Journal: Journal of Veterinary Research

Article Title: Immunoreactivity of p21, MMP-1 and CB2 receptor proteins in cutaneous canine mast cell tumours: an association with the three-tier grading system

doi: 10.2478/jvetres-2023-0066

Figure Lengend Snippet: Immunohistochemistry with primary anti-p21 antibody with positive reaction visible as brown staining. A – weak nuclear reaction in a grade 1 mast cell tumour (MCT); B – moderate nuclear reaction in a grade 2 MCT; C – strong nuclear reaction in a grade 3 MCT

Article Snippet: The primary antibodies used in this study, all canine-specific, were as follows: anti-CB2R (Cat. No. TA317640; OriGene Technologies, Rockville, MD, USA), anti-p21 (Cat. No. M7202; clone SX118, Agilent Dako, Santa Clara, CA, USA) and anti-MMP-1 (Cat. No. ABIN2777120; antibodies-online, Aachen, Germany), diluted 1 : 200, 1 : 50 and 1 : 500, respectively.

Techniques: Immunohistochemistry, Staining

Journal: Journal of Veterinary Research

Article Title: Immunoreactivity of p21, MMP-1 and CB2 receptor proteins in cutaneous canine mast cell tumours: an association with the three-tier grading system

doi: 10.2478/jvetres-2023-0066

Figure Lengend Snippet: Frequency of immunoreactivity for p21 protein in grade 1, 2 and 3 mast cell tumours (MCTs). * – P-value < 0.05; **_ P-value < 0.01; *** P-value < 0.001 compared to grade 1 MCTs; (−) – no reaction; (+) – weak reaction; (++) – moderate reaction; (+++) – strong reaction

Article Snippet: The primary antibodies used in this study, all canine-specific, were as follows: anti-CB2R (Cat. No. TA317640; OriGene Technologies, Rockville, MD, USA), anti-p21 (Cat. No. M7202; clone SX118, Agilent Dako, Santa Clara, CA, USA) and anti-MMP-1 (Cat. No. ABIN2777120; antibodies-online, Aachen, Germany), diluted 1 : 200, 1 : 50 and 1 : 500, respectively.

Techniques:

Journal: bioRxiv

Article Title: Replicative stress in gastroesophageal adenocarcinoma is associated with chromosomal instability and sensitivity to DNA damage response inhibitors

doi: 10.1101/2023.03.27.534412

Figure Lengend Snippet: A. Tabulated summary of IHC stainings indicating the number of premalignant (green) and malignant (blue) gastric lesions scored for each staining criterion. DDR/replication stress markers-53BP1, H2AX, pH2AX in red; Cell cycle regulators-p53, p16, p21 in purple. B. Summary of the stainings (DDR markers and the cell cycle regulation markers) in paired premalignant and malignant gastric lesions from gastric cancer patient8. PM= Premalignant gastric lesion, M= Malignant gastric lesion. C. Representative IHC images of paired premalignant and malignant gastric lesions from a gastric cancer patient (patient8) stained for DDR markers and cell cycle regulation markers, including H&E staining. Insets represent the zoomed area for each image.

Article Snippet: The primary antibodies used for immunohistochemistry were: Human tissue-rabbit anti-ki67 (1:1000, Biocare Medical, #CRM325-5b6), rabbit anti-H2AX (1:1000, CST, #7631), rabbit anti-pH2AX ser139 (1:1000, CST, #9718), mouse anti-53BP1 (1:1000, Millipore, #3802), mouse anti-p53 (1:1000, CST, #48818), mouse anti-P16INK4 (1:1000, Abcam, #54210), rabbit anti-p21 (1:1000, CST, #2947), Murine tissue-rabbit anti-ki67 (1:1000, Biocare Medical, #CRM325-5b6), rabbit anti-pH2AX ser139, (1:1000, CST, #9718), rabbit anti-p53 (1:1000, Abcam, #1431), rabbit anti-p21 (1:1000, Abcam, #188224).

Techniques: Staining

Journal: bioRxiv

Article Title: Replicative stress in gastroesophageal adenocarcinoma is associated with chromosomal instability and sensitivity to DNA damage response inhibitors

doi: 10.1101/2023.03.27.534412

Figure Lengend Snippet: A. Summary of dysplasia grade and staining pattern of proliferation marker ki67, DDR marker pH2AX, and cell cycle regulation markers (p53 and p21) in paired normal (grey), premalignant (green), and malignant (blue) gastric lesions of four MNU treated mice. B. Representative IHC images of the staining pattern of proliferation marker ki67, DDR marker pH2AX, and cell cycle regulation markers (p53 and p21) in paired normal, premalignant, and malignant gastric lesions of MNU treated mouse4 (dysplasia score 5).

Article Snippet: The primary antibodies used for immunohistochemistry were: Human tissue-rabbit anti-ki67 (1:1000, Biocare Medical, #CRM325-5b6), rabbit anti-H2AX (1:1000, CST, #7631), rabbit anti-pH2AX ser139 (1:1000, CST, #9718), mouse anti-53BP1 (1:1000, Millipore, #3802), mouse anti-p53 (1:1000, CST, #48818), mouse anti-P16INK4 (1:1000, Abcam, #54210), rabbit anti-p21 (1:1000, CST, #2947), Murine tissue-rabbit anti-ki67 (1:1000, Biocare Medical, #CRM325-5b6), rabbit anti-pH2AX ser139, (1:1000, CST, #9718), rabbit anti-p53 (1:1000, Abcam, #1431), rabbit anti-p21 (1:1000, Abcam, #188224).

Techniques: Staining, Marker

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: High p21 expression correlates with poor survival in breast cancer patients . A-C , The relationship of higher p21 expression and breast cancer outcome was assessed by Kaplan-Meier survival analysis. The survival rates were analyzed using three different and separate splits of the patients, based on p21 expression levels (A, median; B, upper quartile; C, lower quartile). For each split, the patients were then divided into high and low groups with respect to p21 expression. Overall survival (left panels) and distant metastasis-free survival (right panels) correlate with p21 gene expression. Number of breast cancer patients at risk with higher expression (red) and lower expression (black) of p21 at the indicated time points.

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques: Expressing, Gene Expression

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: Silencing p21 prevents breast tumor local invasion in vivo and cancer cell migration and invasion . A , Total lysates from parental and shRNA p21 SCP2 cells were analyzed by immunoblotting for the protein levels of p21 and β-tubulin. B , Parental and shRNA p21 SCP2 cells were injected into the mammary glands of four- to six-week-old female Balb/c nude mice. The size of mammary tumor was measured from two sets of mice (eight per group; error bars indicate SEM). C , Representative photographs show hematoxylin and eosin staining of the mammary gland (tumor and fat pad) of 12- to 15-month-old mice. D and E , Transwell cell migration (left panel) and GFR-Matrigel invasion assay (right panel) of parental and shRNA p21 SCP2 cells were performed. Graphs show total migrated and invaded cell number counted by Image J (error bars indicate SEM; n = 3 independent experiments). Student's unpaired t -test was used to compare parental vs. shRNA p21 SCP2 groups. Differences were considered significant at * P < 0.05.

