Journal: Cancer medicine

Article Title: Nuclear receptor coactivator 6 (NCoA6) promotes cell proliferation, migration, and invasion in pancreatic cancer.

doi: 10.1002/cam4.6427

Figure Lengend Snippet: FIGURE 3 NCoA6 knockdown decreases the proliferation, cell cycle, and apoptosis of pancreatic cancer cells. CCK-8 assays (A, B) and colony formation assays (C, D) were used to test the proliferation of PDAC cells transfected with NCoA6 shRNAs. The representative images and statistical results of the cell cycle (E, F) and apoptosis (G, H) using flow cytometry. (I, J) Western blotting analysis of p21 and p27 expression in NCoA6-silenced PANC-1 and SW1990 cells. (K, L) Western blotting analysis of CDK4, CDK2, Cyclin D1, Cyclin E1, and Cyclin A2 expression in NCoA6-silenced PANC-1 and SW1990 cells.

Article Snippet: Protein extraction and western blotting were performed as described in our previous research.16 Antibodies against NCoA6 (1:1000), E- cadherin (1:5000), N- cadherin (1:3000), FBW7 (1:1000), CDX2 (1:1000), cyclin- dependent kinase 4 (CDK4, 1:2000), cyclin- dependent kinase 2 (CDK2, 1:5000), Cyclin D1 (1:5000), Cyclin E1 (1:1000), and Cyclin A2 (1:2000) were obtained from Proteintech.

Techniques: Knockdown, CCK-8 Assay, Transfection, Flow Cytometry, Western Blot, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Super enhancer-driven LINC01013 mediates hypoxia-induced mitochondrial dysfunction by HSPA9 to determine pulmonary arterial smooth muscle cell fate

doi: 10.1007/s00018-025-06071-3

Figure Lengend Snippet: Silencing of LINC01013 inhibited hypoxia-induced hPASMC proliferation and inflammation. A , B hPASMC proliferation was determined by CCK8 and EdU incorporation assays ( n = 6). EdU (red), DAPI (blue), scale bar, 50 μm. C Western blotting analysis of PCNA, cyclin A and cyclin D in hPASMCs ( n = 6). D RT‒qPCR analysis showed the mRNA levels of TNF-α and IL-6 after LINC01013 knockdown, β-actin was used as a internal reference gene ( n = 6). E Western blotting analysis of TNF-α and IL-6 in hPASMCs. ( n = 6). All values are presented as the mean ± SEM. Statistical analysis was performed with one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001. Nor, normoxia; Hyp, hypoxia; NC, negative control; si, siRNA

Article Snippet: The antibody against CEBPB (PB9171, BA0670, 1:500, Boster, Wuhan, China), H3K27ac (A7253, 1:500, ABclonal, Wuhan, China), H3K4me1 (A2355, 1:500, Wuhan, China), PCNA (A00125, 1:500, Boster, Wuhan, China), Cyclin A (PB0515, 1:500, Boster, Wuhan, China), Cyclin D (BM4272, 1:500, Boster, Wuhan, China), IL-6 (AF7236, 1:500, Beyotime, Shanghai, China), TNF-α (AF8208, 1:500, Beyotime, Shanghai, China), PKM2 (4053, 1:1000, Cell Signaling, MA, US), HK II (66974-1-Ig, 1:1000, Proteintech, IL, USA), PDH (2784, 1:1000, Cell Signaling, MA, US), HSPA9 (14887-1-AP, 1:5000, Proteintech, IL, USA), VDAC1 (10866-1-AP, 1:5000, Proteintech, IL, USA), and β-actin (TA-09, 1:1000, ZSGB‐BIO, Beijing, China) was incubated at 4 °C overnight, followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h, and proteins were visualized with enhanced chemiluminescence reagents.

Techniques: Western Blot, Knockdown, Negative Control

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: MiR-29c-3p Suppresses the Migration, Invasion and Cell Cycle in Esophageal Carcinoma via CCNA2/p53 Axis

doi: 10.3389/fbioe.2020.00075

Figure Lengend Snippet: Primer sequence.

Article Snippet: In order to investigate the underlying mechanism of CCNA2 on EC cells, cells were classified into four groups: si-NC + DMSO, si-CCNA2 + DMSO, si-NC + PFTβand si-CCNA2 + PFTβ groups [PFTβ, p53 inhibitor, HY-16702, MedChemExpress, 10 μM ( )]. qRT-PCR was firstly performed to test CCNA2 level in each group, finding that CCNA2 was markedly decreased in cells transfected with si-CCNA2 + DMSO ( ).

Techniques: Sequencing

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: MiR-29c-3p Suppresses the Migration, Invasion and Cell Cycle in Esophageal Carcinoma via CCNA2/p53 Axis

doi: 10.3389/fbioe.2020.00075

Figure Lengend Snippet: MiR-29c-3p is decreased in EC tissues accompanied by low survival rate and associated with the increase of CCNA2. TCGA database was utilized to access expression data of miRNAs and mRNAs of ESCA, and (A) the results of differential analysis were plotted in Volcano plots, with red representing high expression and green representing low expression. In panel (B) , miR-29c-3p level in EC tissues were determined as shown in a box plot. (C) Survival analysis of miR-29c-3p in TCGA-ESCA dataset was performed, with the red line as high expression and blue line as low expression. In panel (D) , Venn diagram was made to find the candidate targets of miR-29c-3p, acquiring 10 DEmRNAs. In panel (E) , correlation analysis was conducted between miR-29c-3p and CCNA2 (–0.57) as plotted in a heat map. In panel (F) , CCNA2 expression in EC cells was examined. Clinical tissue samples were used to further explore the (G) expression of miR-29c-3p and CCNA2 mRNA in EC tissues by qRT-PCR, (H) the protein level of CCNA2 (P1, P2, P3 referred to three EC samples) via Western blot and (I) the correlation between miR-29c-3p and CCNA2. * P < 0.05.

