Journal: iScience
Article Title: Multifaceted role of POU5F1P1 in regulating its parental stem cell gene, POU5F1
doi: 10.1016/j.isci.2026.115137
Figure Lengend Snippet: 5′ UTR sequence of PG1 interacts with the 5′ UTR of OCT4A, leading to translational repression (A) Fluorescence intensity of FLAG immunostaining in NIH3T3 cells transfected with OCT4A-FLAG containing UTR, PG1 with UTR (PG1 full), or its 5′ UTR (PG1 5′ UTR) or 3′ UTR (PG1 3′ UTR) sequences. Transfection efficiency was normalized by dividing the fluorescence intensity of the co-transfected pRFP by n = 3. Mean ± SD. One-way ANOVA. ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, and ∗∗∗∗ = p < 0.0001. (B) Immunostaining with a specific OCT4 antibody (C-10; sc5279) was performed when full-length PG1 (PG1 full) and PG1 5′ UTR sequence plasmids were transfected into PA1 cells. To verify the transfected cells, pRFP was co-transfected, and the fluorescence signal intensity of OCT4A immunostaining was quantified in RFP-positive cells. Scale bars, 100 μm. n = 3. Mean ± SD. One-way ANOVA. ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, and ∗∗∗∗ = p < 0.0001. (C) Immunostaining with a specific OCT4A antibody (C-10; sc5279) when the RNA of the PG1 5′ UTR or 3′ UTR sequence was transfected into PA1 cells. n = 10 (calculated views). Mean ± SD. One-way ANOVA. ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, and ∗∗∗∗ = p < 0.0001. Similar results were obtained in three independent experiments. (D) Quantity of OCT4A mRNA when the UTR sequence of PG1 was transfected into PA1 cells. Relative mRNA levels were measured using real-time PCR with specific primers ( D and S1E). Data are presented as relative expression levels normalized to HPRT1, with HPRT1 set to 1. n = 3. Mean ± SD. One-way ANOVA. ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, and ∗∗∗∗ = p < 0.0001; ns = not significant. (E) Western blot analysis of OCT4A protein levels following the transfection of PG1 5′ UTR RNA into PA1 cells. PA1 cells were transfected with the PG1 5′ UTR sequence RNA at two concentrations (1: 5 nM and 2: 10 nM). Protein expression was detected using an OCT4A-specific antibody (C-10; sc5279). The graph on the right displays the quantification of Western blot band intensity from three independent experiments. Band intensities were normalized against the total protein amount visualized by Coomassie Brilliant Blue (CBB) staining of the gel, which served as a loading control. n = 3. Mean ± S.D. One-way ANOVA. ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, and ∗∗∗∗ = p < 0.0001. (F and G) ALP staining images (E) and nestin immunostaining (F) after the introduction of PG1 5′ UTR RNA into PA1 cells. Scale bars, 50 μm. (H) Fluorescence intensity of FLAG immunostaining following forced expression of full-length OCT4A-FLAG and full-length PG1, mutants with the 5′ UTR shortened by 60 nt were transfected into NIH3T3 cells. n = 3. Mean ± SD. (I) Relative positive rate of FLAG immunostaining following forced expression of full-length PG1 and full-length OCT4A-FLAG, 5′ or 3′ UTR deleted OCT4A-FLAG, transfected into NIH3T3 cells. n = 5. Mean ± SD. Unpaired two-tailed Student’s t test. ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, and ∗∗∗∗ = p < 0.0001. (J) Fluorescence intensity of FLAG immunostaining following forced expression of full-length PG1 and full-length OCT4A, and OCT4A mutants with mutations in their 5′ UTR sequence, were transfected into NIH3T3 cells. Upper: Comparison of the sequence information of the 5′ UTRs of OCT4A and PG1, where the orientation of PG1 is complementary to that of OCT4A. The common nucleotide positions between OCT4A and PG1 are marked with an “∗.” The bottom row shows the sequences in which the common nucleotides in OCT4A were substituted with different bases. Left: Fluorescent images of FLAG immunostaining and co-transfection with RFP in both wild-type and mutant cells. Scale bars, 50 μm. Right: Relative positivity rate of FLAG-positive cells in mutant cells transfected with the vector or the full-length PG1 plasmid. The results for the wild type are shown in H n = 5. 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. (K) Fluorescence intensity of FLAG immunostaining following the forced expression of OCT4A wild-type and PG1 5′ UTR or its variants with different 5′ UTR lengths in the complementary region to OCT4A, transfected into NIH3T3 cells. n = 5. Mean ± SD. One-way ANOVA. ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, and ∗∗∗∗ = p < 0.0001. (L) Fluorescence intensity per cell stained with the antibody C-10 against OCT4A. PA1 cells were transfected with three different amounts of synthesized RNA, each containing an equal number of RNA molecules for each type of synthesized RNA (Control RNA, PG1 5′ UTR wild, and PG1-5′ UTR -119 nt). Normalization was performed by dividing the number of DAPI-stained nuclei to account for differences in the number of cells per well. n = 3. Mean ± SD. One-way ANOVA. ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, and ∗∗∗∗ = p < 0.0001. Plasmid vectors were introduced as mock controls in (A, H–K), and control RNA was used in (C–G, and L).
