Fig. 2: Abrogation of ERCC1 triggers cytoplasmic stress responses in Er1F/− macrophages.
From: Tissue-infiltrating macrophages mediate an exosome-based metabolic reprogramming upon DNA damage
a Immunofluorescence detection of GRP78 (n > 250 cells counted per genotype) marking the dilation of ER, LC3 (n > 40 cells counted per genotype) for autophagy, P62 for autophagic activity (n > 15 optical fields per genotype, ~150 cells per field) and b Gm130 for Golgi dispersal (~500 cells per genotype; arrowhead) in Er1F/− and Er1F/+ BMDMs. For GRP78 and LC3, the colored numbers indicate the average percentage of (+) stained cells ± SEM for the indicated, color-matched protein. For p62, the colored numbers indicate the average mean fluorescence intensity ± SEM of p62 signal. For Gm130, the green-colored numbers indicate the average percentage of (+) stained cells ± SEM showing Golgi dispersal. c Western blot levels of GRP78, P62, LC3, and Tubulin in Er1F/− and Er1F+ BMDMs. The graph shows the fold change of indicated protein levels in Er1F/− BMDMs compared to Er1F+ corresponding controls (n = 3 per group). d Immunofluorescence detection of GRP78 (for ER stress) (n > 500 cells counted per genotype), LC3 (for autophagy) (n > 750 cells counted per genotype) and e Gm130 (for Golgi dispersal) in MMC-treated BMDMs (n > 500 cells counted per genotype). Numbers indicate the average percentage of (+) stained cells ± SEM for the indicated, color-matched protein. For Gm130, the green-colored numbers indicate the average percentage of (+) stained cells ± SEM showing Golgi dispersal. f Immunofluorescence detection of LC3 (for autophagy) (~250 cells counted per genotype) and Gm130 (for Golgi dispersal) (~950 cells counted per genotype) in LPS-stimulated BMDMs. g Immunofluorescence detection of Gm130 (for Golgi dispersal) in MMC-treated and control BMDMs exposed to ATM (ATMi) (~200 cells counted) or ATR (ATRi) (500 cells counted) inhibitor (as indicated). The green-colored numbers indicate the average percentage of (+) stained cells ± SEM showing Golgi dispersal. h Western blot levels of GRP78 and LC3 in MMC-treated and control (ctrl) macrophages exposed to ATM (ATMi) or ATR (ATRi) inhibitor (as indicated; Tubl.: tubulin, unt: untreated). The graph represents the fold change in indicated protein levels in MMC-treated macrophages exposed to ATM (ATMi) or ATR (ATRi) inhibitor compared to corresponding controls (n = 4 per group) (i–j). Representative transmission electron micrographs of Er1F+ (i) and Er1F/− (j–l) BMDMs. Arrowheads depict the presence of intracellular vesicles (j left panel), organized in larger vacuolar structures (j right panel), the appearance of cytoplasm-filled projections (k left panel), the convoluted network of pseudopodia-like structures (k right panel) containing vesicles (l left panel) and pseudopodia-associated extracellular vesicles (l right panel). Scale bars are shown separately for each micrograph. The significance set at p-value: *≤0.05, **≤0.01 (two-tailed Student’s t-test). Gray line is set at 5 μm scale.
