TLR7 (A, C, E, G, I, and K) and TLR8 (B, D, F, H, J, and L) protein expression was analyzed in the tumoral area — SCC (A and B) and ADC (C–F) — as well as in the alveolar epithelium (G and H), bronchial epithelium (I and J), and Ti-BALT (K and L) by immunohistochemical labeling of paraffin-embedded lung tumors, as described in Methods. (E and F) The subcellular localization of TLR7 and TLR8 was detected in tumor cells. Original magnification, ×40 (A–D and G–L); ×65 (E and F).

A549 cells were untreated or treated with IL-1β (10 ng/ml; A and B), loxoribine (10 μg/ml; C and D), or poly U (10 μg/ml; E and F) for the indicated periods of time. (A, C, and E) Cell lysates were analyzed by immunoblot with anti–phospho-specific IκBα, anti-IκBα, and anti-actin antibodies. (B, D, and F) Quantification of the bands was realized using Image J software. Histograms represent the intensity of the respective band, expressed as relative intensity normalized to actin, for both phospho-IκBα and IκBα. Results are representative of 3 independent experiments.

(A) A549 cells were unstimulated or stimulated with loxoribine (10 μg/ml), poly U (10 μg/ml), or Gardiquimod (10 μg/ml) and incubated with Alamar blue for 10 days. Reduction of Alamar blue was then determined by spectrophotometry at 570 and 600 nm. The percentage of reduced Alamar blue was calculated as described by the manufacturer. (B) A549 cells were unstimulated or stimulated with loxoribine, poly U, Gardiquimod, etoposide, or cycloheximide for 24 hours. The percentage of dead cells was determined by Trypan blue exclusion. (C) DNA content was measured by IP staining after RNAse A treatment. Percentages indicate the proportion of subdiploid (G₀/G₁) and diploid cells (G₂/M). Data in A–C represent mean ± SD from 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 versus medium, Student’s t test. (D) A549 cells were cultured for 6 hours in the presence of loxoribine, poly U, or Gardiquimod, and the expression of Bcl-2 was assayed using TaqMan Low-Density Array technology. The fold increase (arbitrary units) was obtained by 2^–ΔΔCT. (E and F) A549 cells were cultured for 36 hours in the presence of loxoribine, poly U, or Gardiquimod. (E) Cell lysates were analyzed by immunoblot with anti–Bcl-2 and anti-actin antibodies. (F) Quantification of the bands was realized using Image J software. Histograms represent the intensity of the respective band, expressed as relative intensity normalized to actin. Results are representative of 3 independent experiments.

A549 (A and B) or SK-MES cells (C and D) were cultured in 6-well plates with or without loxoribine (added at days 0, 3, 6 and 9), and were treated at day 12 with carboplatine and cisplatine at doses ranging 0–1,000 μM and 0–10,000 μM, respectively. The colony number was determined after Crystal Violet coloration at the day 15, and cell viability was analyzed by the surviving fraction, calculated as described in Figure 6. Data represent mean ± SD from a duplicate experiment. *P < 0.05; **P < 0.01, ***P < 0.001, Student’s t test.

A549 or SK-MES cells were cultured for 6 hours in the presence of poly I:C, LPS, loxoribine, poly U, or Gardiquimod. Total RNA was extracted and analyzed for the expression of 182 genes using TaqMan Low-Density Array technology. All genes with a CT value greater than 35 were excluded from the analysis. The ΔCT, ΔΔCT, and 2^–ΔΔCT values were calculated, and values clustered, as described in Methods; shown is the fold increase (arbitrary units) obtained by 2^–ΔΔCT.

(A) Expression of TLR1–TLR10 in A549, H1355, and SK-MES cell lines was analyzed by RT-PCR. Shown is 1 representative of 3 independent experiments. (B and C) Expression of TLR7 and TLR8 was determined in the cells by flow cytometry after intracellular staining in lung tumoral cell lines (B) or in nontumoral bronchial epithelial cell lines (C). Results are representative of 3 independent experiments. Gray histograms represent isotype controls; black histograms represent TLR7 or TLR8 Abs.

A549 cells were untreated or treated with IL-1β (10 ng/ml) or loxoribine (10 μg/ml) for the indicated periods of time. (A) Nuclear fractions were analyzed by ELISA, and the relative quantity of p50 and p65 NF-κB subunits in nucleus is expressed in arbitrary units normalized to unstimulated nuclear extracts. (B and C) The intracellular localization of the p65 NF-κB subunit was determined by immunofluorescence staining (B), and NF-κB activation was determined by NF-κB gene reporter assay after transfection with the pNIFTY2 plasmid (C). Data in C represent mean ± SD from 3 independent experiments. *P < 0.05, **P < 0.01.

A549 (A, C, and E) or SK-MES cells (B, D, and F) were cultured in 6-well plates with or without loxoribine (added at days 0, 3, 6, and 9). Cells were then treated or not with cycloheximide, cisplatine, carboplatine, doxorubicine, or Navelbine at day 12 (A–D), or treated or not with cisplatine or carboplatine in association with Navelbine or doxorubicine (E and F). (A and B) The colony number (shown below) was determined after Crystal Violet coloration. (C–F) Cell viability was analyzed at day 15 by the surviving fraction, calculated as [no. colonies after chemotherapy treatment/(no. cells seeded at day 0 × PE)] × 100, where PE is plating efficiency (calculated as no. colonies/no. cells seeded at day 0). Data represent mean ± SD from 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 versus unstimulated, Student’s t test.

A549 cells were transfected or not with TLR7 siRNA, MyD88 siRNA, or scrambled siRNA. (A and B) At 3 and 5 days after transfection, the mRNA expression levels of TLR7 (A) and MyD88 (B) were determined by quantitative RT-PCR. The fold decrease (arbitrary units) was obtained by 2^–ΔΔCT. (C) 2 days after transfection, the cells were stimulated with loxoribine or LPS for 48 hours. Cells were then treated or not with cisplatine for additional 36 hours, and cell viability was analyzed by annexin V staining and flow cytometry analysis. The percentages of dead cells were determined as annexin V–positive cells relative to cells treated with cisplatine only. Values shown are calculated as relative percentage of dead cells subtracted from 100%. Data represent mean ± SD from 3 independent experiments.

Total RNA was extracted from primary tumor cells of 3 ADC patients (patients 33, 84, and 97) and 3 SCC patients (patients 23, 25, and 31) or from SK-MES and A549 cells stimulated with loxoribine, poly U, or Gardiquimod, and the expression of 182 genes was assayed. The 11 genes found to be highly modulated in Figure 9 are shown for comparison. The ΔCT, ΔΔCT, and 2^–ΔΔCT values were calculated, and values clustered, as described in Methods; shown is the fold increase (arbitrary units) obtained by 2^–ΔΔCT.