Journal: Journal of Biological Chemistry

Article Title: Taste of a Pill

doi: 10.1074/jbc.m114.570564

Figure Lengend Snippet: FIGURE 4. Localization of OCT3/Oct3 in salivary gland epithelial cells. A, detection of OCT3 (panel i, green) in human submandibular glands. Staining of Na/K-ATPase (panel ii), a basolateral marker, and nuclei (panel iii) is shown in red and blue, respectively. B, detection of Oct3 (panel i, green) and nuclei (panel ii, blue) in salivary gland sections from Oct3/ (upper panels) and Oct3/ (lower panels) mice. In overlays (A, panel iv, and B, panel iii), the arrow and arrowhead indicate basolateral and apical membranes of salivary gland epithelial cells, respectively.

Article Snippet: The sections were blocked in goat serum in PBS, incu- bated overnight at 4 °C with rabbit anti-OCT3 polyclonal antibody (1:125 dilution; Genway), and co-labeled with anti-human Na /K -ATPase -subunit monoclonal antibody (1:500 dilution; Sigma).

Techniques: Staining, Marker

Journal: Journal of Biological Chemistry

Article Title: Taste of a Pill

doi: 10.1074/jbc.m114.570564

Figure Lengend Snippet: FIGURE 7. Model proposed for OCT3-mediated organic cation (OC) transport in salivary gland epithelial cells. OCT3 on the basolateral membrane of epithelial cells mediates metformin uptake from the blood into the cells. Once metformin is highly concentrated inside the cells, OCT3 on the apical membrane facilitateseffluxofmetforminintothesaliva.Thesolidarrowsindicatethepreferreddirectionofmetformintransportwhendrugconcentrationsinthesystemic circulation are high. The dashed arrows indicate the possible transport direction when systemic drug concentrations decline.

Article Snippet: The sections were blocked in goat serum in PBS, incu- bated overnight at 4 °C with rabbit anti-OCT3 polyclonal antibody (1:125 dilution; Genway), and co-labeled with anti-human Na /K -ATPase -subunit monoclonal antibody (1:500 dilution; Sigma).

Techniques: Membrane

Journal: Molecular Medicine Reports

Article Title: Knockdown of OCT4 suppresses the growth and invasion of pancreatic cancer cells through inhibition of the AKT pathway

doi: 10.3892/mmr.2014.2367

Figure Lengend Snippet: Expression of OCT4 protein in pancreatic cancer tissues (magnification, ×200). Pancreatic cancer tissues and ANCT were immunohistochemically stained with an anti-OCT4 antibody and classified as (−) and (+). (A) Positive expression in pancreatic cancer. (B) Negative expression in pancreatic cancer. (C) Positive expression in ANCT. (D) Negative expression in ANCT. Positive immunostaining of OCT4 was mainly localized in the nucleus of the tumor and ANCT cells. OCT4, octamer-binding transcription factor 4; ANCT, adjacent non-cancer tissues.

Article Snippet: Normal serum or phosphate-buffered saline (PBS; Wuhan Boster Biological Engineering Co., Ltd.) was used to replace anti-OCT4 antibody in the negative controls.

Techniques: Expressing, Staining, Immunostaining, Binding Assay

Journal: Molecular Medicine Reports

Article Title: Knockdown of OCT4 suppresses the growth and invasion of pancreatic cancer cells through inhibition of the AKT pathway

doi: 10.3892/mmr.2014.2367

Figure Lengend Snippet: Expression of OCT4 protein in pancreatic cancer tissues.

Article Snippet: Normal serum or phosphate-buffered saline (PBS; Wuhan Boster Biological Engineering Co., Ltd.) was used to replace anti-OCT4 antibody in the negative controls.

