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

Journal: BMC Oral Health

Article Title: Salvianolic acid B functionalized injectable GelMA hydrogel for pulpitis in a vital pulp therapy: a dual anti-inflammatory property and enhanced reparative dentinogenesis activity material

doi: 10.1186/s12903-026-07880-z

Figure Lengend Snippet: Characterization of hDPSCs. A Light microscopy images of primary hDPSCs on 9th day. B Image of hDPSCs colony forming units at 14 days. C Alizarin red staining of mineralized nodules. D Oil red staining of adipogenic induction. E Alcian bale staining of chondrogenic induction. F Flow cytometric analysis of hDPSCs using multiple markers. hDPSCs were positive for CD73, CD90, and CD105, and negative for CD34 and CD45. G Positive immunofluorescence to nestin and stro-1, and negative to cytokeratin-3 of hDPSCs

Article Snippet: Primary antibodies included rabbit anti-human nestin (Affinity Biosciences, China), mouse anti-human stro−1 (R&D systems, USA) and mouse anti-human cytokeratin (Abcam, USA).

Techniques: Light Microscopy, Staining, Immunofluorescence

Journal: Animals : an Open Access Journal from MDPI

Article Title: Odontoblasts in Equine Hypsodont Teeth—How They Cope with Permanent Occlusal Wear

doi: 10.3390/ani16020341

Figure Lengend Snippet: Antibodies for Western Blot.

Article Snippet: Rabbit anti-human Nestin Clone: aa1295-1344 Cat No.: LS-B5875 , LSBio, Newark, CA, USA , 1:200 , Equine.

Techniques: Western Blot

Journal: Scientific Reports

Article Title: Impact of LIN7A silencing on U87 cell invasion and its clinical significance in glioblastoma

doi: 10.1038/s41598-025-91285-5

Figure Lengend Snippet: The detailed information of antibodies used in this study.

Article Snippet: Primary antibody , Anti-human nestin (rabbit polyclonal) , IF 1:200 , ZRB69 , MilliporeSigma.

Techniques:

Journal: Stem Cell Research & Therapy

Article Title: Transcriptional landscape of the interaction of human Mesenchymal Stem Cells with Glioblastoma in bioprinted co-cultures

doi: 10.1186/s13287-024-04022-6

Figure Lengend Snippet: Bioprinting of MSC and GBM primary cells: morphological aspects A ) Schematic illustration of bioprinting of scaffold. B ) Bioprinted scaffold with (a) confocal image of tumoroid structure in scaffold and (b) labeling of tumoroid with antibodies directed against GFAP (green), nestin (red) and nuclei (blue). C ) Use of transwell to analyze the migration of MSC toward GBM in the presence of defined medium (DM) or conditioned medium (CM) obtained from 72 h GBM cultures. Images were taken 4 h later. D ) Migration of MSC CAF towards tumor cells in scaffold, beginning of tumoroid formation in scaffolds

Article Snippet: For immunocytochemistry, biospheres were fixed with 4% paraformaldehyde for 1 h then permeabilized with 0.1% Triton X-100 for 30 min saturated with 5% BSA and then incubated with rabbit anti-human nestin (Proteintech, Rosemont, IL, USA) and mouse anti-human anti-GFAP (Proteintech).

Techniques: Labeling, Migration

Journal: Molecular neurobiology

Article Title: Neuronal Stem Cells from Late-Onset Alzheimer Patients Show Altered Regulation of Sirtuin 1 Depending on Apolipoprotein E Indicating Disturbed Stem Cell Plasticity.

doi: 10.1007/s12035-023-03633-z

Figure Lengend Snippet: Fig. 2 Profiling of neuronal markers in iPSC-derived NSCs on 7 d. A NSCs from healthy (reference) WISCi004-B ana- lyzed by immunofluorescence (IF) and flow cytometry. IF analysis showed the localization of neural stem cell (NSC) mark- ers SRY-box transcription factor 2 (SOX2), vimentin (VIM), nes- tin (NES), paired box 6 (PAX6), neurogenin 3 (NEUROG3), Musashi RNA-binding protein 1 (MSI1), and the epitope stage- specific embryonic antigen (SSEA1). Proteins of interest are shown by co-staining in green and red versus DNA staining in blue (scale bar, 100 μm). Flow cytometry histo- grams are shown for neuronal markers SOX2 and PAX6 (each in red; isotype controls in green). B Flow cytometry histograms of neuronal markers in NSCs from WICi004-B (ref- erence), MLUi009-A (matched control), and MLUi008-B (AD) on 7 d proving the induction of NSC markers SOX1 and NES (each in red; isotype controls in green). Representative histograms show positive counts versus fluorescence intensity. The percentage of positive cells is indicated

Article Snippet: 1 3 Mouse IgG anti-human ACTB WB: 1:40,000 Merck A5441 Mouse IgG anti-human APOE WB: 1:500; IF: 1:500 Bio-Techne NB110-60531 Mouse IgG anti-human ATG7 WB: 1:1000; IF: 1:100 Bio-Techne MAB6608 Mouse IgG anti-human COL1A1 IF: 1:100 Merck C2456 Rabbit IgG anti-human CDH1 IF: 1:100 Abcam Ab40772 Mouse IgG anti-human FGF2 WB: 1:100 Santa Cruz sc-136255 Mouse IgG anti-human FGF2 IF: 1:100 Bio-Techne NBP1-47749 Rabbit IgG anti-human MSI1 IF: 1:100 Merck AB5977 Mouse IgG anti-human NES IF: 1:100 Santa Cruz Sc-23927 Rabbit IgG anti-human NEUROG3 IF: 1:100 Abcam Ab38548 Mouse IgG anti-human OCT4 (POU5F1) IF: 1:100 Santa Cruz Sc-5279 Mouse IgG anti-human PAX6 IF: 1:100 Santa Cruz Sc-53108 Goat IgG anti-human PRRX1 IF: 1:100 Bio-Techne NBP1-06067 Rabbit IgG anti-human p21m (CDKN1A) WB: 1:1000; IF: 1:800 Cell Signaling Technology 2947 Mouse IgG anti-human p16 (CDKN2A) IF: 1:100 Origene TA500036 Mouse IgM anti-human PTEN WB: 1:100; IF: 1:200 Thermo Fisher Scientific MA5-12278 Mouse IgG anti-human SIRT1 WB: 1:1000; IF: 1:1000 Bio-Techne NBP1-51641 Goat IgG anti-human SOX2 IF: 1:100 Santa Cruz Sc17320 Mouse IgG anti-human SOX17 IF: 1:100 Santa Cruz Sc-130295 Mouse IgM anti-human SSEA1 IF: 1:100 Santa Cruz Sc-21702 Mouse IgG anti-human STAT3 WB: 1:5000; IF: 1:100 Thermo Fisher Scientific MA1-13042 Mouse IgG anti-human VIM IF: 1:100 Merck MAB3400 Goat IgG anti-mouse HRP WB: 1:10,000 Dianova 115-035-003 Goat IgG anti-rabbit HRP WB: 1:3000 Cell Signaling Technology 7074 Goat IgM anti-mouse HRP WB: 1:4000 Thermo Fisher Scientific 62-6820 Donkey IgG anti-goat Alexa FluorTM 488 IF: 1:400 Thermo Fisher Scientific A11055 Goat IgG anti-mouse Alexa FluorTM 488 IF: 1:400 Thermo Fisher Scientific A11001 1 3

Techniques: Derivative Assay, Immunofluorescence, Flow Cytometry, RNA Binding Assay, Staining, Control, Fluorescence