Fig 1: Overexpression of Cyclin T1 in the adult heart facilitates Myc-driven transcription in adult cardiomyocytes.a Immunohistochemistry (left) and quantification (right) of Cyclin T1 in the liver isolated from adult R26CMER/+ or heart isolated from Myh6-Cre; R26LSL-CMER/+ mice systemically infected with adeno-associated virus (serotype 9, 1 × 1011 vg/mouse) encoding either RFP (AAV9-RFP, n = 5) or Ccnt1 (AAV9-Ccnt1, n = 4) 4 weeks post infection. Mean and s.e.m shown. Unpaired t-test; AAV9-RFP vs AAV9-Ccnt1: ***P < 0.0001. Representative images based on analysis of five independent mice. b Immunoblot analysis of the C-terminal domain of phosphorylated RNA polymerase II (p-Rpb1(S2)), CDK9 and Cyclin T1 expression in the heart isolated from adult Myh6-Cre; R26LSL-CMER/+ mice systemically infected with adeno-associated virus encoding either RFP (AAV9-RFP) or Ccnt1 (AAV9-Ccnt1) 4 weeks post infection. Replicate samples are derived from independent mice. c Quantitative RT-PCR analysis of Cad, Bzw2, Pinx1, Polr3d, St6 and Cdc25a expression in control (R26LSL-CMER/+; no cre, n = 5) and Myh6-Cre; R26LSL-CMER/+ (R26LSL-CMER/+; cre+, n = 7) adult mouse heart isolated 4 weeks post systemic infection with an adeno-associated virus encoding Ccnt1 (AAV9-Ccnt1) and 4 h post administration of 4-OHT. Expression is relative to the respective wild type (R26LSL-CMER/+). Mean and s.e.m shown. Unpaired t-test; no cre vs cre+: ***P < 0.0005 (Cad, Bzw2, Pinx1 and St6), **P = 0.0013 (Cdc25a), **P = 0.0035 (Polr3d). Replicate samples are derived from independent mice. d Enrichment of common Myc targets (liver, lung, kidney and heart) in cardiomyocytes isolated from the heart of AAV-CCNT1 infected Myh6-cre; R26LSL-CMER/+ (Myc+Cyclin T1) mice in comparison to uninfected Myh6-cre; R26LSL-CMER/+ mice (Myc) at 4 h post administration of 4-OHT, as determined by RNA sequencing. Including normalised enrichment score (NES) and FDR q-value (FDR).
Fig 2: Inhibition or overexpression of P-TEFb modulates efficiency of Myc-driven transcription.a Immunoblot analysis of the C-terminal domain (CTD) of RNA Polymerase II—total (Rpb1) and phosphorylated (p-Rpb1(S5) and p-Rpb1(S2)), CDK9, Cyclin T1 and Larp7 expression in the heart, liver, lung and kidney isolated from wild-type (R26+/+) mice. The composite figure is generated from the same samples loaded across multiple blots and a representative image for GAPDH is shown. b Immunoblot analysis of the CTD of RNA Polymerase II, phosphorylated (p-Rpb1(S2)), and MycERT2 protein expression in wild type (R26+/+) or R26CMER/+ livers, isolated 4 h post administration of 4-OHT either alone (4-OHT) or in combination with 60 mg/kg AZ5576 (4-OHT + AZ5576). c Quantitative RT-PCR analysis of Smpdl3b, Cad, Gnl3 and Polr3g in wild type (R26+/+) and R26CMER/+ livers, isolated 4 h post administration of 4-OHT (n = 6 R26+/+, n = 3 R26CMER/+) either alone or in combination with 60 mg/kg AZ5576 (4-OHT + AZ5576, n = 4). Expression is relative to the respective wild type (R26+/+). Mean and s.d shown. Two-way ANOVA with Tukey’s multiple comparisons test; R26+/+ vs 4-OHT: ***P = 0.001 (Smpdl3b, Cad, Gnl3 and Polr3g), R26CMER/+ 4-OHT vs 4-OHT + AZ5576: ***P = 0.001 (Smpdl3b, Cad and Gnl3), **P = 0.01 (Polr3g). d Immunoblot analysis of Cyclin T1 and phosphorylated CTD of RNA polymerase II (p-Rpb1(S2)) in R26CMER/+ primary cardiomyocytes infected with an adenovirus encoding either GFP (Ad-GFP) or Ccnt1 (Ad-Ccnt1). Replicate samples are derived from independent primary cardiomyocyte isolations. e Quantitative RT-PCR analysis of Cad, Bzw2, Pinx1, Polr3d, St6 and Cdc25a in wild type (R26+/+, n = 5 except Pinx1 where n = 4 for Ad-GFP control) and R26CMER/+ (n = 5 except St6 where n = 4 for Ad-GFP control) primary cardiomyocytes infected with an adenovirus encoding either GFP (Ad-GFP) or Ccnt1 (Ad-Ccnt1), 4 h post addition of 100 nM 4-OHT. Expression is relative to an individual wild type (R26+/+) Ad-GFP control. Mean and s.d shown. One-way ANOVA with Tukey’s multiple comparisons test; Ad-GFP R26+/+ vs R26CMER/+: *P = 0.05 (Cad), Ad-Ccnt1 R26+/+ vs R26CMER/+: *P = 0.05 (Cad, Pinx1, Polr3d and St6) **P = 0.01 (Bzw2 and Cdc25a). Replicate samples are derived from independent primary cardiomyocyte isolations and independent mice. Source data are provided as a Source Data file.
