Fig 2: The long variant of RPS24 is significantly up-regulated in spheroids of four cell lines. (A) Spheroids from four cell lines were imaged and harvested 5 d following cell seeding. (B) RT-qPCR using variant-specific RPS24 primers were used to confirm the ASPCR data from Figure 4. The ΔΔCt method was used, normalizing to reference genes RPLP0 and RPL13A, and the fold-change made relative to normoxia. Data (n = 8), mean fold-change ± SEM. One-way ANOVA was performed on the ΔCt values. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001. Scale bar, 500 µm.

Fig 3: Endogenous RPS24 short and long transcript variants, and exogenous protein isoforms associate with polysomes. (A) Using transcript variant-specific RPS24 primers, short and long variants were amplified via RT-PCR from polysome fractions with either cycloheximide, or EDTA as a control to dissociate polysomes. (B) Polysomes were isolated from two cell lines each stably expressing one of the FLAG-tagged RPS24 protein isoforms (eS24) using cycloheximide and EDTA. Western blots were performed using anti-FLAG, or β-Actin as a control protein not associated with polysomes. Experiments performed in U87MG glioblastoma in normoxia (21% O2). See Supplemental Table S1 for primer sequences.