Fig 1: CDK7 promotes glucose consumption in H460 cells. a Immunoblots of lysate from H460 cells transfected with control shRNA or shRNA targeted against CDK2, CDK4, CDK7, and TRKA. n = 2. Glucose consumption in H460 cells transfected with control shRNA or b pooled or c individual shRNA separately targeted against CDK2, CDK4, CDK7, and TRKA. n = 4. P values determined by one-way ANOVA tests. d Glucose consumption dose response curves in H460 cells transfected with control shRNA or shRNA targeted against CDK7 and treated with Milciclib. n = 4. P values determined by a two-way ANOVA test. e mRNA levels from H460 cells transfected with control shRNA or pooled or individual shRNA targeted against CDK7. n = 2. f Immunoblots of lysate from H460 cells transfected with control shRNA or pooled or individual shRNA targeted against CDK7. n = 2. ns: not significant. *P < 0.05; ***P < 0.001; ****P < 0.0001. Data are plotted as mean ± SEM.
Fig 2: PKCι activates CDK7 to promote glucose consumption. a Immunoblots from H460 cells transfected with control shRNA or shRNA targeted against PKCι. Seq. 1 and 2 represent different shRNA sequences. n = 2. b Glucose consumption in H460 cells transfected with control shRNA or shRNA targeted against PKCι. Control: n = 8; Seq. 1: n = 7; Seq. 2: n = 4. P value determined by a one-way ANOVA test. c Glucose consumption dose response curves in H460 cells transfected with control shRNA or shRNA targeted against PKCι and treated with Milciclib. Control: n = 8; Seq. 1: n = 7; Seq. 2: n = 4. P values determined by a two-way ANOVA test. d GLUT1 mRNA levels from H460 cells transfected with a control, wild-type (WT) CDK7, or T170A mutant CDK7 overexpression plasmid. n = 2. e Immunoblots from H460 cells transfected with a control, WT CDK7, or T170A mutant CDK7 overexpression plasmid. n = 2. f Glucose consumption in H460 cells transfected with a control, WT CDK7, or T170A mutant CDK7 overexpression plasmid. n = 4. P values determined by a one-way ANOVA test. g Glucose consumption dose response curves in H460 cells transfected with a control, WT CDK7, or T170A mutant CDK7 overexpression plasmid and treated with Milciclib. n = 4. P values determined by a two-way ANOVA test. h Immunoblots from H460 cells treated with vehicle or Milciclib (10 μM). n = 2. i HA-CDK7 levels on the GLUT1 promoter in H460 cells transfected with a control or HA-CDK7 overexpression plasmid. n = 2. j Rpb1 and phospho-Rpb1 levels on the GLUT1 promoter in H460 cells treated with vehicle or Milciclib (10 μM). n = 2. ns: not significant. **P < 0.01; ****P < 0.0001. Data are plotted as mean ± SEM.
