Journal: Cancers

Article Title: CITK Loss Inhibits Growth of Group 3 and Group 4 Medulloblastoma Cells and Sensitizes Them to DNA-Damaging Agents

doi: 10.3390/cancers12030542

Figure Lengend Snippet: CITK knockdown decreases proliferation and induces cytokinesis failure in medulloblastoma cell lines. ( A ) Western blot analysis of total lysate from D283 and D341 cells, 72 h after treatment, with nontargeting (siCtrl) or CITK-specific siRNAs (siCITK1 and siCITK2). The level of CITK was analyzed, and the internal loading control was vinculin (VINC). The numbers below each band express the ratio between its normalized intensity and the average normalized intensity of the siCtrl. ( B ) 10,000 D283 cells were transfected with siCtrl, siCITK1 and siCITK2 and plated in triplicates. Growth curves were obtained by assessing cell number in each well, at 100, 150 and 200 h after transfection. ( C ) 80,000 D341 cells were transfected with siCtrl, siCITK1 and siCITK2 and plated in triplicates. Growth curves were obtained by assessing cell number in each well at 72 and 144 h after transfection. ( D ) Representative image of D283 cells processed for immunofluorescence 100 h after transfection with nontargeting or CITK-specific siRNA and stained with DAPI and anti-Phalloidin antibody. ( E ) Quantification of binucleated D283 cells in the indicated transfections, performed as in ( D ). ( F ) Representative image of D341 processed for immunofluorescence 72 h after transfection with nontargeting or CITK-specific siRNA and stained with DAPI and anti-Phalloidin antibody. ( G ) Quantification of binucleated D341 cells in the indicated transfections, performed as in ( F ). All quantifications were based on three independent biological replicates. Error bars, SEM. *, p < 0.05; **, p < 0.01, ***, p < 0.001; two-tailed Student’s t -test. Scale bars, 10 µm.

Article Snippet: To analyze cell proliferation, 1 × 10 4 D283 cells were seeded into 24-well plates, transfected with siCtrl, siCITK1 and siCITK2 and counted in triplicates, after 100, 150 and 250 h. Then, 8 × 10 4 D341 cells were seeded into 6-well plates, transfected with siCtrl, siCITK1 and siCITK2 and counted, after 72 and 144 h, in triplicates, with Biorad TC20 Automated Cell Counter (Bio-Rad, Hercules, CA, USA).

Techniques: Knockdown, Western Blot, Control, Transfection, Immunofluorescence, Staining, Two Tailed Test

Journal: Cancers

Article Title: CITK Loss Inhibits Growth of Group 3 and Group 4 Medulloblastoma Cells and Sensitizes Them to DNA-Damaging Agents

doi: 10.3390/cancers12030542

Figure Lengend Snippet: CITK knockdown leads to apoptosis and cell-cycle arrest in D283 and D341 cell lines. ( A ) Western blot analysis of total lysates from D283 and D341 cells, 100 and 72 h, respectively, after treatment with nontargeting (siCtrl) or CITK-specific siRNAs (siCITK1 and siCITK2). The level of CITK, pTP53, BAX, P21 and cCASP3 were analyzed, and the internal loading control was vinculin (VINC). ( B , C ) Quantification of the relative density of pTP53, BAX, P21 and cCASP3 in D283 and D341-treated cells, normalized to VINC and siCtrl average values. ( D ) Example of cell-cycle profile of D283 cells transfected with siCtrl and siCITK1 for 100 h. G0/G1, S and G2-M phases are shown. ( E ) Quantification of the percentage of D283 and D341 treated cells in the different cell-cycle phases. ( F ) Representative image of D283 processed for immunofluorescence 100 h after transfection with nontargeting or CITK-specific siRNA and stained with DAPI and TUNEL assay. ( G ) Quantification of the percentage of TUNEL positive nuclei in D283 and D341 treated cells. All quantifications were based on three independent biological replicates. Error bars, SEM. *, p < 0.05; **, p < 0.01, ***, p < 0.001; two-tailed Student’s t -test. Scale bars, 10 µm.

