Structured Review

Cell Signaling Technology Inc nanog
Neuronal and synaptic maturation during differentiation of hiPSC to cortical neurons. Representative images from immunocytochemistry staining during differentiation. (A and B) <t>NANOG</t> and <t>OCT-4</t> were strongly stained on d0 and the stainings became weak on d4, while KI-67 had similar staining intensities on d0 and d4. (C) PAX-6, a primary neuro-progenitor expression decreased from d20 to d40 of differentiation. (D) Staining for neuron specific tubulin, TUJ-1 was weak on d30 but strong staining was observed on d60. (E) SV-2, a pre-synaptic protein, staining was weak on d60. On d120, a strong punctuate staining was observed (arrows). (F) PSD-95, a post-synaptic density protein, staining was weak on d60. On d120, the staining had intensified and became punctate (arrows). Green or red = protein of interest, blue = nuclei (DAPI), Scale bar = 20 μm.
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Images

1) Product Images from "Expression and secretion of synaptic proteins during stem cell differentiation to cortical neurons"

Article Title: Expression and secretion of synaptic proteins during stem cell differentiation to cortical neurons

Journal: Neurochemistry International

doi: 10.1016/j.neuint.2018.10.014

Neuronal and synaptic maturation during differentiation of hiPSC to cortical neurons. Representative images from immunocytochemistry staining during differentiation. (A and B) NANOG and OCT-4 were strongly stained on d0 and the stainings became weak on d4, while KI-67 had similar staining intensities on d0 and d4. (C) PAX-6, a primary neuro-progenitor expression decreased from d20 to d40 of differentiation. (D) Staining for neuron specific tubulin, TUJ-1 was weak on d30 but strong staining was observed on d60. (E) SV-2, a pre-synaptic protein, staining was weak on d60. On d120, a strong punctuate staining was observed (arrows). (F) PSD-95, a post-synaptic density protein, staining was weak on d60. On d120, the staining had intensified and became punctate (arrows). Green or red = protein of interest, blue = nuclei (DAPI), Scale bar = 20 μm.
Figure Legend Snippet: Neuronal and synaptic maturation during differentiation of hiPSC to cortical neurons. Representative images from immunocytochemistry staining during differentiation. (A and B) NANOG and OCT-4 were strongly stained on d0 and the stainings became weak on d4, while KI-67 had similar staining intensities on d0 and d4. (C) PAX-6, a primary neuro-progenitor expression decreased from d20 to d40 of differentiation. (D) Staining for neuron specific tubulin, TUJ-1 was weak on d30 but strong staining was observed on d60. (E) SV-2, a pre-synaptic protein, staining was weak on d60. On d120, a strong punctuate staining was observed (arrows). (F) PSD-95, a post-synaptic density protein, staining was weak on d60. On d120, the staining had intensified and became punctate (arrows). Green or red = protein of interest, blue = nuclei (DAPI), Scale bar = 20 μm.

Techniques Used: Immunocytochemistry, Staining, Expressing

2) Product Images from "Generation of induced pluripotent stem cells from human Tenon's capsule fibroblasts"

Article Title: Generation of induced pluripotent stem cells from human Tenon's capsule fibroblasts

Journal: Molecular Vision

doi:

Expression of human embryonic stem cell markers in human Tenon’s capsule fibroblast derived induced pluripotent stem cells. A : Confocal immunofluorescent images of representative human Tenon’s capsule fibroblast derived induced pluripotent stem cells (TiPS) clones stained with the human embryonic stem (hES) markers SSEA-4 (red), TRA-1–60 (green), Nanog (red), and Oct-4 (red). Nuclei were stained with DAPI (blue). hESCs were used as a control. Scale bars=50 μm. B : Flow cytometry analysis of the equivalent SSEA-4 expression levels in TiPS and hESCs. 99.5% and 99.7% SSEA-4 + cells (in the Q4 area), respectively, whereas 0.3% in HTFs (p
Figure Legend Snippet: Expression of human embryonic stem cell markers in human Tenon’s capsule fibroblast derived induced pluripotent stem cells. A : Confocal immunofluorescent images of representative human Tenon’s capsule fibroblast derived induced pluripotent stem cells (TiPS) clones stained with the human embryonic stem (hES) markers SSEA-4 (red), TRA-1–60 (green), Nanog (red), and Oct-4 (red). Nuclei were stained with DAPI (blue). hESCs were used as a control. Scale bars=50 μm. B : Flow cytometry analysis of the equivalent SSEA-4 expression levels in TiPS and hESCs. 99.5% and 99.7% SSEA-4 + cells (in the Q4 area), respectively, whereas 0.3% in HTFs (p

Techniques Used: Expressing, Derivative Assay, Staining, Flow Cytometry, Cytometry

3) Product Images from "CD55 regulates self-renewal and cisplatin resistance in endometrioid tumors"

Article Title: CD55 regulates self-renewal and cisplatin resistance in endometrioid tumors

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20170438

CD55 regulates self-renewal and cisplatin resistance in endometrioid tumors. CD55 is glycophosphatidylinositol (GPI)–anchored to lipid rafts and via LIME binding signals intracellularly to ROR2 and LCK. ROR2 via JNK signaling regulates pluripotency gene expression, namely NANOG, SOX2, and OCT4 to maintain stemness in CSCs. In parallel, CD55 via the LCK pathway promotes the expression of DNA repair genes (including BRCA1 and MLH1) to drive cisplatin resistance.
Figure Legend Snippet: CD55 regulates self-renewal and cisplatin resistance in endometrioid tumors. CD55 is glycophosphatidylinositol (GPI)–anchored to lipid rafts and via LIME binding signals intracellularly to ROR2 and LCK. ROR2 via JNK signaling regulates pluripotency gene expression, namely NANOG, SOX2, and OCT4 to maintain stemness in CSCs. In parallel, CD55 via the LCK pathway promotes the expression of DNA repair genes (including BRCA1 and MLH1) to drive cisplatin resistance.

Techniques Used: Binding Assay, Expressing

CD55 maintains self-renewal and cisplatin resistance in endometrioid tumors. (A) Cell lysates from cisplatin-naive CSCs silenced using two CD55 shRNA constructs (KD1, KD2) and a nontargeting shRNA (NT) control were immunoblotted for CD55, NANOG, SOX2, and OCT4. Actin was used as a loading control. Data are representative of two or three independent experiments. (B) A2780 CSCs silenced for CD55 and NT controls were flowed for GFP signal intensity, which indicates NANOG promoter activity. (C) Limiting dilution analysis plots of CD55 NT control compared with CD55 KD1 and KD2 silencing constructs in cisplatin-naive CSCs. (D) In vivo tumor initiation studies were performed with five mice per group, and the estimates of stem cell frequencies of CD55 NT control compared with the CD55 KD1 and KD2 silencing constructs are shown. (E) CD55-silenced cisplatin-naive CSCs and their NT controls were treated with 0–50 µM cisplatin, and percentage surviving cells is graphed. Data are representative of three independent experiments. (F and G) In vivo cisplatin sensitivity studies were performed comparing the NT control group with the CD55-silenced group, and the graph shows the growth rate of tumors compared with the first day of cisplatin treatment. (H) Hematoxylin and eosin–stained slides of tumors excised from mice treated with cisplatin and vehicle controls. *, P
Figure Legend Snippet: CD55 maintains self-renewal and cisplatin resistance in endometrioid tumors. (A) Cell lysates from cisplatin-naive CSCs silenced using two CD55 shRNA constructs (KD1, KD2) and a nontargeting shRNA (NT) control were immunoblotted for CD55, NANOG, SOX2, and OCT4. Actin was used as a loading control. Data are representative of two or three independent experiments. (B) A2780 CSCs silenced for CD55 and NT controls were flowed for GFP signal intensity, which indicates NANOG promoter activity. (C) Limiting dilution analysis plots of CD55 NT control compared with CD55 KD1 and KD2 silencing constructs in cisplatin-naive CSCs. (D) In vivo tumor initiation studies were performed with five mice per group, and the estimates of stem cell frequencies of CD55 NT control compared with the CD55 KD1 and KD2 silencing constructs are shown. (E) CD55-silenced cisplatin-naive CSCs and their NT controls were treated with 0–50 µM cisplatin, and percentage surviving cells is graphed. Data are representative of three independent experiments. (F and G) In vivo cisplatin sensitivity studies were performed comparing the NT control group with the CD55-silenced group, and the graph shows the growth rate of tumors compared with the first day of cisplatin treatment. (H) Hematoxylin and eosin–stained slides of tumors excised from mice treated with cisplatin and vehicle controls. *, P

Techniques Used: shRNA, Construct, Activity Assay, In Vivo, Mouse Assay, Staining

LIME is necessary for intracellular CD55 signaling. (A) Immunoprecipitation (IP) experiments with CD55 antibody were performed in cisplatin-naive CSCs, and eluates were probed for lipid raft adaptor proteins LIME and PAG. (B) Cell lysates from LIME-silenced A2780 CSCs and their nontargeted (NT) controls were immunoblotted and probed with LIME, ROR2, pLCK (Y394), and LCK. Actin was used as loading control. (C) IP experiments with CD55 antibody were performed in LIME-silenced and NT control cisplatin-naive CSCs and eluates were probed for ROR2, pLCK (Y394), LCK, LIME, and CD55. (D) Immunoblots of cisplatin-naive CSCs with LIME-silenced and NT controls were immunoblotted for LIME, NANOG, SOX2, and OCT4. Actin was used as a loading control. (E) Limiting dilution analysis of LIME NT control compared with LIME sh1 and sh2 silencing constructs in cisplatin-naive CSCs. (F) LIME-silenced cisplatin-naive CSCs and their NT controls were treated with 0–50 μM cisplatin, and percentage of surviving cells is graphed. All data are representative of two or three independent experiments. **, P
Figure Legend Snippet: LIME is necessary for intracellular CD55 signaling. (A) Immunoprecipitation (IP) experiments with CD55 antibody were performed in cisplatin-naive CSCs, and eluates were probed for lipid raft adaptor proteins LIME and PAG. (B) Cell lysates from LIME-silenced A2780 CSCs and their nontargeted (NT) controls were immunoblotted and probed with LIME, ROR2, pLCK (Y394), and LCK. Actin was used as loading control. (C) IP experiments with CD55 antibody were performed in LIME-silenced and NT control cisplatin-naive CSCs and eluates were probed for ROR2, pLCK (Y394), LCK, LIME, and CD55. (D) Immunoblots of cisplatin-naive CSCs with LIME-silenced and NT controls were immunoblotted for LIME, NANOG, SOX2, and OCT4. Actin was used as a loading control. (E) Limiting dilution analysis of LIME NT control compared with LIME sh1 and sh2 silencing constructs in cisplatin-naive CSCs. (F) LIME-silenced cisplatin-naive CSCs and their NT controls were treated with 0–50 μM cisplatin, and percentage of surviving cells is graphed. All data are representative of two or three independent experiments. **, P

Techniques Used: Immunoprecipitation, Western Blot, Construct

CD55 is sufficient to drive self-renewal and cisplatin-resistance in endometrioid non-CSCs. (A) Immunoblots of cisplatin-naive non-CSCs with CD55 overexpression (OE) and empty vector controls were probed with CD55, NANOG, SOX2, and OCT4. Actin was used as loading control. Data are representative of two independent experiments. (B) mRNA expression was determined by quantitative real-time PCR and compared between CD55-overexpressing A2780 non-CSCs and empty vector control non-CSCs. Actin was used as a control. Three technical replicates were used. (C) Limiting dilution analysis plots of empty vector control compared with CD55 overexpression in cisplatin-naive non-CSCs. The graph compares the estimates of the percentage of self-renewal frequency in sorted populations with the corresponding p-values. Data are representative of three independent experiments. (D) A2780 non-CSCs transduced with CD55 overexpression and empty vector controls were flowed for GFP signal intensity, which indicates NANOG promoter activity. (E) Tumorsphere from A2780 non-CSCs transduced with CD55 and empty vector control were imaged using a digital immunofluorescence microscope. (F) CD55-overexpressing cisplatin-naive non-CSCs and their empty vector controls were treated with 0–50 µM cisplatin, and percentage of surviving cells was graphed. Data are representative of three independent experiments. (G) Relative caspase 3/7 activity of CD55-overexpressing cisplatin-naive cells and empty vector controls after cisplatin treatment. Relative caspase activities in cisplatin treated groups were calculated after normalizing the corrected readings to untreated controls in each group. Data are representative of two independent experiments, and three technical replicates were used in each. *, P
Figure Legend Snippet: CD55 is sufficient to drive self-renewal and cisplatin-resistance in endometrioid non-CSCs. (A) Immunoblots of cisplatin-naive non-CSCs with CD55 overexpression (OE) and empty vector controls were probed with CD55, NANOG, SOX2, and OCT4. Actin was used as loading control. Data are representative of two independent experiments. (B) mRNA expression was determined by quantitative real-time PCR and compared between CD55-overexpressing A2780 non-CSCs and empty vector control non-CSCs. Actin was used as a control. Three technical replicates were used. (C) Limiting dilution analysis plots of empty vector control compared with CD55 overexpression in cisplatin-naive non-CSCs. The graph compares the estimates of the percentage of self-renewal frequency in sorted populations with the corresponding p-values. Data are representative of three independent experiments. (D) A2780 non-CSCs transduced with CD55 overexpression and empty vector controls were flowed for GFP signal intensity, which indicates NANOG promoter activity. (E) Tumorsphere from A2780 non-CSCs transduced with CD55 and empty vector control were imaged using a digital immunofluorescence microscope. (F) CD55-overexpressing cisplatin-naive non-CSCs and their empty vector controls were treated with 0–50 µM cisplatin, and percentage of surviving cells was graphed. Data are representative of three independent experiments. (G) Relative caspase 3/7 activity of CD55-overexpressing cisplatin-naive cells and empty vector controls after cisplatin treatment. Relative caspase activities in cisplatin treated groups were calculated after normalizing the corrected readings to untreated controls in each group. Data are representative of two independent experiments, and three technical replicates were used in each. *, P

Techniques Used: Western Blot, Over Expression, Plasmid Preparation, Expressing, Real-time Polymerase Chain Reaction, Transduction, Activity Assay, Immunofluorescence, Microscopy

CD55 signals via ROR2-JNK pathway to regulate self-renewal. (A) Cell lysates from cisplatin-naive CSCs and non-CSCs were immunoblotted for ROR2, pJNK (T183/Y185), and JNK. Actin was used as a loading control. (B) Immunoprecipitation (IP) analysis with CD55 antibody were performed in cisplatin-naive CSCs, and eluates were immunoblotted for ROR2. (C) Immunoblots of ROR2 silenced using two shRNA constructs and nontargeting constructs in cisplatin-naive CSCs for ROR2, pJNK (T183/Y185), JNK, NANOG, SOX2, and OCT4. Actin was used as a loading control. (D) ROR2 silenced and NT controlled A2780 CSCs analyzed by flow cytometry for GFP intensity, which indicates NANOG promoter activity. (E) Limiting dilution analysis of CD55 NT control compared with ROR2 sh1 and sh2 silencing constructs in cisplatin-naive CSCs. (F) ROR2-silenced cisplatin-naive CSCs and their NT controls were treated with 0–50 µM cisplatin, and percentage surviving cells is graphed. All data are representative of two or three independent experiments. ***, P
Figure Legend Snippet: CD55 signals via ROR2-JNK pathway to regulate self-renewal. (A) Cell lysates from cisplatin-naive CSCs and non-CSCs were immunoblotted for ROR2, pJNK (T183/Y185), and JNK. Actin was used as a loading control. (B) Immunoprecipitation (IP) analysis with CD55 antibody were performed in cisplatin-naive CSCs, and eluates were immunoblotted for ROR2. (C) Immunoblots of ROR2 silenced using two shRNA constructs and nontargeting constructs in cisplatin-naive CSCs for ROR2, pJNK (T183/Y185), JNK, NANOG, SOX2, and OCT4. Actin was used as a loading control. (D) ROR2 silenced and NT controlled A2780 CSCs analyzed by flow cytometry for GFP intensity, which indicates NANOG promoter activity. (E) Limiting dilution analysis of CD55 NT control compared with ROR2 sh1 and sh2 silencing constructs in cisplatin-naive CSCs. (F) ROR2-silenced cisplatin-naive CSCs and their NT controls were treated with 0–50 µM cisplatin, and percentage surviving cells is graphed. All data are representative of two or three independent experiments. ***, P

Techniques Used: Immunoprecipitation, Western Blot, shRNA, Construct, Flow Cytometry, Cytometry, Activity Assay

4) Product Images from "Hedgehog Signals Mediate Anti-Cancer Drug Resistance in Three-Dimensional Primary Colorectal Cancer Organoid Culture"

Article Title: Hedgehog Signals Mediate Anti-Cancer Drug Resistance in Three-Dimensional Primary Colorectal Cancer Organoid Culture

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19041098

Effects of Hedgehog signal inhibitors on expression of stem cell marker proteins in tumor ALI organoids. After the organoids were treated with GANT61 (10 μM) or AY9944 (10 μM) for six days, protein expression was determined by Western blotting: GLI-1 ( n = 4–5) ( A ); c-Myc ( n = 4–5) ( B ); CD44 ( n = 4–5) ( C ); and Nanog ( n = 4–5) ( D ). Equal protein loading was confirmed using total actin antibody. * p
Figure Legend Snippet: Effects of Hedgehog signal inhibitors on expression of stem cell marker proteins in tumor ALI organoids. After the organoids were treated with GANT61 (10 μM) or AY9944 (10 μM) for six days, protein expression was determined by Western blotting: GLI-1 ( n = 4–5) ( A ); c-Myc ( n = 4–5) ( B ); CD44 ( n = 4–5) ( C ); and Nanog ( n = 4–5) ( D ). Equal protein loading was confirmed using total actin antibody. * p

Techniques Used: Expressing, Marker, Western Blot

Summary of the present results. GLI-1-targeted Hedgehog signal inhibitors improved the sensitivity for anti-cancer drug treatment in tumor ALI organoids at least in part through the decrease of CD44, Nanog, and c-Myc expression.
Figure Legend Snippet: Summary of the present results. GLI-1-targeted Hedgehog signal inhibitors improved the sensitivity for anti-cancer drug treatment in tumor ALI organoids at least in part through the decrease of CD44, Nanog, and c-Myc expression.

Techniques Used: Expressing

5) Product Images from "High-throughput fingerprinting of human pluripotent stem cell factor responsiveness and lineage induction bias"

Article Title: High-throughput fingerprinting of human pluripotent stem cell factor responsiveness and lineage induction bias

Journal: Nature methods

doi: 10.1038/nmeth.2684

Single cell protein profiling reveals Oct4 and Sox2 mark early cell fates ( a ) Sample images of control conditions stained for Tra-1-60, Gata4, Nanog, Snail, and Brachyury (Bry). Scale bar 100 μm. ( b ) 2D hierarchical clustering of protein expression levels (%positive) of markers and sets of markers across control conditions. Left panel displays sample similarity tree, with samples clustered using a distance threshold of 0.4. Clusters are indicated in red numerals. ( c ) Sample plots of Oct4 and Sox2 intensity values from controls. Thresholds can be based on these controls to classify cells as positive or negative for each marker. ( d ) Quantification of Oct4 and Sox2 subpopulations in control conditions. Statistically compared to SF control value, * ANOVA p
Figure Legend Snippet: Single cell protein profiling reveals Oct4 and Sox2 mark early cell fates ( a ) Sample images of control conditions stained for Tra-1-60, Gata4, Nanog, Snail, and Brachyury (Bry). Scale bar 100 μm. ( b ) 2D hierarchical clustering of protein expression levels (%positive) of markers and sets of markers across control conditions. Left panel displays sample similarity tree, with samples clustered using a distance threshold of 0.4. Clusters are indicated in red numerals. ( c ) Sample plots of Oct4 and Sox2 intensity values from controls. Thresholds can be based on these controls to classify cells as positive or negative for each marker. ( d ) Quantification of Oct4 and Sox2 subpopulations in control conditions. Statistically compared to SF control value, * ANOVA p

Techniques Used: Staining, Expressing, Marker

6) Product Images from "REAC technology and hyaluron synthase 2, an interesting network to slow down stem cell senescence"

Article Title: REAC technology and hyaluron synthase 2, an interesting network to slow down stem cell senescence

Journal: Scientific Reports

doi: 10.1038/srep28682

HAS2 inhibition blunts the effect of REAC treatment on the transcription of stemness related genes. At each indicated passage, ADhMSCs were left untreated (control) or subjected to REAC treatment for 12 hours, in the absence or presence of 1 mM 4-MU. The mRNA levels of Oct4 ( A ), Sox2 ( B ), or Nanog ( C ) were normalized to HPRT1 and were expressed as fold of change relative to mRNA level at time 0 (unexposed cells at passage 5), defined as 1. At each time point, mRNA levels from REAC-treated ADhMSCs were significantly different from those detected in control untreated cells. mRNA levels from cells that had been treated with REAC in the presence of 4-MU were significantly different from the expression levels detected in REAC-treated cells in the absence of the HAS2 inhibitor. (mean ± S.E.; n = 6; P
Figure Legend Snippet: HAS2 inhibition blunts the effect of REAC treatment on the transcription of stemness related genes. At each indicated passage, ADhMSCs were left untreated (control) or subjected to REAC treatment for 12 hours, in the absence or presence of 1 mM 4-MU. The mRNA levels of Oct4 ( A ), Sox2 ( B ), or Nanog ( C ) were normalized to HPRT1 and were expressed as fold of change relative to mRNA level at time 0 (unexposed cells at passage 5), defined as 1. At each time point, mRNA levels from REAC-treated ADhMSCs were significantly different from those detected in control untreated cells. mRNA levels from cells that had been treated with REAC in the presence of 4-MU were significantly different from the expression levels detected in REAC-treated cells in the absence of the HAS2 inhibitor. (mean ± S.E.; n = 6; P

Techniques Used: Inhibition, Expressing

REAC-mediated rescue of Oct4 and Sox2 and NANOG protein expression is antagonized by HAS2 inhibitor. Total cellular extracts were obtained from ADhMSCs that had been exposed for 12 hours in the absence (−R) or presence of REAC (+R), or from cells that had been subjected to a 12-hour REAC treatment in the presence of 1 mM 4-MU (+R+I). Total lysate from human iPS was used as a control (iPS). Western blot analyses were performed by the aid of polyclonal antibody directed against the indicated target proteins. Representative of six separate experiments.
Figure Legend Snippet: REAC-mediated rescue of Oct4 and Sox2 and NANOG protein expression is antagonized by HAS2 inhibitor. Total cellular extracts were obtained from ADhMSCs that had been exposed for 12 hours in the absence (−R) or presence of REAC (+R), or from cells that had been subjected to a 12-hour REAC treatment in the presence of 1 mM 4-MU (+R+I). Total lysate from human iPS was used as a control (iPS). Western blot analyses were performed by the aid of polyclonal antibody directed against the indicated target proteins. Representative of six separate experiments.

