Journal: Respiratory Research

Article Title: YAP/TAZ activation mediates PQ-induced lung fibrosis by sustaining senescent pulmonary epithelial cells

doi: 10.1186/s12931-024-02832-z

Figure Lengend Snippet: Paraquat treatment induces pulmonary cellular senescence in mice: A HE staining and Masson’s trichrome staining of representative lung sections from control and PQ group (original magnification 100×, scale bar = 400 μm), B SA-β-gal staining of parenchyma and trachea in lung tissues (original magnification 200×, scale bar = 200 μm), C representative images of immunoflourescence staining for p16 (red) and DAPI (blue) in lung sections (original magnification 200×, scale bar = 200 μm), D total lung protein was assessed for p16 and p21, with β-actin as loading control ( N = 8) by Western blotting, E relative mRNA levels of SASP markers Il6 , Il1a and Il8 compared to Actb in total lung tissues ( N = 4 for ctrl group, N = 6 for PQ group) were analyzed by qRT-PCR, F serum levels of SASP markers Il-6, Il-1α and Il-8 ( N = 8) were tested by ELISA assay. Values are shown as mean ± SEM. Data were analyzed by Student’s t test. * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001

Article Snippet: The primary antibodies included anti-p16 Ink4a (for immunofluorescence: ab211542, 1:100, Abcam, UK; for Western blotting: for human: 10883-1, 1:1000, Proteintech, China; for mouse: sc-1661, 1:1000, Santa Cruz Technology, TX, United States), anti-p21 Cip1/Waf1 (sc-6246, 1:1000, Santa Cruz Technology), anti-β-actin (4970, 1:8000, CST, MA, United States), anti-Ki-67 (AG8471, 1:100, Beyotime, China), anti-α-SMA (19245, 1:200, CST), anti-pYAP (Ser127) (13008S, 1:1000, CST), anti-YAP (12395S, 1:1000 for Western blotting and 1:400 for immunohistochemisty, CST; 13584-1-AP, 1:200 for immunofluorescence, Proteintech), anti-pTAZ (Ser89) (59971S, 1:1000, CST), anti-TAZ (83669S, 1:1000 for Western blotting and 1:200 for immunofluorescence, CST), anti-Survivin (10508-1-ap, 1:1000, Proteintech), anti-Bcl-2 (BS1511, 1:1000, BioWorld, China), anti-Bax (50599-2-Ig, 1:1000, Proteintech), anti-Fn1 (sc-8422, 1:1000 for Western blotting, Santa Cruz Technology; ab45688, 1: 500 for immunofluorescence, Abcam), anti-AQP5 (20334-1-AP, 1:100, Proteintech), Sfptc (10774-1-AP, 1:200, Proteintech), anti-Cyk19 (GB12197, 1:500) and anti-Ctgf (23936-1-AP, 1:1000, Proteintech).

Techniques: Staining, Control, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Respiratory Research

Article Title: YAP/TAZ activation mediates PQ-induced lung fibrosis by sustaining senescent pulmonary epithelial cells

doi: 10.1186/s12931-024-02832-z

Figure Lengend Snippet: Paraquat induces pulmonary epithelial cell senescence in vitro: A WST-1 analysis shows dose-dependent decreases of A549 and 16HBE cells after PQ exposure for 72 h, B ROS content in A549 cells after treated with 80 µM PQ for 72 h was analyzed with DHE staining (red) (original magnification 200×, scale bar = 200 μm), C A549 cells were stained with SA-β-gal after treated with 80 µM PQ for 24 h, 48 h and 72 h (original magnification 200×, scale bar = 200 μm), and quantification of positive cells, D A549 cells treated with 80 µM PQ for 24 h, 48 h and 72 h were harvested and assessed for p16 and p21, with β-actin as loading control by Western blotting, E relative mRNA levels of CDKN2A (p16), CDKN1A (p21), and SASP markers IL6 , IL1a and IL8 compared to ACTB in A549 cells treated with 80 µM PQ for 24 h, 48 h and 72 h were analyzed by qRT-PCR. F immunoflourescence staining of A549 cells treated with 80 µM PQ for 72 h for Ki-67 (red), p16 (red) and DAPI (blue) (original magnification 200×, scale bar = 200 μm), and quantification of positive cells. All statistical data were from three independent experiments. Values are shown as mean ± SEM. Data were analyzed by Student’s t test between 2 groups, and one-way ANOVA with the Dunnett’s correction was used for comparisons among multiple groups. * P < 0.05, ** P < 0.01, *** P < 0.005

