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: 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: 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: Cell Death Discovery

Article Title: Nucleosome remodeling and deacetylation complex and MBD3 influence mouse embryonic stem cell naïve pluripotency under inhibition of protein kinase C

doi: 10.1038/s41420-022-01131-0

Figure Lengend Snippet: A qPCR showed that mRNA levels of MBD3 decreased after PKCi-derived mES at passage 5 were transfected with shMBD3, with PKCi and shNC groups used as controls. B MBD3 and β-actin protein levels were evaluated by Western blot in PKCi-derived mES with MBD3 knockdown (upper panel). Quantitative density analysis showed that shMBD3 decreased protein levels of MBD3 (lower panel). C qPCR showed that compared with the control groups, shMBD3 increased the mRNA levels of pluripotency genes NANOG and OCT4 (left panel) but did not affect the mRNA levels of differentiation genes (right panel). D Western blot detection of NANOG, OCT4, and β-actin protein expression in PKCi-derived mES with MBD3 knockdown (left panel). Quantitative density analysis showed that shMBD3 increased protein levels of NANOG and OCT4 (right panel). E Knockdown of MBD3 did not affect the morphology or AP staining of PKCi-derived mES. Scale bar, 200 μm (upper panel). Knockdown of MBD3 did not affect the total number of AP-positive colonies (left panel) but increased the percentage of mixed colonies and decreased the percentage of differentiated colonies (right panel). Data were shown as mean ± SD ( n = 3). The letters a and b indicated significant differences among groups ( P < 0.05).

Article Snippet: Membranes were blocked with 5% non-fat powdered milk (A600669, Sangon Biotech, China) for 1 h, after which membranes were washed with tris-buffered saline containingTween 20 (TBST; 9005-64-5, Sangon Biotech) and incubated with anti-p-β-catenin (1:1000, 9561T, Cell Signaling Technology, CST, USA), anti-β-catenin (1:1000, A19657, ABclonal, China), anti-p-ERK (1:1000, 3510, CST), anti-ERK (1:1000, 4695, CST), anti-p-AKT (1:1000, AP1214, ABclonal), anti-AKT (1:1000, A17909, ABclonal), anti-HDAC5 (A0632, ABclonal), anti-HDAC1 (1:1000, A0238, ABclonal), anti-HDAC2 (1:1000, A2084, ABclonal), anti-MTA1 (1:1000, A16085, ABclonal), anti-MBD3 (1:1000, A2251, Lot 0046180101, ABclonal), anti-CHD3 (1:1000, A2221, ABclonal), anti-RbAP46 (1:1000, A6967, ABclonal), anti-RbAP48 (1:1000, A13934, ABclonal), anti-NANOG (1:1000, Lannuo Biotechnology, China), anti-OCT4 (1:1000, Lannuo Biotechnology), anti-REX1 (1:1000, Lannuo Biotechnology), anti-FGF5 (1:1000, ab88118, Abcam), or anti-cTnT (1:1000, A4914, ABclonal) antibodies overnight at 4 °C.

Techniques: Derivative Assay, Transfection, Western Blot, Expressing, Staining

Journal: Cell Death Discovery

Article Title: Nucleosome remodeling and deacetylation complex and MBD3 influence mouse embryonic stem cell naïve pluripotency under inhibition of protein kinase C

doi: 10.1038/s41420-022-01131-0

Figure Lengend Snippet: A qPCR showed that mRNA levels of MBD3 increased after PKCi-derived mES at passage 5 were transfected with FUW-MBD3, with the PKCi and FUW-M2rtTA plasmid groups used as controls. B MBD3 and β-actin protein levels were evaluated by Western blot after PKCi-derived mES were transfected with FUW-MBD3 (upper panel). Quantitative density analysis showed that protein levels of MBD3 increased after transfection with FUW-MBD3 (lower panel). C qPCR showed decreased mRNA levels of pluripotency genes NANOG and OCT4 and naïve-state marker REX1 , but increased mRNA levels of primed-state marker FGF5 after MBD3 was overexpressed (left panel). MBD3 overexpression also increased mRNA levels of the endoderm marker CK8 , mesoderm markers cTnT , BMP4 , and DESMIN , and ectoderm markers PAX6 and SOX17 (right panel). D Western blot detection of NANOG, OCT4, REX1, FGF5, cTnT, and β-actin protein expression after MBD3 overexpression (left panel). Quantitative density analysis showed that MBD3 overexpression decreased protein levels of NANOG, OCT4, and REX1, but increased protein levels of FGF5 and cTnT (right panel). E MBD3 overexpression induced PKCi-derived mES differentiation and resulted in a loss of AP staining. Scale bar, 200 μm (upper panel). Overexpression of MBD3 reduced the total number of AP-positive colonies (left panel), decreased the percentage of undifferentiated and mixed colonies, and increased the percentage of differentiated colonies (right panel). Data were shown as mean ± SD ( n = 3). The letters a and b indicated significant differences among groups ( P < 0.05).