Article Snippet: In order to investigate the underlying mechanism of CCNA2 on EC cells, cells were classified into four groups: si-NC + DMSO, si-CCNA2 + DMSO, si-NC + PFTβand si-CCNA2 + PFTβ groups [PFTβ, p53 inhibitor, HY-16702, MedChemExpress, 10 μM ( )]. qRT-PCR was firstly performed to test CCNA2 level in each group, finding that CCNA2 was markedly decreased in cells transfected with si-CCNA2 + DMSO ( ).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: MiR-29c-3p Suppresses the Migration, Invasion and Cell Cycle in Esophageal Carcinoma via CCNA2/p53 Axis

doi: 10.3389/fbioe.2020.00075

Figure Lengend Snippet: MiR-29c-3p targets CCNA2 and inhibits its expression. Targeted binding sites of miR-29c-3p and CCNA2 were predicted before as shown in panel (A) . To investigate their targeted relationship, (B) dual-luciferase assay was performed to confirm their targeted binding, and (C) RIP was conducted to describe the effect of miR-29c-3p on CCNA2. Moreover, (D,E) qRT-PCR and Western blot were carried out to determine CCNA2 expression in mRNA and protein levels in miR-29c-3p mimic transfected cells, so as to further verify such relationship. * P < 0.05.

Article Snippet: In order to investigate the underlying mechanism of CCNA2 on EC cells, cells were classified into four groups: si-NC + DMSO, si-CCNA2 + DMSO, si-NC + PFTβand si-CCNA2 + PFTβ groups [PFTβ, p53 inhibitor, HY-16702, MedChemExpress, 10 μM ( )]. qRT-PCR was firstly performed to test CCNA2 level in each group, finding that CCNA2 was markedly decreased in cells transfected with si-CCNA2 + DMSO ( ).

Techniques: Expressing, Binding Assay, Luciferase, Quantitative RT-PCR, Western Blot, Transfection

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: MiR-29c-3p Suppresses the Migration, Invasion and Cell Cycle in Esophageal Carcinoma via CCNA2/p53 Axis

doi: 10.3389/fbioe.2020.00075

Figure Lengend Snippet: CCNA2 silencing regulates the migration, invasion and cell cycle in EC by promoting p53 signaling pathway. si-NC + DMSO, si-CCNA2 + DMSO, si-NC + PFTβ and si-CCNA2 + PFTβ were transfected into cells. qRT-PCR and Western blot were conducted to determine (A) the CCNA2 mRNA and (B) protein levels of CCNA2 as well as p53. MTT, Transwell, and flow cytometry were performed to investigate the effects of silencing CCNA2 on EC cell activities, including (C) cell viability, (D) migration and invasion, (E) cell cycle. * P < 0.05.

Article Snippet: In order to investigate the underlying mechanism of CCNA2 on EC cells, cells were classified into four groups: si-NC + DMSO, si-CCNA2 + DMSO, si-NC + PFTβand si-CCNA2 + PFTβ groups [PFTβ, p53 inhibitor, HY-16702, MedChemExpress, 10 μM ( )]. qRT-PCR was firstly performed to test CCNA2 level in each group, finding that CCNA2 was markedly decreased in cells transfected with si-CCNA2 + DMSO ( ).

Techniques: Migration, Transfection, Quantitative RT-PCR, Western Blot, Flow Cytometry

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: MiR-29c-3p Suppresses the Migration, Invasion and Cell Cycle in Esophageal Carcinoma via CCNA2/p53 Axis

doi: 10.3389/fbioe.2020.00075

Figure Lengend Snippet: MiR-29c-3p mediates the migration, invasion and cell cycle in EC via CCNA2/p53 axis. Cells were treated with inhibitor NC + si-NC, inhibitor NC + si-CCNA2, miR-29c-3p inhibitor + si-NC and miR-29c-3p inhibitor + si-CCNA2, and then harvested for (A) Western blot to detect the protein levels of CCNA2 and p53. (B) MTT was performed to test cell viability, (C) Transwell was conducted to assay the ability of cell migration and invasion, and (D) flow cytometry was carried out to determine the effect of miR-29c-3p on cell cycle. * P < 0.05.

Article Snippet: In order to investigate the underlying mechanism of CCNA2 on EC cells, cells were classified into four groups: si-NC + DMSO, si-CCNA2 + DMSO, si-NC + PFTβand si-CCNA2 + PFTβ groups [PFTβ, p53 inhibitor, HY-16702, MedChemExpress, 10 μM ( )]. qRT-PCR was firstly performed to test CCNA2 level in each group, finding that CCNA2 was markedly decreased in cells transfected with si-CCNA2 + DMSO ( ).

Techniques: Migration, Western Blot, Flow Cytometry

Journal: International immunopharmacology

Article Title: Immunosensitivity mediated by downregulated AKT1-SKP2 induces anti-PD-1-associated thyroid immune injury.

doi: 10.1016/j.intimp.2023.110452

Figure Lengend Snippet: Fig. 5. The downstream regulation of AKT1-SKP2 in NTHY cells.

Article Snippet: SKP2 inhibitor (SKP2i, MCE, 5uM) is solubilized by DMSO.

Techniques:

Journal: International immunopharmacology

Article Title: Immunosensitivity mediated by downregulated AKT1-SKP2 induces anti-PD-1-associated thyroid immune injury.

doi: 10.1016/j.intimp.2023.110452

Figure Lengend Snippet: Fig. 6. Interaction between SKP2 and PD-L1. (A) DAPI is in blue, SKP2 in red, and PD-L1 in green. In NTHY cells, SKP2 is mainly expressed in the nucleus and PD- L1 in non-nuclear area. (B) NTHY cells are co-transfected with pcDNA3.1-SKP2 plasmid and pcDNA3.1-PDL1 plasmid. The interaction between SKP2 and PD-L1 protein is proved by immunoprecipitation.

