Journal: Scientific Reports

Article Title: Cyclin-dependent kinase 9 is required for the survival of adult Drosophila melanogaster glia

doi: 10.1038/s41598-017-07179-8

Figure Lengend Snippet: cdk9 is necessary for glial survival. ( a–h ) Representative images of 7d old adult brains immunostained with anti-Repo to label glia for Fig. 1i,j. The images are maximum projections of optical sections. The outlined area demarcates the region of the central brain where the number of glia were quantified. ( i ) Number of anti-Repo-expressing glia in the central brain of 7d old adult flies in mir-31a mutants (mir-31a KO/KO) and where UAS-GFP , UAS-CG16947 , UAS-cdk9 RNAi or UAS-p53 RNAi lines were expressed in glia using Repo-Gal4 . Glia were represented as a percentage of the control animals, Repo-Gal4 > UAS-GFP (left axis). Raw number of Repo-expressing glia in the central brain of the genotypes (right axis). One-way ANOVA was used for statistical analysis and error bars represent SEM. ( j ) Number of anti-Repo-expressing glia in the central brain of 7d old adult flies. UAS-GFP , UAS-CG16947 and UAS-cdk9 RNAi expression was driven by Repo-Gal4 under the control of tubulin Gal80 ts (tubGal80 ts ). Adult-only expression was achieved by rearing the flies at 18 °C then moving the flies to 29 °C to allow Repo-Gal4 activity after eclosion. Data is represented as a percentage of the number of glia in the UAS-GFP control condition (left axis). Raw number of Repo-expressing glia in the central brain of the genotypes (right axis). One-way ANOVA was used for statistical analysis and error bars represent SEM.

Article Snippet: 2.5 mg of protein was incubated together with 2 μg of rabbit anti-CG16947 (Lifespan Biosciences, LS-B13598 ), or 2 μg of mouse anti-cdk9 (Novus Biologicals, H00001025-M07) overnight on a tube rotator at 4 °C.

Techniques: Expressing, Control, Activity Assay

Journal: Scientific Reports

Article Title: Cyclin-dependent kinase 9 is required for the survival of adult Drosophila melanogaster glia

doi: 10.1038/s41598-017-07179-8

Figure Lengend Snippet: cdk9 is necessary for adult glial survival. ( a–d ) Representative images of 7d old adult brains immunostained with anti-Repo to label glia for Fig. 2e,f. The images are maximum projections of optical sections. The outlined area demarcates the region of the central brain where the number of glia were quantified. ( e ) Number of anti-Repo-expressing cells in the central brain of 7d old mir-31a mutants (mir-31a KO/KO) when either UAS-GFP or UAS-cdk9 was expressed only in adult glia with Repo-Gal4 under the control of tubGal80 ts . Repo-Gal4 > UAS-GFP represent otherwise wildtype control animals to demonstrate the effectiveness of CDK9 at restoring the wildtype levels of glia in the adult brain. Data is represented as a percentage of the number of glia in the UAS-GFP control condition (left axis). Raw number of Repo-expressing glia in the central brain of the genotypes (right axis). One-way ANOVA was used. Error bars represent SEM. ( f ) Number of anti-Repo-expressing cells in the central brain of 7d old adults expressing either UAS-GFP or UAS-cdk9 only in glia with Repo-Gal4 under the control of Gal80 ts . Data is represented as a percentage of the number of glia in the UAS-GF P control condition (left axis). Raw number of Repo-expressing glia in the central brain of the genotypes (right axis). Unpaired Student’s t-test was used. Error bars represent SEM.

Article Snippet: 2.5 mg of protein was incubated together with 2 μg of rabbit anti-CG16947 (Lifespan Biosciences, LS-B13598 ), or 2 μg of mouse anti-cdk9 (Novus Biologicals, H00001025-M07) overnight on a tube rotator at 4 °C.

