Journal: Nature communications

Article Title: Phosphoproteome profiling uncovers a key role for CDKs in TNF signaling.

doi: 10.1038/s41467-021-26289-6

Figure Lengend Snippet: Fig. 4 TNF-induced cell death leads to a strong RNA-processing response and regulation of the phosphorylation status of CDKs. a Experimental scheme of U937 cells that were untreated, treated with TNF (100 ng/ml, red) alone, treated with TNF and Smac mimetic (SM/Birinapant, 1.25 μM, green) to induce apoptosis, or with TNF, SM, and IDN-6556 (Emricasan, 10 μM, blue) to induce necroptosis. b Z-scored phosphosite intensities of CYLD and XIAP that are regulated upon TNF treatment, TNF-induced apoptosis, and TNF-induced necroptosis (±SEM, n = 4 biologically independent experiments). XIAP phosphosites were retrieved from a DDA dataset (Methods). c Heatmap of z-scored phosphosite intensities that are one-way ANOVA significantly regulated upon TNF treatment or TNF-induced cell death (FDR < 0.05). The profiles are color-coded according to their distance from the respective cluster center (red is close to the center, blue is further away from the center). d Fisher’s exact test on kinases with significantly regulated phosphosites upon different treatments (FDR < 0.02). e Heatmap of means of z-scored phosphosite intensities of CDKs that significantly changed upon treatment of U937 cells (one-way ANOVA, FDR < 0.05). f Heatmap of means of z-scored phosphosite intensities of CDKs that changed significantly (one-way ANOVA) upon treatment of BMDMs with TNF (red) alone, TNF, and TAK1 inhibitor (TAKi, 1 μM) to induce apoptosis (green) or TNF, TAK1i, and the caspase inhibitor IDN-6556 to induce necroptosis (blue). Untreated cells (UT) served as controls (FDR < 0.05). g–i Immunoblots of U937 cells stimulated with TNF as indicated or in combination with CDK inhibitors Dinaciclib (6 nM) (g), THZ531 (200 nM) (h), and AZD4573 (6 nM) (i) and probed with antibodies against phosphorylated (S2) and total (N-terminal) RPB1, phosphorylated (T186), and total CDK9, CDK12, and β-actin (n = 2 biologically independent experiments). Source data are provided as source file.

Article Snippet: Antibodies used for immunoblotting were as follows (diluted 1:1000): anti-human caspase-8 (MBL, M058-3), anti-cleaved human caspase-3 (Cell Signaling Technology CST, 9661), anti-human MLKL (Merck Millipore, MABC604), phospho anti-human RPB1 S2 (Millipore, 04-1571), anti-human RPB1 (CST, D8L4Y), phospho anti-human p65 (CST, 3033 P), anti-human IκBα (CST, 9242), phospho anti-human p38 (CST, 9215), anti-human p38 (CST, 9212), anti-human CDK12 (CST, 11973), phospho anti-human CDK9 (CST, 2549), anti-human CDK9 (CST, 2316), anti-human A20 (Santa Cruz, sc-166692), anti-human MCL1 (CST,4572), anti-human XIAP (MBL, M044-3), anti-human FLIP (CST, 56343) and antihuman β-actin (CST, 4967).

Techniques: Phospho-proteomics, Western Blot

Journal: Nature communications

Article Title: Phosphoproteome profiling uncovers a key role for CDKs in TNF signaling.

doi: 10.1038/s41467-021-26289-6

Figure Lengend Snippet: Fig. 5 CDK9 and CDK12/13 inhibitors potently reduce transcription of TNF-target genes. a Heatmap of z-scored protein intensities significantly changed upon TNF treatment for 6 h in U937 cells (FDR < 0.05). Cells were additionally treated with Dinaciclib (6 nM), CDK12-IN3 (60 nM), and THZ531 (400 nM). Fisher’s exact test on up- and downregulated protein clusters (two-sided Student’s t-test, FDR < 0.001). The profiles are color-coded according to their distance from the respective cluster center (red is close to the center, blue is further away from the center). b Z-scored protein levels of selected TNF-target genes and members of the TNF-signaling pathway (±SD, n = 4 biologically independent experiments). c Immunoblot of U937 cells treated with TNF alone and in combination with the pan-CDK inhibitor Dinaciclib. Proteins were blotted for IκBα, phosphorylated p65, phosphorylated, and total p38 and β-actin (loading control) (n = 2 biologically independent experiments). d qPCR of CCL2 of U937 cells treated for 4 h with TNF alone or in combination with CDK inhibitors Dinaciclib (6 nM), CDK12-IN3 (60 nM), SR4835 (160 nM), THZ531 (200 nM) and AZD4573 (6 nM) (±SD, n = 3 biologically independent experiments). e–h U937 cells treated with TNF alone and in combination with CDK inhibitors (concentrations as in d) were blotted for cFLIP and β-actin (n = 2 biologically independent experiments). Source data are provided as source file.