Article Snippet: SKP2 inhibitor (SKP2i, MCE, 5uM) is solubilized by DMSO.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation

Journal: International immunopharmacology

Article Title: Immunosensitivity mediated by downregulated AKT1-SKP2 induces anti-PD-1-associated thyroid immune injury.

doi: 10.1016/j.intimp.2023.110452

Figure Lengend Snippet: Fig. 8. Hypothesis: NIVO increases the immunosensitivity of NTHY cells by downregulating AKT1-SKP2.

Article Snippet: SKP2 inhibitor (SKP2i, MCE, 5uM) is solubilized by DMSO.

Techniques:

Journal: International immunopharmacology

Article Title: Immunosensitivity mediated by downregulated AKT1-SKP2 induces anti-PD-1-associated thyroid immune injury.

doi: 10.1016/j.intimp.2023.110452

Figure Lengend Snippet: Fig. 7. AKT1, SKP2, PD-L1 and IFN-γ in thyroid tissue. (A) The thyroid gland of mice is used for detection by immunohistochemistry. Fewer AKT1, SKP2, PD-L1 and more IFN-γ are observed in the NIVO group than in the CTRL group (10X). (B) Further, levels of various proteins in mouse thyroid are quantified by WB.(C) Human thyroid tissue is used for detection by WB. results revealed that PD-L1 is down-regulated and IFNG is up-regulated in the HT group compared to the non-HT group, however, there is no difference in AKT1 and SKP2.

Article Snippet: SKP2 inhibitor (SKP2i, MCE, 5uM) is solubilized by DMSO.

Techniques: Immunohistochemistry

Journal: Frontiers in Immunology

Article Title: Multi-omics analysis and experimental validation of the value of monocyte-associated features in prostate cancer prognosis and immunotherapy

doi: 10.3389/fimmu.2024.1426474

Figure Lengend Snippet: CCNA2 identified as the best prognostic gene among monocyte-associated genes. (A, B) Functional analysis of monocyte-related prognostic genes. (C, D) GBM and RandomForest algorithms to screen key prognostic genes in the TCGA-PRAD dataset. (E, F) GBM and RandomForest algorithms to screen key prognostic genes in the GSE16560 dataset. (G, H) KM curves of CCNA2 and ACSM3 in the TCGA-PRAD dataset. (I, J) KM curves of CCNA2 and ACSM3 in the GSE16560 dataset.

Article Snippet: Next, CCNA2 antibody (BOSTER, PB9424) was applied dropwise to cover the tissue chip, which was left at room temperature for 2 hours.

Techniques: Functional Assay

Journal: Frontiers in Immunology

Article Title: Multi-omics analysis and experimental validation of the value of monocyte-associated features in prostate cancer prognosis and immunotherapy

doi: 10.3389/fimmu.2024.1426474

Figure Lengend Snippet: CCNA2 positively correlates with monocyte infiltration levels. (A) Correlation analysis of CCNA2 and monocyte infiltration levels. (B, C) Analysis of CCNA2 correlation with monocyte markers. (D) Mendelian randomization analysis of high HLA-DR expressing monocytes in relation to prostate cancer. (E–I) Single-cell analysis of the correlation between CCNA2 and immune cell infiltration.

Article Snippet: Next, CCNA2 antibody (BOSTER, PB9424) was applied dropwise to cover the tissue chip, which was left at room temperature for 2 hours.

Techniques: Expressing, Single-cell Analysis

Journal: Frontiers in Immunology

Article Title: Multi-omics analysis and experimental validation of the value of monocyte-associated features in prostate cancer prognosis and immunotherapy

doi: 10.3389/fimmu.2024.1426474

Figure Lengend Snippet: Functional analysis of CCNA2 in PRAD. (A) KEGG analysis of CCNA2 in PRAD. (B–J) GSEA analysis of CCNA2 in PRAD.

Article Snippet: Next, CCNA2 antibody (BOSTER, PB9424) was applied dropwise to cover the tissue chip, which was left at room temperature for 2 hours.

Techniques: Functional Assay

Journal: Frontiers in Immunology

Article Title: Multi-omics analysis and experimental validation of the value of monocyte-associated features in prostate cancer prognosis and immunotherapy

doi: 10.3389/fimmu.2024.1426474

Figure Lengend Snippet: CCNA2 has a high binding capacity to PRAD-targeted drugs. (A) Analysis of the binding capacity of CCNA2 to PD1 inhibitors. (B) Analysis of the binding capacity of CCNA2 to bicalutamide. (C) Analysis of the binding capacity of CCNA2 to enzalutamide. (D) Analysis of the binding capacity of CCNA2 to abiraterone.

Article Snippet: Next, CCNA2 antibody (BOSTER, PB9424) was applied dropwise to cover the tissue chip, which was left at room temperature for 2 hours.

Techniques: Binding Assay

Journal: Frontiers in Immunology

Article Title: Multi-omics analysis and experimental validation of the value of monocyte-associated features in prostate cancer prognosis and immunotherapy

doi: 10.3389/fimmu.2024.1426474

Figure Lengend Snippet: CCNA2 is highly expressed in PRAD and is associated with poor patient prognosis. (A, B) Differential expression of CCNA2 in PRAD. (C) Diagnostic predictive value of CCNA2 in PRAD. (D) KM curve of overall survival of CCNA2 in PRAD. (E) Prognostic predictive value of CCNA2 in PRAD.

Article Snippet: Next, CCNA2 antibody (BOSTER, PB9424) was applied dropwise to cover the tissue chip, which was left at room temperature for 2 hours.

