detected using qRT-PCR and Western blotting (N = 3). Statistical analysis: one-way ANOVA with Tukey’s post- hoc test. ***P < 0.001. (F) Ten paired PBMCs and tumor tissues were collected from ESCC patients. The percentage of myeloid cells, monocytic MDSCs (M-MDSCs), and polymorphonuclear myeloid derived suppressor cells (PMN-MDSCs) was detected using flow cytometry (N = 10). Statistical analysis: two tailed paired student’s t-test. **P < 0.01 vs. PBMCs

Article Snippet: Subsequently, either an anti-PKN2 antibody or an IgG antibody (Proteintech, Rosemont, IL, USA) was introduced.

Techniques: Immunohistochemistry, Expressing, Staining, Clinical Proteomics, Quantitative RT-PCR, Western Blot, Derivative Assay, Flow Cytometry, Two Tailed Test

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: PKN2 enhances the immunosuppressive activity of polymorphonuclear myeloid-derived suppressor cells in esophageal carcinoma by mediating fatty acid oxidation.

doi: 10.1186/s10020-025-01132-6

Figure Lengend Snippet: Fig. 2 PKN2 is highly expressed in PMN-MDSCs of EC mouse tumor tissues. (A) Spontaneous carcinogenesis was induced by 4-NQO. Mouse spleen tissues and esophageal tissues were collected. The expression of PKN2 mRNA and protein levels were determined using qRT-PCR and Western blotting (N = 5). Statistical analysis: two-way ANOVA with Tukey’s post-hoc test. ***P < 0.001. (B) The percentage of myeloid cells, TAMs, B cells, DCs, M-MDSCs, and PMN-MDSCs in spleen and esophageal tissues of EC mice was detected using flow cytometry (N = 5). Statistical analysis: two tailed paired student’s t-test. **P < 0.01 vs. spleen

Article Snippet: Subsequently, either an anti-PKN2 antibody or an IgG antibody (Proteintech, Rosemont, IL, USA) was introduced.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Flow Cytometry, Two Tailed Test

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: PKN2 enhances the immunosuppressive activity of polymorphonuclear myeloid-derived suppressor cells in esophageal carcinoma by mediating fatty acid oxidation.

doi: 10.1186/s10020-025-01132-6

Figure Lengend Snippet: Fig. 4 PKN2-highly expressed PMN-MDSCs inhibit CTL killing ability to promote ESCC organoid activity. (A) HE staining and immunohistochemistry (anti-PanCK and anti-CK5) of ESCC organoids and their primary tumor tissues. Scale bar = 100 μm. (B) The methodology illustrated the co-culture of ESCC organoids and CTLs. (C) Bright field results of the co-culture system. Scale bar = 100 μm. (D) Cell death was observed by flow cytometry using PI and Violet antibodies (N = 3). Statistical analysis: one-way ANOVA with Tukey’s post-hoc test. *P < 0.05, **P < 0.01, ***P < 0.001. (E) The proliferation of T cells was de tected by CFSE staining and flow cytometry (N = 3). Statistical analysis: one-way ANOVA with Tukey’s post-hoc test. *P < 0.05. (F) Soluble IL-2 and IFN-γ lev els in the supernatants of the co-culture system were determined by ELISA (N = 3). Statistical analysis: one-way ANOVA with Tukey’s post-hoc test. *P < 0.05

Article Snippet: Subsequently, either an anti-PKN2 antibody or an IgG antibody (Proteintech, Rosemont, IL, USA) was introduced.

Techniques: Activity Assay, Staining, Immunohistochemistry, Co-Culture Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: PKN2 enhances the immunosuppressive activity of polymorphonuclear myeloid-derived suppressor cells in esophageal carcinoma by mediating fatty acid oxidation.

doi: 10.1186/s10020-025-01132-6

Figure Lengend Snippet: Fig. 5 PKN2 promotes fatty acid oxidation in PMN-MDSCs. (A) PMN-MDSCs were isolated from EC mouse spleen tissues and subsequently transfected with the PKN2 overexpression lentivirus. The modulation of FAO by PKN2 in PMN-MDSCs was detected using the Fatty Acid Oxidation Assay Kit (N = 3). Sta tistical analysis: two tailed un-paired student’s t-test. *P < 0.05 vs. OE-NC. (B) Following a 24-hour incubation period, the OCR was detected using Oligomy cin (4 µM), FCCP (1.6 µM), rotenone (0.5 µM) + antimycin A (0.5 µM), and etomoxir (18 µM, an FAO inhibitor) (N = 3). Statistical analysis: two tailed un-paired student’s t-test. *P < 0.05 vs. OE-NC. (C) The effect of PKN2 on acetyl-CoA levels in PMN-MDSCs (N = 3). Statistical analysis: two tailed un-paired student’s t-test. **P < 0.01 vs. OE-NC. (D) Arg-1 expression was detected using Western blotting. (E) ROS generation was detected using the DCFH-DA probe and the fluorescence intensity was determined using flow cytometry (N = 3). Statistical analysis: one-way ANOVA with Tukey’s post-hoc test. ***P < 0.001. (F) IL-10 and TGF-β levels in the supernatants were determined by ELISA (N = 3). Statistical analysis: one-way ANOVA with Tukey’s post-hoc test. *P < 0.05. **P < 0.01. (G) PMN-MDSCs were isolated from EC mouse spleen tissues and subsequently transfected with the PKN2 overexpression lentivirus and CPT1A/B/C siRNAs. The modulation of FAO was detected using the Fatty Acid Oxidation Assay Kit (N = 3). Statistical analysis: one-way ANOVA with Tukey’s post-hoc test. *P < 0.05. (H) Following a 24-hour incubation period, the OCR was detected (N = 3). Statistical analysis: one-way ANOVA with Tukey’s post-hoc test. **P < 0.01. ***P < 0.001. (I) Arg-1 expression was detected using Western blotting. (J) ROS generation was detected using the DCFH-DA probe and the fluorescence intensity was determined using flow cytometry (N = 3). Statistical analysis: one-way ANOVA with Tukey’s post-hoc test. ***P < 0.001. (K) IL-10 and TGF-β levels in the supernatants were determined by ELISA (N = 3). Statistical analysis: one-way ANOVA with Tukey’s post-hoc test. *P < 0.05

Article Snippet: Subsequently, either an anti-PKN2 antibody or an IgG antibody (Proteintech, Rosemont, IL, USA) was introduced.

Techniques: Isolation, Transfection, Over Expression, Oxidation Assay, Two Tailed Test, Incubation, Expressing, Western Blot, Fluorescence, Flow Cytometry, Enzyme-linked Immunosorbent Assay