Flow cytometry quantification of HPG Click-iT staining in MB135iDUX4 myoblasts

We used metabolic labeling with methionine analog L-homopropargylglycine (HPG) followed by fixation and Click-iT chemistry to measure levels of nascent protein synthesis 0-92h following a pulse of DUX4. Conclusion: We confirmed DUX4 inhibition of protein synthesis by labeling cells with methionine analog L-homopropargylglycine (HPG) followed by fixation and Click-iT chemistry, wherein fluorescence microscopy and flow cytometry showed a dramatic reduction in HPG-labeled peptides after a pulse of DUX4, but not with transcriptionally inactive DUX4(F67A). Notes: Cells were incubated for 30 minutes in DMEM media depleted for methionine and cysteine, followed by a 1-hour incubation in methionine-depleted media supplemented with 200µM L-HPG HCl (Sigma-Aldrich Cat#900893). Cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized 0.5% TritonX-100, and stained with Click-iT HPG Alexa Fluor 488 Protein Synthesis Assay Kit (Invitrogen/Thermo Fisher Cat#C10428) according to manufacturer’s protocol. Cells were resuspended in FACS buffer for analysis by flow cytometry using BD LSRFortessa X-50 with BD FACSDiva software. Data were analyzed using FlowJo v10.5.3.