Fig. 6

IHT enhances Aβ degradation by microglia via TFEB-mediated autophagy. A Primary microglia were infected with three shTfeb lentivirus (#1, #2, and #3). Interference efficiency were determined by western blot. B, C Cells were co-stained with anti-LC3 and anti-LAMP1 antibodies. Scale bar = 5 μm. The colocalization ratio of LC3 and LAMP1 in single cell was quantified using Manders' colocalization coefficients. n > 30 (Student's t test). D sh-Tfeb #2 lentivirus (sh2)-infected microglia were treated with 1 μM of oAβ for 12 h. Aβ1–42 in the medium were detected with ELISA. n = 4. (Student's t test). E–L sh-Tfeb #2 lentivirus (sh2)-infected microglia were co-treated with oAβ and a single-day IHT. E Aβ1–42 in the medium was measured via ELISA. n = 6. F TFEB expression was determined using qRT-PCR. n = 3. G, H Cells were treated with bafilomycin A1 for 1 h, followed by staining with anti-LC3 and anti-LAMP1 antibodies. Scale bar = 5 μm. The colocalization ratio of LC3 with LAMP1 was evaluated using Manders' colocalization coefficients. n > 40. I–L Cells were treated with Aβ-555 for 30 min, or co-treated with Aβ-555 and bafilomycin A1 for 30 min and chased. Then, cells were stained with anti-LC3 or anti-LAMP1 antibodies. Scale bar = 5 μm. The colocalization ratio of Aβ-555 with LC3 or LAMP 1 in a single cell was determined via Manders' colocalization coefficient. n > 30. *P < 0.05, **P < 0.01, and ***P < 0.001 using two-way ANOVA