Journal: ESC Heart Failure

Article Title: MicroRNA‐494 regulates high glucose‐induced cardiomyocyte apoptosis and autophagy by PI3K/AKT/ mTOR signalling pathway

doi: 10.1002/ehf2.14311

Figure Lengend Snippet: MicroRNA (miR)‐494 regulates cell apoptosis and autophagy via the PI3K/AKT/mTOR signalling pathway. The expression level of PI3K/AKT/mTOR pathway‐related proteins in different groups was detected by western blotting. Data are presented as the mean ± SD, n = 3, with three replicates. Replicates = 3. ** P < 0.01 and *** P < 0.001. HG, high glucose; NC, negative control.

Article Snippet: After blocking with 5% skim milk at room temperature for 2 h, the membrane was incubated with corresponding primary antibodies at 4°C: anti‐Bax rabbit monoclonal antibody (1:2000, Abcam, ab32503), anti‐Bcl‐2 rabbit polyclonal antibody (1:2000, Abcam, ab196495), anti‐LC3B rabbit monoclonal antibody (1:1000, Abcam, ab192890), anti‐p62 rabbit monoclonal antibody (1:15 000, Abcam, ab109012), anti‐Beclin‐1 rabbit monoclonal antibody (1:1600, Abcam, ab137020), anti‐p‐PI3K rabbit monoclonal antibody (1:1000, Abcam, ab40776), anti‐p‐AKT rabbit monoclonal antibody (1:4500, Abcam, ab81283), anti‐p‐mTOR rabbit monoclonal antibody (1:2500, Abcam, ab137133), anti‐PI3K mouse monoclonal antibody (1:1800, Abcam, ab140307), anti‐AKT rabbit monoclonal antibody (1:9000, Abcam, ab179463), anti‐mTOR rabbit monoclonal antibody (1:3000, Abcam, ab2732), and anti‐GAPDH rabbit monoclonal antibody (1:8000, Abcam, ab181602).

Techniques: Expressing, Western Blot, Negative Control

Journal: ESC Heart Failure

Article Title: MicroRNA‐494 regulates high glucose‐induced cardiomyocyte apoptosis and autophagy by PI3K/AKT/ mTOR signalling pathway

doi: 10.1002/ehf2.14311

Figure Lengend Snippet: Schematic representation of microRNA (miR)‐494‐dependent regulation of the PI3K/AKT/mTOR pathway involved in the progression of diabetic cardiomyopathy. This diagram shows that during the process of diabetic cardiomyopathy, miR‐494 is up‐regulated and it inhibits the phosphorylation level of the PI3K/AKT/mTOR pathway, which promotes the progression of cardiomyocyte apoptosis and autophagy.

Article Snippet: After blocking with 5% skim milk at room temperature for 2 h, the membrane was incubated with corresponding primary antibodies at 4°C: anti‐Bax rabbit monoclonal antibody (1:2000, Abcam, ab32503), anti‐Bcl‐2 rabbit polyclonal antibody (1:2000, Abcam, ab196495), anti‐LC3B rabbit monoclonal antibody (1:1000, Abcam, ab192890), anti‐p62 rabbit monoclonal antibody (1:15 000, Abcam, ab109012), anti‐Beclin‐1 rabbit monoclonal antibody (1:1600, Abcam, ab137020), anti‐p‐PI3K rabbit monoclonal antibody (1:1000, Abcam, ab40776), anti‐p‐AKT rabbit monoclonal antibody (1:4500, Abcam, ab81283), anti‐p‐mTOR rabbit monoclonal antibody (1:2500, Abcam, ab137133), anti‐PI3K mouse monoclonal antibody (1:1800, Abcam, ab140307), anti‐AKT rabbit monoclonal antibody (1:9000, Abcam, ab179463), anti‐mTOR rabbit monoclonal antibody (1:3000, Abcam, ab2732), and anti‐GAPDH rabbit monoclonal antibody (1:8000, Abcam, ab181602).

Techniques:

Journal: Journal of Virology

Article Title: GCRV-II invades monocytes/macrophages and induces macrophage polarization and apoptosis in tissues to facilitate viral replication and dissemination

doi: 10.1128/jvi.01469-23

Figure Lengend Snippet: Polarization of M1 and M2 macrophages in vitro. (A) SDS-PAGE analysis of purified recombinant intact CiIFN-γ2 (17.6 kDa), CiIL-4/13A (17.2 kDa), and CiIL-4/13B (17.1 kDa) proteins. (B and C) Measurement of CiIFN-γ2, CiIL-4/13A, and CiIL-4/13B protein activity by determining the phosphorylation levels of STAT5, ERK, and mTOR. (C) Measurement of phosphorylation levels of STAT5, ERK, and mTOR in three separate experiments by determining the gray value of the WB bands of CiIFN-γ2, CiIL-4/13A, and CiIL-4/13B proteins using the ImageJ software. (D and E) Verification of M1 macrophage polarization by mRNA expression levels of IL-1β, CXCR 3.1, and iNOS (D), as well as enzyme activity of iNOS (E). (F and G) Verification of M2 macrophage polarization by mRNA expression levels of IL-10, CXCR 3.2, and arginase-2 (F), as well as arginase-2 enzyme activity (G). Relative mRNA expression level was normalized to that of EF1a (n = 3) (*P < 0.05 and **P < 0.01).

