Fig 2: (A,B,C) 24h incubation with Leptomycin B at 10 ng/ml, results in nuclear accumulation of LC3A, LC3C and Beclin-1 in lung A549,H1299 and glioblastom U87,T98 cell lines. (D) 24h incubation with Ivermectin at 100µg/ml, reduced the nuclear expression of LC3A/Beclin-1 in A549 and H1299 cell lines and increased the perinuclear accumulation of LC3A in the H1299 cell line. (E) Western blots of the nuclear cell fraction confirm accumulation of LC3s and Beclin-1 in lung and glioblastoma cell lines after incubation with Leptomycin B. The reduction of proteins after incubation with Ivermectin was not consistent in all cell lines.

Fig 3: SC75741-induced TDP25 degradation depends on p62 and LC3C. (A). HEK293 WT and p62-/- cells were transfected with GFP-TDP25 for 24 h, treated with SC75741 (5 μM) for 12 and 24 h. Cell lysates were immunoblotted with indicated antibodies. The numbers under the blot represent the gray scale quantification (GFP/Tubulin). (B). HEK293 p62-/- cells were transfected with GFP-TDP25 and Flag-p62 for 24 h, treated with SC75741 (5 μM) for 12 h. Cell lysates were immunoblotted with indicated antibodies. The numbers under the blot represent the gray scale quantification (GFP/Tubulin). (C). HEK293 p62-/- cells were transfected with GFP-TDP25 and Flag-p62 for 24 h, treated with SC75741 (5 μM) for 12 h. The GFP fluorescence was analyzed by fluorescence microscopy. (data represent mean ± SD; n = 8, **** p < 0.0001, two-tailed t test). (D). HEK293 WT cells were transfected with GFP-TDP25 for 24 h, treated with or without SC75741 (5 μM) for another 6 h, the interaction between p62 and TDP25 was analyzed by immunoprecipitation. (E). Hela WT and ATG8-/- cells were transfected with GFP-TDP25 for 24 , treated with SC75741 (5 μM) for 3 and 6 . Cell lysates were immunoblotted with indicated antibodies. The numbers under the blot represent the gray scale quantification (GFP/Tubulin). (F).Hela ATG8-/- cells were transfected with GFP-TDP25 and HA-LC3C for 24 h, treated with SC75741 (5 μM) for 6 h. Cell lysates were immunoblotted with indicated antibodies. The numbers under the blot represent the gray scale quantification (GFP/Tubulin). (G). Hela ATG8-/- cells were transfected with GFP-TDP25 and HA-LC3C for 24 h, treated with SC75741 (5 μM) for 6 h. The GFP fluorescence was analyzed by fluorescence microscopy. (data represent mean ± SD; n = 10, **** p < 0.0001, two-tailed t test). (H). HEK293 p62-/- cells were transfected with GFP, GFP-TDP25, Flag and Flag-p62 for 24 h, treated with or without SC75741 (5 μM) for another 6 h, the interaction of p62 with TDP25 and LC3C were analyzed by immunoprecipitation. (I). HEK293 p62-/- cells were transfected with GFP-TDP25 and Flag-p62 for 24 h, treated with SC75741 (5 μM) for 6 h and the co-localization among TDP25, p62, lamp1 were done using immunofluorescence and confocal microscopy. Arrows indicate the co-localization. Scale bar, 20 μm. (J). HEK293 p62-/- cells were transfected with GFP-TDP25 and Flag-p62 for 24 h, treated with SC75741 (5 μM) for 6h and the co-localization among TDP25, p62, LC3C were done using immunofluorescence and confocal microscopy. Arrows indicate the co-localization. Scale bar, 20 μm.