Figure 2
M. fermentans-mediated necrotic cell death induced by intracellular Aβ1–42 deposition. (a) Representative western blotting images from mock- or M. fermentans-infected differentiated SH-SY5Y cells using the indicated antibodies at 1, 7, 12, and 19 dpi. (b, c) Bar graphs of western blotting analysis data from (a) for the ratio of p-tau (b) and t-tau (c) to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) relative to mock controls at each time point. (d) Representative western blotting images from M. fermentans-infected differentiated SH-SY5Y cells using the indicated antibodies at 19 dpi after calf intestinal phosphatase (CIP) treatment. (e) Representative western blotting images from mock- or M. fermentans-infected differentiated SH-SY5Y cells using the indicated antibodies at 1, 7, 12, and 19 dpi. (f) Bar graph of western blotting analysis data from (e) for the ratio of Aβ1–42 to GAPDH relative to mock controls at each time point. (g) Representative flow cytometry histogram of intracellular Aβ1–42 in mock- or M. fermentans-infected differentiated SH-SY5Y cells at 19 dpi. (h) Bar graph for data from (g). (i) Representative immunocytochemistry images from mock- or M. fermentans-infected differentiated SH-SY5Y cells using the indicated antibodies at 19 dpi. (j) Bar graph of mean fluorescence intensity (MFI) for each protein normalized by Hoechst intensity from (i). (k) Bar graph of hAPP gene expression levels normalized against the GAPDH gene of hAPP knockdown SH-SY5Y cells relative to control knockdown SH-SY5Y cells, determined by qPCR. (l) Representative flow cytometry dot plots from Annexin V/PI apoptosis assays for mock- or M. fermentans-infected indicated gene knockdown SH-SY5Y cells at 19 dpi. (m) Bar graph of Annexin V-negative PI-positive cells from (l). Original blots are presented in Supplementary Fig. S9. Con, control knockdown SH-SY5Y cells; APP, hAPP knockdown SH-SY5Y cells. Bar graphs present mean values ± SD. Scale bars = 20 μm for (i); *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; NS, not significant (unpaired Student’s t-test). Data are averages of three or more independent experiments.
