Journal: Journal for immunotherapy of cancer

Article Title: Eliciting an immune-mediated antitumor response through oncolytic herpes simplex virus-based shared antigen expression in tumors resistant to viroimmunotherapy.

doi: 10.1136/jitc-2021-002939

Figure Lengend Snippet: Figure 1 Vector construction, validation and assessment of EphA2 expression. (A) Schematic of the C170 and C172 EphA2 expression viruses. (B) Southern blot confirming the anticipated NcoI fragments in our new recombinants (1.2 and 3.0 kb, C170; 2.3 and 3.0 kb, C172), (C) Immunofluorescent imaging shows different cellular distributions of the full length and secreted forms of the EphA2. (D) Western blots of CT2A infected cells and supernatants show that C170-expressed EphA2 remains cell associated, whereas C172 expressing the extracellular form of EphA2 secretes the protein into the supernatant. TAA: tumor- associated antigen. UTR: untranslated region. TGN: Trans Golgi Network. DAPI: 4′,6-diamidino-2-phenylindole

Article Snippet: Cells were labeled with the following antibody staining panels for analysis of the adaptive immune cells: CD11b- Violet 421 (M1/70), CD4- BV785 (GK1.5), CD25- PE (7D4/CD25), CD8aBV510 (53–6.7), CD3ε-BV 711 (145–2 C11), CD44- APC (IM7), CD45- BV605 (30- F11), NKp46–PE- Cy7 (29A1.4) and B220- AF488 (RA3- 6B2), and H2Kb/H2Db (28- 8- 6) from Bio- Legend (San Diego, California, USA) and mouse EphA2 (REA579) from Miltenyi Biotec (Auburn, California, USA).

Techniques: Plasmid Preparation, Biomarker Discovery, Expressing, Southern Blot, Imaging, Western Blot, Infection

Journal: Journal for immunotherapy of cancer

Article Title: Eliciting an immune-mediated antitumor response through oncolytic herpes simplex virus-based shared antigen expression in tumors resistant to viroimmunotherapy.

doi: 10.1136/jitc-2021-002939

Figure Lengend Snippet: Figure 2 Viral replication and cytopathic effect in C57BL/6 murine CT2A MG panels show viral replication kinetics, cytopathic effect and EphA2 surface expression in infected CT2A C57BL/6-based MG cells. MG, malignant glioma.

Article Snippet: Cells were labeled with the following antibody staining panels for analysis of the adaptive immune cells: CD11b- Violet 421 (M1/70), CD4- BV785 (GK1.5), CD25- PE (7D4/CD25), CD8aBV510 (53–6.7), CD3ε-BV 711 (145–2 C11), CD44- APC (IM7), CD45- BV605 (30- F11), NKp46–PE- Cy7 (29A1.4) and B220- AF488 (RA3- 6B2), and H2Kb/H2Db (28- 8- 6) from Bio- Legend (San Diego, California, USA) and mouse EphA2 (REA579) from Miltenyi Biotec (Auburn, California, USA).

Techniques: Expressing, Infection

Journal: Journal for immunotherapy of cancer

Article Title: Eliciting an immune-mediated antitumor response through oncolytic herpes simplex virus-based shared antigen expression in tumors resistant to viroimmunotherapy.

doi: 10.1136/jitc-2021-002939

Figure Lengend Snippet: Figure 4 CT2A tumor infiltrate immunophenotypic analysis. (A). Representative summary of TILs and population changes 6 days post saline, C134, and C170 (C134 +Epha2) treatment. Numbers below pie chart represent TILs/ml brain. (B–D). T-cell tumor infiltrate and subset analysis from saline (green), C134 (salmon), and C170 (blue)-treated mice. (E) Representative gating summary of CD8 subsets and CD62L/CD44 staining in treated mice. CD8 subset analysis of (F) CD8, CD25+, (G) CD8+, CD44+, CD62L effector-like population changes, and (H) CD8+, CD44+, CD62L+central memory-like population changes in C170- treated mice. Data were analyzed by one-way analysis of variance; p values were adjusted by Holm’s procedure. TIL: tumor- infiltrating leukocyte, MDSC: myeloid derived suppressor cells.