Techniques: Quantitative Proteomics, Diagnostic Assay

Journal: Molecular Vision

Article Title: Small interfering RNA targeting of S phase kinase-interacting protein 2 inhibits cell proliferation of pterygium fibroblasts

doi:

Figure Lengend Snippet: Down-regulation of Skp2 protein by siRNA in PFC in vitro by immunoflurescence. Immunoflurescence staining indicated high constitutive levels of Skp2 protein (arrow indication) in the nucleolus of PFC and NFC transfected with pSuppressor vehicle ( A , D ) or without transfection ( B , E ). Transfection with Skp2 siRNA dramatically decreased the expression of Skp2 protein in PFC cells ( C , F ). The scale bar is equal to 40 μm.

Article Snippet: Skp2 antibody and the Steptavidin-Alkaline Phosphatase Complex (SABC) kit were purchased from the Boster Company (Wuhan, China).

Techniques: In Vitro, Staining, Transfection, Expressing

Journal: Molecular Vision

Article Title: Small interfering RNA targeting of S phase kinase-interacting protein 2 inhibits cell proliferation of pterygium fibroblasts

doi:

Figure Lengend Snippet: Down-regulation of Skp2 protein by siRNA in PFC in vivo by immunoflurescence. Immunoflurescence staining indicated high constitutive levels of Skp2 protein in the nucleolus of PFC and NFC transfected with pSuppressor vehicle ( A , D ) or without transfection ( B , E ). Transfection with Skp2 siRNA dramatically decreased the expression of Skp2 protein in PFC cells ( C , F ). The scale bar is equal to 40 μm.

Article Snippet: Skp2 antibody and the Steptavidin-Alkaline Phosphatase Complex (SABC) kit were purchased from the Boster Company (Wuhan, China).

Techniques: In Vivo, Staining, Transfection, Expressing

Journal: Molecular Vision

Article Title: Small interfering RNA targeting of S phase kinase-interacting protein 2 inhibits cell proliferation of pterygium fibroblasts

doi:

Figure Lengend Snippet: Skp2 siRNA induced p27 kip1 accumulationin in PFC in vitro by immunofluorescence. Immunofluorescence staining indicated the expression of p27 kip1 dramatically increased in PFC and NFC transfection with Skp2 siRNA ( C , F ) when compared with PFC transfection with pSuppressor vehicle ( A , D ) or without transfection ( B , E ). The scale bar is equal to 40 μm.

Article Snippet: Skp2 antibody and the Steptavidin-Alkaline Phosphatase Complex (SABC) kit were purchased from the Boster Company (Wuhan, China).

Techniques: In Vitro, Immunofluorescence, Staining, Expressing, Transfection

Journal: Molecular Vision

Article Title: Small interfering RNA targeting of S phase kinase-interacting protein 2 inhibits cell proliferation of pterygium fibroblasts

doi:

Figure Lengend Snippet: Skp2 siRNA induced p27 kip1 accumulationin in PFC in vivo by immunofluorescence. Immunofluorescence staining indicated the expression of p27 kip1 dramatically increased in PFC and NFC transfection with Skp2 siRNA ( C , F ) when compared with PFC transfection with pSuppressor vehicle ( A , D ) or without transfection ( B , E ). The scale bar is equal to 40 μm.

Article Snippet: Skp2 antibody and the Steptavidin-Alkaline Phosphatase Complex (SABC) kit were purchased from the Boster Company (Wuhan, China).

Techniques: In Vivo, Immunofluorescence, Staining, Expressing, Transfection

Journal: Molecular Vision

Article Title: Small interfering RNA targeting of S phase kinase-interacting protein 2 inhibits cell proliferation of pterygium fibroblasts

doi:

Figure Lengend Snippet: Cell viability assay by MTT. After 6 to 12 days transfection with Skp2 siRNA, cell viability was significantly decreased in cultured hPFC cells compared with vehicle control and blank control epsecially on the 6th day after transfection. *p<0.01 versus vehicle and blank control; **p<0.05 versus vehicle and blank control.

Article Snippet: Skp2 antibody and the Steptavidin-Alkaline Phosphatase Complex (SABC) kit were purchased from the Boster Company (Wuhan, China).

Techniques: Viability Assay, Transfection, Cell Culture, Control

Journal: Molecular Vision

Article Title: Small interfering RNA targeting of S phase kinase-interacting protein 2 inhibits cell proliferation of pterygium fibroblasts

doi:

Figure Lengend Snippet: Skp2 siRNA inhibited the cell proliferation of PFC in vitro (Brdu). For Brdu incorporation, cells growing on coverslips were incubated with Brdu. Incorporated Brdu was detected with antibodies as described in the Methods. Statistical analysis after cell counting showed that Brdu positive cells decreased after hPFC transfection with Skp2 siRNA compared with control cells. **p<0.01 versus vehicle and blank control.

Article Snippet: Skp2 antibody and the Steptavidin-Alkaline Phosphatase Complex (SABC) kit were purchased from the Boster Company (Wuhan, China).

Techniques: In Vitro, BrdU Incorporation Assay, Incubation, Cell Counting, Transfection, Control

Journal: Molecular Vision

Article Title: Small interfering RNA targeting of S phase kinase-interacting protein 2 inhibits cell proliferation of pterygium fibroblasts

doi:

Figure Lengend Snippet: Skp2 siRNA inhibited the cell proliferation of PFC in vivo (Brdu). For Brdu incorporation, ptergium tissue were incubated with Brdu. Incorporated Brdu was detected with antibodies as described in the Methods. Statistical analysis after cell counting showed that Brdu positive cells decreased after hPFC transfection with Skp2 siRNA compared with control cells. **p<0.01 versus vehicle and blank control.

Article Snippet: Skp2 antibody and the Steptavidin-Alkaline Phosphatase Complex (SABC) kit were purchased from the Boster Company (Wuhan, China).