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques: In Vivo, Migration, shRNA, Western Blot, Injection, Staining, Invasion Assay

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: TGFβ induces p21 expression in migratory and invasive human breast cancer cells . A , Real-time PCR was performed to measure the mRNA level of p21 gene (error bars indicate SD; n = 3 independent experiments) for the indicated cell lines. B , Cells were treated with or without 5 ng/ml TGFβ for the indicated times. Total cell lysates were analyzed for p21 and β-tubulin protein levels by Western blotting. C , Total cell lysates were analyzed for c-myc, p15 and β-tubulin protein levels by Western blotting. D , SCP25 cells were pretreated with 10 µM TGFβ type I receptor (TβRI) inhibitor (SB431542) or vehicle (DMSO) for 30 minutes and then stimulated with TGFβ. Total cell lysates were analyzed for p21 and β-tubulin protein levels by Western blotting. E , MDA cells were transfected with 40 nM Scrambled (Scr), Smad2 or Smad3 siRNAs in response to TGFβ. Total cell lysates were analyzed for Smad2/3, p21 and β-tubulin protein levels by Western blotting.

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transfection

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: p21 expression is required for TGFβ-mediated cell migration . A , Representative images of phase contrast and wound mask of indicated cell lines stimulated with TGFβ in scratch/wound healing assay. The initial wound mask (black) and wound closure (grey) were measured using the Essen Instruments Scratch Wound Module. B , The time course of cell migration for the indicated cell lines was quantified using the relative wound density metrics at two-hour time intervals (error bars indicate SEM; n = 3 independent experiments). C , SCP2 cells were transfected with Scr or p21 siRNAs and then stimulated with or without TGFβ for 24 hrs. Total cell lysates were analyzed for p21 and β-tubulin by Western blotting. D , Representative images of phase contrast (top panels) and wound mask (bottom panels) of transfected SCP2 cells with the indicated siRNAs in scratch/wound healing assay. E , The time course of transfected SCP2 cell migration was quantified using the relative wound density metrics at two-hour time intervals (error bars indicate SEM; n = 3 independent experiments).

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques: Expressing, Migration, Wound Healing Assay, Transfection, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: p21 expression is required for TGFβ-mediated cell invasion . A , SCP2 cells were transfected with the indicated siRNAs and cultured in the presence or absence of TGFβ. GFR-Matrigel coated Transwell invasion assay was performed and images of the invading cells were photographed. B , Total cell number was counted by Image J and fold induction was quantified (error bars indicate SD; n = 3 independent experiments). C , SCP2 cells were transfected with a Scr or p21 siRNA as well as a flag-tagged p21 cDNA in the presence or the absence of TGFβ. p21 protein levels were then analyzed by Western blotting. D , Cell invasion was assessed using the Transwell Invasion assay. The number of invaded cells was counted by Image J (error bars indicate SD; n = 3 independent experiments). E , Cell invasion of transfected SUM159 was assessed using the Transwell Invasion assay. F , Total cell number was counted by Image J and fold induction was quantified (error bars indicate SD; n = 3 independent experiments).

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques: Expressing, Transfection, Cell Culture, Transwell Invasion Assay, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: p21 interacts with Smad3 and modulates TGFβ-induced transcriptional activity . A , HEK293 and SCP2 cells were co-transfected with myc-Smad2, myc-Smad3 and flag-p21. Transfected cells were stimulated TGFβ for 8 hrs. Cell lysates were immunoprecipitated with an anti-flag antibody and analyzed by immunoblotting using Smad2/3 and p21 antibodies. B , Transfected MDA cells were immunoblotting by phospho-Smad3 (p-Smad3), Smad2/3 and β-tubulin antibodies in response to TGFβ for 30 minutes. C , SCP2 cells were co-transfected with either mock, Scr siRNA, p21 siRNA or flag-tagged p21 construct (p21 cDNA) and SBE promoter construct (CAGA12-luc). Transfected cells were stimulated with or without TGFβ for 16 hrs. Luciferase activity of CAGA12-luc was measured and normalized to β-galactosidase (error bars indicate SEM; n = 3 independent experiments). D and E , SUM159 and SCP2 cells were treated with or without TGFβ for the indicated times. The mRNA levels of indicated genes were then analyzed by real-time PCR (error bars indicate SEM; n = 3 independent experiments).

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques: Activity Assay, Transfection, Immunoprecipitation, Western Blot, Construct, Luciferase, Real-time Polymerase Chain Reaction

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: p21 regulates TGFβ-induced downstream genes involved in cell invasion . A , SUM159 cells were transfected with Scr or p21 siRNA as well as p21 cDNA. Cells were then treated with or without TGFβ and the mRNA levels of indicated genes were analyzed by real-time PCR (error bars indicate SEM; n = 3 independent experiments). B , SUM159 cells transfected with Scr siRNA and p21 siRNA were treated with TGFβ for the indicated times. The mRNA level of TGFBI gene was measured by real-time PCR (error bars indicate SEM; n = 3 independent experiments). C , SCP2 cells were transfected with the indicated siRNAs. Cell invasion was assessed using the Transwell Invasion assay. D , Total cell number was counted by Image J and number of invaded cells was quantified (error bars indicate SEM; n = 3 independent experiments).

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques: Transfection, Real-time Polymerase Chain Reaction, Transwell Invasion Assay

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: p21/p/CAF regulates TGFβ transcriptional activity and Smad3 occupancy on SBE . A , SCP2 and SUM159 cells were treated with TGFβ. Cell lysates were analyzed by co-immunoprecipitation using specific antibodies, as indicated. B , HEK293 cells were co-transfected with myc-Smad2, myc-Smad3 and flag-p21 with or without p/CAF siRNA. Transfected cells were stimulated with TGFβ for eight hours. Cell lysates were immunoprecipitated with an anti-flag antibody and analyzed by immunoblotting using Smad2/3 and flag antibodies. C and D , SUM159 cells were transfected with Scr or p/CAF siRNAs as well as a flag-tagged p21 cDNA, treated with or without TGFβ. The mRNA levels of indicated genes were analyzed by real-time PCR (error bars indicate SEM; n = 3 independent experiments). E , SCP2 cells transfected with Scr and p/CAF siRNA were stimulated with or without TGFβ. Cell invasion was quantified by relative TGFβ fold induction (error bars indicate SEM; n = 3 independent experiments). F , Transfected SCP2 cells were subjected to immunoblotting p/CAF and β-tubulin. G , HEK293 cells were co-transfected with myc-Smad3, myc-Smad2 and p/CAF. Immunoprecipitated Smad2/3 using an anti-myc antibody was subjected to Western blotting. H , DNA precipitation (DNA IP) was performed using biotinylated control and 4× CAGA SBE oligonucleotides, following by streptavidin precipitation. Western blotting of Smad3 and p/CAF is shown. I , SCP2 cells were transfected with p21 or p/CAF siRNAs. Samples were subjected to DNA IP and immunoblotting of Smad3. J , Transfected SCP2 cells were stimulated with or without TGFβ for 16 hrs. Luciferase activity of CAGA12-luc was measured and normalized to β-galactosidase (error bars indicated SEM; n =3 independent experiments).