Article Snippet: Antibodies used for immunoblotting were as follows (diluted 1:1000): anti-human caspase-8 (MBL, M058-3), anti-cleaved human caspase-3 (Cell Signaling Technology CST, 9661), anti-human MLKL (Merck Millipore, MABC604), phospho anti-human RPB1 S2 (Millipore, 04-1571), anti-human RPB1 (CST, D8L4Y), phospho anti-human p65 (CST, 3033 P), anti-human IκBα (CST, 9242), phospho anti-human p38 (CST, 9215), anti-human p38 (CST, 9212), anti-human CDK12 (CST, 11973), phospho anti-human CDK9 (CST, 2549), anti-human CDK9 (CST, 2316), anti-human A20 (Santa Cruz, sc-166692), anti-human MCL1 (CST,4572), anti-human XIAP (MBL, M044-3), anti-human FLIP (CST, 56343) and antihuman β-actin (CST, 4967).

Techniques: Western Blot, Control

Journal: Nature communications

Article Title: Phosphoproteome profiling uncovers a key role for CDKs in TNF signaling.

doi: 10.1038/s41467-021-26289-6

Figure Lengend Snippet: Fig. 4 TNF-induced cell death leads to a strong RNA-processing response and regulation of the phosphorylation status of CDKs. a Experimental scheme of U937 cells that were untreated, treated with TNF (100 ng/ml, red) alone, treated with TNF and Smac mimetic (SM/Birinapant, 1.25 μM, green) to induce apoptosis, or with TNF, SM, and IDN-6556 (Emricasan, 10 μM, blue) to induce necroptosis. b Z-scored phosphosite intensities of CYLD and XIAP that are regulated upon TNF treatment, TNF-induced apoptosis, and TNF-induced necroptosis (±SEM, n = 4 biologically independent experiments). XIAP phosphosites were retrieved from a DDA dataset (Methods). c Heatmap of z-scored phosphosite intensities that are one-way ANOVA significantly regulated upon TNF treatment or TNF-induced cell death (FDR < 0.05). The profiles are color-coded according to their distance from the respective cluster center (red is close to the center, blue is further away from the center). d Fisher’s exact test on kinases with significantly regulated phosphosites upon different treatments (FDR < 0.02). e Heatmap of means of z-scored phosphosite intensities of CDKs that significantly changed upon treatment of U937 cells (one-way ANOVA, FDR < 0.05). f Heatmap of means of z-scored phosphosite intensities of CDKs that changed significantly (one-way ANOVA) upon treatment of BMDMs with TNF (red) alone, TNF, and TAK1 inhibitor (TAKi, 1 μM) to induce apoptosis (green) or TNF, TAK1i, and the caspase inhibitor IDN-6556 to induce necroptosis (blue). Untreated cells (UT) served as controls (FDR < 0.05). g–i Immunoblots of U937 cells stimulated with TNF as indicated or in combination with CDK inhibitors Dinaciclib (6 nM) (g), THZ531 (200 nM) (h), and AZD4573 (6 nM) (i) and probed with antibodies against phosphorylated (S2) and total (N-terminal) RPB1, phosphorylated (T186), and total CDK9, CDK12, and β-actin (n = 2 biologically independent experiments). Source data are provided as source file.

Article Snippet: Antibodies used for immunoblotting were as follows (diluted 1:1000): anti-human caspase-8 (MBL, M058-3), anti-cleaved human caspase-3 (Cell Signaling Technology CST, 9661), anti-human MLKL (Merck Millipore, MABC604), phospho anti-human RPB1 S2 (Millipore, 04-1571), anti-human RPB1 (CST, D8L4Y), phospho anti-human p65 (CST, 3033 P), anti-human IκBα (CST, 9242), phospho anti-human p38 (CST, 9215), anti-human p38 (CST, 9212), anti-human CDK12 (CST, 11973), phospho anti-human CDK9 (CST, 2549), anti-human CDK9 (CST, 2316), anti-human A20 (Santa Cruz, sc-166692), anti-human MCL1 (CST,4572), anti-human XIAP (MBL, M044-3), anti-human FLIP (CST, 56343) and antihuman β-actin (CST, 4967).

