Journal: Translational Psychiatry

Article Title: Astrocytic APOE3-Christchurch expression ameliorates brain amyloid-β pathology in 5xFAD mice

doi: 10.1038/s41398-026-04002-9

Figure Lengend Snippet: ( A - C ) Aβ40 and Aβ42 levels in the soluble TBS fractions ( A ), detergent soluble (TBS-X) fractions ( B ), and insoluble neutralized formic acid (FA) fractions ( C ) of cortical brain homogenates in the 5xFAD mice were measured by ELISA at 8 months of age, normalized to protein concentrations of each fraction. ( D ) Soluble oligomeric Aβ (oAβ) levels in the TBS-X fractions of cortical brain homogenates in the 5xFAD mice were measured by ELISA at 8 months of age, normalized to protein concentrations. ( E, F ) Amounts of full-length APP in the TBS-X fractions of mouse cortical lysates were measured by western blot at 8 months of age, normalized to those of β-actin (ACTB). Data are expressed as means ± SEM (GFP: N = 12; APOE3: N = 18; APOE3Ch: N = 13; Open circles: females, closed circles: males). Injection group differences were analyzed using two-way ANOVA, with Tukey’s multiple comparisons test, adjusting for sex. *, P < 0.05; **, P < 0.01.

Article Snippet: Recombinant Aβ42 monomer (1 mg/ml) (Stressmarq, #SPR-485) was incubated at room temperature for 10 min. To dissolve the peptide, 7.5 μl of cold DMSO (Sigma Aldrich) was added, followed by 92.5 μl of cold PBS (PH 7.4) to reach a final concentration of 220 μM monomeric Aβ42.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Injection

Journal: Translational Psychiatry

Article Title: Astrocytic APOE3-Christchurch expression ameliorates brain amyloid-β pathology in 5xFAD mice

doi: 10.1038/s41398-026-04002-9

Figure Lengend Snippet: ( A ) Isogenic iPSC-derived astrocytes (iPSC-ACs) with homozygous APOE3 (left) and APOE3Ch (right) were immunostained for GFAP (green) and S100b (red). DAPI (blue) stains nuclei. Scale bars: 100 µm. ( B ) APOE mRNA levels in the iPSC-ACs were measured by RT-qPCR, normalized to those of β-actin ( ACTB ). ( C, D ) Amounts of APOE in the conditioned medium (CM) from iPSC-ACs were measured by western blot, normalized to protein amounts of cell lysates. ( E ) Effects of the CM from iPSC-ACs on Aβ42 aggregation was assessed by western blot using 6E10 antibody. ( F - G ) Populations of Aβ fibrils (F; > 150 kDa), oligomers (G; 37–150 kDa), and monomers ( H ; < 10 kDa) were quantified. Data expressed as means ± SEM (n = 3–5 independent differentiation batches). Group differences were analyzed using two-tailed student t-test or one-way ANOVA with Tukey’s multiple comparisons test. *, P < 0.05.

Article Snippet: Recombinant Aβ42 monomer (1 mg/ml) (Stressmarq, #SPR-485) was incubated at room temperature for 10 min. To dissolve the peptide, 7.5 μl of cold DMSO (Sigma Aldrich) was added, followed by 92.5 μl of cold PBS (PH 7.4) to reach a final concentration of 220 μM monomeric Aβ42.

Techniques: Derivative Assay, Quantitative RT-PCR, Western Blot, Two Tailed Test

Journal: Autophagy

Article Title: Vitamin K2 induces autophagy and apoptosis simultaneously in leukemia cells.