Article Snippet: Membranes were blocked with 5% non-fat powdered milk (A600669, Sangon Biotech, China) for 1 h, after which membranes were washed with tris-buffered saline containingTween 20 (TBST; 9005-64-5, Sangon Biotech) and incubated with anti-p-β-catenin (1:1000, 9561T, Cell Signaling Technology, CST, USA), anti-β-catenin (1:1000, A19657, ABclonal, China), anti-p-ERK (1:1000, 3510, CST), anti-ERK (1:1000, 4695, CST), anti-p-AKT (1:1000, AP1214, ABclonal), anti-AKT (1:1000, A17909, ABclonal), anti-HDAC5 (A0632, ABclonal), anti-HDAC1 (1:1000, A0238, ABclonal), anti-HDAC2 (1:1000, A2084, ABclonal), anti-MTA1 (1:1000, A16085, ABclonal), anti-MBD3 (1:1000, A2251, Lot 0046180101, ABclonal), anti-CHD3 (1:1000, A2221, ABclonal), anti-RbAP46 (1:1000, A6967, ABclonal), anti-RbAP48 (1:1000, A13934, ABclonal), anti-NANOG (1:1000, Lannuo Biotechnology, China), anti-OCT4 (1:1000, Lannuo Biotechnology), anti-REX1 (1:1000, Lannuo Biotechnology), anti-FGF5 (1:1000, ab88118, Abcam), or anti-cTnT (1:1000, A4914, ABclonal) antibodies overnight at 4 °C.

Techniques: Derivative Assay, Transfection, Plasmid Preparation, Western Blot, Marker, Over Expression, Expressing, Staining

Journal: Cell Death Discovery

Article Title: Nucleosome remodeling and deacetylation complex and MBD3 influence mouse embryonic stem cell naïve pluripotency under inhibition of protein kinase C

doi: 10.1038/s41420-022-01131-0

Figure Lengend Snippet: A MBD3 RNA expression increased in mES at passage 5 when the PKC inhibitor was removed for 48 h and was partially reversed by MBD3 knockdown, with the PKCi and PKCi removal plus shNC transfection groups used as controls. B MBD3 and β-actin protein levels were evaluated by Western blot (upper panel). Quantitative density analysis showed that MBD3 protein levels increased after PKC inhibitor was removed, which was partially reversed by MBD3 knockdown (lower panel). C PKC inhibitor removal decreased mRNA levels of pluripotency genes NANOG , OCT4 , and SOX2 and naïve-state markers KLF4, FGF4, REX1, and NROB1 , whereas MBD3 knockdown partially increased the mRNA levels of pluripotency gene, NANOG, SOX2, KLF4, and NROB1 . PKC inhibitor removal increased mRNA levels of primed-state markers T and FGF5 and the mesoderm gene cTnT , whereas MBD3 knockdown partially decreased mRNA levels of T , FGF5 , and cTnT . D NANOG, KLF4, FGF5, cTnT, and β-actin levels were detected by Western blot (left panel). Quantitative density analysis showed that NANOG and KLF4 protein levels decreased and FGF5 and cTnT protein levels increased after PKC inhibitor removal, whereas NANOG and KLF4 protein levels partially increased, but FGF5 and cTnT levels partially decreased after MBD3 knockdown (right panel). E After PKC inhibitor removal, mES differentiated and did not show AP staining, whereas MBD3 knockdown partially recovered mES morphology and restored AP staining. Scale bar, 200 μm (upper panel). PKC inhibitor removal decreased the total number of AP-positive colonies, which was partially reversed by MBD3 knockdown (left panel). MBD3 knockdown partially reversed the decrease in the percentage of mixed and undifferentiated colonies and the increase in the percentage of differentiated colonies induced by PKC inhibitor removal (right panel). Data were shown as mean ± SD ( n = 3). The letters a, b, and c indicated significant differences among groups ( P < 0.05).