Techniques: Expressing, Control

Journal: Scientific Reports

Article Title: Cyclin-dependent kinase 9 is required for the survival of adult Drosophila melanogaster glia

doi: 10.1038/s41598-017-07179-8

Figure Lengend Snippet: cdk9 interacts directly with CG16947. ( a ) Alignment of the Drosophila melanogaster (fly) and human protein sequences of cdk9. Highlighted portion denotes region that the commercial anti-cdk9 antibody was generated against human cdk9 protein. ( b ) CG16947 (50 kDa) was detected in the lysates from Repo-Gal4 > UAS-GFP (Repo > UAS-GFP) and Repo-Gal4 > UAS-CG16947 (Repo > UAS-CG16947) used for co-immunoprecipitation in 3d, e, f and Supplemental Fig. . Kinesin (120 kDa) was used as a loading control to ensure that similar levels of protein were loaded from both genotypes. ( c ) cdk9 (49 kDa) was detected in the lysates from Repo-Gal4 > UAS-GFP (Repo > UAS-GFP) and Repo-Gal4 > UAS-CG16947 (Repo > UAS-CG16947) used for co-immunoprecipitation in 3d, e, f and Supplemental Fig . Kinesin (120 kDa) was used as a loading control to ensure that similar levels of protein were loaded from both genotypes. ( d ) Western blot of anti-CG16947 co-immunoprecipitation. Flies were of either Repo-Gal4 > UAS-GFP (Repo > UAS-GFP) or Repo > UAS-CG16947 (Repo > UAS-CG16947) genotype. Blot was probed with anti-cdk9. Lysate refers to the input for the co-immunoprecipitation. A control condition where the beads were incubated with the anti-CG16947 antibody in the absence of lysate was done to distinguish anti-CG16947 antibody elution from the beads versus the CG16947 protein itself. A 49 kDa cdk9 band was detected in the lysates and the without DTT elution of the anti-rchy1 pulldown in both Repo-Gal4 > UAS-GFP (Repo > UAS-GFP) and Repo-Gal4 > UAS-CG16947 (Repo > UAS-CG16947) conditions. ( e ) Co-immunoprecipitation with anti-cdk9. Anti-cdk9 pulled down CG16947 protein (50 kDa). An increase in the number and intensity of the bands recognised by anti-CDK9 in the MG132-treated samples compared to when the lysate and co-immunoprecipitation was done without MG132 suggest that CG16947 was actively degraded via the proteasome in vitro . The multiple bands observed below the expected size for CG16947 likely represent degraded CG16947, which can be observed when the proteasome is inhibited in vitro . ( f ) Co-immunoprecipitation with anti-cdk9. anti-cdk9 pulled down cdk9 protein that was ubiquitinated as shown by the bands above the expected size for cdk9, 49 kDa and degraded, as shown by the bands below 49 kDa. An increase in the number and intensity of the bands recognised by anti-ubiquitin in the MG132-treated samples demonstrated that cdk9 was actively ubiquitinated and degraded via the proteasome in vitro . A higher exposure of the blot is shown in Supplemental Fig. where ubiquitinated cdk9 and its degraded products can be detected in the lysates without MG132.

Article Snippet: 2.5 mg of protein was incubated together with 2 μg of rabbit anti-CG16947 (Lifespan Biosciences, LS-B13598 ), or 2 μg of mouse anti-cdk9 (Novus Biologicals, H00001025-M07) overnight on a tube rotator at 4 °C.

Techniques: Generated, Immunoprecipitation, Control, Western Blot, Incubation, In Vitro, Ubiquitin Proteomics

Journal: Scientific Reports

Article Title: Cyclin-dependent kinase 9 is required for the survival of adult Drosophila melanogaster glia

doi: 10.1038/s41598-017-07179-8

Figure Lengend Snippet: Table depicting number of samples, mean and SEM of all genotypes tested.

Article Snippet: 2.5 mg of protein was incubated together with 2 μg of rabbit anti-CG16947 (Lifespan Biosciences, LS-B13598 ), or 2 μg of mouse anti-cdk9 (Novus Biologicals, H00001025-M07) overnight on a tube rotator at 4 °C.