doi: 10.4161/auto.5941

Figure Lengend Snippet: Figure 3. Induction of autophagy in response to VK2 in HL-60neo/HL-60bcl-2 cells. Involvement of LC3B in VK2-induced autophagy: After exposure to 10 μM VK2 for 48 and 72 hr, the cells were processed for fluorescent immunocytostaining with anti-LC3B Ab and counterstaining with DAPI for nucleus as described in Materials ant Methods. (A) HL-60neo/bcl-2 cells (original magnification x 1,000). (B) Quantification of the cells showing the punctuated pattern of LC3B staining, which marks cells with autophagosome forma- tion. One hundred cells were assessed and ratios for the cells showing the punctuated pattern of LC3B staining were expressed. Results shown are the means ± SD from the results of three independent experi- ments. *p < 0.001. Development of acidic vesicular organelles (AVOs) after VK2 treatment: (C) After staining the cells with acridine orange, AVOs were quantified using flowcytometer in HL-60neo (upper) and HL-60bcl-2 (lower) treated with or without VK2 (5 μM, 10 μM or 20 μM) for 72 hr. X-axis, green color intensity; Y-axis, red color intensity. Expression of isoforms of LC3B and caspase-3: (D) Cellular pro- teins were lysed at the indicated time after incubation with or without 10 μM of VK2. Proteins were separated by either 11.25% or 15% SDS-PAGE. Aliquots of 40 μg of protein extracts were used for immunoblot- ting using anti-caspase-3 and anti-LC3B Abs, respectively. The anti-β-actin mAb was used for protein-loading equivalence. Expression of isoforms of LC3B in the pres- ence and absence of protease inhibitors: (E) After treatment with 10 μM of VK2 for 44 hrs in HL-60neo and for 92 hrs in HL-60bcl-2 cells, cells were further cultured with/without protease inhibitors, E-64-d (10 μg/ml) and pepstatin A (10 μg/ml) in the presence of VK2 for 4 hrs. Cellular proteins were lysed and immunoblotted with anti-LC3B Ab.

Article Snippet: The membranes were probed with antibodies (Abs) such as anti-human Bcl-2 monoclonal (m) Ab (BD Biosciences Pharmingen, San Jose, CA), anti-microtubule-associated protein 1 light chain 3B (LC3B) Ab, anti-caspase-3 Ab (Cell Signaling Technology, Danvers, MA), anti-cleaved caspase-3 Ab (Cell Signaling Technology), antiAtg5 Ab (Abgent, San Diego, CA), anti-Atg7 Ab (ProSci, Poway, CA), anti-human catalase rabbit mAb (Epitomics Inc., Burlingame, CA), and anti-β-actin Ab (Sigma-Aldrich), respectively.

Techniques: Staining, Expressing, Incubation, SDS Page, Western Blot, Cell Culture

Journal: Autophagy

Article Title: Vitamin K2 induces autophagy and apoptosis simultaneously in leukemia cells.

doi: 10.4161/auto.5941

Figure Lengend Snippet: Figure 4. Effects of a caspase-3 inhibitor on VK2-induced autophagy in HL-60neo cells. (A) HL- 60neo cells were cultured with Z-DEVD-FMK at various concentrations with/without 10 μM of VK2 for 96 hr. The number of cells was assessed with the WST cell counting kit as described in Materials and Methods. *p < 0.001. (B) HL-60neo cells were treated with 10 μM VK2 with/without 100 μM of Z-DEVD-FMK for 48 hr and 96 hr, respectively. Cellular proteins were lysed and separated by either 11.25% or 15% SDS-PAGE. Aliquots of 40 μg of protein extracts were used for immunoblotting using anti-cleaved caspase-3 Ab, anti-LC3B Ab, and anti-β-actin mAb, respectively.

Article Snippet: The membranes were probed with antibodies (Abs) such as anti-human Bcl-2 monoclonal (m) Ab (BD Biosciences Pharmingen, San Jose, CA), anti-microtubule-associated protein 1 light chain 3B (LC3B) Ab, anti-caspase-3 Ab (Cell Signaling Technology, Danvers, MA), anti-cleaved caspase-3 Ab (Cell Signaling Technology), antiAtg5 Ab (Abgent, San Diego, CA), anti-Atg7 Ab (ProSci, Poway, CA), anti-human catalase rabbit mAb (Epitomics Inc., Burlingame, CA), and anti-β-actin Ab (Sigma-Aldrich), respectively.

