Journal: eLife

Article Title: Pak2 is required for actin cytoskeleton remodeling, TCR signaling, and normal thymocyte development and maturation

doi: 10.7554/eLife.02270

Figure Lengend Snippet: ( A ) Western blot analysis using anti-Pak2 antiserum and cell lysates from the thymus of Pak2 F/F (WT), and Pak2 F/F ;Cd4 -Cre (KO) mice. Anti-Erk1/2 antibody was used as loading control. Shown are representative of two independent experiments. ( B ) Quantitative PCR analysis of Pak2 mRNA expression in DP, semi-mature and mature CD4SP thymocytes. Shown are Pak2 mRNA levels from DP, semi-mature and mature CD4SP thymocytes relative to Pak2 F/F DP thymocytes (error bars; SD). Data are representative of two independent experiments. ( C ) Western blot analysis using anti-PAK2 and cell lysates from the thymi of Pak2 F/F (WT), Pak2 F/+ ;Lck- Cre (HET) and Pak2 F/F ;Lck- Cre (KO) mice. ( D ) Representative flow cytometry analyses of CD4 and CD8 expression on lymphocytes from spleens (n = 5), peripheral lymph nodes (pLn; axillary, brachial and inguinal lymph nodes, n = 4), and mesenteric lymph nodes (mLn, n = 4) from Pak2 F/F (WT) and Pak2 F/F ;Lck -Cre (KO) mice. Numbers in each quadrant represent the percentage of cells in the indicated quadrant. ( E ) Flow cytometry analyses of CD62L and CD44 on CD4+ T cells from Pak2 F/F (WT) and Pak2 F/F ;Lck -Cre (KO) mice. ( F ) Quantification of cell numbers of different lymphocyte subsets from Pak2 F/F and Pak2 F/F ;Lck- Cre mice. Error bars: SD (spleen [ n = 5 mice per genotype], pLn [ n = 4 mice per genotype], and mLn [n = 4 mice per genotype]; blood [n = 2 mice per genotype]). *, 0.01

Article Snippet: Pak2 (TA306346) antibody is from Origene (Rockville, MD).

Techniques: Western Blot, Control, Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry, Two Tailed Test, Generated

Journal: eLife

Article Title: Pak2 is required for actin cytoskeleton remodeling, TCR signaling, and normal thymocyte development and maturation

doi: 10.7554/eLife.02270

Figure Lengend Snippet: ( A ) Quantification of cell numbers of different lymphocyte subsets from Pak2 F/F and Pak2 F/F ;Lck- Cre mice. Error bars: SD (spleen [ n = 5 mice per genotype], pLn [ n = 4 mice per genotype], and mLn [n = 4 mice per genotype]; blood [n = 2 mice per genotype]). *, 0.01

Article Snippet: Pak2 (TA306346) antibody is from Origene (Rockville, MD).

Techniques: Two Tailed Test

Journal: eLife

Article Title: Pak2 is required for actin cytoskeleton remodeling, TCR signaling, and normal thymocyte development and maturation