Article Snippet: The primary antibodies included anti-p16 Ink4a (for immunofluorescence: ab211542, 1:100, Abcam, UK; for Western blotting: for human: 10883-1, 1:1000, Proteintech, China; for mouse: sc-1661, 1:1000, Santa Cruz Technology, TX, United States), anti-p21 Cip1/Waf1 (sc-6246, 1:1000, Santa Cruz Technology), anti-β-actin (4970, 1:8000, CST, MA, United States), anti-Ki-67 (AG8471, 1:100, Beyotime, China), anti-α-SMA (19245, 1:200, CST), anti-pYAP (Ser127) (13008S, 1:1000, CST), anti-YAP (12395S, 1:1000 for Western blotting and 1:400 for immunohistochemisty, CST; 13584-1-AP, 1:200 for immunofluorescence, Proteintech), anti-pTAZ (Ser89) (59971S, 1:1000, CST), anti-TAZ (83669S, 1:1000 for Western blotting and 1:200 for immunofluorescence, CST), anti-Survivin (10508-1-ap, 1:1000, Proteintech), anti-Bcl-2 (BS1511, 1:1000, BioWorld, China), anti-Bax (50599-2-Ig, 1:1000, Proteintech), anti-Fn1 (sc-8422, 1:1000 for Western blotting, Santa Cruz Technology; ab45688, 1: 500 for immunofluorescence, Abcam), anti-AQP5 (20334-1-AP, 1:100, Proteintech), Sfptc (10774-1-AP, 1:200, Proteintech), anti-Cyk19 (GB12197, 1:500) and anti-Ctgf (23936-1-AP, 1:1000, Proteintech).

Techniques: In Vitro, Staining, Control, Western Blot, Quantitative RT-PCR

Journal: Respiratory Research

Article Title: YAP/TAZ activation mediates PQ-induced lung fibrosis by sustaining senescent pulmonary epithelial cells

doi: 10.1186/s12931-024-02832-z

Figure Lengend Snippet: Senescent lung epithelial cells promote lung fibroblast transformation via secreting SASP factors: A Schematic illustration of the establishment of senescent lung epithelial cell model. A549 and 16HBE cells were exposed to 200 µM PQ for 24 h, and then changed with fresh medium for continuous 6-day culture. Cells and supernatant (PQ-conditional medium, PQ-CM) were harvested at the 7th day; medium incubated with untreated cells for 48 h were harvested as control medium, B A549 cells were stained with SA-β-gal after exposed to 200 µM PQ for 24 h and continuously cultured for 6 days (original magnification 200×, scale bar = 200 μm), C A549 cells harvested at the 7th day were assessed for p16 and p21, with β-actin as loading control by Western blotting, D SASP markers IL-6, IL-1α and IL-8 in the supernatant were tested by ELISA assay, E immunoflourescence staining of HLF cells after 72 h incubation with A549 and 16HBE CM for Ki-67 (red), α-SMA (green) and DAPI (blue) (original magnification 200×, scale bar = 200 μm), and quantification of positive cells, F HLF cells incubated with CM for 72 h were assessed for α-SMA, with Tubulin as loading control by Western blotting. All statistical data were from three independent experiments. Values are shown as mean ± SEM. Data were analyzed by Student’s t test. * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001, n.s. no statistical significance, # no statistical significance compared to blank medium group

Article Snippet: The primary antibodies included anti-p16 Ink4a (for immunofluorescence: ab211542, 1:100, Abcam, UK; for Western blotting: for human: 10883-1, 1:1000, Proteintech, China; for mouse: sc-1661, 1:1000, Santa Cruz Technology, TX, United States), anti-p21 Cip1/Waf1 (sc-6246, 1:1000, Santa Cruz Technology), anti-β-actin (4970, 1:8000, CST, MA, United States), anti-Ki-67 (AG8471, 1:100, Beyotime, China), anti-α-SMA (19245, 1:200, CST), anti-pYAP (Ser127) (13008S, 1:1000, CST), anti-YAP (12395S, 1:1000 for Western blotting and 1:400 for immunohistochemisty, CST; 13584-1-AP, 1:200 for immunofluorescence, Proteintech), anti-pTAZ (Ser89) (59971S, 1:1000, CST), anti-TAZ (83669S, 1:1000 for Western blotting and 1:200 for immunofluorescence, CST), anti-Survivin (10508-1-ap, 1:1000, Proteintech), anti-Bcl-2 (BS1511, 1:1000, BioWorld, China), anti-Bax (50599-2-Ig, 1:1000, Proteintech), anti-Fn1 (sc-8422, 1:1000 for Western blotting, Santa Cruz Technology; ab45688, 1: 500 for immunofluorescence, Abcam), anti-AQP5 (20334-1-AP, 1:100, Proteintech), Sfptc (10774-1-AP, 1:200, Proteintech), anti-Cyk19 (GB12197, 1:500) and anti-Ctgf (23936-1-AP, 1:1000, Proteintech).