Article Snippet: Cells were labeled with the following antibody staining panels for analysis of the adaptive immune cells: CD11b- Violet 421 (M1/70), CD4- BV785 (GK1.5), CD25- PE (7D4/CD25), CD8aBV510 (53–6.7), CD3ε-BV 711 (145–2 C11), CD44- APC (IM7), CD45- BV605 (30- F11), NKp46–PE- Cy7 (29A1.4) and B220- AF488 (RA3- 6B2), and H2Kb/H2Db (28- 8- 6) from Bio- Legend (San Diego, California, USA) and mouse EphA2 (REA579) from Miltenyi Biotec (Auburn, California, USA).

Techniques: Saline, Staining, Derivative Assay

Journal: Journal for immunotherapy of cancer

Article Title: Eliciting an immune-mediated antitumor response through oncolytic herpes simplex virus-based shared antigen expression in tumors resistant to viroimmunotherapy.

doi: 10.1136/jitc-2021-002939

Figure Lengend Snippet: Figure 7 Summary of 67C-4-oHSV antitumor response studies. C170 was evaluated in vitro and showed equivalent (A) replication and (B) cytopathic activity as the parent virus C134. (C) 67 C-4 expresses EphA2 on the cell surface in mock and C170 infected cells. (D) Schematic summary of experimental approach. (E&F) Independent studies show C170 virotherapy treatment of established 67 C-4 flank tumors shows that C170 significantly suppresses tumor growth when compared with saline or C134-treated cohorts. Data were analyzed by using analysis of variance with repeated measures. P values were adjusted by Holm’s procedure.

Article Snippet: Cells were labeled with the following antibody staining panels for analysis of the adaptive immune cells: CD11b- Violet 421 (M1/70), CD4- BV785 (GK1.5), CD25- PE (7D4/CD25), CD8aBV510 (53–6.7), CD3ε-BV 711 (145–2 C11), CD44- APC (IM7), CD45- BV605 (30- F11), NKp46–PE- Cy7 (29A1.4) and B220- AF488 (RA3- 6B2), and H2Kb/H2Db (28- 8- 6) from Bio- Legend (San Diego, California, USA) and mouse EphA2 (REA579) from Miltenyi Biotec (Auburn, California, USA).

Techniques: In Vitro, Activity Assay, Virus, Infection, Saline

Journal: Journal for immunotherapy of cancer

Article Title: Eliciting an immune-mediated antitumor response through oncolytic herpes simplex virus-based shared antigen expression in tumors resistant to viroimmunotherapy.

doi: 10.1136/jitc-2021-002939

Figure Lengend Snippet: Figure 8 T-cell function studies from saline and oHSV treated mice shows that C170 treatment induces an antigen-specific T- cell response in the periphery of long-term survivors. Splenocytes from saline (blue column) or oHSV-treated mice (red column, C134; green column, C170) were analyzed. (A) At the start, there was no difference in the populations that used peptide pulsing; however, after pulsing with 10 µm EphA2 or 10µM OVA peptide (negative control), C170-treated mice significantly increase their activated (B) CD25(+), (C) GZMB(+), and (D) CD25+, GZMB + dual staining CD8 + populations indicative of an EphA2-specific population response. (E) Representative flow plot showingCD8(+) GZMB(+) population and gating, and the (F) GZMB CD25 dual-positive populations. oHSV, oncolytic herpes simplex virus; OVA, ovalbumin.