Techniques:

Journal: Kidney international

Article Title: The Smad3/Smad4/CDK9 complex promotes renal fibrosis in mice with unilateral ureteral obstruction.

doi: 10.1038/ki.2015.235

Figure Lengend Snippet: Figure 1| Smad3, Smad4, and cyclin-dependent kinase 9 (CDK9) complex formation in the development and progression of unilateral ureteral obstruction (UUO). (a) Western blotting (WB) demonstrated expression levels of CDK9 in the kidneys 6 h, 12 h, 2 days (d), 4d, and 7d after sham or UUO surgery. (b) Quantitation of relative signal intensities of CDK9/α-tubulin. Data are mean ± s.d., n = 6. *Po0.05, versus sham or UUO 6-h group; #Po0.05, versus UUO 12-h group; $Po0.05, versus UUO 24-h group. (c–l) Confocal microscopy demonstrated the expression of CDK9 (green, d, h), α-smooth muscle actin (α-SMA, red, e, i), merged (f, j, k–m) and DAPI (4,6-diamidino-2-phenylindole; blue, c, g) in the mouse kidney with sham operation (c–f) and kidney with UUO (g–m). c–j, × 600. (k) Area in (j), × 1800. Arrows: samples of CDK9+/α-SMA+ myofibroblasts cells. (l) Quantitation of the percentages of CDK9+/α-SMA+ cells in total DAPI(+) cells. (m) Immunoprecipitation (IP)/WB demonstrated the interactions between Smad3 and Smad4, and Smad3 and CDK9 in the kidneys 6 h, 12 h, 2d, 4d, and 7d after sham or UUO surgery. (n) Quantitation of relative signal intensities of Smad4/Smad3 and CDK9/Smad3. Data are mean ± s.d., n = 6. *Po0.05, versus sham or UUO 6-h group.

Article Snippet: After blocking for 30 min at 4 °C in 5% bovine serum albumin in phosphate-buffered saline with 0.1% Tween 20, the membrane was incubated overnight with rabbit anti-Smad3, CDK9, p-Smad3 Thr179 (Biorbyt, Cambridge, UK), or rabbit antiphospho-Smad3 (Ser423/425) (Cell signaling Technology), or Smad4 (Cell Signalling Technology).

Techniques: Western Blot, Expressing, Quantitation Assay, Confocal Microscopy, Immunoprecipitation

Journal: Kidney international

Article Title: The Smad3/Smad4/CDK9 complex promotes renal fibrosis in mice with unilateral ureteral obstruction.

doi: 10.1038/ki.2015.235

Figure Lengend Snippet: Figure 3| Smad3, Smad4, and cyclin-dependent kinase 9 (CDK9) complex formation in unilateral ureteral obstruction (UUO). (a) Immunoprecipitation (IP)/western blotting (WB) demonstrated the interactions between CDK9 and Smad3 and CDK9 and Smad4 in the kidneys 7 days after sham or UUO surgery. Quantitation of relative signal intensities of (b) Smad3/CDK9 and (c) Smad4/CDK9 7 days after sham or UUO surgery. Data are mean ± s.d., n = 6. *Po0.05 versus WT sham or WT UUO. NS, P40.05 versus WT UUO. (d) IP/WB demonstrated interactions between Smad4 and CDK9 in the kidneys 7 days after sham or UUO surgery in Smad3 wild-type (WT) or Smad3 knockout (KO) mice. (e) WB demonstrated the expression levels of α-smooth muscle actin (α-SMA), fibronectin (FN), collagen I (Col. I) and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) in the kidneys 7 days after sham or UUO surgery in Smad3 WT or Smad3 KO mice. (f) Quantitation of relative signal intensities of α-SMA, FN, and Col. I in the kidneys 7 days after sham or UUO surgery in Smad3 WT or Smad3 KO mice. Data are mean ± s.d., n = 6. *Po0.05; **Po0.01. (g) IP/WB demonstrated interaction between Smad3 and CDK9, phosphorylated Smad3 T179 (p-T179), and phosphorylated Smad3 C-terminus (p-Tail) in the kidneys 7 days after sham or UUO surgery in Smad4 WT or Smad4 KO. (h) WB demonstrated the expression levels of α-SMA, FN, Col. I, and GAPDH in the kidneys 7 days after sham or UUO surgery in Smad4 WT or Smad4 KO mice. (i) Quantitation of relative signal intensities of α-SMA, FN, and Col. I in the kidneys 7 days after sham or UUO surgery in Smad4 WT or Smad4 KO mice. Data are mean ± s.d., n = 6. *Po0.05; ***Po0.001.

