Long non-coding RNA Neat1 and paraspeckle components are translational regulators in hypoxia

HL-1 cells were transduced with Lucky Luke bicistronic lentivector containing the IRES of FGF1 mRNA. Cells were submitted to normoxia or hypoxia 1% O₂ during 4 hr, 8 hr or 24 hr. Renilla and firefly luciferase activities were measured (page 1) and the IRES activities evaluated with the ratio LucF/LucR (page 2). Mann-Whitney test was performed with n=12. **P<0.01. For each hypoxia condition the mean of the LucF/LucR ratio has been calculated with 12 cell culture biological replicates, normalized to normoxia Experiments A, B, C correspond to experiments performed at different dates, while 1, 2, 3, 4 are experiments performed in parallel at the same date, each of them being already the mean of three technical replicates (36 technical replicates in total).

A/ FISH and smiFISH probes. B/ qPCR primers and cloning primers. C/ LNA gapmers used in Neat1 knock-down assays. D/ SiRNA SMARTpools (Dharmacon) used for IRES activity studies. E/ Fluidigm deltagene primers. The corresponding genes are indicated in the left column.

HL-1 cells were transduced with Lucky Luke bicistronic lentivectors containing the IRES of FGF1, FGF2, VEGFA (IRES a or b), VEGFC, VEGFD, IGF1R, MYC or EMCV. Cells were then treated with a pool of gapmers Neat1, or control during normoxia or hypoxia 1% O_2. For the lentivector with FGF1 IRES, cells were also treated with the gapmer Neat1_2 (N1_2, page 2). Renilla and firefly luciferase activities were measured (upper panel) and the IRES activities evaluated with the ratio LucF/LucR (lower panel). Mann-Whitney test was performed with n=9. *P<0.05, **P<0.01, ***<0.001, ****P<0.0001. For each IRES the mean has been calculated with nine cell culture biological replicates Experiments A, B, C correspond to experiments performed at different dates, while 1, 2, 3 are experiments performed in parallel at the same date, each of them being already the mean of three technical replicates (27 technical replicates in total).

HL-1 cells were transduced with a Lucky Luke bicistronic lentivector containing the IRES of FGF1. Cells were then treated with siSfpq (A), siRps2 (B), sihnRNPM (C), siNucleolin (siNcl, D) or siControl (siCtrl) smartpools during normoxia or hypoxia 1% O_2. Renilla and firefly luciferase activities were measured and the IRES activities evaluated with the ratio LucF/LucR. Mann-Whitney test was performed with n=9 (n=12 for FGF1 IRES). *P<0.05, **P<0.01, ***<0.001, ****P<0.0001. For each siRNA the mean has been calculated with nine cell culture biological replicates Experiments A, B, C correspond to experiments performed at different dates, while 1, 2, 3 are experiments performed in parallel at the same date, each of them being already the mean of three technical replicates (27 technical replicates in total).

HL-1 cells were transduced with Lucky Luke bicistronic lentivectors containing the IRES of FGF1, FGF2, VEGFA (IRES a or b), VEGFC, VEGFD, IGF1R, MYC or EMCV. Cells were then treated with siP54^nrb or siControl (siCtrl) smartpool during normoxia or hypoxia 1% O_2. Renilla and firefly luciferase activities were measured (upper panel, or page 1 for FGF1 IRES) and the IRES activities evaluated with the ratio LucF/LucR (lower panel, or page 2 for FGF1 IRES). Mann-Whitney test was performed with n=9 (n=12 for FGF1 IRES). *P<0.05, **P<0.01, ***<0.001, ****P<0.0001. For each IRES the mean has been calculated with nine cell culture biological replicates Experiments A, B, C correspond to experiments performed at different dates, while 1, 2, 3 are experiments performed in parallel at the same date, each of them being already the mean of three technical replicates (27 technical replicates in total).

HL-1 cells were transduced with Lucky Luke bicistronic lentivectors containing the IRES of FGF1, FGF2, VEGFA (IRES a or b), VEGFC, VEGFD, IGF1R, MYC or EMCV. Cells were then treated with siPSPC1 or siControl (siCtrl) smartpool during normoxia or hypoxia 1% O_2. Renilla and firefly luciferase activities were measured (upper panel, or page 1 for FGF1 IRES) and the IRES activities evaluated with the ratio LucF/LucR (lower panel, or page 2 for FGF1 IRES). Mann-Whitney test was performed with n=9 (n=12 for FGF1 IRES). *P<0.05, **P<0.01, ***<0.001, ****P<0.0001. For each IRES the mean has been calculated with nine cell culture biological replicates Experiments A, B, C correspond to experiments performed at different dates, while 1, 2, 3 are experiments performed in parallel at the same date, each of them being already the mean of three technical replicates (27 technical replicates in total).

p54^nrb-HA transduced HL-1 cells were subjected to normoxia or hypoxia, then nucleus and cytoplasm fractionation was performed and extracts were immunoprecipitated using anti-HA antibody. Enriched interacting proteins were identified by using a label-free quantitative mass spectrometry approach. The dataset list of proteins identified by MS/MS is presented in alphabetical order. An unpaired bilateral student t-test with equal variance was used. Enrichment significance thresholds correspond to an absolute log2-transformed fold-change (FC) greater than 1 and a -log10-transformed (p-value) greater than 1.3.

HL-1 cardiomyocytes were transfected with gapmer Neat1, Neat1_2, or control. Polysomes were purified on sucrose gradient as described in Star Methods. RNAs were purified from cytoplasmic extracts and from pooled polysomal fractions and analyzed on a Fluidigm deltagene PCR array from two biologicals replicates (cell culture dishs and cDNAs), each of them measured in three technical replicates (PCR reactions). mRNA levels in polysomes (polysomal RNA/ total RNA) were analyzed for each gene. Relative quantification (RQ) of mRNA level was calculated using the 2–ΔΔCT method with normalization to Gapdh mRNA and to HL-1 tranfected by gapmer control, and is shown as fold change of expression. RQ1 and RQ2 correspond to two independent experiments. To measure the fold change of repression, the mean is expressed as –1/RQ for the values <1 (yellow column).