Structured Review

Santa Cruz Biotechnology anti sox2
Cyclin K expression positively correlates with proliferation. a Analyses of cyclin K protein expression during murine brain development by immunoblotting. Cyclin K protein expression in embryonic (E) and postnatal (P) murine brains correlated with that of <t>Sox2,</t> a marker of neural progenitor cell proliferation. b Analyses of cyclin K protein expression by immunoblotting during murine liver development. c Cyclin K expression detected by immunochemistry during the process of murine liver regeneration in vivo. 2nd, immunochemistry using secondary antibodies alone. 0 h denotes samples collected immediately after partial hepatectomy. Scale bar, 40 μm. d Comparison of cyclin K by immunoblotting in normal and H1299 cancer cells using equal cell numbers as loading control. HFF, neonatal human foreskin fibroblast. e Cyclin K expression detected by immunochemistry in normal and H1299 cancer cells. HFF, neonatal human foreskin fibroblast. Scale bar, 40 μm. f Time course analyses of cyclin K expression by immunoblotting in HCT116 cells treated with protein synthesis inhibitor cycloheximide (CHX, 50 μg/ml). g Time course analyses of cyclin K expression by immunoblotting in cells treated with proteasome inhibitor MG132 (5 μM) in human normal and HCT116 cancer cells. HFF, neonatal human foreskin fibroblast. Experiments were repeated for three times ( a – c ), and more than three times when cell lines were used ( d – g ). Representative results are shown
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1) Product Images from "Cyclin K regulates prereplicative complex assembly to promote mammalian cell proliferation"

Article Title: Cyclin K regulates prereplicative complex assembly to promote mammalian cell proliferation

Journal: Nature Communications

doi: 10.1038/s41467-018-04258-w

Cyclin K expression positively correlates with proliferation. a Analyses of cyclin K protein expression during murine brain development by immunoblotting. Cyclin K protein expression in embryonic (E) and postnatal (P) murine brains correlated with that of Sox2, a marker of neural progenitor cell proliferation. b Analyses of cyclin K protein expression by immunoblotting during murine liver development. c Cyclin K expression detected by immunochemistry during the process of murine liver regeneration in vivo. 2nd, immunochemistry using secondary antibodies alone. 0 h denotes samples collected immediately after partial hepatectomy. Scale bar, 40 μm. d Comparison of cyclin K by immunoblotting in normal and H1299 cancer cells using equal cell numbers as loading control. HFF, neonatal human foreskin fibroblast. e Cyclin K expression detected by immunochemistry in normal and H1299 cancer cells. HFF, neonatal human foreskin fibroblast. Scale bar, 40 μm. f Time course analyses of cyclin K expression by immunoblotting in HCT116 cells treated with protein synthesis inhibitor cycloheximide (CHX, 50 μg/ml). g Time course analyses of cyclin K expression by immunoblotting in cells treated with proteasome inhibitor MG132 (5 μM) in human normal and HCT116 cancer cells. HFF, neonatal human foreskin fibroblast. Experiments were repeated for three times ( a – c ), and more than three times when cell lines were used ( d – g ). Representative results are shown
Figure Legend Snippet: Cyclin K expression positively correlates with proliferation. a Analyses of cyclin K protein expression during murine brain development by immunoblotting. Cyclin K protein expression in embryonic (E) and postnatal (P) murine brains correlated with that of Sox2, a marker of neural progenitor cell proliferation. b Analyses of cyclin K protein expression by immunoblotting during murine liver development. c Cyclin K expression detected by immunochemistry during the process of murine liver regeneration in vivo. 2nd, immunochemistry using secondary antibodies alone. 0 h denotes samples collected immediately after partial hepatectomy. Scale bar, 40 μm. d Comparison of cyclin K by immunoblotting in normal and H1299 cancer cells using equal cell numbers as loading control. HFF, neonatal human foreskin fibroblast. e Cyclin K expression detected by immunochemistry in normal and H1299 cancer cells. HFF, neonatal human foreskin fibroblast. Scale bar, 40 μm. f Time course analyses of cyclin K expression by immunoblotting in HCT116 cells treated with protein synthesis inhibitor cycloheximide (CHX, 50 μg/ml). g Time course analyses of cyclin K expression by immunoblotting in cells treated with proteasome inhibitor MG132 (5 μM) in human normal and HCT116 cancer cells. HFF, neonatal human foreskin fibroblast. Experiments were repeated for three times ( a – c ), and more than three times when cell lines were used ( d – g ). Representative results are shown

Techniques Used: Expressing, Marker, In Vivo

2) Product Images from "Overactivation of Notch1 Signaling Induces Ectopic Hair Cells in the Mouse Inner Ear in an Age-Dependent Manner"

Article Title: Overactivation of Notch1 Signaling Induces Ectopic Hair Cells in the Mouse Inner Ear in an Age-Dependent Manner

Journal: PLoS ONE

doi: 10.1371/journal.pone.0034123

Expression of the sensory epithelium marker Parvalbumin and Sox2 in new HCs, and Sox10 in new SCs. ( A–A′ ) Images of samples double stained with Parvalbumin and EGFP at HC layer (A) and SC layer (A′) of the ectopic sensory patches in the utricle non-sensory region of a CAG CreER+ ; Rosa26-NICD loxp/+ embryo treated with tamoxifen at ∼E13 and analyzed at ∼E19. The arrow points to a new Parvalbumin+ HC. ( B–B′ ) Triple staining of Myosin-VI, Sox2, and EGFP. Both ectopic HCs (B) and SCs (B′) were Sox2+. The arrow points to a new Sox2+/Myosin-VI+ HC. Of note, Myosin-VI was visualized in a pseudo-green color. ( C–C′ ) Triple staining of Myosin-VI, Sox10, and EGFP. The arrow points to a new Myosin-VI+/Sox10−negative HC. The SCs (either EGFP+ or EGFP−negative) were Sox10+. Note that Myosin-VI was also visualized in a pseudo-green color. Scale bars: 20 µm.
Figure Legend Snippet: Expression of the sensory epithelium marker Parvalbumin and Sox2 in new HCs, and Sox10 in new SCs. ( A–A′ ) Images of samples double stained with Parvalbumin and EGFP at HC layer (A) and SC layer (A′) of the ectopic sensory patches in the utricle non-sensory region of a CAG CreER+ ; Rosa26-NICD loxp/+ embryo treated with tamoxifen at ∼E13 and analyzed at ∼E19. The arrow points to a new Parvalbumin+ HC. ( B–B′ ) Triple staining of Myosin-VI, Sox2, and EGFP. Both ectopic HCs (B) and SCs (B′) were Sox2+. The arrow points to a new Sox2+/Myosin-VI+ HC. Of note, Myosin-VI was visualized in a pseudo-green color. ( C–C′ ) Triple staining of Myosin-VI, Sox10, and EGFP. The arrow points to a new Myosin-VI+/Sox10−negative HC. The SCs (either EGFP+ or EGFP−negative) were Sox10+. Note that Myosin-VI was also visualized in a pseudo-green color. Scale bars: 20 µm.

Techniques Used: Expressing, Marker, Staining

3) Product Images from "In vivo Notch Reactivation in Differentiating Cochlear Hair Cells Induces Sox2 and Prox1 Expression but Does Not Disrupt Hair Cell Maturation"

Article Title: In vivo Notch Reactivation in Differentiating Cochlear Hair Cells Induces Sox2 and Prox1 Expression but Does Not Disrupt Hair Cell Maturation

Journal: Developmental Dynamics

doi: 10.1002/dvdy.23754

Sox2 expression in adult HCs with ectopic Notch1 signaling
Figure Legend Snippet: Sox2 expression in adult HCs with ectopic Notch1 signaling

Techniques Used: Expressing

Ectopic NICD causes re-expression of Sox2 and Prox1
Figure Legend Snippet: Ectopic NICD causes re-expression of Sox2 and Prox1

Techniques Used: Expressing

4) Product Images from "Tetraspanin CD9 stabilizes gp130 by preventing its ubiquitin-dependent lysosomal degradation to promote STAT3 activation in glioma stem cells"

Article Title: Tetraspanin CD9 stabilizes gp130 by preventing its ubiquitin-dependent lysosomal degradation to promote STAT3 activation in glioma stem cells

Journal: Cell Death and Differentiation

doi: 10.1038/cdd.2016.110

Tetraspanin CD9 is preferentially expressed in GSCs and is essential for the GSC maintenance. ( a ) The expression heatmap of tetraspanins in GSC lines ( n =12) relative to CGCs ( n =32) from the GEO profiles (GEO: GDS3885). Four candidates, including CD9 , TSPAN7 , TSPAN11 and TSPAN33 were significantly upregulated in GSCs relative to CGCs. Data were visualized using Cluster/Java Treeview. ( b ) Immunoblot analysis showing the preferential expressions of CD9 and the GSC marker SOX2 in GSCs ( n =6) relative to the matched NSTCs ( n =6) isolated from human GBMs. ( c ) Immunofluorescent staining of CD9 (in green) and the GSC marker SOX2 (in red, upper panel), OLIG2 (in red, middle panel) or CD133 (in red, lower panel) in GSC tumorspheres. Scale bar represents 25 μ m. ( d ) Immunoblot analyses of CD9, the GSC marker SOX2 and the neuronal differentiation marker MAP2 during the serum-induced differentiation of GSCs. The levels of CD9 and the GSC marker SOX2 decreased, while the expression of the differentiation marker MAP2 concomitantly increased over a 7-day period. ( e ) In vitro limiting dilution analyses of the secondary tumorsphere formations of GSCs expressing shCD9 (shCD9-1 and -2) or non-targeting shRNA (shNT, control). Disrupting CD9 expression attenuated the self-renewal capacity of GSCs. ** P
Figure Legend Snippet: Tetraspanin CD9 is preferentially expressed in GSCs and is essential for the GSC maintenance. ( a ) The expression heatmap of tetraspanins in GSC lines ( n =12) relative to CGCs ( n =32) from the GEO profiles (GEO: GDS3885). Four candidates, including CD9 , TSPAN7 , TSPAN11 and TSPAN33 were significantly upregulated in GSCs relative to CGCs. Data were visualized using Cluster/Java Treeview. ( b ) Immunoblot analysis showing the preferential expressions of CD9 and the GSC marker SOX2 in GSCs ( n =6) relative to the matched NSTCs ( n =6) isolated from human GBMs. ( c ) Immunofluorescent staining of CD9 (in green) and the GSC marker SOX2 (in red, upper panel), OLIG2 (in red, middle panel) or CD133 (in red, lower panel) in GSC tumorspheres. Scale bar represents 25 μ m. ( d ) Immunoblot analyses of CD9, the GSC marker SOX2 and the neuronal differentiation marker MAP2 during the serum-induced differentiation of GSCs. The levels of CD9 and the GSC marker SOX2 decreased, while the expression of the differentiation marker MAP2 concomitantly increased over a 7-day period. ( e ) In vitro limiting dilution analyses of the secondary tumorsphere formations of GSCs expressing shCD9 (shCD9-1 and -2) or non-targeting shRNA (shNT, control). Disrupting CD9 expression attenuated the self-renewal capacity of GSCs. ** P

Techniques Used: Expressing, Marker, Isolation, Staining, In Vitro, shRNA

5) Product Images from "Complement proteins C7 and CFH control the stemness of liver cancer cells via LSF-1"

Article Title: Complement proteins C7 and CFH control the stemness of liver cancer cells via LSF-1

Journal: Cancer letters

doi: 10.1016/j.canlet.2015.12.005

Direct upregulation of Nanog, Oct4, Sox2, and c-Myc promoters by LSF-1 enhances tumorsphere formation and the in vivo growth of liver cancer cells. (A–B) Dual luciferase reporter assay with the pGL3-Nanog, Oct4, Sox2, and c-Myc promoter reporters.
Figure Legend Snippet: Direct upregulation of Nanog, Oct4, Sox2, and c-Myc promoters by LSF-1 enhances tumorsphere formation and the in vivo growth of liver cancer cells. (A–B) Dual luciferase reporter assay with the pGL3-Nanog, Oct4, Sox2, and c-Myc promoter reporters.

Techniques Used: In Vivo, Luciferase, Reporter Assay

Tumorsphere cells expressed higher level of stemness factors than 2D cultured primary HCC cells. (A–C) Nanog (A), Oct4 (B) and Sox2 (C) RNA measurements from 2D and sphere (S) from primary HCC. (D–F) Nanog (D), Oct4 (E) and Sox2 (F) RNA
Figure Legend Snippet: Tumorsphere cells expressed higher level of stemness factors than 2D cultured primary HCC cells. (A–C) Nanog (A), Oct4 (B) and Sox2 (C) RNA measurements from 2D and sphere (S) from primary HCC. (D–F) Nanog (D), Oct4 (E) and Sox2 (F) RNA

Techniques Used: Cell Culture

6) Product Images from "Cleaved CD44 intracellular domain supports activation of stemness factors and promotes tumorigenesis of breast cancer"

Article Title: Cleaved CD44 intracellular domain supports activation of stemness factors and promotes tumorigenesis of breast cancer

Journal: Oncotarget

doi:

CD44ICD interacts with the stemness factors, Nanog, Sox2, and Oct4 and regulates the nuclear-localization (A) MDA-MB-231 cells were transfected with CD44ICD. The interaction between CD44 and Nanog, Sox2, and Oct4 were detected with an immunoprecipitation assay described in “materials and methods”. (B) HEK293 cells were transfected with CD44ICD or the truncated mutant constructs, ICD_ΔN17, ICD_ΔN35, ICD_ΔC19, and co-transfected with Sox2 and Oct4 expression vectors for 36 hr as described in “materials methods”. Their interaction was detected with an immunoprecipitation assay. (C) Endogenous CD44 stable knockdown MDA-MB-231 and MCF-7 cells were transfected with the control and CD44ICD and C-terminal region truncated CD44 vectors. The changes in the localization of CD44ICD and stemness factors were detected by western blot. GAPDH and Lamin A/C were used as loading controls.
Figure Legend Snippet: CD44ICD interacts with the stemness factors, Nanog, Sox2, and Oct4 and regulates the nuclear-localization (A) MDA-MB-231 cells were transfected with CD44ICD. The interaction between CD44 and Nanog, Sox2, and Oct4 were detected with an immunoprecipitation assay described in “materials and methods”. (B) HEK293 cells were transfected with CD44ICD or the truncated mutant constructs, ICD_ΔN17, ICD_ΔN35, ICD_ΔC19, and co-transfected with Sox2 and Oct4 expression vectors for 36 hr as described in “materials methods”. Their interaction was detected with an immunoprecipitation assay. (C) Endogenous CD44 stable knockdown MDA-MB-231 and MCF-7 cells were transfected with the control and CD44ICD and C-terminal region truncated CD44 vectors. The changes in the localization of CD44ICD and stemness factors were detected by western blot. GAPDH and Lamin A/C were used as loading controls.

Techniques Used: Multiple Displacement Amplification, Transfection, Immunoprecipitation, Mutagenesis, Construct, Expressing, Western Blot

CD44-depletion reduces both the expression and nuclear localization of the stemness factors, Nanog, Sox2, and Oct4 MDA-MB-231 and MCF-7 cells were transfected with scrambled siRNA (scRNA) and CD44 siRNA, and then treated with 5 μM of GSI for 24 hr. (A) mRNA (upper) and protein levels (lower) of CD44 and stemness factors were detected with an RT-PCR and western blot analysis. (B and C) the changes in the localization of cleaved CD44ICD and stemness factors were detected using western blot analysis. The cells were treated with 5 μM of GSI for the indicated times. GAPDH and Lamin A/C were used as loading controls. (D) The changes in the localization of CD44ICD and its co-localization with stemness factors were detected during treatment with 2 μM of GSI for 12 hr in MDA-MB-231 and MCF-7 cells using an immunocytochemical analysis as described in “materials methods”.
Figure Legend Snippet: CD44-depletion reduces both the expression and nuclear localization of the stemness factors, Nanog, Sox2, and Oct4 MDA-MB-231 and MCF-7 cells were transfected with scrambled siRNA (scRNA) and CD44 siRNA, and then treated with 5 μM of GSI for 24 hr. (A) mRNA (upper) and protein levels (lower) of CD44 and stemness factors were detected with an RT-PCR and western blot analysis. (B and C) the changes in the localization of cleaved CD44ICD and stemness factors were detected using western blot analysis. The cells were treated with 5 μM of GSI for the indicated times. GAPDH and Lamin A/C were used as loading controls. (D) The changes in the localization of CD44ICD and its co-localization with stemness factors were detected during treatment with 2 μM of GSI for 12 hr in MDA-MB-231 and MCF-7 cells using an immunocytochemical analysis as described in “materials methods”.

