Journal: bioRxiv

Article Title: ATR enforcement of the S/G2 checkpoint prevents premature S phase shutdown and genome instability

doi: 10.64898/2026.05.07.723638

Figure Lengend Snippet: a.) Scatterplot of DNA content versus mean EdU intensity in MCF10A cells. Colors shown in the key and dotted lines denote gating used for analysis throughout this publication. b.) Scatterplot of DNA content versus median cyclin B1 intensity in MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. Colors shown in the key and dotted lines denote cyclin B1 gating used for analysis throughout this article. c.) Scatterplot of DNA content versus mean EdU intensity in MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. Colors shown in the key and dotted lines denote cyclin B1 gating used for analysis throughout this article. d.) Quantification of mean cyclin B1 intensity in MCF10A cells with non-targeting or cyclin B1 knockdowns to demonstrate cyclin B1 antibody specificity. A Mann-Whitney test was conducted to assess significance. Error bars are representative of 10-90 percentile range. P value: <0.0001. e.) Quantification of median cyclin B1 cytoplasmic intensity across the cell cycle from RPE-1 cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs (n=3 biological replicates). Data are presented as mean ± SEM. f.) Quantification of integrated SLBP intensity in MCF10A cells with non-targeting or SLBP knockdowns to demonstrate SLBP antibody specificity. Error bars are representative of 10-90 percentile range. A Mann-Whitney test was conducted to assess significance. P value: <0.0001. g.) Breakdown of quantification of median SLBP nuclear intensity across the cell cycle from into individual graphs. Data are presented as mean ± SEM. h.) Quantification of median SLBP nuclear intensity across the cell cycle from MCF10A cells treated with DMSO, 5μM AZD6738, 5μM RO-3306, or a combination of 5μM AZD6738 and 5μM RO-3306 for 1hr (n=3 biological replicates). Data are presented as mean ± SEM. i.) Quantification of median SLBP nuclear intensity across the cell cycle from MCF10A cells treated with DMSO, 5μM AZD6738, 1μM NU6140, or a combination of 5μM AZD6738 and 1μM NU6140 for 1hrs (n=3 biological replicates). Data are presented as mean ± SEM.

Article Snippet: Human hTERT RPE-1 cells (ATCC, CRL-4000) were grown in DMEM/F12 medium supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, A5256801), 100U/mL penicillin/streptomycin, and 10μg/mL hygromycin B (Roche, 10843555001).

Techniques: MANN-WHITNEY

Journal: bioRxiv

Article Title: ATR enforcement of the S/G2 checkpoint prevents premature S phase shutdown and genome instability

doi: 10.64898/2026.05.07.723638

Figure Lengend Snippet: a.) Quantification of median cyclin B1 cytoplasmic intensity in RPE-1 cells with low or high yH2AX across the cell cycle. Cells were treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs (n=3 biological replicates). Data are presented as mean ± SEM. b.) Quantification of mean yH2AX nuclear intensities across the cell cycle from control and cyclin B1 knockdown RPE-1 cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs (n=3 biological replicates). Data are presented as mean ± SEM. c.) Quantification of mean yH2AX nuclear intensity across the cell cycle from MCF10A cells treated with DMSO + DMSO, DMSO + 5μM AZD6738, DMSO + 2μM LY2603618, 5μM KU60019 + DMSO, 5μM KU60019 + 5μM AZD6738, 5μM KU60019 + or 2μM LY2603618 + 5μM KU60019 for 7hrs (n=3 biological replicates). Data are presented as mean ± SEM. d.) Quantification of mean yH2AX nuclear intensity across the cell cycle from MCF10A cells treated with DMSO + DMSO, DMSO + 5μM AZD6738, DMSO + 2μM LY2603618, 5μM KU60019 + DMSO, 5μM NU7441 + 5μM AZD6738, 5μM NU7441 + or 2μM LY2603618 + 5μM NU7441 for 7hrs (n=3 biological replicates). Data are presented as mean ± SEM. e.) Representative images of cells undergoing normal mitosis versus mitotic failure. Scale bar is equal to 15μm.

Article Snippet: Human hTERT RPE-1 cells (ATCC, CRL-4000) were grown in DMEM/F12 medium supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, A5256801), 100U/mL penicillin/streptomycin, and 10μg/mL hygromycin B (Roche, 10843555001).