Techniques: Transformation Assay, Incubation, Control, Staining, Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Respiratory Research

Article Title: YAP/TAZ activation mediates PQ-induced lung fibrosis by sustaining senescent pulmonary epithelial cells

doi: 10.1186/s12931-024-02832-z

Figure Lengend Snippet: Co-locolization of Yap/Taz and p16 is observed in type II alveolar epithelial cells and bronchial epithelial cells: A representative images of immunoflourescence staining for Yap or Taz (red), p16 (yellow), type II alveolar epithelial cell marker Sftpc (green) and DAPI (blue) in lung sections of PQ group and control group (original magnification 400×, scale bar = 100 μm), B representative images of immunoflourescence staining for Yap or Taz (red), p16 (yellow), bronchial epithelial cell marker Cytokeratin 19 (Cyk19, green) and DAPI (blue) in lung sections of control and PQ groups (original magnification 400×, scale bar = 100 μm)

Article Snippet: The primary antibodies included anti-p16 Ink4a (for immunofluorescence: ab211542, 1:100, Abcam, UK; for Western blotting: for human: 10883-1, 1:1000, Proteintech, China; for mouse: sc-1661, 1:1000, Santa Cruz Technology, TX, United States), anti-p21 Cip1/Waf1 (sc-6246, 1:1000, Santa Cruz Technology), anti-β-actin (4970, 1:8000, CST, MA, United States), anti-Ki-67 (AG8471, 1:100, Beyotime, China), anti-α-SMA (19245, 1:200, CST), anti-pYAP (Ser127) (13008S, 1:1000, CST), anti-YAP (12395S, 1:1000 for Western blotting and 1:400 for immunohistochemisty, CST; 13584-1-AP, 1:200 for immunofluorescence, Proteintech), anti-pTAZ (Ser89) (59971S, 1:1000, CST), anti-TAZ (83669S, 1:1000 for Western blotting and 1:200 for immunofluorescence, CST), anti-Survivin (10508-1-ap, 1:1000, Proteintech), anti-Bcl-2 (BS1511, 1:1000, BioWorld, China), anti-Bax (50599-2-Ig, 1:1000, Proteintech), anti-Fn1 (sc-8422, 1:1000 for Western blotting, Santa Cruz Technology; ab45688, 1: 500 for immunofluorescence, Abcam), anti-AQP5 (20334-1-AP, 1:100, Proteintech), Sfptc (10774-1-AP, 1:200, Proteintech), anti-Cyk19 (GB12197, 1:500) and anti-Ctgf (23936-1-AP, 1:1000, Proteintech).

Techniques: Staining, Marker, Control

Journal: Respiratory Research

Article Title: YAP/TAZ activation mediates PQ-induced lung fibrosis by sustaining senescent pulmonary epithelial cells

doi: 10.1186/s12931-024-02832-z

Figure Lengend Snippet: Yap and Taz knockdown abrogates paraquat-Induced pulmonary fibrosis in vivo: ( A ) Schematic illustration of the animal experiment. C57BL/6 mice were intratracheal infected with 5 × 10 10 PFU of AAV-Ctrl, AAV-shYap, AAV-shTaz or both AAV-shYap and AAV-shTaz, and intratracheal instillated with 0.02 mg PQ at day 21 postinfection. PBS was intratracheal instillated at day 0 and day 21 as blank control, ( B ) body weight of mice in each group, ( C ) Masson’s trichrome staining (original magnification 100×, scale bar = 400 μm) and ( D ) SA-β-gal staining (original magnification 100×, scale bar = 400 μm) of representative lung sections from each group: (a) blank control group, (b) AAV-Ctrl group, (c) AAV-shYap group, (d) AAV-shTaz group, (e) AAV-Yap + AAV-shTaz group, ( E ) total lung protein was assessed for p16, p21, Fn1, Ctgf, Bcl-2 and Bax, with β-actin as loading control by Western blotting ( N = 4 for PBS group, N = 6 for other groups). All graphs are shown as mean ± SEM. Parametric variables were calculated using two-tailed Student’s t test between 2 groups. * P < 0.05, ** P < 0.01, *** P < 0.005

