Fig 2: Autophagic marker transcript modulation(A) RT-qPCR analysis of TFEB in HepG2 and Hep3B cells after 72 h of treatment with 100 nM panobinostat. (B) MAP1LC3, BECLIN1, AMBRA1, ATG5, ATG12, SQSTM, UVRAG, TP73 were analyzed in HepG2 and Hep3B cells after 6, 24, 48 and 72 h incubation with 100 nM panobinostat. mRNA expression was normalized to GAPDH and results are expressed relative to untreated controls set at 1.0. Shown are means ± SEM of three independent experiments performed in triplicates.

Fig 3: Yeast PI3K complex II affects TDP-43 toxicity and turnover. a Serial dilution growth assay of indicated isogenic null strains expressing vector and TDP-43. b, c Indicated strains expressing TDP-43-YFP were examined/quantified for overall TDP-43-YFP levels as in Fig. 1e (b) or foci intensity (c). Significance was assessed by one-way ANOVA. Repeated data are shown as mean ± s.e.m. d TDP-43-YFP turnover in indicated strains, as in Fig. 1a. e WT strain co-transformed with TDP-43-mRuby2 and Vps34-GFP, Vps38-GFP, or Atg14-GFP. % value indicates co-localization of TDP-43 foci with Vps34, Vps38, or Atg14. Scale bar = 2 µm

Fig 4: Autophagosome dynamic and aggregation(A) Dynamic of autophagosomes in HepG2 and Hep3B cells stably transfected with MAP1LC3B-GFP-RFP-tag. Treatment with 100 nM panobinostat (6 to 72 h) caused a time dependent shift of fluorescence. RFP lightening appeared stronger than green fluorescence at late treatment time. Control cells are shown at time zero of treatment. Panobinostat caused massive cell death after 72 h. (B) Immunoprecipitation of beclin1 in HepG2 and Hep3B after a short time of treatment. Autophagosomes components Atg12, Map1LC3B and UVRAG increased after treatment with 100 nM panobinostat. Ectopic Map1LC3B (GFP) was detected. (C) Quantification of early and late autophagosome vesicles detected by T.E.M. Mean relative amount of vesicles in HepG2 and Hep3B cells ± SD is shown.