Techniques Used: Expressing, Multiple Displacement Amplification, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot

Overexpression of CD44ICD in CD44-depleted cells increases both the expression and nuclear-localization of the stemness factors, Nanog, Sox2, and Oct4 Endogenous CD44 stable knockdown MDA-MB-231 and MCF-7 cells were transfected with the control vector and with the full-length CD44 and CD44ICD expression vectors. CD44ICD transfected cells were treated with 5 μM of GSI for 24 hr. (A) The mRNA (upper) and protein levels (lower) of CD44 and stemness factors were detected with an RT-PCR and western blot analysis. (B) The changes in the localization of CD44ICD and stemness factors were detected with a western blot analysis. GAPDH and Lamin A/C were used as loading controls. (C) Endogenous CD44 stable knockdown MDA-MB-231 and MCF-7 cells were transfected with the control and full-length form of CD44, and with the cleavage site truncated mutant CD44 vector. The changes in the localization of CD44ICD and stemness factors were detected with a western blot analysis.
Figure Legend Snippet: Overexpression of CD44ICD in CD44-depleted cells increases both the expression and nuclear-localization of the stemness factors, Nanog, Sox2, and Oct4 Endogenous CD44 stable knockdown MDA-MB-231 and MCF-7 cells were transfected with the control vector and with the full-length CD44 and CD44ICD expression vectors. CD44ICD transfected cells were treated with 5 μM of GSI for 24 hr. (A) The mRNA (upper) and protein levels (lower) of CD44 and stemness factors were detected with an RT-PCR and western blot analysis. (B) The changes in the localization of CD44ICD and stemness factors were detected with a western blot analysis. GAPDH and Lamin A/C were used as loading controls. (C) Endogenous CD44 stable knockdown MDA-MB-231 and MCF-7 cells were transfected with the control and full-length form of CD44, and with the cleavage site truncated mutant CD44 vector. The changes in the localization of CD44ICD and stemness factors were detected with a western blot analysis.

Techniques Used: Over Expression, Expressing, Multiple Displacement Amplification, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Mutagenesis

Overexpression of CD44ICD increases the transcriptional activation of the stemness factors, Sox2, and Oct4 (A) The transcriptional activation was measured with a reporter assay in wild-type (diagonal bars) and CD44KD MCF-7 (solid bars) cells. Cells were transfected with a Sox2 and Oct4 reporter vector alone or co-transfected with CD44 siRNA, CD44 or CD44ICD expression vectors. Following transfection, the cells were incubated for 12 hr and a vehicle (−) or 5 μM of GSI (+) were added. The cells were incubated for an additional 24 hr. The transcriptional activity was measured by luciferase activity described in “materials and methods”. (B) The expression of CD44ICD or of the truncated mutant constructs, ICD_ΔN17 and ICD_ΔC19 were detected with a western blot. (C) MCF-7 cells were transfected with the reporter vector alone or co-transfected with CD44-ICD or CD44-ICD_ΔN17 expression vectors for 36 hr. The luciferase activity was measured as described in “materials methods”. (D) MCF-7 cells were transfected with the reporter vector alone or co-transfected with CD44ICD or CD44ICD_ΔC19 expression vectors for 36 hr. The luciferase activity was measured. The data are presented as the mean ± SD ( n = 3). Significant differences are indicated by an asterisk (* p
Figure Legend Snippet: Overexpression of CD44ICD increases the transcriptional activation of the stemness factors, Sox2, and Oct4 (A) The transcriptional activation was measured with a reporter assay in wild-type (diagonal bars) and CD44KD MCF-7 (solid bars) cells. Cells were transfected with a Sox2 and Oct4 reporter vector alone or co-transfected with CD44 siRNA, CD44 or CD44ICD expression vectors. Following transfection, the cells were incubated for 12 hr and a vehicle (−) or 5 μM of GSI (+) were added. The cells were incubated for an additional 24 hr. The transcriptional activity was measured by luciferase activity described in “materials and methods”. (B) The expression of CD44ICD or of the truncated mutant constructs, ICD_ΔN17 and ICD_ΔC19 were detected with a western blot. (C) MCF-7 cells were transfected with the reporter vector alone or co-transfected with CD44-ICD or CD44-ICD_ΔN17 expression vectors for 36 hr. The luciferase activity was measured as described in “materials methods”. (D) MCF-7 cells were transfected with the reporter vector alone or co-transfected with CD44ICD or CD44ICD_ΔC19 expression vectors for 36 hr. The luciferase activity was measured. The data are presented as the mean ± SD ( n = 3). Significant differences are indicated by an asterisk (* p

Techniques Used: Over Expression, Activation Assay, Reporter Assay, Transfection, Plasmid Preparation, Expressing, Incubation, Activity Assay, Luciferase, Mutagenesis, Construct, Western Blot

Basal expression levels of CD44 in breast cancer cell lines and mammosphere formation (A) The basal expression levels of Nanog, Sox2, Oct4, and CD44 were detected with an RT-PCR (left panel) and western blot (right panel) analysis, respectively. GAPDH was used as a loading control. (B) CD44 levels were measured using a FACs analysis. (C) The mammosphere forming ability was measured under sphere forming conditions for 15 days as described in “materials methods”. All experiments were performed in the indicated breast cancer cell lines.
Figure Legend Snippet: Basal expression levels of CD44 in breast cancer cell lines and mammosphere formation (A) The basal expression levels of Nanog, Sox2, Oct4, and CD44 were detected with an RT-PCR (left panel) and western blot (right panel) analysis, respectively. GAPDH was used as a loading control. (B) CD44 levels were measured using a FACs analysis. (C) The mammosphere forming ability was measured under sphere forming conditions for 15 days as described in “materials methods”. All experiments were performed in the indicated breast cancer cell lines.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, FACS

7) Product Images from "Dynamic ubiquitylation of Sox2 regulates proteostasis and governs neural progenitor cell differentiation"

Article Title: Dynamic ubiquitylation of Sox2 regulates proteostasis and governs neural progenitor cell differentiation

Journal: Nature Communications

doi: 10.1038/s41467-018-07025-z

COP1 promotes NPCs differentiation in a Sox2-dependent manner. a NPCs were plated in Matrigel-coated six-well plate for neuronal differentiation. For the indicated times, fixed and stained with anti-Sox2 (green), anti-COP1 (red), anti-TUJ1 (red), or CUL4A (green)-specific antibodies. Nuclei were counterstained with Hoechst 33342 (HO, blue). Scale bar, 50 µm. b , c NPCs were infected with lentiviruses expressing COP1 shRNA or negative control (NC) shRNA for 126 h. Immunoblotting ( b ) and immunostaining ( c ) were performed for Sox2, COP1, TUJ1, or Nestin. Scale bar, 50 µm. d NPCs with double knockdown of COP1 and Sox2 were generated by lentivirus infection. Immunostaining were performed for Sox2, TUJ1. Scale bar, 50 µm. The ratio of TUJ1-positive or Sox2-positive cells were quantified. Results are shown as mean ± s.d. Each error bar shows the standard deviation of numbers of positive cells in 10 fields of view. * P
Figure Legend Snippet: COP1 promotes NPCs differentiation in a Sox2-dependent manner. a NPCs were plated in Matrigel-coated six-well plate for neuronal differentiation. For the indicated times, fixed and stained with anti-Sox2 (green), anti-COP1 (red), anti-TUJ1 (red), or CUL4A (green)-specific antibodies. Nuclei were counterstained with Hoechst 33342 (HO, blue). Scale bar, 50 µm. b , c NPCs were infected with lentiviruses expressing COP1 shRNA or negative control (NC) shRNA for 126 h. Immunoblotting ( b ) and immunostaining ( c ) were performed for Sox2, COP1, TUJ1, or Nestin. Scale bar, 50 µm. d NPCs with double knockdown of COP1 and Sox2 were generated by lentivirus infection. Immunostaining were performed for Sox2, TUJ1. Scale bar, 50 µm. The ratio of TUJ1-positive or Sox2-positive cells were quantified. Results are shown as mean ± s.d. Each error bar shows the standard deviation of numbers of positive cells in 10 fields of view. * P

Techniques Used: Staining, Infection, Expressing, shRNA, Negative Control, Immunostaining, Generated, Standard Deviation

COP1 and OTUD7B coordinate to control NPCs differentiation. a , b NPCs were plated in Matrigel-coated six-well plate for neuronal differentiation. For the indicated times, immunoblotting ( a ) and immunofluorescence ( b ) of Sox2, CUL4A, COP1, OTUD7B and TUJ1 were performed. c Cells transfected with HA-Ub and indicated shRNA were treated by MG132 for 8 h, and the lysates were immunoprecipated with anti-Sox2 antibody and immunoblotted with anti-HA. d Summary of our findings. OTUD7B stabilizes Sox2 through deubiquitylation and maintains the stemness of NPCs, while CUL4A DET1-COP1 promotes Sox2 degradation through ubiquitylation and induces differentiation of NPCs. OTUD7B and CUL4A DET1-COP1 exert opposite roles in regulating Sox2 protein stability at the post-translational level. The representative images are shown from three independent experiments. Unprocessed original scans of blots are shown in Supplementary Fig. 9
Figure Legend Snippet: COP1 and OTUD7B coordinate to control NPCs differentiation. a , b NPCs were plated in Matrigel-coated six-well plate for neuronal differentiation. For the indicated times, immunoblotting ( a ) and immunofluorescence ( b ) of Sox2, CUL4A, COP1, OTUD7B and TUJ1 were performed. c Cells transfected with HA-Ub and indicated shRNA were treated by MG132 for 8 h, and the lysates were immunoprecipated with anti-Sox2 antibody and immunoblotted with anti-HA. d Summary of our findings. OTUD7B stabilizes Sox2 through deubiquitylation and maintains the stemness of NPCs, while CUL4A DET1-COP1 promotes Sox2 degradation through ubiquitylation and induces differentiation of NPCs. OTUD7B and CUL4A DET1-COP1 exert opposite roles in regulating Sox2 protein stability at the post-translational level. The representative images are shown from three independent experiments. Unprocessed original scans of blots are shown in Supplementary Fig. 9

Techniques Used: Immunofluorescence, Transfection, shRNA

OTUD7B deubiquitylates and stabilizes Sox2. a The indicated OTU subfamily DUBs were each transfected into HEK293T cells. 48 h later, cell lysates were subjected to western blot. Quantification of relative Sox2 levels is shown. Results are shown as mean ± s.d. Each error bar shows the standard deviation of the value from three independent experiment. *** P
Figure Legend Snippet: OTUD7B deubiquitylates and stabilizes Sox2. a The indicated OTU subfamily DUBs were each transfected into HEK293T cells. 48 h later, cell lysates were subjected to western blot. Quantification of relative Sox2 levels is shown. Results are shown as mean ± s.d. Each error bar shows the standard deviation of the value from three independent experiment. *** P

Techniques Used: Transfection, Western Blot, Standard Deviation

OTUD7B maintains NPCs stemness. a Immunoblotting of Sox2 in MEF with OTUD7B knockout and WT control. b The MEF cells were treated with CHX (10 µg/ml), and collected at the indicated times for western blot. Quantification of Sox2 levels relative to tubulin is shown. Results are shown as mean ± s.d. n = 3 independent experiments. ** P
Figure Legend Snippet: OTUD7B maintains NPCs stemness. a Immunoblotting of Sox2 in MEF with OTUD7B knockout and WT control. b The MEF cells were treated with CHX (10 µg/ml), and collected at the indicated times for western blot. Quantification of Sox2 levels relative to tubulin is shown. Results are shown as mean ± s.d. n = 3 independent experiments. ** P

Techniques Used: Knock-Out, Western Blot

CUL4A contributes to Sox2 ubiquitylation in NPCs differentiation. a NPCs (neural stem/progenitor cells derived from human NPSCs, WA09) were cultured in neural induction medium (NIM) to undergo cellular differentiation for the indicated times. NPCs were treated with 10 µg per ml CHX, and collected at the indicated times for western blot. Quantification of Sox2 levels relative to tubulin is shown. Results are shown as mean ± s.d. n = 3 independent experiments. ** P
Figure Legend Snippet: CUL4A contributes to Sox2 ubiquitylation in NPCs differentiation. a NPCs (neural stem/progenitor cells derived from human NPSCs, WA09) were cultured in neural induction medium (NIM) to undergo cellular differentiation for the indicated times. NPCs were treated with 10 µg per ml CHX, and collected at the indicated times for western blot. Quantification of Sox2 levels relative to tubulin is shown. Results are shown as mean ± s.d. n = 3 independent experiments. ** P

Techniques Used: Derivative Assay, Cell Culture, Cell Differentiation, Western Blot

CUL4A DET1-COP1 interacts with Sox2 and regulates its stability. a Network view of E3–Sox2 interactions (left panel) and the E3 hierarchical tree for Sox2 (right panel). UbiBrowser was employed to explore the E3 ligases for Sox2. The representative predicted E3 ligases surround Sox2. The node colors and characters reflect the E3 type. The edge width, the node size, and the edge shade are corrected with the confidence score. The predicted E3s and their position in the E3 family hierarchical tree was presented. In this tree, texts in each circle (just like “U”, “D” and “SO”) represent the E3 family. The number in the bracket following each E3 family represents the number of corresponding predicted E3–Sox2 interaction. b NPCs cell lysates were subjected to immunoprecipitation with control IgG or anti-Sox2 antibodies and detected CUL4A, COP1, DET1, DDB1, Roc1, and Sox2 protein levels. c The lysates of HEK293T cells transfected with indicated constructs were subjected to immunoprecipitation with anti-Myc or Histidine tag-specific affinity resin (agarose beads). The immunoprecipitates or the eluates were then blotted. d Overview of the structures of COP1 wild type and different truncates. HEK293T cells were co-transfected with Myc-Sox2 and the indicated COP1 truncates. The lysates were collected and subjected to immunoprecipitation with anti-Flag. The immunoprecipitates were then blotted. e Overview of the structure of Sox2 wild type and different VP mutants. Recombinant proteins (His-COP1, GST-Sox2, GST-Sox2-A1, GST-Sox2-A2, and GST-Sox2-AA) were expressed and purified. GST-Sox2 bound to glutathione-Sepharose 4B beads was incubated with His- COP1 for 24 h at 4 °C. Then the beads were washed and proteins were eluted, followed by western blotting. f HEK293T cells were transfected with indicated constructs. The lysates were collected and blotted with anti-Flag and anti-Myc antibody. The representative images are shown from three independent experiments. Unprocessed original scans of blots are shown in Supplementary Fig. 9
Figure Legend Snippet: CUL4A DET1-COP1 interacts with Sox2 and regulates its stability. a Network view of E3–Sox2 interactions (left panel) and the E3 hierarchical tree for Sox2 (right panel). UbiBrowser was employed to explore the E3 ligases for Sox2. The representative predicted E3 ligases surround Sox2. The node colors and characters reflect the E3 type. The edge width, the node size, and the edge shade are corrected with the confidence score. The predicted E3s and their position in the E3 family hierarchical tree was presented. In this tree, texts in each circle (just like “U”, “D” and “SO”) represent the E3 family. The number in the bracket following each E3 family represents the number of corresponding predicted E3–Sox2 interaction. b NPCs cell lysates were subjected to immunoprecipitation with control IgG or anti-Sox2 antibodies and detected CUL4A, COP1, DET1, DDB1, Roc1, and Sox2 protein levels. c The lysates of HEK293T cells transfected with indicated constructs were subjected to immunoprecipitation with anti-Myc or Histidine tag-specific affinity resin (agarose beads). The immunoprecipitates or the eluates were then blotted. d Overview of the structures of COP1 wild type and different truncates. HEK293T cells were co-transfected with Myc-Sox2 and the indicated COP1 truncates. The lysates were collected and subjected to immunoprecipitation with anti-Flag. The immunoprecipitates were then blotted. e Overview of the structure of Sox2 wild type and different VP mutants. Recombinant proteins (His-COP1, GST-Sox2, GST-Sox2-A1, GST-Sox2-A2, and GST-Sox2-AA) were expressed and purified. GST-Sox2 bound to glutathione-Sepharose 4B beads was incubated with His- COP1 for 24 h at 4 °C. Then the beads were washed and proteins were eluted, followed by western blotting. f HEK293T cells were transfected with indicated constructs. The lysates were collected and blotted with anti-Flag and anti-Myc antibody. The representative images are shown from three independent experiments. Unprocessed original scans of blots are shown in Supplementary Fig. 9