Techniques: In Vivo, BrdU Incorporation Assay, Incubation, Cell Counting, Transfection, Control

Journal: Molecular Vision

Article Title: Small interfering RNA targeting of S phase kinase-interacting protein 2 inhibits cell proliferation of pterygium fibroblasts

doi:

Figure Lengend Snippet: Down-regulation of PCNA protein by siRNA in PFC in vitro and in vivo. Immunofluorescence staining indicated the expression of PCNA decreased in PFC and NFC transfection with Skp2 siRNA ( C , F ) when compared with hPFC and hNFC transfection with pSuppressor vehicle ( A , D ) or without transfection ( B , E ). The scale bar is equal to 40 μm.

Article Snippet: Skp2 antibody and the Steptavidin-Alkaline Phosphatase Complex (SABC) kit were purchased from the Boster Company (Wuhan, China).

Techniques: In Vitro, In Vivo, Immunofluorescence, Staining, Expressing, Transfection

Journal: Molecular Vision

Article Title: Small interfering RNA targeting of S phase kinase-interacting protein 2 inhibits cell proliferation of pterygium fibroblasts

doi:

Figure Lengend Snippet: Down-regulation of PCNA protein by siRNA in PFC in vitro and in vivo. Immunofluorescence staining indicated the expression of PCNA decreased in PFC and NFC transfection with Skp2 siRNA ( C , F ) when compared with hPFC and hNFC transfection with pSuppressor vehicle ( A , D ) or without transfection ( B , E ). The scale bar is equal to 40 μm.

Article Snippet: Skp2 antibody and the Steptavidin-Alkaline Phosphatase Complex (SABC) kit were purchased from the Boster Company (Wuhan, China).

Techniques: In Vitro, In Vivo, Immunofluorescence, Staining, Expressing, Transfection

Journal: Molecular Vision

Article Title: Small interfering RNA targeting of S phase kinase-interacting protein 2 inhibits cell proliferation of pterygium fibroblasts

doi:

Figure Lengend Snippet: Skp2 siRNA inhibited the proliferation of PFC and NFC in vivo. Hematoxylin and eosin staining was performed to examine the histological changes 14 days after transfection. Obvious PFC and NFC proliferation was detected in pSuppressor vehicle group or without transfection ( A , D , B , E ). There was little PFC and NFC proliferation in Skp2 siRNA transfection group ( C , F ). Scale bar is equal to 20 μm.

Article Snippet: Skp2 antibody and the Steptavidin-Alkaline Phosphatase Complex (SABC) kit were purchased from the Boster Company (Wuhan, China).

Techniques: In Vivo, Staining, Transfection

Journal: bioRxiv

Article Title: Reciprocal regulation of p21 and Chk1 controls the Cyclin D1-RB pathway to mediate senescence onset after DNA damage-induced G2 arrest

doi: 10.1101/2021.08.17.452482

Figure Lengend Snippet: A. Immunoblots showing RB phosphorylation (red arrow and PS780-RB) and expression of different cyclins in HDF that were released from quiescence (G0) by serum addition (+FCS) for indicated times. LC, loading control. B. Flow cytometry DNA content profiles of the asynchronously growing non-treated (NT) and HDF exposed to bleomycin (Bleo) or ICRF-193 (ICRF) for 48 hours. C. Representative immunofluorescence images (n=3) showing co-expression of Ki67 and p21 in non-treated (NT) and HDF exposed to ICRF-193 (IC) for 16 and 48 hr. Scale bar, 10 μM. D. Quantification of Ki67 intensity in immunofluorescence images from HDF exposed to ICRF-193 (IC) for indicated times (% of NT). Pooled cells from four independent experiments. More than 100 cells were analyzed in each experiment. NT, non-treated cells. Box plot whiskers indicate 10-90% boundary. P values were calculated with two-sided Student’s t test; ***P ≤ 0.001, ****P ≤ 0.0001. E. Immunoblots showing changes in cell cycle regulators in HDF upon g irradiation (IR, 10Gy) or treatment with ICRF-193 (IC) for indicated times. NT, non-treated cells; SEN, senescent cells. LC, loading control. F. Phase-contrast images showing β-galactosidase staining of non-treated (NT) and HDF exposed to ICRF-193 (ICRF) for 2 or 4 weeks (n=2). NT, non-treated cells. G. Quantification of β-galactosidase staining in HDF exposed to γ irradiation (IR, 10Gy), ICRF-193 (IC) or bleomycin (Bl) for two weeks. NT, non-treated cells. Data are mean +/- SEM of at least two independent experiments. P values were calculated with two-sided Student’s t test; ****P ≤ 0.0001. Loading controls (LC) were amido-black stained membranes.

Article Snippet: cyclin E1 (HE12, SCBT sc-247), https://www.scbt.com/fr/p/cyclin-e-antibody-he12 cyclin D1 (CST 2926, Abcam ab16663), https://www.cellsignal.com/products/primary-antibodies/cyclin-d1-dcs6-mouse-mab/2926 https://www.abcam.com/cyclin-d1-antibody-sp4-ab16663.html cyclin A (Novocastra 6E6 and SCBT sc751), https://www.citeab.com/antibodies/725566-ab16726-anti-cyclin-a2-antibody-6e6 https://www.scbt.com/fr/p/cyclin-a-antibody-h-432 p21 (CST 2946, 2947), https://www.cellsignal.com/products/primary-antibodies/p21-waf1-cip1-12d1-rabbit-mab/2947 https://www.cellsignal.com/products/primary-antibodies/p21-waf1-cip1-dcs60-mouse-mab/2946 H2A.X phospho-S139 (Merck Millipore clone JBW301, 05-636), https://www.merckmillipore.com/FR/fr/product/Anti-phospho-Histone-H2A.X-Ser139-Antibody-clone-JBW301,MM_NF-05-636-I?ReferrerURL=https%3A%2F%2Fwww.google.com%2F&bd=1 53BP1 (Novus biologicals NB100-304); https://www.novusbio.com/products/53bp1-antibody_nb100-304 Ki-67 (Abcam ab16667) https://www.abcam.com/ki67-antibody-sp6-ab16667.html Ki-67 (BDTL 610968) https://www.bdbiosciences.com/en-us/products/reagents/microscopy-imaging-reagents/immunofluorescence-reagents/purified-mouse-anti-human-ki-67.610969