doi: 10.7554/eLife.02270

Figure Lengend Snippet: ( A ) Flow cytometry analysis of 1:1 mixed bone marrow chimeras. Chimeras were generated by transferring 1:1 mixed WT Pak2 +/+ (CD45.1 + CD45.2 + ) and KO Pak2 F/F ;Cd4 -Cre (CD45.2 + ) donor bone marrows into lethally irradiated C57BL6 hosts that express CD45.1. Data shown are representative of five bone marrow chimeras. CD4-CD8-double negative (DN) thymocytes generated from donor hematopoietic stem cells were gated using CD45.2 markers, and plotted with CD45.1 expression to distinguish T cells produced from WT (CD45.1 positive) and Pak2 F/F ;Cd4- Cre (CD45.1 negative) donors (first panel). Reconstitution of CD19+ B cells from spleen or pLN by WT (CD45.1 positive) and Pak2 F/F ;Cd4 -Cre (CD45.1 negative) donors showed similar contribution of donor BM cells from WT and Pak2 F/F ;Cd4 -Cre mice (second panel). In contrast, CD4 and CD8 T cells from Pak2 F/F ;Cd4 -Cre (CD45.1 negative) donors were markedly underrepresented compared to those from WT (CD45.1 positive) donors (third and fourth panels). ( B ) Total lymphocytes generated either from Pak2 +/+ or Pak2 F/F ;Cd4 -Cre donor bone marrow cells were gated and plotted as CD19 vs CD3 expression. ( C ) Expression of CD4 and CD8 is shown in CD3 positive cells generated either from Pak2 +/+ or Pak2 F/F ;Cd4 -Cre donor bone marrow cells. DOI: http://dx.doi.org/10.7554/eLife.02270.005

Article Snippet: Pak2 (TA306346) antibody is from Origene (Rockville, MD).

Techniques: Flow Cytometry, Generated, Transferring, Irradiation, Expressing, Produced

Journal: eLife

Article Title: Pak2 is required for actin cytoskeleton remodeling, TCR signaling, and normal thymocyte development and maturation

doi: 10.7554/eLife.02270

Figure Lengend Snippet: ( A ) Flow cytometry of lymphocytes from thymi of Pak2 F/F or Pak2 F/F ;Lck -Cre mice (n = 5). ( B ) Quantification of cell numbers of different thymic subsets from Pak2 F/F and Pak2 F/F ;Lck -Cre mice. Error bars: SEM (n = 5 mice per genotype). **, 0.001

Article Snippet: Pak2 (TA306346) antibody is from Origene (Rockville, MD).

Techniques: Flow Cytometry, Two Tailed Test, Expressing

Journal: eLife

Article Title: Pak2 is required for actin cytoskeleton remodeling, TCR signaling, and normal thymocyte development and maturation

doi: 10.7554/eLife.02270

Figure Lengend Snippet: ( A ) Flow cytometry analyses of CD4 SP thymocytes of Pak2 F/F (WT), Pak2 +/+ ;Cd4- Cre (WT), Pak2 F/+ ;Cd4- Cre (Het), or Pak2 F/F ;Cd4- Cre (KO) mice. ( B ) Quantification of cell numbers of different thymic subsets. Error bars: SEM (n = 10 mice). ( C ) CD4 and CD8 FACS analyses showing decreased percentage of CD4SP thymocytes in OTII + ; Pak2 F/F ;Cd4- Cre mice. Shown are representative data from three mice from each genotype. ( D ) Cell numbers of different thymic subsets from OTII + ; Pak2 F/F or OTII + ; Pak2 F/F ;Cd4- Cre mice. Graphs in this figure show mean ± SEM (n = 3). *, p=0.014. ( E ) Expression of TCR transgene assessed by anti-Vα2 antibody in total thymocytes from OTII + ; Pak2 F/F or OTII + ; Pak2 F/F ;Cd4- Cre mice. Shown are representative data from three mice from each genotype. DOI: http://dx.doi.org/10.7554/eLife.02270.007

Article Snippet: Pak2 (TA306346) antibody is from Origene (Rockville, MD).

Techniques: Flow Cytometry, Expressing

Journal: eLife

Article Title: Pak2 is required for actin cytoskeleton remodeling, TCR signaling, and normal thymocyte development and maturation

doi: 10.7554/eLife.02270

Figure Lengend Snippet: ( A ) CD4SP TCR hi thymocytes were gated and analyzed by expression of CD69 and CD62L. Fraction A (CD69 hi CD62L low ), semi-mature stage; Fraction B (CD69 low CD62L low ); Fraction C (CD69 low CD62L hi ), mature stage. Shown are representative data of ten mice per genotype. ( B ) Quantification of cell numbers of A, B, and C fractions in CD4SP thymocytes. Error bars: SEM (n = 10). *, 0.01

Article Snippet: Pak2 (TA306346) antibody is from Origene (Rockville, MD).