Techniques Used: Immunoprecipitation, Transfection, Construct, Recombinant, Purification, Incubation, Western Blot

CUL4A DET1-COP1 ubiquitylates SOX2. a HEK293T cell line with knockdown of COP1 (upper panel) or DET1 (lower panel) were treated with CHX (10 µg/ml), and collected at the indicated times for western blot. Quantification of Sox2 level relative to tubulin is shown. Results are shown as mean ± s.d. n = 3 independent experiments. ** P
Figure Legend Snippet: CUL4A DET1-COP1 ubiquitylates SOX2. a HEK293T cell line with knockdown of COP1 (upper panel) or DET1 (lower panel) were treated with CHX (10 µg/ml), and collected at the indicated times for western blot. Quantification of Sox2 level relative to tubulin is shown. Results are shown as mean ± s.d. n = 3 independent experiments. ** P

Techniques Used: Western Blot

COP1 interacts with Sox2 directly. a Cells were transfected with indicated constructs and siRNA. Flag-Sox2 was immunoprecipated with anti-Flag and immunoblotted with anti-Myc. b HEK293T cells were transfected with indicated constructs and siRNA, and Sox2 was immunoprecipated with anti-Flag and immunoblotted with anti-Myc. c HEK293T cells were transfected with indicated constructs and western blot was performed to measure the expression of Flag-Sox2, Flag-COP1, and Myc-DET1. d Increasing amounts of Flag-COP1 were co-transfected together with Flag-Sox2 and DET1 into HEK293T cells and ectopic Flag-Sox2 expression was detected. e The predicted work model of CUL4A DET1-COP1 for Sox2 degradation. The representative images are shown from three independent experiments. Unprocessed original scans of blots are shown in Supplementary Fig. 9
Figure Legend Snippet: COP1 interacts with Sox2 directly. a Cells were transfected with indicated constructs and siRNA. Flag-Sox2 was immunoprecipated with anti-Flag and immunoblotted with anti-Myc. b HEK293T cells were transfected with indicated constructs and siRNA, and Sox2 was immunoprecipated with anti-Flag and immunoblotted with anti-Myc. c HEK293T cells were transfected with indicated constructs and western blot was performed to measure the expression of Flag-Sox2, Flag-COP1, and Myc-DET1. d Increasing amounts of Flag-COP1 were co-transfected together with Flag-Sox2 and DET1 into HEK293T cells and ectopic Flag-Sox2 expression was detected. e The predicted work model of CUL4A DET1-COP1 for Sox2 degradation. The representative images are shown from three independent experiments. Unprocessed original scans of blots are shown in Supplementary Fig. 9

Techniques Used: Transfection, Construct, Western Blot, Expressing

8) Product Images from "The FOXM1–ABCC5 axis contributes to paclitaxel resistance in nasopharyngeal carcinoma cells"

Article Title: The FOXM1–ABCC5 axis contributes to paclitaxel resistance in nasopharyngeal carcinoma cells

Journal: Cell Death & Disease

doi: 10.1038/cddis.2017.53

Paclitaxel-resistant cells increased as a sub-population of CD44+ CSCs and underwent EMT. ( a ) CSC sub-population. CNE2TR and CNE2 cells were labeled with fluorescent antibodies against CD44 (APC). CD44+ cells were detected by flow cytometry. ( b ) CNE2 and CNE2TR cells were seeded in soft agar for cell sphere formation. The protein levels of SOX2, SHH and ALDH1 were tested by western blot. ( c ) Cell migration assay. A confluent monolayer of CNE2TR and CNE2 cells was scratched. Displaced cells were moved, and the cell gaps were monitored at 24, 48 and 72 h after scratching. The cell gap was quantified by Image Pro Plus software and the data were presented as the cell gap distance. ( d ) Cell invasion (transwell) assay. CNE2TR/CNE2 cells were starved for 48 h, and re-plated on transwell plate inserts with serum-free media, whereas culture media with 10% FBS was placed in the bottom wells. Invasive cells on the membrane were stained by crystal violet 24 h after cell plating. ( e ) The expression of EMT-associated molecules was tested in CNE2TR/CNE2 and CNE1T/CNE1 cells. ( f ) The expression of E-cadherin proteins in NPC cells was monitored at 24, 48 and 72 h after paclitaxel treatment. *** P
Figure Legend Snippet: Paclitaxel-resistant cells increased as a sub-population of CD44+ CSCs and underwent EMT. ( a ) CSC sub-population. CNE2TR and CNE2 cells were labeled with fluorescent antibodies against CD44 (APC). CD44+ cells were detected by flow cytometry. ( b ) CNE2 and CNE2TR cells were seeded in soft agar for cell sphere formation. The protein levels of SOX2, SHH and ALDH1 were tested by western blot. ( c ) Cell migration assay. A confluent monolayer of CNE2TR and CNE2 cells was scratched. Displaced cells were moved, and the cell gaps were monitored at 24, 48 and 72 h after scratching. The cell gap was quantified by Image Pro Plus software and the data were presented as the cell gap distance. ( d ) Cell invasion (transwell) assay. CNE2TR/CNE2 cells were starved for 48 h, and re-plated on transwell plate inserts with serum-free media, whereas culture media with 10% FBS was placed in the bottom wells. Invasive cells on the membrane were stained by crystal violet 24 h after cell plating. ( e ) The expression of EMT-associated molecules was tested in CNE2TR/CNE2 and CNE1T/CNE1 cells. ( f ) The expression of E-cadherin proteins in NPC cells was monitored at 24, 48 and 72 h after paclitaxel treatment. *** P

Techniques Used: Labeling, Flow Cytometry, Cytometry, Western Blot, Cell Migration Assay, Software, Transwell Assay, Staining, Expressing

9) Product Images from "In Vivo Generation of Immature Inner Hair Cells in Neonatal Mouse Cochleae by Ectopic Atoh1 Expression"

Article Title: In Vivo Generation of Immature Inner Hair Cells in Neonatal Mouse Cochleae by Ectopic Atoh1 Expression

Journal: PLoS ONE

doi: 10.1371/journal.pone.0089377

Heterogeneous Sox2 expression and absence of Glast1 in new HCs. (A–A’’’) Whole-mount triple staining of Atoh1-HA, myosin VI, and Sox2 in cochleae of PLP/CreER T+ ; Atoh1-HA+ mice at P22. Arrows point to new HCs that maintained Sox2 expression. The arrowhead represents the new HC without Sox2 expression. (B) Percentages of Sox2-positive and Sox2-negative new HCs. OHCs: outer hair cells; IHC: inner hair cell. (C–C’’’) Triple staining of Atoh1-HA, parvalbumin, and Glast1 in cochleae of PLP/CreER T+ ; Atoh1-HA+ mice at P22. Arrows show the new HC that did not express Glast1. Arrowheads point to a Glast1+ IB/IPh cell that wraps the new HC. There was no overlap of parvalbumin and Glast1 signals at the top of the new HC membrane. Scale bar: 20 µm.
Figure Legend Snippet: Heterogeneous Sox2 expression and absence of Glast1 in new HCs. (A–A’’’) Whole-mount triple staining of Atoh1-HA, myosin VI, and Sox2 in cochleae of PLP/CreER T+ ; Atoh1-HA+ mice at P22. Arrows point to new HCs that maintained Sox2 expression. The arrowhead represents the new HC without Sox2 expression. (B) Percentages of Sox2-positive and Sox2-negative new HCs. OHCs: outer hair cells; IHC: inner hair cell. (C–C’’’) Triple staining of Atoh1-HA, parvalbumin, and Glast1 in cochleae of PLP/CreER T+ ; Atoh1-HA+ mice at P22. Arrows show the new HC that did not express Glast1. Arrowheads point to a Glast1+ IB/IPh cell that wraps the new HC. There was no overlap of parvalbumin and Glast1 signals at the top of the new HC membrane. Scale bar: 20 µm.

Techniques Used: Expressing, Staining, Plasmid Purification, Mouse Assay, Immunohistochemistry

10) Product Images from "Dopamine D2 receptor and β-arrestin 2 mediate Amyloid-β elevation induced by anti-parkinson’s disease drugs, levodopa and piribedil, in neuronal cells"

Article Title: Dopamine D2 receptor and β-arrestin 2 mediate Amyloid-β elevation induced by anti-parkinson’s disease drugs, levodopa and piribedil, in neuronal cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0173240

Levodopa and piribedil enhance Aβ generation and γ-secretase activity in induced human neuronal cells. (A) Human NSC and its induced neuronal cells stained with DAPI, nestin, Sox2, ki67 or DCX. Scale bar, 20 μm. (B) qPCR quantification of NSC-expressing genes, nestin and Sox2. Data are mean + s.e.m., n = 3. ***p
Figure Legend Snippet: Levodopa and piribedil enhance Aβ generation and γ-secretase activity in induced human neuronal cells. (A) Human NSC and its induced neuronal cells stained with DAPI, nestin, Sox2, ki67 or DCX. Scale bar, 20 μm. (B) qPCR quantification of NSC-expressing genes, nestin and Sox2. Data are mean + s.e.m., n = 3. ***p

Techniques Used: Activity Assay, Staining, Real-time Polymerase Chain Reaction, Expressing

11) Product Images from "Hey1 and Hey2 Control the Spatial and Temporal Pattern of Mammalian Auditory Hair Cell Differentiation Downstream of Hedgehog Signaling"

Article Title: Hey1 and Hey2 Control the Spatial and Temporal Pattern of Mammalian Auditory Hair Cell Differentiation Downstream of Hedgehog Signaling

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.1494-14.2014

Hey1 and Hey2 downregulation in prosensory cells occurs independently of Atoh1 . A–D , Hey1 and Hey2 transcripts are highly expressed in prosensory cells. At E13.5, Hey1 ( C ) and Hey2 ( D ) are coexpressed with Sox2 ( A ) in the prosensory domain (bracket) and Atoh1 ( B ) begins to be upregulated in the cochlear base. A , * Sox2 expression in spiral ganglion neurons. Scale bar, 200 μm. E–L , Downregulation of Hey1 and Hey2 occurs in the absence of HC differentiation. In E15.5, Atoh1 fl/+ (wild-type) cochlear tissue, basal-to-apical upregulation of Atoh1 ( F ) in IHCs (arrowhead) and OHCs (bar) coincides with the basal-to-apical downregulation of Hey1 ( G ) and Hey2 ( H ) in Sox2 -positive HC and SC precursors ( E , bracket). E , * Sox2 expression in ganglion neurons. In E15.5, Atoh1 mutant ( Atoh1 Δ/Δ ) cochlear tissue initial downregulation of Hey1 ( K ) and Hey2 ( L ) in Sox2 -positive HC and SC precursors ( I , bracket) occurs in the absence of Atoh1 ( J ). Red asterisks indicate degenerated sensory epithelia in the Atoh1 mutant cochlear base. Scale bars, 200 μm.
Figure Legend Snippet: Hey1 and Hey2 downregulation in prosensory cells occurs independently of Atoh1 . A–D , Hey1 and Hey2 transcripts are highly expressed in prosensory cells. At E13.5, Hey1 ( C ) and Hey2 ( D ) are coexpressed with Sox2 ( A ) in the prosensory domain (bracket) and Atoh1 ( B ) begins to be upregulated in the cochlear base. A , * Sox2 expression in spiral ganglion neurons. Scale bar, 200 μm. E–L , Downregulation of Hey1 and Hey2 occurs in the absence of HC differentiation. In E15.5, Atoh1 fl/+ (wild-type) cochlear tissue, basal-to-apical upregulation of Atoh1 ( F ) in IHCs (arrowhead) and OHCs (bar) coincides with the basal-to-apical downregulation of Hey1 ( G ) and Hey2 ( H ) in Sox2 -positive HC and SC precursors ( E , bracket). E , * Sox2 expression in ganglion neurons. In E15.5, Atoh1 mutant ( Atoh1 Δ/Δ ) cochlear tissue initial downregulation of Hey1 ( K ) and Hey2 ( L ) in Sox2 -positive HC and SC precursors ( I , bracket) occurs in the absence of Atoh1 ( J ). Red asterisks indicate degenerated sensory epithelia in the Atoh1 mutant cochlear base. Scale bars, 200 μm.

Techniques Used: Expressing, Mutagenesis

Loss of Hey1 and Hey2 does not alter prosensory cell proliferation. A–G , Loss of Hey1 and Hey2 does not alter the apical-to-basal gradient of prosensory cell cycle exit. Incorporation of EdU in prosensory cells was analyzed in Hey2 −/− (control) ( A , C , E ) and Hey1 Δ/Δ Hey2 −/− (DKO) ( B , D , F ) animals. EdU was injected at E13.5, and E15.5 cochlear sections were immunostained for prosensory marker SOX2 (green) and processed for EdU labeling (red). Shown are both merged and EdU only images (′) for the cochlear apex ( A–B′ ), mid ( C–D′ ), and base ( E–F′ ). White bracket indicates prosensory domain. Scale bar, 100 μm. G , Quantification of EdU incorporation in Hey2 −/− control (gray bar) and DKO (red bar) prosensory cells in the cochlear apex, mid, and base. Data are mean ± SEM ( n = 3, two independent experiments). n.s., Not significant. H–T , Loss of Hey1 and Hey2 does not alter proliferation rate of HC and SC precursors. EdU was injected at E12.5 and EdU incorporation in HCs and SCs was analyzed at E18.5 in control ( Hey2 −/+ and Hey1 Δ/Δ Hey2 −/+ ) and DKO cochlear surface preparations. H–Q , EdU incorporation in E18.5 Hey2 −/+ (control) ( H–L ) and DKO ( M–Q ) cochlear sensory epithelium. Shown are high-power confocal images of MYO6-positive HC layer (green) at the cochlear apical tip ( H , M ), apex ( I , N ), mid-apex ( J , O ), mid-base ( K , P ), and base ( L , Q ). Arrows point to IHCs; brackets indicate OHCs. EdU-positive nuclei are shown in red. Scale bar, 50 μm. R–T , Quantification of EdU incorporation in IHCs ( R ), OHCs ( S ), and oSCs ( T ) in E18.5 Hey2 −/+ (light gray), Hey1 Δ/Δ Hey2 −/+ (dark gray), and DKO (red). Data are mean ± SEM ( n = 3, two independent experiments). * p ≤ 0.05. n.s., Not significant.
Figure Legend Snippet: Loss of Hey1 and Hey2 does not alter prosensory cell proliferation. A–G , Loss of Hey1 and Hey2 does not alter the apical-to-basal gradient of prosensory cell cycle exit. Incorporation of EdU in prosensory cells was analyzed in Hey2 −/− (control) ( A , C , E ) and Hey1 Δ/Δ Hey2 −/− (DKO) ( B , D , F ) animals. EdU was injected at E13.5, and E15.5 cochlear sections were immunostained for prosensory marker SOX2 (green) and processed for EdU labeling (red). Shown are both merged and EdU only images (′) for the cochlear apex ( A–B′ ), mid ( C–D′ ), and base ( E–F′ ). White bracket indicates prosensory domain. Scale bar, 100 μm. G , Quantification of EdU incorporation in Hey2 −/− control (gray bar) and DKO (red bar) prosensory cells in the cochlear apex, mid, and base. Data are mean ± SEM ( n = 3, two independent experiments). n.s., Not significant. H–T , Loss of Hey1 and Hey2 does not alter proliferation rate of HC and SC precursors. EdU was injected at E12.5 and EdU incorporation in HCs and SCs was analyzed at E18.5 in control ( Hey2 −/+ and Hey1 Δ/Δ Hey2 −/+ ) and DKO cochlear surface preparations. H–Q , EdU incorporation in E18.5 Hey2 −/+ (control) ( H–L ) and DKO ( M–Q ) cochlear sensory epithelium. Shown are high-power confocal images of MYO6-positive HC layer (green) at the cochlear apical tip ( H , M ), apex ( I , N ), mid-apex ( J , O ), mid-base ( K , P ), and base ( L , Q ). Arrows point to IHCs; brackets indicate OHCs. EdU-positive nuclei are shown in red. Scale bar, 50 μm. R–T , Quantification of EdU incorporation in IHCs ( R ), OHCs ( S ), and oSCs ( T ) in E18.5 Hey2 −/+ (light gray), Hey1 Δ/Δ Hey2 −/+ (dark gray), and DKO (red). Data are mean ± SEM ( n = 3, two independent experiments). * p ≤ 0.05. n.s., Not significant.

