Cell cycle analysis was performed with the Click‐iT Plus EdU Flow Cytometry Assay Kit (Invitrogen) and DAPI as DNA staining reagent (BD Biosciences). Five days after shRNA transduction, cells were incubated with 10 μM EdU solution for 1 h, washed with 3 ml 1% BSA/DPBS, fixed with 4% PFA, permeabilized, and incubated with EdU detection solution and DAPI. Stained cells were acquired on a BD LSR II flow cytometer (BD Biosciences) within 1 h.
The effects of verteporfin on apoptotic and mitotic rates were assessed by flow cytometric detection of cleaved PARP and phosphorylated histone H3S10, respectively. MLS cells grown in medium supplemented with 2% FBS were treated with 0.25 μM verteporfin for 72 h, detached using 0.025% trypsin (Life Technologies), fixed in 2% PFA, washed in PBS, and permeabilized in 0.25% Triton X‐100/PBS for 5 min on ice. After an additional washing step, cells were stained for 60 min with PE Mouse anti‐Cleaved PARP (Asp214) (BD Biosciences) and phospho‐Histone H3S10 (#9716, Alexa Fluor 647 Conjugate, Cell Signaling) antibodies. Fluorescence intensity was measured using a FACSCanto II flow cytometer, and data were analyzed using the FACSDiva software (BD Biosciences).
The effects of verteporfin on apoptotic and mitotic rates were assessed by flow cytometric detection of cleaved PARP and phosphorylated histone H3S10, respectively. MLS cells grown in medium supplemented with 2% FBS were treated with 0.25 μM verteporfin for 72 h, detached using 0.025% trypsin (Life Technologies), fixed in 2% PFA, washed in PBS, and permeabilized in 0.25% Triton X‐100/PBS for 5 min on ice. After an additional washing step, cells were stained for 60 min with PE Mouse anti‐Cleaved PARP (Asp214) (BD Biosciences) and phospho‐Histone H3S10 (#9716, Alexa Fluor 647 Conjugate, Cell Signaling) antibodies. Fluorescence intensity was measured using a FACSCanto II flow cytometer, and data were analyzed using the FACSDiva software (BD Biosciences).