Techniques: Expressing

Journal: Molecular Medicine Reports

Article Title: Knockdown of OCT4 suppresses the growth and invasion of pancreatic cancer cells through inhibition of the AKT pathway

doi: 10.3892/mmr.2014.2367

Figure Lengend Snippet: Expression of OCT4 in pancreatic cancer cells with different degrees of differentiation. The expression of OCT4 in pancreatic cancer cells with different degrees of differentiation (Bxpc3, Panc-1 and Mia PaCa-2) was examined by (A) real-time PCR and (B and C) western blot assays, of which OCT4 was highly expressed in the Panc-1 cell line compared with the other two cell lines (P<0.01). OCT4, octamer binding transcription factor 4.

Article Snippet: Normal serum or phosphate-buffered saline (PBS; Wuhan Boster Biological Engineering Co., Ltd.) was used to replace anti-OCT4 antibody in the negative controls.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Binding Assay

Journal: Molecular Medicine Reports

Article Title: Knockdown of OCT4 suppresses the growth and invasion of pancreatic cancer cells through inhibition of the AKT pathway

doi: 10.3892/mmr.2014.2367

Figure Lengend Snippet: Correlation of OCT4 expression with the clinicopathological characteristics of patients with pancreatic cancer.

Article Snippet: Normal serum or phosphate-buffered saline (PBS; Wuhan Boster Biological Engineering Co., Ltd.) was used to replace anti-OCT4 antibody in the negative controls.

Techniques: Expressing

Journal: Molecular Medicine Reports

Article Title: Knockdown of OCT4 suppresses the growth and invasion of pancreatic cancer cells through inhibition of the AKT pathway

doi: 10.3892/mmr.2014.2367

Figure Lengend Snippet: Effect of OCT4 knockdown on the expression of AKT in pancreatic cancer cells. After pancreatic cancer cells were transfected with the Lv-shOCT4 for 24 h, the expression levels of OCT4 and AKT were detected by (A and B) real-time PCR and (C–F) western blot analysis. The expression of OCT4 and AKT was significantly decreased in the Lv-shOCT4 group compared with the CON and NC groups ( ** P<0.01). OCT4, octamer binding transcription factor 4; Lv-shOCT4, lentivirus-mediated OCT4 shRNA vector; CON, control vector; NC, negative control vector.

Article Snippet: Normal serum or phosphate-buffered saline (PBS; Wuhan Boster Biological Engineering Co., Ltd.) was used to replace anti-OCT4 antibody in the negative controls.

Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Binding Assay, shRNA, Plasmid Preparation, Negative Control

Journal: Molecular Medicine Reports

Article Title: Knockdown of OCT4 suppresses the growth and invasion of pancreatic cancer cells through inhibition of the AKT pathway

doi: 10.3892/mmr.2014.2367

Figure Lengend Snippet: Effect of OCT4 knockdown on cell proliferation. (A) Cell proliferative activity, indicated by MTT assay, markedly decreased in a time-dependent manner in the Lv-shOCT4 group compared with the CON and NC groups ( ** P<0.01). (B and C) Endogenous expression of PCNA, indicated by western blot analysis, was significantly decreased in the Lv-shOCT4 group compared with the NC and CON groups ( ** P<0.01). OCT4, octamer binding transcription factor 4; Lv-shOCT4, lentivirus-mediated OCT4 shRNA vector; PCNA, proliferating cell nuclear antigen; CON, control vector; NC, negative control vector.

Article Snippet: Normal serum or phosphate-buffered saline (PBS; Wuhan Boster Biological Engineering Co., Ltd.) was used to replace anti-OCT4 antibody in the negative controls.

Techniques: Activity Assay, MTT Assay, Expressing, Western Blot, Binding Assay, shRNA, Plasmid Preparation, Negative Control

Journal: Molecular Medicine Reports

Article Title: Knockdown of OCT4 suppresses the growth and invasion of pancreatic cancer cells through inhibition of the AKT pathway

doi: 10.3892/mmr.2014.2367

Figure Lengend Snippet: Effect of OCT4 knockdown on cell invasion (magnification, ×200). (A and B) Cell invasive potential, indicated by Transwell assay, was markedly weakened in the Lv-shOCT4 group compared with the CON and NC groups ( ** P<0.01). (C and D) Endogenous expression of MMP-2, indicated by western blot analysis, was significantly decreased in the Lv-shOCT4 group compared with the NC and CON groups ( ** P<0.01). OCT4, octamer binding transcription factor 4; Lv-shOCT4, lentivirus-mediated OCT4 shRNA vector; CON, control vector; NC, negative control vector; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MMP-2, matrix metalloproteinase-2.