Techniques Used: Expressing, Western Blot

7) Product Images from "MicroRNA-203 inhibits long noncoding RNA HOTAIR and regulates tumorigenesis through epithelial-to-mesenchymal transition pathway in renal cell carcinoma"

Article Title: MicroRNA-203 inhibits long noncoding RNA HOTAIR and regulates tumorigenesis through epithelial-to-mesenchymal transition pathway in renal cell carcinoma

Journal: Molecular cancer therapeutics

doi: 10.1158/1535-7163.MCT-17-0925

Overexpression of miR-203 inhibits epithelial-to-mesenchymal transition and stemness in RCC (A–D) qRT-PCR analysis showing significant increase in the mRNA levels of E-cadherin, claudin and decrease in mRNA levels of vimentin. (E) Western blot analysis showing protein levels of E-cadherin, claudin, vimentin and beta actin (control) in ACHN and Caki-1 cells after overexpression of miR-203. (F) E-cadherin (red) and vimentin (green) immunostaining counterstained with DAPI (blue) in ACHN and Caki1 cells after transfections with miR-Control or miR-203 mimic, scale bar: 500 μm (right bottom). (G) Western blot analysis showing expression of stemness marker protein levels of KLF4, nanog and beta actin (control) in ACHN and Caki-1 cells after overexpression of miR-203.
Figure Legend Snippet: Overexpression of miR-203 inhibits epithelial-to-mesenchymal transition and stemness in RCC (A–D) qRT-PCR analysis showing significant increase in the mRNA levels of E-cadherin, claudin and decrease in mRNA levels of vimentin. (E) Western blot analysis showing protein levels of E-cadherin, claudin, vimentin and beta actin (control) in ACHN and Caki-1 cells after overexpression of miR-203. (F) E-cadherin (red) and vimentin (green) immunostaining counterstained with DAPI (blue) in ACHN and Caki1 cells after transfections with miR-Control or miR-203 mimic, scale bar: 500 μm (right bottom). (G) Western blot analysis showing expression of stemness marker protein levels of KLF4, nanog and beta actin (control) in ACHN and Caki-1 cells after overexpression of miR-203.

Techniques Used: Over Expression, Quantitative RT-PCR, Western Blot, Immunostaining, Transfection, Expressing, Marker

8) Product Images from "Extracellular matrix collagen I promotes the tumor progression of residual hepatocellular carcinoma after heat treatment"

Article Title: Extracellular matrix collagen I promotes the tumor progression of residual hepatocellular carcinoma after heat treatment

Journal: BMC Cancer

doi: 10.1186/s12885-018-4820-9

Sorafenib suppressed the in vivo collagen I-induced tumor progression of heat-treated residual HCC cells. a Mice with the tumors derived from heat-exposed residual MHCC97H cells with collagen I were subjected to treatment. Compared with the control group, sorafenib significantly inhibited tumor growth. b The mRNA expression of Ki-67, twist and Nanog were down-regulated in the sorafenib group. c The changes of PCNA, Nanog, vimentin, E-cadherin, N-cadherin, and ERK activation were detected by Western blot. The p-ERK levels were normalized for ERK. Expression levels of E-cadherin, PCNA, vimentin, N-cadherin and Nanog were normalized to GAPDH. d The immunohistochemical staining of PCNA, E-Cadherin, Nanog and phosphorylated ERK in the tumors (scale bar, 50 μm). **, P
Figure Legend Snippet: Sorafenib suppressed the in vivo collagen I-induced tumor progression of heat-treated residual HCC cells. a Mice with the tumors derived from heat-exposed residual MHCC97H cells with collagen I were subjected to treatment. Compared with the control group, sorafenib significantly inhibited tumor growth. b The mRNA expression of Ki-67, twist and Nanog were down-regulated in the sorafenib group. c The changes of PCNA, Nanog, vimentin, E-cadherin, N-cadherin, and ERK activation were detected by Western blot. The p-ERK levels were normalized for ERK. Expression levels of E-cadherin, PCNA, vimentin, N-cadherin and Nanog were normalized to GAPDH. d The immunohistochemical staining of PCNA, E-Cadherin, Nanog and phosphorylated ERK in the tumors (scale bar, 50 μm). **, P

Techniques Used: In Vivo, Mouse Assay, Derivative Assay, Expressing, Activation Assay, Western Blot, Immunohistochemistry, Staining

Collagen I stimulated the proliferation, motility, and the expression of EMT and progenitor-like markers in heat-treated residual HCC cells. a Compared with the cells cultured on Matrigel, heat-treated residual HCC cells on collagen I displayed a proliferative, protrusive and spindle-like appearance. b Collagen I promoted proliferation of heat-treated residual HCC cells as determined by the WST-1 proliferation assay. The OD (optical density) was measured at 450 nm wavelength. c Compared with Matrigel, collagen I enhanced the motility of heated-exposed residual HCC cells as demonstrated by tracking analysis. d As shown by qRT-PCR, the mRNA expression of Ki-67, twist, and Nanog was increased in heat-exposed residual HCC cells cultured on collagen I versus Matrigel. e The increased expression of PCNA, vimentin, N-cadherin and Nanog protein in heat-exposed residual HCC cell cultured on collagen I was detected by Western blot. Expression levels of target proteins were normalized to the corresponding levels of GAPDH. **, P
Figure Legend Snippet: Collagen I stimulated the proliferation, motility, and the expression of EMT and progenitor-like markers in heat-treated residual HCC cells. a Compared with the cells cultured on Matrigel, heat-treated residual HCC cells on collagen I displayed a proliferative, protrusive and spindle-like appearance. b Collagen I promoted proliferation of heat-treated residual HCC cells as determined by the WST-1 proliferation assay. The OD (optical density) was measured at 450 nm wavelength. c Compared with Matrigel, collagen I enhanced the motility of heated-exposed residual HCC cells as demonstrated by tracking analysis. d As shown by qRT-PCR, the mRNA expression of Ki-67, twist, and Nanog was increased in heat-exposed residual HCC cells cultured on collagen I versus Matrigel. e The increased expression of PCNA, vimentin, N-cadherin and Nanog protein in heat-exposed residual HCC cell cultured on collagen I was detected by Western blot. Expression levels of target proteins were normalized to the corresponding levels of GAPDH. **, P

Techniques Used: Expressing, Cell Culture, Proliferation Assay, Quantitative RT-PCR, Western Blot

Collagen I promoted the in vivo progression of heat-treated residual HCC cells. a The mRNA expression of PCNA, cyclin D1, Ki-67, twist and Nanog was increased in the tumors from heat-exposed residual MHCC97H cells inoculated with collagen I. b The protein expression of PCNA, vimentin, N-cadherin, E-cadherin, Nanog and ERK phosphorylation were detected by Western blot. The p-ERK was normalized for ERK. Expression levels of E-cadherin, PCNA, vimentin, N-cadherin and Nanog were normalized to GAPDH. c The expression of PCNA, E-Cadherin, Nanog and phosphorylated ERK were evaluated using immunohistochemical staining (scale bar, 50 μm). **, P
Figure Legend Snippet: Collagen I promoted the in vivo progression of heat-treated residual HCC cells. a The mRNA expression of PCNA, cyclin D1, Ki-67, twist and Nanog was increased in the tumors from heat-exposed residual MHCC97H cells inoculated with collagen I. b The protein expression of PCNA, vimentin, N-cadherin, E-cadherin, Nanog and ERK phosphorylation were detected by Western blot. The p-ERK was normalized for ERK. Expression levels of E-cadherin, PCNA, vimentin, N-cadherin and Nanog were normalized to GAPDH. c The expression of PCNA, E-Cadherin, Nanog and phosphorylated ERK were evaluated using immunohistochemical staining (scale bar, 50 μm). **, P

Techniques Used: In Vivo, Expressing, Western Blot, Immunohistochemistry, Staining

Collagen I induced the activation of ERK in heat-exposed residual HCC cells. a The up-regulated level of ERK1/2 phosphorylation were induced in heat-exposed residual HCC cells cultured on collagen I versus Matrigel. The p-ERK content was normalized for ERK. b ERK1/2 inhibitor U0126 (25 μM) or sorafenib (5 μM) could reverse the collagen I-promoted proliferative, protrusive and spindle-like appearance of heat-treated residual HCC cells. c ERK1/2 inhibitor (U0126, 25 μM) or sorafenib (5 μM) reversed collagen I-mediated upregulation of ERK1/2 in heat-exposed residual HCC cells. Collagen I-induced upregulation of proliferation (PCNA), EMT (vimentin and N-cadherin), cancer stem cell marker Nanog was markedly reduced in heat-exposed residual HCC cells pretreated with ERK1/2 inhibitor (U0126, 25 μM) or sorafenib (5 μM). The p-ERK content was normalized for ERK. Expression levels of PCNA, vimentin, N-cadherin and Nanog were normalized to Tubulin
Figure Legend Snippet: Collagen I induced the activation of ERK in heat-exposed residual HCC cells. a The up-regulated level of ERK1/2 phosphorylation were induced in heat-exposed residual HCC cells cultured on collagen I versus Matrigel. The p-ERK content was normalized for ERK. b ERK1/2 inhibitor U0126 (25 μM) or sorafenib (5 μM) could reverse the collagen I-promoted proliferative, protrusive and spindle-like appearance of heat-treated residual HCC cells. c ERK1/2 inhibitor (U0126, 25 μM) or sorafenib (5 μM) reversed collagen I-mediated upregulation of ERK1/2 in heat-exposed residual HCC cells. Collagen I-induced upregulation of proliferation (PCNA), EMT (vimentin and N-cadherin), cancer stem cell marker Nanog was markedly reduced in heat-exposed residual HCC cells pretreated with ERK1/2 inhibitor (U0126, 25 μM) or sorafenib (5 μM). The p-ERK content was normalized for ERK. Expression levels of PCNA, vimentin, N-cadherin and Nanog were normalized to Tubulin

Techniques Used: Activation Assay, Cell Culture, Marker, Expressing

9) Product Images from "Genistein inhibits stemness of SKOV3 cells induced by macrophages co-cultured with ovarian cancer stem-like cells through IL-8/STAT3 axis"

Article Title: Genistein inhibits stemness of SKOV3 cells induced by macrophages co-cultured with ovarian cancer stem-like cells through IL-8/STAT3 axis

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/s13046-018-1010-1

GEN alleviated stemness of SKOV3 cells induced by Co-CM. SKOV3 cells with Co-CM from the co-culture of SKOV3-derived OCSLCs with THP-1 macrophages and were treated with or without different concentrations of GEN (10, 20, and 40 μM). The sphere and colony formation rate ( a and b , scale bar, 100 μm) and expression levels of CD133 and CD44 ( c ) as well as Nanog and Oct4 ( d ) in SKOV3 cells were shown. *P
Figure Legend Snippet: GEN alleviated stemness of SKOV3 cells induced by Co-CM. SKOV3 cells with Co-CM from the co-culture of SKOV3-derived OCSLCs with THP-1 macrophages and were treated with or without different concentrations of GEN (10, 20, and 40 μM). The sphere and colony formation rate ( a and b , scale bar, 100 μm) and expression levels of CD133 and CD44 ( c ) as well as Nanog and Oct4 ( d ) in SKOV3 cells were shown. *P

Techniques Used: Co-Culture Assay, Derivative Assay, Expressing

10) Product Images from "Epithelial cell adhesion molecule overexpression regulates epithelial-mesenchymal transition, stemness and metastasis of nasopharyngeal carcinoma cells via the PTEN/AKT/mTOR pathway"

Article Title: Epithelial cell adhesion molecule overexpression regulates epithelial-mesenchymal transition, stemness and metastasis of nasopharyngeal carcinoma cells via the PTEN/AKT/mTOR pathway

Journal: Cell Death & Disease

doi: 10.1038/s41419-017-0013-8

EpCAM overexpression mediates stem-like properties of NPC cells. a Western blot analysis showed that the expression levels of the CSC biomarkers CD44, OCT4, Nanog and ABCG2, as well as the EMT regulatory factor Slug were significantly enhanced in the EpCAM-expressing S-18 and 6–10B cells but suppressed in EpCAM-depleted HONE1 cells compared with control cells. b Representative light microscopy images of stem-like spheres after ectopic EpCAM expression in S-18 and 6–10B cells. The number of stem-like spheres formed in each group was determined in triplicate plates. All values are expressed as the mean ± SEM. Overexpression of EpCAM significantly enhanced sphere formation of NPC cells (** P
Figure Legend Snippet: EpCAM overexpression mediates stem-like properties of NPC cells. a Western blot analysis showed that the expression levels of the CSC biomarkers CD44, OCT4, Nanog and ABCG2, as well as the EMT regulatory factor Slug were significantly enhanced in the EpCAM-expressing S-18 and 6–10B cells but suppressed in EpCAM-depleted HONE1 cells compared with control cells. b Representative light microscopy images of stem-like spheres after ectopic EpCAM expression in S-18 and 6–10B cells. The number of stem-like spheres formed in each group was determined in triplicate plates. All values are expressed as the mean ± SEM. Overexpression of EpCAM significantly enhanced sphere formation of NPC cells (** P

Techniques Used: Over Expression, Western Blot, Expressing, Light Microscopy

11) Product Images from "Variant U1 snRNAs are implicated in human pluripotent stem cell maintenance and neuromuscular disease"

Article Title: Variant U1 snRNAs are implicated in human pluripotent stem cell maintenance and neuromuscular disease

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw711

vU1s participate in early cell fate decisions. ( A ) Steady state levels of pluripotent stem cell marker mRNAs, including OCT4, NANOG and SOX2, were measured following transfection of human fibroblasts (NHDF) cells with increasing doses (0.0375, 0.0625, 0.125 and 0.25 μg) of a mixed pool of vU1-expressing plasmids (vU1.2, vU1.3, vU1.8, vU1.13 and vU1.20), by qRT-PCR analysis. Changes in pluripotent mRNA levels ( left graph ), and vU1 levels (that could be specifically amplified) ( right graph ), are expressed as fold-difference over levels quantitated in cells transfected with control vector alone, which is set to 1.0. Error bars represent SEM of three independent transfection experiments (Two-way ANOVA analysis; ** = P > 0.05, *** = P > 0.001, **** = P > 0.0001). ( B ) FACS analysis of NANOG expression in human fibroblasts transfected with decreasing doses of the pooled vU1 plasmids, including 0.5, 0.25 and 0.125 μg. iPSCs and pGEM4 transfected human fibroblast (NDHF-1) cells were used as positive and negative controls, respectively. Histograms represent NANOG fluorescence (black line) compared to isotype control (shaded gray). The % of NANOG positive cells is noted in each histogram.
Figure Legend Snippet: vU1s participate in early cell fate decisions. ( A ) Steady state levels of pluripotent stem cell marker mRNAs, including OCT4, NANOG and SOX2, were measured following transfection of human fibroblasts (NHDF) cells with increasing doses (0.0375, 0.0625, 0.125 and 0.25 μg) of a mixed pool of vU1-expressing plasmids (vU1.2, vU1.3, vU1.8, vU1.13 and vU1.20), by qRT-PCR analysis. Changes in pluripotent mRNA levels ( left graph ), and vU1 levels (that could be specifically amplified) ( right graph ), are expressed as fold-difference over levels quantitated in cells transfected with control vector alone, which is set to 1.0. Error bars represent SEM of three independent transfection experiments (Two-way ANOVA analysis; ** = P > 0.05, *** = P > 0.001, **** = P > 0.0001). ( B ) FACS analysis of NANOG expression in human fibroblasts transfected with decreasing doses of the pooled vU1 plasmids, including 0.5, 0.25 and 0.125 μg. iPSCs and pGEM4 transfected human fibroblast (NDHF-1) cells were used as positive and negative controls, respectively. Histograms represent NANOG fluorescence (black line) compared to isotype control (shaded gray). The % of NANOG positive cells is noted in each histogram.

Techniques Used: Marker, Transfection, Expressing, Quantitative RT-PCR, Amplification, Plasmid Preparation, FACS, Fluorescence

12) Product Images from "Icaritin enhances mESC self-renewal through upregulating core pluripotency transcription factors mediated by ERα"

Article Title: Icaritin enhances mESC self-renewal through upregulating core pluripotency transcription factors mediated by ERα

Journal: Scientific Reports

doi: 10.1038/srep40894

CDX2 regulates the expression of Cyclin E/CDK2 and pluripotency transcription factors in mESCs. mESCs were transfected with control (Coni) or Cdx2 (Cdx2i) siRNA. ( a ) Protein levels of Cyclin E and CDK2 in response to Cdx2 gene knockdown were detected by Western blot analysis. Protein ( b ) and mRNA ( c ) expression of OCT4, NANOG, SOX2, KLF4 following Cdx2 knockdown was examined by Western blot and quantitative real-time PCR respectively. Western blot shown are cropped from the original blots. ( d ) Representative images of ALP + colonies at day 5 following siRNA mediated Cdx2 knockdown in mESCs. ( e ) Quantitation of ALP + colonies at day 5 following Cdx2 knockdown. ( f) BrdU incorporation assay for cell proliferation following siRNA mediated Cdx2 knockdown in mESCs. Each treatment was performed in triplicate. Values were the mean ± SD (n = 3). * P
Figure Legend Snippet: CDX2 regulates the expression of Cyclin E/CDK2 and pluripotency transcription factors in mESCs. mESCs were transfected with control (Coni) or Cdx2 (Cdx2i) siRNA. ( a ) Protein levels of Cyclin E and CDK2 in response to Cdx2 gene knockdown were detected by Western blot analysis. Protein ( b ) and mRNA ( c ) expression of OCT4, NANOG, SOX2, KLF4 following Cdx2 knockdown was examined by Western blot and quantitative real-time PCR respectively. Western blot shown are cropped from the original blots. ( d ) Representative images of ALP + colonies at day 5 following siRNA mediated Cdx2 knockdown in mESCs. ( e ) Quantitation of ALP + colonies at day 5 following Cdx2 knockdown. ( f) BrdU incorporation assay for cell proliferation following siRNA mediated Cdx2 knockdown in mESCs. Each treatment was performed in triplicate. Values were the mean ± SD (n = 3). * P

Techniques Used: Expressing, Transfection, Western Blot, Real-time Polymerase Chain Reaction, ALP Assay, Quantitation Assay, BrdU Incorporation Assay

p130 modulates the expression of Cyclin E and pluripotency transcription factors. mESCs were transfected with control (Coni) or p130 (p130i) siRNA followed by further analysis. ( a ) Protein levels of Cyclin E and CDK2 in response to p130 gene knockdown was detected by Western blot analysis. Protein ( b ) and mRNA ( c ) expression of OCT4, NANOG, SOX2, KLF4 in mESCs following p130 knockdown was examined by Western blot and quantitative real-time PCR respectively. Western blot shown are cropped from the original blots. ( d ) Representative images of ALP + colonies at day 5 following siRNA mediated p130 knockdown in mESCs. ( e) Quantitation of ALP + colonies at day 5 following p130 knockdown. ( f ) BrdU incorporation assay for cell proliferation following siRNA mediated p130 knockdown in mESCs. Each treatment was performed in triplicate. Values were the mean ± SD (n = 3). * P
Figure Legend Snippet: p130 modulates the expression of Cyclin E and pluripotency transcription factors. mESCs were transfected with control (Coni) or p130 (p130i) siRNA followed by further analysis. ( a ) Protein levels of Cyclin E and CDK2 in response to p130 gene knockdown was detected by Western blot analysis. Protein ( b ) and mRNA ( c ) expression of OCT4, NANOG, SOX2, KLF4 in mESCs following p130 knockdown was examined by Western blot and quantitative real-time PCR respectively. Western blot shown are cropped from the original blots. ( d ) Representative images of ALP + colonies at day 5 following siRNA mediated p130 knockdown in mESCs. ( e) Quantitation of ALP + colonies at day 5 following p130 knockdown. ( f ) BrdU incorporation assay for cell proliferation following siRNA mediated p130 knockdown in mESCs. Each treatment was performed in triplicate. Values were the mean ± SD (n = 3). * P

Techniques Used: Expressing, Transfection, Western Blot, Real-time Polymerase Chain Reaction, ALP Assay, Quantitation Assay, BrdU Incorporation Assay

Icaritin promotes mESCs self-renewal. ( a ) The chemical structure of Icaritin. ( b ) mESCs were exposed to 5 nM or 10 nM Icaritin for 5 days, undifferentiated cell colonies were visualized by positive staining with ALP. ( c ) Quantitation of ALP + colony numbers in ( b) . ( d ) The mESCs proliferation was examined by BrdU incorporation assay following Icaritin exposure for 48 h. ( e ) mRNA expression of pluripotency transcription factors Oct4, Nanog, Sox2, Klf4 upon Icaritin treatment detected by quantitative real-time PCR. Expression levels were normalized to β-actin. ( f ) Protein levels of OCT4, NANOG, SOX2, KLF4 in mESCs after Icaritin treatment were detected by Western blot. β-actin was used as a loading control. Shown are cropped from the original blots. Values were the mean ± SD (n = 3). * P
Figure Legend Snippet: Icaritin promotes mESCs self-renewal. ( a ) The chemical structure of Icaritin. ( b ) mESCs were exposed to 5 nM or 10 nM Icaritin for 5 days, undifferentiated cell colonies were visualized by positive staining with ALP. ( c ) Quantitation of ALP + colony numbers in ( b) . ( d ) The mESCs proliferation was examined by BrdU incorporation assay following Icaritin exposure for 48 h. ( e ) mRNA expression of pluripotency transcription factors Oct4, Nanog, Sox2, Klf4 upon Icaritin treatment detected by quantitative real-time PCR. Expression levels were normalized to β-actin. ( f ) Protein levels of OCT4, NANOG, SOX2, KLF4 in mESCs after Icaritin treatment were detected by Western blot. β-actin was used as a loading control. Shown are cropped from the original blots. Values were the mean ± SD (n = 3). * P

