Fig. 5: Depletion of the ER stress transcriptional regulator Jumonji KDM4A upregulates ER stress and mTOR signaling, decreases cell proliferation in vitro, and blunts tumor growth in vivo.

A Immunoblot demonstrating decrease of KDM4A (top) in H446 cells compared to parental and Cas9-only control cells. GAPDH (bottom) was used as the loading control. B Loss of KDM4A in H446 decreases proliferation compared to parental and Cas9-only H446 cells. See the methods section for experimental details. Error bars represent SD from the mean across 4–5 biological replicates. ** = p ≤ 0.01, * = p ≤ 0.05, by two-tailed t-test, unequal variance. C Depletion of KDM4A in H446 decreases colony formation potential compared to parental and Cas9-only H446 cells. Each colony is defined by a cluster of approximately 100 cells. Error bars are SEM across six replicates. **** = p ≤ 0.0001 by two-tailed t-test, unequal variance. D Upregulation of ER stress pathway genes measured by RNA-sequencing in KDM4A knockdown compared to parental and Cas9-only H446 cells. Data are shown as mean ± SD, n = 3. *** = p ≤ 0.001, ** = p ≤ 0.01, * = p ≤ 0.05, by two-tailed t-test, unpaired with Welch’s correction. E ER stress proteins p-elF2α, DDIT3, and CHAC1 are upregulated in H446 KDM4A knockdown cells vs controls, as shown by Western blot. Tubulin is a loading control. F MTS viability curves of H446 Cas9 vs KDM4A knockdown cells in response to mTOR inhibitors. Data are average ± SD for each dose, n = 4. P-values for curve comparisons calculated by GraphPad Prism (top, bottom, IC₅₀, Hill coefficient). G Growth of cell line-derived xenografts in nude mice demonstrates suppressed tumor progression in H446 tumors with KDM4A knockdown compared to controls. Data are shown as mean ± SEM. H446 parental (n = 5), H446 Cas9 (n = 6), H446 Cas9 KDM4A (n = 7) and * = p ≤ 0.05, by one-tailed t-test, unequal variance. H Bar graph representing final tumor volumes in H446 xenografts. H446 KDM4A knockdown xenograft volumes were significantly lower compared to parental and Cas9-only controls. Data are shown as means ± SEM. ** = p ≤ 0.01, * = p < 0.05, by one-tailed t-test, unpaired with Welch’s correction.