Article Snippet: Normal serum or phosphate-buffered saline (PBS; Wuhan Boster Biological Engineering Co., Ltd.) was used to replace anti-OCT4 antibody in the negative controls.

Techniques: Transwell Assay, Expressing, Western Blot, Binding Assay, shRNA, Plasmid Preparation, Negative Control

Journal: Nature Communications

Article Title: Imprinting fidelity in mouse iPSCs depends on sex of donor cell and medium formulation

doi: 10.1038/s41467-022-33013-5

Figure Lengend Snippet: A Schematic representation of the reprogramming protocol; briefly, a transgenic “ reprogrammable ” female mouse on a C57BL/6J genetic background (i4F-BL6) was crossed with a Mus musculus castaneus (CAST) male mouse to generate E13.5 F1 hybrid embryos from which mouse embryonic fibroblasts (MEFs) were obtained. MEFs were reprogrammed by induction of the polycistronic Yamanaka cassette ( Oct4 / Sox2 / Klf4 / c-Myc - OSKM) in the presence of doxycycline (DOX) for 12 days. Individual clones of mouse induced pluripotent stem cells reprogrammed in Knockout Serum Replacement medium (KSR-iPSCs) were picked on day 12 and expanded until approximately day 50. B Clustering analysis of the normalised RNAseq counts for all the biological triplicates of female MEFs, female (F KSR2 and F KSR4) and male (M KSR3 and M KSR5) iPSCs and TX 2i ESCs. C Expression analysis by RNAseq of a panel of pluripotent genes in female MEFs, female (F KSR2, F KSR4), male (M KSR3 and M KSR5) iPSCs and TX 2i ESCs. The graph shows the average Log2 Reads Per Kilobase per Million mapped reads (RPKM) expression values ± Standard Deviation (SD) from biological triplicates of each sample. Source data are provided as a Source Data file. D Table and representative H&E staining of teratomas after subcutaneous injection of 2 × 10 6 cells into the flanks of NSG mice. iPSCs efficiently contribute to ectoderm, mesoderm, endoderm and occasionally trophectoderm. i Low magnification of a mature teratoma, scale bar represents 250 µm. ii Trophectoderm-derived trophoblast giant cells (black arrowhead), associated with large vascular spaces (blue arrowhead), characteristic of placental tissue. iii Ectodermal components corresponding to squamous epithelium (black arrowhead). iv Endodermal components corresponding to respiratory-type epithelium, including ciliated (black arrowhead), and mucin-producing goblet cells (blue arrowhead). v, vi, vii , Mesodermal components (black arrowhead) corresponding to muscle, cartilage, and bone, respectively; ii-vii scale bar represents 100 µm. Table summarises the successful generation of teratomas with tissues from the three germ layers from F KSR2, F KSR4, M KSR3 and M KSR5 iPSCs. Two teratomas per cell line were generated and analysed by H&E staining.

Article Snippet: A blocking step was performed by incubation with 1% Bovine Serum Albumin (BSA; Cat# 05470-5 G, Sigma-Aldrich) in PBS for 15 min and subsequently the cells were incubated with primary antibodies for OCT4 (monoclonal clone 7F9.2 m, Cat# MAB4419, Merck Millipore, 1:200 dilution), SSEA-1 (monoclonal clone MC-480, Cat# MAB4301, Merck Millipore, 1:100 dilution) and NANOG (polyclonal, Cat# RCAB002P-F, Reprocell, 1:150 dilution) diluted in 1% BSA/PBS for 45 min. After three washes with PBS, cells were incubated for 45 min with the secondary antibody CyTM3 AffiniPure F(ab’) 2 Fragment Goat Anti-Mouse IgG (H + L) (polyclonal, Cat# 115-166-003, Jackson ImmunoResearch Laboratories Inc., 1:200 dilution).