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques: Activity Assay, Immunoprecipitation, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Control, Luciferase

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: High expression of p/CAF/p21/pSmad3 is associated with lymph node positivity . A , Representative immunohistochemistry images of pSmad3, p21 and p/CAF in breast cancer tissue microarray samples. B , Overall pSmad3, p21 and p/CAF immunohistochemistry staining intensity between lymph node negative (LN-) and positive (LN+) tissues. C , Percentage of case distribution according to immunoreactivity of pSmad3, p21 and p/CAF in tumor cells, the score for negative (0), low (1 to 2) and high (3 to 4).

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques: Expressing, Immunohistochemistry, Microarray, Staining

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: Model of pro-invasive function for the cell cycle regulator p21 in human breast cancer . The role of p21 as both a direct transcriptional target of TGFβ and a co-stimulatory factor of p/CAF/Smad3 in regulation of pro-invasive genes in triple negative breast cancer cells.

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques:

Journal: British Journal of Cancer

Article Title: Defining the molecular response to trastuzumab, pertuzumab and combination therapy in ovarian cancer

doi: 10.1038/bjc.2012.176

Figure Lengend Snippet: Effect of single or combination therapy on proliferation, cell cycle, apoptosis and protein markers over time. Total and phosphoprotein levels were determined by immunohistochemistry of TMAs for Ki67, phopho-histone H3, cleaved caspase 3, p21 and p27. See for comparable AQUA quantitative immunofluorescence results.

Article Snippet: Primary antibody incubations were Ki67 (DAKO; M7240) at 1 : 50; phospho-Histone H3 (Cell Signaling Technology; 9701) at 1 : 100; p27 (Cell Signaling Technology; 2552) at 1 : 100 for 1 h at room temperature; p21 (Cell Signaling Technology; 2946) at 1 : 100; anti-phosphorylated ERK (pERK) 1/2 (Cell Signaling Technology; 9101) at 1 : 25; anti-phosphorylated Akt (pAkt; Ser 473 ; Cell Signaling Technology; 4060) at 1 : 100; cleaved caspase 3, (#9661 Cell Signaling Technology; 1 : 200) were used overnight at 4°C.

Techniques: Immunohistochemistry, Immunofluorescence

Journal: British Journal of Cancer

Article Title: Defining the molecular response to trastuzumab, pertuzumab and combination therapy in ovarian cancer

doi: 10.1038/bjc.2012.176

Figure Lengend Snippet: Combination therapy is more effective than either agent alone in a primary tumour. The primary ovarian xenograft HOX424 shows significantly greater growth inhibition to combined trastuzumab and pertuzumab treatment than to either agent alone. Total and phosphoprotein levels were determined by AQUA quantitative immunofluorescence of TMAs for Ki67, phospho-histone H3, p21, p27 and phospho-AKT after 24 days. * P <0.05 Student’s t -test of treatment relative to control.

Article Snippet: Primary antibody incubations were Ki67 (DAKO; M7240) at 1 : 50; phospho-Histone H3 (Cell Signaling Technology; 9701) at 1 : 100; p27 (Cell Signaling Technology; 2552) at 1 : 100 for 1 h at room temperature; p21 (Cell Signaling Technology; 2946) at 1 : 100; anti-phosphorylated ERK (pERK) 1/2 (Cell Signaling Technology; 9101) at 1 : 25; anti-phosphorylated Akt (pAkt; Ser 473 ; Cell Signaling Technology; 4060) at 1 : 100; cleaved caspase 3, (#9661 Cell Signaling Technology; 1 : 200) were used overnight at 4°C.

Techniques: Inhibition, Immunofluorescence, Control

Journal: Aging Cell

Article Title: Virus‐Induced Cellular Senescence Causes Pulmonary Sequelae Post‐Influenza Infection

doi: 10.1111/acel.70140

Figure Lengend Snippet: Time course of pulmonary pathological manifestations and p16 and p21 expression following IAV infection. (A) Left panels. Representative micrographs of H&E‐stained lung sections showing bronchi (upper panel) and bronchial wall (lower panel) of mock‐infected mice and IAV‐infected mice. The zoomed areas are indicated by rectangles. Right panel. Scatter‐plot graph showing bronchial wall thickness. (B) Left panels, Representative micrographs showing lung parenchyma. Right panel. Scatter plot showing mean liner intercept (MLI) measurements. (C) Left panels, Representative micrographs showing lung parenchyma stained with Sirius Red used to visualize collagen deposition (hallmark of lung fibrosis). Zoomed area are indicated by squares. Right panel, Scatter‐plot graph showing parenchymal fibrosis quantification according to Aschcroft score. (D) Left panel. Representative micrographs showing immunofluorescence of p16 (white) in lung cells. Blue—DAPI nuclear staining, green—elastin autofluorescence. The zoomed areas (lower panels) are indicated. (E) Left panel. Representative micrographs showing p21 expression by immunohistochemistry. (D, E) Right panel, Scatter‐plot graphs representing the percentage of p16‐positive (D) and p21‐positive (E) cells in the different groups of mice. (A–E) Scales are indicated (bar = 50 or 100 μm). Graphs represent individual values per mice and the mean ± SEM ( n = 3–11). Significant differences were determined using a one‐way ANOVA followed by Bonferroni post hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: The following primary antibodies were used: rabbit anti‐p21 polyclonal antibody (28248‐1‐AP, Proteintech, 1:200), rabbit anti‐53BP1 polyclonal antibody (NB100‐304, Novusbio, 1:200) and rabbit monoclonal anti‐phospho‐Histone H2A.X antibody (20E3, #9718, 1:500, Cell signaling Technology).

Techniques: Expressing, Infection, Staining, Immunofluorescence, Immunohistochemistry

Journal: Aging Cell

Article Title: Virus‐Induced Cellular Senescence Causes Pulmonary Sequelae Post‐Influenza Infection

doi: 10.1111/acel.70140

Figure Lengend Snippet: p16 and viral antigen and ©H2AX and p21 co‐expression in lungs during influenza. (A) Representative micrograph showing immunofluorescence of viral hemagglutinin (IAV, red) and p16 (white) in lung sections collected from IAV‐infected mice at 4 and 7 dpi. Bleu—DAPI nuclear staining, green, elastin autofluorescence). Scales are indicated. (B) Expression of gamma‐H2A.X and p21 protein in IAV‐infected whole lung homogenates as assessed by western blotting. The relative protein levels are normalized to β Actin. Graphs represent individual values per mice and the mean ± SD ( n = 4–7). Significant differences were determined using a one‐way ANOVA followed by Bonferroni post hoc test (** p < 0.01, **** p < 0.0001). (C) Expression of gammaH2AX in the lungs from mock‐infected and IAV‐infected (7 dpi) mice by immunohistochemistry. Blue–Nuclear hematoxylin staining (bar = 100 μm).