Article Snippet: After blocking for 30 min at 4 °C in 5% bovine serum albumin in phosphate-buffered saline with 0.1% Tween 20, the membrane was incubated overnight with rabbit anti-Smad3, CDK9, p-Smad3 Thr179 (Biorbyt, Cambridge, UK), or rabbit antiphospho-Smad3 (Ser423/425) (Cell signaling Technology), or Smad4 (Cell Signalling Technology).

Techniques: Immunoprecipitation, Western Blot, Quantitation Assay, Knock-Out, Expressing

Journal: Kidney international

Article Title: The Smad3/Smad4/CDK9 complex promotes renal fibrosis in mice with unilateral ureteral obstruction.

doi: 10.1038/ki.2015.235

Figure Lengend Snippet: Figure 4| Knockdown of cyclin-dependent kinase 9 (CDK9) decreases transforming growth factor-β1 (TGF-β1)-induced Smad3 linker phosphorylation and fibrotic response in renal fibroblasts. (a) Primary cultured mouse renal fibroblasts were transfected with CDK9 siRNA or a scrambled siRNA control. Two days after transfection, renal fibroblasts were stimulated with recombinant TGF-β1 for (a) 2 days or (b) 30 min. (a) Western blotting (WB) shows the expression of CDK9, α-smooth muscle actin (α-SMA), fibronectin, collagen I, and α-tubulin in renal fibroblasts. (b) Immunoprecipitation (IP)/WB or WB shows the interaction between Smad3 and Smad4, Smad3 p-Tail and nuclear p-T179, and the internal control Histone 2A in renal fibroblasts. Quantification of the relative signal intensities of (c) α-SMA/α-tubulin, fibronectin/α-tubulin, collagen I/α-tubulin, (d) Smad4/Smad3, and p-Tail Smad3/Smad3. Data are mean ± s.d., n = 4. *Po0.05;***Po0.001; NS, not significant.

Article Snippet: After blocking for 30 min at 4 °C in 5% bovine serum albumin in phosphate-buffered saline with 0.1% Tween 20, the membrane was incubated overnight with rabbit anti-Smad3, CDK9, p-Smad3 Thr179 (Biorbyt, Cambridge, UK), or rabbit antiphospho-Smad3 (Ser423/425) (Cell signaling Technology), or Smad4 (Cell Signalling Technology).

Techniques: Knockdown, Phospho-proteomics, Cell Culture, Transfection, Control, Recombinant, Western Blot, Expressing, Immunoprecipitation

Journal: Kidney international

Article Title: The Smad3/Smad4/CDK9 complex promotes renal fibrosis in mice with unilateral ureteral obstruction.