Techniques: Cell Culture, Cell Counting, SDS Page, Western Blot

Journal: Autophagy

Article Title: Vitamin K2 induces autophagy and apoptosis simultaneously in leukemia cells.

doi: 10.4161/auto.5941

Figure Lengend Snippet: Figure 5. Capsulated fragmented nuclei in HL-60bcl-2 cells after treatment with VK2. (A) Morphological features of HL-60neo after 72 hr-treatment with VK2 (10 μM) with/without Z-DEVD-FMK (100 μM) and of HL-60bcl-2 cells after 96 hr-treatment with VK2. May-Grünwald-Giemsa staining, original magnification x 1,000. (B) Percentages of HL-60 cells with apoptotic bodies containing either capsulated nuclear fragments or uncapsulated nuclear. After treatment HL- 60neo/bcl-2 cells with 10 μM of VK2 for various length of time, morphologic changes were assessed in 100 cells after May-Grünwald Giemsa stain as well as Figure 4A. (This is one of representative result from 3 separate experiments.) (C) Electron microscopy of HL-60bcl-2 cells after treatment with 10 μM VK2 for 96 hr. (D) Fluorescent immunocytostaining with anti-LC3B Ab in HL-60bcl-2 cells after 96 hr-treatment with 10 μM VK2. Fluorescent immunocytostaining with anti-LC3B Ab (original magnification x 1,000) was performed as described in Figure 3A and C, respectively. The slides stained with anti-LC3B Ab were monitored using Zeiss LSM510 confocal microscope (Original magnification x 600).

Article Snippet: The membranes were probed with antibodies (Abs) such as anti-human Bcl-2 monoclonal (m) Ab (BD Biosciences Pharmingen, San Jose, CA), anti-microtubule-associated protein 1 light chain 3B (LC3B) Ab, anti-caspase-3 Ab (Cell Signaling Technology, Danvers, MA), anti-cleaved caspase-3 Ab (Cell Signaling Technology), antiAtg5 Ab (Abgent, San Diego, CA), anti-Atg7 Ab (ProSci, Poway, CA), anti-human catalase rabbit mAb (Epitomics Inc., Burlingame, CA), and anti-β-actin Ab (Sigma-Aldrich), respectively.

Techniques: Staining, Giemsa Stain, Electron Microscopy, Microscopy

Journal: Autophagy

Article Title: Vitamin K2 induces autophagy and apoptosis simultaneously in leukemia cells.

doi: 10.4161/auto.5941

Figure Lengend Snippet: Figure 7. Effects of inhibition of VK2-inducing autophagy in a Atg5-/- mouse embryonic fibroblast (MEF) cell line with the Atg5 Tet-off system. Tet-off Atg5 MEF cells were incubated with 10 ng/ml doxycycline hydrochloride (Dox) for 120 hrs, and thereafter cultured in the presence or absence of 1 to 50 μM of VK2 for 72 hrs. (A) Cell growth inhibition in response to VK2 was assessed using WST assay kit. (B) MEF cells treated with or without Dox were cultured in the presence or absence of 10 μM of VK2 for 72 hr. Then, cellular proteins were separated by 11.25% SDS-PAGE and immunoblotted with either anti-Atg5, anti-LC3B, or anti-β-actin Abs.

Article Snippet: The membranes were probed with antibodies (Abs) such as anti-human Bcl-2 monoclonal (m) Ab (BD Biosciences Pharmingen, San Jose, CA), anti-microtubule-associated protein 1 light chain 3B (LC3B) Ab, anti-caspase-3 Ab (Cell Signaling Technology, Danvers, MA), anti-cleaved caspase-3 Ab (Cell Signaling Technology), antiAtg5 Ab (Abgent, San Diego, CA), anti-Atg7 Ab (ProSci, Poway, CA), anti-human catalase rabbit mAb (Epitomics Inc., Burlingame, CA), and anti-β-actin Ab (Sigma-Aldrich), respectively.