Techniques: Expressing, Two Tailed Test

Journal: eLife

Article Title: Pak2 is required for actin cytoskeleton remodeling, TCR signaling, and normal thymocyte development and maturation

doi: 10.7554/eLife.02270

Figure Lengend Snippet: ( A ) CD4SP TCR hi thymocytes were gated and analyzed by expression of CD69 and CD62L. Semi-mature stage (CD69 hi CD62L low ) and mature stage (CD69 low CD62L hi ) are shown. Note lack of the cells in the mature stage. ( B ) Upper panels: expression of TCR transgene assessed by anti-Vα2 antibody in CD4SP thymocytes from OTII + ; Pak2 F/F or OTII + ; Pak2 F/F ;Lck- Cre mice. Lower panels: Vα2 hi thymocytes in CD4SP thymocytes were gated and analyzed by expression of CD69 and CD62L. Shown are representative data from three mice from each genotype. DOI: http://dx.doi.org/10.7554/eLife.02270.009

Article Snippet: Pak2 (TA306346) antibody is from Origene (Rockville, MD).

Techniques: Expressing

Journal: eLife

Article Title: Pak2 is required for actin cytoskeleton remodeling, TCR signaling, and normal thymocyte development and maturation

doi: 10.7554/eLife.02270

Figure Lengend Snippet: ( A ) Apoptosis detected using 7AAD/Annexin V staining in freshly isolated Pak2 F/F or Pak2 F/F ;Cd4 -Cre CD4SP thymocytes. Semi-mature (CD69 hi CD62L low , Fraction A in ), mature (CD69 low CD62L hi , Fraction C in ) and CD69 low CD62L low (Fraction B in ) CD4SP thymocytes were gated and analyzed. Data are representative of two independent experiments. ( B ) Intracellular staining of active caspase 3 following 0, 1, and 2 hr of incubation in 10% FBS serum containing media. Data are representative of two independent experiments. ( C ) Apoptosis detected using 7AAD/Annexin V staining following 24 hr of incubation in 10% FBS serum containing media. Semi-mature and mature CD4SP thymocytes were gated and analyzed. Data are representative of two independent experiments. DOI: http://dx.doi.org/10.7554/eLife.02270.010

Article Snippet: Pak2 (TA306346) antibody is from Origene (Rockville, MD).

Techniques: Staining, Isolation, Incubation

Journal: eLife

Article Title: Pak2 is required for actin cytoskeleton remodeling, TCR signaling, and normal thymocyte development and maturation

doi: 10.7554/eLife.02270

Figure Lengend Snippet: ( A ) Flow cytometry analysis of 1:1 mixed bone marrow chimeras generated by transferring WT ( Pak2 +/+ , CD45.1 + CD45.2 + ) and Pak2 F/F ;Cd4- Cre mice (CD45.2 + ) donor bone marrow cells that contain hematopoietic stem cells (HSCs) into lethally irradiated C57BL6 hosts that express CD45.1+. Thymocytes generated either from Pak2 +/+ or Pak2 F/F ;Cd4- Cre donor bone marrow cells were identified using CD45 congenic markers and CD4 vs CD8 expression was shown. TCR hi CD4SP thymocytes were gated and expression of CD69 and CD62L was examined. Data shown are representative of five bone marrow chimeras. ( B ) Aberrant expression of trafficking molecules and maturation markers in CD4 SP thymocytes generated from Pak2 F/F ;Cd4- Cre donor. ( C ) Impaired expression of IL-7Rα in CD4SP T cells generated from Pak2 F/F ;Cd4- Cre donor bone marrow cells. ( D ) Block in the semi-mature to mature transition of Pak2-deficient CD4 SP thymocytes in fetal thymic organ culture. CD4 vs CD8 (top panels) expression of total culture and CD69 vs CD62L expression within TCR hi CD4SP population (bottom panels). Shown are data representative FTOC experiments of six embryos per each genotype. ( E ) Altered expression of maturation markers in TCR hi CD4SP thymocytes from FTOC. ( F ) IL-7Rα expression in DP, CD4SP, and semi-mature (CD69 hi CD62L low ) CD4SP thymocytes from FTOC. DOI: http://dx.doi.org/10.7554/eLife.02270.011

