plasmid pcdna 3 rrm2  (Addgene inc)

Journal: bioRxiv

Article Title: KDM5 histone-demethylases contribute to replication stress response and tolerance

doi: 10.1101/2019.12.16.877399

Figure Lengend Snippet: A- Relative mRNA expression levels of KDM5A, KDM5B, and the E2F target genes RRM2, CDC6 and CCNE1 upon transfection of the indicated siRNA in U2OS cells (siCtle corresponds to a non-targeting pool of siRNA). Expression levels were normalized to the reference gene P0 (ribosomal phosphoprotein P0) and calculated relative to 1 for the siCtle sample. The mean and standard deviation from 3 independent experiments are shown. The star (*) indicates significant difference between the siCtle and the K5A/B siRNA treated cells (pvalue <0.05 calculated using a paired t-test). B- Western-blot analysis of KDM5A, KDM5B, RRM2 and GAPDH as a loading control from U2OS cells transfected with siRNA directed against KDM5A and KDM5B. Two distinct couples of siRNA (siK5A+B-1 and -2) were used. C- Cell cycle distribution of U2OS cells depleted for KDM5A and KDM5B using siK5A+B-1 compared to siCtle treated cells, analyzed by the high content imaging system Operetta following EdU labeling and DAPI staining. D- Percentage of living cells following depletion of KDM5A and B using siK5A+B-1 siRNAs. The mean and standard deviation from 3 independent experiments are shown, following normalization to 100 for siCtle treated cells. Paired t-tests indicate a pvalue <0.05 between the first couple of siK5A/B siRNA and siCtle treated cells (*) but not for the second one with pvalue=0.054. E- ChIP analysis of KDM5A on the RRM2 and CDC6 promoter (Prom.) and coding (Cod.) regions. The myogenin gene is not expressed in U2OS cells and its promoter serves as a negative control. A representative experiment out of 4 is shown. F- Western-blot analysis of KDM5A, KDM5B, RRM2 and GAPDH from U2OS cells treated each 24 hours or not with KDM5 inhibitor CPI-455 for 48 hours. G- Relative mRNA expression levels of KDM5A, KDM5B and RRM2 in cells treated, each 24 hours, with 12.5 mM KDM5 inhibitor CPI-455 (+) or DMSO (-) for 48 hours. Expression levels were normalized to the reference gene P0 (ribosomal phosphoprotein P0) and calculated relative to 1 for the siCtle sample. The mean and standard deviation from 3 independent experiments are shown. A paired t-test indicated no significant difference for all tested genes between CPI treated and untreated cells. H- Percentage of living cells following treatment each 24 hours with CPI-455 for 72 hours (+) or DMSO (-). The mean and standard deviation from 3 independent experiments are shown, following normalization to 100 for DMSO treated cells. A paired t-test indicated no significant difference between CPI treated and untreated cells.

Article Snippet: The plasmid pCDNA 3 -RRM2 was purchased from Addgene.

Techniques: Expressing, Transfection, Standard Deviation, Western Blot, Control, Imaging, Labeling, Staining, Negative Control

Journal: bioRxiv

Article Title: KDM5 histone-demethylases contribute to replication stress response and tolerance

doi: 10.1101/2019.12.16.877399

Figure Lengend Snippet: A -­-Viability of U2OS, H25 and H50, measured by WST assay, 72 hours following treatment with increasing doses of HU, as indicated. B -­-Western-­-blot analysis of CHK1 and S354-­-phospho CHK1 (P-­-CHK1), in U2OS, H25 and H50 cells before and following 1 hour treatment with 1mM HU. C-­- mRNA expression levels of KDM5A, KDM5B and RRM2, in U2OS, H25 and H50 cell lines. mRNA expression are normalized with the reference gene P0, and calculated relative to 1 for the siCtle. The mean and standard deviation from 3 independent experiments are shown. * pvalue<0.05 using a paired t-­-test. D -­-levels of KDM5A, KDM5B and RRM2 were analyzed by western-­-blot in the parental U2OS cells and its HU tolerant derivatives H25 and H50, grown in the presence of HU at 0, 0.25, and 0.5 mM respectively. GAPDH is used as a loading control. A representative experiment is shown.

Article Snippet: The plasmid pCDNA 3 -RRM2 was purchased from Addgene.

Techniques: WST Assay, Western Blot, Expressing, Standard Deviation, Control

Journal: bioRxiv

Article Title: KDM5 histone-demethylases contribute to replication stress response and tolerance

doi: 10.1101/2019.12.16.877399

Figure Lengend Snippet: A- Relative mRNA expression levels of KDM5A, KDM5B, and RRM2, upon transfection of the indicated siRNA in H50 cells. Expression levels were normalized to the reference gene P0 and calculated relative to 1 for the siCtle sample. The mean and standard deviation from 4 independent experiments are shown, following normalization to 100 for siCtle treated cells. A paired t-test indicates significant difference between siK5A+B or siRRM2 and siCtle treated cells (*: pvalue<0.05, **: pvalue<0.01) B- Western-blot analysis of KDM5A, KDM5B, RRM2 and GAPDH as a loading control from H50 cells transfected with the indicated siRNA. C -Percentage of living cells following transfection of the indicated siRNA. The mean and standard deviation from 3 independent experiments are shown, following normalization to 100 for siCtle treated cells. A paired t-test indicates significant difference between siK5A+B or siRRM2 and siCtle treated cells (*: pvalue=0.013, **: pvalue=0.011) D- Western-blot analysis of KDM5A, KDM5B, RRM2, GAPDH, H3K4me3 and histone H3 from U2OS cells treated each 24 hours with 12.5 μM KDM5 inhibitor CPI-455 (+) or DMSO (-) for 48 hours. E- Relative mRNA expression levels of KDM5A, KDM5B and RRM2 in cells treated, each 24 hours, with 12.5 μM KDM5 inhibitor CPI-455 (+) or DMSO (-) for 48 hours. Expression levels were normalized to the reference gene P0 and calculated relative to 1 for the siCtle sample. The mean and standard deviation from 3 independent experiments are shown. The star * indicates significant difference between siK5A+B and siCtle treated cells (pvalue < 0.05, paired t test). F- Percentage of living cells following treatment of H50 cells each 24 hours with 12.5 μM CPI-455 for 72 hours (+) or DMSO (-). The mean and standard deviation from 3 independent experiments are shown, following normalization to 100 for DMSO-treated cells. G- H50 cells were treated with the indicated siRNA and 24 hours later transfected with pCDNA 3 -RRM2 p(RRM2) (+) or the empty vector (-). Expression of KDM5A, KDM5B, RRM2 and GAPDH were analyzed by western-blot 24 hours after plasmids transfection. H -Percentage of living cells 72 hours following electroporation of the indicated siRNA, combined to transfection of pCDNA 3 -RRM2 (pRRM2: +) or the empty vector (pCDNA 3: -) 24 hours later. To ensure efficient knockdown of KDM5A/B, cells were transfected once more with siRNA 24 hours following plasmids transfection. The mean and standard deviation from 4 independent experiments are shown, following normalization to 100 for siCtle/pCDNA 3 transfected cells. * Statistical analysis with a paired t-test indicated that RRM2 surexpression significantly rescue the viability of K5A/B depleted cells with a pvalue <0.05.

Article Snippet: The plasmid pCDNA 3 -RRM2 was purchased from Addgene.

Techniques: Expressing, Transfection, Standard Deviation, Western Blot, Control, Plasmid Preparation, Electroporation, Knockdown