Techniques: Western Blot, Phospho-proteomics, Expressing, Control, Flow Cytometry, Immunofluorescence, Irradiation, Staining

Journal: bioRxiv

Article Title: Reciprocal regulation of p21 and Chk1 controls the Cyclin D1-RB pathway to mediate senescence onset after DNA damage-induced G2 arrest

doi: 10.1101/2021.08.17.452482

Figure Lengend Snippet: A. Immunoblots showing G1 cyclins, p21 and RB/p130 phosphorylation after DNA damage- induced cell cycle exit (left panel) and in senescent cells (right panel). HDF were exposed to bleomycin (Bl) or ICRF-193 (IC) for indicated times and for 12 hr in right panel. Sen: population doubling (PD) 74. NT, non-treated cells. Arrow shows CycA band. LC, loading control. B. Violin plots showing nuclear size in non-treated (NT) and HDF exposed to ICRF for 48 hr and 12 days. More than 200 cells were analysed in each experiment (n=2). Insert: phase contrast images showing β-galactosidase staining of HDF exposed to ICRF-193 (IC) for two weeks. C. Representative immunofluorescence images (n=3) showing co-expression of CycD1 and CycA (upper panel) or p21 (lower panel) in non-treated (NT) and HDF and exposed to ICRF- 193 (IC) for 16 hours. Bar, 10 μM. D. Quantification of Cyclin D1 intensity in Cyclin A-positive nuclei in non-treated (NT) and HDF exposed to ICRF-193 (IC) for 8 or 16 hours (% of NT). Pooled cells (>200) from three independent experiments. Box plot whiskers indicate 10-90% boundary. E. Quantification of p21 intensity in Cyclin D1-positive nuclei in non-treated (NT) and HDF exposed to ICRF-193 (IC) for indicated times (% of NT). Pooled cells (>200) from three independent experiments. Box plot whiskers indicate 10-90 % boundary. F. Immunoblots of the indicated proteins in p21 immunoprecipitates (IP) from extracts of non-treated (NT) and HDF exposed to bleomycin (Bl) or ICRF-193 (IC) for indicated times. G. Immunoblots of the indicated proteins in CycD1 immunoprecipitates (IP) from extracts of non-treated (NT) and HDF exposed to bleomycin (Bl) or ICRF-193 (IC) for 16 hr. H. Immunoblots showing effects of p21 KD on Cyclin D1-specific RB phosphorylation (P S780 ) in extracts from non-treated (NT) or HDF and HMEC exposed to ICRF-193 (IC) for 16 hr. Arrows shows increased P S780 -RB after p21 KD (+). LC, loading control. I. Immunoblots of the indicated proteins in CycD1 immunoprecipitates (IP) from extracts of non-treated (NT) or HDF exposed to ICRF-193 (IC) for 16 hr that were previously depleted (+) or not (-) for p21 (sip21). J. Immunoblots showing effects of CycD1 and CycE1 knockdown on p130 and RB phosphorylation (arrows). HDF were depleted for CycD1 (cD1) or CycE1 (cE1) for 24 hr. Ct, siRNA control. LC, loading control. K. Quantification of Ki67 intensity in T 121 -expressing fibroblasts exposed to bleomycin (Bl) or ICRF-193 (IC) for 48 hr (% of NT). Doxycycline (Do) was added 12 hr before exposure to genotoxic agents. Pooled cells (>100) from two independent experiments. Box plot whiskers indicate 10-90 % boundary. NT, non-treated cells. Loading controls (LC) were amido-black stained membranes. Data are representative or mean +/- SEM of at least two independent experiments. P values were calculated with two-sided Student’s t test; ***P ≤ 0.001, ****P ≤ 0.0001.

Article Snippet: cyclin E1 (HE12, SCBT sc-247), https://www.scbt.com/fr/p/cyclin-e-antibody-he12 cyclin D1 (CST 2926, Abcam ab16663), https://www.cellsignal.com/products/primary-antibodies/cyclin-d1-dcs6-mouse-mab/2926 https://www.abcam.com/cyclin-d1-antibody-sp4-ab16663.html cyclin A (Novocastra 6E6 and SCBT sc751), https://www.citeab.com/antibodies/725566-ab16726-anti-cyclin-a2-antibody-6e6 https://www.scbt.com/fr/p/cyclin-a-antibody-h-432 p21 (CST 2946, 2947), https://www.cellsignal.com/products/primary-antibodies/p21-waf1-cip1-12d1-rabbit-mab/2947 https://www.cellsignal.com/products/primary-antibodies/p21-waf1-cip1-dcs60-mouse-mab/2946 H2A.X phospho-S139 (Merck Millipore clone JBW301, 05-636), https://www.merckmillipore.com/FR/fr/product/Anti-phospho-Histone-H2A.X-Ser139-Antibody-clone-JBW301,MM_NF-05-636-I?ReferrerURL=https%3A%2F%2Fwww.google.com%2F&bd=1 53BP1 (Novus biologicals NB100-304); https://www.novusbio.com/products/53bp1-antibody_nb100-304 Ki-67 (Abcam ab16667) https://www.abcam.com/ki67-antibody-sp6-ab16667.html Ki-67 (BDTL 610968) https://www.bdbiosciences.com/en-us/products/reagents/microscopy-imaging-reagents/immunofluorescence-reagents/purified-mouse-anti-human-ki-67.610969

Techniques: Western Blot, Phospho-proteomics, Control, Staining, Immunofluorescence, Expressing, Knockdown