Techniques Used: Staining, ALP Assay, Quantitation Assay, BrdU Incorporation Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot

13) Product Images from "Synergistic antitumor effect of a γ-secretase inhibitor PF-03084014 and sorafenib in hepatocellular carcinoma"

Article Title: Synergistic antitumor effect of a γ-secretase inhibitor PF-03084014 and sorafenib in hepatocellular carcinoma

Journal: Oncotarget

doi: 10.18632/oncotarget.26209

Enhanced Notch1 and Snail1 expression and EMT-mediated stemness in sorafenib resistant HCC spheroids ( A ) 97H spheroids were treated with high doses of sorafenib (10–15 μM) for over two weeks to generate sorafenib resistant cells. Western blot analysis of phospho-Erk1/2 and phospho-Akt in sorafenib-resistant (Sor+) cells compared with control (Sor−). ( B ) Western blot analysis of Snail1 and pStat3 in sorafenib-resistant (Sor+) cells compared with control (Sor−) (left panel). mRNA levels of NOTCH1 and its ligands JAG1 in sorafenib-resistant spheroids versus control (right panel). ( C ) mRNA levels of the EMT related genes SNAIL1, SNAIL2, CDH2 (N-CADHERIN), VIM (VIMENTIN) (left panel), and CDH1 (E-CADHERIN) (right panel) in sorafenib-resistant 97H spheroids compared to control (non-sorafenib resistance). ( D ) Phase contrast images of the cell morphologies of sorafenib-resistant cells compared with control. ( E ) mRNA levels of the multidrug resistant genes, ABCG2 and ABCB1, in sorafenib-resistant 97H spheroids versus control. ( F ) mRNA levels of the stemness genes, NANOG, OCT4, SOX2, and KLF4, in sorafenib-resistant and control spheroids. qPCR data are represented as the mean ± SD, n = 2 (from different sorafenib-resistant populations). An independent t test was used for statistical comparison. * p
Figure Legend Snippet: Enhanced Notch1 and Snail1 expression and EMT-mediated stemness in sorafenib resistant HCC spheroids ( A ) 97H spheroids were treated with high doses of sorafenib (10–15 μM) for over two weeks to generate sorafenib resistant cells. Western blot analysis of phospho-Erk1/2 and phospho-Akt in sorafenib-resistant (Sor+) cells compared with control (Sor−). ( B ) Western blot analysis of Snail1 and pStat3 in sorafenib-resistant (Sor+) cells compared with control (Sor−) (left panel). mRNA levels of NOTCH1 and its ligands JAG1 in sorafenib-resistant spheroids versus control (right panel). ( C ) mRNA levels of the EMT related genes SNAIL1, SNAIL2, CDH2 (N-CADHERIN), VIM (VIMENTIN) (left panel), and CDH1 (E-CADHERIN) (right panel) in sorafenib-resistant 97H spheroids compared to control (non-sorafenib resistance). ( D ) Phase contrast images of the cell morphologies of sorafenib-resistant cells compared with control. ( E ) mRNA levels of the multidrug resistant genes, ABCG2 and ABCB1, in sorafenib-resistant 97H spheroids versus control. ( F ) mRNA levels of the stemness genes, NANOG, OCT4, SOX2, and KLF4, in sorafenib-resistant and control spheroids. qPCR data are represented as the mean ± SD, n = 2 (from different sorafenib-resistant populations). An independent t test was used for statistical comparison. * p

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

Combination therapy inverted EMT and CSC stemness ( A ) HCC spheroids treated with vehicle, PF-03084014, sorafenib, and PF-03084014 + sorafenib for 48–72 hrs followed by qRT-PCR analysis. The statistical comparison is of mRNA levels of SNAIL1, SNAIL2, CDH2, VIMENTIN, and CDH1 in drug treated cells versus control. ( B ) mRNA levels of stemness genes NANOG and OCT4 in drug treated cells versus control. ( C ) mRNA levels of ABCG2 and ABCB1. qPCR data are the mean ± SD, n = 2. * p
Figure Legend Snippet: Combination therapy inverted EMT and CSC stemness ( A ) HCC spheroids treated with vehicle, PF-03084014, sorafenib, and PF-03084014 + sorafenib for 48–72 hrs followed by qRT-PCR analysis. The statistical comparison is of mRNA levels of SNAIL1, SNAIL2, CDH2, VIMENTIN, and CDH1 in drug treated cells versus control. ( B ) mRNA levels of stemness genes NANOG and OCT4 in drug treated cells versus control. ( C ) mRNA levels of ABCG2 and ABCB1. qPCR data are the mean ± SD, n = 2. * p

Techniques Used: Quantitative RT-PCR, Real-time Polymerase Chain Reaction

14) Product Images from "High-Frequency Repetitive Transcranial Magnetic Stimulation Mediates Autophagy Flux in Human Bone Mesenchymal Stromal Cells via NMDA Receptor–Ca2+–Extracellular Signal-Regulated Kinase–Mammalian Target of Rapamycin Signaling"

Article Title: High-Frequency Repetitive Transcranial Magnetic Stimulation Mediates Autophagy Flux in Human Bone Mesenchymal Stromal Cells via NMDA Receptor–Ca2+–Extracellular Signal-Regulated Kinase–Mammalian Target of Rapamycin Signaling

Journal: Frontiers in Neuroscience

doi: 10.3389/fnins.2019.01225

Repetitive transcranial magnetic stimulation (rTMS) does not affect the stemness of bone mesenchymal stromal cells (BMSCs). (A) Cell viability was assessed by Cell Counting Kit-8 assays. n = 3–5. All data were analyzed with a one-way ANOVA followed by Dunnett’s multiple comparison test. NS, not significant. (B) Nanog, Oct4, and Sox2 were assessed by western blotting. (C–E) Quantification of western blotting for Nanog, Oct4, and Sox2. Data were analyzed with a one-way ANOVA followed by Dunnett’s multiple comparison test. NS, not significant. n = 3. Error bar = SD. (F) phosphorylated epidermal growth factor receptor (p-EGFR), EGFR, p-PKA, and PKA were assessed by western blotting. (G,H) Quantification of western blotting for p-EGFR/EGFR and p-PKA/PKA. Data were analyzed with a one-way ANOVA followed by Dunnett’s multiple comparison test. NS, not significant. n = 3. Error bar = SD. (I) Schematic diagram of the autophagy pathway induced by rTMS in BMSCs.
Figure Legend Snippet: Repetitive transcranial magnetic stimulation (rTMS) does not affect the stemness of bone mesenchymal stromal cells (BMSCs). (A) Cell viability was assessed by Cell Counting Kit-8 assays. n = 3–5. All data were analyzed with a one-way ANOVA followed by Dunnett’s multiple comparison test. NS, not significant. (B) Nanog, Oct4, and Sox2 were assessed by western blotting. (C–E) Quantification of western blotting for Nanog, Oct4, and Sox2. Data were analyzed with a one-way ANOVA followed by Dunnett’s multiple comparison test. NS, not significant. n = 3. Error bar = SD. (F) phosphorylated epidermal growth factor receptor (p-EGFR), EGFR, p-PKA, and PKA were assessed by western blotting. (G,H) Quantification of western blotting for p-EGFR/EGFR and p-PKA/PKA. Data were analyzed with a one-way ANOVA followed by Dunnett’s multiple comparison test. NS, not significant. n = 3. Error bar = SD. (I) Schematic diagram of the autophagy pathway induced by rTMS in BMSCs.

Techniques Used: Cell Counting, Western Blot

15) Product Images from "Cx26 drives self-renewal in triple-negative breast cancer via interaction with NANOG and focal adhesion kinase"

Article Title: Cx26 drives self-renewal in triple-negative breast cancer via interaction with NANOG and focal adhesion kinase

Journal: Nature Communications

doi: 10.1038/s41467-018-02938-1

The Cx26/NANOG/FAK interaction is enriched in MDA-MB-231 CSCs, and Cx26 regulates NANOG protein stability in MDA-MB-231 CSCs. a Immunoprecipitation with anti-Cx26 antibody was performed in MDA-MB-231 CSCs and b non-CSCs transduced with either empty vector or Cx26 overexpression vector. The precipitates were probed for pFAK (Y397), total FAK, NANOG, and Cx26 by immunoblotting. c Immunoblots of the cell lysates of MDA-MB-231 NANOG-GFP CSCs silenced for Cx26 using two shRNA constructs each (sh1 and sh2) and a non-targeting (NT) control were probed with Cx26, NANOG, pFAK (Y397), and total FAK antibodies. Actin was used as a loading control. d Fold difference in mRNA expression of Cx26 and NANOG in Cx26 -silenced MDA-MB-231 CSCs compared with NT control was determined by qPCR. Actin was used as a normalization control (* p
Figure Legend Snippet: The Cx26/NANOG/FAK interaction is enriched in MDA-MB-231 CSCs, and Cx26 regulates NANOG protein stability in MDA-MB-231 CSCs. a Immunoprecipitation with anti-Cx26 antibody was performed in MDA-MB-231 CSCs and b non-CSCs transduced with either empty vector or Cx26 overexpression vector. The precipitates were probed for pFAK (Y397), total FAK, NANOG, and Cx26 by immunoblotting. c Immunoblots of the cell lysates of MDA-MB-231 NANOG-GFP CSCs silenced for Cx26 using two shRNA constructs each (sh1 and sh2) and a non-targeting (NT) control were probed with Cx26, NANOG, pFAK (Y397), and total FAK antibodies. Actin was used as a loading control. d Fold difference in mRNA expression of Cx26 and NANOG in Cx26 -silenced MDA-MB-231 CSCs compared with NT control was determined by qPCR. Actin was used as a normalization control (* p

Techniques Used: Multiple Displacement Amplification, Immunoprecipitation, Transduction, Plasmid Preparation, Over Expression, Western Blot, shRNA, Construct, Expressing, Real-time Polymerase Chain Reaction

Cx26 mutants failing to complex with NANOG or FAK/NANOG disrupt the self-renewal capacity of TNBC CSCs. a , b After transfection of plasmids expressing GFP-fused Cx26 wild-type and mutant proteins, immunoprecipitation was performed using a GFP antibody in MDA-MB-231 and PDX TN-1 cells, and the resulting immunoprecipitates were probed with FAK, NANOG, and GFP antibodies. c Stem cell frequencies determined by limiting dilution sphere-forming assays indicate that the expression of the D66H-RFP mutant Cx26 in MDA-MB-231 CSCs significantly reduced the stem cell frequency compared with the expression of the wild-type Cx26-RFP and empty vector control. d D66H-GFP or G59A-GFP mutant Cx26 expression in MDA-MB-231 parental cells failed to increase stem cell frequency compared with the expression of the wild-type Cx26-GFP (* p
Figure Legend Snippet: Cx26 mutants failing to complex with NANOG or FAK/NANOG disrupt the self-renewal capacity of TNBC CSCs. a , b After transfection of plasmids expressing GFP-fused Cx26 wild-type and mutant proteins, immunoprecipitation was performed using a GFP antibody in MDA-MB-231 and PDX TN-1 cells, and the resulting immunoprecipitates were probed with FAK, NANOG, and GFP antibodies. c Stem cell frequencies determined by limiting dilution sphere-forming assays indicate that the expression of the D66H-RFP mutant Cx26 in MDA-MB-231 CSCs significantly reduced the stem cell frequency compared with the expression of the wild-type Cx26-RFP and empty vector control. d D66H-GFP or G59A-GFP mutant Cx26 expression in MDA-MB-231 parental cells failed to increase stem cell frequency compared with the expression of the wild-type Cx26-GFP (* p

Techniques Used: Transfection, Expressing, Mutagenesis, Immunoprecipitation, Multiple Displacement Amplification, Plasmid Preparation

Model of Cx26, NANOG, and FAK interaction in luminal breast cancer and TNBC cells. Schematic depicting the interaction between Cx26, NANOG, and FAK. In luminal breast cancer cells, Cx26 interacts with FAK and NANOG individually, but the Cx26/NANOG/FAK complex does not form. In TNBC non-CSCs, FAK interacts with Cx26 but is not phosphorylated and NANOG is not present in a complex with Cx26 and FAK. In TNBC CSCs, all three proteins are expressed and form a complex, driving self-renewal
Figure Legend Snippet: Model of Cx26, NANOG, and FAK interaction in luminal breast cancer and TNBC cells. Schematic depicting the interaction between Cx26, NANOG, and FAK. In luminal breast cancer cells, Cx26 interacts with FAK and NANOG individually, but the Cx26/NANOG/FAK complex does not form. In TNBC non-CSCs, FAK interacts with Cx26 but is not phosphorylated and NANOG is not present in a complex with Cx26 and FAK. In TNBC CSCs, all three proteins are expressed and form a complex, driving self-renewal

Techniques Used:

Elevated Cx26 expression in TNBC tissue samples and in TNBC cancer stem cells. a Gene expression profiles of 20 different connexins in 2408 TNBC tissue samples were compared with those of 250 normal breast tissue samples across seven different TNBC-normal datasets using the Oncomine TM ). b Cell lysates from CSCs enriched from two TNBC cell lines of different subtypes and from a TNBC PDX were probed with anti-Cx26 and anti-Cx43 antibodies. The NANOG-GFP reporter was used to enrich CSCs from MDA-MB-231 (mesenchymal-like) and HCC70 (basal-like) cells, and PDX TN-1 CSCs were enriched according to ALDH activity. Actin was used as a loading control. c mRNA expression was determined by qPCR and compared between cancer stem cells (CSCs) and non-CSCs enriched from MDA-MB-231 and HCC70 cells using the NANOG-GFP reporter system and between those enriched from PDX TN-1 cells using ALDH activity sorting (ALDEFLUOR assay). Actin was used as a normalization control (* p
Figure Legend Snippet: Elevated Cx26 expression in TNBC tissue samples and in TNBC cancer stem cells. a Gene expression profiles of 20 different connexins in 2408 TNBC tissue samples were compared with those of 250 normal breast tissue samples across seven different TNBC-normal datasets using the Oncomine TM ). b Cell lysates from CSCs enriched from two TNBC cell lines of different subtypes and from a TNBC PDX were probed with anti-Cx26 and anti-Cx43 antibodies. The NANOG-GFP reporter was used to enrich CSCs from MDA-MB-231 (mesenchymal-like) and HCC70 (basal-like) cells, and PDX TN-1 CSCs were enriched according to ALDH activity. Actin was used as a loading control. c mRNA expression was determined by qPCR and compared between cancer stem cells (CSCs) and non-CSCs enriched from MDA-MB-231 and HCC70 cells using the NANOG-GFP reporter system and between those enriched from PDX TN-1 cells using ALDH activity sorting (ALDEFLUOR assay). Actin was used as a normalization control (* p

Techniques Used: Expressing, Multiple Displacement Amplification, Activity Assay, Real-time Polymerase Chain Reaction

Cx26 is necessary and sufficient for the maintenance of self-renewal, in vivo tumor initiation, and NANOG expression. a Cell lysates from MDA-MB-231 and HCC70 CSCs silenced using three Cx26 shRNA constructs (sh1, sh2, and sh3) and a non-targeting shRNA (NT) control were probed with Cx26, Cx43, NANOG, and OCT4 antibodies. Actin was used as a loading control. b In vivo tumor initiation studies were performed in Cx26 -silenced MDA-MB-231 and HCC70 CSCs with at least four mice per group, and the p value was calculated using a log-rank analysis. The graphs show the estimates of stem cell frequencies of NT control compared with the Cx26 shRNA silencing constructs and their corresponding p values. c MDA-MB-231 and HCC70 non-CSCs containing Cx26 overexpression vector or empty vector were probed with anti-Cx26, NANOG, OCT4, and SOX2 antibodies. Actin was used as a loading control. d In vivo tumor initiation studies were performed comparing the empty vector group with the Cx26 overexpression group, and the p value was calculated using a log-rank analysis. The graphs show the estimates of stem cell frequencies with the corresponding p values for the empty vector compared with Cx26 overexpression in MDA-MB-231 and HCC70 non-CSCs. (* p
Figure Legend Snippet: Cx26 is necessary and sufficient for the maintenance of self-renewal, in vivo tumor initiation, and NANOG expression. a Cell lysates from MDA-MB-231 and HCC70 CSCs silenced using three Cx26 shRNA constructs (sh1, sh2, and sh3) and a non-targeting shRNA (NT) control were probed with Cx26, Cx43, NANOG, and OCT4 antibodies. Actin was used as a loading control. b In vivo tumor initiation studies were performed in Cx26 -silenced MDA-MB-231 and HCC70 CSCs with at least four mice per group, and the p value was calculated using a log-rank analysis. The graphs show the estimates of stem cell frequencies of NT control compared with the Cx26 shRNA silencing constructs and their corresponding p values. c MDA-MB-231 and HCC70 non-CSCs containing Cx26 overexpression vector or empty vector were probed with anti-Cx26, NANOG, OCT4, and SOX2 antibodies. Actin was used as a loading control. d In vivo tumor initiation studies were performed comparing the empty vector group with the Cx26 overexpression group, and the p value was calculated using a log-rank analysis. The graphs show the estimates of stem cell frequencies with the corresponding p values for the empty vector compared with Cx26 overexpression in MDA-MB-231 and HCC70 non-CSCs. (* p

Techniques Used: In Vivo, Expressing, Multiple Displacement Amplification, shRNA, Construct, Mouse Assay, Over Expression, Plasmid Preparation

Cx26 forms a TNBC-specific complex with focal adhesion kinase (FAK) and NANOG. Cell lysates from bulk cell cultures of a mesenchymal-like TNBC (MDA-MB-231), basal-like TNBC (HCC70), TNBC (PDX TN-1), and b non-TNBC (MCF7 and MCF10A) were subjected to immunoprecipitation with anti-Cx26, anti-FAK, and anti-NANOG antibodies. pFAK (Y397), FAK, Cx26, and NANOG proteins in the precipitated complex were detected by western blotting using specific antibodies. Fifteen percent of the lysate used for immunoprecipitation was loaded as the input control. As a negative control, immunoprecipitation with the corresponding non-immune IgG was performed. c Schematic summarizing the interactions detected in TNBC versus mammary epithelial and luminal breast cancer cells
Figure Legend Snippet: Cx26 forms a TNBC-specific complex with focal adhesion kinase (FAK) and NANOG. Cell lysates from bulk cell cultures of a mesenchymal-like TNBC (MDA-MB-231), basal-like TNBC (HCC70), TNBC (PDX TN-1), and b non-TNBC (MCF7 and MCF10A) were subjected to immunoprecipitation with anti-Cx26, anti-FAK, and anti-NANOG antibodies. pFAK (Y397), FAK, Cx26, and NANOG proteins in the precipitated complex were detected by western blotting using specific antibodies. Fifteen percent of the lysate used for immunoprecipitation was loaded as the input control. As a negative control, immunoprecipitation with the corresponding non-immune IgG was performed. c Schematic summarizing the interactions detected in TNBC versus mammary epithelial and luminal breast cancer cells

Techniques Used: Multiple Displacement Amplification, Immunoprecipitation, Western Blot, Negative Control

16) Product Images from "Single cell transcriptome analysis of human, marmoset and mouse embryos reveals common and divergent features of preimplantation development"

Article Title: Single cell transcriptome analysis of human, marmoset and mouse embryos reveals common and divergent features of preimplantation development

Journal: Development (Cambridge, England)

doi: 10.1242/dev.167833

OTX2 protein localisation in primate embryos. (A) Schematic of Otx2 expression over preimplantation development. (B,C) Confocal microscopy immunofluorescence images of (B) NANOG, GATA6 and DAPI, and (C) NANOG, OTX2 and DAPI in marmoset late blastocysts. (D) Confocal sections, 3D reconstruction and single-plane image of NANOG, GATA2, OTX2 and DAPI localisation in an early human blastocyst. (E) Confocal sections of the indicated markers in a representative late human blastocyst. White arrowheads indicate PrE cells.
Figure Legend Snippet: OTX2 protein localisation in primate embryos. (A) Schematic of Otx2 expression over preimplantation development. (B,C) Confocal microscopy immunofluorescence images of (B) NANOG, GATA6 and DAPI, and (C) NANOG, OTX2 and DAPI in marmoset late blastocysts. (D) Confocal sections, 3D reconstruction and single-plane image of NANOG, GATA2, OTX2 and DAPI localisation in an early human blastocyst. (E) Confocal sections of the indicated markers in a representative late human blastocyst. White arrowheads indicate PrE cells.