Article Snippet: Pak2 (TA306346) antibody is from Origene (Rockville, MD).

Techniques: Flow Cytometry, Generated, Transferring, Irradiation, Expressing, Blocking Assay, Organ Culture

Journal: eLife

Article Title: Pak2 is required for actin cytoskeleton remodeling, TCR signaling, and normal thymocyte development and maturation

doi: 10.7554/eLife.02270

Figure Lengend Snippet: ( A ) Defects in mRNA expression of KLF2 in mature CD4SP thymocytes from Pak2 F/F ;Cd4 -Cre mice. KLF2 mRNA levels in semi-mature and mature CD4SP thymocytes relative to Pak2 F/F DP thymocytes (left panel, mean ± SD of triplicates, results are representative of three independent experiment); KLF2 mRNA levels in mature Pak2 F/F ;Cd4 -Cre CD4SP thymocytes relative to mature Pak2 F/F CD4SP thymocytes (middle panel, mean ± SEM; each dot represents one mouse, n = 3); KLF2 mRNA levels in semi-mature Pak2 F/F ;Cd4 -Cre CD4SP thymocytes relative to semi-mature Pak2 F/F CD4SP thymocytes (right panel, mean ± SEM; each dot represents one mouse, n = 3). ***, p=0.0001; **, 0.001

Article Snippet: Pak2 (TA306346) antibody is from Origene (Rockville, MD).

Techniques: Expressing, Phospho-proteomics, Western Blot, Control

Journal: eLife

Article Title: Pak2 is required for actin cytoskeleton remodeling, TCR signaling, and normal thymocyte development and maturation

doi: 10.7554/eLife.02270

Figure Lengend Snippet: ( A ) Increased apoptosis in the Qa2 hi CD4SP thymocyte subset from Pak2 F/F ;Cd4 -Cre mice following 24 hr of incubation in media or in plate-bound CD3 or CD3/CD28 stimulation. Data are representative of two independent experiments. ( B ) Increased apoptosis in CD4SP thymocytes from Pak2 F/F ;Cd4 -Cre mice following 24 hr of incubation in media or in plate-bound CD3 or CD3/CD28 stimulation. Apoptosis detected using 7AAD/Annexin V staining. Data are representative of three independent experiments. ( C ) Increased apoptosis in resting semi-mature (CD62L low ) CD4SP thymocytes from Pak2 F/F ;Cd4 -Cre mice detected using Annexin V staining or active caspase 3 intracellular staining following 24 hr of incubation in 10% FBS serum containing media. Data are representative of three (annexin V) and two (active caspase 3) independent experiments. ( D ) Increased apoptosis in semi-mature (CD62L low ) CD4SP thymocytes from Pak2 F/F ;Cd4 -Cre mice detected using Annexin V staining following 24 hr of incubation in media or in plate-bound CD3 or CD3/CD28 stimulation. Data are representative of four independent experiments. DOI: http://dx.doi.org/10.7554/eLife.02270.013

Article Snippet: Pak2 (TA306346) antibody is from Origene (Rockville, MD).