Fig 3: c-MYC rescues general transcription and prevents senescence in the absence of MITF. a Immunoblot analysis of 501 mel cells under retrovirus-driven c-MYC expression compared to EV and subsequent siMITF1 vs. siSCR transfection. b MITF, c-MYC, and CDK7 mRNA expression in 501 mel cells under conditions analogous to a. Relative expression was measured by qRT-PCR, normalized to GAPDH and given as mean ± SD from technical triplicates (two-tailed unpaired t-test). c Transcriptional activity measured as EU incorporation in 501 mel cells in analogy to a and b. Graphs indicate mean ±SEM of fluorescence intensity in ≥250 nuclei (two-tailed unpaired t-test). d Proliferation of 501 mel cells in analogy to a–c. Graphs represent mean ± SD of crystal violet absorbance from technical triplicates (two-tailed unpaired t-test). e SA-β-gal positivity in 501 mel cells at day 3 in analogy to d. Data represent mean ± SD from technical triplicates (two-tailed unpaired t-test). Micrographs display SA-β-gal signals and Hoechst 33342 nuclear staining under corresponding conditions (scale: 50 µm). f Regression analysis of MITF and MYC single-cell mRNA expression from a panel of metastatic melanomas (GSE72056). g ChIP‐seq tracks of MITF-binding signals at FUBP2/KHSRP gene locus in primary melanocytes, and 501 mel and COLO829 BT168F melanoma cell lines. Green arrowheads indicate MITF-binding consensus sequences (E box) at the promoter and first intronic region of the FUBP2/KHSRP gene. GEO accession numbers are listed under Materials and Methods. h MITF, c-MYC, and FUBP2 mRNA expression in 501 mel cells after siSCR, siMITF1, or siFUBP2 transfection. Relative expression was measured by qRT-PCR, normalized to GAPDH and given as mean ± SD from technical triplicates (two-tailed unpaired t-test). i Immunofluorescence and immunoblot analyses of MITF, MYC, and FUBP2 protein expression in analogy to h. DRAQ5 used for nuclear staining and actin used as loading control (two-tailed unpaired t-test of c-MYC fluorescence intensity in 100 nuclei is indicated). a–e, h, i Experiments were repeated twice with comparable results
Fig 4: PIK3CA activates CDK7 to promote glucose consumption. a Immunoblots from HCC827 cells transfected with a control or PIK3CA overexpression plasmid. n = 2. b Glucose consumption dose response curves of HCC827 cells transfected with a control or PIK3CA overexpression plasmid and treated with Milciclib. n = 4. P values determined by a two-way ANOVA test. c Immunoblots from H460 cells transfected with a control or PTEN overexpression plasmid. n = 2. d Glucose consumption dose response curves of H460 cells transfected with a control or PTEN overexpression plasmid and treated with Milciclib. Control: n = 4, PTEN overexpression: n = 6. P values determined by a two-way ANOVA test. e Glucose consumption dose response curves in H1975 cells transfected with control shRNA or shRNA targeted against CDK7 and treated with Milciclib. n = 4 except n = 3 for CDK7 shRNA (individual). P values determined by a two-way ANOVA test. f Immunoblots of lysate from H1975 cells transfected with a control or PTEN overexpression plasmid. n = 2. g Glucose consumption dose response curves of H1975 cells transfected with a control or PTEN overexpression plasmid and treated with Milciclib. n = 4. P values determined by a two-way ANOVA test. *P < 0.05; **P < 0.01; ****P < 0.0001.
Fig 5: CDK7 is a direct transcriptional target of MITF and c-MYC. a Time course of CDK7 mRNA expression in 501 mel cells transfected with siMITF1 vs. control siRNA under treatment with phorbol ester (TPA). Data represent mean ± SD from technical triplicates (two-tailed paired t-test). b Immunoblot analysis of MITF, c-MYC and CDK7 protein expression in 501 mel cells under TPA treatment. Lower and upper arrowheads indicate non-phosphorylated (54 kd) and phosphorylated (60 kd) MITF forms, respectively. Actin used as loading control. c ChIP-seq tracks of MITF (marked red) and c-MYC (marked blue) binding signals at CDK7 gene locus in primary melanocytes and primary melanomas, melanoma cell lines and non-melanocytic cancer cells, respectively. Green arrowhead indicates E box consensus sequence −86 base pairs upstream of the transcriptional start site of CDK7. GEO accession numbers of corresponding data sets are listed under Materials and methods. d ChIP, in vivo occupancy of MITF at CDK7 promoter in 501 mel cells in the presence or absence of forced c-MYC expression (OE overexpression) compared to empty vector (EV) including intron sequence and IgG controls. Mean ± SD from technical triplicates. e ChIP, in vivo occupancy of exogenous c-MYC (OE) at CDK7 promoter in 501 mel cells under siSCR or siMITF1 transfection. Intron sequence and IgG were used as controls. Mean ± SD is presented from technical triplicates. d, e Two-tailed paired t-test. a, b, d, e Experiments were performed twice and biological replicates revealed very similar results
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