Figure 5.

PG1 proteins are easily degraded and do not have tumorigenic or proliferative activity, unlike parent OCT4A

(A) Tumorigenic activity of NIH3T3 cells was evaluated using a soft agar colony formation assay. n = 3. Mean ± SD. One-way ANOVA. ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, and ∗∗∗∗ = p < 0.0001. Scale bars, 50 μm.

(B) The intracellular localization of C-terminal FLAG-tagged PG1 and OCT4A was confirmed by immunostaining with an anti-FLAG antibody following forced expression of these proteins in HEK293A, HEK293T, MCF7, Ishikawa, or HCT116 cells. Scale bars, 50 μm.

(C) Growth curves of stable mix lines stably expressing OCT4A or PG1 (and EGFP control) in HCT116 colon cancer cells. n = 3. Mean ± SD.

(D) Immune positivity rates following forced expression of OCT4A and PG1 proteins with FLAG tags compared to RFP control. Left: Fluorescent antibody staining of FLAG and RFP in normal human and mouse cells (HEK293A, NIH3T3, IMR-90) with forced expression. Right: Relative percentage of FLAG-positive cells normalized to RFP transfection efficiency. n = 3. Mean ± SD.

(E) Western blot analysis of OCT4A and PG1 expression in COS7 and HEK293A cells using anti-FLAG antibodies. An acrylamide gel stained with Coomassie Brilliant Blue (CBB) was used as the loading control.

(F) Fluorescence intensity of cells expressing EGFP-fusion proteins. Left: Fluorescence images at 12, 24, and 36 h after HEK293A cell transfection. Right: Time-course analysis of fluorescence intensity using time-lapse microscopy.

(G) Detection of EGFP and FLAG-tagged OCT4A and PG1 by the P2A-based polycistronic expression Left: Fluorescent antibody staining of FLAG and EGFP fluorescence images in HEK293A cells with forced expression. Middle: Average EGFP fluorescence intensity. Sixteen fields of view were captured per well, and the mean fluorescence intensity was determined. Right: Relative percentage of FLAG-positive cells normalized to EGFP transfection efficiency. n = 3. Mean ± SD. Unpaired two-tailed Student’s t test. ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, and ∗∗∗∗ = p < 0.0001; ns = not significant. Scale bars, 50 μm.

(H) Half-lives of OCT4A and PG1 proteins evaluated by western blotting in COS7 cells. CHX: cycloheximide (minutes after cycloheximide treatment). The acrylamide gel stained with CBB is shown under the panel as a loading control. n = 3. Mean ± SD.

(I) Effects of the proteasome inhibitor MG132 and autophagy inhibitor bafilomycin A1 on PG1 stability in COS7 cells. Left: Immunostaining using FLAG-tagged fluorescent antibodies. RFP indicated that transfection was performed uniformly. Scale bars, 50 μm. Middle: Proportion of FLAG-positive cells among the transfected cells. n = 3. Mean ± SD. One-way ANOVA. ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, and ∗∗∗∗ = p < 0.0001. Right: Quantification of PG1-FLAG protein levels by western blotting after varying the drug dose and in the presence or absence of treatment. An acrylamide gel stained with CBB was used as the loading control.