Journal: Oncogene

Article Title: Activated p53 suppresses the histone methyltransferase EZH2 gene.

doi: 10.1038/sj.onc.1207706

Figure Lengend Snippet: Figure 1 EZH2 expression is downregulated in senescent cells. (a) Morphology and senescence-associated b-galactosidase (SA-b-Gal) staining (pH 6.0) of young (p21, left panel), near-senescent (p29, middle panel), and hTERT-immortalized (T31, right panel) WI-38 cells. (b) Telomerase activity determined by TRAPeze assay. Lane 1, empty vector-infected WI-38 cells (V); lane 2, hTERT-infected WI-38 cells (TERT). (c) Semiquantitative RT–PCR analysis. Total RNA samples were isolated from young (p21), near-senescent (p29), and hTERT-immortalized (T31) WI-38 cells, respectively. GAPDH gene expression was used as a normalization control. (d) Quantitative real-time PCR of p21Waf1 (empty bar) and EZH2 (solid bar) gene expressions in young (p21), near-senescent (p29), and hTERT-immortalized (T31) WI-38 cells. Values shown are means7s.d. of duplicate readings and represent expression patterns normalized to GAPDH. (e) Immunoblot analysis of nuclear extract protein (Nuc.) or whole-cell lysate (WCL) from young (p21), near- senescent (p29), and hTERT-immortalized (T31) WI-38 cells under normal condition. The asterisk indicates that the EZH2 antibody crossreacted with the unspecific cytoplasmic protein. Antibodies used are indicated in Materials and methods. Anti-b-tubulin was used as a control for protein loading

Article Snippet: Mouse monoclonal anti-p53 (DO-1 and 1801; kindly provided by Dr D Lane), rabbit polyclonal anti-p53 (produced in our laboratory), mouse monoclonal anti-MDM2 (4B2, 2A9, 2A10; kindly provided by Dr M Oren, Weizmann Institute of Science), rabbit polyclonal anti-p21 (C-19; Santa Cruz), rabbit polyclonal anti-EZH2 (Upstate Biochemistry), and mouse monoclonal anti-b-tubulin (Sigma) were used for immunoblotting.

Techniques: Expressing, Staining, Activity Assay, Plasmid Preparation, Infection, Reverse Transcription Polymerase Chain Reaction, Isolation, Gene Expression, Control, Real-time Polymerase Chain Reaction, Western Blot

Journal: Oncogene

Article Title: Activated p53 suppresses the histone methyltransferase EZH2 gene.

doi: 10.1038/sj.onc.1207706

Figure Lengend Snippet: Figure 2 Downregulation of EZH2 in senescent cells is partially p53 dependent. (a) Semiquantitative RT–PCR analysis of total RNA samples, which were isolated from young (p21) and near- senescent (p29) WI-38 cells, stably infected with empty vector (V) or GSE56 (G). GAPDH gene expression was used as a normal- ization control. (b) Lifespan of WI-38 empty vector (V) and GSE56 (G)-infected cells. The mean PDLs were calculated as detailed in Materials and methods. (c) Quantitative real-time PCR of EZH2 (solid bar) and p21Waf1 (empty bar) gene expressions in young (p21) and near-senescent (p29) WI-38 cells, stably infected with empty vector (V) or GSE56 (G), respectively. Values shown are means7s.d. of duplicate readings and represent expression patterns normalized to GAPDH. (d) Immunoblot analysis of total protein extracts from young (p21) and near-senescent (p29) WI-38 cells, stably infected with empty vector (V) or GSE56 (G). Anti-b- tubulin was used as a control for protein loading

Article Snippet: Mouse monoclonal anti-p53 (DO-1 and 1801; kindly provided by Dr D Lane), rabbit polyclonal anti-p53 (produced in our laboratory), mouse monoclonal anti-MDM2 (4B2, 2A9, 2A10; kindly provided by Dr M Oren, Weizmann Institute of Science), rabbit polyclonal anti-p21 (C-19; Santa Cruz), rabbit polyclonal anti-EZH2 (Upstate Biochemistry), and mouse monoclonal anti-b-tubulin (Sigma) were used for immunoblotting.

Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Stable Transfection, Infection, Plasmid Preparation, Gene Expression, Control, Real-time Polymerase Chain Reaction, Expressing, Western Blot

Journal: Oncology Reports

Article Title: Suppression of CDCA3 inhibits prostate cancer progression via NF-κB/cyclin D1 signaling inactivation and p21 accumulation

doi: 10.3892/or.2021.8253

Figure Lengend Snippet: Knockdown of CDCA3 induces G0/G1-phase arrest in DU145 and PC-3 cells. (A) Flow cytometry was used to analyze the cell cycle following CDCA3 knockdown in DU145 and PC-3 cells. (B) Cell cycle-associated protein levels, including cyclin D1 and p21, were analyzed via western blotting. GAPDH was used as a loading control. Data are presented as the mean ± SD of three independent experiments. **P<0.01 and ***P<0.001. CDCA3, cell division cycle-associated 3; sh-, short hairpin; NC, negative control.

