Hypoxic response of sea-level, non-CMS, and CMS cells: Marked response of CMS samples. (A) Flow cytometric analysis using CD235a (glycophorin A) as a marker after culturing as detailed in Fig. 1 at the EB stage at day 28. Sendai virus results: Representative FACS plots of sea level, non-CMS, and CMS in normoxia (left) and in hypoxia (right). The dot plot represents the live cells as gated through propidium iodide. CD235a⁺ cells are shown in red along the y axis, and CD235a⁻ cells are shown in blue. The percentage in each figure represents the relative proportion of CD235a cells. There are major differences between CMS (bottom) versus the non-CMS (middle) and sea-level (top) samples. The FACS plots are representative of one experiment. Similar results were obtained in all the experimental repeats. (B) Summary of hypoxic response of CMS patients (n = 5 subjects) and non-CMS (n = 4 subjects) and sea-level (n = 3 subjects) control subjects. The graph depicts the relative proportion of CD235a quantified 3 wk after the administration of hypoxia (5% O₂). There is a significantly striking difference between sea level, non-CMS, and CMS under hypoxia. ***, P < 0.001. Error bars represent the mean ± SEM of at least two to three measurements. The experiment was repeated at least three times. (C) Summary of interclonal variability among the subjects: three clones (clones 1, 2, and 3) were tested for three subjects (subjects 1, 2, and 3) for each group: CMS, non-CMS, and sea level. The y axis depicts the relative proportion of CD235a under hypoxia for different clones. Error bars represent the mean ± SEM of at least two to three measurements. (D) Dose response. The graph represents the response (as measured by proportion of CD235a [y-axis]) of CMS, non-CMS, and sea-level cells to 21%, 10%, 5%, and 1.5% O₂ levels (x axis). Each point depicts the mean ± SEM of at least two to three measurements. CMS shows hyperresponsiveness at 10, 5, and 1.5% O₂. The experiment was repeated at least three times.