Fig. 2

Kdm6a is differentially expressed in XX vs. XY splenocytes and B cells isolated from HKSP-immunized FCG mice. One-week post-HKSP immunization, RNA-Sequencing was performed on splenocytes isolated from male and female FCG mice. Levels of Kdm6a expression are represented as relative expression using EdgeR values (A). Differential expression of Kdm6a was confirmed by qRT-PCR using both splenocytes and B cells isolated from HKSP-immunized females (B) and males (C). KDM6a protein levels were assessed by western blot using lysates generated from HKSP-immunized FCG splenocytes (D, E). Relative levels of protein expression were quantified as KDM6a:beta-tubulin ratios (E). Kdm6a expression was also assessed in B cells from HKSP-immunized gonadectomized (Gdx) and sham-operated (Sham) females (F) and males (G, Additional file 9: Table S4). RNA-FISH was utilized to determine if Kdm6a was expressed from the inactivate X chromosome in B cells isolated from female XX FCG mice previously immunized with HKSP. Representative images of individual DAPI, Xist, and Kdm6a, as well as merged images, are presented (H). Colocalization of Kdm6a and Xist signals was considered indicative Kdm6a expression from the inactive X chromosome. Data are represented as the mean ± SEM with each data point representing one mouse. Statistical analyses by two-way ANOVA followed by Sidak’s multiple comparisons test (A, E), unpaired t-tests (B, C), or by three-way ANOVA followed by Tukey’s multiple comparisons test (F, G). Comparisons in graphs are representative of multiple comparisons tests; *p < 0.05; **p < 0.01; ****p < 0.0001. ANOVA main and interactive effects for A & E are provided in Additional file 9: Table S3