Techniques Used: Injection, Marker, Labeling

Loss of Hey1 and Hey2 results in complex HC and SC patterning defects. A–W′ , HC and SC phenotype was analyzed in the early postnatal control ( Hey2 −/− and Hey2 −/+ ) and DKO cochlea in whole mounts (surface preparations) and sections. HCs were labeled using MYO6 and phalloidin staining, and SCs were labeled using SOX2 staining. HC and SC subtypes were identified by morphology and their relative location within the sensory epithelium. Arrowheads indicate IHC domain; brackets indicate OHC domain. Dashed line indicates missing OHCs (white) and missing oSCs (yellow). Asterisks label ectopic IHCs (white) and ectopic iSCs (yellow). Arrows indicate ectopic OHCs (white) and ectopic oSCs (yellow). bc, inner border cell; ph, inner phalangeal cell; ip, inner pillar cell; op, outer pillar cell; d1–3, Deiters cells. A–F , HC phenotype in Hey2 −/− ( A , C , E ) and DKO ( B , D , F ) cochlear surface preparations, Stage P0. Shown are high-power confocal images of MYO6 and phalloidin-positive HC layer at apical ( A , B ), mid ( C , D ), and basal ( E , F ) positions. Scale bar, 50 μm. G , H , HC phenotype in Hey2 −/− ( G ) and DKO ( H ) cochlear surface preparations, Stage P4. Shown are high-power confocal images of the HC layer (phalloidin, white) at an apical location. Scale bar, 50 μm. I–K , Quantification of ectopic IHCs ( I ), ectopic OHCs ( J ), and missing OHCs ( K ) in DKO (red diamonds) and Hey2 −/− (green diamonds) and Hey2 −/+ (black diamonds) cochlea Stage P0–P2. Cochlear surface preparations were divided into three segments (base, mid, apex), and ectopic and missing HCs were counted for each segment per 1 mm. Data are mean ± SEM ( n = 7, three independent experiments). * p ≤ 0.05. L–O′ , HC and SC phenotype in DKO ( M , M′ , O , O′ ) and Hey2 −/− (control) ( L , L′ , N , N′ ) cochlear surface preparations, Stage P0. Shown are confocal images of the HC layer (phalloidin, white) and corresponding (′) SC layer (SOX2, green) of apical ( L–M′ ) and basal segments ( N–O′ ). Scale bar, 50 μm. P–U , SC phenotype in Hey2 −/− (control) ( P , R , T ) and DKO ( Q , S , U ) cochlear sections, Stage P0. Shown are confocal images of apical ( P , Q ), mid ( R , S ), and basal ( T , U ) cochlear sections immunostained with HC marker MYO6 (red) and SC marker SOX2 (green). Scale bar, 50 μm. V–W′ , Pillar cell phenotype in Hey2 −/+ (control) ( V , V′ ) and DKO ( W , W′ ) cochlear surface preparations, Stage P0. Shown are merged and single (′) channel confocal images of HC layer in cochlear apex. Phalloidin-positive HC bundles are shown in red, and p75 (NGFR)-positive pillar cell heads are shown in green and white (′). The pillar cell-specific p75 staining is disrupted by an ectopic HC (white asterisk) in the DKO cochlea ( W , W′ ). Scale bar, 50 μm.
Figure Legend Snippet: Loss of Hey1 and Hey2 results in complex HC and SC patterning defects. A–W′ , HC and SC phenotype was analyzed in the early postnatal control ( Hey2 −/− and Hey2 −/+ ) and DKO cochlea in whole mounts (surface preparations) and sections. HCs were labeled using MYO6 and phalloidin staining, and SCs were labeled using SOX2 staining. HC and SC subtypes were identified by morphology and their relative location within the sensory epithelium. Arrowheads indicate IHC domain; brackets indicate OHC domain. Dashed line indicates missing OHCs (white) and missing oSCs (yellow). Asterisks label ectopic IHCs (white) and ectopic iSCs (yellow). Arrows indicate ectopic OHCs (white) and ectopic oSCs (yellow). bc, inner border cell; ph, inner phalangeal cell; ip, inner pillar cell; op, outer pillar cell; d1–3, Deiters cells. A–F , HC phenotype in Hey2 −/− ( A , C , E ) and DKO ( B , D , F ) cochlear surface preparations, Stage P0. Shown are high-power confocal images of MYO6 and phalloidin-positive HC layer at apical ( A , B ), mid ( C , D ), and basal ( E , F ) positions. Scale bar, 50 μm. G , H , HC phenotype in Hey2 −/− ( G ) and DKO ( H ) cochlear surface preparations, Stage P4. Shown are high-power confocal images of the HC layer (phalloidin, white) at an apical location. Scale bar, 50 μm. I–K , Quantification of ectopic IHCs ( I ), ectopic OHCs ( J ), and missing OHCs ( K ) in DKO (red diamonds) and Hey2 −/− (green diamonds) and Hey2 −/+ (black diamonds) cochlea Stage P0–P2. Cochlear surface preparations were divided into three segments (base, mid, apex), and ectopic and missing HCs were counted for each segment per 1 mm. Data are mean ± SEM ( n = 7, three independent experiments). * p ≤ 0.05. L–O′ , HC and SC phenotype in DKO ( M , M′ , O , O′ ) and Hey2 −/− (control) ( L , L′ , N , N′ ) cochlear surface preparations, Stage P0. Shown are confocal images of the HC layer (phalloidin, white) and corresponding (′) SC layer (SOX2, green) of apical ( L–M′ ) and basal segments ( N–O′ ). Scale bar, 50 μm. P–U , SC phenotype in Hey2 −/− (control) ( P , R , T ) and DKO ( Q , S , U ) cochlear sections, Stage P0. Shown are confocal images of apical ( P , Q ), mid ( R , S ), and basal ( T , U ) cochlear sections immunostained with HC marker MYO6 (red) and SC marker SOX2 (green). Scale bar, 50 μm. V–W′ , Pillar cell phenotype in Hey2 −/+ (control) ( V , V′ ) and DKO ( W , W′ ) cochlear surface preparations, Stage P0. Shown are merged and single (′) channel confocal images of HC layer in cochlear apex. Phalloidin-positive HC bundles are shown in red, and p75 (NGFR)-positive pillar cell heads are shown in green and white (′). The pillar cell-specific p75 staining is disrupted by an ectopic HC (white asterisk) in the DKO cochlea ( W , W′ ). Scale bar, 50 μm.

Techniques Used: Labeling, Staining, Immunohistochemistry, Marker

Loss of Hey1 and Hey2 results in accelerated auditory HC differentiation. A , qPCR analysis of relative Atoh1 , Sox2 , Hey1 , and Hey2 mRNA levels in Hey1 Δ/Δ Hey2 −/− (DKO) and control ( Hey1 Δ/Δ Hey2 −/+ , Hey1 +/+ Hey2 −/+ and Hey1 +/+ Hey2 −/− ) cochlear epithelia, Stage E13.5. Data are mean ± SEM. B–E , ISH-based analysis of Atoh1 expression pattern in Hey2 −/− (control) ( B , D ) and Hey1 Δ/Δ Hey2 −/− (DKO) ( C , E ) cochlear sections, Stage E14.5 ( B , C ) and Stage E15.0 ( D , E ). At both E14.5 and E15.0, Atoh1 expression extends further apically in DKO ( C , E ) than control ( B , D ) cochlear sections. Scale bar, 100 μm. F–G″ , Low-power ( F , G ) and high-power ( F′ , F″ , G′ , G″ ) confocal images of HC-specific Atoh1/nGFP reporter expression (green) in E14.0 Hey2 −/− (control; F , F′ , F″ ) and E14.0 Hey1 Δ/Δ Hey2 −/− (DKO; G , G′ , G″ ) cochlear sections. F , G , Yellow boxes represent location of the high-power images ( F′ , F″ , G′ , G″ ). Scale bar, 100 μm. H–I , Low-power images of E15.0 Hey2 −/− ( I ) and E15.0 DKO ( H ) cochlear epithelial ducts immunostained for myosin VI (MYO6, white). Yellow dotted line indicates MYO6-positive sensory domains; red arrows indicate beginning and end. Sale bar, 100 μm. J , K , Quantification of cochlear length and extent of HC differentiation in E15.0 Hey1 Δ/Δ Hey2 −/− (DKO) and Hey2 −/+ and Hey2 −/− (control) littermates. Graphs represent cochlear duct length ( J ) and length of MYO6-positive sensory domain ( K ) for control (gray bar) and DKO (red bar) cochlear ducts. Data are mean ± SEM. n = 4 or 5 cochlear explants from three independent experiments. * p ≤ 0.05. n.s., Not significant.
Figure Legend Snippet: Loss of Hey1 and Hey2 results in accelerated auditory HC differentiation. A , qPCR analysis of relative Atoh1 , Sox2 , Hey1 , and Hey2 mRNA levels in Hey1 Δ/Δ Hey2 −/− (DKO) and control ( Hey1 Δ/Δ Hey2 −/+ , Hey1 +/+ Hey2 −/+ and Hey1 +/+ Hey2 −/− ) cochlear epithelia, Stage E13.5. Data are mean ± SEM. B–E , ISH-based analysis of Atoh1 expression pattern in Hey2 −/− (control) ( B , D ) and Hey1 Δ/Δ Hey2 −/− (DKO) ( C , E ) cochlear sections, Stage E14.5 ( B , C ) and Stage E15.0 ( D , E ). At both E14.5 and E15.0, Atoh1 expression extends further apically in DKO ( C , E ) than control ( B , D ) cochlear sections. Scale bar, 100 μm. F–G″ , Low-power ( F , G ) and high-power ( F′ , F″ , G′ , G″ ) confocal images of HC-specific Atoh1/nGFP reporter expression (green) in E14.0 Hey2 −/− (control; F , F′ , F″ ) and E14.0 Hey1 Δ/Δ Hey2 −/− (DKO; G , G′ , G″ ) cochlear sections. F , G , Yellow boxes represent location of the high-power images ( F′ , F″ , G′ , G″ ). Scale bar, 100 μm. H–I , Low-power images of E15.0 Hey2 −/− ( I ) and E15.0 DKO ( H ) cochlear epithelial ducts immunostained for myosin VI (MYO6, white). Yellow dotted line indicates MYO6-positive sensory domains; red arrows indicate beginning and end. Sale bar, 100 μm. J , K , Quantification of cochlear length and extent of HC differentiation in E15.0 Hey1 Δ/Δ Hey2 −/− (DKO) and Hey2 −/+ and Hey2 −/− (control) littermates. Graphs represent cochlear duct length ( J ) and length of MYO6-positive sensory domain ( K ) for control (gray bar) and DKO (red bar) cochlear ducts. Data are mean ± SEM. n = 4 or 5 cochlear explants from three independent experiments. * p ≤ 0.05. n.s., Not significant.

Techniques Used: Real-time Polymerase Chain Reaction, In Situ Hybridization, Expressing

12) Product Images from "Combinatorial effects of an epigenetic inhibitor and ionizing radiation contribute to targeted elimination of pancreatic cancer stem cell"

Article Title: Combinatorial effects of an epigenetic inhibitor and ionizing radiation contribute to targeted elimination of pancreatic cancer stem cell

Journal: Oncotarget

doi: 10.18632/oncotarget.21642

5-aza-dC treatment in combination with IR reduced the regulatory factors of self-renewal and cell surface markers of CSCs in pancreatic cancer cells (A) Immunoblot analysis was performed to measure the expression pattern for the regulatory factors of self-renewal (Oct4, Nanog, Sox2, and ALDH1) and cell surface markers (CD44, CD24, and CD133) in in MIA PaCa-2 and PANC-1 cells treated with 5-aza-dC alone or with irradiation (2 and 4 Gy). (B-C) Right: FACS for CD44 and CD24 cells of MIA PaCa-2 (B) and PANC-1 (C) cells treated with 5-aza-dC or IR, both alone and in combination. Orange indicated CD44+/CD24+ population. Red indicated CD44-/CD24- population. Left: % of CD44+, CD44- or CD44-/CD24- pancreatic cancer cells after irradiation and 5-aza-dC treatment in pancreatic cancer cells. A1, A2, A3, and A4 indicate CD44+/CD24-, CD44+/CD24+, CD44-/CD24-, CD44-/CD24+ populations, respectively. Data are means ± standard deviation from 3 independent experiments. P -values were calculated using Student’s t -test. * P
Figure Legend Snippet: 5-aza-dC treatment in combination with IR reduced the regulatory factors of self-renewal and cell surface markers of CSCs in pancreatic cancer cells (A) Immunoblot analysis was performed to measure the expression pattern for the regulatory factors of self-renewal (Oct4, Nanog, Sox2, and ALDH1) and cell surface markers (CD44, CD24, and CD133) in in MIA PaCa-2 and PANC-1 cells treated with 5-aza-dC alone or with irradiation (2 and 4 Gy). (B-C) Right: FACS for CD44 and CD24 cells of MIA PaCa-2 (B) and PANC-1 (C) cells treated with 5-aza-dC or IR, both alone and in combination. Orange indicated CD44+/CD24+ population. Red indicated CD44-/CD24- population. Left: % of CD44+, CD44- or CD44-/CD24- pancreatic cancer cells after irradiation and 5-aza-dC treatment in pancreatic cancer cells. A1, A2, A3, and A4 indicate CD44+/CD24-, CD44+/CD24+, CD44-/CD24-, CD44-/CD24+ populations, respectively. Data are means ± standard deviation from 3 independent experiments. P -values were calculated using Student’s t -test. * P

Techniques Used: Expressing, Irradiation, FACS, Standard Deviation

13) Product Images from "Melanoma Spheroids Grown Under Neural Crest Cell Conditions Are Highly Plastic Migratory/Invasive Tumor Cells Endowed with Immunomodulator Function"

Article Title: Melanoma Spheroids Grown Under Neural Crest Cell Conditions Are Highly Plastic Migratory/Invasive Tumor Cells Endowed with Immunomodulator Function

Journal: PLoS ONE

doi: 10.1371/journal.pone.0018784

Melanoma spheroid cells display higher expression of pluripotency transcription factors. A . Expression of pluripotency transcription factors OCT4, NANOG, SOX2 and KLF4 by spheroid (MS) and adherent (MA) cells from SLM8 and Mela1 was determined by quantitative PCR. Results are presented as mean values of relative mRNA expression ± SD from three independent experiments; *p
Figure Legend Snippet: Melanoma spheroid cells display higher expression of pluripotency transcription factors. A . Expression of pluripotency transcription factors OCT4, NANOG, SOX2 and KLF4 by spheroid (MS) and adherent (MA) cells from SLM8 and Mela1 was determined by quantitative PCR. Results are presented as mean values of relative mRNA expression ± SD from three independent experiments; *p

