Figure 7.

Downregulation of HMGB1 expression rescued HG-induced LMP via a CTSB- but not a CTSL-dependent pathway. (a) After exposure to NG or HG for 24 h, the total protein of HsRPE and ARPE-19 cells was subjected to western blot analysis using the indicated antibodies (CTSB, CTSL, and ACTB). ACTB was used as a protein loading control. Representative images from three independent experiments with biological and technical replicates are shown, n = 6. (b) HsRPE and ARPE-19 cells were pretreated with complete medium containing CA-074Me (10 ng/mL) or Z-FF-FMK (40 μM) for 1 h and then exposed to HG for 24 h. Immunofluorescence signals after double-staining for LGALS3 (red) and LAMP1 (green) in cell samples are shown, and Manders’ coefficients for colocalization of LGALS3 with LAMP1 were calculated. Representative images from three independent experiments with biological and technical replicates are shown. Scale bars: 10 μm, n = 6. (c) ARPE-19 cells were transfected with siRNA-HMGB1 and Scr-siRNA and then exposed to HG for 24 h. The mRNA expression of CTSB and CTSL in ARPE-19 cells was measured by real-time PCR, n = 6. (d) HsRPE and ARPE-19 cells were transfected with siRNA-HMGB1 and Scr-siRNA and then exposed to HG for 24 h. Cell samples were subjected to western blot analysis using the indicated antibodies (CTSB, CTSL, and ACTB). ACTB was used as a protein loading control. Representative images from three independent experiments with biological and technical replicates are shown, n = 6. ^#P > 0.05; **P < 0.01; ***P < 0.001.