Fig 3: Cardiomyocyte HRasG12V expression enables Myc-driven transcription. (A) Scheme of experimental work. (B) Immunoblot analysis of the C-terminal domain of phosphorylated RNA Polymerase II [p-Rpb1(S2)], Cyclin T1 and CDK9 protein expression in hearts isolated from control (R26+/+; TetO-HRas, R26+/+; Myh6-tTA or R26+/+) and TetO-HRas; Myh6-tTA; R26 CMER/+(HRas/Myc) mice 4 weeks post withdrawal of doxycycline and at 24 h post administration of 4-OHT. Expression of Gapdh is included as a loading control. Replicate samples are derived from independent mice. (C) Quantitative RT-PCR analysis of Cad, Bzw2, Pinx1, Polr3d, St6, and Cdc25a in hearts from control (R26+/+; TetO-HRas, R26+/+; Myh6-tTA or R26+/+, n ≥ 14), TetO-HRas; Myh6-tTA; R26+/+(HRas, n = 4), R26CMER/+ (Myc, n = 6) and TetO-HRas; Myh6-tTA; R26 CMER/+ (MycHRas, n = 5) mice 4 weeks post withdrawal of doxycycline and 4 h post-administration of 4-OHT. Expression is normalised to Actin and Gapdh and relative to the respective control. Error bars show s.e.m. Two Way ANOVA with multiple comparisons test; control vs R26CMER/+: p*** = 0.001 (Cad), control vs TetO-HRas; Myh6-tTA; R26 CMER/+: p** = 0.01 (Bzw2, Pinx1, Polr3d, Cdc25a) p*** = 0.001 (St6). Replicate samples are derived from independent mice. (D) Heat map showing the union of DEGs called in adult liver and adult heart. Shown are mRNA expression fold changes (Log2) upon MycERT2 activation relative to wild-type, as determined by RNA sequencing of R26CMER/+ (n ≥ 3) mice in comparison to wild-type (R26+/+, n ≥ 3) and TetO-HRas; Myh6-tTA; R26 CMER/+ mice (Myc/HRas, n = 3) in comparison to TetO-HRas; Myh6-tTA; R26+/+ mice (n = 3) at 4 h post administration of 4-OHT. (E) Venn diagrams of overlap in the number of genes showing an increase (top) and decrease (bottom) in expression in response to supraphysiological Myc expression. The most significant GO Biological Process and ASCHS4 gene lists that overlap are shown. Comparisons included the liver of R26CMER/+ (Myc liver, n = 3) compared to wild type. The adult mouse heart was isolated 4 weeks post systemic infection with an adeno associated virus encoding Ccnt1 (AAV9-Ccnt1) compared to control and heart from TetO-HRas; Myh6-tTA; R26 CMER/+ (n = 3) 4 weeks after HRasG12D expression and 4 h post Myc activation compared to control, as determined by RNA sequencing (FDR < 0.05 and abs(log2FC) > 0.5).