doi: 10.1038/ki.2015.235

Figure Lengend Snippet: Figure 5| Cyclin-dependent kinase 9 (CDK9) promotes transforming growth factor-β1 (TGF-β1)-induced collagen I promoter activity. Western blotting (WB) demonstrated expression levels of (a) nuclear or (b) cytoplasm (Cyto) phosphorylated Smad3 T179 (p-T179) in the kidneys after sham or unilateral ureteral obstruction (UUO) surgery. (c) Immunoprecipitation (IP)/WB demonstrated interactions between CDK9 and Smad3 and CDK9 and Smad4 and the levels of p-T179 after TGF-β1 stimulation in mouse renal fibroblasts. (d) Collagen I promoter luciferase assay demonstrated the effects of CDK9 on Smad3, Smad4 and Smad3, and Smad4 enhancement on collagen I promoter activity with or without TGF-β1 stimulation in mouse renal fibroblasts. Data are mean ± s.d. Experiments were repeated three times. Data were analyzed by two- way analysis of variance for different vector transfections and with or without TGF-β1 treatment. Control versus TGF-β1, Po0.05; a–f represent different levels of collagen I promoter luciferase activity. (e) Collagen I luciferase activity assay demonstrated the effects of CDK9 on Smad3 WT or Smad3 linker-mutated enhancement on collagen I promoter activity in mouse renal fibroblasts. Data are mean ± s.d. Experiments were repeated three times. *Po0.05; **Po0.01. (f–h) Collagen I lunciferase activity assay demonstrated the effects of double knockout of Smad3 and Smad4 (Smad3/4 / ) on CDK9 enhancement (f), kinase-dead CDK9 (dnCDK9) on Smad4 enhancement (g), and 3 serine at Smad3 C-terminus replaced with arginine (3S/A) on CDK9 enhancement (h) on collagen I promoter activity in mouse renal fibroblasts. Data are mean ± s.d. Experiments were repeated three times. *Po0.05; **Po0.01; ***Po0.001.

Article Snippet: After blocking for 30 min at 4 °C in 5% bovine serum albumin in phosphate-buffered saline with 0.1% Tween 20, the membrane was incubated overnight with rabbit anti-Smad3, CDK9, p-Smad3 Thr179 (Biorbyt, Cambridge, UK), or rabbit antiphospho-Smad3 (Ser423/425) (Cell signaling Technology), or Smad4 (Cell Signalling Technology).

Techniques: Activity Assay, Western Blot, Expressing, Immunoprecipitation, Luciferase, Plasmid Preparation, Transfection, Control, Double Knockout

Journal: Kidney international

Article Title: The Smad3/Smad4/CDK9 complex promotes renal fibrosis in mice with unilateral ureteral obstruction.

doi: 10.1038/ki.2015.235

Figure Lengend Snippet: Figure 6| Cyclin-dependent kinase 9 (CDK9) inhibitor inhibits transforming growth factor-β1 (TGF-β1)-induced fibrotic response in renal fibroblasts. (a) Collagen I promoter luciferase assay demonstrated effects of CDK9 inhibitor (CDK9i) and Smad3 inhibitor (SIS3) on collagen I promoter activity with or without TGF-β1 stimulation in mouse renal fibroblasts. Data are mean ± s.d. Experiments were repeated three times. Data were analyzed by one-way analysis of variance for different treatments. *Po0.05 versus control; #Po0.05 versus TGF-β1; $Po0.05 versus TGF-β1+CDK9i 50 nM. (b) Western blotting (WB) demonstrated the expression levels of α-smooth muscle actin (α-SMA), collagen I (Col. I), and fibronectin (FN), and internal control α-tubulin 3 days after different treatments in mouse renal fibroblasts. (c) WB demonstrated nuclear phosphorylated Smad3 T179 (Smad3 p-T179) and phosphorylated RNAPII Ser5 (RNAPII p-Ser5) for the indicated period of time of treatments in mouse renal fibroblasts.

Article Snippet: After blocking for 30 min at 4 °C in 5% bovine serum albumin in phosphate-buffered saline with 0.1% Tween 20, the membrane was incubated overnight with rabbit anti-Smad3, CDK9, p-Smad3 Thr179 (Biorbyt, Cambridge, UK), or rabbit antiphospho-Smad3 (Ser423/425) (Cell signaling Technology), or Smad4 (Cell Signalling Technology).

