Skip to main content
. 2019 Dec 9;8:e50094. doi: 10.7554/eLife.50094

Figure 4. IRESs from (lymph)angiogenic factor mRNAs are activated in early hypoxia.

Figure 4.

(A–D) To measure IRES-dependent translation during hypoxia, HL-1 cardiomyocytes were transduced with bicistronic dual luciferase lentivectors (termed ‘Lucky-Luke’) containing different IRESs cloned between the genes of renilla (LucR) and firefly (LucF) luciferase (A). In bicistronic vectors, the translation of the first cistron LucR is cap-dependent, whereas translation of the second cistron LucF is IRES-dependent (Créancier et al., 2000). Cardiomyocytes transduced by the CRF1AL+ lentivector Lucky-Luke reporter containing FGF1 IRES were submitted to a hypoxia time-course (0 hr, 1 hr, 2 hr, 4 hr, 6 hr, 8 hr, 16 hr and 24 hr) and data from each time point were compared to those from normoxia with a non-parametric Mann-Whitney test (B). Endogenous FGF1 protein expression was measured by Jess capillary Simple Western of extracts of cardiomyocytes in normoxia or those submitted to 8 hr of hypoxia (normalized to Jess quantification of total proteins as described in 'Materials and methods'). Three independent experiments were performed; a representative experiment is shown (C). HL-1 cardiomyocytes transduced by different Lucky-Luke constructs were submitted to 4 hr, 8 hr or 24 hr of hypoxia and their luciferase activities were measured. IRES activities during hypoxia, expressed as LucF/LucR ratio, are normalized to those during normoxia. Histograms correspond to means ± standard deviation of the mean, data from hypoxic cardiomyocytes compared to those from normoxic cardiomyocytes with a non-parametric Mann-Whitney (M-W) test: *p<0.05, **p<0.01, ***<0.001, ****p<0.0001. For each IRES, the mean was calculated from nine cell culture biological replicates, each of them being the mean of three technical replicates (27 technical replicates in total but the M-W test was performed with n = 9). Detailed values of biological replicates are presented in Supplementary file 3. A no-IRES control was also used and values are presented in Supplementary file 3J.