Journal: The Plant Journal

Article Title: The Arabidopsis cyclin-dependent kinase-activating kinase CDKF;1 is a major regulator of cell proliferation and cell expansion but is dispensable for CDKA activation

doi: 10.1111/j.1365-313x.2009.03884.x

Figure Lengend Snippet: Figure 5. The activities of cyclin-dependent kinase activating kinases CDKA and CDKB are not reduced in the cdkf;1-1 mutants. (a), (b) Protein levels and kinase activities of CDKA and CDKB in the wild-type and the cdkf;1-1 mutants. Protein extracts (10 lg for CDKA, 40 lg for CDKB and 5 lg for actin) from 10-day-old seedlings (a) or calli (b) were subjected to immunoblotting with specific antibodies. A kinase assay was conducted with immunoprecipitates by using histone H1 as a substrate. (c) Detection of the phosphorylated form of CDKA;1. Protein extracts (60 lg) from calli were incubated with or without lambda protein phosphatase (left), or depleted with anti-CDKA;1 antibody (right). Then the samples were subjected to immunoblotting with anti-CDKA;1 or anti-phospho-Cdc2 (Thr161) antibody. CDKA-P indicates CDKA;1 phosphorylated within the T-loop. (d) Protein extracts (60 lg) from calli of wild-type plants and the cdkf;1-1 mutants were subjected to immunoblotting with anti-phospho-Cdc2 (Thr161) antibody. Bands with an asterisk representing non-specific cross-reactions with the antibody.

Article Snippet: Commercial antibodies used include anti-actin antibody (MP Biomedicals, http:// www.mpbio.com/) and anti-phospho-Cdc2 (Thr161) antibody (Cell Signaling Technology, http://www.cellsignal.com/).

Techniques: Western Blot, Kinase Assay, Incubation

Journal: Research Square

Article Title: Preventing recurrence in Sonic Hedgehog Subgroup Medulloblastoma using the OLIG2 inhibitor CT-179

doi: 10.21203/rs.3.rs-2949436/v1

Figure Lengend Snippet: (A) High OLIG2 expression correlates with poor OS in SHH-MB α subtype patients which harboring TP53 mutation. Kaplan-Meier curves of the SHH-driven MB α subtype patients based on OLIG2 expression. (B) OLIG2 expression in PDX models, primary cell lines and ATCC pediatric MB cell lines (data are shown as means ± SD, n=6). (C) OLIG2 expression at protein level in MB cell lines. (D) OLIG2 immunostaining in a MB xenograft model D425. Oligodendroglioma patient tissue was used as a positive control. Arrowheads, OLIG2 + cells. Scale bars are indicated in (D) (E) Representative western blots show expression of OLIG2 expression 72 hours after siRNA OLIG2 knockdown (KD) in Daoy cells. (F) Representative western blots show expression of cleaved caspase-3, total caspase-3 and cleaved PARP and total PARP in Daoy 72 hours after siRNA OLIG2 knockdown. (G) Representative western blots show expression of cyclin B1, p-CDK1, p-HH3, total CDK1 and total HH3 in Daoy 72 hours after siRNA OLIG2 knockdown. (H) Daoy cells stalled in G 2 /M phase 72 hours after siRNA OLIG2 knockdown (means ± SD, **p < 0.01, n=6). The p value was determined by one-way ANOVA. (I) Top panel shows a Daoy cell in anaphase with normal nucleus morphology and spindle alignment. siRNA OLIG2 knockdown results in abnormal nucleus phenotypes including satellite micronuclei. Scale bars are indicated in (I). (J) Bright field images of Daoy cells transfected with scrambled control sequence and siRNA OLIG2 KD Seq #2 over 48 hours. Cells labelled with cleaved Caspase-3/7 were shown in green. (K) Cell confluence (left) and cleaved caspase-3 confluence of Daoy after siRNA knockdown compared to scrambled control, wild-type Daoy with or without lipofectamine.

Article Snippet: Rabbit polyclonal anti-phospho-cdc2 (clone Thr161) , Cell Signalling Technology , Cat# 9114.