Article Snippet: Membranes were blocked with 5% non-fat powdered milk (A600669, Sangon Biotech, China) for 1 h, after which membranes were washed with tris-buffered saline containingTween 20 (TBST; 9005-64-5, Sangon Biotech) and incubated with anti-p-β-catenin (1:1000, 9561T, Cell Signaling Technology, CST, USA), anti-β-catenin (1:1000, A19657, ABclonal, China), anti-p-ERK (1:1000, 3510, CST), anti-ERK (1:1000, 4695, CST), anti-p-AKT (1:1000, AP1214, ABclonal), anti-AKT (1:1000, A17909, ABclonal), anti-HDAC5 (A0632, ABclonal), anti-HDAC1 (1:1000, A0238, ABclonal), anti-HDAC2 (1:1000, A2084, ABclonal), anti-MTA1 (1:1000, A16085, ABclonal), anti-MBD3 (1:1000, A2251, Lot 0046180101, ABclonal), anti-CHD3 (1:1000, A2221, ABclonal), anti-RbAP46 (1:1000, A6967, ABclonal), anti-RbAP48 (1:1000, A13934, ABclonal), anti-NANOG (1:1000, Lannuo Biotechnology, China), anti-OCT4 (1:1000, Lannuo Biotechnology), anti-REX1 (1:1000, Lannuo Biotechnology), anti-FGF5 (1:1000, ab88118, Abcam), or anti-cTnT (1:1000, A4914, ABclonal) antibodies overnight at 4 °C.

Techniques: RNA Expression, Transfection, Western Blot, Staining

Journal: Cell Death Discovery

Article Title: Nucleosome remodeling and deacetylation complex and MBD3 influence mouse embryonic stem cell naïve pluripotency under inhibition of protein kinase C

doi: 10.1038/s41420-022-01131-0

Figure Lengend Snippet: Sequences of primers used for qRT-PCR.

Article Snippet: Membranes were blocked with 5% non-fat powdered milk (A600669, Sangon Biotech, China) for 1 h, after which membranes were washed with tris-buffered saline containingTween 20 (TBST; 9005-64-5, Sangon Biotech) and incubated with anti-p-β-catenin (1:1000, 9561T, Cell Signaling Technology, CST, USA), anti-β-catenin (1:1000, A19657, ABclonal, China), anti-p-ERK (1:1000, 3510, CST), anti-ERK (1:1000, 4695, CST), anti-p-AKT (1:1000, AP1214, ABclonal), anti-AKT (1:1000, A17909, ABclonal), anti-HDAC5 (A0632, ABclonal), anti-HDAC1 (1:1000, A0238, ABclonal), anti-HDAC2 (1:1000, A2084, ABclonal), anti-MTA1 (1:1000, A16085, ABclonal), anti-MBD3 (1:1000, A2251, Lot 0046180101, ABclonal), anti-CHD3 (1:1000, A2221, ABclonal), anti-RbAP46 (1:1000, A6967, ABclonal), anti-RbAP48 (1:1000, A13934, ABclonal), anti-NANOG (1:1000, Lannuo Biotechnology, China), anti-OCT4 (1:1000, Lannuo Biotechnology), anti-REX1 (1:1000, Lannuo Biotechnology), anti-FGF5 (1:1000, ab88118, Abcam), or anti-cTnT (1:1000, A4914, ABclonal) antibodies overnight at 4 °C.

Techniques:

Journal: Research

Article Title: The Mechanics of Tumor Cells Dictate Malignancy via Cytoskeleton-Mediated APC/Wnt/β-Catenin Signaling

doi: 10.34133/research.0224

Figure Lengend Snippet: Cell mechanics regulate Wnt signaling and self-renewal through APC–β-catenin interaction. (A and B) Colocalization between APC and β-catenin stained by immunofluorescence in softened and stiffened tumor cells. The representative images were shown in (A), and the Pearson correlation coefficient was calculated in (B). n = 30 cells per condition. Scale bar, 25 μm. (C) Coimmunoprecipitation analysis of the interaction between APC and β-catenin. Representative of 2 independent experiments. (D and E) Immunoblotting of APC in softened and stiffened tumor cells. n = 3. (F) Immunoblotting of the total and phosphorylated β-catenin when APC was silenced or overexpressed. Representative of 2 independent experiments. (G) Expressions of Wnt downstream genes and stemness genes measured by qRT-PCR. n = 3. (H) Expressions of Oct4 and Bmi1 measured by Western blotting. Representative of 2 independent experiments. (I and J) Formation and growth of tumor spheroids in soft agar and fibrin when stiffened Huh-7 cells were transfected with APC siRNA. n = 3. (K and L) Formation and growth of tumor spheroids in soft agar and fibrin when softened cells were transfected with APC plasmids. n = 3. (M and N) Formation of tumor spheroids in soft agar when Huh-7 cells were transfected with APC siRNA or plasmids and then treated with IWR or LiCl. n = 3. ANOVA was used for the statistical analysis together with the post hoc Bonferroni test. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The following primary antibodies were used: mouse anti-Oct4 at 1:200 (Zenbio, catalog no. 220487), mouse anti-β-catenin at 1:200 (Proteintech, catalog no. 66379-1-Ig), rabbit anti-APC at 1:200 (ABclonal, catalog no. A2818), and rabbit anti-MYLK at 1:200 (Proteintech, catalog no. 21642-1-AP).