Techniques Used: Expressing, Confocal Microscopy, Immunofluorescence

17) Product Images from "Chchd10 or Chchd2 are not Required for Human Motor Neuron Differentiation In Vitro but Modify Synaptic Transcriptomes"

Article Title: Chchd10 or Chchd2 are not Required for Human Motor Neuron Differentiation In Vitro but Modify Synaptic Transcriptomes

Journal: bioRxiv

doi: 10.1101/828376

Human iPSC are viable and pluripotent in absence of CHCHD2 or CHCHD10 and have normal mitochondrial ultrastructure. (A) Schematic representation of genome editing strategy. CRISPR-Cas9 system was used to target 5’UTR and exon 1. (B) Pluripotency was validated with immunocytochemical analysis of pluripotency markers Nanog and TRA-1-81. Dapi indicates nuclear staining. Scale bar 50 μ m. (C) qRT-PCR of pluripotency markers OCT4 and SOX2 , normalized to GAPDH (n=3). (D) Expression of CHCHD2 and CHCHD10 in knockout iPSCs by qRT-PCR, normalized to GAPDH (n=3 per clone). (E) Immunocytochemical analysis verifies knockout of CHCHD2 and CHCHD10. Scale bar 20 μ m. (F) Immunoblot and quantification of CHCHD10 and CHCHD2 levels from whole cell lysates. Quantification shows the average of three independent experiments, normalized to mitochondrial HSP60. (G) Blue-native PAGE shows unaltered mitochondrial respiratory chain complexes. Quantifications shown relative to average control levels. (H) mtDNA copy number was analyzed by qRT-PCR (n=3 per clone). (I) Electron micrograph showing intact mitochondrial ultrastructure in CHCHD2 and CHCHD10 knockout iPSC. Scale bar 0.5 μ m. Quantifications of D10 KOs clones are shown as pooled results from 3 clones F3, G4 and F2. See also Figure S1 for results of individual D10KO clones and full immunoblots. Data are shown as mean ± SD, * P
Figure Legend Snippet: Human iPSC are viable and pluripotent in absence of CHCHD2 or CHCHD10 and have normal mitochondrial ultrastructure. (A) Schematic representation of genome editing strategy. CRISPR-Cas9 system was used to target 5’UTR and exon 1. (B) Pluripotency was validated with immunocytochemical analysis of pluripotency markers Nanog and TRA-1-81. Dapi indicates nuclear staining. Scale bar 50 μ m. (C) qRT-PCR of pluripotency markers OCT4 and SOX2 , normalized to GAPDH (n=3). (D) Expression of CHCHD2 and CHCHD10 in knockout iPSCs by qRT-PCR, normalized to GAPDH (n=3 per clone). (E) Immunocytochemical analysis verifies knockout of CHCHD2 and CHCHD10. Scale bar 20 μ m. (F) Immunoblot and quantification of CHCHD10 and CHCHD2 levels from whole cell lysates. Quantification shows the average of three independent experiments, normalized to mitochondrial HSP60. (G) Blue-native PAGE shows unaltered mitochondrial respiratory chain complexes. Quantifications shown relative to average control levels. (H) mtDNA copy number was analyzed by qRT-PCR (n=3 per clone). (I) Electron micrograph showing intact mitochondrial ultrastructure in CHCHD2 and CHCHD10 knockout iPSC. Scale bar 0.5 μ m. Quantifications of D10 KOs clones are shown as pooled results from 3 clones F3, G4 and F2. See also Figure S1 for results of individual D10KO clones and full immunoblots. Data are shown as mean ± SD, * P

Techniques Used: CRISPR, Staining, Quantitative RT-PCR, Expressing, Knock-Out, Blue Native PAGE, Clone Assay, Western Blot

18) Product Images from "Suppressing Nodal Signaling Activity Predisposes Ectodermal Differentiation of Epiblast Stem Cells"

Article Title: Suppressing Nodal Signaling Activity Predisposes Ectodermal Differentiation of Epiblast Stem Cells

Journal: Stem Cell Reports

doi: 10.1016/j.stemcr.2018.05.019

Characterization of EpiSC S/F (A) Expression of SOX2 and OCT4 (upper panels), NANOG, FGF5, and T (middle panels), and OTX2, SIX3, and E-CADHERIN (lower panels) in EpiSCs S/F and EpiSCs. Immunofluorescence and DAPI counterstaining. ∗ p
Figure Legend Snippet: Characterization of EpiSC S/F (A) Expression of SOX2 and OCT4 (upper panels), NANOG, FGF5, and T (middle panels), and OTX2, SIX3, and E-CADHERIN (lower panels) in EpiSCs S/F and EpiSCs. Immunofluorescence and DAPI counterstaining. ∗ p

Techniques Used: Expressing, Immunofluorescence

19) Product Images from "Functional Characterization of IPSC-Derived Brain Cells as a Model for X-Linked Adrenoleukodystrophy"

Article Title: Functional Characterization of IPSC-Derived Brain Cells as a Model for X-Linked Adrenoleukodystrophy

Journal: PLoS ONE

doi: 10.1371/journal.pone.0143238

Morphological and specific marker characterization of fibroblast-derived IPSC. (A) Fibroblasts from a male healthy or patient with AMN or cALD disease were transduced with retroviral vectors expressing reprogramming factors OCT4, SOX2, NANOG, LIN28, KLF4, and c-MYC as described under methods. IPSC colony before isolation. (B) A putative control IPSC line was isolated and expanded under feeder-free maintenance medium for human IPSC, colonies growth was observed for 5 days by phase contrast image. (C-D) Control, AMN or cALD IPSC expressed the SOX2 and SSEA4 markers of pluripotency. (E) Summary chart depicts the markers for IPSC lines that were characterized. Scale bars represent 200 μm.
Figure Legend Snippet: Morphological and specific marker characterization of fibroblast-derived IPSC. (A) Fibroblasts from a male healthy or patient with AMN or cALD disease were transduced with retroviral vectors expressing reprogramming factors OCT4, SOX2, NANOG, LIN28, KLF4, and c-MYC as described under methods. IPSC colony before isolation. (B) A putative control IPSC line was isolated and expanded under feeder-free maintenance medium for human IPSC, colonies growth was observed for 5 days by phase contrast image. (C-D) Control, AMN or cALD IPSC expressed the SOX2 and SSEA4 markers of pluripotency. (E) Summary chart depicts the markers for IPSC lines that were characterized. Scale bars represent 200 μm.

Techniques Used: Marker, Derivative Assay, Transduction, Expressing, Isolation

20) Product Images from "Reprogramming of Adult Peripheral Blood Cells into Human Induced Pluripotent Stem Cells as a Safe and Accessible Source of Endothelial Cells"

Article Title: Reprogramming of Adult Peripheral Blood Cells into Human Induced Pluripotent Stem Cells as a Safe and Accessible Source of Endothelial Cells

Journal: Stem Cells and Development

doi: 10.1089/scd.2017.0132

hiPSC reprogramming and characterization. (A) Experimental timeline for the reprogramming of PBMCs and ECs into hiPSCs. Cell morphology observed during the reprogramming of PBMCs at days 4, 9, and 17 (scale bar = 100 μm). (B) Immunofluorescence staining for the pluripotency markers Oct3/4, Sox2, and Nanog in hiPSCs (scale bar = 200 μm). (C) Flow cytometry-based detection of the pluripotency markers SSEA-4, Tra-1-60, and Tra-1-81 in hiPSCs. (D) Teratoma formation in immunodeficient mice after transplantation of hiPSCs; teratomas contained tissues from all three germ layers. ECs, endothelial cells; hiPSC, human induced pluripotent stem cell; PBMCs, peripheral blood mononuclear cells. Color images are available online at www.libeberpub.com/scd
Figure Legend Snippet: hiPSC reprogramming and characterization. (A) Experimental timeline for the reprogramming of PBMCs and ECs into hiPSCs. Cell morphology observed during the reprogramming of PBMCs at days 4, 9, and 17 (scale bar = 100 μm). (B) Immunofluorescence staining for the pluripotency markers Oct3/4, Sox2, and Nanog in hiPSCs (scale bar = 200 μm). (C) Flow cytometry-based detection of the pluripotency markers SSEA-4, Tra-1-60, and Tra-1-81 in hiPSCs. (D) Teratoma formation in immunodeficient mice after transplantation of hiPSCs; teratomas contained tissues from all three germ layers. ECs, endothelial cells; hiPSC, human induced pluripotent stem cell; PBMCs, peripheral blood mononuclear cells. Color images are available online at www.libeberpub.com/scd

Techniques Used: Immunofluorescence, Staining, Flow Cytometry, Cytometry, Mouse Assay, Transplantation Assay

21) Product Images from "Melatonin Treatment Improves Mesenchymal Stem Cells Therapy by Preserving Stemness during Long-term In Vitro Expansion"

Article Title: Melatonin Treatment Improves Mesenchymal Stem Cells Therapy by Preserving Stemness during Long-term In Vitro Expansion

Journal: Theranostics

doi: 10.7150/thno.15412

Melatonin treatment decreases ROS levels, inhibits p53 pathway, and preserves NANOG expression in long-term passaged BMMSCs. (A-F) In vitro passaged rat BMMSCs of P1, P4, P15 treated with DMSO or 10 nM melatonin were used in following analysis. (A) ROS levels of BMMSCs were measured by flow cytometer (n=3). (B) Sod2 mRNA levels of BMMSCs were detected by real-time RT-PCR. β-actin was used as the loading control for quantification (n=3). (C) Expression of SOD2 protein was detected by western blot analysis. β-actin was used as the loading control (n=3). (D and E) Real-time RT-PCR was performed to measure p53 , p16 (D) and Nanog (E) mRNA levels in BMMSCs (n=3). (F) Expression of p53, p16 and NANOG protein was detected by western blotting (n=3). (G-P) BMMSCs were treated with DMSO, melatonin or melatonin plus LUZ from the 1 st passage to the 15 th passage. (G) Mel-1A-R and Mel-1B-R mRNA levels in BMMSCs were analysis by realtime RT-PCR. (n=3). (H) CFU-F formation of BMMSCs was detected by toluidine blue staining and calculated (n=3). (I) Proliferation of BMMSCs was detected from day 0 to day 6 (n=3). (J) ALP staining and ELISA analysis were performed to detect the activity of ALP of BMMSCs after 7-day osteogenic induction (n=3). (K) Mineralized nodules formed by BMMSCs were tested by alizarin red staining after 28-day osteogenic induction and quantified (n=3). (L-N) Runx2 , Ocn (L) , p53 , p16 (M) and Nanog (N) mRNA levels in BMMSCs were analyzed by Realtime RT-PCR (n=3). (O) ROS levels and Sod2 mRNA levels in BMMSCs (n=3). (P) Expression of Mel-1A-R, Mel-1B-R, SOD2, p53, p16 and NANOG protein was detected by western blot analysis. (n=3). Data are shown as mean±SD. P value is presented in each graph.
Figure Legend Snippet: Melatonin treatment decreases ROS levels, inhibits p53 pathway, and preserves NANOG expression in long-term passaged BMMSCs. (A-F) In vitro passaged rat BMMSCs of P1, P4, P15 treated with DMSO or 10 nM melatonin were used in following analysis. (A) ROS levels of BMMSCs were measured by flow cytometer (n=3). (B) Sod2 mRNA levels of BMMSCs were detected by real-time RT-PCR. β-actin was used as the loading control for quantification (n=3). (C) Expression of SOD2 protein was detected by western blot analysis. β-actin was used as the loading control (n=3). (D and E) Real-time RT-PCR was performed to measure p53 , p16 (D) and Nanog (E) mRNA levels in BMMSCs (n=3). (F) Expression of p53, p16 and NANOG protein was detected by western blotting (n=3). (G-P) BMMSCs were treated with DMSO, melatonin or melatonin plus LUZ from the 1 st passage to the 15 th passage. (G) Mel-1A-R and Mel-1B-R mRNA levels in BMMSCs were analysis by realtime RT-PCR. (n=3). (H) CFU-F formation of BMMSCs was detected by toluidine blue staining and calculated (n=3). (I) Proliferation of BMMSCs was detected from day 0 to day 6 (n=3). (J) ALP staining and ELISA analysis were performed to detect the activity of ALP of BMMSCs after 7-day osteogenic induction (n=3). (K) Mineralized nodules formed by BMMSCs were tested by alizarin red staining after 28-day osteogenic induction and quantified (n=3). (L-N) Runx2 , Ocn (L) , p53 , p16 (M) and Nanog (N) mRNA levels in BMMSCs were analyzed by Realtime RT-PCR (n=3). (O) ROS levels and Sod2 mRNA levels in BMMSCs (n=3). (P) Expression of Mel-1A-R, Mel-1B-R, SOD2, p53, p16 and NANOG protein was detected by western blot analysis. (n=3). Data are shown as mean±SD. P value is presented in each graph.

Techniques Used: Expressing, In Vitro, Flow Cytometry, Cytometry, Quantitative RT-PCR, Western Blot, Reverse Transcription Polymerase Chain Reaction, Staining, ALP Assay, Enzyme-linked Immunosorbent Assay, Activity Assay

22) Product Images from "Sulforaphane and TRAIL induce a synergistic elimination of advanced prostate cancer stem-like cells"

Article Title: Sulforaphane and TRAIL induce a synergistic elimination of advanced prostate cancer stem-like cells

Journal: International Journal of Oncology

doi: 10.3892/ijo.2014.2335

TRAIL and sulforaphane inhibit tumor growth in vivo and reduce CSC marker expression, with the strongest effects after their combination. (A) Untreated PC3 cells in Matrigel were transplanted into a plastic ring on the chorioallantoic membrane of fertilized chicken eggs at day 9 of embryonic development. At day 11, a 1-cm 2 Whatman paper saturated with 10 μ M sulforaphane was placed directly adjacent to the tumor plastic ring. At day 12, a 5 μ g/ml TRAIL solution was dropped onto the Whatman paper until saturation. The tumor xenografts were resected at day 18 of embryonic development, and the volumes were determined as described in Materials and methods. The volumes of the individual tumors per group are presented as black dots and the bars indicate the average tumor size of each group. Representative images of the resected tumors are shown in the upper panel. (B) After tumor resection, the morphology and the body weights of the chicken embryos were evaluated. The average body weights of the embryos per group are presented in the diagram, and images of representative chicken embryos are shown. (C) The livers of the embryos were sectioned, and H E staining was performed and visualized using microscopy. (D) Slices of the tumor tissue were immunohistochemically stained with an antibody for the detection of the cleaved fragment of active caspase-3 and the signal was detected using microscopy. (E) Slices of tumor tissue were double-immunofluorescence stained with antibodies for the detection of CD133/CXCR4, Nanog/c-Met, Ki67/EpCAM, and ALDH1/CD44 and positive signals were detected by fluorescence microscopy. The bar indicates 50 μ m.
Figure Legend Snippet: TRAIL and sulforaphane inhibit tumor growth in vivo and reduce CSC marker expression, with the strongest effects after their combination. (A) Untreated PC3 cells in Matrigel were transplanted into a plastic ring on the chorioallantoic membrane of fertilized chicken eggs at day 9 of embryonic development. At day 11, a 1-cm 2 Whatman paper saturated with 10 μ M sulforaphane was placed directly adjacent to the tumor plastic ring. At day 12, a 5 μ g/ml TRAIL solution was dropped onto the Whatman paper until saturation. The tumor xenografts were resected at day 18 of embryonic development, and the volumes were determined as described in Materials and methods. The volumes of the individual tumors per group are presented as black dots and the bars indicate the average tumor size of each group. Representative images of the resected tumors are shown in the upper panel. (B) After tumor resection, the morphology and the body weights of the chicken embryos were evaluated. The average body weights of the embryos per group are presented in the diagram, and images of representative chicken embryos are shown. (C) The livers of the embryos were sectioned, and H E staining was performed and visualized using microscopy. (D) Slices of the tumor tissue were immunohistochemically stained with an antibody for the detection of the cleaved fragment of active caspase-3 and the signal was detected using microscopy. (E) Slices of tumor tissue were double-immunofluorescence stained with antibodies for the detection of CD133/CXCR4, Nanog/c-Met, Ki67/EpCAM, and ALDH1/CD44 and positive signals were detected by fluorescence microscopy. The bar indicates 50 μ m.

Techniques Used: In Vivo, Marker, Expressing, Staining, Microscopy, Immunofluorescence, Fluorescence

23) Product Images from "Pluripotent stem cells secrete Activin A to improve their epiblast competency after injection into recipient embryos"

Article Title: Pluripotent stem cells secrete Activin A to improve their epiblast competency after injection into recipient embryos

Journal: Protein & Cell

doi: 10.1007/s13238-017-0470-y

Secretions from ESCs and iPSCs affect EPI development . (A) Schematic of the method used to collect the condition medium. (B) Experimental design. Zona-free embryos at 4-cell stage were treated in the mixed medium containing KSOM and CM and then immunostained at E4.5 to test the effect of the condition medium on early embryo development fate. CM, condition medium. (C) Nanog immunostaining in E4.5 embryos treated with condition medium from feeder, R1 ESCs and iPSCs. Nuclei were stained with DAPI (Blue). Scale bars, 20 μm. (D) Average numbers of EPI cells (Nanog-positive cells) in condition medium-treated embryos at E4.5. Error bars indicate SD. * P
Figure Legend Snippet: Secretions from ESCs and iPSCs affect EPI development . (A) Schematic of the method used to collect the condition medium. (B) Experimental design. Zona-free embryos at 4-cell stage were treated in the mixed medium containing KSOM and CM and then immunostained at E4.5 to test the effect of the condition medium on early embryo development fate. CM, condition medium. (C) Nanog immunostaining in E4.5 embryos treated with condition medium from feeder, R1 ESCs and iPSCs. Nuclei were stained with DAPI (Blue). Scale bars, 20 μm. (D) Average numbers of EPI cells (Nanog-positive cells) in condition medium-treated embryos at E4.5. Error bars indicate SD. * P

Techniques Used: Immunostaining, Staining

Activin A represses EPI lineage . (A) Nanog immunostaining in Activin A-treated and untreated embryos at E4.5. Nuclei were stained with DAPI (Blue). Scale bars, 20 μm. (B) Average numbers of EPI cells (Nanog-positive cells) in Activin A-treated and untreated embryos at E4.5. Error bars indicate SD. ** P
Figure Legend Snippet: Activin A represses EPI lineage . (A) Nanog immunostaining in Activin A-treated and untreated embryos at E4.5. Nuclei were stained with DAPI (Blue). Scale bars, 20 μm. (B) Average numbers of EPI cells (Nanog-positive cells) in Activin A-treated and untreated embryos at E4.5. Error bars indicate SD. ** P

Techniques Used: Immunostaining, Staining

Activin A enhances ESC contribution to chimeras generated by blastocyst injection . (A) Experimental design. We treated 4-cell embryos without zona in KSOM including Activin A and then performed the blastocyst injection to test whether Activin A can affect the contribution of ESCs to chimeras. (B) The ESC colonies (left panel, bright field; right panel, DsRed). Scale bar, 20 μm. (C) ESC-injected embryos at E4.5. Scale bar, 20 µm. (D) Nanog immunostaining in E4.5 chimeric embryos after blastocyst injection. Nuclei were stained with DAPI (Blue). Scale bars, 20 μm. (E) Summary of ESC (DsRed) contribution to E4.5 embryos. P
Figure Legend Snippet: Activin A enhances ESC contribution to chimeras generated by blastocyst injection . (A) Experimental design. We treated 4-cell embryos without zona in KSOM including Activin A and then performed the blastocyst injection to test whether Activin A can affect the contribution of ESCs to chimeras. (B) The ESC colonies (left panel, bright field; right panel, DsRed). Scale bar, 20 μm. (C) ESC-injected embryos at E4.5. Scale bar, 20 µm. (D) Nanog immunostaining in E4.5 chimeric embryos after blastocyst injection. Nuclei were stained with DAPI (Blue). Scale bars, 20 μm. (E) Summary of ESC (DsRed) contribution to E4.5 embryos. P

Techniques Used: Generated, Injection, Immunostaining, Staining

24) Product Images from "Glioblastoma Cancer Stem Cells Evade Innate Immune Suppression of Self-Renewal through Reduced TLR4 Expression"

Article Title: Glioblastoma Cancer Stem Cells Evade Innate Immune Suppression of Self-Renewal through Reduced TLR4 Expression

Journal: Cell stem cell

doi: 10.1016/j.stem.2016.12.001

Targeting RBBP5 Mimics TLR4 Overexpression in CSCs (A) Validation of two shRNA constructs targeting RBBP5 at the protein level compared with non-targeting vector (NT). Western blots were also stained for the pluripotency factors SOX2, NANOG, and OCT4. CSCs were infected with lentivirus containing one of two different RBBP5 shRNA constructs, selected for stable expression, and assayed within five passages for all experiments. Actin was used as a loading control. (B) The proliferation of CSCs stably expressing RBBP5 shRNA was measured and compared with an NT vector. The growth of the cells over 7 days as determined by CellTiter Glo was normalized to the growth of NT cells. (C and D) Limiting dilution analysis of the effect of RBBP5 knockdown on two different specimens. Cells were plated in a limiting dilution manner, and the number of wells containing spheres was counted after 10 days to generate stem cell frequencies using the online algorithm detailed in the STAR Methods. (E) Mice (n = 7 for KD, n = 6 for NT) were intracranially injected with 1000 T4121 NT or RBBP5-knockdown CSCs, and the time until endpoint was recorded. Kaplan-Meier survival plots are shown, and the log rank p value for significance between groups is shown next to the survival curve. (F) Proliferation was analyzed after RBBP5 overexpression over 7 days in CSCs. Growth was assessed using CellTiter Glo and normalized to that of cells containing an empty vector. Cells were infected with lentivirus, selected for stable expression, and used within five passages for all experiments. (G) Limiting dilution analyses of both CSCs and non-CSCs overexpressing RBBP5 compared with a control vector. Cells were plated in a limiting dilution manner, and the number of wells containing spheres was counted after 10 days to generate stem cell frequencies using the online algorithm detailed in the STAR Methods. .
Figure Legend Snippet: Targeting RBBP5 Mimics TLR4 Overexpression in CSCs (A) Validation of two shRNA constructs targeting RBBP5 at the protein level compared with non-targeting vector (NT). Western blots were also stained for the pluripotency factors SOX2, NANOG, and OCT4. CSCs were infected with lentivirus containing one of two different RBBP5 shRNA constructs, selected for stable expression, and assayed within five passages for all experiments. Actin was used as a loading control. (B) The proliferation of CSCs stably expressing RBBP5 shRNA was measured and compared with an NT vector. The growth of the cells over 7 days as determined by CellTiter Glo was normalized to the growth of NT cells. (C and D) Limiting dilution analysis of the effect of RBBP5 knockdown on two different specimens. Cells were plated in a limiting dilution manner, and the number of wells containing spheres was counted after 10 days to generate stem cell frequencies using the online algorithm detailed in the STAR Methods. (E) Mice (n = 7 for KD, n = 6 for NT) were intracranially injected with 1000 T4121 NT or RBBP5-knockdown CSCs, and the time until endpoint was recorded. Kaplan-Meier survival plots are shown, and the log rank p value for significance between groups is shown next to the survival curve. (F) Proliferation was analyzed after RBBP5 overexpression over 7 days in CSCs. Growth was assessed using CellTiter Glo and normalized to that of cells containing an empty vector. Cells were infected with lentivirus, selected for stable expression, and used within five passages for all experiments. (G) Limiting dilution analyses of both CSCs and non-CSCs overexpressing RBBP5 compared with a control vector. Cells were plated in a limiting dilution manner, and the number of wells containing spheres was counted after 10 days to generate stem cell frequencies using the online algorithm detailed in the STAR Methods. .

