Fig 2: CDK16 inhibition is involved in multiple cancer-related signaling pathways. A-B GSEA analysis using RNA-seq data of MDA-MB-231 cells treated with low (1 µM) or high concentrations (5 µM) of Reb for 48 h. The GSEA plots showed mitotic spindle (A) and G2/M checkpoint signatures (B) were enriched in the control group. C-D GSEA analysis using RNA-seq data of MDA-MB-231 cells with Reb treatment (C) or with CDK16-KD (D). The GSEA plots showed E2F targets were enriched in the control groups. E-F Heat map shows the Z-score normalized expression of the most affected E2F target genes in MDA-MB-231 cells upon Reb treatment (E) or with CDK16-KD (F) according to RNA-seq data. The displayed genes are the intersection of the core enrichment genes from the GSEA analysis shown in (C) and (D). G-H Immunoblot analysis of total Rb and p-Rb expression in MDA-MB-231 cells treated with indicated concentrations of Reb for 48 h (G) or with CDK16-KD (H). I-J qPCR analysis of Rb-E2F direct targets expression in MDA-MB-231 cells treated with Reb_low (1 µM) or Reb_high (5 µM) for 48 h (I) or with CDK16-KD (J). Data are presented as mean ± SD and p values were obtained by two-tailed Student’s t-test (I and J). *p < 0.05, ** p < 0.01, ***p < 0.001, **** p < 0.0001, ns, not significant

Fig 3: ULK1 and Beclin1 are required for Cyclin Y/CDK16-induced autophagy.a HeLa cells were co-transfected with siRNA against ULK1, Beclin1 or a control and vectors expressing GFP-CDK16 and Cyclin Y-Flag for 72 h. Lysates were immunoblotted with the indicated antibodies. (n = 3). b Representative confocal images of HeLa cells treated as in panel a. GFP-CDK16 identified transfected cells. Endogenous LC3 staining (red) with the 4E12 antibody monitored autophagy. Scale bar: 50 µm. c Quantification of the LC3 dots shown in panel b. Statistical significance was measured via unpaired and two-tailed Student’s t-tests and is presented as follows: ****p < 0.0001. All error bars indicate SD. (n = 1; 250 cells were analyzed for each treatment; siControl vs. siControl Cyclin Y/CDK16: t = 13.49, df = 8; siControl Cyclin Y/CDK16 vs. siULK1 Cyclin Y/CDK16: t = 13.53, df = 8; siControl Cyclin Y/CDK16 vs. siBeclin1 Cyclin Y/CDK16: t = 13.15, df = 8). n biological independent replicate. SD standard deviation. Source data are provided as a Source Data file.

Fig 4: Knockdown of CDK16 inhibits tumor growth of TNBC in vitro and in vivo. A 2D-colony formation assay of indicated TNBC cells transfected with scramble-shRNA or CDK16-shRNAs. Shown are representative images (left panel) and quantification of colonies (right panel). B 3D-colony formation assay of indicated CDK16-KD TNBC cells. Shown are representative images (left panel, scale bar: 100 µm) and quantification of colonies (right panel). C-E 2D-colony formation assay of MDA-MB-231 cells stably expressing control vector (vector), wild-type CDK16 (CDK16) or kinase-inactive CDK16D304A mutant (D304A) followed with transfection of scramble-shRNA or shRNA targeting the 3'-UTR of CDK16. Shown are immunoblot analysis for CDK16 expression (C), representative colony images (D), and quantification of colonies (E). F-I Tumorigenesis assay of MDA-MB-231 cells with CDK16-KD (n = 4 mice per group). Shown are tumor-free survival curve (F), tumor growth curve (G), statistics of tumor weight (H), and image of tumors (I). J-M Tumorigenesis assay of MDA-MB-468 cells with CDK16-KD (n = 3 mice per group). Shown are tumor-free survival curve (J), tumor growth curve (K), statistics of tumor weight (L), and image of tumors (M). Data are presented as mean ± SD (A, B, and E), mean ± SEM (G and K) or boxplot (H and L). p values were obtained by repeated measures two-way ANOVA (G and K), or two-tailed Student’s t-test (others). All *p < 0.05, ** p < 0.01, ***p < 0.001, **** p < 0.0001, ns, not significant

Fig 5: AMPK-induced autophagy requires Cyclin Y/CDK16.a Immortalized Cdk16+/+ and Cdk16-/- MEFs were treated with 1 mM AICAR for 1 or 16 h. Lysates were immunoblotted with the indicated antibodies (n = 3). b Representative confocal images of the Cdk16+/+ and Cdk16-/- MEFs treated as in panel a. Endogenous LC3 Staining with the 4E12 antibody monitored autophagy. Scale bar: 50 µm. c LC3 dots of experiments as shown in panel b were quantified. Statistical significance was measured via unpaired and two-tailed Student’s t-tests and is presented as follows: **p < 0.01. All error bars indicate SD. (n = 3; 100 cells counted for each replicate; Cdk16+/+ + AICAR vs. Cdk16-/- + AICAR: t = 4.964; df = 4). d Immortalized Cdk16+/+ and Cdk16-/- MEFs were treated with 1 mM AICAR for 1 h and/or with 200 nM Bafilomycin A1 (Baf. A1) for 6 h as indicated. For the combination, AICAR was added during the last hour of Baf. A1 treatment. Lysates were immunoblotted with the indicated antibodies (n = 1). e NIH3T3 cells were transfected with siRNA against Cyclin Y or control siRNA and stimulated with 0.5 mM AICAR/50 µM A769662 (A769) or with 200 nM Baf. A1 or a combination. Proteins were analyzed as indicated. (n = 3). f Representative confocal images of the NIH3T3 cells treated as in panel e. Endogenous LC3 was stained with the 4E12 antibody to depict autophagy. Scale bar: 50 µm. g LC3 dots as shown in panel f were quantified. Statistical significance was measured via unpaired and two-tailed Student’s t-tests and is presented as follows: **p < 0.01, ***p < 0.001. All error bars indicate SD. (n = 3; 100 cells counted for each replicate; AICAR/A769: t = 9.494, df = 6; Baf. A1: t = 6.549, df = 6; AICAR/A769 + Baf. A1: t = 9.484, df = 4). n biological independent replicate. SD standard deviation. Source data are provided as a Source Data file.