Article Snippet: Other antibodies used in the study included β-actin mouse mAb (1:10,000, AC004, ABclonal), arginase-2 rabbit pAb (1:500, A6355, ABclonal), iNOS rabbit mAb (1:500, A3774, ABclonal), Bcl-2 rabbit pAb (1:5,000, A0208, ABclonal), HRP rabbit anti-goat IgG (1:5,000, AS029, ABclonal), p-STAT5 rabbit mAb (1:2,000, AP0758, ABclonal), p-ERK rabbit pAb (1:500, AP0472, ABclonal), and p-mTOR rabbit pAb (1:500, AP0094, ABclonal).

Techniques: In Vitro, SDS Page, Purification, Recombinant, Activity Assay, Software, Expressing

Journal: iScience

Article Title: Inhibition of CD44 suppresses the formation of fibrotic scar after spinal cord injury via the JAK2/STAT3 signaling pathway

doi: 10.1016/j.isci.2024.108935

Figure Lengend Snippet: The JAK2/STAT3 signaling pathway participated in the CD44-mediated functional changes in fibroblasts (A) Western blotting analyzed the expression of p -Smad2, p -JAK2, JAK2, p-STAT3, and STAT3 in the fibroblasts after IM7 treatment. (B‒F) Quantification of Western blotting results in A. N = 3 of each group by one-way ANOVA. (G) Western blotting determined the expression of p -JAK2, p-STAT3, and p -Smad2 in the fibroblasts after WP1066 treatment. (H‒J) Quantification of western blotting results in G. N = 3 of each group by one-way ANOVA. (K) Western blotting analyzed the expression of α-SMA, collagen-I, FN, laminin, and CD44 in the fibroblasts after WP1066 treatment. (L‒P) Quantification of western blotting results in K. N = 3 of each group by one-way ANOVA. (Q and R) Quantification analysis of the rate of proliferation (%) (Q) per 10 6 μm 2 (n = 5) and cell numbers of migration (R) per 10 6 μm 2 (n = 5) in different groups by one-way ANOVA. All data were presented as mean ± SEM. (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). (See also Figure S3 ).

Article Snippet: Rabbit monoclonal anti- p -JAK2 , Abmart , Cat#T56570; RRID: AB_2936394.

Techniques: Functional Assay, Western Blot, Expressing, Migration

Journal: iScience

Article Title: Inhibition of CD44 suppresses the formation of fibrotic scar after spinal cord injury via the JAK2/STAT3 signaling pathway

doi: 10.1016/j.isci.2024.108935

Figure Lengend Snippet: STAT3 was the downstream of JAK2 in CD44-mediated fibrotic effect in the inflammatory microenvironment (A) Western blotting analyzed the expression of p -JAK2, p-STAT3, STAT3, and p -Smad2 in the fibroblasts stimulated with IS after inhibition of AG490. (B‒E) Quantification of western blotting results in A. N = 3 of each group by one-way ANOVA. (F) Western blotting analyzed the expression of CD44, α-SMA, collagen-I, FN, and laminin in the fibroblasts stimulated with IS after inhibition of AG490. (G‒K) Quantification of western blotting results in F. N = 3 of each group by one-way ANOVA. (L and M) Quantification analysis of the rate of proliferation (%) (L) per 10 6 μm 2 (n = 5) and cell numbers of migration (M) per 10 6 μm 2 (n = 5) in different groups by one-way ANOVA. All data were presented as mean ± SEM. (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). (See also Figure S3 ).

Article Snippet: Rabbit monoclonal anti- p -JAK2 , Abmart , Cat#T56570; RRID: AB_2936394.

Techniques: Western Blot, Expressing, Inhibition, Migration

Journal: iScience

Article Title: Inhibition of CD44 suppresses the formation of fibrotic scar after spinal cord injury via the JAK2/STAT3 signaling pathway

doi: 10.1016/j.isci.2024.108935

Figure Lengend Snippet: Diagram of molecule mechanism of CD44-JAK2-STAT3 in the fibroblasts following SCI, macrophages are activated and secrete varieties of cytokines and inflammatory factors, and recruit fibroblasts to participate in the subsequent repair process Then CD44 on the fibroblasts would be activated by these proinflammatory factors, which could be inhibited by IM7. The signal is transferred into cells, and activates the JAK2 and STAT3. Activated STAT3 enters into nucleus and regulates the expression of fibrosis-related genes at the transcriptional level by binding specific DNA promoter sequences, which ultimately promotes the proliferation, differentiation, and migration of fibroblasts, leading to the deposition of excessive ECM proteins and finally forms dense fibrotic scar tissue.

Article Snippet: Rabbit monoclonal anti- p -JAK2 , Abmart , Cat#T56570; RRID: AB_2936394.

Techniques: Expressing, Binding Assay, Migration

Journal: iScience

Article Title: Inhibition of CD44 suppresses the formation of fibrotic scar after spinal cord injury via the JAK2/STAT3 signaling pathway

doi: 10.1016/j.isci.2024.108935

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti- p -JAK2 , Abmart , Cat#T56570; RRID: AB_2936394.

Techniques: Recombinant, In Vivo, Lysis, Acid Assay, Software