Techniques: Transgenic Assay, Clone Assay, Knock-Out, Expressing, Standard Deviation, Staining, Injection, Derivative Assay, Generated

Journal: Nature Communications

Article Title: Imprinting fidelity in mouse iPSCs depends on sex of donor cell and medium formulation

doi: 10.1038/s41467-022-33013-5

Figure Lengend Snippet: A Schematic representation of the reprogramming protocol; briefly, a transgenic “ reprogrammable ” female mouse on a C57BL/6J genetic background (i4F-BL6) was crossed with a Mus musculus castaneus (CAST) male mouse to generate E13.5 F1 hybrid embryos from which mouse embryonic fibroblasts (MEFs) were collected. MEFs were reprogrammed by induction of the polycistronic Yamanaka cassette ( Oct4 / Sox2 / Klf4 / c-Myc - OSKM) in the presence of doxycycline (DOX) for 12 days. Individual mouse induced pluripotent stem cells reprogrammed in Foetal Bovine Serum medium (FBS-iPSCs) clones were picked at day 12 and expanded until approximately day 50. B Methylation analysis of Peg3 , Dlk1-Dio3 , Igf2-H19 and PWS/AS ICRs in male and female MEFs (note: same data as in Fig. for MEFs), female (F FBS1-5) and male (M FBS1-5) FBS-iPSCs; Each graph represents the mean percentage ± SD methylation levels measured at each CpG within different genomic regions per parental allele for each sample (number of CpG per locus - Peg3 : n = 24; Dlk1-Dio3 : n = 27; Igf2-H19 : n = 16; PWS/AS: n = 15); Scheme on the bottom of each graph represents the normal methylation status of each ICR in the correspondent regions (white circle – unmethylated ICR; black circle – methylated ICR; Mat – maternal allele; Pat – paternal allele; orange rectangles – expressed genes; grey rectangles – silenced genes; regions are not drawn to scale. Source data are provided as Supplementary Data . C Allelic expression of H19 and Snrpn genes assayed by RT-PCR followed by Sanger sequencing. Chromatograms are shown for female MEFs, F FBS5 and M FBS5 iPSCs; Table summarises the allele-specific expression for all the FBS-iPSCs as well as female and male MEFs, F KSR2 and M KSR3 iPSCs. Schemes on the left of the female MEFs chromatograms represent the normal imprinting profile of both H19 and Snrpn and the associated single nucleotide polymorphism (SNP) for each allele (white circle – unmethylated ICR; black circle – methylated ICR; Mat – maternal allele; Pat – paternal allele; pink rectangle – maternally H19 expressed gene; blue rectangle – paternally Snrpn expressed gene; grey rectangles – silenced genes; regions are not drawn to scale).

Article Snippet: A blocking step was performed by incubation with 1% Bovine Serum Albumin (BSA; Cat# 05470-5 G, Sigma-Aldrich) in PBS for 15 min and subsequently the cells were incubated with primary antibodies for OCT4 (monoclonal clone 7F9.2 m, Cat# MAB4419, Merck Millipore, 1:200 dilution), SSEA-1 (monoclonal clone MC-480, Cat# MAB4301, Merck Millipore, 1:100 dilution) and NANOG (polyclonal, Cat# RCAB002P-F, Reprocell, 1:150 dilution) diluted in 1% BSA/PBS for 45 min. After three washes with PBS, cells were incubated for 45 min with the secondary antibody CyTM3 AffiniPure F(ab’) 2 Fragment Goat Anti-Mouse IgG (H + L) (polyclonal, Cat# 115-166-003, Jackson ImmunoResearch Laboratories Inc., 1:200 dilution).