Techniques Used: Expressing, Mass Spectrometry, Real-time Polymerase Chain Reaction

14) Product Images from "Knockdown of Foxg1 in supporting cells increases the trans-differentiation of supporting cells into hair cells in the neonatal mouse cochlea"

Article Title: Knockdown of Foxg1 in supporting cells increases the trans-differentiation of supporting cells into hair cells in the neonatal mouse cochlea

Journal: Cellular and Molecular Life Sciences

doi: 10.1007/s00018-019-03291-2

Lineage tracing of Sox2+ SCs. a Tamoxifen was injected at P3, and Sox2+ SCs were traced by following the expression of tdTomato fluorescent protein. b , c Lineage tracing images of cochlear Sox2+ SCs in Sox2 CreER/+ Foxg1 loxp/loxp Rosa26-tdTomato mice ( b ) and Sox2 CreER/+ Rosa26-tdTomato mice ( c ). tdTomato+/Myo7a+ IHCs and OHCs are indicated by arrows and arrowheads, respectively. Scale bar, 20 µm. d Quantification of tdTomato+ (Tom+) IHCs and OHCs per cochlea and per turn. The n refers to the number of mice. * p
Figure Legend Snippet: Lineage tracing of Sox2+ SCs. a Tamoxifen was injected at P3, and Sox2+ SCs were traced by following the expression of tdTomato fluorescent protein. b , c Lineage tracing images of cochlear Sox2+ SCs in Sox2 CreER/+ Foxg1 loxp/loxp Rosa26-tdTomato mice ( b ) and Sox2 CreER/+ Rosa26-tdTomato mice ( c ). tdTomato+/Myo7a+ IHCs and OHCs are indicated by arrows and arrowheads, respectively. Scale bar, 20 µm. d Quantification of tdTomato+ (Tom+) IHCs and OHCs per cochlea and per turn. The n refers to the number of mice. * p

Techniques Used: Injection, Expressing, Mouse Assay

Foxg1 cKD in Sox2+ SCs results in increased HC number and decreased SC number. a Tamoxifen was I.P. injected into P1 Sox2 CreER/+ Foxg1 loxp/loxp Rosa26-tdTomato mice to knock down Foxg1 in Sox2+ SCs, and the mice were sacrificed at P3 for FAC sorting of Sox2+ SCs for real-time qPCR. b FAC sorting data for Sox2+ SCs. c Quantification of Foxg1 mRNA expression based on four independent qPCR experiments. *** p
Figure Legend Snippet: Foxg1 cKD in Sox2+ SCs results in increased HC number and decreased SC number. a Tamoxifen was I.P. injected into P1 Sox2 CreER/+ Foxg1 loxp/loxp Rosa26-tdTomato mice to knock down Foxg1 in Sox2+ SCs, and the mice were sacrificed at P3 for FAC sorting of Sox2+ SCs for real-time qPCR. b FAC sorting data for Sox2+ SCs. c Quantification of Foxg1 mRNA expression based on four independent qPCR experiments. *** p

Techniques Used: Injection, Mouse Assay, Real-time Polymerase Chain Reaction, Expressing

The extra IHCs could survive to P30. a Tamoxifen was I.P. injected at P1, and the mice were sacrificed at P7, P14, and P30. b , c Extra IHCs (arrows) and OHCs (square brackets) are seen in the apical (Apex), middle (Middle), and basal (Base) turns of P7 Sox2 CreER/+ Foxg1 loxp/loxp mice cochleae. Sox2 CreER/+ mice were used as controls. Myo7a was used as the HC marker. Scale bar, 50 µm. ( d , e ) Quantification of the total IHCs and OHCs per 100 µm cochlea length at P14 and P30 ( d ) and the comparison between the three ages in control and Foxg1 cKD mice ( e ). The n refers to the number of mice. * p
Figure Legend Snippet: The extra IHCs could survive to P30. a Tamoxifen was I.P. injected at P1, and the mice were sacrificed at P7, P14, and P30. b , c Extra IHCs (arrows) and OHCs (square brackets) are seen in the apical (Apex), middle (Middle), and basal (Base) turns of P7 Sox2 CreER/+ Foxg1 loxp/loxp mice cochleae. Sox2 CreER/+ mice were used as controls. Myo7a was used as the HC marker. Scale bar, 50 µm. ( d , e ) Quantification of the total IHCs and OHCs per 100 µm cochlea length at P14 and P30 ( d ) and the comparison between the three ages in control and Foxg1 cKD mice ( e ). The n refers to the number of mice. * p

Techniques Used: Injection, Mouse Assay, Marker

The proliferation of Sox2+ SCs and Lgr5+ progenitors has no change in Foxg1 cKD mice. a EdU (50 mg/kg body weight) was injected at P3, P4, and P5 to label proliferating cells. b EdU was stained (blue) in Sox2 CreER/+ Foxg1 loxp/loxp , Foxg1 loxp/loxp , and Sox2 CreER/+ mice. Myo7a and Sox2 were used as HC and SC markers, respectively. Scale bar, 20 µm. c Quantification of EdU+ SCs per cochlea. n = 3 mice per group. n.s . not significant. d Tamoxifen was injected into Lgr5-EGFP CreER/+ Foxg1 loxp/loxp mice to conditionally knockdown Foxg1 in Lgr5+ progenitors. After 2 days, Lgr5+ progenitors were isolated by FAC sorting and cultured in vitro for 5 days to form spheres. e Spheres formed by Lgr5+ progenitors from Lgr5-EGFP CreER/+ Foxg1 loxp/loxp and Lgr5-EGFP CreER/+ mice. Scale bar, 50 µm. f Quantification of sphere number per well and sphere diameter of each passage. At least three wells of spheres were quantified. n.s . not significant
Figure Legend Snippet: The proliferation of Sox2+ SCs and Lgr5+ progenitors has no change in Foxg1 cKD mice. a EdU (50 mg/kg body weight) was injected at P3, P4, and P5 to label proliferating cells. b EdU was stained (blue) in Sox2 CreER/+ Foxg1 loxp/loxp , Foxg1 loxp/loxp , and Sox2 CreER/+ mice. Myo7a and Sox2 were used as HC and SC markers, respectively. Scale bar, 20 µm. c Quantification of EdU+ SCs per cochlea. n = 3 mice per group. n.s . not significant. d Tamoxifen was injected into Lgr5-EGFP CreER/+ Foxg1 loxp/loxp mice to conditionally knockdown Foxg1 in Lgr5+ progenitors. After 2 days, Lgr5+ progenitors were isolated by FAC sorting and cultured in vitro for 5 days to form spheres. e Spheres formed by Lgr5+ progenitors from Lgr5-EGFP CreER/+ Foxg1 loxp/loxp and Lgr5-EGFP CreER/+ mice. Scale bar, 50 µm. f Quantification of sphere number per well and sphere diameter of each passage. At least three wells of spheres were quantified. n.s . not significant

Techniques Used: Mouse Assay, Injection, Staining, Isolation, Cell Culture, In Vitro

15) Product Images from "Toll-Like Receptor 4 Deficiency Impairs Motor Coordination"

Article Title: Toll-Like Receptor 4 Deficiency Impairs Motor Coordination

Journal: Frontiers in Neuroscience

doi: 10.3389/fnins.2016.00033

TLR4 is highly expressed in cerebellar PCs. (A) TLR4 expression was detected in the PL. (B) Immunohistochemical analysis of calbindin revealed colocalization with TLR4 in PCs. (C) TLR4 expression was not detected in Sox2-positive BG in the PL. (D) NeuN-positive GCs located in the GCL and PL (dashed lines) did not express TLR4. Scar bar = 50 μm in (A) , 25 μm in (B,C,D) . ML, molecular layer; PL, Purkinje cell layer; GCL, granule cell layer.
Figure Legend Snippet: TLR4 is highly expressed in cerebellar PCs. (A) TLR4 expression was detected in the PL. (B) Immunohistochemical analysis of calbindin revealed colocalization with TLR4 in PCs. (C) TLR4 expression was not detected in Sox2-positive BG in the PL. (D) NeuN-positive GCs located in the GCL and PL (dashed lines) did not express TLR4. Scar bar = 50 μm in (A) , 25 μm in (B,C,D) . ML, molecular layer; PL, Purkinje cell layer; GCL, granule cell layer.

Techniques Used: Expressing, Immunohistochemistry

16) Product Images from "Accumulation of low-dose BIX01294 promotes metastatic potential of U251 glioblastoma cells"

Article Title: Accumulation of low-dose BIX01294 promotes metastatic potential of U251 glioblastoma cells

Journal: Oncology Letters

doi: 10.3892/ol.2017.5626

Sequential treatment with Bix increases the expression of cancer stem cell markers and neurosphere formation in U251 cells. (A) KLF4, CD133, SOX2 and OCT4 mRNA expression patterns under single or sequential treatment with Bix were assessed by reverse
Figure Legend Snippet: Sequential treatment with Bix increases the expression of cancer stem cell markers and neurosphere formation in U251 cells. (A) KLF4, CD133, SOX2 and OCT4 mRNA expression patterns under single or sequential treatment with Bix were assessed by reverse

Techniques Used: Expressing

17) Product Images from "Dormant glioblastoma cells acquire stem cell characteristics and are differentially affected by Temozolomide and AT101 treatment"

Article Title: Dormant glioblastoma cells acquire stem cell characteristics and are differentially affected by Temozolomide and AT101 treatment

Journal: Oncotarget

doi: 10.18632/oncotarget.22514

(Co)-Expression of EphA5, IGFBP5 and H2BK with the stem cell markers KLF4, OCT4, MSI1 or SOX2 in solid human GBM samples Solid human GBM sections were stained by double-immunohistochemistry for IGFBP5, EphA5 and H2BK in combinations with KLF4, OCT4, MSI1 or SOX2 (white bars indicate 20 μm).
Figure Legend Snippet: (Co)-Expression of EphA5, IGFBP5 and H2BK with the stem cell markers KLF4, OCT4, MSI1 or SOX2 in solid human GBM samples Solid human GBM sections were stained by double-immunohistochemistry for IGFBP5, EphA5 and H2BK in combinations with KLF4, OCT4, MSI1 or SOX2 (white bars indicate 20 μm).

Techniques Used: Expressing, Staining, Immunohistochemistry

Induction and (co)-expression of dormancy- and stemness-associated genes during TMZ treatment in glioma cell lines ( A and C ) Non-stem GBM cell lines were stimulated with 500 μM TMZ or 0.2% DMSO (control) for 10 days and EphA5, IGFBP5 and H2BK (A) and OCT4, KLF4, MSI1 or SOX2 (C) expression was analysed in TMZ-stimulated compared to control samples using qRT-PCR ( * p
Figure Legend Snippet: Induction and (co)-expression of dormancy- and stemness-associated genes during TMZ treatment in glioma cell lines ( A and C ) Non-stem GBM cell lines were stimulated with 500 μM TMZ or 0.2% DMSO (control) for 10 days and EphA5, IGFBP5 and H2BK (A) and OCT4, KLF4, MSI1 or SOX2 (C) expression was analysed in TMZ-stimulated compared to control samples using qRT-PCR ( * p

Techniques Used: Expressing, Quantitative RT-PCR

18) Product Images from "In Vivo Visualization of Notch1 Proteolysis Reveals the Heterogeneity of Notch1 Signaling Activity in the Mouse Cochlea"

Article Title: In Vivo Visualization of Notch1 Proteolysis Reveals the Heterogeneity of Notch1 Signaling Activity in the Mouse Cochlea

Journal: PLoS ONE

doi: 10.1371/journal.pone.0064903

Characterization of the Notch1 Cre (Low)/+ mouse line. ( A ) Schematic illustration of Notch1 Cre/+ mice. The NICD was replaced by 6×Myc–tagged Cre recombinase. The blue arrow represents the cleavage site. ( B–D’ ) Comparison between cochleae from Notch1 Cre/+ mice and control mice ( Notch1 +/+ ). (B, B’) Myosin-VI+ OHCs (three rows; red) and IHCs (one row; red) sit above Sox2+ SCs (green) in a control ( Notch1 +/+ ) cochlea at P6. (C, C’) In most regions across the entire cochlea, Notch1 Cre (Low)/+ mice is indistinguishable from controls. (D, D’) An extra row of DCs always appear underneath the fourth row of OHCs (white dotted rectangular area) in Notch1 Cre (Low)/+ cochleae. Although extra DCs and OHCs are frequently observed, each of them spans only a short stretch. The Sox2+ cells outside the dotted line (B’, C’, and D’) are Hensen cells (h). ( E–G ) Morphology of HCs at P30 in control (E) and Notch1 Cre (Low)/+ mice (F–G). The distance between OHCs and IHCs is extended. The extra row of OHCs (arrow in G) in Notch1 Cre (Low)/+ mice survive and align well with surrounding HCs. D1–D4: three or four rows of Deiters’ cell; OPC: outer pillar cell; IPC: inner pillar cell; IPH: inner phalangeal cell; h: Hensen’s cell. ECD: extracellular domain; TM: transmembrane domain; NICD: Notch1 intracellular domain. Bars: 20 µm. Bar in (B) also applies to C–D’. Bar in (E) also applies to (F).
Figure Legend Snippet: Characterization of the Notch1 Cre (Low)/+ mouse line. ( A ) Schematic illustration of Notch1 Cre/+ mice. The NICD was replaced by 6×Myc–tagged Cre recombinase. The blue arrow represents the cleavage site. ( B–D’ ) Comparison between cochleae from Notch1 Cre/+ mice and control mice ( Notch1 +/+ ). (B, B’) Myosin-VI+ OHCs (three rows; red) and IHCs (one row; red) sit above Sox2+ SCs (green) in a control ( Notch1 +/+ ) cochlea at P6. (C, C’) In most regions across the entire cochlea, Notch1 Cre (Low)/+ mice is indistinguishable from controls. (D, D’) An extra row of DCs always appear underneath the fourth row of OHCs (white dotted rectangular area) in Notch1 Cre (Low)/+ cochleae. Although extra DCs and OHCs are frequently observed, each of them spans only a short stretch. The Sox2+ cells outside the dotted line (B’, C’, and D’) are Hensen cells (h). ( E–G ) Morphology of HCs at P30 in control (E) and Notch1 Cre (Low)/+ mice (F–G). The distance between OHCs and IHCs is extended. The extra row of OHCs (arrow in G) in Notch1 Cre (Low)/+ mice survive and align well with surrounding HCs. D1–D4: three or four rows of Deiters’ cell; OPC: outer pillar cell; IPC: inner pillar cell; IPH: inner phalangeal cell; h: Hensen’s cell. ECD: extracellular domain; TM: transmembrane domain; NICD: Notch1 intracellular domain. Bars: 20 µm. Bar in (B) also applies to C–D’. Bar in (E) also applies to (F).

Techniques Used: Mouse Assay

Notch1 Cre/+ -mediated reporter expression is difficult to detect in the cochlear prosensory domain at embryonic day (E) 14.5. (A–A’) A single slice of confocal image demonstrating that tdTomato reporter expression (red) was undetectable in Sox2 positive (green) sensory precursor cells in cochleae of Notch1 Cre (low)/+ ; Rosa26-CAG-tdTomato loxp/+ mice at E14.5. (A’) is the high magnification image of the rectangular region in (A) taken in the organ of Corti region. (B–B’) A single slice of confocal image taken in cochleae of Notch1 Cre (High)/+ ; Rosa26-CAG-tdTomato loxp/+ mice. (B’) is the high magnification image of the rectangular region in (B) taken in the organ of Corti region, showing that a few cells were Sox2+/tdTomato+ (arrows), whereas the majority were Sox2+ only. Scale bar is 200 µm (A, B), 20 µm (A’, B’).
Figure Legend Snippet: Notch1 Cre/+ -mediated reporter expression is difficult to detect in the cochlear prosensory domain at embryonic day (E) 14.5. (A–A’) A single slice of confocal image demonstrating that tdTomato reporter expression (red) was undetectable in Sox2 positive (green) sensory precursor cells in cochleae of Notch1 Cre (low)/+ ; Rosa26-CAG-tdTomato loxp/+ mice at E14.5. (A’) is the high magnification image of the rectangular region in (A) taken in the organ of Corti region. (B–B’) A single slice of confocal image taken in cochleae of Notch1 Cre (High)/+ ; Rosa26-CAG-tdTomato loxp/+ mice. (B’) is the high magnification image of the rectangular region in (B) taken in the organ of Corti region, showing that a few cells were Sox2+/tdTomato+ (arrows), whereas the majority were Sox2+ only. Scale bar is 200 µm (A, B), 20 µm (A’, B’).

