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. 2024 Apr 8;84(9):1426–1442. doi: 10.1158/0008-5472.CAN-23-3334

Figure 4.

Figure 4. Palbociclib acts through the CCND-CDK4/6-RB axis. A, Western blot analysis of RB1 and ACTIN protein expression in shRB1 cell lines with (+) or without (−) dox (representative blot, n = 2). Average protein expression was quantified relative to ACTIN, normalized to the -dox condition, and is shown underneath each blot. B, Colony formation assays of shRB1 cell lines treated with or without dox in combination with vehicle control, 1 μmol/L palbociclib, or 1 μmol/L abemaciclib. C, Microarray expression analysis of 28 DSRCT tumors comparing CCND and CDK4/6 RNA levels. D, Heatmap of log2-fold change in expression of CCND–CDK4/6–RB pathway members when EWSR1::WT1 is depleted from shWT1 DSRCT cell lines. E, Western blot of CCND1 and ACTIN protein expression in shCCND1 cell lines with (+) or without (−) dox (representative blot, n = 2). Average protein expression was quantified relative to ACTIN, normalized to the -dox condition, and is shown underneath each blot. F, Colony-formation assays of shCCND1 DSRCT cells with or without dox treatment (n = 3). G, Cell-cycle analysis of shCCND1 DSRCT cells with or without dox treatment (n = 3). H, Western blot analysis of RB phosphorylation in BER-DSRCT shCCND1 cells with (+) or without (−) dox (representative blot, n = 2). Phosphorylated RB fraction was determined by quantifying pRB S708 relative to total RB and normalizing to the -dox condition. Average protein expression of CDK4 and CDK6 was quantified relative to ACTIN and normalized to the -dox condition. Quantifications are shown underneath each blot. ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant.

Palbociclib acts through the CCND-CDK4/6-RB axis. A, Western blot analysis of RB1 and ACTIN protein expression in shRB1 cell lines with (+) or without (−) dox (representative blot, n = 2). Average protein expression was quantified relative to ACTIN, normalized to the -dox condition, and is shown underneath each blot. B, Colony formation assays of shRB1 cell lines treated with or without dox in combination with vehicle control, 1 μmol/L palbociclib, or 1 μmol/L abemaciclib. C, Microarray expression analysis of 28 DSRCT tumors comparing CCND and CDK4/6 RNA levels. D, Heatmap of log2-fold change in expression of CCND–CDK4/6–RB pathway members when EWSR1::WT1 is depleted from shWT1 DSRCT cell lines. E, Western blot of CCND1 and ACTIN protein expression in shCCND1 cell lines with (+) or without (−) dox (representative blot, n = 2). Average protein expression was quantified relative to ACTIN, normalized to the -dox condition, and is shown underneath each blot. F, Colony-formation assays of shCCND1 DSRCT cells with or without dox treatment (n = 3). G, Cell-cycle analysis of shCCND1 DSRCT cells with or without dox treatment (n = 3). H, Western blot analysis of RB phosphorylation in BER-DSRCT shCCND1 cells with (+) or without (−) dox (representative blot, n = 2). Phosphorylated RB fraction was determined by quantifying pRB S708 relative to total RB and normalizing to the -dox condition. Average protein expression of CDK4 and CDK6 was quantified relative to ACTIN and normalized to the -dox condition. Quantifications are shown underneath each blot. ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant.