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Fig. 4 | Journal of Experimental & Clinical Cancer Research

Fig. 4

From: Mitochondrial ROS drive resistance to chemotherapy and immune-killing in hypoxic non-small cell lung cancer

Fig. 4

C/EBP-β LAP mediates chemo-immuno-resistance in hypoxic non-small cell lung cancer and is a negative prognostic factor. NCI-H2228 cells, cultured as indicated in Fig. 1a, were treated with a non-targeting sequence (scr) or with two shRNAs (sh1, sh2) targeting C/EBP-β. a C/EBP-β LAP mRNA level, measured by RT-PCR in technical triplicates. Data are means±SD (n = 4 biological replicates). ***p < 0.001: H, H/N, H/N/H versus N cells; °°°p < 0.001: sh1/sh2-cells versus scr-cells. b Immunoblot of C/EBP-β LAP. Actin is included as control of equal protein loading. The image is representative of 1 out of 3 experiments. c C/EBP-β-silenced cells (sh1) were treated for 48 h in normoxia (20% O2) with increasing concentrations (from 1 × 10− 9 to 1 × 10− 5 M) of cisplatin (Pt) and docetaxel (Dx). Cells treated with a non-targeting sequence (scr) were included as control. Cell viability was measured by a chemiluminescence-based assay, in technical quadruplicates (n = 3 biological replicates). Representative (inhibitor) vs. normalized dose-response curves and relative IC50, obtained with the GraphPad Prism 9 software. d Amount of released [14C]-IPP, considered an index of efflux, measured by liquid scintillation, in technical triplicates. Data are means±SD (n = 3 biological replicates). ***p < 0.001: H, H/N, H/N/H versus N cells; °°°p < 0.001: sh1/sh2-cells versus scr cells. e. Experimental scheme of the co-cultures of NCI-H2228 cells and Vγ9Vδ2 T cells. f Percentage of Ki67+IFN-γ+ Vγ9Vδ2 T cells collected after the co-cultures with the NCI-H2228 cells, evaluated by flow cytometry, in technical duplicates. Data are means±SD (n = 5 biological replicates). **p < 0.01,***p < 0.001: H, H/N, H/N/H versus N cells; °°°p < 0.001: sh1/sh2-cells versus scr-cells. g Percentage of annexin V+PI+ NCI-H2228 cells, as index of tumor cells killed by Vγ9Vδ2 T-cells, evaluated by flow cytometry, in technical duplicates. Data are means±SD (n = 5 biological replicates). ***p < 0.001: H, H/N, H/N/H versus N cells; °°°p < 0.001: sh1/sh2-cells versus scr-cells. h Representative immunohistochemistry images of intratumor hypoxic regions, measured with a pimonidazole-based probe, and C/EBP-β LAP-positive regions within 3 UPN NSCLC samples (63× objective, 20× ocular). Bar: 50 μm. i Patients were classified as LAPlow (n = 29) and LAPhigh (n = 31) according to the median value of staining. PFS and OS probability were calculated using the Kaplan-Meier method. ***p < 0.001

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