Techniques Used: Over Expression, shRNA, Construct, Plasmid Preparation, Western Blot, Staining, Infection, Expressing, Stable Transfection, Mouse Assay, Injection

TLR4 Expression Negatively Correlates with Stemness (A and B) Experimental paradigm to assess the effect of LPS on tumor growth in vivo (A). Mice (n = 7 per arm) were injected with cells from xenograft T3832. After 14 days, tumors were injected with LPS (150 or 300 μg), and tumor volume (B) was measured using electronic calipers 3 days later. Tumor volume at day 3 was normalized to the volume of the tumor at day 0 when LPS was injected. (C) Bulk tumors derived from patient specimens were sorted for TLR4 surface expression using fluorescence-activated cell sorting (FACS). The 20% of cells with the highest expression and the 20% of cells with the lowest expression were plated in a limiting dilution manner, and the number of wells containing spheres was counted after 10 days to generate stem cell frequencies using the online algorithm detailed in the STAR Methods. (D) CSCs were differentiated in DMEM containing 10% fetal bovine serum (FBS) for the indicated lengths of time, and protein levels of TLR4, SOX2, OCT4, NANOG, GFAP, and Actin were assessed by western blotting. .
Figure Legend Snippet: TLR4 Expression Negatively Correlates with Stemness (A and B) Experimental paradigm to assess the effect of LPS on tumor growth in vivo (A). Mice (n = 7 per arm) were injected with cells from xenograft T3832. After 14 days, tumors were injected with LPS (150 or 300 μg), and tumor volume (B) was measured using electronic calipers 3 days later. Tumor volume at day 3 was normalized to the volume of the tumor at day 0 when LPS was injected. (C) Bulk tumors derived from patient specimens were sorted for TLR4 surface expression using fluorescence-activated cell sorting (FACS). The 20% of cells with the highest expression and the 20% of cells with the lowest expression were plated in a limiting dilution manner, and the number of wells containing spheres was counted after 10 days to generate stem cell frequencies using the online algorithm detailed in the STAR Methods. (D) CSCs were differentiated in DMEM containing 10% fetal bovine serum (FBS) for the indicated lengths of time, and protein levels of TLR4, SOX2, OCT4, NANOG, GFAP, and Actin were assessed by western blotting. .

Techniques Used: Expressing, In Vivo, Mouse Assay, Injection, Derivative Assay, Fluorescence, FACS, Western Blot

TLR4 Overexpression Decreases Proliferation and Reduces CSC Maintenance (A and B) Transient TLR4 overexpression in CSCs was measured at both the mRNA (A) and protein (B) levels using qRT-PCR and western blotting, respectively. Western blots were also stained for the pluripotency factors SOX2, NANOG, and OCT4. CSCs were nucleofected with pcDNA3-TLR4-YFP and used for experiments 3 days later. Actin was used as an internal control for qRT-PCR and a loading control for western blotting. (C) Proliferation over 7 days was assessed in CSCs derived from the shown specimens using CellTiter Glo after overexpression of TLR4. Growth was normalized to cells containing an empty vector. (D) CSCs overexpressing TLR4 were treated with LPS at 500 ng/mL, and proliferation was assessed for 7 days relative to cells containing a control vector. (E) Limiting dilution analysis was used to estimate stem cell frequencies in CSCs after nucleofection with control or TLR4 overexpression vectors. Cells were plated in a limiting dilution manner, and the number of wells containing spheres was counted after 10 days to generate stem cell frequencies using the online algorithm detailed in the STAR Methods. .
Figure Legend Snippet: TLR4 Overexpression Decreases Proliferation and Reduces CSC Maintenance (A and B) Transient TLR4 overexpression in CSCs was measured at both the mRNA (A) and protein (B) levels using qRT-PCR and western blotting, respectively. Western blots were also stained for the pluripotency factors SOX2, NANOG, and OCT4. CSCs were nucleofected with pcDNA3-TLR4-YFP and used for experiments 3 days later. Actin was used as an internal control for qRT-PCR and a loading control for western blotting. (C) Proliferation over 7 days was assessed in CSCs derived from the shown specimens using CellTiter Glo after overexpression of TLR4. Growth was normalized to cells containing an empty vector. (D) CSCs overexpressing TLR4 were treated with LPS at 500 ng/mL, and proliferation was assessed for 7 days relative to cells containing a control vector. (E) Limiting dilution analysis was used to estimate stem cell frequencies in CSCs after nucleofection with control or TLR4 overexpression vectors. Cells were plated in a limiting dilution manner, and the number of wells containing spheres was counted after 10 days to generate stem cell frequencies using the online algorithm detailed in the STAR Methods. .

Techniques Used: Over Expression, Quantitative RT-PCR, Western Blot, Staining, Derivative Assay, Plasmid Preparation

25) Product Images from "Suppression of pancreatic adenocarcinoma upregulated factor (PAUF) increases the sensitivity of pancreatic cancer to gemcitabine and 5FU, and inhibits the formation of pancreatic cancer stem like cells"

Article Title: Suppression of pancreatic adenocarcinoma upregulated factor (PAUF) increases the sensitivity of pancreatic cancer to gemcitabine and 5FU, and inhibits the formation of pancreatic cancer stem like cells

Journal: Oncotarget

doi: 10.18632/oncotarget.19458

PAUF overexpression in pancreatic cancer spheres (A) RT-PCR showed that the overexpression of Oct4, Nanog, Stat3, and Sox2 genes in sphere formed from CFPAC-1 cells compare with adherent cells. (B) Up-regulation of secretory PAUF was determined by western blot in CFPAC-1, CAPAN-1 and HPAC spheres (Sp) than adherent (Ad) cells.
Figure Legend Snippet: PAUF overexpression in pancreatic cancer spheres (A) RT-PCR showed that the overexpression of Oct4, Nanog, Stat3, and Sox2 genes in sphere formed from CFPAC-1 cells compare with adherent cells. (B) Up-regulation of secretory PAUF was determined by western blot in CFPAC-1, CAPAN-1 and HPAC spheres (Sp) than adherent (Ad) cells.

Techniques Used: Over Expression, Reverse Transcription Polymerase Chain Reaction, Western Blot

26) Product Images from "Extracellular matrix collagen I promotes the tumor progression of residual hepatocellular carcinoma after heat treatment"

Article Title: Extracellular matrix collagen I promotes the tumor progression of residual hepatocellular carcinoma after heat treatment

Journal: BMC Cancer

doi: 10.1186/s12885-018-4820-9

Sorafenib suppressed the in vivo collagen I-induced tumor progression of heat-treated residual HCC cells. a Mice with the tumors derived from heat-exposed residual MHCC97H cells with collagen I were subjected to treatment. Compared with the control group, sorafenib significantly inhibited tumor growth. b The mRNA expression of Ki-67, twist and Nanog were down-regulated in the sorafenib group. c The changes of PCNA, Nanog, vimentin, E-cadherin, N-cadherin, and ERK activation were detected by Western blot. The p-ERK levels were normalized for ERK. Expression levels of E-cadherin, PCNA, vimentin, N-cadherin and Nanog were normalized to GAPDH. d The immunohistochemical staining of PCNA, E-Cadherin, Nanog and phosphorylated ERK in the tumors (scale bar, 50 μm). **, P
Figure Legend Snippet: Sorafenib suppressed the in vivo collagen I-induced tumor progression of heat-treated residual HCC cells. a Mice with the tumors derived from heat-exposed residual MHCC97H cells with collagen I were subjected to treatment. Compared with the control group, sorafenib significantly inhibited tumor growth. b The mRNA expression of Ki-67, twist and Nanog were down-regulated in the sorafenib group. c The changes of PCNA, Nanog, vimentin, E-cadherin, N-cadherin, and ERK activation were detected by Western blot. The p-ERK levels were normalized for ERK. Expression levels of E-cadherin, PCNA, vimentin, N-cadherin and Nanog were normalized to GAPDH. d The immunohistochemical staining of PCNA, E-Cadherin, Nanog and phosphorylated ERK in the tumors (scale bar, 50 μm). **, P

Techniques Used: In Vivo, Mouse Assay, Derivative Assay, Expressing, Activation Assay, Western Blot, Immunohistochemistry, Staining

Collagen I stimulated the proliferation, motility, and the expression of EMT and progenitor-like markers in heat-treated residual HCC cells. a Compared with the cells cultured on Matrigel, heat-treated residual HCC cells on collagen I displayed a proliferative, protrusive and spindle-like appearance. b Collagen I promoted proliferation of heat-treated residual HCC cells as determined by the WST-1 proliferation assay. The OD (optical density) was measured at 450 nm wavelength. c Compared with Matrigel, collagen I enhanced the motility of heated-exposed residual HCC cells as demonstrated by tracking analysis. d As shown by qRT-PCR, the mRNA expression of Ki-67, twist, and Nanog was increased in heat-exposed residual HCC cells cultured on collagen I versus Matrigel. e The increased expression of PCNA, vimentin, N-cadherin and Nanog protein in heat-exposed residual HCC cell cultured on collagen I was detected by Western blot. Expression levels of target proteins were normalized to the corresponding levels of GAPDH. **, P
Figure Legend Snippet: Collagen I stimulated the proliferation, motility, and the expression of EMT and progenitor-like markers in heat-treated residual HCC cells. a Compared with the cells cultured on Matrigel, heat-treated residual HCC cells on collagen I displayed a proliferative, protrusive and spindle-like appearance. b Collagen I promoted proliferation of heat-treated residual HCC cells as determined by the WST-1 proliferation assay. The OD (optical density) was measured at 450 nm wavelength. c Compared with Matrigel, collagen I enhanced the motility of heated-exposed residual HCC cells as demonstrated by tracking analysis. d As shown by qRT-PCR, the mRNA expression of Ki-67, twist, and Nanog was increased in heat-exposed residual HCC cells cultured on collagen I versus Matrigel. e The increased expression of PCNA, vimentin, N-cadherin and Nanog protein in heat-exposed residual HCC cell cultured on collagen I was detected by Western blot. Expression levels of target proteins were normalized to the corresponding levels of GAPDH. **, P

Techniques Used: Expressing, Cell Culture, Proliferation Assay, Quantitative RT-PCR, Western Blot

Collagen I promoted the in vivo progression of heat-treated residual HCC cells. a The mRNA expression of PCNA, cyclin D1, Ki-67, twist and Nanog was increased in the tumors from heat-exposed residual MHCC97H cells inoculated with collagen I. b The protein expression of PCNA, vimentin, N-cadherin, E-cadherin, Nanog and ERK phosphorylation were detected by Western blot. The p-ERK was normalized for ERK. Expression levels of E-cadherin, PCNA, vimentin, N-cadherin and Nanog were normalized to GAPDH. c The expression of PCNA, E-Cadherin, Nanog and phosphorylated ERK were evaluated using immunohistochemical staining (scale bar, 50 μm). **, P
Figure Legend Snippet: Collagen I promoted the in vivo progression of heat-treated residual HCC cells. a The mRNA expression of PCNA, cyclin D1, Ki-67, twist and Nanog was increased in the tumors from heat-exposed residual MHCC97H cells inoculated with collagen I. b The protein expression of PCNA, vimentin, N-cadherin, E-cadherin, Nanog and ERK phosphorylation were detected by Western blot. The p-ERK was normalized for ERK. Expression levels of E-cadherin, PCNA, vimentin, N-cadherin and Nanog were normalized to GAPDH. c The expression of PCNA, E-Cadherin, Nanog and phosphorylated ERK were evaluated using immunohistochemical staining (scale bar, 50 μm). **, P

Techniques Used: In Vivo, Expressing, Western Blot, Immunohistochemistry, Staining

Collagen I induced the activation of ERK in heat-exposed residual HCC cells. a The up-regulated level of ERK1/2 phosphorylation were induced in heat-exposed residual HCC cells cultured on collagen I versus Matrigel. The p-ERK content was normalized for ERK. b ERK1/2 inhibitor U0126 (25 μM) or sorafenib (5 μM) could reverse the collagen I-promoted proliferative, protrusive and spindle-like appearance of heat-treated residual HCC cells. c ERK1/2 inhibitor (U0126, 25 μM) or sorafenib (5 μM) reversed collagen I-mediated upregulation of ERK1/2 in heat-exposed residual HCC cells. Collagen I-induced upregulation of proliferation (PCNA), EMT (vimentin and N-cadherin), cancer stem cell marker Nanog was markedly reduced in heat-exposed residual HCC cells pretreated with ERK1/2 inhibitor (U0126, 25 μM) or sorafenib (5 μM). The p-ERK content was normalized for ERK. Expression levels of PCNA, vimentin, N-cadherin and Nanog were normalized to Tubulin
Figure Legend Snippet: Collagen I induced the activation of ERK in heat-exposed residual HCC cells. a The up-regulated level of ERK1/2 phosphorylation were induced in heat-exposed residual HCC cells cultured on collagen I versus Matrigel. The p-ERK content was normalized for ERK. b ERK1/2 inhibitor U0126 (25 μM) or sorafenib (5 μM) could reverse the collagen I-promoted proliferative, protrusive and spindle-like appearance of heat-treated residual HCC cells. c ERK1/2 inhibitor (U0126, 25 μM) or sorafenib (5 μM) reversed collagen I-mediated upregulation of ERK1/2 in heat-exposed residual HCC cells. Collagen I-induced upregulation of proliferation (PCNA), EMT (vimentin and N-cadherin), cancer stem cell marker Nanog was markedly reduced in heat-exposed residual HCC cells pretreated with ERK1/2 inhibitor (U0126, 25 μM) or sorafenib (5 μM). The p-ERK content was normalized for ERK. Expression levels of PCNA, vimentin, N-cadherin and Nanog were normalized to Tubulin

Techniques Used: Activation Assay, Cell Culture, Marker, Expressing

27) Product Images from "Involvement of c-Fos in the promotion of cancer stem-like cell properties in head and neck squamous cell carcinoma"

Article Title: Involvement of c-Fos in the promotion of cancer stem-like cell properties in head and neck squamous cell carcinoma

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

doi: 10.1158/1078-0432.CCR-16-2811

Exogenous expression of c-Fos enhances the expression stemness related markers. Cal27-control, Cal27-c-Fos, MDA1386Tu-control and MDA1386Tu-c-Fos cells lysates were subjected to Western blot analysis for (A) Notch1 and Sox2, (B) Nanog and c-Myc, and (C) c-Met expression using specific antibodies. The blots were reprobed with an antibody to actin for comparison of protein loading in each lane. Densitometric analysis were done by using Image J software and shown on the right.
Figure Legend Snippet: Exogenous expression of c-Fos enhances the expression stemness related markers. Cal27-control, Cal27-c-Fos, MDA1386Tu-control and MDA1386Tu-c-Fos cells lysates were subjected to Western blot analysis for (A) Notch1 and Sox2, (B) Nanog and c-Myc, and (C) c-Met expression using specific antibodies. The blots were reprobed with an antibody to actin for comparison of protein loading in each lane. Densitometric analysis were done by using Image J software and shown on the right.

Techniques Used: Expressing, Western Blot, Software

28) Product Images from "High NRF2 level mediates cancer stem cell-like properties of aldehyde dehydrogenase (ALDH)-high ovarian cancer cells: inhibitory role of all-trans retinoic acid in ALDH/NRF2 signaling"

Article Title: High NRF2 level mediates cancer stem cell-like properties of aldehyde dehydrogenase (ALDH)-high ovarian cancer cells: inhibitory role of all-trans retinoic acid in ALDH/NRF2 signaling

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-0903-4

ATRA treatment inhibits NRF2 activation in ALDH-H cells. a After ATRA incubation for 24 h under the indicated concentration (0–20 μM), the protein level of ALDH1A1 was measured in ALDH-H cells. b Transcript levels of ALDH1A1 were assessed in ALDH-H cells after ATRA treatment (10 μM, 24 h). Data represent the mean ± SD from three experiments. c Protein levels of KLF4, NANOG, BCRP, and MDR1 were monitored in ATRA-treated ALDH-H cells. d Transcript levels of KLF4, NANOG , and BCRP were measured in ATRA-treated ALDH-H. Data represent the mean ± SD from three experiments. a P
Figure Legend Snippet: ATRA treatment inhibits NRF2 activation in ALDH-H cells. a After ATRA incubation for 24 h under the indicated concentration (0–20 μM), the protein level of ALDH1A1 was measured in ALDH-H cells. b Transcript levels of ALDH1A1 were assessed in ALDH-H cells after ATRA treatment (10 μM, 24 h). Data represent the mean ± SD from three experiments. c Protein levels of KLF4, NANOG, BCRP, and MDR1 were monitored in ATRA-treated ALDH-H cells. d Transcript levels of KLF4, NANOG , and BCRP were measured in ATRA-treated ALDH-H. Data represent the mean ± SD from three experiments. a P

Techniques Used: Activation Assay, Incubation, Concentration Assay

ALDH-H cells display CSC-like properties. a Transcript levels for KLF4, NANOG, OCT4, SOX2 , and BCRP were assessed in ALDH-L and ALDH-H cells using RT-PCR analysis. Values represent the mean ± SD of three experiments. b Western analysis of KLF4, NANOG, BCRP, and MDR1 was carried out in ALDH-L and ALDH-H cells. Similar blots were obtained in three independent experiments. c , d Cell viability was monitored after incubation with doxorubicin ( c ) or paclitaxel ( d ) for 24 h in ALDH-L and ALDH-H cells. Values represent the mean ± SD from 8 to 10 sampled wells. a P
Figure Legend Snippet: ALDH-H cells display CSC-like properties. a Transcript levels for KLF4, NANOG, OCT4, SOX2 , and BCRP were assessed in ALDH-L and ALDH-H cells using RT-PCR analysis. Values represent the mean ± SD of three experiments. b Western analysis of KLF4, NANOG, BCRP, and MDR1 was carried out in ALDH-L and ALDH-H cells. Similar blots were obtained in three independent experiments. c , d Cell viability was monitored after incubation with doxorubicin ( c ) or paclitaxel ( d ) for 24 h in ALDH-L and ALDH-H cells. Values represent the mean ± SD from 8 to 10 sampled wells. a P

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Western Blot, Incubation

NRF2 activation is involved in CSC-like properties of ALDH-H cells. a Protein levels of NRF2, NQO1, and AKR1C1 were measured in established control (sc) and NRF2 -silenced ALDH-H cells (iNRF2). b ALDH1A1 protein level was assessed in sc and iNRF2 ALDH-H cells. c Protein levels of KLF4, NANOG, BCRP, and MDR1 were determined in sc and iNRF2 ALDH-H cells. Similar blots were obtained in three independent experiments ( a − c ). d Cell viability was monitored after incubation with doxorubicin for 48 h in sc and iNRF2 ALDH-H cells. Values represent the mean ± SD from six sampled wells. a P
Figure Legend Snippet: NRF2 activation is involved in CSC-like properties of ALDH-H cells. a Protein levels of NRF2, NQO1, and AKR1C1 were measured in established control (sc) and NRF2 -silenced ALDH-H cells (iNRF2). b ALDH1A1 protein level was assessed in sc and iNRF2 ALDH-H cells. c Protein levels of KLF4, NANOG, BCRP, and MDR1 were determined in sc and iNRF2 ALDH-H cells. Similar blots were obtained in three independent experiments ( a − c ). d Cell viability was monitored after incubation with doxorubicin for 48 h in sc and iNRF2 ALDH-H cells. Values represent the mean ± SD from six sampled wells. a P

Techniques Used: Activation Assay, Incubation

29) Product Images from "Transcriptional Repression by the BRG1-SWI/SNF Complex Affects the Pluripotency of Human Embryonic Stem Cells"

Article Title: Transcriptional Repression by the BRG1-SWI/SNF Complex Affects the Pluripotency of Human Embryonic Stem Cells

Journal: Stem Cell Reports

doi: 10.1016/j.stemcr.2014.07.004

Unique BRG1-SWI/SNF Composition Is Important to Maintain the Stem Cell Phenotype of hESCs (A) Morphology and AP staining of hESC colonies upon knockdown of BAF155 or BAF170 in hESCs. (B) Expression of BAF components, OCT4, and NANOG was examined by western blotting upon knockdown of BAF155 or BAF170 in hESCs. (C) Flow-cytometry analyses of the surface marker Tra-1-81 on hESCs upon depletion of BAF155 or BAF170. (D) Morphology of hESC colonies upon BAF170 knockdown and/or expression of V5-tagged BAF155 or BAF170 cDNAs. OE, overexpression; Ctrl, empty vector control for overexpression analyses. (E) Morphology of colonies from mESCs and mEpiSCs upon depletion of individual SWI/SNF components. Scale bars in (A), (D), and (E), 400 μm. See also Figures S2 and S3 .
Figure Legend Snippet: Unique BRG1-SWI/SNF Composition Is Important to Maintain the Stem Cell Phenotype of hESCs (A) Morphology and AP staining of hESC colonies upon knockdown of BAF155 or BAF170 in hESCs. (B) Expression of BAF components, OCT4, and NANOG was examined by western blotting upon knockdown of BAF155 or BAF170 in hESCs. (C) Flow-cytometry analyses of the surface marker Tra-1-81 on hESCs upon depletion of BAF155 or BAF170. (D) Morphology of hESC colonies upon BAF170 knockdown and/or expression of V5-tagged BAF155 or BAF170 cDNAs. OE, overexpression; Ctrl, empty vector control for overexpression analyses. (E) Morphology of colonies from mESCs and mEpiSCs upon depletion of individual SWI/SNF components. Scale bars in (A), (D), and (E), 400 μm. See also Figures S2 and S3 .