Techniques: Incubation, Staining

Journal: eLife

Article Title: Pak2 is required for actin cytoskeleton remodeling, TCR signaling, and normal thymocyte development and maturation

doi: 10.7554/eLife.02270

Figure Lengend Snippet: ( A ) Inhibition of MAPK pathway by UO126, a MEK inhibitor. Effects of UO126 (a MEK inhibitor) and rapamycin (an inhibitor of mTOR) on phosphorylation of Erk1/2 and RSK were demonstrated by immunoblotting analysis of phosphorylation status of Erk1/2 (T202/Y204) and RSK (T359/S363) in total thymocytes from Pak2 F/F or Pak2 F/F ;Cd4 -Cre mice. Cells were preincubated in the presence of DMSO, rapamycin (25 nM), and UO126 (10 μM) at the stated concentration for 30 min and treated with plate-bound anti-CD3 stimulation for 30 and 60 min. UO126 completely abrogated activation of MAPK and RSK, but rapamycin did not substantially inhibit activation of MAPK and RSK. ( B ) Inhibition of mTORC1-mediated pathway by rapamycin. Effect of rapamycin was examined by immunoblotting analysis of phosphorylation status of p70S6K (T389). Phosphorylation of p70S6K at T389 was completely inhibited by rapamycin (25 nM). ( C ) Phosphorylation of S6 at S235/S236 and p70S6K at T389 was completely dependent upon mTOR-mediated pathway. Cells were preincubated in the presence of DMSO, rapamycin (25 nM), and UO126 (10 μM) at the stated concentration for 30 min. Cells were rested in media or treated with plate-bound anti-CD3 stimulation for 30 and 60 min. Immunoblotting analysis of phosphorylation status of S6 (S235/S236) and p70S6K (T389) in total thymocytes from Pak2 F/F or Pak2 F/F ;Cd4 -Cre mice in media (resting) or following plate-bound anti-CD3 stimulation for 30 and 60 min are shown. ( D ) Intensity of each band shown in C was measured, normalized by intensity of GAPDH (as loading control), and shown in the graph as fold increase. Shown are data representative of three independent experiments. ( E ) Phosphorylation of S6 at S235/S236 and p70S6K at T389 were dependent upon activation of mTOR. Relative intensity of inhibitor-treated samples compared to DMSO-treated samples at 30 min or 60 min after stimulation are shown. Intensity of each band was normalized by DMSO-treated sample at each time point and displayed as relative intensity. Graphs in this figure show mean ± SEM (n = 3). DOI: http://dx.doi.org/10.7554/eLife.02270.014

Article Snippet: Pak2 (TA306346) antibody is from Origene (Rockville, MD).

Techniques: Inhibition, Phospho-proteomics, Western Blot, Concentration Assay, Activation Assay, Control

Journal: eLife

Article Title: Pak2 is required for actin cytoskeleton remodeling, TCR signaling, and normal thymocyte development and maturation

doi: 10.7554/eLife.02270

Figure Lengend Snippet: ( A ) Increased actin polymerization in resting DP, CD4 and CD8 SP thymocytes from Pak2 F/F Cd4 -Cre mice. The mean fluorescence intensity of phalloidin-Alexa488 in the DP, CD4SP and CD8SP thymocyte populations was normalized to the value of WT DP thymocytes at rest. (error bars; SEM, n = 3). ****, p<0.0001 ( B ) Increased activation of Rac1 at resting from Pak2 F/F ;Cd4 -Cre thymocytes using Rac1 pull-down assay. WCL; whole cell lysates. Shown are data representative of three independent experiments. ( C ) Spreading areas of CD4SP thymocytes on the cover slips. Cells were allowed to spread for 60 min on coverslips coated with anti-CD3. Z-stack images of F-actin staining of the cells were collected and spreading areas where cells contact the cover slips were analyzed. Shown are data representative of three independent experiments. (error bars; SEM, Pak2 F/F [n = 21], Pak2 F/F ;Cd4 -Cre [n = 25]). ****, p<0.0001. ( D ) T cell spreading and actin polymerization triggered by plate-bound TCR stimulation. Shown are z-stack images of the cells from the contact sites where T cells touch the cover slides to the top of the cells. Left panels; maximal projection of z-stack images of F-actin staining (F-actin staining at the contact site [green] and F-actin staining of the rest of the z-stacks [red]). Scale bar: 5 μm. Middle panels: F-actin staining on the contact site to visualize cell spreading. Right panels: z stack images of F-actin were complied and reconstructed as a 3D image. Shown is the orthogonal image viewed from the z-axis. Shown are data representative of three independent experiments. DOI: http://dx.doi.org/10.7554/eLife.02270.015

Article Snippet: Pak2 (TA306346) antibody is from Origene (Rockville, MD).