Techniques: Phospho-proteomics, Western Blot

Journal: Nature communications

Article Title: Phosphoproteome profiling uncovers a key role for CDKs in TNF signaling.

doi: 10.1038/s41467-021-26289-6

Figure Lengend Snippet: Fig. 5 CDK9 and CDK12/13 inhibitors potently reduce transcription of TNF-target genes. a Heatmap of z-scored protein intensities significantly changed upon TNF treatment for 6 h in U937 cells (FDR < 0.05). Cells were additionally treated with Dinaciclib (6 nM), CDK12-IN3 (60 nM), and THZ531 (400 nM). Fisher’s exact test on up- and downregulated protein clusters (two-sided Student’s t-test, FDR < 0.001). The profiles are color-coded according to their distance from the respective cluster center (red is close to the center, blue is further away from the center). b Z-scored protein levels of selected TNF-target genes and members of the TNF-signaling pathway (±SD, n = 4 biologically independent experiments). c Immunoblot of U937 cells treated with TNF alone and in combination with the pan-CDK inhibitor Dinaciclib. Proteins were blotted for IκBα, phosphorylated p65, phosphorylated, and total p38 and β-actin (loading control) (n = 2 biologically independent experiments). d qPCR of CCL2 of U937 cells treated for 4 h with TNF alone or in combination with CDK inhibitors Dinaciclib (6 nM), CDK12-IN3 (60 nM), SR4835 (160 nM), THZ531 (200 nM) and AZD4573 (6 nM) (±SD, n = 3 biologically independent experiments). e–h U937 cells treated with TNF alone and in combination with CDK inhibitors (concentrations as in d) were blotted for cFLIP and β-actin (n = 2 biologically independent experiments). Source data are provided as source file.

Article Snippet: Antibodies used for immunoblotting were as follows (diluted 1:1000): anti-human caspase-8 (MBL, M058-3), anti-cleaved human caspase-3 (Cell Signaling Technology CST, 9661), anti-human MLKL (Merck Millipore, MABC604), phospho anti-human RPB1 S2 (Millipore, 04-1571), anti-human RPB1 (CST, D8L4Y), phospho anti-human p65 (CST, 3033 P), anti-human IκBα (CST, 9242), phospho anti-human p38 (CST, 9215), anti-human p38 (CST, 9212), anti-human CDK12 (CST, 11973), phospho anti-human CDK9 (CST, 2549), anti-human CDK9 (CST, 2316), anti-human A20 (Santa Cruz, sc-166692), anti-human MCL1 (CST,4572), anti-human XIAP (MBL, M044-3), anti-human FLIP (CST, 56343) and antihuman β-actin (CST, 4967).

Techniques: Western Blot, Control

Journal: Oncology reports

Article Title: Targeting CDK9: A novel biomarker in the treatment of endometrial cancer.

doi: 10.3892/or.2020.7746

Figure Lengend Snippet: Figure 1. Different CDK9 staining intensities and H&E staining of endometrial cancer tissues. According to the CDK9 staining in the tumor samples, the staining patterns were divided into 5 groups: i) l<10% positive cells (1+); ii) 10‑25% positive cells (2+); iii) 26‑50% positive cells (3+); iv) 51‑75% positive cells (4+); v) >75% positive cells (5+). (Original magnification, x400). CDK9, cyclin‑dependent kinase 9; H&E, hematoxylin and eosin.

Article Snippet: Monoclonal rabbit anti-human CDK9 antibody (cat. no. 2316; Cell Signaling Technology, Inc.) was purchased from Cell Signaling Technology, Inc..

Techniques: Staining

Journal: Oncology reports

Article Title: Targeting CDK9: A novel biomarker in the treatment of endometrial cancer.

doi: 10.3892/or.2020.7746

Figure Lengend Snippet: Figure 3. CDK9 expression in endometrial cancer cell lines. (A) Expression levels of CDK9 in endometrial cancer cell lines (AN3CA, ARK‑2, HEC‑1A, HEC‑1B, lshikawa, RL95‑2 and SPAC1S) as determined by western blotting. (B) Relative expression of CDK9 and α‑tubulin in the endometrial cancer cell lines. CDK9, cyclin‑dependent kinase 9.

