anti pim1  (Bioss)

Journal: PLOS One

Article Title: PIM1 induces hypoxia-related fibroblast senescence in a mouse model of stress urinary incontinence

doi: 10.1371/journal.pone.0335501

Figure Lengend Snippet: (A) Relative mRNA expression of PIM1 in vaginal wall tissues (n = 3). (B) Protein expression of PIM1 in vaginal wall tissues and quantitative analysis, quantitative analysis is expressed as a relative value to control (n = 3). (C,D) Representative images of immunohistochemical staining of PIM1 in vaginal wall tissues and quantitative analysis, quantitative analysis is expressed as a relative value to control (scale bar: 20μm, n = 3). e: Pattern image of cellular hypoxia model. 300μM CoCl 2 was treated with L929 cells for 12 weeks respectively, = 3). (E) Schematic representation of the chemical hypoxia induction model in cultured cells. 300 μM CoCl 2 treated L929 cells for 12 h and 24 h, respectively. con: control cells, 12h: CoCl 2 treated L929 cells for 12 h group, 24h: CoCl 2 treated L929 cells for 24 h group. (F, G) representative images and statistical analysis of hypoxia probe (Hypoxyprobe-1) staining representative images and statistical graphs. Statistical graphs are represented as relative quantification compared to control (scale bar: 20 μm, n = 3). (H,I)Representative images and statistical graphs of PIM1 immunofluorescence staining of different subgroups of cells. Statistical graphs are represented as relative quantification compared to control (scale bar: 20 μm, n = 3). (J)Relative mRNA expression of PIM1 in different subgroups of cells (n = 3). (K) Representative images and statistical graphs of immunofluorescence staining of PIM1and HIF-1α in different subgroups of cells. Statistical graphs are represented as relative quantification compared to control (n = 3). Statistical significance was determined, with **P < 0.01,***P < 0.001 and ****P < 0.0001 compared to the control group.

Article Snippet: These membranes were blocked with 5% skim milk in TBST for 1 hour and incubated overnight at 4°C with primary antibodies at the following dilutions: PIM1 (1:1000, Abclonal, #A19695), P21 (1:1000, Wanleibio, #WL0362), P16 (1:3000, ELK Biotechnology, #ES8349), LaminB1 (1:1000, Abclonal, #A1910), γH2A.X (1:1000, Wanleibio, #WL00616a), HIF-1α (1:1500, Abclonal, #A26889), and β-actin (1:20000, Proteintech, #66009–1-Ig).

Techniques: Expressing, Control, Immunohistochemical staining, Staining, Cell Culture, Quantitative Proteomics, Immunofluorescence

Journal: PLOS One

Article Title: PIM1 induces hypoxia-related fibroblast senescence in a mouse model of stress urinary incontinence

doi: 10.1371/journal.pone.0335501

Figure Lengend Snippet: (A) Schematic diagram of the hypoxia chamber system for establishing cellular hypoxia (1% O₂) in L929 cells. Experimental groups: normoxic control (21% O₂), 24-h hypoxia, and 48-h hypoxia. (B) Representative immunofluorescence images and quantitative analysis of Hypoxyprobe-1 staining (red) demonstrating progressive hypoxia development (scale bar: 20 μm; n = 3 independent experiments). Data normalized to normoxic control. (C) Western blot analysis with densitometric quantification showing time-dependent upregulation of PIM1 and HIF-1α protein expression (n = 3 biological replicates). (D) Immunofluorescence staining and quantitative assessment of PIM1 expression (green) under different oxygen conditions (scale bar: 20 μm; n = 3 technical replicates). Statistical significance was determined, with ****P < 0.0001 compared to the control group.

Article Snippet: These membranes were blocked with 5% skim milk in TBST for 1 hour and incubated overnight at 4°C with primary antibodies at the following dilutions: PIM1 (1:1000, Abclonal, #A19695), P21 (1:1000, Wanleibio, #WL0362), P16 (1:3000, ELK Biotechnology, #ES8349), LaminB1 (1:1000, Abclonal, #A1910), γH2A.X (1:1000, Wanleibio, #WL00616a), HIF-1α (1:1500, Abclonal, #A26889), and β-actin (1:20000, Proteintech, #66009–1-Ig).

Techniques: Control, Immunofluorescence, Staining, Western Blot, Expressing

Journal: PLOS One

Article Title: PIM1 induces hypoxia-related fibroblast senescence in a mouse model of stress urinary incontinence

doi: 10.1371/journal.pone.0335501

Figure Lengend Snippet: (A)PIM1 mRNA expression in L929 cells transfected with the PIM1 overexpression plasmid was detected using RT-qPCR. (B)Expression and quantitative analysis of PIM1 protein in L929 cells transfected with the PIM1 overexpression plasmid were determined by Western blot. (C) Representative images (left) and quantitative analysis (right) of wound healing assays in cells with PIM1 overexpression (scale bar: 100μm). (D) Transwell migration assay showing representative images (right) and quantification (left) of migrated cells(scale bar: 100μm). (E)Proliferation capacity of L929 cells transfected with the PIM1 overexpression plasmid was assessed using EDU staining and quantitatively analyzed (scale bar: 20μm). (F)SA-β-gal staining and quantitative analysis of L929 cells transfected with the PIM1 overexpression plasmid were performed (scale bar: 50μm). (G)Expression and quantitative analysis of LaminB1, P21, P16, γH2A.X, and β-actin proteins in L929 cells transfected with the PIM1 overexpression plasmid were determined by Western blot. (H)Laser confocal immunofluorescence images of HP1γ (green), H3K9me3 (red), and DAPI (blue) in L929 cells transfected with the PIM1 overexpression plasmid and quantification using ImageJ software (scale bar: 10μm). (I) Cell cycle analysis by flow cytometry in L929 cells transfected with PIM1 overexpression plasmid. Representative DNA histograms show cell distribution in G1, S, and G2 phases (analyzed by FlowJo software using Watson model for curve fitting).All results represent data from three independent experiments. Control group: CON, empty vector group: pVector, PIM1-expressing plasmid group: pPIM1. **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: non-significant.