Journal: bioRxiv

Article Title: Reciprocal regulation of p21 and Chk1 controls the Cyclin D1-RB pathway to mediate senescence onset after DNA damage-induced G2 arrest

doi: 10.1101/2021.08.17.452482

Figure Lengend Snippet: A. Model: Expected effects of Chk1 and Chk2 knockdown on RB pathway and DNA damage-induced G2 arrest/exit switch. B. Flow cytometry DNA content profiles of non-treated (NT-siCtl) and siCtl, siChk1 and siChk2 U2OS cells exposed to bleomycin (Bleo) and ICRF-193 (ICRF) for 48 hr. See Fig.5SA for NT-siChk1 and NT-siChk2. C. Phase contrast images from video-microscopy sequences showing binuclear daughter cell after mitosis and cytokinesis failure (arrowheads) in ICRF- treated U2OS Chk1 KD cells. Time after the addition of the drug is indicated. D. Immunoblots showing effects of Chk1 (siCh1-black arrow) and Ckh2 (siCh2-white arrow) knockdown on Ki67 and the indicated cell cycle regulators in non-treated (NT) and U2OS cells exposed to ICRF-193 (ICRF) or bleomycin (Bleo) for 48 hr. LC, loading control. See for 16 hr time-point. E. Immunoblot analysis of Cyclin D1 and Cyclin E1 immunoprecipitates (IP) showing effects of Chk1 (Ch1-black arrow) and Chk2 (Ch2-white arrow) knockdown on cyclin-CDK-p21 complexes in U2OS exposed to ICRF-193 (ICRF) and bleomycin (Bleo) for 48 hr. Arrows: reduction of CDK6 in CycD1 complexes. F. Immunoblots showing effects of CDK4 (K4), CDK6 (K6) or double CDK4 / CDK6 (K4/K6) knockdown on Cyclin D1-specific RB phosphorylation (P S795 , P S780 ) in U2OS cells. Loading controls (LC) are amido-black stained membranes.

Article Snippet: cyclin E1 (HE12, SCBT sc-247), https://www.scbt.com/fr/p/cyclin-e-antibody-he12 cyclin D1 (CST 2926, Abcam ab16663), https://www.cellsignal.com/products/primary-antibodies/cyclin-d1-dcs6-mouse-mab/2926 https://www.abcam.com/cyclin-d1-antibody-sp4-ab16663.html cyclin A (Novocastra 6E6 and SCBT sc751), https://www.citeab.com/antibodies/725566-ab16726-anti-cyclin-a2-antibody-6e6 https://www.scbt.com/fr/p/cyclin-a-antibody-h-432 p21 (CST 2946, 2947), https://www.cellsignal.com/products/primary-antibodies/p21-waf1-cip1-12d1-rabbit-mab/2947 https://www.cellsignal.com/products/primary-antibodies/p21-waf1-cip1-dcs60-mouse-mab/2946 H2A.X phospho-S139 (Merck Millipore clone JBW301, 05-636), https://www.merckmillipore.com/FR/fr/product/Anti-phospho-Histone-H2A.X-Ser139-Antibody-clone-JBW301,MM_NF-05-636-I?ReferrerURL=https%3A%2F%2Fwww.google.com%2F&bd=1 53BP1 (Novus biologicals NB100-304); https://www.novusbio.com/products/53bp1-antibody_nb100-304 Ki-67 (Abcam ab16667) https://www.abcam.com/ki67-antibody-sp6-ab16667.html Ki-67 (BDTL 610968) https://www.bdbiosciences.com/en-us/products/reagents/microscopy-imaging-reagents/immunofluorescence-reagents/purified-mouse-anti-human-ki-67.610969

Techniques: Knockdown, Flow Cytometry, Microscopy, Western Blot, Control, Phospho-proteomics, Staining

Journal: bioRxiv

Article Title: Reciprocal regulation of p21 and Chk1 controls the Cyclin D1-RB pathway to mediate senescence onset after DNA damage-induced G2 arrest

doi: 10.1101/2021.08.17.452482

Figure Lengend Snippet: A. Flow cytometry DNA content profiles of control (siCtl) and U2OS cells depleted for Chk1 (siCh1), p21 (sip21) or both (siCh1/21) proteins exposed to bleomycin (Bleo) for 48 hours. Arrow indicates sub-G1 population representing death cells. See for effects of respective knockdowns on non-treated and U2OS exposed to bleomycin for 16 hours. B. Immunoblots showing effects of Chk1 (Ch1), p21 or double p21/Chk1 (DKD) knockdown on Ki67 and indicated cell cycle regulators and DNA damage signalling effectors in U2OS cells exposed to bleomycin for 48 hr. LC, loading control. See for effects of respective knockdowns on non-treated and U2OS exposed to bleomycin for 16 hr. C. Immunoblots showing effects of caffeine (Caf) and ATM inhibitor KU-55933 (Ku) on RB, P T68 -Chk2, P S317 -Chk1 and p21 in non-treated (NT) and U2OS cells exposed to bleomycin for 48 hr (arrows). LC, loading control. D. Representative phase contrast images from a video-microscopy sequence showing fragmented nuclei after mitotic catastrophe in bleomycin-treated double Ckh1/p21 KD cells. Time (days, hours) after drug addition is indicated. Arrows show mitotic cell and resulting daughter cells. Most right-hand image (upper panel) shows cell debris from the same sequence. Complete field is shown in . E. Immunoblots showing effects of Chk1 (Ch1), p21 or double p21/Chk1 (DKD) knockdowns on Ki67 and the indicated cell cycle regulators in HCT-116 cells exposed to bleomycin (Bleo) for 8 and 48 hours. LC, loading control. F. Immunoblots of CycD1 immunoprecipitates (IP) showing effects of Chk1 (Ch1), p21 or double p21/Chk1 (DKD) knockdowns on cyclin-CDK-CKI complexes in HCT-116 cells exposed to bleomycin (Bl) for 48 hr. Loading controls (LC) were amido-black stained membranes. Black arrow: Chk1 KD; White arrow; p21 KD; asterisk: DKD. G. Model: Proposed roles of Chk1, Chk2 and p21-CycD-RB axis in DNA damage-induced senescence onset after permanent G2 arrest (G2 exit). See text for details.