Article Snippet: The following primary antibodies were used: rabbit anti‐p21 polyclonal antibody (28248‐1‐AP, Proteintech, 1:200), rabbit anti‐53BP1 polyclonal antibody (NB100‐304, Novusbio, 1:200) and rabbit monoclonal anti‐phospho‐Histone H2A.X antibody (20E3, #9718, 1:500, Cell signaling Technology).

Techniques: Expressing, Immunofluorescence, Infection, Staining, Western Blot, Immunohistochemistry

Journal: Aging Cell

Article Title: Virus‐Induced Cellular Senescence Causes Pulmonary Sequelae Post‐Influenza Infection

doi: 10.1111/acel.70140

Figure Lengend Snippet: Consequences of genetic elimination of senescent cells on lung sequelae post‐influenza. p16‐ATTAC mice were intraperitoneally inoculated with AP20187 (0.5 mg/kg, twice weekly) starting the day before infection. Lungs from vehicle‐treated and AP20187‐treated mice were collected on 28 dpi. (A) Representative micrographs showing immunofluorescence of p16 (white) and CD68 (red) in lung cells. Blue–DAPI nuclear staining, green–Elastin autofluorescence. Bar—100 or 25 μm. (B) Effect of AP20187 treatment on macrophages infiltration in lung. Left panel, Representative whole lung scan showing CD68‐immunofluorescence (red). Blue–Dapi nucler staining. Right panel: Scatter plot showing the abundance of CD68 positive cells (% from total). Graphs represent individual values per mice and the mean ± SEM ( n = 6–8). (C) Expression of p16, p21 protein and gammaH2AX in IAV‐infected whole lung homogenates as assessed by western blotting. The relative protein levels are normalized to β‐Actin ( n = 5–8). (D) Effect of AP20187 treatment on lung emphysema. Left panel: Representative hematoxylin/eosin sections of lung from different group of mice. Bar = 200 μ. Right panel. Scatter plot showing mean liner intercept (MLI). (E) Effect of AP treatment on bronchial regeneration. Left panel: Representative micrographs showing bronchial wall. Hematoxylin/eosin staining. Bar 25 μ. Right panel. Scatter‐plot graph showing bronchial wall thickness. (F) Effect of AP20187 treatment on pulmonary fibrosis. Left panel: Representative Sirus‐Red stained sections of lung from different group of mice. Bar = 200 μ. Right panel. Scatter plot showing Ashcroft score. (G) Relative expression of the pulmonary fibrosis markers Coll3 and pSmad3 in whole lung homogenates as assessed by western blotting. The protein level of Coll3 was normalized to beta Actin and thatt of pSmad3 was normalized to Smad3. (B–G) Graphs represent individual values per mice and the mean ± SEM ( n = 7–8). One of two representative experiments is shown. Significant differences was determined using the Mann–Whitney U test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.001).

Article Snippet: The following primary antibodies were used: rabbit anti‐p21 polyclonal antibody (28248‐1‐AP, Proteintech, 1:200), rabbit anti‐53BP1 polyclonal antibody (NB100‐304, Novusbio, 1:200) and rabbit monoclonal anti‐phospho‐Histone H2A.X antibody (20E3, #9718, 1:500, Cell signaling Technology).

Techniques: Infection, Immunofluorescence, Staining, Expressing, Western Blot, MANN-WHITNEY

Journal: Aging Cell

Article Title: Virus‐Induced Cellular Senescence Causes Pulmonary Sequelae Post‐Influenza Infection

doi: 10.1111/acel.70140

Figure Lengend Snippet: Consequences of ABT‐263 treatment on lung sequelae post‐influenza. (A) Expression of p16, p21 protein and ♥ ‐H2AX in IAV‐infected whole lung homogenates as assessed by western blotting (28 dpi). The relative protein levels are normalized to beta‐Actin ( n = 5–6). (B) Viral load as assessed by quantitative RT‐PCR (7 dpi). (C, D) Left panel. Lung sections (28 dpi) were stained with hematoxylin/eosin (C) or Sirius red (D) (bar = 200 μ). Right panel. Scatter plot showing mean liner intercept (MLI) and parenchymal fibrosis quantification. (E, F) Effect of senescent cells elimination on bronchial regeneration (bar = 100 μ) (28 dpi). (E) Representative micrographs showing immunofluorescence of p16 (white) in lung cells of vehicle‐treated and ABT‐263‐treated mice. Blue–DAPI nuclear staining. The zoomed areas are indicated by rectangles. Bar—100 μm. (F) Left panel, Representative micrographs of H&E‐stained lung sections. Right panel, Scatter‐plot graph showing bronchial wall thickness. (A–F) Graphs represent individual values per mice and the mean ± SD ( n = 4–8). Significant differences was determined using the Mann–Whitney U test (* p < 0.05, ** p < 0.01).

Article Snippet: The following primary antibodies were used: rabbit anti‐p21 polyclonal antibody (28248‐1‐AP, Proteintech, 1:200), rabbit anti‐53BP1 polyclonal antibody (NB100‐304, Novusbio, 1:200) and rabbit monoclonal anti‐phospho‐Histone H2A.X antibody (20E3, #9718, 1:500, Cell signaling Technology).

Techniques: Expressing, Infection, Western Blot, Quantitative RT-PCR, Staining, Immunofluorescence, MANN-WHITNEY

Journal: Aging Cell

Article Title: Treatment with the senolytics dasatinib/quercetin reduces SARS‐CoV ‐2‐related mortality in mice

doi: 10.1111/acel.13771

Figure Lengend Snippet: Immunohistochemistry (IHC) images of senescence markers in lung tissue from SARS‐CoV‐2 infected K18‐hACE2 mice. (a) Representative IHC of p21 CIP1 ‐positive cells in the lungs of the indicated groups. Top row: Staining in isolated areas of lymphocyte accumulation (mock group), and in dense inflammation foci (SARS‐CoV‐2‐infected mice). Bottom row: Staining in the lung tissue (excluding inflammation foci), indicated as parenchyma. Arrows indicate p21 CIP1 ‐positive cells in mock samples. Scale bars: 100 μm (top row) and 50 μm (bottom row). (b) Quantification of p21 CIP1 ‐positive cells in whole lung sections (left graphic), only in inflammation foci (middle) and parenchyma (right graphic), using ImageJ/FIJI software. In the mock‐infected group, two of four animals displayed a few minor inflammation foci, as shown in the IHC. In the SARS‐CoV‐2 group, four of seven animals showed dense inflammation foci. Bottom numbers: Numerical data of the number of cells analyzed. p Values determined by one‐way ANOVA, Fisher's LSD test. * p ≤ 0.05; ** p ≤ 0.005; *** p ≤ 0.0005. (c) Representative IHC of p19 ARF ‐positive cells in dense inflammation foci in lungs of the indicated mice groups. Arrows indicate p19 ARF ‐positive cell in mock sample. Scale bars: 100 μm (20 μm in the magnified square). (d) Quantification of p19 ARF ‐positive cells in dense inflammation foci of lung samples from individually analyzed mice, as described in b. p value determined by one‐way ANOVA, Fisher's LSD test. * p ≤ 0.05. Data from one out of two independent experiments.

