CDK9 regulates mTORC1 targets independently of mTOR and regulates mRNA translation. (A) U937 and MV4-11 cells were treated with atuveciclib for 0, 1, 2, and 4 hours and lysed, and proteins were resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies. The immunoblots with antibodies against the phosphorylated forms of the proteins or against the total proteins were from lysates from the same experiments analyzed in parallel by SDS-PAGE. (B) Lysates from cytoplasmic fractions of U937 and MV4-11 cells were immunoprecipitated by CDK9- or Rabbit IgG preconjugated beads. Immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. (C) Lysates from U937 and MV4-11 cells were immunoprecipitated using an anti-LARP1 antibody or Rabbit IgG. Immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. U937 (D) and MV4-11 (E) cells were treated with control (DMSO) or atuveciclib for 2 hours, and cell lysates were separated on 10% to 50% sucrose gradients. Gradients were subjected to ultracentrifugation, and fractions were collected by continuous monitoring of optical density (OD) at 254 nm. The OD 254 nm is shown as a function of gradient depth. A representative profile of 1 of 3 independent experiments is shown. Monosomal and polysomal fractions were resolved by SDS-PAGE and immunoblotted with the indicated antibodies (lower panels). (F) The areas under the polysomal and monosomal peaks were quantified using ImageJ software. The ratio of area under the polysomal over the monosomal peaks was calculated for atuveciclib and is represented as percent control (DMSO). Shown are means + SE of 3 independent experiments. **P < .01 using a paired Student t test.