Techniques: Inhibition, Incubation, Cell Culture, WST Assay, SDS Page

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 deficiency protects against Ang II‐induced myocardial hypertrophy and fibrosis. WT and TRIM40 −/− mice were infused with saline or Ang II for 4 weeks. (A) Mouse systolic blood pressure was measured weekly using a non‐invasive tail‐cuff method (n = 6). (B) Serum Ang II concentration was detected in mice (n = 6). (C) Representative echocardiographic images of mice from each group (n = 6). (D–F) Cardiac function parameters showing EF (D), FS (E), and IVRT (F) (n = 6). (G) Serum CK‐MB levels were measured using an ELISA kit (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 cross‐sectional area was assessed by dual immunofluorescence staining for cTnT (red) to mark cardiomyocytes and fluorescein‐labeled WGA (green) to delineate cell membranes (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) Representative western blot analyses of MYH7, ANP, and BNP in heart tissue, with GAPDH as a loading control (n = 6). (O) Densitometric quantification of the blots in N (n = 6). (P, Q) mRNA expression levels of hypertrophy‐associated genes (P) and inflammation‐related genes (Q) in heart tissues of mice. Data was normalized to Actb (n = 6). 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. 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, Enzyme-linked Immunosorbent Assay, Isolation, Immunofluorescence, Staining, Labeling, Western Blot, Control, Expressing, 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 deficiency prevents TAC‐induced myocardial hypertrophy and fibrosis. WT and TRIM40 −/− mice were subjected to TAC to model pressure overload‐induced cardiac hypertrophy and remodeling. (A) Representative echocardiographic images of mice from each group (n = 6). (B–D) Cardiac function parameters showing EF (B), FS (C), and IVRT (D) (n = 6). (E) Serum CK‐MB levels were measured using an ELISA kit (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 cross‐sectional area was assessed by dual immunofluorescence staining for cTnT (red) to mark cardiomyocytes and fluorescein‐labeled WGA (green) to delineate cell membranes (scale bar = 50 µm) (n = 6). (I) Quantitative analysis of cardiomyocyte cross‐sectional area. A minimum of 100 cells were measured from different fields across at least four samples per group (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) Representative western blot analyses of MYH7, ANP, and BNP in heart tissue, with GAPDH as a loading control (n = 6). (N) Densitometric quantification of the blots in M (n = 6). (O, P) mRNA expression levels of hypertrophy‐associated genes (O) and inflammation‐related genes (P) in heart tissues. Data were normalized to Actb (n = 6). 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: Enzyme-linked Immunosorbent Assay, Isolation, Immunofluorescence, Staining, Labeling, Western Blot, Control, Expressing, 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: Cardiomyocyte‐specific knockdown of TRIM40 alleviates Ang II‐induced cardiac remodeling. (A) Mouse systolic blood pressure was measured weekly using a non‐invasive tail‐cuff method (n = 6). (B) Serum Ang II concentration was detected in mice (n = 6). (C) Representative echocardiographic images from each group of mice (n = 6). (D‐F) Cardiac function parameters: EF (D), FS (E), and IVRT (F) (n = 6). (G) Serum CK‐MB levels (n = 6). (H) Heart weight/body weight ratio (n = 6). (I) Representative images of freshly isolated heart specimens (n = 6). (J) Quantitative analysis of the cross‐sectional area of heart specimens shown in (I) (n = 6). (K) Cardiomyocyte cross‐sectional area was assessed by dual immunofluorescence staining for cTnT (red) to mark cardiomyocytes and fluorescein‐labeled WGA (green) to delineate cell membranes (scale bar = 50 µm) (n = 6). (L) Quantitative analysis of cardiomyocyte cross‐sectional area based on K. A minimum of 100 cells were measured from different fields across at least four samples per group (n = 6). (M) H&E staining of heart tissue sections (scale bar = 50 µm) (n = 6). (N, O) Myocardial fibrosis evaluated by Picrosirius Red (N) and Masson's trichrome (O) staining (scale bar = 50 µm) (n = 6). (P) Representative Western blots of MYH7, ANP, and BNP protein expression in heart tissue, with GAPDH as a loading control (n = 6). (Q) Densitometric quantification of the blots shown in (P) (n = 6). (R, S) mRNA expression levels of hypertrophy‐associated (R) and inflammation‐related (S) genes in heart tissues (n = 6). Data were normalized to Actb (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. ns indicates not statistically significant; ** 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: Knockdown, Concentration Assay, Isolation, Immunofluorescence, Staining, Labeling, Western Blot, Expressing, Control, 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 regulates cardiomyocyte hypertrophic and fibrotic responses in culture. (A) After transfection with either a negative control (NC) or TRIM40 siRNA (si‐TRIM40), NRVMs were treated with Ang II (1 µ m for 24 h). Cardiomyocyte size was assessed by dual immunofluorescence staining for cTnT (green) and Rhodamine‐phalloidin (red). Nuclei were counterstained with DAPI (blue) (scale bar = 50 µm) (n = 3). (B) Changes in NRVMs size in response to Ang II. At least 100 cells from each of three independent samples were analyzed per group (n = 3). (C) Representative western blot of MYH7, ANP, and BNP proteins in NRVMs, with GAPDH as loading control (n = 3). (D, E) The mRNA levels of hypertrophy‐associated genes (D) and inflammation‐related genes (E) in NRVMs treated as indicated. Data was normalized to Actb (n = 3). (F) Following transfection with Flag‐TRIM40 or an empty vector (EV), NRVMs were treated with Ang II (1 µ m for 24 h). Cardiomyocyte size was assessed by the dual immunofluorescence staining method described in (A) (scale bar = 50 µm) (n = 3). (G) Changes in NRVMs size in response to Ang II. At least 100 cells from each of three independent samples were analyzed per group (n = 3). (H) Representative western blot of MYH7, ANP, and BNP proteins in NRVMs, with GAPDH as loading control (n = 3). (I, J) mRNA expression levels of hypertrophy‐associated genes (I) and inflammation‐related genes (J) in NRVMs. Data were normalized to Actb (n = 3). 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 < 0.05, ** 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: Transfection, Negative Control, Immunofluorescence, Staining, Western Blot, Control, Plasmid Preparation, Expressing, 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 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: Molecular and Cellular Biology

