Fig 2: Increase in nuclear NF-?B sub-units p65 and p50 expression in hESC-CMs following IH: (A) quantification of the NF-?B subunits p65 and p50 expression in the nucleus following normoxia or IH only on CMs. Normoxia (21% O2, 37 °C), intermittent hypoxia (IH) (1% O2, 37 °C) for 12 h (60 Cycles). Results are averages of 3 separate experiments performed in 10 replicates Student’s t-test *** p < 0.001, n = 39,852; (B) immunostaining of p65, p50 and cardiac troponin T (cardiomyocytes specific marker) following normoxia or IH. Differentiated CMs were detected with antibodies to cardiac troponin T (green) and p65 or p50 (pink), followed by the appropriate secondary antibody. Nuclei were stained with DAPI (blue); (B) normoxia conditions and p65; (C) IH conditions and p65; (D) normoxia conditions and p50; and (E) IH conditions and p50. 40× magnification.

Fig 3: NF-?B heterodimer expression: High grade urothelial carcinoma.(A) High grade non invasive urothelial carcinoma H&E × 200. (B) Immunostaining with RelA (p65) showed nuclear positivity (brown colour) indicated by arrow IHC × 200. (C) NF-?B p50 showed moderate cytoplasmic positivity IHC × 200. Note: adjacent lymphocytes (in-built control) have taken up nuclear staining indicated by arrows. (D) High grade papillary invasive urothelial carcinoma with squamous differentiation H&E × 200. (E) Immunostaining with RelA (p65) showed nuclear positivity (brown colour) as indicated by arrows IHC × 200. (F) NF-?B p50 showed few nuclear and cytoplasmic positivity in squamous regions which are indicated by arrows IHC × 200. (G) High grade invasive urothelial carcinoma H&E × 100. (H) Immunostaining with RelA (p65) showed nuclear positivity (brown colour) as indicated by arrows IHC × 200. (I) NF-?B p50 showed extensive cytoplasmic positivity as indicated by arrows IHC × 200. Scale bar indicates 50 µM.

Fig 4: NF-?B is activated by stimulation with sera from OSAS patients. (A) A representative picture of a cluster of cells is presented. Cardiomyocytes are identified by staining with anti-cardiac troponin (green). NF-?B subunits: Anti p50 or anti p65 (pink). Arrows point to nuclei expressing p50 or p65. (B) The total average intensity of NF-?B subunits p50 and p65 staining was measured in the nuclei of cardiomyocytes. The cells were incubated each time with 12 different OSAS sera and compared to 10 different control sera (5%). The results presented are an average of 3 separate experiments done in duplicates, showing a significant increase of nuclear p50 and p65 following incubation with OSAS sera. Squares and circles represent individual sera. Statistical significance was determined by the Student’s t-test comparing cells incubated with control or OSAS sera (p50 p = 0.01, p65 p = 0.001). * p < 0.05, *** p < 0.01.