DOI: 10.6084/m9.figshare.14936196.v1
title: Additional file 1 of mRNP granule proteins Fmrp and Dcp1a differentially regulate mRNP complexes to contribute to control of muscle stem cell quiescence and activation

description: Additional file 1: Table 1. Bio-informatic analysis of transcripts encoding mRNP components. To assess whether changes in expression of mRNP proteins resulted from changes in expression of their mRNAs, we used the recent RNA seq analysis derived from muscle satellite cells fixed by perfusion of adult mice (to prevent cell activation that results from disruption of the niche during isolation [42]. These fixed satellite cells are thought to more accurately represent the quiescent (G0 state) and have a transcriptome profile distinct from MuSC isolated without fixation, which are now understood to represent cells in an early activation state. Activated satellite cells (ASC) represent proliferating primary myoblasts 2.5 days post isolation from the animal. Transcripts encoding P body genes were selected from the RNAseq data and grouped according to their function as outlined [43, 44]. We calculated fold changes from FPKM values (Fragments Per Kilobase of transcript per Million mapped reads) RNA seq data comparing fixed (quiescent) satellite cells and activated satellite cells [44] and used a cut-off of 1.5 +/- (for up regulation and down regulation). False Discovery Rate approach: Two stage step-up method of Benjamini, Krieger and Yekutieli was used and 10% FDR was set up for generating p values for the analysis. Figure S1. Differential association of decay complex proteins in different cellular states. Immuno-staining of Dcp1a/Edc4/Pat1 (left) and Dcp1a/Ago2 (right) in muscle cells in culture: quiescent (G0), 3 hr reactivated (R3), proliferative (MB), and differentiated (MT). Blue arrows indicate co-localization of Dcp1a/Edc4/Pat1 in puncta. Red arrows indicate co-localization of Dcp1a/Ago2 in puncta. Note the absence of Dcp1a or Pat1 puncta in G0, and the rapid reassembly in R3. Also note prominent nuclear staining for Edc4 in G0. Figure S2. (A) Supplementary to Figure 4A Additional representative immunofluorescence images showing Fmrp (green) and Dcp1a (red) puncta in G0, MB and MT, as well as cells reactivated for 3 hr from G0 (R3). Arrows indicate prominent puncta. (B). Corrected Mean Fluorescence intensities (CMI) of Fmrp and Dcp1a respectively in MB, G0, R3 and MT. For quantification, more than 3 cells per group was used and CMI intensities from more than 12 puncta were analysed. Corrected mean intensity was calculated using CMI= Total intensity of signal – (Area of signal x Mean background signal). For quantification, more than 3 cells per group was used and MFI intensities from more than 10 puncta was analysed. Figure S3. Knockdown of Fmrp leads to reduced cell renewability and is not accompanied by apoptosis. (A) Colony formation assay shows that reduced EdU incorporation in Fmrp knockdown cells correlates with compromised self-renewal. Bar graph represents mean ± sd from n=3 biological replicates. Two tailed paired Student’s t-test is indicated as ***p&lt;0.001. (B) Proliferating myoblasts (MB) were treated with siRNAs (Scr, siFmr1) for 18 hr and harvested at 24 hrs for FACS analysis of 10,000 cells performed after staining for apoptosis markers. X-axis represents Annexin V and Y-axis represents propidium iodide. Upper Panel: Flow cytometric profile for MB. Lower Panel: Quantification of FACS plots shows that Fmrp knockdown cells do not undergo apoptosis. n=3, mean ± sd. Figure S4. Knockdown of Fmrp is not accompanied by senescence. (A). SA β-galactosidase assay performed in MB cells treated with siRNAs (Scr, siFmr1) for 24 h or reactivated from quiescence for proliferation for 24 hrs (R24) does not show any significant difference in X-gal staining between control and Fmr1 Knock down cells. (B). Analysis of DNA damage-induced foci of γH2AX in cells reactivated from quiescence for 24h (R24) does not reveal any increase in Fmr1 knock down cells. (C). qRT-PCR analysis for p21 did not reveal any significant change in Fmr1 knocked down condition in MB, G0 or R24. All bar graphs represent mean ± sd from n =2. Two tailed paired Student’s t-test is indicated as ***p &lt;0.001. Figure S5. (A): Phenotyping of 3 cellular conditions: A replicate blot is shown of the data depicted in Figure 4C. Myogenin is exclusively expressed by MT, Cyclin D1 is enriched in MB, and G0 cells lack both proteins, confirming their quiescence by the absence of both proliferation and differentiation programs. (B) Global Transcript status: The global transcript levels of Cyclin D1, p27, MyoD1, Myf5, Gapdh in MB, G0, MT were analysed using data sets from Venugopal et.al., [49] 2020. GEO database (Series GSE110742). *p &lt; 0.05. **p &lt; 0.01, ***p &lt;0.001. Figure S6. Dcp1a and Fmrp Knockdowns have opposing effects on cell proliferation but compromise differentiation. This data represents quantification of western blots shown in Figure 8. (A to D): Densitometry of western blots of Dcp1a, Fmrp , Cyclin A2 , Cyclin E proteins normalized with Gapdh as internal control in MB , MT, G0 and R3 (E): Western blots of Myogenin and Myosin Heavy chain proteins in G0 and R3 with Gapdh as internal control. All bar graphs represent mean ± sd from n ≥ 3 ,Two tailed paired Student’s t-test is indicated is indicated as *p &lt; 0.05. *p &lt; 0.01, ***p &lt;0.001. Figure S7. Altered expression of cell cycle and myogenic transcripts in knockdown cells held in G0-inducing conditions. Cells were transfected with siFmr1 and siDcp1a pools for 18 hours, then placed in suspension culture and 48 hours later RNA was isolated for qRT-PCR of Dcp1a, Fmr1, Ki67, Cyclin A2, Cyclin D1, Cyclin E1, Cyclin B, p27, p21, MyoD1, and Myf 5 Loss of Dcp1a leads to increased abundance of transcripts encoding positive regulators of the cell cycle (Ki67, Cyclins), along with suppression of Cdk inhibitor p21 mRNA levels, consistent with increased EdU incorporation. Transcripts encoding Pax7 and MyoD were suppressed. Knockdown of Fmr1 leads to reduced abundance of Cyclin A2, B and D1 mRNAs, consistent with decreased EdU incorporation. Gapdh was used as internal control and normalized to Scr G0 condition. All bar graphs represent mean ± sd from n =3. Two tailed paired Student’s t-test is indicated is indicated as *p &lt; 0.05. **p &lt; 0.01, ***p &lt;0.001.