Supporting materials information captions (Bi-allelic MARVELD2 Variant Identified with Exome Sequencing in a Consanguineous Multiplex Ghanaian Family Segregating Non-Syndromic Hearing Impairment)

Supporting materials information captions Table S1. HEK-293 glass bottom dish culture transfection reagents composition Table S2. HEK-293 cells cover slip culture transfection reagents composition Table S3. Western blot densitometric analysis Figure S1. Sanger validation of site-directed mutagenesis: thymine duplication circled in red. Figure S2. pCMV10 and pcDNA-3 constructs backbone and components Figure S3. CADD Phred scores against MARVELD2 reported variant classification. Figure retrieved from ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) CADD = Combined Annotation Dependent Depletion Figure S4. Mutant MARVELD2 Fluorescence and immunocytochemistry. HEK 293 Cells 24- and 48-hours post-transfection confocal imaging. Panel A) Wild type and Mutant EYFP expression. Panel B) Wild type and Mutant EYFP localization Bars = 10 µm Figure S5. Mutant MARVELD2 Immunocytochemistry. Panel A) Wild type and Mutant EYFP expression. Panel B) Wild type and Mutant EYFP localization. Bars = 5 µm Figure S6. Wild type or Mutant MARVELD2-N-EYFP, zonula occluden-1 (ZO-1), and phalloidin staining along cellular margins and tricellular junctions. HEK-293 cells 48 hours post-transfection localization of MARVELD2-p.Val354SerfsTerfs*5 protein (WT or MT) were stained for ZO-1 and phalloidin, and viewed using confocal microscopy. MARVELD2 proteins were viewed using YFP channel, ZO-1 by Texas-red (depicted as violet), phalloidin actin stain using Oregon green (green) and Hoechst stain for DNA in the nuclei (blue). Wild-type Panel). Wild-type transfected HEK-293 cells; localized expressed MARVELD2-N-EYFP is condensed and consistent (EYFP panel and white arrows in merged panel). Phalloidin F-actin stained; accumulation demonstrated by observed positive-spots along cell margins. Mutant Panel). In mutant transfected HEK-293 cells, localization of expressed tagged MARVELD2-p.Val354SerfsTerfs*5 seems to be diffused (EYFP panel). Phalloidin F-actin stained; accumulation also revealing relatively less stress fibres distribution seen as positive spots along membrane barrier compared to the wild type. Bars = 10 µm Figure S7. Uncropped Western Blots Images Figure S8. Uncropped Protein Blots Images: WT; MT; Empty Vector, Flag; ZO-1; MW ladder, and IgG (WT and MT) Figure S9. Uncropped Western Blots Images: Second co-immunoprecipitation