Article Snippet: Antigen retrieval was performed with CC1 32 min, only for p21 and High pH buffer, Dako, Agilent (p19 and SARS‐CoV‐2 nucleocapsid); endogenous peroxidase was blocked (hydrogen peroxide at 3%) and slides were then incubated with the appropriate primary antibody as detailed: rat monoclonal anti‐p21 CIP1 (291H; 1/10; CNIO, Monoclonal Core Unit); rat monoclonal anti‐p19 ARF (5‐C3‐1; 1/50; Santa Cruz sc‐32748); rat monoclonal anti‐CDKN2A/p16 INK4a antibody (33B; CNIO, Monoclonal Core Unit) and rabbit monoclonal anti‐SARS‐CoV‐2 nucleocapsid (N) protein (019; 1/15,000; Sino Biological 40143‐R019).

Techniques: Immunohistochemistry, Infection, Staining, Isolation, Software

Journal: Aging Cell

Article Title: Treatment with the senolytics dasatinib/quercetin reduces SARS‐CoV ‐2‐related mortality in mice

doi: 10.1111/acel.13771

Figure Lengend Snippet:

Article Snippet: Antigen retrieval was performed with CC1 32 min, only for p21 and High pH buffer, Dako, Agilent (p19 and SARS‐CoV‐2 nucleocapsid); endogenous peroxidase was blocked (hydrogen peroxide at 3%) and slides were then incubated with the appropriate primary antibody as detailed: rat monoclonal anti‐p21 CIP1 (291H; 1/10; CNIO, Monoclonal Core Unit); rat monoclonal anti‐p19 ARF (5‐C3‐1; 1/50; Santa Cruz sc‐32748); rat monoclonal anti‐CDKN2A/p16 INK4a antibody (33B; CNIO, Monoclonal Core Unit) and rabbit monoclonal anti‐SARS‐CoV‐2 nucleocapsid (N) protein (019; 1/15,000; Sino Biological 40143‐R019).

Techniques:

Journal: Oncotarget

Article Title: CAMK2N1 inhibits prostate cancer progression through androgen receptor-dependent signaling

doi:

Figure Lengend Snippet: (A) CAMK2N1, AR and p21 protein levels were determined by Western blot in LNCaP cells with stably knockdown of CAMK2N1. LNCaP cells were treated with 10 nM R1881 for 10 hrs. (B-F) CAMK2N1, AR, PSA, TMPRSS2 and p21 mRNA levels were determined by qRT-PCR in LNCaP cells with stably knockdown of CAMK2N1. LNCaP cells were treated with 10 nM R1881 for 10 hrs. (G-J) Androgen-responsive luciferase reporter genes (PSA-Luc, MMTV-Luc) were assessed for AR activity. LNCaP cells with CAMK2N1 overexpression or knockdown were treated with R1881 for 24 hrs. (K) CHIP analysis of AR for PSA promoter region in LNCaP cells with stable knockdown of CAMK2N1. LNCaP cells were treated with R1881 or vehicle for 10 hrs. CHIP assay was performed using an anti-AR antibody.

Article Snippet: Tumor tissues were stained with primary antibody including Ki67 (RM-9106-S1, Thermo), Bcl-2 (SC-7382, Santa cruz), Bax (SC-7480, Santa cruz), p21 (SC-6246, Santa cruz).

Techniques: Western Blot, Stable Transfection, Knockdown, Quantitative RT-PCR, Luciferase, Activity Assay, Over Expression

Journal: Oncotarget

Article Title: CAMK2N1 inhibits prostate cancer progression through androgen receptor-dependent signaling

doi:

Figure Lengend Snippet: (A) Expression levels of pAKT ser473 , AKT, AR, Bcl-2, BAX, and p21 were determined by Western blot in LNCaP cells with stable CAMK2N1 knockdown. (B) Expression levels of pAKT ser473 , AKT and AR were determined by Western blot in LNCaP cells with stable CAMK2N1 overexpression. (C-D) CAMK2N1 knockdown cells treated with 10 nM R1881 and/or 20μM KN-93 inhibitor. PSA-Luc was assessed for AR activity. CAMKIIβ and AR protein levels were determined by Western blot. (E-F) LNCaP cells transiently transfected with CAMK2N1 and/or CAMKIIβ while treated with 10 nM R1881. PSA-Luc was assessed for AR activity. CAMK2N1 and CAMKIIβ protein levels were determined by Western blot. (G-H) CAMK2N1 knockdown cells treated with 10 nM R1881 and/or 20 μM AKT VIII. PSA-Luc was assessed for AR activity. pAKT ser473 , AKT and AR β protein levels were determined by Western blot. (I-J) LNCaP cells transiently transfected with CAMK2N1 and/or m-AKT. PSA-Luc was assessed for AR activity. CAMK2N1 and CAMKIIβ protein levels were determined by Western blot.

Article Snippet: Tumor tissues were stained with primary antibody including Ki67 (RM-9106-S1, Thermo), Bcl-2 (SC-7382, Santa cruz), Bax (SC-7480, Santa cruz), p21 (SC-6246, Santa cruz).

Techniques: Expressing, Western Blot, Knockdown, Over Expression, Activity Assay, Transfection

Journal: Oncotarget

Article Title: CAMK2N1 inhibits prostate cancer progression through androgen receptor-dependent signaling

doi:

Figure Lengend Snippet: (A) Expression levels of pAKT ser473 , AKT, AR, Bcl-2, BAX, and p21 were determined by Western blot in C4-2 cells with stable CAMK2N1 knockdown. (B) Expression levels of pAKT ser473 , AR Bcl-2, BAX, and p21 were determined by Western blot in C4-2 cells with stable CAMK2N1 overexpression. (C) C4-2 cells with stable overexpression of CAMK2N1 and AR knockdown. Cells were analyzed for cell proliferation by MTT assay. CAMK2N1 and AR protein levels were determined by Western blot. (D) C4-2 cells with stable overexpression of CAMK2N1 followed by treatment with or without Casodex (10 μM) were analyzed for cell proliferation by MTT. (E) C4-2 cells with stable knockdown of CAMK2N1 were analyzed for cell cycle by flow cytometry. (F) C4-2 cells with stable overexpression of CAMK2N1 and AR knockdown. Cells were analyzed for cell apoptosis by Annexin V staining.

Article Snippet: Tumor tissues were stained with primary antibody including Ki67 (RM-9106-S1, Thermo), Bcl-2 (SC-7382, Santa cruz), Bax (SC-7480, Santa cruz), p21 (SC-6246, Santa cruz).