Fig 4: Overexpression of Cyclin T1 in the adult heart facilitates Myc-driven proliferation of adult cardiomyocytes.a Immunofluorescent staining of cardiac troponin, p-H3,Ki67, PCM1, wheat germ agglutinin (WGA) and aurora B in control (R26LSL-CMER/+; no cre) and Myh6-Cre; R26LSL-CMER/+ (R26LSL-CMER/+; cre+) adult mouse heart isolated 4 weeks post systemic infection with an adeno-associated virus encoding Ccnt1 (AAV9-Ccnt1) and 48 h post administration of tamoxifen. Representative images based on analysis of five independent mice. Scale bars represents 10 µm (aurora B) and 50 µm (all others). b Quantification of p-H3-positive nuclei percentage in control (R26LSL-CMER/+; no cre, n = 9) and Myh6-Cre; R26LSL-CMER/+ (R26LSL-CMER/+; cre+, n = 7) adult mouse heart isolated 4 weeks post systemic infection with an adeno-associated virus encoding Ccnt1 (AAV9-Ccnt1) and 48 h post administration of tamoxifen. Means are taken from five images per mouse; Mean and s.e.m shown. Unpaired t-test; no cre vs cre+ P < 0.0001. c The weight (mg) of hearts isolated from control (R26LSL-CMER/+; no cre, n = 9 48 h, n = 5 72 h) and Myh6-Cre; R26LSL-CMER/+ (R26LSL-CMER/+; cre+, n = 7 48 h, n = 6 72 h) adult mouse heart 4 weeks post systemic infection with an adeno-associated virus encoding Ccnt1 (AAV9-Ccnt1) and 48 and 72 h post administration of tamoxifen, expressed as fold change over the length (mm) of a tibia isolated from the same mouse. Mean and s.e.m shown. One-way ANOVA with Tukey’s multiple comparisons test; no cre vs cre+: *P = 0.05 (48 h), ***P = 0.001 (72 h). Replicate samples are derived from independent mice. d Image of the whole heart and a tibia from control (R26LSL-CMER/+; no cre) and Myh6-Cre; R26LSL-CMER/+ (R26LSL-CMER/+; cre+) adult mouse heart isolated 4 weeks post systemic infection with an adeno-associated virus encoding Ccnt1 (AAV9-Ccnt1) and 72 h post administration of tamoxifen. Scale bar represents 10 mm. Representative images based on analysis of 11 independent mice. e Quantification of the number and size of cardiomyocytes from control (R26LSL-CMER/+; no cre, n = 5) and Myh6-Cre; R26LSL-CMER/+ (R26LSL-CMER/+; cre+, n = 6) adult mouse heart isolated 4 weeks post systemic infection with an adeno-associated virus encoding Ccnt1 (AAV9-Ccnt1) and 72 h post administration of tamoxifen. Mean and s.d shown. One-way Mann–Whitney test; no cre vs cre+ **P = 0.0043. Replicate samples are derived from independent mice. Source data are provided as a Source Data file.
Fig 5: Cardiac HRasG12V and MycERT2 expression results in cardiomyocyte apoptosis. (A) Left- immunofluorescent staining of cleaved caspase 3 (green) and cardiac troponin (pink) in heart from control, TetO-HRas; Myh6-tTA; R26+/+ (HRas), R26CMER/+ (Myc) and TetO-HRas; Myh6-tTA; R26 CMER/+ (Myc/HRas) mice 4 weeks post withdrawal of doxycycline and 24 h post administration of tamoxifen. Representative images based on analysis of at least 3 independent mice. Right- quantification of CC3 positive cardiomyocytes in hearts isolated from control (R26+/+; TetO-HRas, R26+/+; Myh6-tTA or R26+/+, black, n = 8), TetO-HRas; Myh6-tTA; R26+/+ (HRas, red, n = 4), R26CMER/+ (Myc, blue, n = 4) and TetO-HRas; Myh6-tTA; R26 CMER/+ (Myc/HRas, purple, n = 3) mice 4 weeks post withdrawal of doxycycline and 24 h post administration of tamoxifen. Bars are 100 µm Representative images based on analysis of 5 images per mouse; error bars show s.e.m. One-Way ANOVA with multiple comparisons test; Myc/HRas vs control: p = 0.0003, Myc/HRas vs Myc: P = 0.0006, Myc/HRas vs HRas; Myh6-tTA; R26+/+: p = 0.0295. (B) Left- immunofluorescent staining of heart from Control and Myh6-Cre; R26LSL– CMER/+ (Myc/Cyclin T1) mice 4 weeks post systemic infection with an AAV-Cyclin T1 adeno-associated virus and 48 post administration of tamoxifen (tam) at adulthood. Top: Control is R26+/+, cleaved caspase 3 (green) and cardiac troponin (pink), Bottom: Control is Myh6-Cre; R26LSL– CMER/+ without tam treatment. TUNEL (green). Representative images based on analysis of 5 independent mice. Right- quantification of TUNEL positive cells in hearts isolated from R26+/+ (WT) mice infected with AAV-LacZ and or AAV-Cyclin T1 and Myh6-Cre; R26LSL– CMER/+ (Myc) mice 4 weeks post systemic infection with an adeno-associated virus and 48 post administration of tamoxifen (tam). Bars are 100 µm. Representative images based on analysis of 5 images per mouse; error bars show s.e.m. Thymus is shown as a positive control. One-Way ANOVA with multiple comparisons test. ns = not significant. (C) Venn diagram of the overlap of the GSEA hallmark signatures obtained from the upregulated gene expression profile of Myc/HRas expressing hearts or Myc/Cyclin T1 expressing hearts. Adult mouse hearts were either isolated 4 weeks post systemic infection with an adeno associated virus encoding Ccnt1 (AAV9-Ccnt1) compared to control and heart from TetO-HRas; Myh6-tTA; R26 CMER/+ (n = 3) 4 weeks after HRasG12D expression and 4 h post Myc activation compared to control, as determined by RNA sequencing (FDR < 0.05 and abs(log2FC) > 0.5). The significant non-overlapping GSEA hallmark signatures are shown below.
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