Article Snippet: Mouse monoclonal anti-FLAG antibody (1/500; clone M2; #F1804, Sigma), mouse monoclonal anti-OCT3/4 antibody (1/100; C-10; #sc-5279, Santa Cruz), and rabbit anti-human Nestin antibody (1/100; N1602; #18741, Immuno-Biological Laboratories) were used as primary antibodies.
Techniques: Sequencing, Fluorescence, Immunostaining, Transfection, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Staining, Control, Two Tailed Test, Comparison, Cotransfection, Mutagenesis, Plasmid Preparation, Synthesized
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![McERV-PTLVs specifically transduce cells of brain tumors induced by GSCs injection, leaving normal brain tissues untouched (A) Mice with intracranial tumor induced by GSCs were transduced with concentrated McERV-PTLVs encoding marker EGFP. Seven days after transduction, the brain was isolated, and first cut in coronal section by the injection holes was performed. (B) Fluorescence microscopy images representing the area of the first cut demonstrate the distribution of transduced cells expressing marker EGFP. Magnified images with signed white scale bar provided. White doted lines represent the EGFP-positive area. The small image at the left upper corner of the first picture is the coronal slice stained immunohistochemically with <t>anti-nestin</t> antibodies representing the tumor engraftment. (C) Control group of mice was injected with 1×PBS instead of GSCs and then with McERV-PTLVs. The brain of control mice was analyzed as written above. (D) Fluorescence microscopy images of the area of the first cut represent the absence of EGFP-positive cells. The scheme of the coronal section of caudate-putamen area, where injection holes were performed (marked with red-dotted square). The small image left from the first picture is the coronal slice stained immunohistochemically with anti-nestin antibodies representing the absence of tumor. (E) Mice with intracranial tumor induced by GSCs were transduced with concentrated VSVG-PTLVs encoding marker EGFP. Black-dotted arrow below represents the timescale. (F) The fluorescence microscopy images represent the more disseminated areas of EGFP-positive cells provided on several magnifications (pointed by white arrows). The small image at the left bottom corner of the first picture is the coronal slice stained immunohistochemically, with anti-nestin antibodies representing the tumor engraftment. (G) Images of hematoxylin/eosin staining (right column) <t>and</t> <t>immunohistochemical</t> staining of slices with anti-(human) nestin antibody. Black dotted line represents the area of tumor growth. The upper row samples obtained from mice injected with GSCs and McERV-PTLVs; middle row:1×PBS and McERV-PTLVs; bottom row: GSCs and VSVG-PTLVs. Black scale bars, 100 μm. (H) Fluorescence microscopy images of IF-stained slices obtained from mice with GSC-induced tumor and injected with McERV-PTLVs. DAPI: nuclei staining (blue); anti-(human) nestin mAb stained with Alexa 555 (Red) fluor-conjugated secondary Ab, EGFP: marker fluorescent protein indicates the cells transduced with McERV-PTLVs (the merged image of EGFP [green] and anti-nestin [red] pictures are represented). The white dotted line represents the nestin-positive area of tumor growth. (I) Fluorescence microscopy images of IF-stained slices obtained from mice of control group injected with 1×PBS and McERV PTLVs, representing the absence of nestin and EGFP-positive cells. (J) Fluorescence microscopy images of IF-stained slices obtained from mice with GSC-induced tumor and injected with VSVG-PTLVs. White scale bars on H, I, and J: 100 μm.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_1216/pmc13011216/pmc13011216__gr6.jpg)