Techniques: Expressing, Mutagenesis, Immunostaining, Positive Control, Western Blot, Knockdown, Transfection, Control, Sequencing

Journal: Research Square

Article Title: Preventing recurrence in Sonic Hedgehog Subgroup Medulloblastoma using the OLIG2 inhibitor CT-179

doi: 10.21203/rs.3.rs-2949436/v1

Figure Lengend Snippet: (A) MB cell lines were treated with CT-179 for 7 days and a median inhibitory concentration (IC 50 ) determined experimentally for each cell line (data are shown as means ± SD, n=3). (B) Daoy were treated with 1 μM CT-179 for 17, 24, 72 or 96 hours, after which total protein were analyzed. OLIG2 expression peaked at 17 and 24 hours after treatment, then decreased to basal and completely diminished after 72 hours. (C) Quantification of OLIG2 expression in Daoy in response of CT-179 (data are shown as means ± SD, n=2). (D) Percentage of Annexin V + cells in Daoy cells and Med-813 treated with CT-179 (1 μM) or RT (2 Gy) alone, or in combination (means ± SD, ***p < 0.001, n=6). The p value was determined by one-way ANOVA. (E) Representative western blots show expression of cleaved caspase-3, caspase-3 and cleaved PARP in Daoy after treated with CT-179 (1 μM). (F) Representative western blots show expression of MCL-1, BCL-1 and BCL-xL in Daoy after treated with CT-179 (1 μM). (G) Cell cycle occupation analysis of Daoy stained with propodium iodide after 24 hours treatment with vehicle or 1 μM CT-179 or RT (2 Gy) alone, or in combination (means ± SD, ***p < 0.001, n=6). The p value was determined by two-way ANOVA. (H) Representative western blots show expression of cyclin B1, p-CDK1, p-HH3, total CDK1 and total HH3 in Med-813 cells after CT-179 (1 μM) treatment. (I) Top panel shows a Daoy cell in anaphase with normal nucleus morphology and spindle alignment. CT-179 treatment (1 μM) results in abnormal nucleus phenotypes including satellite micronuclei (white arrow) and ancillary nucleus lobe formation (green arrows), suggesting cells undergo a mitotic slippage response.

Article Snippet: Rabbit polyclonal anti-phospho-cdc2 (clone Thr161) , Cell Signalling Technology , Cat# 9114.

Techniques: Concentration Assay, Expressing, Western Blot, Staining

Journal: Research Square

Article Title: Preventing recurrence in Sonic Hedgehog Subgroup Medulloblastoma using the OLIG2 inhibitor CT-179

doi: 10.21203/rs.3.rs-2949436/v1

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-phospho-cdc2 (clone Thr161) , Cell Signalling Technology , Cat# 9114.

Techniques: Staining, Imaging, Sequencing, Over Expression, Biomarker Discovery, Knockdown, Control, Software

Journal: PLoS ONE

Article Title: Trichostatin A Targets the Mitochondrial Respiratory Chain, Increasing Mitochondrial Reactive Oxygen Species Production to Trigger Apoptosis in Human Breast Cancer Cells

doi: 10.1371/journal.pone.0091610

Figure Lengend Snippet: ( A ) Histograms depict cell cycle distributions in MCF-10A, MDA-MB-231 and MCF-7 cells after a 24 h treatment with DMSO (control) or TSA (1000 nmol/L, 500 nmol/L or 500 nmol/L). ( B ) Western blot of CyclinB1, Phospho-cdc2 (Thr161) and H3P in MCF-10A, MDA-MB-231 and MCF-7. Representative of 3 experiments. ( C ) Cell cycle distribution in MDA-MB-231 cells after exposure to 500 nmol/L of TSA at the indicated time points. ( D ) A time-course study of the effects of 1000 nmol/L, 500 nmol/L and 500 nmol/L TSA on the percentages of apoptotic cells in the MCF-10A, MDA-MB-231 and MCF-7 cell lines, respectively. Apoptosis was determined by flow cytometric analysis of the PI-positive and Annexin-V-positive cells. Values were shown as the means ± SD, n = 3. **P<0.01 versus the 0 h group using Student's t-test. A pan-caspase inhibitor, zVAD-fmk, was used to protect MDA-MB-231 cells from apoptosis; graphs showed the percentage of apoptotic cells under different treatments ( E ) and the cell cycle distributions ( F ). **P<0.01 compared to the TSA-treated alone group using Student's t-test.

Article Snippet: Gels were analyzed by immunoblotting with antibodies against Cytochrome C, COX IV, Bcl-xL, Bcl-2, NDUFS1, NDUFS6, UQCRFS1, CYC1 (10993-1-AP, 11242-1-AP, 10783-1-AP, 12789-1-AP, 19532-1-AP, 12444-1-AP, 14417-1-AP, 18443-1-AP, 10242-1-AP, respectively; Proteintech; 1∶1000 dilutions), GAPDH, Bax, cleaved PARP, CyclinB1, Phospho-cdc2(Thr161) (2118S, 5023, 9541, 4138, 9114 respectively; Cell Signaling; 1∶1000 dilutions), H3P (ab5176, 1∶2000 dilutions) and acetylated histone H3 (06-599; Millipore; 1∶1000 dilutions).

Techniques: Control, Western Blot