Article Snippet: Monoclonal rabbit anti-human CDK9 antibody (cat. no. 2316; Cell Signaling Technology, Inc.) was purchased from Cell Signaling Technology, Inc..

Techniques: Expressing, Western Blot

Journal: Oncology reports

Article Title: Targeting CDK9: A novel biomarker in the treatment of endometrial cancer.

doi: 10.3892/or.2020.7746

Figure Lengend Snippet: Figure 2. Higher expression of CDK9 is present in metastatic and recurrent endometrial cancer tissues compared with that found in the patient matched primary tumors and CDK9 is correlated with poor patient prognosis. (A) Distribution of CDK9 immunohistochemical staining scores among primary, metastatic, and recurrent endometrial cancer tissues. (B and C) Correlation between expression of CDK9 in the primary endometrial cancer tissues (Low, CDK9 staining ≤2+; High, CDK9 staining ≥3+) and PFS (B) or OS (C) in endometrial cancer patients by Kaplan‑Meier survival curve analysis. CDK9, cyclin‑dependent kinase 9; PFS, progression‑free survival; OS, overall survival.

Article Snippet: Monoclonal rabbit anti-human CDK9 antibody (cat. no. 2316; Cell Signaling Technology, Inc.) was purchased from Cell Signaling Technology, Inc..

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Oncology reports

Article Title: Targeting CDK9: A novel biomarker in the treatment of endometrial cancer.

doi: 10.3892/or.2020.7746

Figure Lengend Snippet: Figure 5. CDK9 inhibitor reduces endometrial cancer cell proliferation by suppressing transcription elongation and inducing apoptosis in endometrial cancer cells. (A and B) Relative cell viability of AN3CA and SPAC1S cells after exposure to different concentrations of the CDK9 inhibitor LDC067 for 5 days. **P<0.01 compared with the lowest concentration group (1x10‑3 µM). (C and D) Expression levels of CDK9 and related signaling pathway proteins involved in transcription and apoptosis after treatment with LDC067 in cells by western blot analysis. CDK9, cyclin‑dependent kinase 9; Mcl‑1, myeloid cell leukemia‑1; Bax, proapoptotic protein BCL2 associated X, apoptosis regulator; PARP, poly(ADP‑ribose) polymerase.

Article Snippet: Monoclonal rabbit anti-human CDK9 antibody (cat. no. 2316; Cell Signaling Technology, Inc.) was purchased from Cell Signaling Technology, Inc..

Techniques: Concentration Assay, Expressing, Western Blot

Journal: Oncology reports

Article Title: Targeting CDK9: A novel biomarker in the treatment of endometrial cancer.

doi: 10.3892/or.2020.7746

Figure Lengend Snippet: Figure 4. CDK9 knockdown by siRNA transfection suppresses endometrial cancer cell proliferation. (A and B) MTT assay revealed significant dose‑dependent inhibition of cell proliferation after CDK9 siRNA treatment. **P<0.01 compared with the cell only control group. (C and D) Expression levels of CDK9 and related signaling pathway proteins involved in transcription and apoptosis after transfection of CDK9 siRNA and nonspecific siRNA (NC siRNA) in AN3CA and SPAC1S cell lines by western blot analysis. CDK9, cyclin‑dependent kinase 9; Mcl‑1, myeloid cell leukemia‑1; Bax, proapoptotic protein BCL2 associated X, apoptosis regulator.

Article Snippet: Monoclonal rabbit anti-human CDK9 antibody (cat. no. 2316; Cell Signaling Technology, Inc.) was purchased from Cell Signaling Technology, Inc..

Techniques: Knockdown, Transfection, MTT Assay, Inhibition, Control, Expressing, Western Blot

Journal: Oncology reports

Article Title: Targeting CDK9: A novel biomarker in the treatment of endometrial cancer.

doi: 10.3892/or.2020.7746

Figure Lengend Snippet: Figure 7. Inhibition of CDK9 reduces endometrial cancer cell migration. (A and B) Representative images of AN3CA and SPAC1S cell migration after CDK9 inhibitor LDC067 treatment for 0, 24, and 48 h. (C and D) Quantification of cell migration distance of AN3CA and SPAC1S cells after LDC067 treatment. **P<0.01 compared with the Cell only group. CDK9, cyclin‑dependent kinase 9.

