Application software x

HeLa cells were cultured in 12-well plates on 18 mm coverslips and co-transfected with either pFLAG-HERC5 and pVP40-EGFP (10:1 ratio) or pGL3 and pVP40-EGFP (10:1 ratio). Twenty-four hours after transfection, the coverslips containing the cells were washed twice with PF buffer (1× PBS + 1% FBS), fixed for 10 min in 1× PBS containing 4% formaldehyde and 2% sucrose, permeabilized in 1× PBS containing 0.1% Triton X 100 (Sigma) and then washed twice more with PF buffer. Coverslips were incubated with primary antibody rabbit anti-FLAG (1:500 dilution) for 1 h, washed 3× with PF buffer and incubated with either secondary antibody anti-rabbit 594 (1:1000) for 1 h. Coverslips were washed 3×, incubated in Hoechst 33342 (1:10,000 dilution) (Life Technologies) for 5 min and washed 6× with PF buffer. Coverslips were then mounted on glass slides with 10 µL Vectashield mounting media (Vector Laboratories Inc., Burlingame, CA, USA) and sealed with nail polish. Confocal micrographs were obtained using a Leica TCS SP8 (Leica Microsystems) microscope, and Leica Application Software X was used for image acquisition.

Cells on coverslips were exposed to heme in serum- and antibiotic-free DMEM for 60 min, rinsed twice and then cells were incubated for 3 h in DMEM containing 10% FBS and antibiotics. Cells were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) pH 7.4 for 15 min. Coverslips were washed with PBS and samples were blocked with 5% goat serum in PBS supplemented with 0.3% Triton X-100 for 60 min. Samples were then incubated with primary antibody against CHOP (Proteintech Group Manchester M3 3WF, United Kingdom) at a 1:500 dilution or against ATF4 (Cell Signaling Technologies, Danvers, MA, United States) at a 1:500 dilution overnight at 4°C in antibody dilution buffer (1% BSA in PBS supplemented with 0.3% Triton X-100). The secondary antibody was a goat anti-rabbit IgG conjugated to Alexa Fluor^® 532 (Thermo Fisher Scientific, Waltham, MA, United States) used at a 1:1000 dilution in antibody dilution buffer and incubated for 60 min at room temperature. Nuclei were visualized with Hoechst. Cells treated with 1 μM of thapsigargin for 3 h was used as positive control. Nuclear translocation of CHOP and ATF4 was investigated with TCS SP8 STED microscope using Leica Application Software X (Leica, Mannheim, Germany).

Cells were treated as described above with RANKL in the presence or absence of FHb. Cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) pH 7.4 for 15 minutes. Coverslips were washed with PBS and samples were blocked with 5% goat serum in PBS supplemented with 0.3% Triton X-100 for 60 min. Samples were then incubated with primary antibody against NFATc1 (Novus Biologicals, Littleton, CO, USA) at a 1 : 250 dilution overnight at 4°C in antibody dilution buffer (1% BSA in PBS supplemented with 0.3% Triton X-100). The secondary antibody was a goat anti-mouse IgG conjugated to Alexa Fluor® 488 (Thermo Scientific) used at a 1 : 500 dilution in antibody dilution buffer and incubated for 60 min at room temperature. Nuclei were visualized with Hoechst. Nuclear translocation was investigated with TCS SP8 STED microscope using the Leica Application Software X (Leica, Mannheim, Germany).