Fig. 5.

Hypoxia enhances interaction between hnRNPM and FGF9 mRNA and promotes translation. (a) Gel image of FGF9 RT-PCR from hnRNPM-immunoprecipitation assay. RNase treatment was performed to indicate that PCR products were amplified from RNA origin. (b) The quantitative measurement of FGF9 TaqMan assays from hnRNPM-immunoprecipitation assay. The relative FGF9 mRNA level was shown as the amount measured in hypoxia normalized to the amount in normoxia. (c) Representative Western blot image shows levels of hnRNPM pulldown by full length FGF9 RNA 5’UTR (FL) or just IRES sequences from cells cultured under normoxia or hypoxia conditions. (d) Immunofluorescent images showed cellular distribution of hnRNPM (red) in HEK293 cells cultured in normoxia or hypoxia conditions. The arrowheads indicate cytosolic hnRNPM. Nuclei were marked with DAPI (blue). (e) Number of cytosolic hnRNPM positive cells in normoxia or hypoxia (A total of 100 cells per independent experiment were counted). (f) Representative Western blot image shows the expression of hnRNPM in the cytosolic and nuclear fractions of indicated cells treated with normoxia or hypoxia for 9 h (n = 3 independent experiments). The levels of α-tubulin and lamin A/C are used as markers for cytosolic and nuclear fractions, respectively. (g) The 18S and 28S rRNAs were resolved on a 1% formaldehyde/agarose gel and visualized by ethidium bromide staining (Upper panel). Immunoblotting analysis of gradient fractions was performed using antibodies against hnRNPM, PTBP1, eIF3, eIF4A1, and ribosomal protein RPS6 (Lower panel). (h) The quantitative measurement of mRNAs from hnRNPM-knockdown cytoplasmic extracts. Bar graphs represent the proportion of actively translated mRNA of FGF9 (left panel) and β-actin (right panel) from 3 independent experiments. ^⁎P < .05; ^⁎⁎P < .01; ^⁎⁎⁎P < .001 (Student's t-test). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)