Article Snippet: These membranes were blocked with 5% skim milk in TBST for 1 hour and incubated overnight at 4°C with primary antibodies at the following dilutions: PIM1 (1:1000, Abclonal, #A19695), P21 (1:1000, Wanleibio, #WL0362), P16 (1:3000, ELK Biotechnology, #ES8349), LaminB1 (1:1000, Abclonal, #A1910), γH2A.X (1:1000, Wanleibio, #WL00616a), HIF-1α (1:1500, Abclonal, #A26889), and β-actin (1:20000, Proteintech, #66009–1-Ig).

Techniques: Expressing, Transfection, Over Expression, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Transwell Migration Assay, Staining, Immunofluorescence, Software, Cell Cycle Assay, Flow Cytometry, Control

Journal: PLOS One

Article Title: PIM1 induces hypoxia-related fibroblast senescence in a mouse model of stress urinary incontinence

doi: 10.1371/journal.pone.0335501

Figure Lengend Snippet: (A-B) Knockdown efficiency of PIM1 siRNA (si-PIM1) in L929 cells assessed by RT-qPCR (A) and Western blot (B) at 24h post-transfection, compared with negative control siRNA (si-NC). (C) EdU incorporation assay evaluating proliferative capacity of L929 cells following PIM1 knockdown and subsequent treatment with 300μM CoCl₂ for 24h or 1% O₂ for 48h (scale bar: 20μm). (D) Wound healing assay measuring migratory ability under different treatment conditions (scale bar: 100μm).(E) Transwell migration assay showing representative images (left) and quantification (right) of migrated cells(scale bar: 100μm). (F) SA-β-gal staining and quantification of senescent cells across experimental groups (scale bar: 50μm). (G) Western blot analysis of senescence-associated markers (Lamin B1, PIM1, P21, P16, γH2A.X) with β-actin loading control, including densitometric quantification. (H) Flow cytometric analysis of cell cycle distribution (G1, S, G2 phases) using Watson model-based curve fitting in FlowJo software. (I) Confocal immunofluorescence images showing HP1γ (green), H3K9me3 (red), and DAPI (blue) localization in PIM1-knockdown cells (scale bar: 10μm), with quantitative analysis by ImageJ. All results represent data from three independent experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: non-significant.

Article Snippet: These membranes were blocked with 5% skim milk in TBST for 1 hour and incubated overnight at 4°C with primary antibodies at the following dilutions: PIM1 (1:1000, Abclonal, #A19695), P21 (1:1000, Wanleibio, #WL0362), P16 (1:3000, ELK Biotechnology, #ES8349), LaminB1 (1:1000, Abclonal, #A1910), γH2A.X (1:1000, Wanleibio, #WL00616a), HIF-1α (1:1500, Abclonal, #A26889), and β-actin (1:20000, Proteintech, #66009–1-Ig).

Techniques: Knockdown, Quantitative RT-PCR, Western Blot, Transfection, Negative Control, Wound Healing Assay, Transwell Migration Assay, Staining, Control, Software, Immunofluorescence

Journal: PLOS One

Article Title: PIM1 induces hypoxia-related fibroblast senescence in a mouse model of stress urinary incontinence

doi: 10.1371/journal.pone.0335501

Figure Lengend Snippet: (A) Dose-dependent inhibition of PIM1 by AZD-1208 (10μM and 20μM for 24h) assessed by Western blot, with vehicle-treated cells as control. The 20μM concentration was selected for subsequent experiments. (B) EdU incorporation assay demonstrating proliferative capacity of L929 cells following AZD-1208 treatment combined with either 300μM CoCl₂ (24h) or 1% O₂ (48h) exposure (scale bar: 20μm). (C) Wound healing assay quantifying cell migration under different treatment conditions (scale bar: 200μm). (D) Transwell migration assay showing representative images (left) and quantification (right) of migrated cells(scale bar: 100μm). (E) SA-β-gal staining and quantitative analysis of cellular senescence (scale bar: 50μm). (F) Western blot analysis of senescence-associated markers (Lamin B1, PIM1, P21, P16, γH2A.X) with β-actin loading control, including densitometric quantification. (G) Flow cytometric cell cycle profiling (G1, S, G2 phases) analyzed using FlowJo software with Watson model-based curve fitting. (H) Confocal microscopy images showing subcellular localization of HP1γ (green), H3K9me3 (red), and DAPI (blue) in AZD-1208-treated cells (scale bar: 10μm), with ImageJ-based quantification. All results represent data from three independent experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: non-significant.

Article Snippet: These membranes were blocked with 5% skim milk in TBST for 1 hour and incubated overnight at 4°C with primary antibodies at the following dilutions: PIM1 (1:1000, Abclonal, #A19695), P21 (1:1000, Wanleibio, #WL0362), P16 (1:3000, ELK Biotechnology, #ES8349), LaminB1 (1:1000, Abclonal, #A1910), γH2A.X (1:1000, Wanleibio, #WL00616a), HIF-1α (1:1500, Abclonal, #A26889), and β-actin (1:20000, Proteintech, #66009–1-Ig).

Techniques: Inhibition, Western Blot, Control, Concentration Assay, Wound Healing Assay, Migration, Transwell Migration Assay, Staining, Software, Confocal Microscopy