Fig. 8 | Nature Communications

Fig. 8

From: A RASSF1A-HIF1α loop drives Warburg effect in cancer and pulmonary hypertension

Fig. 8

RASSF1A-HIF-1α axis in primary lung tumor cells. a Representative western blots for RASSF1A protein expression as analyzed in proteins isolated from tumor (T) and non-tumor (N) areas of human non-small cell lung cancer lungs. b Paraffin lung tissue sections from lung tumor patients were subjected to immunohistochemical staining of RASSF1 and HIF1α (HIF1A). Scale bar: 20 μm. c RNA was isolated from tumor and matched non-tumor samples, followed by real time PCRs for indicated genes. The Ct values of tumor samples were divided by the Ct values of respective non-tumor samples to obtain the fold change of RASSF1A expression. n = 9 RASSF1Ahigh and n = 10 RASSF1Alow tumors. d Fold change in RASSF1A expression in various lung tumor tissues plotted vs the pathological stages (I, II, III) of the respective tumors. n = 15 stage I, n = 14 stage II, and n = 27 stage III human lung tumor tissues. *P < 0.05 compared to RASSF1Alow (c) or stage I (d), unpaired Student’s t-test. el Primary cancer cells isolated from two patients with RASSF1A-positive lung tumor were transfected with RASSF1 siRNA (si-RASSF1), HIF1α siRNA (si-HIF1A), and control siRNA (si-Control). 24 h after transfection, cells were exposed to normoxia or hypoxia for 24 h. e, i Lysates obtained were subjected to western blotting for above-mentioned proteins. si-RASSF1 or si-control transfected lysates were further subjected to f, j real time PCRs for LDHA, HK2, g, k western blotting for LDHA, HK2 and h, l lactate assay. f, h, j, l *P < 0.05, **P < 0.01, ***P < 0.001 compared to si-control (hypoxia), one-way ANOVA followed by SNK multiple comparison test. Data represent mean ± s.e.m. For primary tumor cells, n = 2–3 independent experiments from 2 biological replicates (represented as separate)

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