Fig. 3

Apoptosis caused by GO, MCLR and GO-MCLR in HaCaT and L02 cells. HaCaT and L02 cells were cultured in 12-well culture dishes to confluence and exposed to GO (5 µg/mL and 50 µg/mL), MCLR (0.06 µg/mL and 0.6 µg/mL) and GO-MCLR (5 µg/mL + 0.06 µg/mL, 50 µg/mL + 0.6 µg/mL) up to 24 h. Then harvested cells were gently mixed with 300 μL Annexin V-FITC binding solution and 5 µL Annexin V-FITC, and incubated for 15 min at room temperature, away from light. 5 min before detection, 5 µL PI and 200 μL Annexin V-FITC binding solution were added. a After materials treatments and Annexin V-FITC/PI staining, cells apoptotic proportion were determined by flow cytometry. FITC (FL1) and PI (FL3) channels were selected, and 10,000 cells were analyzed per sample. b Quantitative analysis of proportion by flow cytometry, *P < 0.05 versus Control, #P < 0.05 versus GO group. c Confocal microscopy images showing GO, MCLR and GO-MCLR-induced caspases 3/7 expression in HaCaT and L02 cells cells. HaCaT/L02 cells were seeded on 24-well slides with a cell density of 4 × 10⁴ cells/mL,and then were treated with GO, MCLR and GO-MCLR of different concentrations for 24 h and washed with PBS. Cells were incubated with the prepared FLICA caspase 3/7 substrates for 1 h in the dark and rinsed 3 times with the buffer solution. Cell nuclei were stained with Hoechst 33,342