Article Snippet: Cells were labeled with the following antibody staining panels for analysis of the adaptive immune cells: CD11b- Violet 421 (M1/70), CD4- BV785 (GK1.5), CD25- PE (7D4/CD25), CD8aBV510 (53–6.7), CD3ε-BV 711 (145–2 C11), CD44- APC (IM7), CD45- BV605 (30- F11), NKp46–PE- Cy7 (29A1.4) and B220- AF488 (RA3- 6B2), and H2Kb/H2Db (28- 8- 6) from Bio- Legend (San Diego, California, USA) and mouse EphA2 (REA579) from Miltenyi Biotec (Auburn, California, USA).

Techniques: Cell Function Assay, Saline, Negative Control, Staining, Virus

Journal: Stem Cell Research & Therapy

Article Title: Comparative evaluation of the therapeutic efficacy between human amniotic epithelial cells and human umbilical cord mesenchymal stem cells in premature ovarian insufficiency

doi: 10.1186/s13287-025-04881-7

Figure Lengend Snippet: Characterization of hAECs and hUC-MSCs. A Schematic illustration of the experimental design for comparing the effects of hAECs and hUC-MSCs on ovarian function. B , C Morphological feature and flow cytometric analysis of hAECs and hUC-MSCs. D , E Representative images of double immunofluorescence staining for OCT4 and CK18 or N-cadherin in hAECs and hUC-MSCs. F Multilineage differentiation potential of hUC-MSCs into osteoblasts, adipocytes, and chondrocytes, as assessed by Alizarin Red, Oil Red O, and Alcian Blue staining, respectively. Scale bars represent 25, 50, 100 and 200 μm

Article Snippet: After permeabilization with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA), cell were incubated with the following primary antibodies at 4 °C for overnight: OCT4 (1:200, Boster), CK18 (1:200, Boster) and N-cadherin (1:200, Boster).

Techniques: Double Immunofluorescence Staining, Staining

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: Scientific Reports

Article Title: Tousled-like kinase 1 is a negative regulator of core transcription factors in murine embryonic stem cells

doi: 10.1038/s41598-017-18628-9

Figure Lengend Snippet: Tlk1 is not required for mESC self-renewal or pluripotency. ( A ) The efficiency of Tlk1 knockdown (KD) in control (shLuc) and Tlk1- KD mESCs (shTlk1 #1 and #2) was confirmed by RT-qPCR analysis. Data are mean (n = 3) ± SEM. ** Р < 0.01 and *** Р < 0.001. ( B ) The morphology of control (shLuc) and Tlk1 -KD (shTlk1 #1 and #2) mESCs was evaluated using phase-contrast microscopic images and AP staining. Scale bars represent 500 µm. ( C and D ) The mRNA expression of pluripotency-associated and development-associated genes were analyzed by RT-qPCR in control (shLuc) and Tlk1 -KD (shTlk1 #1 and #2) mESCs cultured under undifferentiated self-renewal conditions. All data were normalized to Gapdh and plotted relative to the expression level in control cells. Data are means (n = 3) ± SEM. * Р < 0.05, ** Р < 0.01, and *** Р < 0.001. ( E ) The protein levels of pluripotency factors in control (shLuc) and Tlk1 -KD (shTlk1 #1 and #2) mESCs was analyzed by immunoblotting using antibodies specific to Oct4, Sox2, and Nanog. ( F ) Quantification based on densitometry of Western blotting data from ( E ). All data were normalized to α-tubulin. Data are means (n = 3) ± SEM. * Р < 0.05, ** Р < 0.01, and *** Р < 0.001.

Article Snippet: Primary antibodies were Oct4 (Cat. #sc-5279; Santa Cruz Biotechnology, Inc., Dallas, TX; 1:2000), Sox2 (Cat. #2748; Cell signaling; 1:1000), Nanog (Cat. #NB100-58842; Novus Biologicals; 1:5000), Tlk1 (Cat. #4125; Cell signaling; 1:5000), alpha tubulin (Cat. #LF-PA0146; Ab Frontier; 1:10000), Flag (Cat. #F3165; Sigma-Aldrich; 1:10000) and anti-p-Oct4 (S229) antibody (GenScript; 1:200).