Techniques: Expressing, Western Blot, Knockdown, Over Expression, MTT Assay, Flow Cytometry, Staining

Journal: Oncotarget

Article Title: CAMK2N1 inhibits prostate cancer progression through androgen receptor-dependent signaling

doi:

Figure Lengend Snippet: (A-C) C4-2 tumors with stable CAMK2N1 knockdown were injected into nude mice. Tumor size was measured every 5 days. The data was shown as mean ± SEM for N > 6 separate tumors for each group. (A) Images of tumors dissected from the mice. (B) The tumor size (mm 3 ) versus days of post injection. (C) Tumor was weighted after resection at the end of experiment. (D) mRNA levels of AR, PSA, P21, BAX and BCL2 were determined by qRT-PCR in tumors. (E) IHC staining detected the protein expression of CAMK2N1, AR, p21, Ki67, pAKT ser473 and BCL2 in C4-2 tumor tissues derived from mice. Data for quantified IHC was shown as mean ± SEM for N = 4 tumors in each group.

Article Snippet: Tumor tissues were stained with primary antibody including Ki67 (RM-9106-S1, Thermo), Bcl-2 (SC-7382, Santa cruz), Bax (SC-7480, Santa cruz), p21 (SC-6246, Santa cruz).

Techniques: Knockdown, Injection, Quantitative RT-PCR, Immunohistochemistry, Expressing, Derivative Assay

Journal: Oncogene

Article Title: Inactivation of EGLN3 hydroxylase facilitates Erk3 degradation via autophagy and impedes lung cancer growth

doi: 10.1038/s41388-022-02203-2

Figure Lengend Snippet: A – E LLC stable cell line expressing hydroxylase-inactive R205K or harboring control vector ( B , inset) were subcutaneously inoculated into mice. LLC tumors were dissected on day 21 after implantation. Shown are tumor formation rate ( A ), tumor growth rate ( B ), representative photograph of LLC tumors ( C ), tumor volume ( D ) and tumor mass ( E ) ( n = 6). F , G Immunohistochemistry analysis and quantification of PCNA expression ( F ) and cleaved caspase 3 expression ( G ) in tumors. H β-galactosidase staining and quantification of senescence-associated β-galactosidase (SA-β-Gal). I – N Immunohistochemistry analysis and quantification of p16 expression ( I ), VEGF expression ( J ), CD31 + cells ( K ), macrophage content ( L ), CD25 + cells ( M ), and CD8 + cells ( N ) in tumors. O , P IB analysis of tumor proteins prepared from the control and R205K tumors for the expression of proteins (p53, p21, p16, and HIF1α). Q IB analysis of HIF1α expression in control and R205K LLC cells ( n = 3). Arrow indicates the specific band. HPF high power field; IB immunoblotting; Casp3 Caspase 3; β-Gal β-galactosidase; SA-β-Gal senescence-associated β-galactosidase; HIF1α hypoxia-inducible factor 1α; * p < 0.05; ** p < 0.01; **** p < 0.0001 by unpaired 2-tailed Student’s t test.

Article Snippet: anti-FLAG M2 (Sigma, F1804), anti-HA.11 (BioLegend, 901501), anti-Myc (clone 9E10, Santa Cruz Biotechnology, sc-40), anti-GST (Yeasen, 30902ES60), anti-NRP1 (Cell Signaling Technology, #3725), anti-NRP2 (Cell Signaling Technology, #3366), anti-EGLN3 (Novus, NB100–303), anti-Erk3 (Santa Cruz Biotechnology, sc-365234), anti-arginase1 (Cell Signaling Technology, #9819), anti-α-tubulin (Sigma, T6074), anti-β-actin (Yeasen, 30101ES60), anti-GAPDH (Immunoway, YM3029), anti-CD31 (Abcam, ab28364), anti-Mac3 (BD Phmarmingen, 550292), anti-CD8 (InVitrogen, #14–0081–82), anti-F4/80 (Abcam, ab6640), anti-CD25 (Servicebrio, GB11612), anti-VEGF (Novus Biologicals, NB100–664), anti-PCNA (Servicebrio, GB11010), anti-cleaved caspase-3 (Cell Signaling Technology, #9661), anti-ubiquitin (Santa Cruz Biotechnology, sc-8017), anti-MMP2 (Abcam, ab37150), anti-phospho-IкBα (Cell Signaling Technology, #2859), anti-iNOS (Cell Signaling Technology, #2982), anti-p53 (Santa Cruz Biotechnology, sc-126), anti-LAMP2 (Santa Cruz Biotechnology, sc-18822), anti-p16 (Santa Cruz Biotechnology, sc-1661), anti-p21 (Santa Cruz Biotechnology, sc-817), normal mouse IgG1 (Santa Cruz Biotechnology, sc-3877), and anti-mouse IgG(H + L) (Jackson ImmunoResearch, 715-585-151).

Techniques: Stable Transfection, Expressing, Control, Plasmid Preparation, Immunohistochemistry, Staining, Western Blot

Journal: Nature Communications

Article Title: Diphthamide deficiency promotes association of eEF2 with p53 to induce p21 expression and neural crest defects

doi: 10.1038/s41467-024-47670-1

Figure Lengend Snippet: a Comparison of a Dph1 E237Q/Q41X p21 +/− embryo with wild-type (WT), p21 +/− , and Dph1 E237Q/Q41X littermates at E15.5. While all Dph1 E237Q/Q41X embryos show greatly reduced body size ( n = 4), 67% of Dph1 E237Q/Q41X p21 +/− embryos ( n = 9) exhibited enlarged body size as compare with their Dph1 E237Q/Q41X littermates. Scale bar, 1000 μm. b-e At E15.5, palate was fused in 100% of wild-type ( n = 4) or p21 +/− ( n = 5) embryos, 0% of Dph1 E237Q/Q41X ( n = 4) embryos, and 80% of Dph1 E237Q/Q41X p21 +/− embryos ( n = 5). Scale bar, 250 μm. f A schematic diagram illustrates how diphthamide modification affects cell proliferation.

Article Snippet: Antibodies used for western blotting (WB), immunohistochemistry (IHC) and immunoprecipitation (IP) include rabbit anti-OVCA1/DPH1 (Abcam ab185960, 1:3000 for WB), rabbit anti-DPH1 for Xenopus embryos (Lifespan Bioscience LS-C116434, 1:3000 for WB, 1:200 for IHC), rabbit anti-eEF2 (Cell Signaling Technology 2332, 1:3000 for WB, 1:200 for IHC), rabbit anti-eEF2 (Proteintech 20107-1-AP, 1:60 for IP), rabbit anti-phospho-eEF2 (Thr56)(Cell Signaling Technology 2331, 1:3000 for WB), mouse anti-HA (Abclonal AE008, 1:50 for IP), rabbit anti-HA (Cell Signaling Technology 3724, 1:3000 for WB), rabbit anti-Sox10 (Cell Signaling Technology 89356, 1:200 for IHC), HRP-conjugated anti-beta-actin (Biodragon BD-PB3508, 1:10,000 for WB), rabbit anti-phospho histone3 (Cell Signaling Technology 53348, 1:3,000 for WB, 1:200 for IHC), rabbit anti-histone H3 (Ser10)(Proteintech 17168-1-AP, 1:5000 for WB), rabbit anti-p53 (Proteintech 10442-1-AP, 1:3,000 for WB), mouse anti-p53 (Cell Signaling Technology 488185, 1:2000 for WB), rabbit anti-p21(Proteintech 103551-1-AP, 1:3000 for WB with U251 cell lysate), rabbit anti-p21(Abclonal A19094, 1:3000 for WB with mouse embryo), rabbit anti-PUMA (Proteintech 55120-1-AP, 1:3000 for WB), rabbit anti-caspase3 (Proteintech 19677-1-AP, 1:3,000 for WB, 1:200 for IHC), rabbit anti-cleaved caspase3 (Asp175)(Cell Signaling Technology 9661, 1:3000 for WB, 1:200 for IHC), rabbit anti-lamin B1 (Proteintech 12987-1-AP, 1:5000 for WB), rabbit anti-S6 ribosome protein (Cell Signaling Technology 2217, 1:3000 for WB), HRP-conjugated goat anti-rabbit IgG (Beyotime A0208, 1:10,000 for WB), and Cy3-conjugated goat anti-rabbit IgG (Beyotime A0516, 1:200 for IHC). siRNA duplexes were designed and synthesized by GenePharma, and the sequences are shown in Supplementary Table .