Techniques: Transgenic Assay, Clone Assay, Methylation, Expressing, Reverse Transcription Polymerase Chain Reaction, Sequencing

Journal: Oncotarget

Article Title: Graded expression of microRNA-371a-3p in tumor tissues, contralateral testes, and in serum of patients with testicular germ cell tumor

doi: 10.18632/oncotarget.27565

Figure Lengend Snippet: ( A ) In situ hybridization with a probe against miR-371a-3p causes blue staining in cells. ( B ) Section from A. ( C ) Immunohistochemical staining of the same area with an OCT4 antibody for identification of EC cells. ( D ) H&E staining of the same area.

Article Snippet: OCT4 staining for identification of embryonal carcinomas (EC), PLAP staining for seminomas (SE) and glypican 3 for yolk-sac tumors (YST) (Diagnostic BioSystems, Pleasanton, CA, USA) were then conducted according to institutional standard operating procedures [ , ].

Techniques: In Situ Hybridization, Staining, Immunohistochemical staining

Journal: Development (Cambridge, England)

Article Title: Genetic mosaics and time-lapse imaging identify functions of histone H3.3 residues in mouse oocytes and embryos

doi: 10.1242/dev.141390

Figure Lengend Snippet: H3.3K27 regulates the first cell lineage specification. (A) To generate mosaic blastocysts, HISRainbow male mice were mated with CreER female mice to obtain 1C zygotes. Early 4C embryos were collected and treated with 0.2 µM 4-hydroxytamoxifen for 12 h. This led to nuclear import of the Cre enzyme and genetic recombination within the HISRainbow construct. Embryos were further cultured to early blastocysts for examination. (B) Representative confocal (full projection) images of mosaic blastocysts with individual blastomeres expressing either H3.3eGFP or H3.3K27RmCherry. DNA was stained with DRAQ5. Scale bar: 10 µm. (C) Representative confocal (full projection) images of mosaic blastocysts stained with anti-CDX2 antibody. Scale bar: 10 µm. (D) Quantification of H3.3eGFP- and H3.3K27RmCherry-expressing blastomeres in the inner cells of early blastocysts. Mean±s.e.m. of 30 blastocysts; **P<0.01. (E) Immunofluorescence with anti-H3K27me1 antibody of cells expressing WT H3.3 (n=11) and H3.3K27R (n=8). Mean±s.e.m.; **P<0.01. (F) Immunofluorescence with anti-OCT4 of cells expressing WT H3.3 (n=16) and H3.3K27R (n=8). Arrows indicate co-expression of OCT4 in H3.3- and H3.3K27R-expressing cells. Mean±s.e.m.; *P<0.05. Scale bar: 20 µm.

Article Snippet: The following primary antibodies were used for immunofluorescence: H3K27me1 (Active Motif, 39377, 1:100), H3K27me3 (Abcam, ab6147, 1:100), H3R26me1 (Abcam, ab130898, 1:100), OCT4 (Active Motif, 39811, 1:100), CDX2 (BioGenex, Clone CDX2-88, 1:200) and laminB (Santa Cruz, sc-6217, 1:150).

Techniques: Construct, Cell Culture, Expressing, Staining, Immunofluorescence

Journal: Oncology Letters

Article Title: S100A16 up-regulates Oct4 and Nanog expression in cancer stem-like cells of Yumoto human cervical carcinoma cells

doi: 10.3892/ol.2018.8568

Figure Lengend Snippet: The expression levels of Oct4, Nanog, and S100A16 in the sphere formation of Yumoto cells. The cells were cultured under the non-adherence and serum-free culture conditions of the sphere formation assay for 3 days. (A) Total RNA was extracted, and the expressions of Oct4, Nanog, and S100A16 were evaluated by reverse transcription-polymerase chain reaction. (B) The cell lysates were prepared from these cells, and the expressions of Oct4, Nanog, and S100A16 were detected by immunoblotting using antibodies against Oct4, Nanog, and S100A16. Oct4, octamer-binding transcription factor 4; Nanog, homeobox protein NANOG.

