Fig. 4: TRAIL death receptors mediate IL-8 secretion.

A–C A549 cells were transfected for 24 h with nontargeting siRNA (labeled as “NT”) or siRNA for DR4 (“DR4#1” for first sequence and “DR4#2” for second sequence) or siRNA for DR5 (“DR5#1” for first sequence and “DR5#2” for second sequence). Media was changed for fresh glucose-free media and supernatants were collected 16 h later. IL-8 secretion (A), IL-6 (B), and LIF (C) was measured via ELISA. Results are expressed as Mean of fold change ± SEM (N = 3). D As in A, but media was changed for fresh rich-glucose media. Mean of fold change ± SEM (N = 3). E A549 cells were transfected for 24 h with nontargeting siRNA (“NT”) or siRNA for DR4 (“DR4#1”) or siRNA for DR5 (“DR5#2”). Media was changed and left it for 16 more hours. Cells were collected 16 h after medium replacement, and IL-8 expression was measured via qPCR, shown as Mean of fold change ± SEM (N = 3). F, G DR4, DR5 were CRISPRed out in A549 cells. After plating the cells, media was changed and cells were incubated for 24 h. Supernatants were collected and IL-8 secretion was measured (F). Results are shown normalized to basal mock. Statistic comparison was made vs their own mock sample. Results are represented as Mean of fold change ± SEM (N = 4). A representative WB is shown in G. H–K H2126, H460, and SW900 cells were transfected for 24 h with nontargeting siRNA (labeled as “NT”) or siRNA for DR4 (labeled “DR4#1”) or siRNA for DR5 (labeled “DR5#2”). Media was changed for fresh one and left it for 24 more hours replaced, and supernatants were collected and 24 h later. IL-8 secretion was measured via ELISA. Results shown as Mean of fold change ± SEM (N = 3) for H2126 cells (H), H460 (I), and SW900 (J). Representative western blots of DR4 and DR5 upon silencing of DR4 and DR5 are shown in K. L A549 cells were treated with TRAIL (50 ng/ml), antibody anti-TRAIL (2 ng/ml) or their combination for 24 h. Supernatants were collected and IL-8 secretion was measured. Results are expressed as Mean of fold change ± SEM (N at least of 3). M A549 WT (called “W”), Mock (called “M”), DR4 KO (called “4”) and DR5 KO (called “5”) cells were plated for 24 h and media was replaced with DMEM, DMEM containing 20 µM Thapsigargin as positive control of cell death (Tg) or DMEM containing 50 ng/ml TRAIL. Cells were incubated for 24 h and then collected for PI staining. Cell viability was measured by FACS and indicated as percentage of cells which are PI⁻. Results are expressed as Mean ± SEM (N = 3). N A549 WT, DR4 KO and DR5 KO cells were plated for 24 h. Media was changed to fresh DMEM or fresh DMEM with TRAIL (50 ng/ml) as indicated for an additional 24 h. Cells were collected and stained for surface or total DR4. Cells were measured by FACS and intracellular staining was calculated by subtracting the gMFI of the surface staining to the gMFI of the total staining. Results are shown as gMFI ± SEM (N = 3). O Same as N but for DR5. Statistical analysis performed using Ratio paired t-test compared to NT, or to Mock basal level for F. p-value: *p < 0.05, **p < 0.01, ***p < 0.001.