Techniques: Staining, Immunofluorescence, Western Blot, Quantitative RT-PCR, Transfection

Journal: Research

Article Title: The Mechanics of Tumor Cells Dictate Malignancy via Cytoskeleton-Mediated APC/Wnt/β-Catenin Signaling

doi: 10.34133/research.0224

Figure Lengend Snippet: Cell mechanics-mediated Wnt signaling regulates self-renewal through β-catenin-induced Oct4 transcription. (A) Immunoblotting of Oct4 in softened and stiffened tumor cells. n = 3. (B and C) Formation and growth of tumor spheroids when softened Huh-7 cells were transfected with Oct4 siRNA. n = 3. (D and E) Formation and growth of tumor spheroids when stiffened Huh-7 cells were transfected with Oct4 plasmids. n = 3. (F and G) Formation and growth of tumor spheroids when Huh-7 cells were treated with LiCl and then transfected with Oct4 siRNA. n = 3. (H and I) Formation and growth of tumor spheroids when Huh-7 cells were treated with IWR and then transfected with Oct4 plasmids. n = 3. (J and K) mRNA expression of Oct4 analyzed by qRT-PCR in softened/stiffened Huh-7 cells that were treated with IWR/LiCl. n = 3. (L and M) Interaction between nuclear β-catenin and the promoter of Oct4 analyzed by the cleavage under targets and tagmentation (CUT&Tag) assay. n = 3. ANOVA was used for the statistical analysis together with the post hoc Bonferroni test. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The following primary antibodies were used: mouse anti-Oct4 at 1:200 (Zenbio, catalog no. 220487), mouse anti-β-catenin at 1:200 (Proteintech, catalog no. 66379-1-Ig), rabbit anti-APC at 1:200 (ABclonal, catalog no. A2818), and rabbit anti-MYLK at 1:200 (Proteintech, catalog no. 21642-1-AP).

Techniques: Western Blot, Transfection, Expressing, Quantitative RT-PCR

Journal: Research

Article Title: The Mechanics of Tumor Cells Dictate Malignancy via Cytoskeleton-Mediated APC/Wnt/β-Catenin Signaling

doi: 10.34133/research.0224

Figure Lengend Snippet: The cytoskeleton–APC–Wnt/β-catenin–Oct4 signaling is correlated with tumor differentiation and patient survival. (A to E) Expressions of MLCK, APC, and Oct4 and Pearson correlation coefficient between β-catenin and DAPI in clinical HCC samples with different differentiation levels analyzed by immunofluorescence staining. Patient samples were histologically graded into low, medium, and high differentiation level according to the Edmondson–Steiner grading system. The expressions of the indicated genes and the correlation between β-catenin and DAPI in (A) were quantified in (B) to (E) accordingly based on their differentiation state. Scale bar, 80 μm. n = 10, 25, and 14 for low, medium, and high differentiation level. ANOVA was used for the statistical analysis together with the post hoc Bonferroni test. (F to K) Correlation of the cytoskeleton–APC–Wnt/β-catenin–Oct4 signaling in clinical samples analyzed by linear regression. (L to R) Correlation between the expressions of the cytoskeleton–APC–Wnt/β-catenin–Oct4 signaling and patient survival. The Cancer Genome Atlas (TCGA) databases were utilized to analyze the relationship between the expressions of the indicated genes and patient survival. The statistics were conducted using multivariate Cox regression analysis.

Article Snippet: The following primary antibodies were used: mouse anti-Oct4 at 1:200 (Zenbio, catalog no. 220487), mouse anti-β-catenin at 1:200 (Proteintech, catalog no. 66379-1-Ig), rabbit anti-APC at 1:200 (ABclonal, catalog no. A2818), and rabbit anti-MYLK at 1:200 (Proteintech, catalog no. 21642-1-AP).

Techniques: Immunofluorescence, Staining

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