Techniques Used: Expressing, Mouse Assay

19) Product Images from "RYK promotes the stemness of glioblastoma cells via the WNT/β-catenin pathway"

Article Title: RYK promotes the stemness of glioblastoma cells via the WNT/β-catenin pathway

Journal: Oncotarget

doi: 10.18632/oncotarget.14564

RYK silencing affects neurosphere formation GBM patient-derived stem-like cells (#1, #83) or GBM cell lines (AM38, U87MG, and U251MG) were transfected with Ryk siRNA or a control siRNA sequence. The ability to grow as neurospheres and the expression of stem markers was then analyzed. Knock-down of RYK expression reduced sphere number in both patients analyzed ( A ) as well as in the GBM cell lines ( C ). Data representative of three independent experiments. RYK knockdown also decreased the GSC markers NANOG, OCT3/4, and SOX2 at mRNA and protein levels ( D – F ). In (D) and (E) mRNA expression was assessed by real-time PCR and normalized against β-actin. Experiments were repeated at least twice. In ( B ) and (F), Western blots from representative experiments; β-actin was used as loading control. In (A, C, D, and F), statistical significance calculated using Student's t -test ( p
Figure Legend Snippet: RYK silencing affects neurosphere formation GBM patient-derived stem-like cells (#1, #83) or GBM cell lines (AM38, U87MG, and U251MG) were transfected with Ryk siRNA or a control siRNA sequence. The ability to grow as neurospheres and the expression of stem markers was then analyzed. Knock-down of RYK expression reduced sphere number in both patients analyzed ( A ) as well as in the GBM cell lines ( C ). Data representative of three independent experiments. RYK knockdown also decreased the GSC markers NANOG, OCT3/4, and SOX2 at mRNA and protein levels ( D – F ). In (D) and (E) mRNA expression was assessed by real-time PCR and normalized against β-actin. Experiments were repeated at least twice. In ( B ) and (F), Western blots from representative experiments; β-actin was used as loading control. In (A, C, D, and F), statistical significance calculated using Student's t -test ( p

Techniques Used: Derivative Assay, Transfection, Sequencing, Expressing, Real-time Polymerase Chain Reaction, Western Blot

RYK overexpression promotes neurosphere formation Adherent GBM (U87MG and U251MG) and stem-like derived cells (AM38, U87MG, and U251MG) were transfected with h- RYK cDNA or a control vector. The ability to grow as neurospheres and the expression of stem markers were then analyzed. RYK overexpression increased sphere number in GBM cell lines ( A , left panel) as well as in stem-like GBM cells (A, right panel). Data representative of three independent experiments. RYK knockdown also decreased the GSC markers NANOG, OCT3/4, and SOX2 at mRNA and protein levels in all continuous cell lines analyzed ( B – C ). In (B), mRNA expression was assessed by real-time PCR and normalized against β-actin. Experiments were repeated at least twice. In (C), Western blots are from representative experiments, and β-actin was used as loading control. In (A) and (B), statistical significance calculated using Student's t -test ( p
Figure Legend Snippet: RYK overexpression promotes neurosphere formation Adherent GBM (U87MG and U251MG) and stem-like derived cells (AM38, U87MG, and U251MG) were transfected with h- RYK cDNA or a control vector. The ability to grow as neurospheres and the expression of stem markers were then analyzed. RYK overexpression increased sphere number in GBM cell lines ( A , left panel) as well as in stem-like GBM cells (A, right panel). Data representative of three independent experiments. RYK knockdown also decreased the GSC markers NANOG, OCT3/4, and SOX2 at mRNA and protein levels in all continuous cell lines analyzed ( B – C ). In (B), mRNA expression was assessed by real-time PCR and normalized against β-actin. Experiments were repeated at least twice. In (C), Western blots are from representative experiments, and β-actin was used as loading control. In (A) and (B), statistical significance calculated using Student's t -test ( p

Techniques Used: Over Expression, Derivative Assay, Transfection, Plasmid Preparation, Expressing, Real-time Polymerase Chain Reaction, Western Blot

RYK is overexpressed in GBMs and GSCs ( A ) A significant increase in RYK expression was identified in GBM tissues ( n = 77) compared to normal brains ( n = 23). RYK expression data was obtained from the GEO Profiles database. ( B ) RYK's mRNA expression is greater in patient-derived GSCs ( n = 6) compared to patient-derived GSCs induced to differentiate ( n = 6). RYK expression was assessed by real-time PCR and normalized against β-actin. In ( C ), RYK, NANOG, SOX2, and OCT3/4 protein levels were assessed by Western Blot in three patient-derived GSCs (#1, #83 and #169) and their differentiated counterpart. Real-time PCR ( D , E ) and/or Western blotting ( F ) were performed to analyze RYK, NANOG, SOX2, and OCT3/4 mRNA and/or protein levels in differentiated and stem-like GBM cell lines (AM38, U87MG, U251MG, A172 and LN18). In c and f, Western blots from representative experiments; β-actin was used as loading control. In (C) the experiments were repeated at least twice. In (A, B, D and E), statistical significance calculated using Student's t -test ( p
Figure Legend Snippet: RYK is overexpressed in GBMs and GSCs ( A ) A significant increase in RYK expression was identified in GBM tissues ( n = 77) compared to normal brains ( n = 23). RYK expression data was obtained from the GEO Profiles database. ( B ) RYK's mRNA expression is greater in patient-derived GSCs ( n = 6) compared to patient-derived GSCs induced to differentiate ( n = 6). RYK expression was assessed by real-time PCR and normalized against β-actin. In ( C ), RYK, NANOG, SOX2, and OCT3/4 protein levels were assessed by Western Blot in three patient-derived GSCs (#1, #83 and #169) and their differentiated counterpart. Real-time PCR ( D , E ) and/or Western blotting ( F ) were performed to analyze RYK, NANOG, SOX2, and OCT3/4 mRNA and/or protein levels in differentiated and stem-like GBM cell lines (AM38, U87MG, U251MG, A172 and LN18). In c and f, Western blots from representative experiments; β-actin was used as loading control. In (C) the experiments were repeated at least twice. In (A, B, D and E), statistical significance calculated using Student's t -test ( p

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

20) Product Images from "FOXM1 activates AGR2 and causes progression of lung adenomas into invasive mucinous adenocarcinomas"

Article Title: FOXM1 activates AGR2 and causes progression of lung adenomas into invasive mucinous adenocarcinomas

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1007097

FOXM1 increases cellular proliferation in epiFoxM1-ΔN tumors. (A) Increased number of Ki67-positive cells in epiFoxM1-ΔN tumors is shown by immuno staining (left panels). Numbers of Ki67-positive cells were counted in ten random fields of control and epiFoxM1-ΔN tumors at 200x magnification (right graph). (B) No changes in apoptosis were found in epiFoxM1-ΔN tumors compared to controls. Tumors were stained with antibodies specific to cleaved caspase 3 (arrows, left panels) and the number of positive cells were counted (right graph). The number of cleaved caspase 3-positive cells was counted using ten random fields at 200x magnification. (C) Expression of FOXM1 in lung adenomas increased the number of SOX2-positive cells. The increased SOX2 protein is shown with immunohistochemistry using antibodies against SOX2 (left panels). Increased Sox2 mRNA is demonstrated by qRT-PCR (right graph). β-actin mRNA was used for normalization. A p-value
Figure Legend Snippet: FOXM1 increases cellular proliferation in epiFoxM1-ΔN tumors. (A) Increased number of Ki67-positive cells in epiFoxM1-ΔN tumors is shown by immuno staining (left panels). Numbers of Ki67-positive cells were counted in ten random fields of control and epiFoxM1-ΔN tumors at 200x magnification (right graph). (B) No changes in apoptosis were found in epiFoxM1-ΔN tumors compared to controls. Tumors were stained with antibodies specific to cleaved caspase 3 (arrows, left panels) and the number of positive cells were counted (right graph). The number of cleaved caspase 3-positive cells was counted using ten random fields at 200x magnification. (C) Expression of FOXM1 in lung adenomas increased the number of SOX2-positive cells. The increased SOX2 protein is shown with immunohistochemistry using antibodies against SOX2 (left panels). Increased Sox2 mRNA is demonstrated by qRT-PCR (right graph). β-actin mRNA was used for normalization. A p-value

Techniques Used: Immunostaining, Staining, Expressing, Immunohistochemistry, Quantitative RT-PCR

21) Product Images from "Distinct Expression Pattern of a Deafness Gene, KIAA1199, in a Primate Cochlea"

Article Title: Distinct Expression Pattern of a Deafness Gene, KIAA1199, in a Primate Cochlea

Journal: BioMed Research International

doi: 10.1155/2016/1781894

Expression of KIAA1199 in the organ of Corti. (a) KIAA1199 expression is observed in the organ of Corti. (b) KIAA1199 expression is observed in MYOSIN7a-positive hair cells and supporting cells between the outer and inner sulcus cells, including SOX2-positive supporting cells. (c) Whole-mount immunofluorescence also showed broad expressions of KIAA1199 in the organ of Corti. ISC: inner sulcus cells, OSC: outer sulcus cells, IHC: inner hair cells, and OHC: outer hair cells. The nuclei were counterstained with Hoechst (blue). Scale bar: (a) and (b): 100 μ m, (c): 50 μ m.
Figure Legend Snippet: Expression of KIAA1199 in the organ of Corti. (a) KIAA1199 expression is observed in the organ of Corti. (b) KIAA1199 expression is observed in MYOSIN7a-positive hair cells and supporting cells between the outer and inner sulcus cells, including SOX2-positive supporting cells. (c) Whole-mount immunofluorescence also showed broad expressions of KIAA1199 in the organ of Corti. ISC: inner sulcus cells, OSC: outer sulcus cells, IHC: inner hair cells, and OHC: outer hair cells. The nuclei were counterstained with Hoechst (blue). Scale bar: (a) and (b): 100 μ m, (c): 50 μ m.

Techniques Used: Expressing, Immunofluorescence, Immunohistochemistry

22) Product Images from "Wnt/β-catenin pathway is required for epithelial to mesenchymal transition in CXCL12 over expressed breast cancer cells"

Article Title: Wnt/β-catenin pathway is required for epithelial to mesenchymal transition in CXCL12 over expressed breast cancer cells

Journal: International Journal of Clinical and Experimental Pathology

doi:

Overexpression of CXCL12 confers CSC-like phenotype on MCF-7 cells. A. Colorimetry was applied to assess ALDH activity in mammosphere cells. The absorbance was determined at 450 nm; B. The relative mRNA levels of OCT-4, Nanog, and SOX2 were analyzed by
Figure Legend Snippet: Overexpression of CXCL12 confers CSC-like phenotype on MCF-7 cells. A. Colorimetry was applied to assess ALDH activity in mammosphere cells. The absorbance was determined at 450 nm; B. The relative mRNA levels of OCT-4, Nanog, and SOX2 were analyzed by

Techniques Used: Over Expression, Colorimetric Assay, Activity Assay

23) Product Images from "The microRNA-183/96/182 Cluster is Essential for Stereociliary Bundle Formation and Function of Cochlear Sensory Hair Cells"

Article Title: The microRNA-183/96/182 Cluster is Essential for Stereociliary Bundle Formation and Function of Cochlear Sensory Hair Cells

Journal: Scientific Reports

doi: 10.1038/s41598-018-36894-z

Immunostaining of cochlear sensory epithelium with anti- Sox2 (red) and Myo7a (green) antibodies in P1 WT ( a , c , e ) and KO mice ( b , d , f ). White arrows in e and f show Sox2 staining in the nuclei of OHCs; yellow arrows show that in the nuclei of IHCs. White arrowheads in b and d point to the “ectopic” staining of Myo7a in a subgroup of cells in the GER and LER regions. Similar results were observed in the cochleae from 3 KO and 2 WT mice.
Figure Legend Snippet: Immunostaining of cochlear sensory epithelium with anti- Sox2 (red) and Myo7a (green) antibodies in P1 WT ( a , c , e ) and KO mice ( b , d , f ). White arrows in e and f show Sox2 staining in the nuclei of OHCs; yellow arrows show that in the nuclei of IHCs. White arrowheads in b and d point to the “ectopic” staining of Myo7a in a subgroup of cells in the GER and LER regions. Similar results were observed in the cochleae from 3 KO and 2 WT mice.

Techniques Used: Immunostaining, Mouse Assay, Staining

24) Product Images from "Timely Inhibition of Notch Signaling by DAPT Promotes Cardiac Differentiation of Murine Pluripotent Stem Cells"

Article Title: Timely Inhibition of Notch Signaling by DAPT Promotes Cardiac Differentiation of Murine Pluripotent Stem Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0109588

Derivation of iPSCs using an inducible lentiviral system. ( A ) Schematic representation of iPSC generation. ( B ) RT-PCR analysis of exogenous Yamanaka factors, ES pluripotent markers in fully and partially reprogrammed iPSCs (iPS-Dox6d, iPS-Dox12d). Parental MEFs and ESCs were used as negative and positive controls. ( C ) Immunofluorescence analysis of pluripotent markers, Nanog and SSEA-1 (Red). Oct4-GFP + cells (Green) represented fully reprogramming iPSCs. Scale bar 50 µm. ( D ) Western blot analysis confirmed the pluripotent properties of iPSCs expressing Oct4, Nanog and Sox2. Parental MEF and ES cells were used as negative and positive controls. ( E ) Teratoma formation of iPSCs transplanted into immunodeficient mice. After 4 weeks, iPSC-derived tumors were sectioned and stained with hematoxylin and eosin. Shown were neural (ectoderm), muscle (mesoderm) and glandular (endoderm) tissues from left to right. Scale bar 100 µm. ( F ) Karyotyping analysis showing normal karyotyping of iPSCs after passage 10.
Figure Legend Snippet: Derivation of iPSCs using an inducible lentiviral system. ( A ) Schematic representation of iPSC generation. ( B ) RT-PCR analysis of exogenous Yamanaka factors, ES pluripotent markers in fully and partially reprogrammed iPSCs (iPS-Dox6d, iPS-Dox12d). Parental MEFs and ESCs were used as negative and positive controls. ( C ) Immunofluorescence analysis of pluripotent markers, Nanog and SSEA-1 (Red). Oct4-GFP + cells (Green) represented fully reprogramming iPSCs. Scale bar 50 µm. ( D ) Western blot analysis confirmed the pluripotent properties of iPSCs expressing Oct4, Nanog and Sox2. Parental MEF and ES cells were used as negative and positive controls. ( E ) Teratoma formation of iPSCs transplanted into immunodeficient mice. After 4 weeks, iPSC-derived tumors were sectioned and stained with hematoxylin and eosin. Shown were neural (ectoderm), muscle (mesoderm) and glandular (endoderm) tissues from left to right. Scale bar 100 µm. ( F ) Karyotyping analysis showing normal karyotyping of iPSCs after passage 10.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Western Blot, Expressing, Mouse Assay, Derivative Assay, Staining

25) Product Images from "Dental Epithelial Stem Cells Express the Developmental Regulator Meis1"

Article Title: Dental Epithelial Stem Cells Express the Developmental Regulator Meis1

Journal: Frontiers in Physiology

doi: 10.3389/fphys.2019.00249

Meis1 and Sox2 expression patterns in developing molar and incisor. Frontal (A–C,E) and sagittal (D,F–H) sections of E12.5-E18.5 wild type mice. Sox2 is expressed in the dental placode, mostly on the lingual side, its expression partially coincides with that of Meis1 (A’,E’) . Meis1 and Sox2 expression partially overlaps in the dental epithelium at the bud stage. (B’,F’) . During cap stage, Meis1 and Sox2 are expressed in the developing cervical loops. Some cells from the vestibular lamina and the labial cervical loop express both MEIS1 and SOX2 (C’,G’) . Toward the end of tooth morphogenesis Meis1 expression is confined to the cervical loops, in a fashion similar to that of Sox2 (D’–H’) . Scale bars 100 μm. Dashed line labels epithelial tissue.
Figure Legend Snippet: Meis1 and Sox2 expression patterns in developing molar and incisor. Frontal (A–C,E) and sagittal (D,F–H) sections of E12.5-E18.5 wild type mice. Sox2 is expressed in the dental placode, mostly on the lingual side, its expression partially coincides with that of Meis1 (A’,E’) . Meis1 and Sox2 expression partially overlaps in the dental epithelium at the bud stage. (B’,F’) . During cap stage, Meis1 and Sox2 are expressed in the developing cervical loops. Some cells from the vestibular lamina and the labial cervical loop express both MEIS1 and SOX2 (C’,G’) . Toward the end of tooth morphogenesis Meis1 expression is confined to the cervical loops, in a fashion similar to that of Sox2 (D’–H’) . Scale bars 100 μm. Dashed line labels epithelial tissue.

