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. 2023 Feb 8;8(3):e164324. doi: 10.1172/jci.insight.164324

Figure 3. SOX2 represses KISS1 transcription in immortalized kisspeptin cell lines via direct DNA binding.

Figure 3

(A) Relative mRNA levels of Sox2 in NIH 3T3, KTaR-1, and KTaV-3 cells using real-time quantitative reverse transcription. N = 6–7. hKiss-luc was cotransfected with Sox2 shRNA or a scrambled sequence in an shRNA vector into (B) KTaR-1 or (C) KTaV-3 cells. Luciferase expression in each condition was normalized to the scrambled vector. N = 4–6. (D) Biotin-labeled 30 bp double-stranded oligonucleotides from the indicated regions of the human KISS1 promoter were used in DNA pulldown of protein from NIH 3T3 cells transfected with a hSOX2 expression vector with an HA tag. Following precipitation of DNA/protein complexes with streptavidin magnetic beads, proteins were eluted and analyzed by Western blot with an α-HA antibody. The consensus oligonucleotide contains a 5× multimer of the full SOX2 binding sequence. The scrambled oligonucleotide contains a 5× multimer of a sequence unrelated to SOX2 binding. All conditions were run on same blot and with same exposure. The consensus and scramble lanes were moved to the left side for clarity with the schematic. Representative image from N = 3 biological replicates. Data were analyzed using Student’s t test or 1-way ANOVA with Dunnett’s multiple-comparison post hoc test. Significance indicated by *P < 0.05, ****P < 0.0001.