Alexa fluor plus 568

HT1080 cells (1.5 × 10⁵) were cultured on 35-mm glass bottom dishes (Matsunami) for 12 h at 37 °C. After the indicated treatment, cells were then fixed with 4% (w/v) PFA for 10 min/RT, washed 3 times using PBS, incubated with 0.1% (v/v) Triton X-100 and blocked for 1 h at RT with 3% (w/v) BSA. The cells were then incubated overnight at 4 °C with a primary antibody, either HNEJ-1 (5 μg/ml), anti-ACSL4 (Cat. # PA5-27137, 1:1000, Invitrogen) or anti-cleaved caspase-3 (Cat. #5A1E; 1:500 dilution; Cell signaling), washed 3 times with PBS and incubated with a secondary antibody, either Alexa Fluor® Plus 488 or Alexa Fluor® Plus 568 (Invitrogen). LSM880 was used to analyze intensity and localization. More than fifty cells were quantified with ZEN 3.3 software for integration of each fluorescence (wavelength) area via excluding the cellular background. The relative intensity per cell in arbitrary units is shown.

After H290 and 8A cells were treated with PAL, they were then incubated for 8 h/37 °C. The cells were then fixed with 4% (w/v) paraformaldehyde for 10 min/RT, washed 3 times using PBS, incubated with 0.1% (v/v) Triton X100 and blocked for 1 h at RT with 3% (w/v) bovine serum albumin. The cells were then incubated overnight at 4 °C with primary antibodies against iNOS (Cat. #CXNFT; 1/200 dilution; Invitrogen), p65 (Cat. #D14E12; 1/400 dilution; Cell Signaling Technology), LAMP1 (Cat. #ab25630; 1/500 dilution; Abcam), LC3B (Cat. #D11; 1/1000 dilution; Cell Signaling Technology), SQSTM1/p62 (Cat. #D5L7G, 1/1000 dilution; Cell Signaling Technology), TFEB (Cat. #1337-I-AP, 1/100 dilution; Proteintech) and NF-κB (Cat. #PA5-88084, 1/500 dilution; Thermofisher), washed 3 times with PBS and incubated with the secondary antibodies, Alexa Fluor® Plus 488 or Alexa Fluor® Plus 568 (Invitrogen). A Zeiss confocal microscope (LSM880, Carl Zeiss) was used to observe cellular morphology. The fluorescence intensity and Mander's overlap for image co-localization were measured using ImageJ 4.7v software. Fifty cells were quantified with ImageJ software for integration of each fluorescence (wavelength) area via excluding the cellular background. Relative intensities per cell in arbitrary units are shown.