Techniques Used: Staining, Expressing, Western Blot, Flow Cytometry, Cytometry, Marker, Over Expression, Plasmid Preparation

30) Product Images from "Surface PD-L1, E-cadherin, CD24, and VEGFR2 as markers of epithelial cancer stem cells associated with rapid tumorigenesis"

Article Title: Surface PD-L1, E-cadherin, CD24, and VEGFR2 as markers of epithelial cancer stem cells associated with rapid tumorigenesis

Journal: Scientific Reports

doi: 10.1038/s41598-017-08796-z

Calcium signaling links stemness and tumorigenicity to the transforming phenotype of epithelial UCAPe cells. ( a ) cDNA microarray analysis showing that stemness and calcium signaling regulators were upregulated in UCAPe cells. See Supplemental Tables 2 and 3 for references on selected genes and their links to stemness and calcium signaling. ( b ) Surface immunofluorescence (IF) FACS showing that surface c-Kit ligand (SCF/KL) but not surface c-Kit discriminated UCAPe cells from mesenchymal cells (n = 3). ( c ) Western blotting (WB) showing that constitutive caspase-3 activation and generation of Nanog-D (cleaved fragment related to cellular d ifferentiation) and Nanog-S (cleaved fragment related to s phere formation) discriminated UCAPe cells from mesenchymal cells (top panel), whereas Oct4 and Sox-2 were not expressed in any of the three cell lines (bottom panel). ( d ) Constitutive caspase-3 activation did not lead to DNA fragmentation evaluated by PI-FACS at 24 hours (n = 3). ( e ) Only UCAPe cells were able to transform in response to calcium signaling inhibition by verapamil plus dasatinib (n = 3). ( f,g ) CD24 but not ABCB1 cell surface expression as evaluated by surface IF-FACS marked transforming UCAPe cells (n = 3).
Figure Legend Snippet: Calcium signaling links stemness and tumorigenicity to the transforming phenotype of epithelial UCAPe cells. ( a ) cDNA microarray analysis showing that stemness and calcium signaling regulators were upregulated in UCAPe cells. See Supplemental Tables 2 and 3 for references on selected genes and their links to stemness and calcium signaling. ( b ) Surface immunofluorescence (IF) FACS showing that surface c-Kit ligand (SCF/KL) but not surface c-Kit discriminated UCAPe cells from mesenchymal cells (n = 3). ( c ) Western blotting (WB) showing that constitutive caspase-3 activation and generation of Nanog-D (cleaved fragment related to cellular d ifferentiation) and Nanog-S (cleaved fragment related to s phere formation) discriminated UCAPe cells from mesenchymal cells (top panel), whereas Oct4 and Sox-2 were not expressed in any of the three cell lines (bottom panel). ( d ) Constitutive caspase-3 activation did not lead to DNA fragmentation evaluated by PI-FACS at 24 hours (n = 3). ( e ) Only UCAPe cells were able to transform in response to calcium signaling inhibition by verapamil plus dasatinib (n = 3). ( f,g ) CD24 but not ABCB1 cell surface expression as evaluated by surface IF-FACS marked transforming UCAPe cells (n = 3).

Techniques Used: Microarray, Immunofluorescence, FACS, Western Blot, Activation Assay, Inhibition, Expressing

31) Product Images from "The influence of retinoic acid on the human oligodendrocyte precursor cells by RNA-sequencing"

Article Title: The influence of retinoic acid on the human oligodendrocyte precursor cells by RNA-sequencing

Journal: Biochemistry and Biophysics Reports

doi: 10.1016/j.bbrep.2016.12.004

Characterization of human ESCs, NSCs and OPCs cell populations. Immunofluorescence staining of specific genes and lineage markers expressed in ESC (A~C), NSC (D–F) and non-sorted OPC (G–H). (A) Oct 3/4 (95±4.3%) and (B) Nanog (89±5.4%) were expressed by all ESC. NSC lineage was confirmed with (D) Nestin (98±2.7%) and (E) Pax6 (87±8.9%) expression. OPC expressed lineage specific markers (G) PDGFRa (65±7.3%) and (H) NG2 (63±7.3%) with less than 2% of cells expressing the immature marker Nestin (H). Cells were counterstained with the nuclear stain DAPI (C, F, G and H). Lineage marker expression values were quantified as the percentage of total DAPI + cells in each culture field of view that expressed the lineage-specific marker of interest. Data are expressed as the mean percent positive cells±SEM from at least 5 random fields of view from at least 3 independent cultures. Representative western blots of lineage-specific protein expression (I), confirming immunofluorescence staining. (J) The relative expression level was measured against GAPDH. Data is expressed as the mean relative expression of protein±standard deviation of three independent experiments. Analysis of statistical significance was calculated by using t-test from Graph pad. Statistical comparisons of significance are shown with adjoining lines: **p
Figure Legend Snippet: Characterization of human ESCs, NSCs and OPCs cell populations. Immunofluorescence staining of specific genes and lineage markers expressed in ESC (A~C), NSC (D–F) and non-sorted OPC (G–H). (A) Oct 3/4 (95±4.3%) and (B) Nanog (89±5.4%) were expressed by all ESC. NSC lineage was confirmed with (D) Nestin (98±2.7%) and (E) Pax6 (87±8.9%) expression. OPC expressed lineage specific markers (G) PDGFRa (65±7.3%) and (H) NG2 (63±7.3%) with less than 2% of cells expressing the immature marker Nestin (H). Cells were counterstained with the nuclear stain DAPI (C, F, G and H). Lineage marker expression values were quantified as the percentage of total DAPI + cells in each culture field of view that expressed the lineage-specific marker of interest. Data are expressed as the mean percent positive cells±SEM from at least 5 random fields of view from at least 3 independent cultures. Representative western blots of lineage-specific protein expression (I), confirming immunofluorescence staining. (J) The relative expression level was measured against GAPDH. Data is expressed as the mean relative expression of protein±standard deviation of three independent experiments. Analysis of statistical significance was calculated by using t-test from Graph pad. Statistical comparisons of significance are shown with adjoining lines: **p

Techniques Used: Immunofluorescence, Staining, Expressing, Marker, Western Blot, Standard Deviation

32) Product Images from "Galectin‐3 promotes CXCR2 to augment the stem‐like property of renal cell carcinoma. Galectin‐3 promotes CXCR2 to augment the stem‐like property of renal cell carcinoma"

Article Title: Galectin‐3 promotes CXCR2 to augment the stem‐like property of renal cell carcinoma. Galectin‐3 promotes CXCR2 to augment the stem‐like property of renal cell carcinoma

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.13860

Enrichment of tumour spheres and galectin‐3 was highly expressed in the tumour spheres of renal cancer cell lines. (A) RCC cells were cultured in a defined serum‐free selection tumour sphere medium for 21 days. (B) The ratio of sphere formation (%) in the RCC cells was measured. (C) The mRNA levels of stemness‐related genes were evaluated in the parental and tumour spheres of kidney cancer cells using RT ‐ qPCR . (D) The protein levels of stemness‐related genes Nanog, Sox2 and Oct4 were analysed using western blotting. (E) The mRNA levels of the galectin family were detected in parental A‐498 (P) and A‐498 spheres (S) using RT ‐ qPCR . (F) The mRNA levels of galectin‐3 were also analysed in the parental and tumour spheres of Caki‐1 and ACHN cells. The protein levels of galectin‐3 (Gal‐3) in both the parental and spheres of A‐498 and Caki cells were analysed using Western blotting. The reported results are representative of three independent experiments. * P
Figure Legend Snippet: Enrichment of tumour spheres and galectin‐3 was highly expressed in the tumour spheres of renal cancer cell lines. (A) RCC cells were cultured in a defined serum‐free selection tumour sphere medium for 21 days. (B) The ratio of sphere formation (%) in the RCC cells was measured. (C) The mRNA levels of stemness‐related genes were evaluated in the parental and tumour spheres of kidney cancer cells using RT ‐ qPCR . (D) The protein levels of stemness‐related genes Nanog, Sox2 and Oct4 were analysed using western blotting. (E) The mRNA levels of the galectin family were detected in parental A‐498 (P) and A‐498 spheres (S) using RT ‐ qPCR . (F) The mRNA levels of galectin‐3 were also analysed in the parental and tumour spheres of Caki‐1 and ACHN cells. The protein levels of galectin‐3 (Gal‐3) in both the parental and spheres of A‐498 and Caki cells were analysed using Western blotting. The reported results are representative of three independent experiments. * P

Techniques Used: Cell Culture, Selection, Quantitative RT-PCR, Western Blot

33) Product Images from "Nanog Signaling Mediates Radioresistance in ALDH-Positive Breast Cancer Cells"

Article Title: Nanog Signaling Mediates Radioresistance in ALDH-Positive Breast Cancer Cells

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms20051151

Nanog promotes ALDH activity and radioresistance through Akt and Notch1 proteins. ( A1 ) siRNA knockdown of Nanog or ( A2 ) Nanog overexpression was performed as described in the Materials and Methods Section in MCF-7, HBL-100 and SKBR3 cells. The transfection efficiency was tested after 48 h by Western blotting. ( B ) ALDH activity as a function of Nanog expression under Notch downregulation and Akt inhibition. Twenty-four hours after Nanog overexpression, cells were transfected with Notch1 siRNA. Forty-eight hours later, ALDH activity was measured with an Aldefluor assay ( B1 ). Twenty-four hours after Nanog overexpression, cells were treated with the Akt inhibitor MK-2206 (250 nM for MCF-7 and 1µM for HBL-100), and ALDH activity was measured 24 h later ( B2 ). Bars represent relative ALDH activity ± the standard deviation (SD) of three independent experiments ( n = 6; ** p
Figure Legend Snippet: Nanog promotes ALDH activity and radioresistance through Akt and Notch1 proteins. ( A1 ) siRNA knockdown of Nanog or ( A2 ) Nanog overexpression was performed as described in the Materials and Methods Section in MCF-7, HBL-100 and SKBR3 cells. The transfection efficiency was tested after 48 h by Western blotting. ( B ) ALDH activity as a function of Nanog expression under Notch downregulation and Akt inhibition. Twenty-four hours after Nanog overexpression, cells were transfected with Notch1 siRNA. Forty-eight hours later, ALDH activity was measured with an Aldefluor assay ( B1 ). Twenty-four hours after Nanog overexpression, cells were treated with the Akt inhibitor MK-2206 (250 nM for MCF-7 and 1µM for HBL-100), and ALDH activity was measured 24 h later ( B2 ). Bars represent relative ALDH activity ± the standard deviation (SD) of three independent experiments ( n = 6; ** p

Techniques Used: Activity Assay, Over Expression, Transfection, Western Blot, Expressing, Inhibition, Standard Deviation

34) Product Images from "Large-scale expansion of Wharton’s jelly-derived mesenchymal stem cells on gelatin microbeads, with retention of self-renewal and multipotency characteristics and the capacity for enhancing skin wound healing"

Article Title: Large-scale expansion of Wharton’s jelly-derived mesenchymal stem cells on gelatin microbeads, with retention of self-renewal and multipotency characteristics and the capacity for enhancing skin wound healing

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-015-0031-3

Expression of pluripotency factors in 2D and 3D cultured cells. Expression of embryonic markers (green) in 2D and 3D cultured cells, as determined by immunocytochemistry. Cell nuclei were stained with Hoechst 33342 (blue). Scale bar: 20 μm (A) . Flow cytometry analysis of C-MYC, OCT4, NANOG, and SOX2 expression in 2D and 3D cultured cells (B) . Percentage of cells positive for the expression of embryonic markers as assayed by flow cytometry (n = 4, P > 0.01) (C) . Quantitative real-time polymerase chain reaction assay and Western blot assay for the four factors (D to G ). (n = 4, P > 0.01). 2D, plate cultures; 3D, spinning bottle cultures.
Figure Legend Snippet: Expression of pluripotency factors in 2D and 3D cultured cells. Expression of embryonic markers (green) in 2D and 3D cultured cells, as determined by immunocytochemistry. Cell nuclei were stained with Hoechst 33342 (blue). Scale bar: 20 μm (A) . Flow cytometry analysis of C-MYC, OCT4, NANOG, and SOX2 expression in 2D and 3D cultured cells (B) . Percentage of cells positive for the expression of embryonic markers as assayed by flow cytometry (n = 4, P > 0.01) (C) . Quantitative real-time polymerase chain reaction assay and Western blot assay for the four factors (D to G ). (n = 4, P > 0.01). 2D, plate cultures; 3D, spinning bottle cultures.

Techniques Used: Expressing, Cell Culture, Immunocytochemistry, Staining, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Western Blot

35) Product Images from "Cancer-associated fibroblasts promote stem cell-like properties of hepatocellular carcinoma cells through IL-6/STAT3/Notch signaling"

Article Title: Cancer-associated fibroblasts promote stem cell-like properties of hepatocellular carcinoma cells through IL-6/STAT3/Notch signaling

Journal: American Journal of Cancer Research

doi:

IL-6-neutralizing antibody inhibited the CAFs-induced promotion of stem cell-like properties in HCC cells. (A-C) IL-6-neutralizing antibody inhibited the effects of CAFs on the sphere-forming (A), colony-forming (B), and migration and invasion (C) abilities of MHCC-97H cells. Scale bar, 100 μm. (D) IL-6-neutralizing antibody abolished CAFs-induced overexpression of Nanog, Sox2, and Oct4 in MHCC-97H cells. Data are shown as means ± SD from at least three independent experiments. (*P
Figure Legend Snippet: IL-6-neutralizing antibody inhibited the CAFs-induced promotion of stem cell-like properties in HCC cells. (A-C) IL-6-neutralizing antibody inhibited the effects of CAFs on the sphere-forming (A), colony-forming (B), and migration and invasion (C) abilities of MHCC-97H cells. Scale bar, 100 μm. (D) IL-6-neutralizing antibody abolished CAFs-induced overexpression of Nanog, Sox2, and Oct4 in MHCC-97H cells. Data are shown as means ± SD from at least three independent experiments. (*P

Techniques Used: Migration, Over Expression

IL-6 secreted from CAFs enhances the stem cell-like properties of HCC cells. (A) Cytokine profiles of the CM from MHCC-97H cells and MHCC-97H-stimulated CAFs were screened using the RayBio human cytokine antibody array. IL-6 was the most significantly secreted cytokine from CAFs. (B) There was dramatically more soluble IL-6 in the CM from MHCC-97H-stimulated CAFs compared with that of MHCC-97H cells as assessed by ELISA assay. (C) Nanog, Sox2, and Oct4 were overexpressed in MHCC-97H cells treated with IL6. (D-F) IL-6 enhanced the sphere-forming (D), colony-forming (E), and migration and invasion (F) abilities of MHCC-97H cells. Scale bar, 100 μm. Data are shown as the means ± SD from at least three independent experiments. (*P
Figure Legend Snippet: IL-6 secreted from CAFs enhances the stem cell-like properties of HCC cells. (A) Cytokine profiles of the CM from MHCC-97H cells and MHCC-97H-stimulated CAFs were screened using the RayBio human cytokine antibody array. IL-6 was the most significantly secreted cytokine from CAFs. (B) There was dramatically more soluble IL-6 in the CM from MHCC-97H-stimulated CAFs compared with that of MHCC-97H cells as assessed by ELISA assay. (C) Nanog, Sox2, and Oct4 were overexpressed in MHCC-97H cells treated with IL6. (D-F) IL-6 enhanced the sphere-forming (D), colony-forming (E), and migration and invasion (F) abilities of MHCC-97H cells. Scale bar, 100 μm. Data are shown as the means ± SD from at least three independent experiments. (*P

Techniques Used: Ab Array, Enzyme-linked Immunosorbent Assay, Migration

36) Product Images from "Demarcation of Stable Subpopulations within the Pluripotent hESC Compartment"

Article Title: Demarcation of Stable Subpopulations within the Pluripotent hESC Compartment

Journal: PLoS ONE

doi: 10.1371/journal.pone.0057276

Distribution of pluripotent markers in undifferentiated REX1 Ven/w hPSCs. A) Immunocytochemistry for OCT4 (red), NANOG (blue, bottom row) or p21 (blue, top row) in REX1 Ven/w cells. Scale bar = 120 microns. B) Quantification of REX1Venus, OCT4 and NANOG expression by high content imaging and automated cell level analysis in undifferentiated cultures (Day 0) and during a time course of retinoic acid induced differentiation (n = 4). V = REX1Venus, O = OCT4, N = NANOG, + = positive, − = negative.
Figure Legend Snippet: Distribution of pluripotent markers in undifferentiated REX1 Ven/w hPSCs. A) Immunocytochemistry for OCT4 (red), NANOG (blue, bottom row) or p21 (blue, top row) in REX1 Ven/w cells. Scale bar = 120 microns. B) Quantification of REX1Venus, OCT4 and NANOG expression by high content imaging and automated cell level analysis in undifferentiated cultures (Day 0) and during a time course of retinoic acid induced differentiation (n = 4). V = REX1Venus, O = OCT4, N = NANOG, + = positive, − = negative.

Techniques Used: Immunocytochemistry, Expressing, Imaging

Co-incidence of pluripotency markers in undifferentiated REX1 Ven/w hPSC cultures. A) Output of imaging analysis measuring the co-expression of REX1Venus (VEN), OCT4 or NANOG (NAN) pluripotency markers in undifferentiated (Undiff) hESCs and cells treated with retinoic acid (RA) for 2 days, n = 4. B) Output of cell level analysis of p21 co-expression with REX1Venus (VEN) or OCT4 positive cells, n = 4.
Figure Legend Snippet: Co-incidence of pluripotency markers in undifferentiated REX1 Ven/w hPSC cultures. A) Output of imaging analysis measuring the co-expression of REX1Venus (VEN), OCT4 or NANOG (NAN) pluripotency markers in undifferentiated (Undiff) hESCs and cells treated with retinoic acid (RA) for 2 days, n = 4. B) Output of cell level analysis of p21 co-expression with REX1Venus (VEN) or OCT4 positive cells, n = 4.

Techniques Used: Imaging, Expressing

37) Product Images from "RASSF1A uncouples Wnt from Hippo signalling and promotes YAP mediated differentiation via p73"

Article Title: RASSF1A uncouples Wnt from Hippo signalling and promotes YAP mediated differentiation via p73

Journal: Nature Communications

doi: 10.1038/s41467-017-02786-5

Premature activation of RASSF1A impairs embryogenesis via p73. a Indicated gene expression levels in published GEO data sets GDS3599 and GDS2156. b Temporal expression of Oct4 and Rassf1A mRNA in the pre-implantation embryo (% of maximum expression) from published GEO data sets GDS752 (black colour) and GDS814 (red colour). c Nuclear localisation of YAP during early stages of pre-implantation development. d Nanog immunofluorescence and e representative images of embryos microinjected with either control (zsCtrl) or RASSF1A-expressing (zsR1A) vectors stained for stem cell marker expression. Bar graph showing total OCT4 protein levels across all embryos in zsR1A versus zsCtrl. f 'Kill curve' to determine lethal RASSF1A concentration in pre-implantation embryos. The graph expresses percentage (%) of blastocyst-forming embryos at the indicated RASSF1A concentration. g Viability of embryos in response to RASSF1A expression and/or sip73 microinjection, n = 15. BF bright field channel. Scale bars: 10–50 μm. *P
Figure Legend Snippet: Premature activation of RASSF1A impairs embryogenesis via p73. a Indicated gene expression levels in published GEO data sets GDS3599 and GDS2156. b Temporal expression of Oct4 and Rassf1A mRNA in the pre-implantation embryo (% of maximum expression) from published GEO data sets GDS752 (black colour) and GDS814 (red colour). c Nuclear localisation of YAP during early stages of pre-implantation development. d Nanog immunofluorescence and e representative images of embryos microinjected with either control (zsCtrl) or RASSF1A-expressing (zsR1A) vectors stained for stem cell marker expression. Bar graph showing total OCT4 protein levels across all embryos in zsR1A versus zsCtrl. f 'Kill curve' to determine lethal RASSF1A concentration in pre-implantation embryos. The graph expresses percentage (%) of blastocyst-forming embryos at the indicated RASSF1A concentration. g Viability of embryos in response to RASSF1A expression and/or sip73 microinjection, n = 15. BF bright field channel. Scale bars: 10–50 μm. *P

Techniques Used: Activation Assay, Expressing, Immunofluorescence, Staining, Marker, Concentration Assay

38) Product Images from "HSA21 Single-Minded 2 (Sim2) Binding Sites Co-Localize with Super-Enhancers and Pioneer Transcription Factors in Pluripotent Mouse ES Cells"

Article Title: HSA21 Single-Minded 2 (Sim2) Binding Sites Co-Localize with Super-Enhancers and Pioneer Transcription Factors in Pluripotent Mouse ES Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0126475

Overlapping SIM2 occupancy with master transcription factor binding sites. a. Frequency distribution of OCT4, SOX2 and NANOG DNA binding sites in a 40kb window centered to the newly identified SIM2 DNA binding sites. Plots show a significant enrichment for the OSN binding sites at the SIM2 peak localization in SIM2 A6 expressing cells. Pie charts show the proportion of SIM2 DNA binding sites overlapping with the OCT4, SOX2 or NANOG binding sites (in grey) (100bp window). p = Fisher’s exact test p-value; F score: measure of the significance of the association (1 = perfect match). b. Protein co-immunoprecipitation experiments of SIM2-FLAG with endogenous OCT4, SOX2, KLF4 (left panel) and NANOG (right panel). Cellular protein extracts from Sim2 expressing cells (A6) or EB3 cells were immunoprecipitated by using antibodies directed against each of the pluripotency factors (N-terminal and C-terminal part of NANOG) or IgG as a negative control for co-immunoprecipitation. Associated proteins were immunoblotted using an anti-FLAG antibody. Red star shows the SIM2-FLAG protein, blue star the signal given by the recognition of the IgG heavy chains. Ø: Beads only; kDa: kilodaltons; protein lysat: protein lysat was loaded as an input control for the immunoblot.
Figure Legend Snippet: Overlapping SIM2 occupancy with master transcription factor binding sites. a. Frequency distribution of OCT4, SOX2 and NANOG DNA binding sites in a 40kb window centered to the newly identified SIM2 DNA binding sites. Plots show a significant enrichment for the OSN binding sites at the SIM2 peak localization in SIM2 A6 expressing cells. Pie charts show the proportion of SIM2 DNA binding sites overlapping with the OCT4, SOX2 or NANOG binding sites (in grey) (100bp window). p = Fisher’s exact test p-value; F score: measure of the significance of the association (1 = perfect match). b. Protein co-immunoprecipitation experiments of SIM2-FLAG with endogenous OCT4, SOX2, KLF4 (left panel) and NANOG (right panel). Cellular protein extracts from Sim2 expressing cells (A6) or EB3 cells were immunoprecipitated by using antibodies directed against each of the pluripotency factors (N-terminal and C-terminal part of NANOG) or IgG as a negative control for co-immunoprecipitation. Associated proteins were immunoblotted using an anti-FLAG antibody. Red star shows the SIM2-FLAG protein, blue star the signal given by the recognition of the IgG heavy chains. Ø: Beads only; kDa: kilodaltons; protein lysat: protein lysat was loaded as an input control for the immunoblot.