Article Snippet: The primary antibodies included anti-p16 Ink4a (for immunofluorescence: ab211542, 1:100, Abcam, UK; for Western blotting: for human: 10883-1, 1:1000, Proteintech, China; for mouse: sc-1661, 1:1000, Santa Cruz Technology, TX, United States), anti-p21 Cip1/Waf1 (sc-6246, 1:1000, Santa Cruz Technology), anti-β-actin (4970, 1:8000, CST, MA, United States), anti-Ki-67 (AG8471, 1:100, Beyotime, China), anti-α-SMA (19245, 1:200, CST), anti-pYAP (Ser127) (13008S, 1:1000, CST), anti-YAP (12395S, 1:1000 for Western blotting and 1:400 for immunohistochemisty, CST; 13584-1-AP, 1:200 for immunofluorescence, Proteintech), anti-pTAZ (Ser89) (59971S, 1:1000, CST), anti-TAZ (83669S, 1:1000 for Western blotting and 1:200 for immunofluorescence, CST), anti-Survivin (10508-1-ap, 1:1000, Proteintech), anti-Bcl-2 (BS1511, 1:1000, BioWorld, China), anti-Bax (50599-2-Ig, 1:1000, Proteintech), anti-Fn1 (sc-8422, 1:1000 for Western blotting, Santa Cruz Technology; ab45688, 1: 500 for immunofluorescence, Abcam), anti-AQP5 (20334-1-AP, 1:100, Proteintech), Sfptc (10774-1-AP, 1:200, Proteintech), anti-Cyk19 (GB12197, 1:500) and anti-Ctgf (23936-1-AP, 1:1000, Proteintech).

Techniques: Knockdown, In Vivo, Infection, Control, Staining, Western Blot, Two Tailed Test

Journal: Frontiers in Medicine

Article Title: P16 expression and recurrent cervical intraepithelial neoplasia after cryotherapy among women living with HIV

doi: 10.3389/fmed.2023.1277480

Figure Lengend Snippet: Baseline demographic and clinical characteristics by P16 status ( n = 160).

Article Snippet: In brief, 4 μm tissue sections were subjected to the following processes: deparaffinization, antigen retrieval with citrate at pH 9, endogenous peroxidase blocking, application of prediluted mouse anti-p16/INK4a (Medaysis, California, United States), and finally, application of horseradish peroxidase (HRP), specific 3,3′-diaminobenzidine (DAB), and counterstaining.

Techniques: Clinical Proteomics

Journal: Frontiers in Medicine

Article Title: P16 expression and recurrent cervical intraepithelial neoplasia after cryotherapy among women living with HIV

doi: 10.3389/fmed.2023.1277480

Figure Lengend Snippet: Prevalence of high-risk HPV (hrHPV) genotypes by p16 expression at baseline treatment.

Article Snippet: In brief, 4 μm tissue sections were subjected to the following processes: deparaffinization, antigen retrieval with citrate at pH 9, endogenous peroxidase blocking, application of prediluted mouse anti-p16/INK4a (Medaysis, California, United States), and finally, application of horseradish peroxidase (HRP), specific 3,3′-diaminobenzidine (DAB), and counterstaining.

Techniques: Expressing

Journal: Frontiers in Medicine

Article Title: P16 expression and recurrent cervical intraepithelial neoplasia after cryotherapy among women living with HIV

doi: 10.3389/fmed.2023.1277480

Figure Lengend Snippet: Factors associated with positive p16 status at baseline cryotherapy treatment (n = 160).

Article Snippet: In brief, 4 μm tissue sections were subjected to the following processes: deparaffinization, antigen retrieval with citrate at pH 9, endogenous peroxidase blocking, application of prediluted mouse anti-p16/INK4a (Medaysis, California, United States), and finally, application of horseradish peroxidase (HRP), specific 3,3′-diaminobenzidine (DAB), and counterstaining.

Techniques: Cell Counting, Clinical Proteomics

Journal: Frontiers in Medicine

Article Title: P16 expression and recurrent cervical intraepithelial neoplasia after cryotherapy among women living with HIV

doi: 10.3389/fmed.2023.1277480

Figure Lengend Snippet: Kaplan–Meier cumulative incidence by p16 expression at baseline. CI, confidence interval; HR, hazard ratio.

Article Snippet: In brief, 4 μm tissue sections were subjected to the following processes: deparaffinization, antigen retrieval with citrate at pH 9, endogenous peroxidase blocking, application of prediluted mouse anti-p16/INK4a (Medaysis, California, United States), and finally, application of horseradish peroxidase (HRP), specific 3,3′-diaminobenzidine (DAB), and counterstaining.

Techniques: Expressing

Journal: Frontiers in Medicine

Article Title: P16 expression and recurrent cervical intraepithelial neoplasia after cryotherapy among women living with HIV

doi: 10.3389/fmed.2023.1277480

Figure Lengend Snippet: Risk of cervical disease recurrence (CIN2+) by combined p16 expression and any one hrHPV genotype and simultaneous multiple hrHPV genotypes at baseline cryotherapy treatment.

Article Snippet: In brief, 4 μm tissue sections were subjected to the following processes: deparaffinization, antigen retrieval with citrate at pH 9, endogenous peroxidase blocking, application of prediluted mouse anti-p16/INK4a (Medaysis, California, United States), and finally, application of horseradish peroxidase (HRP), specific 3,3′-diaminobenzidine (DAB), and counterstaining.

Techniques: Expressing