Techniques: Comparison, Modification

Journal: Oncology Letters

Article Title: PAK6 promotes cervical cancer progression through activation of the Wnt/β-catenin signaling pathway

doi: 10.3892/ol.2020.11797

Figure Lengend Snippet: Primers for amplifying PAK6 and GSK3β.

Article Snippet: Following centrifugation (12,000 × g; 4°C; 5 min), the lysates were incubated with 2 μg anti-PAK6 rabbit polyclonal antibody (cat. no. 13539-1-AP; ProteinTech Group, Inc.) or negative control rabbit IgG (cat. no. A7016; Beyotime Institute of Biotechnology) at 4°C overnight and then rotated at 4°C with a mixture of protein A/G sepharose beads (20 μl/ml) for 4 h. The beads were then washed 3 times with RIPA buffer, and the bound proteins were boiled in 2X Laemmli buffer and further analyzed using western blotting.

Techniques: Sequencing

Journal: Oncology Letters

Article Title: PAK6 promotes cervical cancer progression through activation of the Wnt/β-catenin signaling pathway

doi: 10.3892/ol.2020.11797

Figure Lengend Snippet: Expression levels of PAK6 in cervical carcinoma and paracarcinoma tissues.

Article Snippet: Following centrifugation (12,000 × g; 4°C; 5 min), the lysates were incubated with 2 μg anti-PAK6 rabbit polyclonal antibody (cat. no. 13539-1-AP; ProteinTech Group, Inc.) or negative control rabbit IgG (cat. no. A7016; Beyotime Institute of Biotechnology) at 4°C overnight and then rotated at 4°C with a mixture of protein A/G sepharose beads (20 μl/ml) for 4 h. The beads were then washed 3 times with RIPA buffer, and the bound proteins were boiled in 2X Laemmli buffer and further analyzed using western blotting.

Techniques: Expressing

Journal: Oncology Letters

Article Title: PAK6 promotes cervical cancer progression through activation of the Wnt/β-catenin signaling pathway

doi: 10.3892/ol.2020.11797

Figure Lengend Snippet: Association between PAK6 expression levels and clinicopathological parameters in cervical cancer.

Article Snippet: Following centrifugation (12,000 × g; 4°C; 5 min), the lysates were incubated with 2 μg anti-PAK6 rabbit polyclonal antibody (cat. no. 13539-1-AP; ProteinTech Group, Inc.) or negative control rabbit IgG (cat. no. A7016; Beyotime Institute of Biotechnology) at 4°C overnight and then rotated at 4°C with a mixture of protein A/G sepharose beads (20 μl/ml) for 4 h. The beads were then washed 3 times with RIPA buffer, and the bound proteins were boiled in 2X Laemmli buffer and further analyzed using western blotting.

Techniques: Expressing

Journal: Oncology Letters

Article Title: PAK6 promotes cervical cancer progression through activation of the Wnt/β-catenin signaling pathway

doi: 10.3892/ol.2020.11797

Figure Lengend Snippet: PAK6 expression in cervical cancer tissues and in C33A and HeLa cells. (A) Immunohistochemistry was used to analyze the expression levels of PAK6 in cervical cancer tissues. Scale bars, 200 µm. (B) Expression levels of PAK6 in C33A and HeLa cells were analyzed using western blotting. (C) Semi-quantification of the PAK6 protein expression levels presented in part (B) using ImageJ software. **P<0.01 vs. HeLa cells. PAK6, p21-activated kinase 6.

Article Snippet: Following centrifugation (12,000 × g; 4°C; 5 min), the lysates were incubated with 2 μg anti-PAK6 rabbit polyclonal antibody (cat. no. 13539-1-AP; ProteinTech Group, Inc.) or negative control rabbit IgG (cat. no. A7016; Beyotime Institute of Biotechnology) at 4°C overnight and then rotated at 4°C with a mixture of protein A/G sepharose beads (20 μl/ml) for 4 h. The beads were then washed 3 times with RIPA buffer, and the bound proteins were boiled in 2X Laemmli buffer and further analyzed using western blotting.

Techniques: Expressing, Immunohistochemistry, Western Blot, Software

Journal: Oncology Letters

Article Title: PAK6 promotes cervical cancer progression through activation of the Wnt/β-catenin signaling pathway

doi: 10.3892/ol.2020.11797

Figure Lengend Snippet: Effect of the knockdown of PAK6 expression levels on the proliferation, migration and invasion of HeLa cells. (A) PAK6 mRNA expression levels were analyzed in stably shPAK6-transfected HeLa cells. (B) PAK6 protein expression levels were analyzed in stably shPAK6-transfected HeLa cells using western blotting. (C) Semi-quantification of PAK6 expression levels presented in part (B). (D) Cell Counting Kit-8 assays and (E) colony formation assays were used to analyze the proliferative rate of shPAK6-transfected HeLa cells. (F) Semi-quantification of the number of colonies formed from part (E). (G) Cell migration and invasion were determined in stably shPAK6-transfected HeLa cells, (magnification ×200). (H) Semi-quantification of the number of invasive cells from part (G). (I) Semi-quantification of the number of migratory cells from part (G). **P<0.01 vs. shPAK6 NC. PAK6, p21-activated kinase 6; sh, short hairpin RNA; NC, negative control.

Article Snippet: Following centrifugation (12,000 × g; 4°C; 5 min), the lysates were incubated with 2 μg anti-PAK6 rabbit polyclonal antibody (cat. no. 13539-1-AP; ProteinTech Group, Inc.) or negative control rabbit IgG (cat. no. A7016; Beyotime Institute of Biotechnology) at 4°C overnight and then rotated at 4°C with a mixture of protein A/G sepharose beads (20 μl/ml) for 4 h. The beads were then washed 3 times with RIPA buffer, and the bound proteins were boiled in 2X Laemmli buffer and further analyzed using western blotting.