Article Snippet: cyclin E1 (HE12, SCBT sc-247), https://www.scbt.com/fr/p/cyclin-e-antibody-he12 cyclin D1 (CST 2926, Abcam ab16663), https://www.cellsignal.com/products/primary-antibodies/cyclin-d1-dcs6-mouse-mab/2926 https://www.abcam.com/cyclin-d1-antibody-sp4-ab16663.html cyclin A (Novocastra 6E6 and SCBT sc751), https://www.citeab.com/antibodies/725566-ab16726-anti-cyclin-a2-antibody-6e6 https://www.scbt.com/fr/p/cyclin-a-antibody-h-432 p21 (CST 2946, 2947), https://www.cellsignal.com/products/primary-antibodies/p21-waf1-cip1-12d1-rabbit-mab/2947 https://www.cellsignal.com/products/primary-antibodies/p21-waf1-cip1-dcs60-mouse-mab/2946 H2A.X phospho-S139 (Merck Millipore clone JBW301, 05-636), https://www.merckmillipore.com/FR/fr/product/Anti-phospho-Histone-H2A.X-Ser139-Antibody-clone-JBW301,MM_NF-05-636-I?ReferrerURL=https%3A%2F%2Fwww.google.com%2F&bd=1 53BP1 (Novus biologicals NB100-304); https://www.novusbio.com/products/53bp1-antibody_nb100-304 Ki-67 (Abcam ab16667) https://www.abcam.com/ki67-antibody-sp6-ab16667.html Ki-67 (BDTL 610968) https://www.bdbiosciences.com/en-us/products/reagents/microscopy-imaging-reagents/immunofluorescence-reagents/purified-mouse-anti-human-ki-67.610969

Techniques: Flow Cytometry, Control, Western Blot, Knockdown, Microscopy, Sequencing, Staining

Journal: bioRxiv

Article Title: Reciprocal regulation of p21 and Chk1 controls the Cyclin D1-RB pathway to mediate senescence onset after DNA damage-induced G2 arrest

doi: 10.1101/2021.08.17.452482

Figure Lengend Snippet: A. Flow cytometry DNA content profiles of non-treated control (siCtl) and bleomycin-treated U2OS cells depleted for Chk1 (siCh1), p21 (sip21) or both proteins (siCh1/21) after 16 hr. B. Immunoblots showing effects of siRNA-mediated Chk1 (Ch1), p21 or double p21/Chk1 (DKD) knockdown on p130 and RB phosphorylation and expression of Ki67 and different cell cycle regulators in non-treated (NT) and U2OS cells exposed to bleomycin (Bleo) for 16 hours. Arrows show diminished Ki67 levels and pRb/p130 phosphorylation, accumulation of D-type cyclins and downregulation of Cdk6 upon Chk1 KD that are prevented by p21 knockdown. LC, loading control. C. Immunoblots showing effects of Chk1 (Ch1) or p21 knockdown on pocket protein phosphorylation and expression of different cell cycle regulators in non-treated (NT) and U2OS cells exposed to bleomycin (Bleo) for 7 days (7d). Black arrows: elevated p130 and pRb hyper-phosphorylation after p21 depletion. Red arrowhead: strong P S317 -Chk1 signal in the absence of p21. Blue arrowhead: low Cdk6 levels. LC, loading control. D. Flow cytometry DNA content profiles of non-treated (NT) and U2OS cells exposed to bleomycin for 24 hr. Caffeine (Caf) and KU-55933 (Ku) were added one hour before treatment. E. Representative phase contrast images from a single video-microscopy sequence showing bleomycin-treated control (siCtl) and double Chk1/p21 knock down (siChk1/p21) U2OS cells at different times after exposure to the drug. Red rectangles show the inserts presented in . Loading controls (LC) were amido-black stained membranes. Black arrow: Chk1 KD; White arrow; p21 KD; asterisk: DKD

Article Snippet: cyclin E1 (HE12, SCBT sc-247), https://www.scbt.com/fr/p/cyclin-e-antibody-he12 cyclin D1 (CST 2926, Abcam ab16663), https://www.cellsignal.com/products/primary-antibodies/cyclin-d1-dcs6-mouse-mab/2926 https://www.abcam.com/cyclin-d1-antibody-sp4-ab16663.html cyclin A (Novocastra 6E6 and SCBT sc751), https://www.citeab.com/antibodies/725566-ab16726-anti-cyclin-a2-antibody-6e6 https://www.scbt.com/fr/p/cyclin-a-antibody-h-432 p21 (CST 2946, 2947), https://www.cellsignal.com/products/primary-antibodies/p21-waf1-cip1-12d1-rabbit-mab/2947 https://www.cellsignal.com/products/primary-antibodies/p21-waf1-cip1-dcs60-mouse-mab/2946 H2A.X phospho-S139 (Merck Millipore clone JBW301, 05-636), https://www.merckmillipore.com/FR/fr/product/Anti-phospho-Histone-H2A.X-Ser139-Antibody-clone-JBW301,MM_NF-05-636-I?ReferrerURL=https%3A%2F%2Fwww.google.com%2F&bd=1 53BP1 (Novus biologicals NB100-304); https://www.novusbio.com/products/53bp1-antibody_nb100-304 Ki-67 (Abcam ab16667) https://www.abcam.com/ki67-antibody-sp6-ab16667.html Ki-67 (BDTL 610968) https://www.bdbiosciences.com/en-us/products/reagents/microscopy-imaging-reagents/immunofluorescence-reagents/purified-mouse-anti-human-ki-67.610969

Techniques: Flow Cytometry, Control, Western Blot, Knockdown, Phospho-proteomics, Expressing, Microscopy, Sequencing, Staining