Article Snippet: The following antibodies were used: Mouse monoclonal anti-GAPDH (1:2,000; cat. no. 33033M; BIOSS), rabbit polyclonal anti-CDCA3 (1:1,000; cat. no. YT0819), rabbit polyclonal anti-cleaved caspase-3 (1:1,000; cat. no. YC0006), rabbit polyclonal anti-pro-caspase-3 (1:1,000; cat. no. YT6113), and rabbit polyclonal anti-cyclin-dependent kinase inhibitor 1 (p21; 1:1,000; cat. no. YT3497; all from Immunoway Biotechnology Company), rabbit polyclonal anti-cyclin D1 (1:1,000; cat. no. 0623R; BIOSS), mouse monoclonal anti-NFκB-p65 (1:1,000; cat. no. YM311), rabbit polyclonal anti-phosphorylated (p)-NFκB-p65 (1:1,000; cat. no. YP0192), rabbit polyclonal anti-IKKα/β (1:1,000; cat. no. YT2302), and rabbit polyclonal anti-NFκB-p105/p50 (1:1,000; cat. no. YT3101; all from Immunoway Biotechnology Company), HRP-labeled goat anti-rabbit secondary antibody (1:5,000; cat. no. 40295G-HRP; BIOSS) and HRP-labeled goat anti-mouse secondary antibody (1:5,000; cat. no. 0368G-HRP; BIOSS).

Techniques: Flow Cytometry, Western Blot, Negative 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: 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: iScience

Article Title: Discovery of SOCS7 as a versatile E3 ligase for protein-based degraders

doi: 10.1016/j.isci.2024.109802

Figure Lengend Snippet: SOCS7-based KRAS degrader inhibits pancreatic cancer cells proliferation (A) Efficacy of SOCS7-based KRAS degrader to deplete its target in MIA PaCa-2 compared to a negative degrader (K19dm-SOCS7). KRAS, HRAS and NRAS protein levels were analyzed by western blot after 72 h of dox treatment of the cells at 0.5 μg mL −1 (+) or not induced (−). Expression of the degraders is shown with the FLAG antibody. α-tubulin is the loading control. (B) Effect of SOCS7-based KRAS degrader on RAS downstream signaling pathways of MIA PaCa-2 analyzed by western blot. HSP90 is the loading control. Cells were induced with dox with the same conditions indicated in (A). (C) Quantification of pAKT S473 /AKT and pERK/ERK signals from (B). The signals were normalized to the no dox condition of K19dm-SOCS7. (D and E) Assessment of the effect of SOCS7-based KRAS degrader on 2D-adherent (D) and 3D spheroids (E) proliferation of MIA PaCa-2 cells. These proliferation assays (2D and 3D) were normalized to the no dox condition for each cell line. The dotted lines represent the no dox condition and the plain lines show the dox-treated cells. Statistical analyses were performed using an unpaired t-test (C) or a one-way ANOVA followed by Tukey post-hoc tests (D and E) (∗ p < 0.05; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, not significant). Each experiment was performed three (A–C) or four (D and E) times. Error bars in (C–E) are mean ± SD from at least three biological repeats. See also Figure S9 .

Article Snippet: Primary antibodies include anti-GFP (1/1000, Novus, Cat#NB100-1770), anti-FLAG (1/2000, Sigma, Cat#F1804), anti-actin (1/50000, Sigma, Cat#MAB1501), anti-α-tubulin (1/2000, Abcam, Cat#ab4074), anti-HSP90 (1/4000, CST, Cat#4874S), anti-KRAS (1/100, Santa Cruz Biotechnologies, Cat#sc-30), anti-NRAS (1/1000, Abcam, Cat#ab77392), anti-HRAS (1/500, Proteintech, Cat#18295-1-AP), anti-ALFA HRP-linked (1/4000, NanoTag Biotechnologies, Cat#N1505), anti-Elongin-C (1/1000, Abcam, Cat#ab226831), anti-Elongin-B (1/1000, Abcam, Cat#ab151743), anti-Cullin 5 (1/1000, Abcam, Cat#ab184177), anti-RBX2 (1/1000, Abcam, Cat#ab181986), anti-phospho-p44/22 MAPK (ERK1/2) (1/1000, CST, Cat#4370S), anti-p44/42 MAPK (total ERK1/2) (1/1000, CST, Cat#4695S), anti-phospho-AKT S473 (1/1000, CST, Cat#4058S) and anti-AKT (1/1000, CST, Cat#9272S).

Techniques: Western Blot, Expressing

Journal: iScience

Article Title: Discovery of SOCS7 as a versatile E3 ligase for protein-based degraders

doi: 10.1016/j.isci.2024.109802

Figure Lengend Snippet:

Article Snippet: Primary antibodies include anti-GFP (1/1000, Novus, Cat#NB100-1770), anti-FLAG (1/2000, Sigma, Cat#F1804), anti-actin (1/50000, Sigma, Cat#MAB1501), anti-α-tubulin (1/2000, Abcam, Cat#ab4074), anti-HSP90 (1/4000, CST, Cat#4874S), anti-KRAS (1/100, Santa Cruz Biotechnologies, Cat#sc-30), anti-NRAS (1/1000, Abcam, Cat#ab77392), anti-HRAS (1/500, Proteintech, Cat#18295-1-AP), anti-ALFA HRP-linked (1/4000, NanoTag Biotechnologies, Cat#N1505), anti-Elongin-C (1/1000, Abcam, Cat#ab226831), anti-Elongin-B (1/1000, Abcam, Cat#ab151743), anti-Cullin 5 (1/1000, Abcam, Cat#ab184177), anti-RBX2 (1/1000, Abcam, Cat#ab181986), anti-phospho-p44/22 MAPK (ERK1/2) (1/1000, CST, Cat#4370S), anti-p44/42 MAPK (total ERK1/2) (1/1000, CST, Cat#4695S), anti-phospho-AKT S473 (1/1000, CST, Cat#4058S) and anti-AKT (1/1000, CST, Cat#9272S).

Techniques: Recombinant, Transfection, Magnetic Beads, Fluorescence, Expressing, Software, Sterility, Suction Filtration