Phospho-TFEB (Ser142) Antibody - #AF3845

Fig. 3. LPS impaired autophagic flux in macrophages. (A, B) Representative immunofluorescence and quantification of iNOS-F4/80 double positive macrophages in liver from mice in indicated groups (n = 5). DAPI (blue), iNOS (red), and F4/80 (green). White arrows indicate iNOS-F4/80 double positive macrophages. Scale bars: 50 μm. (C, D) Representative immunofluorescence and quantification of LC3 puncta in F4/80-positive macrophages in liver from mice in indicated group. DAPI (blue), LC3β (red), and F4/80 (green) (n = 5). Scale bars: 50 μm. (E, F) Representative immunofluorescence and quantification of p62-F4/80 double positive macrophages in liver from mice in indicated groups. DAPI (blue), p62 (red), and F4/80 (green) (n = 5). White triangles indicate p62-F4/80 double positive macrophages. Scale bars: 50 μm. (G) The expression of LC3 and p62 in lysates of BMDMs in “control-group” and “LPS-group”, and normalized to β-actin (n = 4). (H) The expression of LC3 and p62 in lysates of BMDMs in indicated groups, and normalized to β-actin (n = 4). And Bafilomycin A1 was added for last 6 h. (I, J) Representative confocal images and quantification of Raw264.7 transduced with Lenti-GFP-mCherry-LC3B (n = 10). Scale bars: 20 μm. (K) The expression of LC3 and p62 in lysates of BMDMs in indicated groups, and normalized to β-actin (n = 4). 3-MA was given as pretreatment for 12 h before LPS administration. (L, M) Representative confocal images and quantification of Raw264.7 transduced with Lenti-GFP-LC3B in indicated groups (n = 8). Scale bars: 10 μm. (N) The expression of LC3 and p62 in lysates of BMDMs in indicated groups, and normalized to β-actin (n = 4). U0126, SP600125 and SB203580 were pretreated to BMDMs for 12 h. (O, P) Representative confocal images and quantification of Raw264.7 transduced with Lenti-GFP-LC3B in indicated groups (n = 8). Scale bars: 20 μm. (Q, R) Representative confocal images and quantification of pHLys Red in indicated groups (n = 5). Scale bars: 50 μm. (J) Real-time PCR analysis for lamp1, ctsa, ctsb, and ctsf from BMDMs in indicated group. All values are presented as mean ± SEM. Statistical significance was measured using the unpaired 2-tailed Student t test for two experimental groups and one way ANOVA test for multiple groups. ns = no significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Tat-Beclin1-induced autosis is regulated by Tfeb modulation in NRCMs. (A–C) NRCMs were treated with 5 µM Scrambled or Tat-Beclin 1 for 6 h and analyzed via Western blotting using anti-Tfeb, anti-pTfeb (Ser142) and anti-Gapdh antibodies (A) The relative ratios of Tfeb to Gapdh (B) and pTfeb to Tfeb (C) were quantified (mean values ± S.E., n = 6 for Tfeb, n = 3 for p-Tfeb; * p < 0.05, *** p < 0.001). (D,E) NRCMs were transduced with either Ad-lacZ or Ad-Tfeb for 24 h (D) or Ad-lacZ or Ad-shTfeb for 48 h (E) and then treated with 5 µM Scrambled or 0.5, 1 or 5 µM Tat-Beclin 1 for 3 h. Cell death was quantified via CellTiter-Blue assays (mean values ± S.D., n = 16; * p < 0.05, ** p < 0.01, not significant (n.s.)) (D,E). (F,G) Knockdown of Tfeb by Ad-shTfeb was confirmed via immunoblot analyses, using anti-Tfeb and anti-Gapdh antibodies (F). Knockdown of Atg7 by Ad-shAtg7 was confirmed via immunoblot analyses, using anti-Atg7 and anti-Gapdh antibodies (G). (H) NRCMs were transduced with either Ad-lacZ or Ad-Tfeb with or without Ad-shAtg7 as indicated for 48 h and then treated with 5 µM Scrambled or Tat-Beclin 1 for 3 h. Cell death was quantified via CellTiter-Blue assays (mean values ± S.D., n = 16; * p < 0.05, ** p < 0.01, not significant (n.s.)).