Fig. 7

Cross-regulation of Fmrp and Dcp1a in knockdown myoblasts. a Following transfection with either Dcp1a or Fmr1 siRNA pools or the control pool (Scr), knockdown myoblasts were incubated in growth medium for 18 h. Left: Immunoblot analysis shows that in Fmrp knockdown, Fmrp abundance is reduced but Dcp1a expression is enhanced. Likewise, Fmrp protein levels are increased in Dcp1a knockdown. Values depicted under each lane represent protein levels from normalized densitometric scans, relative to level in Scr. Right panel: Densitometry of western blots of Dcp1a, and Fmrp proteins normalized relative to Scr with Gapdh as internal control. Bar graphs represent mean ± SD from n = 3. Two-tailed paired Student’s t tests are indicated as * p < 0.05. ** p < 0.01. b Knockdown effects on Dcp1a and Fmrp by their respective targeting siRNAs are detectable at the subcellular level. Consistent with changes at the level of protein abundance, immunofluorescence analysis shows that increased Dcp1a expression in Fmr1 knockdown is accompanied by enhanced Dcp1a puncta assembly, while compromising Dcp1a expression leads to enhanced Fmrp puncta assembly. Scale bars represent 15 μm except in zoomed panels where scale bars represent 8 μm. c Quantitative image analysis of b: The fluorescence intensities of Fmrp and Dcp1a following immunostaining of Scr, siFmr1, and siDcp1a samples were calculated and represented as box and whisker plots. Significant increases in Dcp1a puncta were observed in Fmrp knockdown and vice versa increased Fmrp puncta were observed in Dcp1a knockdown. Data were obtained from triplicate samples for each condition and graph shows scoring of at least n = 75 cells each for Scr (Scrambled), siFmr1 (Fmrp knockdown), siDcp1a (Dcp1a knockdown). Two-tailed paired Student’s t test results are indicated as *** p < 0.001