Fig. 4: Active Cyclin Y/CDK16 complexes induce autophagy.

a NIH3T3 cells stably expressing mCherry-GFP-LC3 were transfected with HA-CDK16 and Cyclin Y-Flag as indicated or treated for 2 h with EBSS or for 4 h with 200 nM Bafilomycin A1 (Baf. A1) and lysates were immunoblotted as indicated. KR kinase-deficient CDK16 mutant, AA CDK16 binding deficient Cyclin Y mutant (n = 3). b Representative confocal images of the NIH3T3-mCherry-GFP-LC3 cells treated as in panel a. Staining of the HA-CDK16 in purple identified transfected cells. Autophagosomes (yellow dots) and autolysosomes (red dots) were detected by an overlay of the GFP and mCherry fluorescent signals. Scale bar: 20 µm. c Quantification of autophagosomes (yellow dots) and autolysosomes (red dots) of cells shown in panel b. 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; 50 cells counted for each replicate; wt vs. KR: t = 5.707, df = 4; wt vs. AA: t = 5.557, df = 4). d GFP-tagged CDK14, CDK15 or CDK16 were expressed with or without Cyclin Y-Flag in HeLa cells. Cyclin Y was immunoprecipitated with a Flag antibody (IP). Lysates were immunoblotted with the indicated antibodies (WCL). (n = 4) e Representative confocal images of HeLa cells treated as in panel d. Fluorescent GFP signals identified CDK expressing cells. Endogenous LC3 (red) was used to measure autophagy with the 4E12 antibody. Scale bar: 50 µm. f Quantification of the LC3 dots shown in panel e. 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; 100 cells were counted for each treatment; control vs. Cyclin Y/CDK16: t = 6.771, df = 14; Cyclin Y/CDK14 vs. Cyclin Y/CDK16: t = 6.855, df = 14; Cyclin Y/CDK15 vs. Cyclin Y/CDK16: t = 7.139, df = 15). n biological independent replicate. SD standard deviation. Source data are provided as a Source Data file.