Article Title: NR4A1 Antagonists Inhibit β1-Integrin-Dependent Breast Cancer Cell Migration

doi: 10.1128/mcb.00912-15

Figure Lengend Snippet: Fig. 1. NR4A1 regulates β1-integrin expression in breast cancer cells and tumors. (A) Breast 598

Article Snippet: NR4A1 antibody was purchased from Novus Biologicals (Littleton, CO).

Techniques: Expressing

Journal: Molecular and Cellular Biology

Article Title: NR4A1 Antagonists Inhibit β1-Integrin-Dependent Breast Cancer Cell Migration

doi: 10.1128/mcb.00912-15

Figure Lengend Snippet: Fig. 3. Role of NR4A1/p300/Sp1 in regulation of β1- and β3-integrin. (A) Analysis of polII, 620

Article Snippet: NR4A1 antibody was purchased from Novus Biologicals (Littleton, CO).

Techniques:

Journal: Molecular and Cellular Biology

Article Title: NR4A1 Antagonists Inhibit β1-Integrin-Dependent Breast Cancer Cell Migration

doi: 10.1128/mcb.00912-15

Figure Lengend Snippet: Fig. 5. Role of NR4A1 on TGFβ-induced migration of MDA-MB-231 cells. (A) MDA-MB-231 647

Article Snippet: NR4A1 antibody was purchased from Novus Biologicals (Littleton, CO).

Techniques: Migration