Techniques: Knockdown, Control, Quantitative RT-PCR, Staining, Expressing, Cell Culture, Western Blot

Journal: Scientific Reports

Article Title: Tousled-like kinase 1 is a negative regulator of core transcription factors in murine embryonic stem cells

doi: 10.1038/s41598-017-18628-9

Figure Lengend Snippet: Tlk1 -deficiency in mESCs causes a delay in the downregulation of core pluripotency factors upon differentiation. ( A , C and E ) Representative immunoblotting images showing Tlk1, Oct4, Sox2, and Nanog levels in Tlk1 -KD cells upon differentiation. Differentiation was induced three different ways as previously described in Fig. . Alpha-tubulin was used as the loading control. ( B , D and F ) Quantification of the relative expression of the target proteins in panels (A,C, and E). The target proteins levels were normalized to that of α-tubulin. The protein expression levels of shLuc KD cells were normalized to 1. The biological data are presented as mean (n = 4) ± SEM for LIF- and EB and for RA (n = 3). * Р < 0.05, ** P < 0.01, and *** P < 0.001. ( G ) Immunofluorescence analysis of Oct4, Nanog and Tlk1 in control (shLuc) and Tlk1 -deficient mESCs. Scale bars represent 100 µm.

Article Snippet: Primary antibodies were Oct4 (Cat. #sc-5279; Santa Cruz Biotechnology, Inc., Dallas, TX; 1:2000), Sox2 (Cat. #2748; Cell signaling; 1:1000), Nanog (Cat. #NB100-58842; Novus Biologicals; 1:5000), Tlk1 (Cat. #4125; Cell signaling; 1:5000), alpha tubulin (Cat. #LF-PA0146; Ab Frontier; 1:10000), Flag (Cat. #F3165; Sigma-Aldrich; 1:10000) and anti-p-Oct4 (S229) antibody (GenScript; 1:200).

Techniques: Western Blot, Control, Expressing, Immunofluorescence

Journal: Scientific Reports

Article Title: Tousled-like kinase 1 is a negative regulator of core transcription factors in murine embryonic stem cells

doi: 10.1038/s41598-017-18628-9

Figure Lengend Snippet: The forced expression of Tlk1 results in the aberrant downregulation of core pluripotency factors and attenuates self-renewal. ( A ) Immunoblot analysis of Oct4, Sox2, and Nanog levels in control mESCs (empty vector or doxycycline depletion) and Tlk1-overexpressing mESCs. The mESCs expressing an empty vector or the Tet-On-Tlk1 or Tet-On-Tlk1-D607A expression vector were cultured in the absence or presence of doxycycline (Dox; 100 ng/ml) for 24 hrs under undifferentiated self-renewal conditions. ( B ) Quantification of results from ( A ). The protein levels of the target genes were normalized to α-tubulin levels. The protein expression levels of each mESC line not treated with doxycycline were normalized to 1. The biological data are presented as mean (n = 6) ± SEM. * Р < 0.05, and ** P < 0.01. ( C ) The morphology and AP staining of Tet-On-inducible Tlk1-expressing cell lines cultured in mock (Dox−) or doxycycline (Dox+) for 48 hrs. Scale bar, 500 µm.

Article Snippet: Primary antibodies were Oct4 (Cat. #sc-5279; Santa Cruz Biotechnology, Inc., Dallas, TX; 1:2000), Sox2 (Cat. #2748; Cell signaling; 1:1000), Nanog (Cat. #NB100-58842; Novus Biologicals; 1:5000), Tlk1 (Cat. #4125; Cell signaling; 1:5000), alpha tubulin (Cat. #LF-PA0146; Ab Frontier; 1:10000), Flag (Cat. #F3165; Sigma-Aldrich; 1:10000) and anti-p-Oct4 (S229) antibody (GenScript; 1:200).

Techniques: Expressing, Western Blot, Control, Plasmid Preparation, Cell Culture, Staining