Techniques Used: Binding Assay, Expressing, Immunoprecipitation, Negative Control

39) Product Images from "Reversal of Aberrant Cancer Methylome and Transcriptome upon Direct Reprogramming of Lung Cancer Cells"

Article Title: Reversal of Aberrant Cancer Methylome and Transcriptome upon Direct Reprogramming of Lung Cancer Cells

Journal: Scientific Reports

doi: 10.1038/srep00592

Generation and characterization of iPC from NSCLC lines. (a) Morphology of parental IMR90 fibroblasts, H358 and H460 cancer cells. Formation of ES-like colonies in IMR90, H358 and H460 cells on day 8 post-infection of four factors. Upon seeding onto feeder layer on day 15 post-infection, iPS and iPCs formed flat and round edged colonies like (b) ES cells i.e. H1 and HES-3. Similar to H1, iPSIMR90, iPCH358 and iPCH460 colonies stained positive for AP. Representative images of individual colonies derived from IMR90, H358 and H460 showed positive staining for pluripotency markers i.e. TRA-1-60 (green) and Nanog (red) respectively. Nuclei were stained with Hoechst 33342 (blue). Scale bars: 20 - 500μm. (c) iPS and iPC expressed high levels of ES cell markers namely SOX2 , NANOG , FGF4 and OCT3/4 as compared to their parental cells. The mRNA expression was normalized to GAPDH mRNA expression. (d) iPS and iPC depicted higher TA when compared to their respective parental cells. Data are presented as mean ± SD.
Figure Legend Snippet: Generation and characterization of iPC from NSCLC lines. (a) Morphology of parental IMR90 fibroblasts, H358 and H460 cancer cells. Formation of ES-like colonies in IMR90, H358 and H460 cells on day 8 post-infection of four factors. Upon seeding onto feeder layer on day 15 post-infection, iPS and iPCs formed flat and round edged colonies like (b) ES cells i.e. H1 and HES-3. Similar to H1, iPSIMR90, iPCH358 and iPCH460 colonies stained positive for AP. Representative images of individual colonies derived from IMR90, H358 and H460 showed positive staining for pluripotency markers i.e. TRA-1-60 (green) and Nanog (red) respectively. Nuclei were stained with Hoechst 33342 (blue). Scale bars: 20 - 500μm. (c) iPS and iPC expressed high levels of ES cell markers namely SOX2 , NANOG , FGF4 and OCT3/4 as compared to their parental cells. The mRNA expression was normalized to GAPDH mRNA expression. (d) iPS and iPC depicted higher TA when compared to their respective parental cells. Data are presented as mean ± SD.

Techniques Used: Infection, Staining, Derivative Assay, Expressing

40) Product Images from "miR-612 suppresses stem cell-like property of hepatocellular carcinoma cells by modulating Sp1/Nanog signaling"

Article Title: miR-612 suppresses stem cell-like property of hepatocellular carcinoma cells by modulating Sp1/Nanog signaling

Journal: Cell Death & Disease

doi: 10.1038/cddis.2016.282

Sp1 suppresses the stemness of HCC in vitro . ( a ) Western blot analysis to evaluate the expression levels of Sp1 in HepG2, HCCLM3 wild-type (WT), negative control (NC) and Sp1 knockdown (shRNA1, shRNA 2, shRNA 3, shRNA4) cells. ( b and c ) Representative images and statistical results of HepG2 and HCCLM3 tumor spheres ( > 50 μ m) after indicated treatments ( n =3; scale bar: 50 μ m). ( d ) Western blot analysis of Nanog, EpCAM, CD133 in HepG2, HCCLM3 wild-type (WT), negative control (NC) and Sp1 knockdown (shRNA4) cells. Statistical analysis by paired t -test (* P
Figure Legend Snippet: Sp1 suppresses the stemness of HCC in vitro . ( a ) Western blot analysis to evaluate the expression levels of Sp1 in HepG2, HCCLM3 wild-type (WT), negative control (NC) and Sp1 knockdown (shRNA1, shRNA 2, shRNA 3, shRNA4) cells. ( b and c ) Representative images and statistical results of HepG2 and HCCLM3 tumor spheres ( > 50 μ m) after indicated treatments ( n =3; scale bar: 50 μ m). ( d ) Western blot analysis of Nanog, EpCAM, CD133 in HepG2, HCCLM3 wild-type (WT), negative control (NC) and Sp1 knockdown (shRNA4) cells. Statistical analysis by paired t -test (* P

Techniques Used: In Vitro, Western Blot, Expressing, Negative Control, shRNA

Related Articles

Marker:

Article Title: Generation of Human‐Induced Pluripotent Stem Cells From Anterior Cruciate Ligament
Article Snippet: .. Cells were then incubated with primary antibodies against markers of pluripotency; NANOG (1:400, cat. no. 4903; Cell Signaling Technology, London, UK), OCT‐4 (1:100, cat. no. 611202; BD Biosciences, Oxford, UK), SOX2 (1:400, cat. no. 3579; Cell Signaling Technology), SSEA‐3 (1:200, cat. no MAB1434; R & D Systems, Abingdon, UK), SSEA‐4 (1:200, cat. no MAB1435; R & D Systems), TRA‐1‐60 (1:200, cat. no. Ab16288; Abcam, Cambridge, UK), TRA‐1‐81 (1:200, cat. no. Ab16289; Abcam), marker of early differentiation; SSEA‐1 (1:200, cat. no. MAB2155; R & D Systems), marker of mesoderm; α‐smooth muscle actin (αSMA) (1:100, cat. no. MAB1420; R & D Systems), marker of endoderm; GATA6 (1:1600, cat. no. 5851; Cell Signaling Technology) and marker of ectoderm; Neurofilament (1:100, cat. no. 2837; Cell Signaling Technology), in the presence of 1% goat serum, followed by Alexa Fluor secondary antibodies (1:200; Thermo Fisher Scientific) and nuclei stained using 4′,6‐diamidino‐2‐phenylindole (DAPI) (cat no. D1306; Thermo Fisher Scientific). .. Images were captured using BX51 fluorescence microscope (Olympus, Southend‐on‐Sea, UK).

Staining:

Article Title: Demarcation of Stable Subpopulations within the Pluripotent hESC Compartment
Article Snippet: .. Cells were stained with Hoechst 33342 and primary antibodies for OCT4 (mouse monoclonal 1∶200, BD #611203), NANOG (rabbit monoclonal 1∶400, Cell Signaling #4903), GATA4 (rabbit polyclonal 1∶300, Sana Cruz #sc-9053) and p21 (rabbit monoclonal 1∶400, Cell Signaling #2947) in 1% BSA in PBS for 2 hours at room temperature or 4°C overnight, washed with PBS and stained with secondary antibodies (Goat anti Mouse AF546 1∶500, Invitrogen # A-11030; Donkey anti Rabbit AF647 1∶500, Invitrogen #A-31573). .. Analysis was performed as previously described , briefly: plates were imaged on a Cellomics ArrayScan HCS reader (Thermo Scientific) or an Operatta High Content Screening System (Perkin Elmer) and images uploaded to a Columbus database (Perkin Elmer) and image analysis of immunofluorescence and reporter fluorescence was performed using Acapella high content and analysis software (Perkin Elmer).

Article Title: Generation of Human‐Induced Pluripotent Stem Cells From Anterior Cruciate Ligament
Article Snippet: .. Cells were then incubated with primary antibodies against markers of pluripotency; NANOG (1:400, cat. no. 4903; Cell Signaling Technology, London, UK), OCT‐4 (1:100, cat. no. 611202; BD Biosciences, Oxford, UK), SOX2 (1:400, cat. no. 3579; Cell Signaling Technology), SSEA‐3 (1:200, cat. no MAB1434; R & D Systems, Abingdon, UK), SSEA‐4 (1:200, cat. no MAB1435; R & D Systems), TRA‐1‐60 (1:200, cat. no. Ab16288; Abcam, Cambridge, UK), TRA‐1‐81 (1:200, cat. no. Ab16289; Abcam), marker of early differentiation; SSEA‐1 (1:200, cat. no. MAB2155; R & D Systems), marker of mesoderm; α‐smooth muscle actin (αSMA) (1:100, cat. no. MAB1420; R & D Systems), marker of endoderm; GATA6 (1:1600, cat. no. 5851; Cell Signaling Technology) and marker of ectoderm; Neurofilament (1:100, cat. no. 2837; Cell Signaling Technology), in the presence of 1% goat serum, followed by Alexa Fluor secondary antibodies (1:200; Thermo Fisher Scientific) and nuclei stained using 4′,6‐diamidino‐2‐phenylindole (DAPI) (cat no. D1306; Thermo Fisher Scientific). .. Images were captured using BX51 fluorescence microscope (Olympus, Southend‐on‐Sea, UK).

Chloramphenicol Acetyltransferase Assay:

Article Title: Cancer-associated fibroblasts promote stem cell-like properties of hepatocellular carcinoma cells through IL-6/STAT3/Notch signaling
Article Snippet: .. Primary antibodies for Notch1 (cat3608), Hes1 (cat#11988), Nanog (cat#4903), Sox2 (cat#3579), Oct4 (cat#2750), STAT3 (cat#8768), and p-STAT3 (cat#9145) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). .. Monoclonal mouse anti-β-actin (cat#ab8226; Abcam) was used as an internal control.

other:

Article Title: Isolation of cancer cells with augmented spheroid-forming capability using a novel tool equipped with removable filter
Article Snippet: Polyclonal antibodies raised against the following proteins were purchased from the indicated commercial sources: ARHGEF3 (Abcam, #ab154263), HHAT (ABGENT, #AP5503a for Figures , ; and Sigma, #SAB2105163 for Figure ), OCT-4A (Cell Signaling, #2840), SOX2 (Cell Signaling, #3579), NANOG (Cell Signaling, #4903), mTOR (Cell Signaling, #2983), mTOR-pS2448 (Cell Signaling, #5536), AKT (Cell Signaling, #75692), and AKT-pS308 (Cell Signaling, #13038).

Incubation:

Article Title: Generation of Human‐Induced Pluripotent Stem Cells From Anterior Cruciate Ligament
Article Snippet: .. Cells were then incubated with primary antibodies against markers of pluripotency; NANOG (1:400, cat. no. 4903; Cell Signaling Technology, London, UK), OCT‐4 (1:100, cat. no. 611202; BD Biosciences, Oxford, UK), SOX2 (1:400, cat. no. 3579; Cell Signaling Technology), SSEA‐3 (1:200, cat. no MAB1434; R & D Systems, Abingdon, UK), SSEA‐4 (1:200, cat. no MAB1435; R & D Systems), TRA‐1‐60 (1:200, cat. no. Ab16288; Abcam, Cambridge, UK), TRA‐1‐81 (1:200, cat. no. Ab16289; Abcam), marker of early differentiation; SSEA‐1 (1:200, cat. no. MAB2155; R & D Systems), marker of mesoderm; α‐smooth muscle actin (αSMA) (1:100, cat. no. MAB1420; R & D Systems), marker of endoderm; GATA6 (1:1600, cat. no. 5851; Cell Signaling Technology) and marker of ectoderm; Neurofilament (1:100, cat. no. 2837; Cell Signaling Technology), in the presence of 1% goat serum, followed by Alexa Fluor secondary antibodies (1:200; Thermo Fisher Scientific) and nuclei stained using 4′,6‐diamidino‐2‐phenylindole (DAPI) (cat no. D1306; Thermo Fisher Scientific). .. Images were captured using BX51 fluorescence microscope (Olympus, Southend‐on‐Sea, UK).

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    Cell Signaling Technology Inc nanog
    Neuronal and synaptic maturation during differentiation of hiPSC to cortical neurons. Representative images from immunocytochemistry staining during differentiation. (A and B) <t>NANOG</t> and <t>OCT-4</t> were strongly stained on d0 and the stainings became weak on d4, while KI-67 had similar staining intensities on d0 and d4. (C) PAX-6, a primary neuro-progenitor expression decreased from d20 to d40 of differentiation. (D) Staining for neuron specific tubulin, TUJ-1 was weak on d30 but strong staining was observed on d60. (E) SV-2, a pre-synaptic protein, staining was weak on d60. On d120, a strong punctuate staining was observed (arrows). (F) PSD-95, a post-synaptic density protein, staining was weak on d60. On d120, the staining had intensified and became punctate (arrows). Green or red = protein of interest, blue = nuclei (DAPI), Scale bar = 20 μm.
    Nanog, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 89 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rat anti nanog
    Expression of <t>β-catenin</t> and <t>Nanog</t> in EpSCs treated with Wnt antagonist, Dkk-1. Real-time PCR analysis of β-catenin mRNA (A) and Nanog mRNA (B) were obtained at different time points (day 1- day 12) in the control, SP treated group and SP with Dkk-1 treated group. The result showed that there were significant differences between the SP group and SP with Dkk-1 group on or after day 3 (*P
    Rat Anti Nanog, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 89/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Neuronal and synaptic maturation during differentiation of hiPSC to cortical neurons. Representative images from immunocytochemistry staining during differentiation. (A and B) NANOG and OCT-4 were strongly stained on d0 and the stainings became weak on d4, while KI-67 had similar staining intensities on d0 and d4. (C) PAX-6, a primary neuro-progenitor expression decreased from d20 to d40 of differentiation. (D) Staining for neuron specific tubulin, TUJ-1 was weak on d30 but strong staining was observed on d60. (E) SV-2, a pre-synaptic protein, staining was weak on d60. On d120, a strong punctuate staining was observed (arrows). (F) PSD-95, a post-synaptic density protein, staining was weak on d60. On d120, the staining had intensified and became punctate (arrows). Green or red = protein of interest, blue = nuclei (DAPI), Scale bar = 20 μm.

    Journal: Neurochemistry International

    Article Title: Expression and secretion of synaptic proteins during stem cell differentiation to cortical neurons

    doi: 10.1016/j.neuint.2018.10.014

    Figure Lengend Snippet: Neuronal and synaptic maturation during differentiation of hiPSC to cortical neurons. Representative images from immunocytochemistry staining during differentiation. (A and B) NANOG and OCT-4 were strongly stained on d0 and the stainings became weak on d4, while KI-67 had similar staining intensities on d0 and d4. (C) PAX-6, a primary neuro-progenitor expression decreased from d20 to d40 of differentiation. (D) Staining for neuron specific tubulin, TUJ-1 was weak on d30 but strong staining was observed on d60. (E) SV-2, a pre-synaptic protein, staining was weak on d60. On d120, a strong punctuate staining was observed (arrows). (F) PSD-95, a post-synaptic density protein, staining was weak on d60. On d120, the staining had intensified and became punctate (arrows). Green or red = protein of interest, blue = nuclei (DAPI), Scale bar = 20 μm.

    Article Snippet: Primary antibodies, OCT-4 (1:400; Cell Signaling, D73G4), NANOG (1:800, Cell Signaling, C30A3), TUJ-1 (1:2000; Abcam ab14545), SV-2 (1:500; DSHB), PSD-95 (1:100; NeuroMab, P78352), KI-67 (1:600; BD Pharmingen™, 550609), PAX-6 (1:600; BioLegend, 901301), nestin (1:50; R & D Systems, MAB1259), CTIP-2 (1:300; Abcam, ab18465), TBR-1 (1:300; Abcam, ab31940), BRN-2 (1:400; Santa Cruz, sc-6029), CUX-1 (1:300; Santa Cruz, sc-13024), GAP-43 (1:1000; Abcam, ab75810), tau (1:1000; Biorbyt, orb175815), NRGN (1:100; Upstate Biotechnologies, 07–425), SNAP-25 (1:400; Sigma Aldrich, S9684-100UL) and SYT-1 antibody (1:200; Synaptic systems, 105,011) were diluted in block buffer and incubated at 4 °C overnight.

    Techniques: Immunocytochemistry, Staining, Expressing

    Expression of human embryonic stem cell markers in human Tenon’s capsule fibroblast derived induced pluripotent stem cells. A : Confocal immunofluorescent images of representative human Tenon’s capsule fibroblast derived induced pluripotent stem cells (TiPS) clones stained with the human embryonic stem (hES) markers SSEA-4 (red), TRA-1–60 (green), Nanog (red), and Oct-4 (red). Nuclei were stained with DAPI (blue). hESCs were used as a control. Scale bars=50 μm. B : Flow cytometry analysis of the equivalent SSEA-4 expression levels in TiPS and hESCs. 99.5% and 99.7% SSEA-4 + cells (in the Q4 area), respectively, whereas 0.3% in HTFs (p

    Journal: Molecular Vision

    Article Title: Generation of induced pluripotent stem cells from human Tenon's capsule fibroblasts

    doi:

    Figure Lengend Snippet: Expression of human embryonic stem cell markers in human Tenon’s capsule fibroblast derived induced pluripotent stem cells. A : Confocal immunofluorescent images of representative human Tenon’s capsule fibroblast derived induced pluripotent stem cells (TiPS) clones stained with the human embryonic stem (hES) markers SSEA-4 (red), TRA-1–60 (green), Nanog (red), and Oct-4 (red). Nuclei were stained with DAPI (blue). hESCs were used as a control. Scale bars=50 μm. B : Flow cytometry analysis of the equivalent SSEA-4 expression levels in TiPS and hESCs. 99.5% and 99.7% SSEA-4 + cells (in the Q4 area), respectively, whereas 0.3% in HTFs (p

    Article Snippet: Primary antibodies TRA-1–60 (Millipore), OCT-4 (Millipore), and NANOG (Cell Signaling, Danvers, MA) were used at 1:200 dilution, and Cy3- or Alexa 488–conjugated goat anti-mouse immunoglobulin G (Invitrogen) was used as the secondary antibody.

    Techniques: Expressing, Derivative Assay, Staining, Flow Cytometry, Cytometry

    CD55 regulates self-renewal and cisplatin resistance in endometrioid tumors. CD55 is glycophosphatidylinositol (GPI)–anchored to lipid rafts and via LIME binding signals intracellularly to ROR2 and LCK. ROR2 via JNK signaling regulates pluripotency gene expression, namely NANOG, SOX2, and OCT4 to maintain stemness in CSCs. In parallel, CD55 via the LCK pathway promotes the expression of DNA repair genes (including BRCA1 and MLH1) to drive cisplatin resistance.

    Journal: The Journal of Experimental Medicine

    Article Title: CD55 regulates self-renewal and cisplatin resistance in endometrioid tumors

    doi: 10.1084/jem.20170438

    Figure Lengend Snippet: CD55 regulates self-renewal and cisplatin resistance in endometrioid tumors. CD55 is glycophosphatidylinositol (GPI)–anchored to lipid rafts and via LIME binding signals intracellularly to ROR2 and LCK. ROR2 via JNK signaling regulates pluripotency gene expression, namely NANOG, SOX2, and OCT4 to maintain stemness in CSCs. In parallel, CD55 via the LCK pathway promotes the expression of DNA repair genes (including BRCA1 and MLH1) to drive cisplatin resistance.

    Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against CD55 (1:1,000; Santa Cruz), CD59 (1:1,000; Abcam), CD46 (1:1,000; Santa Cruz), NANOG (1:500; Cell Signaling), SOX2 (1:500; Cell Signaling), OCT4 (1:500; Cell Signaling), ROR2 (1:1,000; BD Biosciences), pJNK (1:1,000; T183/Y185; Cell Signaling), JNK (1:1,000; Cell Signaling), pLCK (Y394; 1:1,000; BD Biosciences), LCK (1:1,000; Santa Cruz), LIME (1:1,000; Invitrogen), PAG (1:1,000; Genetex), and β-actin (1:1,000; Cell Signaling).