Techniques: Fluorescence, Activation Assay, Pull Down Assay, Staining

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: Scientific reports

Article Title: Mechanical forces trigger invasive behavior in synovial fibroblasts through N-cadherin/ADAM15 -dependent modulation of LncRNA H19.

doi: 10.1038/s41598-025-94012-2

Figure Lengend Snippet: Fig. 1. ADAM15-dependent mechano-induced downregulation of lncRNA H19 is affected by inhibition of p21-activated kinases (PAKs). (A, B) Synovial fibroblasts either silenced with an ADAM15 siRNA or a non-silencing negative siRNA were mechanically strained (1 Hz and 15% elongation) for 1–9 h and H19 levels determined by RT-qPCR. (A) Fold changes of H19 in ADAM15-expressing versus non-expressing cells were calculated using the 2−ΔΔCt. Shown is the mean ± SD of 6 different RASFs. (B) time course of GAPDH- normalized Ct values for H19 upon mechanical strain, showing increase of Ct values (i.e. lower H19 amounts) in ADAM15 expressing cells (black dots) compared to ADAM15 non-expressing cells (open circle) in one representative RASF cell line. (C) RT-qPCR for H19 of RASFs stimulated for 1 and 3 h in the presence of either DMEM medium, or inhibitors for JNK (SP600125), Src family kinases (dasatinib), CAMKII (KN- 93), calmodulin (TFP) and PAKs (IPA-3). **p < 0.005, ***p < 0.0005, Student’s t-test, comparing ADAM15- expressing versus non-expressing cells or inhibitor versus DMEM.

Article Snippet: Slices were glued with mounting medium to a glass slide and cells fixed with ice cold methanol for 5 min and aceton for 45 s. After blocking with 1% horse serum in PBS for 1 h, cells were incubated with sheep-anti N-cadherin (R&D Systems, #AF6426, 1:100) and rabbit anti-Nck (Cell Signaling Technology, #2319, 1:100), phospho-PAK1 (Ser144)/PAK2 (Ser141) (Cell Signaling Technology, #2606, 1:100), anti-phosphoPAK2 PAK2 (R&D Systems, #MAB6849; 1:50), antibodies overnight and visualized with Alexa Fluor 488 anti-goat and Alexa Fluor 594 anti-rabbit conjugated antibodies (1:500; Invitrogen).

Techniques: Inhibition, Quantitative RT-PCR, Expressing

Journal: Scientific reports

Article Title: Mechanical forces trigger invasive behavior in synovial fibroblasts through N-cadherin/ADAM15 -dependent modulation of LncRNA H19.

doi: 10.1038/s41598-025-94012-2

Figure Lengend Snippet: Fig. 2. ADAM15- and N-cadherin (NCAD) dependent activation of PAK2 upon mechanical strain. (A–D) Immunoblots for pPAK2. (A) of RASFs seeded at different cell densities and mechanically strained for 30 and 60 min. (B) from strained RASFs with PAK2 silencing (si) and treated with a negative control siRNA (neg). (C, D) immunoblots for pPAK2, ADAM15 and NCAD from RASFs treated with ADAM15 and NCAD siRNA; right panels, showing the mean ± SD of densitometric analysis of 6 different RASF cell lines. ***p < 0.0005, Student’s t-test. GAPDH served as a loading control.

