Fig. 6: Lack of SETD2 decreases autophagic flux by inhibition of autophagosome-lysosome fusion.

a, b HeLa cells were transfected with a control non-targeting siRNAs pool or SETD2 siRNAs pool for 48 h and thereafter transfected with the mRFP-GFP-LC3 plasmid for 24 h prior to autophagy induction with torin1 for 2 h. The mRFP-GFP-LC3 tandem reporter allows distinction between autophagosomes (GFP⁺ RFP⁺; yellow puncta) and autolysosomes (GFP⁻ RFP⁺; red puncta) and was used to assess the impact of SETD2 knockdown on autophagic flux; the histogram in panel b represents the data of two independent experiments. c Immunobloting analysis of SQSTM1/p62 expression levels as a measurement of autophagic flux under baseline conditions or upon autophagy induction with torin1 for 2 h shows an increase of SQSTM1/p62 levels, corresponding to a decreased autophagy flux, in SETD2-deficient HeLa cells (siRNA SETD2; Line 2 and 4) compared to the control (siRNA Control; Lanes 1 and 3). Treatment with bafilomycin A₁ (BafA1, 40 nM) for 4 h, which inhibits the fusion of autophagosomes with lysosomes, was used to block the autophagy flux. d Quantification of SQSTM1 immunoblotting. e, f HeLa cells were transfected with a control non-targeting siRNA pool or SETD2 siRNA pool for 48 h and thereafter increased expression levels for LAMP2, a lysosomal membrane component, demonstrated by immunofluorescence imaging using phagophore bound-MAP1LC3B counterstaining (e) or immunoblotting using ACTB as a loading control (f). g Immunofluorescence analysis and h flow cytometry analysis performed on HeLa cells transfected with an siRNAs pool targeting SETD2 expression or non-targeting siRNAs control and stained with LysoTracker^TM Red-fluorescent dye for labeling and tracking of acidic organelles such as lysosomes in live cells, further provided support for an accumulation of lysosomes in SETD2-deficient cells. Bars display the mean of three independent experiments (n = 3), error bars represent SEM; *P < 0.05, **P < 0.01, ***P < 0.001, ns: non-significant.