Figure 7.

Gln259 is essential for OCT4A transformation, transcriptional regulatory activity, and protein stability

(A) Schematic diagram of OCT4A, PG1OCT4A, and PG1 protein mutants with forced expression. All constructs contained a FLAG tag added at the C-terminus.

(B) Western blot detection of OCT4A and PG1 protein mutants expressed in COS7 cells. The graph displays the quantification of Western blot band intensity. Band intensities were normalized against the total protein amount, as visualized by Coomassie Brilliant Blue (CBB) staining of the gel. n = 3. Mean ± SD.

(C) Soft agar colony formation by cells stably expressing OCT4A and its mutants in NIH3T3 cells. Left: mixed stable expression lines; right: results of cloned stable expression for each line. n = 3–5. Mean ± SD. One-way ANOVA. ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, and ∗∗∗∗ = p < 0.0001.

(D) Growth curves for the stable expression of amino acid mutants OCT4A and PG1 in NIH3T3 cells. n = 3. Mean ± SD.

(E) Immunofluorescence and western blotting revealed differences in the protein expression of the OCT4A, PG1, and Gln259 amino acid mutants in HEK293A cells. Scale bars, 100 μm. For western blotting, an acrylamide gel stained with CBB was used as the loading control.

(F) Differences in the transcriptional regulatory activities of OCT4A and PG1 mutants in the dual-luciferase assay in COS7 cells. n = 3. Mean ± SD.

(G) Impact of Gln259 on the predicted protein tertiary structure. We compared the predicted structures of OCT4, PG1, and PG1insQ using AlphaFold2. Upper: Indication of the protein domains and α-helix structures of OCT4A.The arrow indicates the position of Q259. (lower) plots represent the accuracy of the prediction model for each protein. The Q259 position is highlighted in pink.