egfr wild type egfrwt  (ATCC)

Journal: Molecular cancer

Article Title: rs822336 binding to C/EBPβ and NFIC modulates induction of PD-L1 expression and predicts anti-PD-1/PD-L1 therapy in advanced NSCLC.

doi: 10.1186/s12943-024-01976-2

Figure Lengend Snippet: Fig. 3 Characterization of human EGFRmut and EGFRwt NSCLC cell lines. HCC827, H1975, PC-9, H460, A549, H1299, H1703 and H1437 cells were seeded into 6-well plates at the density of 2 × 106 per well. Following a 24 h incubation at 37 °C in a 5% CO2 atmosphere, cells were harvested. DNA was extracted and genotyped for rs822336 PD-L1 SNP utilizing PCR

Article Snippet: Human NSCLC cell lines carrying EGFR mutations (EGFRmut) (HCC827, H1975, PC-9) and EGFR wild type (EGFRwt) (H1299, H1703, H1437, H460 and A549) were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Incubation

Journal: Molecular cancer

Article Title: rs822336 binding to C/EBPβ and NFIC modulates induction of PD-L1 expression and predicts anti-PD-1/PD-L1 therapy in advanced NSCLC.

doi: 10.1186/s12943-024-01976-2

Figure Lengend Snippet: Fig. 4 Modulation by IFN-ɣ of PD-L1 expression in NSCLC cell lines carrying different rs822336 genotype. EGFRmut HCC827G/G, H1975G/G, PC-9G/G and EGFRwt H460G/G, A549G/G, H1299C/C, H1703C/C and H1437C/C cells were seeded into 6-well plates at a density of 2 × 106 cells per well and incubated with IFN-ɣ (100ng/ml). Untreated cells were used as a control. Following a 48 h incubation at 37 °C in a 5% CO2 atmosphere, cells were harvested and cell surface stained with Allophycocyanin (APC) anti-PD-L1 IgG2b Ab. APC anti-mouse IgG2b was used as a specificity control. Data, expressed as mean fluorescence intensity (MFI), are representative of the results obtained in three independent experiments. No changes in MFI were observed in specificity control following IFN-ɣ incubation (data not shown)

Article Snippet: Human NSCLC cell lines carrying EGFR mutations (EGFRmut) (HCC827, H1975, PC-9) and EGFR wild type (EGFRwt) (H1299, H1703, H1437, H460 and A549) were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Expressing, Incubation, Control, Staining, Fluorescence

Journal: Molecular cancer

Article Title: rs822336 binding to C/EBPβ and NFIC modulates induction of PD-L1 expression and predicts anti-PD-1/PD-L1 therapy in advanced NSCLC.

doi: 10.1186/s12943-024-01976-2

Figure Lengend Snippet: Fig. 5 Modulation by rs822336 allele-specificity of the in vitro activity of anti-PD-1 nivolumab on NSCLC cells co-cultured with HLA-matched PBMCs. EGFRmut HCC827G/G, H1975G/G and EGFRwt H460G/G, A549G/G, H1299C/C and H1437C/C cells were seeded into 24-well plates at a density of 2 × 105 cells per well. Following a 24 h incubation at 37 °C in a 5% CO2 atmosphere, cells were co-cultured with HLA-matched PBMCs in a 1:10 ratio and incubated with nivolumab (10 µg/ml). HLA-A*11, HLA-A*01, HLA-A*24, HLA-A*25, HLA-B*40 and HLA-A*03 haplotypes were used for matching EGFRmut HCC827G/G, H1975G/G and EGFRwt H460G/G, A549G/G, H1299C/C and H1437C/C cells, respectively, with PBMCs. HLA-matched PBMCs were activated utilizing an anti-CD3 (1 µg/mL) and an anti-CD28 (1 µg/mL) T Cell TransAct (T- Act). Non-activated HLA-matched PBMCs were used as controls. Purified human IgG4 was used as a control for nivolumab. (A) Following a 48 h incubation, cancer cell viability was determined by cell counting kit-8 (CCK-8) assay. Data are expressed as mean percentages of the viability of nivolumab treated cells ± SD as compared to cells incubated with purified human IgG4. The mean percentage of cell viability and SD were calculated from three independent experiments; each of them was performed in triplicate. (B) Following a 48 h incubation, apoptosis induction was determined by Annexin V/PI assay by flow cytometry. Data are expressed as mean percentages of apoptotic cell population of nivolumab treated cells ± SD as compared to cells incubated with purified human IgG4. The mean percentage of apoptotic cells and SD were calculated from three independent experiments; each of them was performed in triplicate. (C-E) Following a 48 h incubation, LDH (C), IFN-γ (D) and TNFα (E) levels in the medium harvested from cultures of HLA-matched PBMCs with cancer cells were measured by LDH assay kit, ELISA Max Deluxe Set Human IFN-γ kit and human TNFα ELISA, respectively. Data are expressed as means of LDH, IFN-γ and TNFα levels ± SD of the results obtained in three independent experiments; each of them performed in triplicate. (ns: not significant; *P ≤ 0.05; ***P ≤ 0.001)

Article Snippet: Human NSCLC cell lines carrying EGFR mutations (EGFRmut) (HCC827, H1975, PC-9) and EGFR wild type (EGFRwt) (H1299, H1703, H1437, H460 and A549) were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: In Vitro, Activity Assay, Cell Culture, Incubation, Purification, Control, Cell Counting, CCK-8 Assay, Flow Cytometry, Lactate Dehydrogenase Assay, Enzyme-linked Immunosorbent Assay

Journal: Molecular cancer

Article Title: rs822336 binding to C/EBPβ and NFIC modulates induction of PD-L1 expression and predicts anti-PD-1/PD-L1 therapy in advanced NSCLC.

doi: 10.1186/s12943-024-01976-2

Figure Lengend Snippet: Fig. 8 Regulation by C/EBPβ and NFIC silencing of PD-L1 expression in NSCLC cell lines carrying different rs822336 genotype under basal conditions or following IFN-ɣ incubation. EGFRmut H1975G/G and EGFRwt H460G/G cells (upper panel) as well as EGFRwt H1299C/C and EGFRwt H1437C/C cells (lower panel), transduced with C/EBPβ- and NFIC-specific siRNAs or siRNA-controls, were seeded into 6-well plates at a density of 2 × 106 cells per well and incubated with IFN-ɣ (100ng/ml). Untreated cells were used as a control. Following a 48 h incubation at 37 °C in a 5% CO2 atmosphere, cells were harvested and lysed. Cell lysates were analyzed by western blot with NFIC, C/EBPβ and PD-L1-specific Abs. Representative results are shown. The levels of NFIC and C/ EBPβ, normalized to GAPDH and relative to untreated siRNA-control as well as levels of PD-L1, normalized to GAPDH and relative to treated siRNA-control, are plotted on the right, and expressed as mean ± SD of the results obtained in three independent experiments

Article Snippet: Human NSCLC cell lines carrying EGFR mutations (EGFRmut) (HCC827, H1975, PC-9) and EGFR wild type (EGFRwt) (H1299, H1703, H1437, H460 and A549) were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Expressing, Incubation, Transduction, Control, Western Blot