    Techniques: Binding Assay, Expressing

    CD55 maintains self-renewal and cisplatin resistance in endometrioid tumors. (A) Cell lysates from cisplatin-naive CSCs silenced using two CD55 shRNA constructs (KD1, KD2) and a nontargeting shRNA (NT) control were immunoblotted for CD55, NANOG, SOX2, and OCT4. Actin was used as a loading control. Data are representative of two or three independent experiments. (B) A2780 CSCs silenced for CD55 and NT controls were flowed for GFP signal intensity, which indicates NANOG promoter activity. (C) Limiting dilution analysis plots of CD55 NT control compared with CD55 KD1 and KD2 silencing constructs in cisplatin-naive CSCs. (D) In vivo tumor initiation studies were performed with five mice per group, and the estimates of stem cell frequencies of CD55 NT control compared with the CD55 KD1 and KD2 silencing constructs are shown. (E) CD55-silenced cisplatin-naive CSCs and their NT controls were treated with 0–50 µM cisplatin, and percentage surviving cells is graphed. Data are representative of three independent experiments. (F and G) In vivo cisplatin sensitivity studies were performed comparing the NT control group with the CD55-silenced group, and the graph shows the growth rate of tumors compared with the first day of cisplatin treatment. (H) Hematoxylin and eosin–stained slides of tumors excised from mice treated with cisplatin and vehicle controls. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: CD55 regulates self-renewal and cisplatin resistance in endometrioid tumors

    doi: 10.1084/jem.20170438

    Figure Lengend Snippet: CD55 maintains self-renewal and cisplatin resistance in endometrioid tumors. (A) Cell lysates from cisplatin-naive CSCs silenced using two CD55 shRNA constructs (KD1, KD2) and a nontargeting shRNA (NT) control were immunoblotted for CD55, NANOG, SOX2, and OCT4. Actin was used as a loading control. Data are representative of two or three independent experiments. (B) A2780 CSCs silenced for CD55 and NT controls were flowed for GFP signal intensity, which indicates NANOG promoter activity. (C) Limiting dilution analysis plots of CD55 NT control compared with CD55 KD1 and KD2 silencing constructs in cisplatin-naive CSCs. (D) In vivo tumor initiation studies were performed with five mice per group, and the estimates of stem cell frequencies of CD55 NT control compared with the CD55 KD1 and KD2 silencing constructs are shown. (E) CD55-silenced cisplatin-naive CSCs and their NT controls were treated with 0–50 µM cisplatin, and percentage surviving cells is graphed. Data are representative of three independent experiments. (F and G) In vivo cisplatin sensitivity studies were performed comparing the NT control group with the CD55-silenced group, and the graph shows the growth rate of tumors compared with the first day of cisplatin treatment. (H) Hematoxylin and eosin–stained slides of tumors excised from mice treated with cisplatin and vehicle controls. *, P

    Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against CD55 (1:1,000; Santa Cruz), CD59 (1:1,000; Abcam), CD46 (1:1,000; Santa Cruz), NANOG (1:500; Cell Signaling), SOX2 (1:500; Cell Signaling), OCT4 (1:500; Cell Signaling), ROR2 (1:1,000; BD Biosciences), pJNK (1:1,000; T183/Y185; Cell Signaling), JNK (1:1,000; Cell Signaling), pLCK (Y394; 1:1,000; BD Biosciences), LCK (1:1,000; Santa Cruz), LIME (1:1,000; Invitrogen), PAG (1:1,000; Genetex), and β-actin (1:1,000; Cell Signaling).

    Techniques: shRNA, Construct, Activity Assay, In Vivo, Mouse Assay, Staining

    LIME is necessary for intracellular CD55 signaling. (A) Immunoprecipitation (IP) experiments with CD55 antibody were performed in cisplatin-naive CSCs, and eluates were probed for lipid raft adaptor proteins LIME and PAG. (B) Cell lysates from LIME-silenced A2780 CSCs and their nontargeted (NT) controls were immunoblotted and probed with LIME, ROR2, pLCK (Y394), and LCK. Actin was used as loading control. (C) IP experiments with CD55 antibody were performed in LIME-silenced and NT control cisplatin-naive CSCs and eluates were probed for ROR2, pLCK (Y394), LCK, LIME, and CD55. (D) Immunoblots of cisplatin-naive CSCs with LIME-silenced and NT controls were immunoblotted for LIME, NANOG, SOX2, and OCT4. Actin was used as a loading control. (E) Limiting dilution analysis of LIME NT control compared with LIME sh1 and sh2 silencing constructs in cisplatin-naive CSCs. (F) LIME-silenced cisplatin-naive CSCs and their NT controls were treated with 0–50 μM cisplatin, and percentage of surviving cells is graphed. All data are representative of two or three independent experiments. **, P

    Journal: The Journal of Experimental Medicine

    Article Title: CD55 regulates self-renewal and cisplatin resistance in endometrioid tumors

    doi: 10.1084/jem.20170438

    Figure Lengend Snippet: LIME is necessary for intracellular CD55 signaling. (A) Immunoprecipitation (IP) experiments with CD55 antibody were performed in cisplatin-naive CSCs, and eluates were probed for lipid raft adaptor proteins LIME and PAG. (B) Cell lysates from LIME-silenced A2780 CSCs and their nontargeted (NT) controls were immunoblotted and probed with LIME, ROR2, pLCK (Y394), and LCK. Actin was used as loading control. (C) IP experiments with CD55 antibody were performed in LIME-silenced and NT control cisplatin-naive CSCs and eluates were probed for ROR2, pLCK (Y394), LCK, LIME, and CD55. (D) Immunoblots of cisplatin-naive CSCs with LIME-silenced and NT controls were immunoblotted for LIME, NANOG, SOX2, and OCT4. Actin was used as a loading control. (E) Limiting dilution analysis of LIME NT control compared with LIME sh1 and sh2 silencing constructs in cisplatin-naive CSCs. (F) LIME-silenced cisplatin-naive CSCs and their NT controls were treated with 0–50 μM cisplatin, and percentage of surviving cells is graphed. All data are representative of two or three independent experiments. **, P

    Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against CD55 (1:1,000; Santa Cruz), CD59 (1:1,000; Abcam), CD46 (1:1,000; Santa Cruz), NANOG (1:500; Cell Signaling), SOX2 (1:500; Cell Signaling), OCT4 (1:500; Cell Signaling), ROR2 (1:1,000; BD Biosciences), pJNK (1:1,000; T183/Y185; Cell Signaling), JNK (1:1,000; Cell Signaling), pLCK (Y394; 1:1,000; BD Biosciences), LCK (1:1,000; Santa Cruz), LIME (1:1,000; Invitrogen), PAG (1:1,000; Genetex), and β-actin (1:1,000; Cell Signaling).

    Techniques: Immunoprecipitation, Western Blot, Construct

    CD55 is sufficient to drive self-renewal and cisplatin-resistance in endometrioid non-CSCs. (A) Immunoblots of cisplatin-naive non-CSCs with CD55 overexpression (OE) and empty vector controls were probed with CD55, NANOG, SOX2, and OCT4. Actin was used as loading control. Data are representative of two independent experiments. (B) mRNA expression was determined by quantitative real-time PCR and compared between CD55-overexpressing A2780 non-CSCs and empty vector control non-CSCs. Actin was used as a control. Three technical replicates were used. (C) Limiting dilution analysis plots of empty vector control compared with CD55 overexpression in cisplatin-naive non-CSCs. The graph compares the estimates of the percentage of self-renewal frequency in sorted populations with the corresponding p-values. Data are representative of three independent experiments. (D) A2780 non-CSCs transduced with CD55 overexpression and empty vector controls were flowed for GFP signal intensity, which indicates NANOG promoter activity. (E) Tumorsphere from A2780 non-CSCs transduced with CD55 and empty vector control were imaged using a digital immunofluorescence microscope. (F) CD55-overexpressing cisplatin-naive non-CSCs and their empty vector controls were treated with 0–50 µM cisplatin, and percentage of surviving cells was graphed. Data are representative of three independent experiments. (G) Relative caspase 3/7 activity of CD55-overexpressing cisplatin-naive cells and empty vector controls after cisplatin treatment. Relative caspase activities in cisplatin treated groups were calculated after normalizing the corrected readings to untreated controls in each group. Data are representative of two independent experiments, and three technical replicates were used in each. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: CD55 regulates self-renewal and cisplatin resistance in endometrioid tumors

    doi: 10.1084/jem.20170438

    Figure Lengend Snippet: CD55 is sufficient to drive self-renewal and cisplatin-resistance in endometrioid non-CSCs. (A) Immunoblots of cisplatin-naive non-CSCs with CD55 overexpression (OE) and empty vector controls were probed with CD55, NANOG, SOX2, and OCT4. Actin was used as loading control. Data are representative of two independent experiments. (B) mRNA expression was determined by quantitative real-time PCR and compared between CD55-overexpressing A2780 non-CSCs and empty vector control non-CSCs. Actin was used as a control. Three technical replicates were used. (C) Limiting dilution analysis plots of empty vector control compared with CD55 overexpression in cisplatin-naive non-CSCs. The graph compares the estimates of the percentage of self-renewal frequency in sorted populations with the corresponding p-values. Data are representative of three independent experiments. (D) A2780 non-CSCs transduced with CD55 overexpression and empty vector controls were flowed for GFP signal intensity, which indicates NANOG promoter activity. (E) Tumorsphere from A2780 non-CSCs transduced with CD55 and empty vector control were imaged using a digital immunofluorescence microscope. (F) CD55-overexpressing cisplatin-naive non-CSCs and their empty vector controls were treated with 0–50 µM cisplatin, and percentage of surviving cells was graphed. Data are representative of three independent experiments. (G) Relative caspase 3/7 activity of CD55-overexpressing cisplatin-naive cells and empty vector controls after cisplatin treatment. Relative caspase activities in cisplatin treated groups were calculated after normalizing the corrected readings to untreated controls in each group. Data are representative of two independent experiments, and three technical replicates were used in each. *, P

    Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against CD55 (1:1,000; Santa Cruz), CD59 (1:1,000; Abcam), CD46 (1:1,000; Santa Cruz), NANOG (1:500; Cell Signaling), SOX2 (1:500; Cell Signaling), OCT4 (1:500; Cell Signaling), ROR2 (1:1,000; BD Biosciences), pJNK (1:1,000; T183/Y185; Cell Signaling), JNK (1:1,000; Cell Signaling), pLCK (Y394; 1:1,000; BD Biosciences), LCK (1:1,000; Santa Cruz), LIME (1:1,000; Invitrogen), PAG (1:1,000; Genetex), and β-actin (1:1,000; Cell Signaling).

    Techniques: Western Blot, Over Expression, Plasmid Preparation, Expressing, Real-time Polymerase Chain Reaction, Transduction, Activity Assay, Immunofluorescence, Microscopy

    CD55 signals via ROR2-JNK pathway to regulate self-renewal. (A) Cell lysates from cisplatin-naive CSCs and non-CSCs were immunoblotted for ROR2, pJNK (T183/Y185), and JNK. Actin was used as a loading control. (B) Immunoprecipitation (IP) analysis with CD55 antibody were performed in cisplatin-naive CSCs, and eluates were immunoblotted for ROR2. (C) Immunoblots of ROR2 silenced using two shRNA constructs and nontargeting constructs in cisplatin-naive CSCs for ROR2, pJNK (T183/Y185), JNK, NANOG, SOX2, and OCT4. Actin was used as a loading control. (D) ROR2 silenced and NT controlled A2780 CSCs analyzed by flow cytometry for GFP intensity, which indicates NANOG promoter activity. (E) Limiting dilution analysis of CD55 NT control compared with ROR2 sh1 and sh2 silencing constructs in cisplatin-naive CSCs. (F) ROR2-silenced cisplatin-naive CSCs and their NT controls were treated with 0–50 µM cisplatin, and percentage surviving cells is graphed. All data are representative of two or three independent experiments. ***, P

    Journal: The Journal of Experimental Medicine

    Article Title: CD55 regulates self-renewal and cisplatin resistance in endometrioid tumors

    doi: 10.1084/jem.20170438

    Figure Lengend Snippet: CD55 signals via ROR2-JNK pathway to regulate self-renewal. (A) Cell lysates from cisplatin-naive CSCs and non-CSCs were immunoblotted for ROR2, pJNK (T183/Y185), and JNK. Actin was used as a loading control. (B) Immunoprecipitation (IP) analysis with CD55 antibody were performed in cisplatin-naive CSCs, and eluates were immunoblotted for ROR2. (C) Immunoblots of ROR2 silenced using two shRNA constructs and nontargeting constructs in cisplatin-naive CSCs for ROR2, pJNK (T183/Y185), JNK, NANOG, SOX2, and OCT4. Actin was used as a loading control. (D) ROR2 silenced and NT controlled A2780 CSCs analyzed by flow cytometry for GFP intensity, which indicates NANOG promoter activity. (E) Limiting dilution analysis of CD55 NT control compared with ROR2 sh1 and sh2 silencing constructs in cisplatin-naive CSCs. (F) ROR2-silenced cisplatin-naive CSCs and their NT controls were treated with 0–50 µM cisplatin, and percentage surviving cells is graphed. All data are representative of two or three independent experiments. ***, P

    Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against CD55 (1:1,000; Santa Cruz), CD59 (1:1,000; Abcam), CD46 (1:1,000; Santa Cruz), NANOG (1:500; Cell Signaling), SOX2 (1:500; Cell Signaling), OCT4 (1:500; Cell Signaling), ROR2 (1:1,000; BD Biosciences), pJNK (1:1,000; T183/Y185; Cell Signaling), JNK (1:1,000; Cell Signaling), pLCK (Y394; 1:1,000; BD Biosciences), LCK (1:1,000; Santa Cruz), LIME (1:1,000; Invitrogen), PAG (1:1,000; Genetex), and β-actin (1:1,000; Cell Signaling).

    Techniques: Immunoprecipitation, Western Blot, shRNA, Construct, Flow Cytometry, Cytometry, Activity Assay

    Expression of β-catenin and Nanog in EpSCs treated with Wnt antagonist, Dkk-1. Real-time PCR analysis of β-catenin mRNA (A) and Nanog mRNA (B) were obtained at different time points (day 1- day 12) in the control, SP treated group and SP with Dkk-1 treated group. The result showed that there were significant differences between the SP group and SP with Dkk-1 group on or after day 3 (*P

    Journal: Cell & Bioscience

    Article Title: Nanog down-regulates the Wnt signaling pathway via β-catenin phosphorylation during epidermal stem cell proliferation and differentiation

    doi: 10.1186/2045-3701-5-5

    Figure Lengend Snippet: Expression of β-catenin and Nanog in EpSCs treated with Wnt antagonist, Dkk-1. Real-time PCR analysis of β-catenin mRNA (A) and Nanog mRNA (B) were obtained at different time points (day 1- day 12) in the control, SP treated group and SP with Dkk-1 treated group. The result showed that there were significant differences between the SP group and SP with Dkk-1 group on or after day 3 (*P

    Article Snippet: The proteins were separated by SDS-PAGE, transferred to nitrocellulose, blocked with bovine serum albumin and incubated with the following primary antibodies: rat anti-β-catenin (CST, USA) diluted 1:1000, rat anti-p-β-catenin (CST, USA) diluted 1:1000, rat anti-c-myc (CST, USA) diluted 1:1000, rat anti-Nanog (CST, USA) diluted 1:2000, rat anti-GSK3β (CST, USA) diluted 1:1000, rat anti-p-GSK3β (CST, USA) diluted 1:1000 or rat anti-GAPDH (Beijing Biosynthesis Biotechnology, China) diluted 1:1000 that was used as a loading control.

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Overexpression of Nanog promotes β-catenin phosphorylation. The protein of total and phosphorylated GSK-3β,total and phosphorylated β-catenin were detected (A) . The ratio of phosphorylated GSK-3β to total GSK-3β was an indicator to inactivation of β-catenin (B) . The bands of β-catenin protein in each group were analyzed quantitatively (C and D) . The ratio of phosphorylated β-catenin to total β-catenin was regarded as the inactive β-catenin (E) . (*P

    Journal: Cell & Bioscience

    Article Title: Nanog down-regulates the Wnt signaling pathway via β-catenin phosphorylation during epidermal stem cell proliferation and differentiation

    doi: 10.1186/2045-3701-5-5

    Figure Lengend Snippet: Overexpression of Nanog promotes β-catenin phosphorylation. The protein of total and phosphorylated GSK-3β,total and phosphorylated β-catenin were detected (A) . The ratio of phosphorylated GSK-3β to total GSK-3β was an indicator to inactivation of β-catenin (B) . The bands of β-catenin protein in each group were analyzed quantitatively (C and D) . The ratio of phosphorylated β-catenin to total β-catenin was regarded as the inactive β-catenin (E) . (*P

    Article Snippet: The proteins were separated by SDS-PAGE, transferred to nitrocellulose, blocked with bovine serum albumin and incubated with the following primary antibodies: rat anti-β-catenin (CST, USA) diluted 1:1000, rat anti-p-β-catenin (CST, USA) diluted 1:1000, rat anti-c-myc (CST, USA) diluted 1:1000, rat anti-Nanog (CST, USA) diluted 1:2000, rat anti-GSK3β (CST, USA) diluted 1:1000, rat anti-p-GSK3β (CST, USA) diluted 1:1000 or rat anti-GAPDH (Beijing Biosynthesis Biotechnology, China) diluted 1:1000 that was used as a loading control.

    Techniques: Over Expression

    Expression of β-catenin and Nanog in EpSCs treated with SP. β-catenin and Nanog were measured at different time points (day 0-day 12) at the mRNA (q-PCR) and protein (western blot) level (A and B) . The bands of each protein were quantitatively analyzed (C and D) . Both q-PCR and western blot analysis showed that β-catenin expression was significantly higher on or after day 3 than at day 0. Nanog and β-catenin expression had an inverse relationship (*P

    Journal: Cell & Bioscience

    Article Title: Nanog down-regulates the Wnt signaling pathway via β-catenin phosphorylation during epidermal stem cell proliferation and differentiation

    doi: 10.1186/2045-3701-5-5

    Figure Lengend Snippet: Expression of β-catenin and Nanog in EpSCs treated with SP. β-catenin and Nanog were measured at different time points (day 0-day 12) at the mRNA (q-PCR) and protein (western blot) level (A and B) . The bands of each protein were quantitatively analyzed (C and D) . Both q-PCR and western blot analysis showed that β-catenin expression was significantly higher on or after day 3 than at day 0. Nanog and β-catenin expression had an inverse relationship (*P

    Article Snippet: The proteins were separated by SDS-PAGE, transferred to nitrocellulose, blocked with bovine serum albumin and incubated with the following primary antibodies: rat anti-β-catenin (CST, USA) diluted 1:1000, rat anti-p-β-catenin (CST, USA) diluted 1:1000, rat anti-c-myc (CST, USA) diluted 1:1000, rat anti-Nanog (CST, USA) diluted 1:2000, rat anti-GSK3β (CST, USA) diluted 1:1000, rat anti-p-GSK3β (CST, USA) diluted 1:1000 or rat anti-GAPDH (Beijing Biosynthesis Biotechnology, China) diluted 1:1000 that was used as a loading control.

    Techniques: Expressing, Polymerase Chain Reaction, Western Blot

    Expression of β-catenin and Nanog in EpSCs infected with lentivirus. Real-time PCR analysis of Nanog mRNA (A) and β-catenin mRNA (B) were obtained at different time points (day 1- day 12) in the control, control lentivirus vector infected group and transduced group. The results showed that there were no significant differences between the control and control lentivirus vector infected group. However, significant differences existed in the transduced group with the control on or after day 3(*P

    Journal: Cell & Bioscience

    Article Title: Nanog down-regulates the Wnt signaling pathway via β-catenin phosphorylation during epidermal stem cell proliferation and differentiation

    doi: 10.1186/2045-3701-5-5

    Figure Lengend Snippet: Expression of β-catenin and Nanog in EpSCs infected with lentivirus. Real-time PCR analysis of Nanog mRNA (A) and β-catenin mRNA (B) were obtained at different time points (day 1- day 12) in the control, control lentivirus vector infected group and transduced group. The results showed that there were no significant differences between the control and control lentivirus vector infected group. However, significant differences existed in the transduced group with the control on or after day 3(*P

    Article Snippet: The proteins were separated by SDS-PAGE, transferred to nitrocellulose, blocked with bovine serum albumin and incubated with the following primary antibodies: rat anti-β-catenin (CST, USA) diluted 1:1000, rat anti-p-β-catenin (CST, USA) diluted 1:1000, rat anti-c-myc (CST, USA) diluted 1:1000, rat anti-Nanog (CST, USA) diluted 1:2000, rat anti-GSK3β (CST, USA) diluted 1:1000, rat anti-p-GSK3β (CST, USA) diluted 1:1000 or rat anti-GAPDH (Beijing Biosynthesis Biotechnology, China) diluted 1:1000 that was used as a loading control.

    Techniques: Expressing, Infection, Real-time Polymerase Chain Reaction, Plasmid Preparation

    Overexpression of Nanog inhibits the Wnt signaling pathway. Real-time PCR analysis of gene expression ( (A) Nanog, (B) β-catenin, and (C) c-myc) of the EpSCs in the control, SP-treated group, transduced group, and the combination treatment group. At the protein level, Nanog, c-myc, and total β-catenin were also detected (D) . The bands of Nanog and c-myc protein in each group were analyzed quantitatively (E and F) . (*P

    Journal: Cell & Bioscience

    Article Title: Nanog down-regulates the Wnt signaling pathway via β-catenin phosphorylation during epidermal stem cell proliferation and differentiation

    doi: 10.1186/2045-3701-5-5

    Figure Lengend Snippet: Overexpression of Nanog inhibits the Wnt signaling pathway. Real-time PCR analysis of gene expression ( (A) Nanog, (B) β-catenin, and (C) c-myc) of the EpSCs in the control, SP-treated group, transduced group, and the combination treatment group. At the protein level, Nanog, c-myc, and total β-catenin were also detected (D) . The bands of Nanog and c-myc protein in each group were analyzed quantitatively (E and F) . (*P

    Article Snippet: The proteins were separated by SDS-PAGE, transferred to nitrocellulose, blocked with bovine serum albumin and incubated with the following primary antibodies: rat anti-β-catenin (CST, USA) diluted 1:1000, rat anti-p-β-catenin (CST, USA) diluted 1:1000, rat anti-c-myc (CST, USA) diluted 1:1000, rat anti-Nanog (CST, USA) diluted 1:2000, rat anti-GSK3β (CST, USA) diluted 1:1000, rat anti-p-GSK3β (CST, USA) diluted 1:1000 or rat anti-GAPDH (Beijing Biosynthesis Biotechnology, China) diluted 1:1000 that was used as a loading control.

    Techniques: Over Expression, Real-time Polymerase Chain Reaction, Expressing