Techniques Used: Expressing, Mouse Assay

Meis1 and Sox2 expression in the adult incisor labial cervical loop. Meis1 and Sox2 are coexpressed in the incisor labial cervical loop (A–A”) . Black arrowheads point at cells expressing both Meis1 and Sox2 transcripts. In the adult labial cervical loop MEIS1 and SOX2 mark the same cells (yellow arrowhead) (B) . Scalebars (A,B) , 100 μm; (A’,A”) , 10 μm.
Figure Legend Snippet: Meis1 and Sox2 expression in the adult incisor labial cervical loop. Meis1 and Sox2 are coexpressed in the incisor labial cervical loop (A–A”) . Black arrowheads point at cells expressing both Meis1 and Sox2 transcripts. In the adult labial cervical loop MEIS1 and SOX2 mark the same cells (yellow arrowhead) (B) . Scalebars (A,B) , 100 μm; (A’,A”) , 10 μm.

Techniques Used: Expressing

Tooth formation in Meis1 KO embryos. Histological analysis show the presence of normal looking lower incisors and molars in Meis1 KO embryos, similar to control littermates (A–D) . 3D reconstructions from micro-CT scans confirm that the forming incisors have a normal morphology (E, F). RNAscope in situ hybridization shows the complete absence of Meis1 expression in Meis1 KO embryos, Sox2 expression pattern remains unaltered in the absence of Meis1 expression (G–J). Scale bars 100 μm. Dashed line labels epithelial tissue.
Figure Legend Snippet: Tooth formation in Meis1 KO embryos. Histological analysis show the presence of normal looking lower incisors and molars in Meis1 KO embryos, similar to control littermates (A–D) . 3D reconstructions from micro-CT scans confirm that the forming incisors have a normal morphology (E, F). RNAscope in situ hybridization shows the complete absence of Meis1 expression in Meis1 KO embryos, Sox2 expression pattern remains unaltered in the absence of Meis1 expression (G–J). Scale bars 100 μm. Dashed line labels epithelial tissue.

Techniques Used: Micro-CT, In Situ Hybridization, Expressing

26) Product Images from "Video image-based analysis of single human induced pluripotent stem cell derived cardiomyocyte beating dynamics using digital image correlation"

Article Title: Video image-based analysis of single human induced pluripotent stem cell derived cardiomyocyte beating dynamics using digital image correlation

Journal: BioMedical Engineering OnLine

doi: 10.1186/1475-925X-13-39

Characterization of iPS cells for their pluripotency. Number 1 represents the cell line UTA.04602.WT and 2 the line UTA.04607.WT. A : iPS cells formed typical colonies for pluripotent stem cells that are rather compact and round in shape. B : The iPS cell colonies typically had well defined edges and distinct cell borders, and the iPS cells had a high nucleus to cytoplasm -ratio and a large nucleoli characteristic for stem cells. C : Endogenous pluripotency gene expression was studied using RT-PCR. Nanog, Oct4, Sox2 and Rex1 were all expressed at mRNA level in the iPS cells. β-actin and GAPDH were used as housekeeping control genes for both endogenous and exogenous markers. D : The expression of pluripotency genes was also studied at the protein level by immunocytochemical staining. The iPS cell expressed several markers for the pluripotent state: Nanog, Oct4, Sox2, SSEA4, TRA-1-60, and TRA-1-81 (all in red). Pictures in the left panel are from the line UTA.04602.WT and the ones on the right side are from UTA.04607.WT. Blue in all pictures indicates the DAPI staining of nuclei. E : Using RT-PCR, it was shown that all the transgenes were silenced in the iPS cells. Negative control is marked with “-” and positive control with “+”. F : Embryoid body (EB) -assay was used to define the pluripotency of the iPS cells in vitro . Markers for all three germ layers were detected from the EBs formed from both cell lines. Alpha - fetoprotein (AFP) was used as a marker for endoderm, kinase insert domain receptor (KDR, also known as vascular endothelial growth factor receptor 2 (VEGFR-2) was used as a marker for mesoderm and nestin was used as an ectoderm marker. GAPDH was used as an endogenous control gene.
Figure Legend Snippet: Characterization of iPS cells for their pluripotency. Number 1 represents the cell line UTA.04602.WT and 2 the line UTA.04607.WT. A : iPS cells formed typical colonies for pluripotent stem cells that are rather compact and round in shape. B : The iPS cell colonies typically had well defined edges and distinct cell borders, and the iPS cells had a high nucleus to cytoplasm -ratio and a large nucleoli characteristic for stem cells. C : Endogenous pluripotency gene expression was studied using RT-PCR. Nanog, Oct4, Sox2 and Rex1 were all expressed at mRNA level in the iPS cells. β-actin and GAPDH were used as housekeeping control genes for both endogenous and exogenous markers. D : The expression of pluripotency genes was also studied at the protein level by immunocytochemical staining. The iPS cell expressed several markers for the pluripotent state: Nanog, Oct4, Sox2, SSEA4, TRA-1-60, and TRA-1-81 (all in red). Pictures in the left panel are from the line UTA.04602.WT and the ones on the right side are from UTA.04607.WT. Blue in all pictures indicates the DAPI staining of nuclei. E : Using RT-PCR, it was shown that all the transgenes were silenced in the iPS cells. Negative control is marked with “-” and positive control with “+”. F : Embryoid body (EB) -assay was used to define the pluripotency of the iPS cells in vitro . Markers for all three germ layers were detected from the EBs formed from both cell lines. Alpha - fetoprotein (AFP) was used as a marker for endoderm, kinase insert domain receptor (KDR, also known as vascular endothelial growth factor receptor 2 (VEGFR-2) was used as a marker for mesoderm and nestin was used as an ectoderm marker. GAPDH was used as an endogenous control gene.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Negative Control, Positive Control, In Vitro, Marker

27) Product Images from "Oct4/Sox2 Binding Sites Contribute to Maintaining Hypomethylation of the Maternal Igf2/H19 Imprinting Control Region"

Article Title: Oct4/Sox2 Binding Sites Contribute to Maintaining Hypomethylation of the Maternal Igf2/H19 Imprinting Control Region

Journal: PLoS ONE

doi: 10.1371/journal.pone.0081962

Sox2 and Oct4 binding to the ICR. EMSAs were performed using WT or mutant ICR probes as indicated with either F9 NE ( A, B ) or recombinant Oct4 and Sox2 ( C, D, F ). ( A ) The effect of octamer specific point mutations on Oct4 and Sox2 binding was assessed by addition of nonspecific (N) and specific unlabeled DNA competitors - oct (O), sox (S), ICR Sox-OctA and B (A and B), or mutant ICR Sox-OctA and B (mA and mB). ( B ) Oct4 and Sox2 supershift assays. Sox2 and Oct4 protein-DNA complexes (white and black triangles respectively) were identified by their respective antibodies (Ab). Presumptive Oct1 complexes are indicated by a grey triangle. ( C ) Recombinant Sox2 binding to Sox-OctA, B and SoxC. ICR probes with mutations to the Sox elements (mSA, mSB, and mSC) were used to confirm Sox2 binding sites. ( D ) Methylation-independent binding of Oct4 and Sox2 to the ICR Sox-Oct motifs Wild type ICR Sox-OctA and B probes (A* and B*) were methylated at all CpGs. ( E ) Oct4 and Sox2 ChIP assays. Enrichment of endogenous F9 or WT and mutant transgenic ICR octamers by Oct4 and Sox2 ChIP was determined by qRT-PCR and normalized to serum controls. Error bars represent standard deviation of the mean (n = 3). Statistical significance of Oct4 and Sox2 enrichments was shown using Student's t-test (*p
Figure Legend Snippet: Sox2 and Oct4 binding to the ICR. EMSAs were performed using WT or mutant ICR probes as indicated with either F9 NE ( A, B ) or recombinant Oct4 and Sox2 ( C, D, F ). ( A ) The effect of octamer specific point mutations on Oct4 and Sox2 binding was assessed by addition of nonspecific (N) and specific unlabeled DNA competitors - oct (O), sox (S), ICR Sox-OctA and B (A and B), or mutant ICR Sox-OctA and B (mA and mB). ( B ) Oct4 and Sox2 supershift assays. Sox2 and Oct4 protein-DNA complexes (white and black triangles respectively) were identified by their respective antibodies (Ab). Presumptive Oct1 complexes are indicated by a grey triangle. ( C ) Recombinant Sox2 binding to Sox-OctA, B and SoxC. ICR probes with mutations to the Sox elements (mSA, mSB, and mSC) were used to confirm Sox2 binding sites. ( D ) Methylation-independent binding of Oct4 and Sox2 to the ICR Sox-Oct motifs Wild type ICR Sox-OctA and B probes (A* and B*) were methylated at all CpGs. ( E ) Oct4 and Sox2 ChIP assays. Enrichment of endogenous F9 or WT and mutant transgenic ICR octamers by Oct4 and Sox2 ChIP was determined by qRT-PCR and normalized to serum controls. Error bars represent standard deviation of the mean (n = 3). Statistical significance of Oct4 and Sox2 enrichments was shown using Student's t-test (*p

Techniques Used: Binding Assay, Mutagenesis, Recombinant, Methylation, Chromatin Immunoprecipitation, Transgenic Assay, Quantitative RT-PCR, Standard Deviation

28) Product Images from "Uner Tan syndrome caused by a homozygous TUBB2B mutation affecting microtubule stability"

Article Title: Uner Tan syndrome caused by a homozygous TUBB2B mutation affecting microtubule stability

Journal: Human Molecular Genetics

doi: 10.1093/hmg/ddw383

Tubb2b is expressed at high levels in the embryonic cerebellum. ( A–C ) Overview scan of a sagittal section of an embryonic day (E) 16.5 head of the Tg(Tubb2b-eGFP)GlbDAK line. eGFP is expressed under the control of the endogenous Tubb2b locus on a bacterial artificial chromosome (BAC). (B ) shows a magnification of the dashed rectangle encircling the cerebellar anlage in (A); in (C), the GFP channel is shown as a grey-scale image of (B). Hoechst staining for nuclear DNA is shown in white in A and B. ( D,E ) Confocal images of a control (D) and a transgenic animal (E). Hoechst staining for nuclear DNA is shown in blue in (D) and (E). PCC: Purkinje cell cluster area; EGZ: External Granular Zone. ( F ) Quantification of the average fluorescence intensity found in the EGZ and the PCC for control ( n = 1) and transgenic mice ( n = 3). For transgenic mice, mean ± SEM are shown. ( G–Z ) Fluorescent images of the developing cerebellum of the Tg(Tubb2b-eGFP)GlbDAK line at E16.5 stained for Pax6 (G–J), pH3 (K–N), NeuN (O–R), Calbindin (S–V) and Sox2 (W–Z). (H, L, P, T and X) show magnifications of the dashed rectangle in (G,K, O,S and W) respectively. (I, M, Q, U and Y), as well as (J, N, R, V and Z) show grey-scale images of the GFP and immunostaining channels, respectively. Scale bars show 1000 µm in A, 200 µm in B, 100 µm in (D) and (G) and 20 µm in (H).
Figure Legend Snippet: Tubb2b is expressed at high levels in the embryonic cerebellum. ( A–C ) Overview scan of a sagittal section of an embryonic day (E) 16.5 head of the Tg(Tubb2b-eGFP)GlbDAK line. eGFP is expressed under the control of the endogenous Tubb2b locus on a bacterial artificial chromosome (BAC). (B ) shows a magnification of the dashed rectangle encircling the cerebellar anlage in (A); in (C), the GFP channel is shown as a grey-scale image of (B). Hoechst staining for nuclear DNA is shown in white in A and B. ( D,E ) Confocal images of a control (D) and a transgenic animal (E). Hoechst staining for nuclear DNA is shown in blue in (D) and (E). PCC: Purkinje cell cluster area; EGZ: External Granular Zone. ( F ) Quantification of the average fluorescence intensity found in the EGZ and the PCC for control ( n = 1) and transgenic mice ( n = 3). For transgenic mice, mean ± SEM are shown. ( G–Z ) Fluorescent images of the developing cerebellum of the Tg(Tubb2b-eGFP)GlbDAK line at E16.5 stained for Pax6 (G–J), pH3 (K–N), NeuN (O–R), Calbindin (S–V) and Sox2 (W–Z). (H, L, P, T and X) show magnifications of the dashed rectangle in (G,K, O,S and W) respectively. (I, M, Q, U and Y), as well as (J, N, R, V and Z) show grey-scale images of the GFP and immunostaining channels, respectively. Scale bars show 1000 µm in A, 200 µm in B, 100 µm in (D) and (G) and 20 µm in (H).