Techniques: Control, Knockdown

Journal: Journal of cell science

Article Title: MTCL2 promotes asymmetric microtubule organization by crosslinking microtubules on the Golgi membrane.

doi: 10.1242/jcs.259374

Figure Lengend Snippet: Fig. 7. MTCL2 depletion results in defective cell migration. (A) Confluent monolayers of HeLa-K cells subjected to control or MTCL2 RNAi were fixed and stained with the indicated antibodies 6 h after wounding. Cells facing the wound edges (white dotted lines) are shown. Scale bar: 50 µm. Note that MTCL2- depleted cells did not polarize MT arrays toward the wound. The right panel indicates the percentage of wound-edge cells with correctly oriented Golgi, defined as those falling in the indicated quadrant (white line) concerning the wound edge. Data represent the means±s.d. for the indicated number (n) of cells from two independent experiments. The P-value was estimated using an unpaired Student’s t-test assuming the two-tailed distribution and two-sample unequal variance. (B) Differential interference contrast images of wound healing RPE1 cells at 0 min and 7 h 20 min after wounding. White dotted line delineates the wound edges. Scale bar: 200 µm. Right panel indicates quantified data on the areas newly buried by cells after wounding. Data represent the mean±s.d. of 44 fields taken from two independent experiments. The P-value was estimated using an unpaired Student’s t-test assuming the two-tailed distribution and two-sample unequal variance. (C) RPE1 cells subjected to wound healing analysis in B were fixed and stained with the indicated antibodies. Cells facing the wound edges are shown. Right panels show an enlarged view. Arrowheads indicate the positions of the centrosomes. Note that MTCL2-depleted cells exhibit separation of the centrosome and Golgi. The centrosomes frequently show significant detachment from the perinuclear region (see yellow arrowhead). White and yellow arrows indicate MTs emanating from the centrosome and the Golgi, respectively. Scale bars: 50 µm and 20 µm (enlarged right panels). (D) Golgi orientation was quantified for wound healing RPE1 cells, as indicated in A. Data represent the means±s.d. for the indicated number (n) of cells from two independent experiments. The P-value was estimated using an unpaired Student’s t-test assuming the two-tailed distribution and two-sample unequal variance. (E) Percentage of wound-edge cells with Golgi detached from the centrosome. Data represent the means±s.d. for the indicated number (n) of cells from two independent experiments. The P-value was estimated using an unpaired Student’s t-test assuming the two-tailed distribution and two-sample unequal variance.

Article Snippet: HEK293T and the hTERT-immortalized human retinal pigment epithelial (RPE1) cells were purchased from ATCC (American Type Culture Collection).

Techniques: Migration, Control, Staining, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: ROS-dependent activation of JNK converts p53 into an efficient inhibitor of oncogenes leading to robust apoptosis

doi: 10.1038/cdd.2013.186

Figure Lengend Snippet: ROS-mediated activation of JNK contributes to the p53-mediated apoptosis, DDR and transcriptional repression of oncogenes. ( a ) Dose-dependent induction of p-JNK, p-Ser33-p53, p-Ser15-p53, γ H2AX, PARP cleavage and inhibition of Wip1, Mcl1 and MdmX by RITA as assessed by western blotting. ( b ) HCT116 and HCT116 p53−/− cells were treated with 1 μ M RITA; analyzed as in ( a ). ( c ) Pretreatment with NAC for 6 h prevented the induction of p-JNK, γ H2AX, PARP cleavage and inhibition of Wip1, Mcl1 and MdmX by RITA as analyzed by immunoblotting. ( d ) JNK inhibitor SP600125 prevented the induction of p-JNK, p-Ser33-p53, γ H2AX, PARP cleavage and inhibition of Wip1, Mcl1 and MdmX by RITA, as assessed by western blotting. ( e ) SP600125 blocked the induction of p-JNK, p-Ser33-p53, γ H2AX, PARP cleavage and inhibition of MdmX and Wip1 by combination treatment with 0.05 μ M RITA and 1 μ M Aura for 24 h, as assessed by western blotting.( f , g ) Depletion of JNK by siRNA prevented the induction of γ H2AX ( f ), p-Ser33-p53 and inhibition of Wip1 and MdmX ( g ), analyzed by immunoblotting. ( h ) Inhibition of JNK by siRNA prevented apoptosis induction by RITA, as measured by FACS of PI-stained cells. ( i ) SP600125 blocked the repression of MCL1, PPM1D, PIK3CA, PIK3CB, EIF4E and MDM4 (MdmX) mRNA upon RITA treatment as assessed by qPCR (mean±S.E.M., n =3) in HCT116 (12 h treatment) and MCF7 (8 h treatment) cells

Article Snippet: Plasmids encoding FLAG-Wip1 and shRNA for Wip1 were kindly provided by René H. Medema, Utrecht, Netherlands.