Article Snippet: Monoclonal rabbit anti-human CDK9 antibody (cat. no. 2316; Cell Signaling Technology, Inc.) was purchased from Cell Signaling Technology, Inc..

Techniques: Inhibition, Migration

Journal: Oncology reports

Article Title: Targeting CDK9: A novel biomarker in the treatment of endometrial cancer.

doi: 10.3892/or.2020.7746

Figure Lengend Snippet: Figure 6. Inhibition of CDK9 suppresses endometrial cancer cell colony formation. (A) Representative images of endometrial cancer cell colony formation after incubation with different concentrations of LDC067 (0, 2.5, 5.0, and 10 µM) for 14 days. (B and C) Quantification of clonogenicity formation of AN3CA (B) and SPAC1S (C) cells after LDC067 treatment. **P<0.01 compared with the Cell only group. CDK9, cyclin‑dependent kinase 9.

Article Snippet: Monoclonal rabbit anti-human CDK9 antibody (cat. no. 2316; Cell Signaling Technology, Inc.) was purchased from Cell Signaling Technology, Inc..

Techniques: Inhibition, Incubation

Journal: Oncology reports

Article Title: Targeting CDK9: A novel biomarker in the treatment of endometrial cancer.

doi: 10.3892/or.2020.7746

Figure Lengend Snippet: Figure 1. Different CDK9 staining intensities and H&E staining of endometrial cancer tissues. According to the CDK9 staining in the tumor samples, the staining patterns were divided into 5 groups: i) l<10% positive cells (1+); ii) 10‑25% positive cells (2+); iii) 26‑50% positive cells (3+); iv) 51‑75% positive cells (4+); v) >75% positive cells (5+). (Original magnification, x400). CDK9, cyclin‑dependent kinase 9; H&E, hematoxylin and eosin.

Article Snippet: Thereafter, the slides were sealed with goat serum for 1 h, and then polyclonal rabbit antibodies against human CDK9 (cat. no. 2316; 1:50 dilution in 1% bovine serum albumin; Cell Signaling Technology, Inc.) were added and incubated overnight.

Techniques: Staining

Journal: Oncology reports

Article Title: Targeting CDK9: A novel biomarker in the treatment of endometrial cancer.

doi: 10.3892/or.2020.7746

Figure Lengend Snippet: Figure 3. CDK9 expression in endometrial cancer cell lines. (A) Expression levels of CDK9 in endometrial cancer cell lines (AN3CA, ARK‑2, HEC‑1A, HEC‑1B, lshikawa, RL95‑2 and SPAC1S) as determined by western blotting. (B) Relative expression of CDK9 and α‑tubulin in the endometrial cancer cell lines. CDK9, cyclin‑dependent kinase 9.

Article Snippet: Thereafter, the slides were sealed with goat serum for 1 h, and then polyclonal rabbit antibodies against human CDK9 (cat. no. 2316; 1:50 dilution in 1% bovine serum albumin; Cell Signaling Technology, Inc.) were added and incubated overnight.

Techniques: Expressing, Western Blot

Journal: Oncology reports

Article Title: Targeting CDK9: A novel biomarker in the treatment of endometrial cancer.

doi: 10.3892/or.2020.7746

Figure Lengend Snippet: Figure 2. Higher expression of CDK9 is present in metastatic and recurrent endometrial cancer tissues compared with that found in the patient matched primary tumors and CDK9 is correlated with poor patient prognosis. (A) Distribution of CDK9 immunohistochemical staining scores among primary, metastatic, and recurrent endometrial cancer tissues. (B and C) Correlation between expression of CDK9 in the primary endometrial cancer tissues (Low, CDK9 staining ≤2+; High, CDK9 staining ≥3+) and PFS (B) or OS (C) in endometrial cancer patients by Kaplan‑Meier survival curve analysis. CDK9, cyclin‑dependent kinase 9; PFS, progression‑free survival; OS, overall survival.