Article Snippet: Slices were glued with mounting medium to a glass slide and cells fixed with ice cold methanol for 5 min and aceton for 45 s. After blocking with 1% horse serum in PBS for 1 h, cells were incubated with sheep-anti N-cadherin (R&D Systems, #AF6426, 1:100) and rabbit anti-Nck (Cell Signaling Technology, #2319, 1:100), phospho-PAK1 (Ser144)/PAK2 (Ser141) (Cell Signaling Technology, #2606, 1:100), anti-phosphoPAK2 PAK2 (R&D Systems, #MAB6849; 1:50), antibodies overnight and visualized with Alexa Fluor 488 anti-goat and Alexa Fluor 594 anti-rabbit conjugated antibodies (1:500; Invitrogen).

Techniques: Activation Assay, Western Blot, Negative Control, Control

Journal: Scientific reports

Article Title: Mechanical forces trigger invasive behavior in synovial fibroblasts through N-cadherin/ADAM15 -dependent modulation of LncRNA H19.

doi: 10.1038/s41598-025-94012-2

Figure Lengend Snippet: Fig. 4. Mechano-induced recruitment of PAK2 together with Nck to the cell membrane. (A, B) RASFs grown on Bioflex plates and either unstimulated or strained for 30 min were fluorescently stained for (A) NCAD and Nck and (B) NCAD and PAK2. Adjacent to images: quantification of the pixel density of NCAD /Nck or NCAD /PAK2 along the white arrow. (C) double staining of NCAD and PAK2 in RASFs with prior silencing of Nck, right, pixel density along arrow. Cell nuclei were counterstained with DAPI. Objective 40x, scale bar = 10 μm. (D) immunoblots for PAK2, pPAK2, Nck and NCAD. RASFs pre-treated with Nck siRNA (I) and a nonsilencing siRNA (N) were mechanically strained for 15 and 30 min, the cell surface biotinylated with non-membrane-permeable biotin reagent and purified on streptavidin-conjugated magnetic beads. Contr: non biotinylated cell lysates enriched on streptavidin beads served as background control. TL = total lysates from 30 min time point. GAPDH was used to control the purity of the plasma membrane fractions.

Article Snippet: Slices were glued with mounting medium to a glass slide and cells fixed with ice cold methanol for 5 min and aceton for 45 s. After blocking with 1% horse serum in PBS for 1 h, cells were incubated with sheep-anti N-cadherin (R&D Systems, #AF6426, 1:100) and rabbit anti-Nck (Cell Signaling Technology, #2319, 1:100), phospho-PAK1 (Ser144)/PAK2 (Ser141) (Cell Signaling Technology, #2606, 1:100), anti-phosphoPAK2 PAK2 (R&D Systems, #MAB6849; 1:50), antibodies overnight and visualized with Alexa Fluor 488 anti-goat and Alexa Fluor 594 anti-rabbit conjugated antibodies (1:500; Invitrogen).

Techniques: Membrane, Staining, Double Staining, Western Blot, Purification, Magnetic Beads, Control, Clinical Proteomics

Journal: Scientific reports

Article Title: Mechanical forces trigger invasive behavior in synovial fibroblasts through N-cadherin/ADAM15 -dependent modulation of LncRNA H19.

doi: 10.1038/s41598-025-94012-2

Figure Lengend Snippet: Fig. 5. Binding of PAK2 to ADAM15/NCAD complex at cell membrane is crucially dependent on ADAM15. (A) IPs of strained RASFs using ADAM15 or NCAD antibodies and detection of PAK2 and Nck. TL = total lysate. IgG, mouse IgG served as background control. (B) cell surface biotinylation and enrichment of membrane fractions on streptavidin beads of strained cells with ADAM15-silenced or nonsilenced (N). Contr: non biotinylated cell lysates enriched on streptavidin beads served as background control. (C, D) IPs using ADAM15 antibodies in (C) RASFs with silenced and nonsilenced ADAM15 expression (N) and (D) in chondrocyte cell lines transfected with full length ADAM15 (full-A15) or ADAM15 lacking the cytoplasmic domain (Δcyto). (E, F) analogous to (C, D) IPs using N-cadherin antibodies, showing precipitates of PAK2 and Nck in ADAM15-expressing cells only. (E) right, densitometric evaluation of PAK2 from experiments obtained from 4 donors.