Techniques: Activation Assay, Inhibition, Western Blot, Staining

Journal: Cell Death and Differentiation

Article Title: ROS-dependent activation of JNK converts p53 into an efficient inhibitor of oncogenes leading to robust apoptosis

doi: 10.1038/cdd.2013.186

Figure Lengend Snippet: Inhibition of Wip1 promotes the induction of γ H2AX upon RITA treatment. ( a ) Wip1 mRNA was repressed after 8 h treatment with 1 μ M RITA, but not 0.1 μ M RITA, as assessed by qPCR (mean±S.E.M., n =3). ( b ) Downregulation of Wip1 protein level correlated with the induction of γ H2AX upon RITA treatment as analyzed by immunoblotting. ( c ) MCF7 and U2OS cells stably transfected with empty vector shRNA or Wip1 shRNA were treated with 0.1 and 1 μ M RITA for indicated periods and γ H2AX was assessed as in ( b ). ( d ) HCT116 and U2OS cells transfected with either empty vector or FLAG-Wip1 were treated with 1 μ M RITA for indicated times. Proteins were detected by western blotting

Article Snippet: Plasmids encoding FLAG-Wip1 and shRNA for Wip1 were kindly provided by René H. Medema, Utrecht, Netherlands.

Techniques: Inhibition, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, shRNA

Journal: Cell Death and Differentiation

Article Title: ROS-dependent activation of JNK converts p53 into an efficient inhibitor of oncogenes leading to robust apoptosis

doi: 10.1038/cdd.2013.186

Figure Lengend Snippet: Depletion of Wip1 confers a sustained transcriptional activation of p53 target genes, but does not facilitate transrepression. ( a and b ) Microarray analysis of MCF7 cells with (indicated in violet) or without (indicated in gray) Wip1 depletion by shRNA, treated with 0.1 μ M RITA or DMSO for indicated time points revealed that p53-mediated transactivation was enhanced by Wip1 silencing. ( c ) Wip1 downregulation led to the increased induction of p53-activated genes (upper panel) but did not augment the repression of pro-survival genes by p53 (lower panel) upon low dose of RITA as analyzed by qPCR (mean±S.E.M., n =3). Insert demonstrates the efficiency of Wip1 depletion, as assessed by immunoblotting. ( d ) MCF7 cells transfected with either empty vector or FLAG-Wip1 were treated with 1 μ M RITA or DMSO for 8 h and mRNA levels of PIK3CA , PIK3CB and IGF-1R were assessed by qPCR (mean±S.E.M., n =3). ( e ) MCF7 cells stably transfected with shWip1 or control shVector were treated with 0.1 and 1 μ M RITA or DMSO for 24 h, and cells were stained with Annexin V followed by FACS analysis (mean±S.E.M., n =3, * P <0.05, ** P <0.01, by two-tailed t -test). ( f ) Model of the synthetic lethality upon activation of p53 and inhibition of TrxR. Inhibition of TrxR lead to the accumulation of ROS and activation of JNK, facilitating p53 function upon its release from Mdm2. In turn, activated p53 induces pro-oxidant genes, which increases the level of ROS, further activating JNK, and thus, p53. Activated JNK converts p53 to an inhibitor of Wip1 and MdmX, therefore amplifying p53 activity. Transcriptional repression of Mcl1, eIF4E and PI3K abolishes survival signaling, contributing to apoptosis induction. Thus, the dual targeting of p53 and TrxR (i.e., by RITA) leads to the robust apoptosis

Article Snippet: Plasmids encoding FLAG-Wip1 and shRNA for Wip1 were kindly provided by René H. Medema, Utrecht, Netherlands.

Techniques: Activation Assay, Microarray, shRNA, Western Blot, Transfection, Plasmid Preparation, Stable Transfection, Control, Staining, Two Tailed Test, Inhibition, Activity Assay