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

Journal: SLAS discovery : advancing life sciences R & D

Article Title: Application of Integrated Drug Screening/Kinome Analysis to Identify Inhibitors of Gemcitabine-Resistant Pancreatic Cancer Cell Growth

doi: 10.1177/2472555218773045

Figure Lengend Snippet: Thermo Fisher SelectScreen kinase assay results and modeling of UNC-721A within the CDK9 active site. (A) The Thermo Fisher SelectScreen kinase assay was used to validate CDK9 as a target of UNC-721A. The kinase assay was performed as described in Methods and kinase inhibition plotted as a function of UNC-721A concentration. The IC50 value was determined to be 0.603 nM (n = 3). (B) Chemical structure of UNC-721A. The predicted fit of this molecule in the ATP binding site of CDK9 was determined by molecular modeling as described in Materials and Methods. This model indicates that the UNC-721A structure fits with high confidence within the CDK9 active site.

Article Snippet: The inhibitors used were CDK9-IN-2 (MedChem Express, Monmouth Junction, NJ, cat. HY-16462), ML167 (Selleckchem, Houston, TX, cat. S7509), PF 670462 (Tocris, Minneapolis, MN, cat. 3316), and Alisertib (ApexBio, Hsinchu City, Taiwan, cat. A4110), respectively.

Techniques: Kinase Assay, Inhibition, Concentration Assay, Binding Assay

Journal: SLAS discovery : advancing life sciences R & D

Article Title: Application of Integrated Drug Screening/Kinome Analysis to Identify Inhibitors of Gemcitabine-Resistant Pancreatic Cancer Cell Growth

doi: 10.1177/2472555218773045

Figure Lengend Snippet: CDK-IN-2 2D and 3D cell viability responses. CDK-IN-2, a selective CDK9 inhibitor, was used to compare the effects on cell growth to that observed with UNC-721A. Cell viability assays were done in both 2D and 3D spheroid format as described above (CellTiter-Glo 3D). The cells consisted of a 1:1 ratio (5000:5000 cells/well) of MiaS or MiaR cells combined with GFP-CAF cells, and they were treated for 72 h and analyzed as above. (A) Results from the 2D analysis of this compound in triplicate. (B) Results from the 3D analysis of this compound in triplicate. Shown are representative figures for n = 3 independent experiments.

Article Snippet: The inhibitors used were CDK9-IN-2 (MedChem Express, Monmouth Junction, NJ, cat. HY-16462), ML167 (Selleckchem, Houston, TX, cat. S7509), PF 670462 (Tocris, Minneapolis, MN, cat. 3316), and Alisertib (ApexBio, Hsinchu City, Taiwan, cat. A4110), respectively.

Techniques: