Journal: JCI Insight

Article Title: CDK12 regulates cellular metabolism to promote glioblastoma growth

doi: 10.1172/jci.insight.190780

Figure Lengend Snippet: ( A ) Schematic experimental design and RNA-seq sample information. ( B ) This plot shows the normalized enrichment score (NES) of Reactome_Citric acid cycle (TCA cycle) and Respiratory Electron_Transport gene sets derived from GSEA. ( C and D ) GSEA and heatmap of Oxidative Phosphorylation genes in RNA-seq data for DMSO and SR treatment. ( E and F ) The qPCR and Western blot results of oxidative phosphorylation– and fatty acid oxidation–related genes after SR treatment in GBM12, GBM22, and U251 cells. The qPCR data are presented as mean ± SD. ( G ) Western blot results of oxidative phosphorylation– and fatty acid oxidation–related genes after loss of CDK12 in GBM12, GBM22, and U251 cells. ( H ) PPARD mRNA expression (log 2 ) in non-tumor and GBM tissues. Data are presented as box-and-whisker plots; the horizontal line within each box represents the median value, and the whiskers denote the minimum and maximum values. Statistical significance was determined using a Student’s t test (* P < 0.05). ( I ) Viability of GBM12 cells expressing shPPARD ( – ) compared to shNTS was assessed for 0, 2, 4, or 6 days. Data are presented as mean ± SD. ( J ) GBM12 cells were transfected with scrambled siRNA (scRNA) or siRNAs against PPARD and PGC1A. Thereafter, cells were exposed to increasing concentrations of SR-4835 and analyzed for cellular viability. Data are presented as mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 by unpaired, 2-tailed t test ( E and H ), 1-way ANOVA with Dunnett’s multiple-comparison test ( I ), or 2-way ANOVA with Tukey’s multiple-comparison test ( J ).

Article Snippet: Antibodies against the following proteins were used: CDK12 (Cell Signaling Technology [CST], 11973), Mcl-1 (CST, 5453), Bcl-2 (clone D55G8, CST, 4223), Bcl-xL (clone 54H6, CST, 2764), Noxa (clone 114C307, Calbiochem, OP180), PARP (clone 46D11, CST, 9532), CCP9 (clone D2D4, CST, 7237), CCP3 (Asp175, CST, 9661), CPT2 (Invitrogen, PA5-12217), PGC1A (Novus Bio, NBP1-04676), PPARD (Abcam, ab23673), GSK3β (clone 27C10, CST, 9315), p-GSK3β (Ser9) (clone D85E12, CST, 5558), HA (CST, 3724S), FLAG (Sigma-Aldrich, F3165), β-actin (Sigma-Aldrich, A1978), anti–rabbit IgG (H+L), HRP (Thermo Fisher Scientific, 31460), and anti–mouse IgG (H+L), HRP (Thermo Fisher Scientific, 31430).

Techniques: RNA Sequencing, Derivative Assay, Phospho-proteomics, Western Blot, Expressing, Whisker Assay, Transfection, Comparison

Journal: JCI Insight

Article Title: CDK12 regulates cellular metabolism to promote glioblastoma growth

doi: 10.1172/jci.insight.190780

Figure Lengend Snippet: ( A ) The Kaplan-Meier survival curves were employed to determine overall survival between GBM12 shNTS and shCDK12 (no. 1798) mice. The median survival in the shNTS ( n = 10) and shCDK12 ( n = 8) groups was 22.5 and 59 days, respectively. A log-rank test was utilized to assess the statistical significance of the observed differences ( P < 0.0001). ( B ) Representative H&E staining images and MRI of GBM12 shNTS and shCDK12 mouse brains at 22 days (Bruker BioSpec, 9.4 Tesla). ( C ) The bar graph shows mouse tumor volumes of brains in B , which were analyzed by Analyze 14.0 ( www.analyzedirect.com ). Data are presented as mean ± SD. ** P < 0.01 by unpaired, 2-tailed t test. ( D ) Tumors were fixed and stained with TUNEL and for Ki67, Mcl-1, Noxa, and PPARD. Scale bar: 30 μm. ( E ) Kaplan-Meier survival curves were employed to determine overall survival between GBM22 shNTS and shCDK12 (no. 1798) groups. The median survival in shNTS ( n = 6) and shCDK12 (no. 1798) ( n = 8) groups was 19 and 29 days, respectively (log-rank, P = 0.0001). ( F ) The median survival in the shNTS+DMSO ( n = 7, blue line), shNTS+TMZ ( n = 5, red line), shCDK12+DMSO ( n = 7, green line), and shCDK12+TMZ ( n = 7, black line) groups were 13, 13, 35, and not-reached days, respectively (log-rank, P < 0.0001). ( G ) GBM12 PDX cells were intracranially injected into the brains of mice and then treated with vehicle (DMSO) or SR-4835 by intraperitoneal injection for 2 weeks. The median survival in DMSO ( n = 6) and SR ( n = 6) groups was 18.5 and 23.5 days, respectively (log-rank, P < 0.0014). Representative H&E staining images of GBM12 DMSO and SR group mouse brains. ( H ) Tumors from the experiment in G were fixed and stained with TUNEL and for Mcl-1, Noxa, PPARD, and PGC1A. Scale bar: 30 μm. ( I ) Quantification of IHC staining intensity for TUNEL, PPARD, and PGC1A presented as a bar graph. Data are presented as mean ± SD. * P < 0.05; ** P < 0.01 by unpaired, 2-tailed t test.

Article Snippet: Antibodies against the following proteins were used: CDK12 (Cell Signaling Technology [CST], 11973), Mcl-1 (CST, 5453), Bcl-2 (clone D55G8, CST, 4223), Bcl-xL (clone 54H6, CST, 2764), Noxa (clone 114C307, Calbiochem, OP180), PARP (clone 46D11, CST, 9532), CCP9 (clone D2D4, CST, 7237), CCP3 (Asp175, CST, 9661), CPT2 (Invitrogen, PA5-12217), PGC1A (Novus Bio, NBP1-04676), PPARD (Abcam, ab23673), GSK3β (clone 27C10, CST, 9315), p-GSK3β (Ser9) (clone D85E12, CST, 5558), HA (CST, 3724S), FLAG (Sigma-Aldrich, F3165), β-actin (Sigma-Aldrich, A1978), anti–rabbit IgG (H+L), HRP (Thermo Fisher Scientific, 31460), and anti–mouse IgG (H+L), HRP (Thermo Fisher Scientific, 31430).

Techniques: Staining, TUNEL Assay, Injection, Immunohistochemistry