SiRNAs (50 pmol) against CAMK1D and other indicated serine-threonine kinases were reverse transfected into HeLa cells. Cell lysates were prepared and analyzed in immunoblots for Cdk9 T-loop phosphorylation, total Cdk9 and β-actin (loading control).

(A) HeLa cells were treated with 100 nM KN-93 or negative control, KN-92. Samples were collected at one hour and four hours post-treatment and cell lysates analyzed by immunoblots for level of Cdk2 T-loop phosphorylation. Inhibition of CamK by KN-93 reduces the level of phosphorylated Cdk2 compared to control KN-92 treated cells.
(B) HeLa cells were treated with 100 nM KN-93 (CaMK inhibitor) or KN-92 (negative control). Cell lysates prepared after one hour and four hours of treatment were analyzed in an immunoblot for effects on Cdk9 T-loop phosphorylation. After normalization to the Cdk7 loading control and compared to the negative control KN-92, quantification of the immunoblot showed a ~ 55% and ~59% reduction of Cdk9 T-loop phosphorylation at one and four hours, respectively, when CaMK was inhibited by KN-93. Total Cdk9 levels were reduced ~15% at one and four hours by treatment with KN-93.
(C) HeLa cells were treated with 20 µM and 50 µM Calmodulin inhibitor, W-7, for seven hours. Cell lysates were immunoblotted and probed for Cdk2 T-loop phosphorylation. There is a dose dependent reduction in the level of phosphorylated Cdk2 upon W-7 treatment compared to control untreated cells.
(D) HeLa cells were treated with the indicated concentration of W-7 for seven hours. Cell extracts were prepared and immunoblots were performed to examine Cdk9 T-loop phosphorylation. After normalization to the β-actin loading control, quantification of the immunoblots showed a ~ 54% reduction of Cdk9 T-loop phosphorylation and ~ 29% reduction in total Cdk9 levels after treatment with 50 µM W-7 for seven hours relative to the untreated control.

(A) Resting primary CD4⁺ T lymphocytes were treated with Thapsigargin (100 µM) or DMSO vehicle control and activated with either Ca²⁺ ionophore, ionomycin or PMA + ionomycin for one hour. Immunoblots were carried out on the cell lysates and examined for induction of Cdk9 T-loop phosphorylation. Immunoblots were quantified by normalizing to Cdk7 loading control and comparing to DMSO treated activated cells. In ionomycin and PMA + ionomycin treated cells, inhibition of Ca²⁺ signaling by Thapsigargin repressed Cdk9 T-loop phosphorylation by 80% and 60% respectively.
(B) Resting primary CD4⁺ T lymphocytes were activated with PMA + ionomycin for 60 hrs. The activated cells were treated with a final concentration of 50 µM W-7. Cells lysates were prepared at the indicated times and immunoblots were performed. Cdk7 was used as loading and specificity control. When compared to activated cells without W-7 treatment and normalized to loading control, quantification of immunoblots showed 84% reduction of Cdk9 T-loop phosphorylation in 4 hours and total Cdk9 levels were reduced by 38% in 4 hours.

Two culture dishes of HeLa cells were treated with 50 µM W-7 for seven hours. The proteasomal inhibitor MG101 (50 µM) was then added to one culture dish for one hour. As an additional control, a culture dish of HeLa cells was treated with only MG101 for one hour. Cell lysates were prepared and immunoblots performed to examine Cdk9 T-loop phosphorylation and total Cdk9 protein levels. When compared to DMSO vehicle control treated cells and normalized to β-actin (loading control), quantification of the immunoblots showed ~ 50% decrease in Cdk9 T-loop phosphorylation and ~ 30% decrease in total Cdk9 levels upon W-7 treatment. Addition of MG101 resulted in significant increases of both phosphorylated and total Cdk9 protein level.

HeLa cells were treated with 50 µM W-7. Samples were collected every hour for three hours. Cell lysates were prepared and immunoblots performed to examine levels of Cdk9 T-loop phosphorylation, overall Cdk9 and Cyclin T1. There was a reduction in the level of Cdk9 T-loop phosphorylation two hours post treatment with no change in the overall Cdk9 protein and Cyclin T1 expression.

(A) HeLa cells were transfected with either 1µg pNL4-3-Tat-Luc or pPCNA-Luc overnight and treated with 50 µM W7 for eight hours. Cell lysates were prepared and Firefly Luciferase activities were measured and normalized to total protein amounts in cell extracts. The normalized Luciferase activity in untreated wells was 28,540 and 450 for pNL4-3-Tat-Luc and pPCNA-Luc, respectively, and was defined as 1.0 to calculate fold-change in the W7 treated wells. (** represents p value of <0.005 and * represents p value of < 0.05 in a paired t-test).
(B) HeLa cells were transfected with either 250 ng pNL4-3-Tat-Luc + 50 ng pcDNA empty vector or 250 ng pHTLV-LTR-Luc + 50 ng pTax overnight and treated with 50 µM W-7 for seven hours. The cells were lysed and Firefly Luciferase assays were carried out. The Luciferase values were normalized to total protein in the cell lysates and set at 1.0 in untreated wells. (** represents p value of < 0.005 in a paired t-test).

HeLa cells were cotransfected with either 250 ng pNL4-3-Tat-Luc + 500 ng Flag-wild type (Wt) Cdk9 or 250 ng pNL4-3-Tat-Luc + 500 ng Flag-T186D Cdk9 overnight and treated with 50 µM W7 for seven hours. Cell lysates were prepared and Firefly Luciferase activities were measured and normalized to total protein amounts in cell extracts. The normalized Luciferase activity in untreated wells was defined as 1.0 to calculate fold-change in the W7 treated wells. Data shown is the average ± SD from three independent experiments (* represents p value of ≤ 0.05 in a paired t-test).