Article Snippet: Slices were glued with mounting medium to a glass slide and cells fixed with ice cold methanol for 5 min and aceton for 45 s. After blocking with 1% horse serum in PBS for 1 h, cells were incubated with sheep-anti N-cadherin (R&D Systems, #AF6426, 1:100) and rabbit anti-Nck (Cell Signaling Technology, #2319, 1:100), phospho-PAK1 (Ser144)/PAK2 (Ser141) (Cell Signaling Technology, #2606, 1:100), anti-phosphoPAK2 PAK2 (R&D Systems, #MAB6849; 1:50), antibodies overnight and visualized with Alexa Fluor 488 anti-goat and Alexa Fluor 594 anti-rabbit conjugated antibodies (1:500; Invitrogen).

Techniques: Binding Assay, Membrane, Control, Expressing, Transfection

Journal: Scientific reports

Article Title: Mechanical forces trigger invasive behavior in synovial fibroblasts through N-cadherin/ADAM15 -dependent modulation of LncRNA H19.

doi: 10.1038/s41598-025-94012-2

Figure Lengend Snippet: Fig. 8. CDH11-mediated increased cell invasion can be blocked by CDH11 silencing and transfection with miR-130a-3p mimics. (A, B) RASFs were treated with H19 siRNA or negative nonsilencing siRNA (N) and (A) MTT-based cell viability assays and (B) cells transmigrated through matrigel were stained with DAPI, the whole transwell photographed and all cells counted. (C) upper panel, invaded cells prior transfected with 130a- 3p mimics, CDH11 siRNA or scramble control, shown is the mean ± SD from 5 different RASFs. (C) lower panel, immunoblots for CDH11 from cells transfected and strained as in upper panel. *p < 0.05, **p < 0.005, ***p < 0.0005, Student’s test. (D) diagram of summarized results: mechanical strain results in NCAD/ADAM15- mediated phosphorylation of PAK2, resulting in the downregulation of lncRNA H19 and miR-130a-3p and subsequent upregulation of CDH11, which in turn promotes an aggressive phenotype: increased cell invasion. Inhibition of PAK signaling by PAK inhibitor IPA-3 blocks mechano-induced H19 downregulation and subsequently CDH11 upregulation. In addition, mechano-induced PAK2 phosphorylation is accompanied by recruitment of SH2/SH3 adapter Nck and PAK2 to the NCAD/ADAM15 complex, but binding of Nck/PAK2 is restricted to the cytoplasmic domain of ADAM15. The mechano-induced redistribution of PAK2 to the cell membrane does not occur when Nck is silenced.

Article Snippet: Slices were glued with mounting medium to a glass slide and cells fixed with ice cold methanol for 5 min and aceton for 45 s. After blocking with 1% horse serum in PBS for 1 h, cells were incubated with sheep-anti N-cadherin (R&D Systems, #AF6426, 1:100) and rabbit anti-Nck (Cell Signaling Technology, #2319, 1:100), phospho-PAK1 (Ser144)/PAK2 (Ser141) (Cell Signaling Technology, #2606, 1:100), anti-phosphoPAK2 PAK2 (R&D Systems, #MAB6849; 1:50), antibodies overnight and visualized with Alexa Fluor 488 anti-goat and Alexa Fluor 594 anti-rabbit conjugated antibodies (1:500; Invitrogen).

Techniques: Transfection, Staining, Control, Western Blot, Phospho-proteomics, Inhibition, Binding Assay, Membrane