Fig. 2

Loss of CAMKKβ attenuates autophagy activity in EOC spheroids. a Western blot analysis and densitometric quantification of p62 abundance and LC3BII: I ratio in OVCAR8 parental, OVCAR8 CAMKK2 KO, HeyA8 parental, and HeyA8 CAMKK2 KO adherent cells (Adh) and spheroids (Sph). b Representative images and SPoRTS analysis of mCherry, EGFP, and mCherry/EGFP ratio from mCherry-EGFP-LC3B (autoR)-expressing OVCAR8 (OVCAR8-autoR) and HeyA8 (HeyA8-autoR) spheroids under control (siNT) and CAMKKβ knockdown (siCAMKKβ) conditions. c Western blot analysis and densitometric quantification of p62 abundance and LC3BII: I ratio in OVCAR8-autoR and HeyA8-autoR adherent cells and spheroids under control and CAMKKβ knockdown conditions. # indicates a non-specific band. Quantified western blot data represent mean normalized p62 abundance or mean LC3BII: I ratio ± SEM for n = 3 independent experiments. α-tubulin was used for normalization. For p62, data are expressed relative to the parental adherent sample, for which the mean was set to 1. Data were analyzed via two-way ANOVA and groups with shared lineage (e.g., parental Adh vs. parental Sph) or shared culture condition (e.g., parental Adh vs. CAMKK2 KO Adh) were compared using the Holm-Šídák method (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). For b, SPoRTS data represent mean ratio of integrated mCherry signal to integrated EGFP signal ± SEM for n = 3 independent experiments, each comprising 5 individual spheroids (15 spheroids in total). Data were analyzed via two-way repeated measures ANOVA (⁺⁺p < 0.01, ⁺⁺⁺⁺p < 0.0001). Scale bars: 500 μm.