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  • br RT qPCR expression analysis br The RNA extraction from


    2.3. RT-qPCR expression analysis
    The RNA extraction from FFPET biopsies was performed using the “High Pure FFPET RNA isolation Kit” (Roche). The RNA from GS9620 was extracted using the “RNeasy mini Kit with DNAse” (Qiagen) and the cDNA was synthetized using the “NZY First-Strand cDNA Synthesis Kit” (NZYtech).
    2.4. In silico analysis of TCGA patients
    73 stage IIIA lung adenocarcinoma patients were analyzed using R2: Genomics Analysis and Visualization Platform (, da-taset id “Tumor lung adenocarcinoma – TCGA – 515 –rsem-tcgars“).
    A549 and H1299 were obtained from the ATCC, Control Fibroblasts 1, 2 and 3 (CF1, CF2, CF3) were primary cultures derived from healthy donors and the Affected Fibroblast (AF) is the commonly used BJ h-tert immortalized fibroblast. The word "affected" indicates that this fibro-blast has a reduced OXPHOS function of unknown origin, as described in results section.
    The H1229 cell line was transfected with the plasmid pKatushka2S (K2S from now) [22] using lipofectamine 2000 reagents (Thermo Fisher Scientific) following manufacturer instructions. The efficiency was evaluated by flow cytometry and 48 h after transfection the cells were selected for one month in DMEM containing G418 0,5 mg/mL. Several clones were isolated by limit dilution and evaluated for their growing capabilities in glucose/galactose, fluorescence intensity, and transfec-tion stability (data not shown). The K2S positive clone, with more si-milar properties to the parental H1299 cell line, named H1299 K2S, was used to carry out all the following experiments.
    All cells were cultured routinely in DMEM, with 10% Fetal Bovine Serum (FBS) and Penicillin Streptomycin (Gibco).
    2.6. Galactose and glucose growth rates
    Cell growth was assayed as described previously [23]. Briefly, cell growth was assayed after growing the cells for 4 days in DMEM con-taining either 4.5 g/L glucose or 0.9 g/L galactose as carbon source. The cells were harvested and counted every 24 h and the growth rates were calculated using Doubling Time Software v3.1.0 ( Mean was calculated from at least three different experi-ments.
    2.7. Co-culture experiments
    Cells were co-cultivated, without refreshing the media, for 4 days. Cells reached approximately a 90% confluence and were analyzed by flow cytometry or confocal microscopy.
    The experiments involving fibroblast cells were seeded at 1:3 ratio (tumor cell line: fibroblast). The day of the analysis, cell lines propor-tions were near 1:1 due to their diverse growth rates. For the effect between different tumor cell lines (A549 and H1299), cells were seeded accordingly to their growth rate to reach approximately a final ratio of 1:1.
    To study the involvement of the basal fibroblast OXPHOS function in this process, fibroblasts were treated with 50 µg/mL of EtBr prior the experiments. After four days of drug exposure, the EtBr was removed and the fibroblasts were co-cultivated and analyzed as described above.
    The mitochondrial inner membrane potential (MIMP) and Cytoplasmic ROS were assessed using tetramethyl rhodamine ester (TMRE, Invitrogen) and 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA, Invitrogen) respectively.
    After addition of the fluorophores (30 µM H2DCF-DA, 100 nM TMRE) and incubation at 37 °C for 30 min in the dark, cells were col-lected in DMEM and analyzed immediately with a MACSQuant® (Miltenyi-Biotec) flow cytometer. Forward and side scatter were used to gate the viable population of cells and the mean fluorescence intensity was determined with FLOWJO (TreeStar). K2S or EpCAM (CD326, Miltenyi-Biotec) positivity in the 655–730 nm bandpass channel was used to distinguish between tumor and fibroblast cells in co-culture.
    For cell sorting, 4 × 106 co-cultured cells were analyzed and sorted using a FASCAria II sorter in two populations (K2S positive and K2S negative) corresponding to H1299 cells or fibroblasts respectively. Monocultured cells mixed just before sorting were used as control. Cells were centrifuged after cell sorting and the pellets were immediately frozen until RNA extraction.
    For the effect of TGF-β on MIMP and ROS levels, 0.75 × 106 cells were grown on MW6 plates and TGF-β was added to a final con-centration of 10 ng/µL and refreshed every day. After 4 days, cells were harvested and MIMP and ROS were evaluated as described previously.
    2.9. Immunofluorescence analysis
    After 4 days of co-culture, immunofluorescences using specific an-tibodies for MCT4 (sc-50329, Santa Cruz Biotechnology), TOM20 (sc-17764, Santa Cruz Biotechnology) and α-SMA (1 A4; Abcam) were carried out as described previously [24].
    Results were analyzed using a 2-tailed Student's t-test to assess statistical significance. Differences in PGC-1α and GAPDH/MT-CO1 ratio levels between Low and High fibrosis groups were evaluated using ANOVA. Bivariate correlations studies between gene expressions were analyzed using the Pearson correlation test. Values of P < 0.05 were considered statistically significant.