Article Snippet: The membranes were washed in Tris-buffered saline with Tween 20 (TBS-T, composed of 10 mM Tris-HCl, pH 8, 150 mM NaCl, and 0.05% Tween 20), blocked for 1 h at room temperature with 5% nonfat milk in TBS-T, and probed overnight at 4°C with anti-S100A16 (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), anti-Oct4 (Bioworld Technology, Inc., St. Louis Park, MN, USA), anti-Nanog (Abcam, Cambridge, UK), anti-p53 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and anti-GAPDH (Cell Signaling Technology, Inc., Danvers, MA, USA) antibodies.

Techniques: Expressing, Cell Culture, Tube Formation Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Binding Assay

Journal: Oncology Letters

Article Title: S100A16 up-regulates Oct4 and Nanog expression in cancer stem-like cells of Yumoto human cervical carcinoma cells

doi: 10.3892/ol.2018.8568

Figure Lengend Snippet: Effect of S100A16-targeting siRNA on the expression of Oct4, Nanog, and p53 in the sphere formation of Yumoto cells. The cells were transfected with S100A16-targeting or control siRNA. After these cells were incubated for 24 h, these cells were cultured under the conditions of the sphere formation assay for 3 days. (A) Total RNA was extracted, and the expression levels of Oct4, Nanog, p53, and S100A16 were evaluated by reverse transcription-polymerase chain reaction. (B) The cell lysates were prepared from these cells, and the expression levels of Oct4 Nanog, p53, and S100A16 were detected by immunoblotting using antibodies against Oct4, Nanog, p53, and S100A16. Oct4, octamer-binding transcription factor 4; Nanog, homeobox protein NANOG; siRNA, short interfering RNA.

Article Snippet: The membranes were washed in Tris-buffered saline with Tween 20 (TBS-T, composed of 10 mM Tris-HCl, pH 8, 150 mM NaCl, and 0.05% Tween 20), blocked for 1 h at room temperature with 5% nonfat milk in TBS-T, and probed overnight at 4°C with anti-S100A16 (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), anti-Oct4 (Bioworld Technology, Inc., St. Louis Park, MN, USA), anti-Nanog (Abcam, Cambridge, UK), anti-p53 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and anti-GAPDH (Cell Signaling Technology, Inc., Danvers, MA, USA) antibodies.

Techniques: Expressing, Transfection, Incubation, Cell Culture, Tube Formation Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Binding Assay, Small Interfering RNA

Journal: Oncology Letters

Article Title: S100A16 up-regulates Oct4 and Nanog expression in cancer stem-like cells of Yumoto human cervical carcinoma cells

doi: 10.3892/ol.2018.8568

Figure Lengend Snippet: (A) Effect of proteasome inhibition on the expressions of Oct4, Nanog, p53, and S100A16 in the sphere formation of Yumoto cells. After the cells were cultured under the conditions of the sphere formation assay for 3 days, these cells were treated with proteasome inhibitor lactacystin (10 µM) under the conditions of the sphere formation assay for 24 h. The cell lysates were prepared from these cells, and the expression levels of Oct4, Nanog, p53, and S100A16 were detected by immunoblotting using antibodies against Oct4, Nanog, p53, and S100A16. (B) Potential schematic diagram of the effect of S100A16 on the expression of Oct4 and Nanog expression in CSCs of Yumoto human cervical carcinoma cells. Oct4, octamer-binding transcription factor 4; Nanog, homeobox protein NANOG.

Article Snippet: The membranes were washed in Tris-buffered saline with Tween 20 (TBS-T, composed of 10 mM Tris-HCl, pH 8, 150 mM NaCl, and 0.05% Tween 20), blocked for 1 h at room temperature with 5% nonfat milk in TBS-T, and probed overnight at 4°C with anti-S100A16 (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), anti-Oct4 (Bioworld Technology, Inc., St. Louis Park, MN, USA), anti-Nanog (Abcam, Cambridge, UK), anti-p53 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and anti-GAPDH (Cell Signaling Technology, Inc., Danvers, MA, USA) antibodies.

Techniques: Inhibition, Cell Culture, Tube Formation Assay, Expressing, Western Blot, Binding Assay