Techniques: Knockdown, Expressing, Migration, Stable Transfection, Transfection, Western Blot, Cell Counting, shRNA, Negative Control

Journal: Oncology Letters

Article Title: PAK6 promotes cervical cancer progression through activation of the Wnt/β-catenin signaling pathway

doi: 10.3892/ol.2020.11797

Figure Lengend Snippet: Effects of the overexpression of PAK6 on the proliferation, migration and invasion of HeLa cells. (A) PAK6 mRNA expression levels in stable PAK6 overexpressing HeLa cells were analyzed. (B) PAK6 protein expression levels were analyzed in stable PAK6 overexpressing HeLa cells using western blotting. (C) Semi-quantification of PAK6 expression levels presented in part (B). (D) Cell Counting Kit-8 assays and (E) colony formation assays were used to analyze the proliferative rate of stable PAK6 overexpressing HeLa cells. (F) Semi-quantification of the number of colonies formed from part (E). (G) Cell migration and invasion were determined in stable overexpressing PAK6 HeLa cells, (magnification ×200). (H) Semi-quantification of the number of invasive cells from part (G). (I) Semi-quantification of the migratory cell number from part (G). *P<0.05, **P<0.01 vs. PAK6 NC. PAK6, p21-activated kinase 6; NC, negative control.

Article Snippet: Following centrifugation (12,000 × g; 4°C; 5 min), the lysates were incubated with 2 μg anti-PAK6 rabbit polyclonal antibody (cat. no. 13539-1-AP; ProteinTech Group, Inc.) or negative control rabbit IgG (cat. no. A7016; Beyotime Institute of Biotechnology) at 4°C overnight and then rotated at 4°C with a mixture of protein A/G sepharose beads (20 μl/ml) for 4 h. The beads were then washed 3 times with RIPA buffer, and the bound proteins were boiled in 2X Laemmli buffer and further analyzed using western blotting.

Techniques: Over Expression, Migration, Expressing, Western Blot, Cell Counting, Negative Control

Journal: Oncology Letters

Article Title: PAK6 promotes cervical cancer progression through activation of the Wnt/β-catenin signaling pathway

doi: 10.3892/ol.2020.11797

Figure Lengend Snippet: Effect of PAK6 knockdown or overexpression on the Wnt/β-catenin signaling pathway in HeLa cells. Western blotting was used to analyze the expression levels of (A) β-catenin, p-β-catenin, GSK3β and p-GSK3β, and (B) E-cadherin and Cyclin D1 in stably shPAK6-transfected HeLa cells. (C) Semi-quantification of the expression levels of proteins in parts (A) and (B). *P<0.05, **P<0.01 vs. shPAK6 NC. Western blotting was used to analyze the expression levels of (D) β-catenin, p-β-catenin, GSK3β and p-GSK3β, and (E) E-cadherin and cyclin D1 in stable PAK6 overexpressing HeLa cells. (F) Semi-quantification of the expression levels of proteins in parts (D) and (E). *P<0.05, **P<0.01 vs. PAK6 NC. (G) Immunofluorescence was used to demonstrate the co-localization of PAK6 and GSK3β. Scale bars, 10 µm. (H) Co-IP was used to analyze the interaction between PAK6 and GSK3β. PAK6, p21-activated kinase 6; sh, short hairpin RNA; NC, negative control; p-, phosphorylated; GSK3β, glycogen synthase kinase 3β; IP, immunoprecipitation.

Article Snippet: Following centrifugation (12,000 × g; 4°C; 5 min), the lysates were incubated with 2 μg anti-PAK6 rabbit polyclonal antibody (cat. no. 13539-1-AP; ProteinTech Group, Inc.) or negative control rabbit IgG (cat. no. A7016; Beyotime Institute of Biotechnology) at 4°C overnight and then rotated at 4°C with a mixture of protein A/G sepharose beads (20 μl/ml) for 4 h. The beads were then washed 3 times with RIPA buffer, and the bound proteins were boiled in 2X Laemmli buffer and further analyzed using western blotting.

Techniques: Knockdown, Over Expression, Western Blot, Expressing, Stable Transfection, Transfection, Immunofluorescence, Co-Immunoprecipitation Assay, shRNA, Negative Control, Immunoprecipitation

Journal: bioRxiv

Article Title: Canonical Wnt signalling is activated during BEC-to-hepatocyte conversion in vivo and modulates liver epithelial cell plasticity in hepatic organoids

doi: 10.1101/2020.11.09.374462

Figure Lengend Snippet: (A) Treatment time for the flow cytometry isolation of BECs (tdTom+ Epcam+ cells) from MCD-injured livers following administration of either AAV8.p21 or AAV8.null viral particles via tail injection. (B) Representative images of MCD-injured livers following 6-day recovery. Dash line highlights the presence of HNF4a tdTomBEC-derived hepatocytes. (n=4 animals) (C and D) RT-qPCR gene expression analysis of tdTom+ Epcam+ cells isolated at various time points from the AAV8.null (non-transdifferentiation, panel C) and the AAV8.p21 (BEC-to-hepatocyte transdifferentiation, panel D) MCD injury models. (n=4 animals per condition). (E) Axin2 RNAscope analysis of serial sections of AAV8.p21 MCD-injured livers at day recovery 6 (top). Immunohistochemistry for tdTom labels cells of ductal origin (bottom). Arrowheads point to BECs in ducts. Asterisks point to BECs Axin2+ cells showing an invasive phenotype. (n=3 animals).

Article Snippet: Animals received a single dose of AAV8.TBG.null or AAV8.TBG.p21 (5*10^12 units/ml) (Addgene) intravenously allowed 1 week wash-out period on normal diet.

Techniques: Flow Cytometry, Isolation, Injection, Derivative Assay, Quantitative RT-PCR, Gene Expression, RNAscope, Immunohistochemistry

Journal: bioRxiv

Article Title: Canonical Wnt signalling is activated during BEC-to-hepatocyte conversion in vivo and modulates liver epithelial cell plasticity in hepatic organoids

doi: 10.1101/2020.11.09.374462

Figure Lengend Snippet: Scheme integrating the dynamics of BEC-to-hepatocyte transdifferentation and the expression patterns of Axin2, Lrp6 and Lgr5 obtained by RT-qPCR analysis of BECs (tdTom+ Epcam+) isolated at different time points during the recovery period of the p21 MCD mouse model. BEC-to-hepatocyte conversion occurs predominantly between day 3 and day 7 of the recovery period . Lrp6 gene expression (blue line) gradually declines between day 3 and day 9. By contrast, Lgr5 gene expression (green line) gradually increases between day 3 and day 9 of the recovery period. Expression of the Wnt/β-catenin target gene Axin2 (magenta line) is significantly increased at day 6 of recovery. Sections represented with dash lines represent predicted gene expression levels.

Article Snippet: Animals received a single dose of AAV8.TBG.null or AAV8.TBG.p21 (5*10^12 units/ml) (Addgene) intravenously allowed 1 week wash-out period on normal diet.

Techniques: Expressing, Quantitative RT-PCR, Isolation, Gene Expression