Article Snippet: Thereafter, the slides were sealed with goat serum for 1 h, and then polyclonal rabbit antibodies against human CDK9 (cat. no. 2316; 1:50 dilution in 1% bovine serum albumin; Cell Signaling Technology, Inc.) were added and incubated overnight.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Oncology reports

Article Title: Targeting CDK9: A novel biomarker in the treatment of endometrial cancer.

doi: 10.3892/or.2020.7746

Figure Lengend Snippet: Figure 5. CDK9 inhibitor reduces endometrial cancer cell proliferation by suppressing transcription elongation and inducing apoptosis in endometrial cancer cells. (A and B) Relative cell viability of AN3CA and SPAC1S cells after exposure to different concentrations of the CDK9 inhibitor LDC067 for 5 days. **P<0.01 compared with the lowest concentration group (1x10‑3 µM). (C and D) Expression levels of CDK9 and related signaling pathway proteins involved in transcription and apoptosis after treatment with LDC067 in cells by western blot analysis. CDK9, cyclin‑dependent kinase 9; Mcl‑1, myeloid cell leukemia‑1; Bax, proapoptotic protein BCL2 associated X, apoptosis regulator; PARP, poly(ADP‑ribose) polymerase.

Article Snippet: Thereafter, the slides were sealed with goat serum for 1 h, and then polyclonal rabbit antibodies against human CDK9 (cat. no. 2316; 1:50 dilution in 1% bovine serum albumin; Cell Signaling Technology, Inc.) were added and incubated overnight.

Techniques: Concentration Assay, Expressing, Western Blot

Journal: Oncology reports

Article Title: Targeting CDK9: A novel biomarker in the treatment of endometrial cancer.

doi: 10.3892/or.2020.7746

Figure Lengend Snippet: Figure 4. CDK9 knockdown by siRNA transfection suppresses endometrial cancer cell proliferation. (A and B) MTT assay revealed significant dose‑dependent inhibition of cell proliferation after CDK9 siRNA treatment. **P<0.01 compared with the cell only control group. (C and D) Expression levels of CDK9 and related signaling pathway proteins involved in transcription and apoptosis after transfection of CDK9 siRNA and nonspecific siRNA (NC siRNA) in AN3CA and SPAC1S cell lines by western blot analysis. CDK9, cyclin‑dependent kinase 9; Mcl‑1, myeloid cell leukemia‑1; Bax, proapoptotic protein BCL2 associated X, apoptosis regulator.

Article Snippet: Thereafter, the slides were sealed with goat serum for 1 h, and then polyclonal rabbit antibodies against human CDK9 (cat. no. 2316; 1:50 dilution in 1% bovine serum albumin; Cell Signaling Technology, Inc.) were added and incubated overnight.

Techniques: Knockdown, Transfection, MTT Assay, Inhibition, Control, Expressing, Western Blot

Journal: Oncology reports

Article Title: Targeting CDK9: A novel biomarker in the treatment of endometrial cancer.

doi: 10.3892/or.2020.7746

Figure Lengend Snippet: Figure 7. Inhibition of CDK9 reduces endometrial cancer cell migration. (A and B) Representative images of AN3CA and SPAC1S cell migration after CDK9 inhibitor LDC067 treatment for 0, 24, and 48 h. (C and D) Quantification of cell migration distance of AN3CA and SPAC1S cells after LDC067 treatment. **P<0.01 compared with the Cell only group. CDK9, cyclin‑dependent kinase 9.

Article Snippet: Thereafter, the slides were sealed with goat serum for 1 h, and then polyclonal rabbit antibodies against human CDK9 (cat. no. 2316; 1:50 dilution in 1% bovine serum albumin; Cell Signaling Technology, Inc.) were added and incubated overnight.

Techniques: Inhibition, Migration

Journal: Oncology reports

Article Title: Targeting CDK9: A novel biomarker in the treatment of endometrial cancer.

doi: 10.3892/or.2020.7746

Figure Lengend Snippet: Figure 6. Inhibition of CDK9 suppresses endometrial cancer cell colony formation. (A) Representative images of endometrial cancer cell colony formation after incubation with different concentrations of LDC067 (0, 2.5, 5.0, and 10 µM) for 14 days. (B and C) Quantification of clonogenicity formation of AN3CA (B) and SPAC1S (C) cells after LDC067 treatment. **P<0.01 compared with the Cell only group. CDK9, cyclin‑dependent kinase 9.

Article Snippet: Thereafter, the slides were sealed with goat serum for 1 h, and then polyclonal rabbit antibodies against human CDK9 (cat. no. 2316; 1:50 dilution in 1% bovine serum albumin; Cell Signaling Technology, Inc.) were added and incubated overnight.

Techniques: Inhibition, Incubation