Techniques: Luciferase, Activity Assay, Control, Western Blot, Expressing

Journal: Kidney international

Article Title: The Smad3/Smad4/CDK9 complex promotes renal fibrosis in mice with unilateral ureteral obstruction.

doi: 10.1038/ki.2015.235

Figure Lengend Snippet: Figure 7| Smad3, Smad4, and cyclin-dependent kinase 9 (CDK9) complex formation in 2-day unilateral ureteral obstruction (UUO) after administration of CDKi or/and SiS3. (a) Western blotting (WB) demonstrated the expression levels of nuclear phosphorylated Smad3 T-179 (p-T179) and Smad3 C-terminus (p-Tail Smad3) in the kidneys 2d after sham or UUO surgery. (b) Quantitation of relative signal intensities of p-T179/Histone 2A and p-Tail Smad3/Smad3. Data are mean ± s.d., n = 6. *Po0.05, versus sham group; #Po0.05, versus UUO vehicle or UUO CDK9i 1 μg/g/day group. (c) Immunoglobulin (IP)/WB demonstrated the interactions between Smad3 and Smad4, Smad3 and CDK9, and Smad4 and CDK9 in the kidneys 2d after sham or UUO surgery. (d) Quantitation of relative signal intensities of Smad4/Smad3, Smad3/CDK9, and Smad4/CDK9. Data are mean ± s.d., n = 6. *Po0.05, versus sham group; #Po0.05, versus UUO vehicle group; $Po0.05, versus UUO CDK9i 4 μg/g/day group or UUO SIS3 2.5 μg/g/day group.

Article Snippet: After blocking for 30 min at 4 °C in 5% bovine serum albumin in phosphate-buffered saline with 0.1% Tween 20, the membrane was incubated overnight with rabbit anti-Smad3, CDK9, p-Smad3 Thr179 (Biorbyt, Cambridge, UK), or rabbit antiphospho-Smad3 (Ser423/425) (Cell signaling Technology), or Smad4 (Cell Signalling Technology).

Techniques: Western Blot, Expressing, Quantitation Assay

Journal: Kidney international

Article Title: The Smad3/Smad4/CDK9 complex promotes renal fibrosis in mice with unilateral ureteral obstruction.

doi: 10.1038/ki.2015.235

Figure Lengend Snippet: Figure 8| The effects of cyclin-dependent kinase 9 inhibitor (CDK9i) and SiS3 on renal fibrosis and inflammation in unilateral ureteral obstruction (UUO). (a) Western blotting (WB) demonstrated the expression levels of α-smooth muscle actin (α-SMA), collagen I (Col. I), and fibronectin (FN) and internal control α-tubulin in the kidneys 7d after sham or UUO surgery with different treatments. (b) Quantitation of relative signal intensities of α-SMA/α-tubulin, Col. I/α-tubulin, and FN/α-tubulin. Data are mean ± s.d., n = 6. *Po0.05, versus sham group; #Po0.05, versus UUO vehicle group; $Po0.05, versus UUO CDK9i 4 μg/g/day group or UUO SiS3 2.5 μg/g/day group. (c) Quantitation of the number of F4/80+ macrophages in the kidneys 7d after sham or UUO surgery with different treatments. Data are mean ± s.d., n = 6. *Po0.05, versus sham group; #Po0.05, versus UUO vehicle group; $Po0.05, versus UUO CDK9i 4 μg/g/day group or UUO SiS3 2.5 μg/g/day group.

Article Snippet: After blocking for 30 min at 4 °C in 5% bovine serum albumin in phosphate-buffered saline with 0.1% Tween 20, the membrane was incubated overnight with rabbit anti-Smad3, CDK9, p-Smad3 Thr179 (Biorbyt, Cambridge, UK), or rabbit antiphospho-Smad3 (Ser423/425) (Cell signaling Technology), or Smad4 (Cell Signalling Technology).

Techniques: Western Blot, Expressing, Control, Quantitation Assay