Article Snippet: To analyze cell proliferation, 1 × 10 4 D283 cells were seeded into 24-well plates, transfected with siCtrl, siCITK1 and siCITK2 and counted in triplicates, after 100, 150 and 250 h. Then, 8 × 10 4 D341 cells were seeded into 6-well plates, transfected with siCtrl, siCITK1 and siCITK2 and counted, after 72 and 144 h, in triplicates, with Biorad TC20 Automated Cell Counter (Bio-Rad, Hercules, CA, USA).

Techniques: Knockdown, Western Blot, Control, Transfection, Immunofluorescence, Staining, TUNEL Assay, Two Tailed Test

Journal: eLife

Article Title: Inhibitors of Rho kinases (ROCK) induce multiple mitotic defects and synthetic lethality in BRCA2-deficient cells

doi: 10.7554/eLife.80254

Figure Lengend Snippet: ( A ) Quantitative real-time PCR of CITK in shBRCA2 HCT116 p21-/- cells transfected with 150 μM of siCITK (N=2). ( B ) Relative cell number (%) of PEO4 and PEO1 after 6 days of being transfected with siROCK (1+2), siCITK or siROCK (1+2)/siCITK and representative images of the transfected cells (N=2). ( C ) Percent of binucleated PEO1 cells transfected with siROCK (1+2), CITK or siROCK (1+2)/siCITK (N=2). ( D ) Quantitative real-time PCR of EMI1 in shBRCA2 HCT116 p21-/- cells transfected with 150 μM of siEMI1 (N=2). ( E ) Relative cell number (%) of PEO4 and PEO1 after 6 days of being transfected with siEMI1 and treated with fasudil (N=2). Representative images of the transfected and treated cells. ( F ) Cell cycle analysis of PEO1 cells following transfection with siEMI1 for 48hs (N=2). Cells were stained with propidium iodide and DNA content was analyzed via FACS (10,000 events per sample). ( G ) Model depicting the events leading to BRCA2-deficient cell death after fasudil treatment. The inhibition or depletion of ROCK in BRCA2-deficient cells leads to cytokinesis failure. As a result, the daughter cells are binucleated (4N) and have extra centrosomes (two instead of one). We speculate that after a subsequent DNA duplication, these cells can attempt mitosis. Mitosis entry with increased DNA content and extra centrosomes may frequently give rise to abnormal and multipolar spindles, leading to misaligned chromosomes and mitotic failure due to multipolar spindle formation. Alternatively, cytokinesis may fail again, and cells may temporarily survive as multinucleated cells, possibly facing cell death during subsequent mitotic attempts.

Article Snippet: Unless otherwise stated, cells were transfected for a total of 48 hr. siROCK1 (#sc-29473 Santa Cruz Biotechnology) and siROCK2 (#sc-29474, Santa Cruz Biotechnology) were used at 100 nM. siEMI1 (#sc-37611 Santa Cruz Biotechnology) and siCITK (#sc-39214 Santa Cruz Biotechnology) were both used at 100 nM.

Techniques: Real-time Polymerase Chain Reaction, Transfection, Cell Cycle Assay, Staining, Inhibition

Journal: eLife

Article Title: Inhibitors of Rho kinases (ROCK) induce multiple mitotic defects and synthetic lethality in BRCA2-deficient cells

doi: 10.7554/eLife.80254

Figure Lengend Snippet: ( A ) Relative cell number (%) of shScramble and shBRCA2 HCT116 p21-/- cells at 6 days after transfection with siCITK or siROCK (1+2) (N=2). Representative images are shown on the right. ( B ) Relative cell number (%) of shScramble and shBRCA2 HCT116 p21-/- cells at 6 days after transfection with siEMI1. Samples were treated with fasudil when indicated (N=2). Representative images are shown on the right. Statistical analysis was performed using a two-way ANOVA test followed by a Bonferroni post-test (*p<0.05, **p<0.01, ***p<0.001). Data are shown as the average of independent experiments with the standard error of the mean.

Article Snippet: Unless otherwise stated, cells were transfected for a total of 48 hr. siROCK1 (#sc-29473 Santa Cruz Biotechnology) and siROCK2 (#sc-29474, Santa Cruz Biotechnology) were used at 100 nM. siEMI1 (#sc-37611 Santa Cruz Biotechnology) and siCITK (#sc-39214 Santa Cruz Biotechnology) were both used at 100 nM.

Techniques: Transfection