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. 2019 Jul 26;218(9):2896–2918. doi: 10.1083/jcb.201903125

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© 2019 Palmer et al.

This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).

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Figure 6. — NRF1 target mRNA expression is increased upon Cdk2 deletion. (A) qPCR analysis of spermatocyte cDNA isolated from corn oil treated (black triangles) and tamoxifen treated (white circles) Cdk2-iKO mice—referred to as noninduced and induced Cdk2-iKO, respectively. Expression is displayed as fold change normalized to the expression of the eEF2 housekeeping gene. STA-PUT spermatocyte isolation (as described in Materials and methods) for this experiment was performed at P68, 12 d after corn oil/tamoxifen treatment (started at P56). 17/25 NRF1 target genes showed significant increases in expression. Three biological replicates were tested for each group for each gene. Gene expression of each biological replicate was assumed to be normally distributed, but this was not formally tested. Error bars are representative of the SD of normalized fold change values from at least three biological replicates as compared to noninduced Cdk2-iKO controls. Significance was determined via unpaired t test (****, P ≤ 0.0001; ***, P < 0.001; **, P < 0.005; *, P < 0.05). (B) Western blotting of NRF1, EHMT1, and H3K9me2 in corn oil–treated Cdk2-iKO (lanes 1–3) or tamoxifen-treated Cdk2-iKO (lanes 4–6) whole testis lysates. For Cdk2-iKO animals, testis isolation for this experiment was performed at P68, 12 d after corn oil/tamoxifen treatment (started at P56). γ-Tubulin and pan-histone H3 (PAN H3) are shown as loading controls.