Fig. 2. GB111-NH₂ tampers with lysosomal activity and impairs autophagic degradation. (A) Primary BMDMs were treated with GB111-NH₂ or DMSO for 16 h followed by stimulation with 7-Keto for indicated times. Immunoblot of p62, LC3-A/B and beta actin from total cell lysate is presented. (B, C) Gel densitometry analyses of LC-3B and p62 normalized to beta actin from four independent trials. Two tailed Student's t-test was used to evaluate the statistical difference between GB111-NH₂ and DMSO for each time point, with the False Discovery Rate (FDR) post hoc correction for multiple comparisons. (D) Primary BMDMs were treated as described in panel A, fixed in cold methanol and stained with anti-p62 antibody (shown in purple) and DAPI (blue). (E) Quantification of microscopy data from two independent experiments. Statistical evaluation performed as described in panels B and C. (F) A representative confocal micrograph of immunofluorescence stains of LAMP-1 (green) and LC-3B (red) in BMDMs treated with GB111-NH₂ or DMSO, as described in (A) or after 4 h of 7-Keto treatment. (G, H) Data were quantified from three independent experiments presenting LC-3B colocalized with LAMP-1 without 7-Keto (G) or after 7-Keto stimulation (H). Bar graphs present the mean ± SEM of DMSO (black bars) or GB111-NH₂ (gray bars) treated cells. Two tailed Student's t-test was used to determine statistical significance. The scale bar is 5μm and 2.5μm for magnified images.