Techniques Used: BAC Assay, Staining, Transgenic Assay, Periodic Counter-current Chromatography, Fluorescence, Mouse Assay, Immunostaining

29) Product Images from "Therapeutic Potential of Induced Neural Stem Cells for Spinal Cord Injury *"

Article Title: Therapeutic Potential of Induced Neural Stem Cells for Spinal Cord Injury *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.588871

Direct reprogramming of fibroblasts into iNSCs. A and B , immunofluorescence microscopy images of cNSCs and iNSCs, using antibodies against Sox2/Nestin ( A ) and SSEA1/Olig2 ( B ). Scale bars , 100 μm. C , expression of NSC and fibroblast markers in
Figure Legend Snippet: Direct reprogramming of fibroblasts into iNSCs. A and B , immunofluorescence microscopy images of cNSCs and iNSCs, using antibodies against Sox2/Nestin ( A ) and SSEA1/Olig2 ( B ). Scale bars , 100 μm. C , expression of NSC and fibroblast markers in

Techniques Used: Immunofluorescence, Microscopy, Expressing

30) Product Images from "GATA3 Promotes the Neural Progenitor State but Not Neurogenesis in 3D Traumatic Injury Model of Primary Human Cortical Astrocytes"

Article Title: GATA3 Promotes the Neural Progenitor State but Not Neurogenesis in 3D Traumatic Injury Model of Primary Human Cortical Astrocytes

Journal: Frontiers in Cellular Neuroscience

doi: 10.3389/fncel.2019.00023

(A) Immunocytochemical (ICC) staining for TUBB3, GFAP, and BrdU in 2D cultures of primary human astrocytes (pHA). (A’–A”’) Individual images of each fluorescence channel. (B–B”) Morphologies of GFAP and TUBB3-positive cells. (C) Percent distribution of individual cell types. (D) Graph quantifying all of the cells for BrdU incorporation. (E) Graph quantifying glia and neurons for BrdU incorporation. Abundance (relative number) of cells are shown in a relative scale. (F) Expression heat-map for selected stem cell and cortical neuronal markers. Red: high expression, green: low/no expression. (G) ICC for TUBB3 and SOX2. (H–H”’) SOX2 and BrdU staining as composite and individual channels. (I) ICC for TUBB3 and PAX6. (J–J”’) PAX6 and BrdU staining as composite and individual channels. (K–N) ICC for TUBB3 with SATB2 (K) , TBR1 (L) , CTIP2 (M) , ER81 (N) . (O) ICC for synaptophysin and acetylated tubulin. (P) ICC for SV2 and MAPT. (Q–T) ICC for TUBB3 and C-FOS in untreated cells (Q) and in cells treated with NMDA (R) , glutamate (S) and kynurenic acid (T) . (U–Y) GCaMP imaging in neurons after addition of the calcium ionophore A23187 (U) , glutamic acid (W) , GABA (X) , and NMDA (Y) . All recordings are performed at the end of culture period. (Z) Green fluorescence histograms for (U–Y) . Scale bars: 50 μm (A–A”’) and 20 μm elsewhere. At least 1,000 cells were counted in every well. Percentages under the panels of (B–B”) indicate the ratios of depicted cell types among the whole set of cells in 2D.
Figure Legend Snippet: (A) Immunocytochemical (ICC) staining for TUBB3, GFAP, and BrdU in 2D cultures of primary human astrocytes (pHA). (A’–A”’) Individual images of each fluorescence channel. (B–B”) Morphologies of GFAP and TUBB3-positive cells. (C) Percent distribution of individual cell types. (D) Graph quantifying all of the cells for BrdU incorporation. (E) Graph quantifying glia and neurons for BrdU incorporation. Abundance (relative number) of cells are shown in a relative scale. (F) Expression heat-map for selected stem cell and cortical neuronal markers. Red: high expression, green: low/no expression. (G) ICC for TUBB3 and SOX2. (H–H”’) SOX2 and BrdU staining as composite and individual channels. (I) ICC for TUBB3 and PAX6. (J–J”’) PAX6 and BrdU staining as composite and individual channels. (K–N) ICC for TUBB3 with SATB2 (K) , TBR1 (L) , CTIP2 (M) , ER81 (N) . (O) ICC for synaptophysin and acetylated tubulin. (P) ICC for SV2 and MAPT. (Q–T) ICC for TUBB3 and C-FOS in untreated cells (Q) and in cells treated with NMDA (R) , glutamate (S) and kynurenic acid (T) . (U–Y) GCaMP imaging in neurons after addition of the calcium ionophore A23187 (U) , glutamic acid (W) , GABA (X) , and NMDA (Y) . All recordings are performed at the end of culture period. (Z) Green fluorescence histograms for (U–Y) . Scale bars: 50 μm (A–A”’) and 20 μm elsewhere. At least 1,000 cells were counted in every well. Percentages under the panels of (B–B”) indicate the ratios of depicted cell types among the whole set of cells in 2D.

Techniques Used: Immunocytochemistry, Staining, Fluorescence, BrdU Incorporation Assay, Expressing, BrdU Staining, Imaging

(A,B) Immunocytochemistry for GFAP and SOX2 on pLV-EGFP (A) and pLV-GATA3-transduced (B) cell cultures. (C,D) Immunocytochemistry for TUBB3 and BrdU on pLV-EGFP (C) and pLV-GATA3-transduced (D) cell cultures. (E) Quantification of the percentage of cells expressing GFAP and SOX2. (F) Quantification of the percentage of BrdU+ cells. (G) Quantification of the percentage of neurons. (H) Schematic view and experimental setup for the scratch wound injury. (I) Immunocytochemical staining for BrdU, GFP, and TUBB3 on pLV-EGFP-transduced cultures. (J) High-magnification image from (I) . (K) Immunocytochemical staining for GFAP, SOX2, and GFP on pLV-EGFP-transduced cultures. Inset: Individual fluorescence channel for SOX2. (L) Immunocytochemical staining for BrdU, GFP, and TUBB3 on pLV-GATA3-transduced cultures. (M) High-magnification image from (L) . (N) Immunocytochemical staining for GFAP, SOX2, and GFP on pLV-GATA3-transduced cultures. Inset: Individual fluorescence channel for SOX2. (O) Quantification graphs for percentage of GFAP and SOX2-positive neural progenitors, BrdU-positive cells, and neurons. (P) GO-term analysis for neurogenesis-related biological process compares lesioned samples of pLV-EGFP and pLV-GATA3. Listed categories are enriched in GATA3-transduced samples. Scale bars: 100 μm.
Figure Legend Snippet: (A,B) Immunocytochemistry for GFAP and SOX2 on pLV-EGFP (A) and pLV-GATA3-transduced (B) cell cultures. (C,D) Immunocytochemistry for TUBB3 and BrdU on pLV-EGFP (C) and pLV-GATA3-transduced (D) cell cultures. (E) Quantification of the percentage of cells expressing GFAP and SOX2. (F) Quantification of the percentage of BrdU+ cells. (G) Quantification of the percentage of neurons. (H) Schematic view and experimental setup for the scratch wound injury. (I) Immunocytochemical staining for BrdU, GFP, and TUBB3 on pLV-EGFP-transduced cultures. (J) High-magnification image from (I) . (K) Immunocytochemical staining for GFAP, SOX2, and GFP on pLV-EGFP-transduced cultures. Inset: Individual fluorescence channel for SOX2. (L) Immunocytochemical staining for BrdU, GFP, and TUBB3 on pLV-GATA3-transduced cultures. (M) High-magnification image from (L) . (N) Immunocytochemical staining for GFAP, SOX2, and GFP on pLV-GATA3-transduced cultures. Inset: Individual fluorescence channel for SOX2. (O) Quantification graphs for percentage of GFAP and SOX2-positive neural progenitors, BrdU-positive cells, and neurons. (P) GO-term analysis for neurogenesis-related biological process compares lesioned samples of pLV-EGFP and pLV-GATA3. Listed categories are enriched in GATA3-transduced samples. Scale bars: 100 μm.

Techniques Used: Immunocytochemistry, Expressing, Staining, Fluorescence

(A) Scheme for lesion experiment. (B,B′) Immunohistochemistry (IHC) for GFAP in control and GATA3-expressing 3D human astrocyte cultures. (C,C′) IHC for SOX2 and GFAP in control and GATA3-expressing 3D human astrocyte cultures. (D,D′) IHC for TUBB3 and GFAP in control and GATA3-expressing 3D human astrocyte cultures. Smaller panels to the right show individual fluorescence channels for TUBB3 and GFAP. (E) Quantification graphs. (F,G) van Gieson’s staining for collagen deposition and connective tissue in pLV-EGFP-transduced lesioned samples (F) and pLV-GATA3-transduced lesioned samples. Arrows indicate dense collagen depositions, which are more numerous in EGFP-transduced samples. On average more than 100 cells were counted per stack (upper limit 608 cells). Scale bars: 100 μm.
Figure Legend Snippet: (A) Scheme for lesion experiment. (B,B′) Immunohistochemistry (IHC) for GFAP in control and GATA3-expressing 3D human astrocyte cultures. (C,C′) IHC for SOX2 and GFAP in control and GATA3-expressing 3D human astrocyte cultures. (D,D′) IHC for TUBB3 and GFAP in control and GATA3-expressing 3D human astrocyte cultures. Smaller panels to the right show individual fluorescence channels for TUBB3 and GFAP. (E) Quantification graphs. (F,G) van Gieson’s staining for collagen deposition and connective tissue in pLV-EGFP-transduced lesioned samples (F) and pLV-GATA3-transduced lesioned samples. Arrows indicate dense collagen depositions, which are more numerous in EGFP-transduced samples. On average more than 100 cells were counted per stack (upper limit 608 cells). Scale bars: 100 μm.

Techniques Used: Immunohistochemistry, Expressing, Fluorescence, Staining

31) Product Images from "Characterization of Lgr5+ progenitor cell transcriptomes in the apical and basal turns of the mouse cochlea"

Article Title: Characterization of Lgr5+ progenitor cell transcriptomes in the apical and basal turns of the mouse cochlea

Journal: Oncotarget

doi: 10.18632/oncotarget.8636

Re-sort analysis, immunostaining, and quantitative PCR of flow-sorted Lgr5+ cells from the apical and basal turns of the postnatal cochlea A. Lgr5-EGFP-CreER cochleae were dissected and separated into apical and basal fractions, and GFP+ and GFP− cells from each fraction were sorted by flow cytometry. B. Re-sort analysis of ALPs and BLPs demonstrated > 90% purity. C. Immediate immunostaining after sorting of Lgr5+ cells from the apex showed a high percentage of Sox2+ (95.6%) and GFP+ (93%) cells but no Myo7a+ cells (0.0%). D. Immunostaining of Lgr5+ cells from the base also showed a high percentage of Sox2+ (94.2%) and GFP+ (94.6%) cells, and no Myo7a+ (0.0%) cells were found in the sorted cells. E. Quantitative PCR results showed the relative expression of Lgr5, Sox2, and Brn3.1 in ALPs and BLPs.
Figure Legend Snippet: Re-sort analysis, immunostaining, and quantitative PCR of flow-sorted Lgr5+ cells from the apical and basal turns of the postnatal cochlea A. Lgr5-EGFP-CreER cochleae were dissected and separated into apical and basal fractions, and GFP+ and GFP− cells from each fraction were sorted by flow cytometry. B. Re-sort analysis of ALPs and BLPs demonstrated > 90% purity. C. Immediate immunostaining after sorting of Lgr5+ cells from the apex showed a high percentage of Sox2+ (95.6%) and GFP+ (93%) cells but no Myo7a+ cells (0.0%). D. Immunostaining of Lgr5+ cells from the base also showed a high percentage of Sox2+ (94.2%) and GFP+ (94.6%) cells, and no Myo7a+ (0.0%) cells were found in the sorted cells. E. Quantitative PCR results showed the relative expression of Lgr5, Sox2, and Brn3.1 in ALPs and BLPs.

Techniques Used: Immunostaining, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Expressing

32) Product Images from "Methanol fixed fibroblasts serve as feeder cells to maintain stem cells in the pluripotent state in vitro"

Article Title: Methanol fixed fibroblasts serve as feeder cells to maintain stem cells in the pluripotent state in vitro

Journal: Scientific Reports

doi: 10.1038/s41598-018-26238-2

Human and porcine iPS cells were maintained on feeders derived from methanol fixed fibroblasts. ( A ) Human iPS cells were cultured on MT-MEF (a), MT-3T3 (b), MT-hMSC (c), and MMC-MEF (d) in KSR medium, and on MT-MEF (e) and Matrigel (f) in mTeSR medium. ( B ) Immunofluorescence analysis of OCT4 and SOX2 expressions in human iPS cells. ( C ) Flow cytometry analysis of SSEA-4 in human iPS cells. ( D ) Porcine iPS cells were cultured on MT-MEF, MT-3T3, and MMC-MEF. ( E ) RT-PCR analysis of OCT4 , SOX2 , NANOG , and ESRRB expressions in porcine iPS cells. Scale bar, 200 μm.
Figure Legend Snippet: Human and porcine iPS cells were maintained on feeders derived from methanol fixed fibroblasts. ( A ) Human iPS cells were cultured on MT-MEF (a), MT-3T3 (b), MT-hMSC (c), and MMC-MEF (d) in KSR medium, and on MT-MEF (e) and Matrigel (f) in mTeSR medium. ( B ) Immunofluorescence analysis of OCT4 and SOX2 expressions in human iPS cells. ( C ) Flow cytometry analysis of SSEA-4 in human iPS cells. ( D ) Porcine iPS cells were cultured on MT-MEF, MT-3T3, and MMC-MEF. ( E ) RT-PCR analysis of OCT4 , SOX2 , NANOG , and ESRRB expressions in porcine iPS cells. Scale bar, 200 μm.

Techniques Used: Derivative Assay, Cell Culture, Immunofluorescence, Flow Cytometry, Cytometry, Reverse Transcription Polymerase Chain Reaction

Culture of mouse ES on fibroblasts fixed by methanol. J1 mES cells were cultured on MEF cells fixed by MT (MT-MEF) and GA (GA-MEF), respectively. ( A ) Morphology and AP staining of J1 mES cells. ( B ) Growth curve of J1 cells. ( C ) qRT-PCR analysis of Oct4 , Nanog , and Sox2 expressions in J1 cells. ( D ) Flow cytometry analysis of SSEA-1 expression in J1 cells. ( E ) Scanning electron microscope analysis of MT-MEF, GA-MEF, and MMC-MEF. ( F ) J1 cells were cultured on MT fixed C2C12, PEF, and PK-15 cells. Phase 1, MT fixed cells; Phase 2, morphology of J1 cells cultured on MT fixed cells. Scale bar, 200 μm. Data indicate mean ± SD, *P
Figure Legend Snippet: Culture of mouse ES on fibroblasts fixed by methanol. J1 mES cells were cultured on MEF cells fixed by MT (MT-MEF) and GA (GA-MEF), respectively. ( A ) Morphology and AP staining of J1 mES cells. ( B ) Growth curve of J1 cells. ( C ) qRT-PCR analysis of Oct4 , Nanog , and Sox2 expressions in J1 cells. ( D ) Flow cytometry analysis of SSEA-1 expression in J1 cells. ( E ) Scanning electron microscope analysis of MT-MEF, GA-MEF, and MMC-MEF. ( F ) J1 cells were cultured on MT fixed C2C12, PEF, and PK-15 cells. Phase 1, MT fixed cells; Phase 2, morphology of J1 cells cultured on MT fixed cells. Scale bar, 200 μm. Data indicate mean ± SD, *P

Techniques Used: Cell Culture, Staining, Quantitative RT-PCR, Flow Cytometry, Cytometry, Expressing, Microscopy

33) Product Images from "High Expression of SOX2 and OCT4 Indicates Radiation Resistance and an Independent Negative Prognosis in Cervical Squamous Cell Carcinoma"

Article Title: High Expression of SOX2 and OCT4 Indicates Radiation Resistance and an Independent Negative Prognosis in Cervical Squamous Cell Carcinoma

Journal: Journal of Histochemistry and Cytochemistry

doi: 10.1369/0022155414532654

SOX2 and OCT4 Expression and their Relationship to Clinicopathological Parameters
Figure Legend Snippet: SOX2 and OCT4 Expression and their Relationship to Clinicopathological Parameters

Techniques Used: Expressing

Representative examples of SOX2 and OCT4 staining of tumors in the radiation-resistant group and radiation-sensitive group (400×). (A) Strong positive staining of SOX2 in the radiation-resistant group. (B) Strong positive staining of OCT4 in the
Figure Legend Snippet: Representative examples of SOX2 and OCT4 staining of tumors in the radiation-resistant group and radiation-sensitive group (400×). (A) Strong positive staining of SOX2 in the radiation-resistant group. (B) Strong positive staining of OCT4 in the

Techniques Used: Staining

Relationship between Expression of SOX2 and OCT4
Figure Legend Snippet: Relationship between Expression of SOX2 and OCT4

Techniques Used: Expressing

SOX2 and OCT4 Expression and Response to Radiotherapy
Figure Legend Snippet: SOX2 and OCT4 Expression and Response to Radiotherapy

Techniques Used: Expressing

SOX2 and OCT4 Expression and Patient Survival
Figure Legend Snippet: SOX2 and OCT4 Expression and Patient Survival

Techniques Used: Expressing

Kaplan-Meier survival curves for LACSCC patients. (A) The 5-year progression-free survival (PFS) rates were 84.58% and 55.48% in patients with low SOX2 expression ( n =49) and high SOX2 expression ( n =83), respectively. There was a significant difference
Figure Legend Snippet: Kaplan-Meier survival curves for LACSCC patients. (A) The 5-year progression-free survival (PFS) rates were 84.58% and 55.48% in patients with low SOX2 expression ( n =49) and high SOX2 expression ( n =83), respectively. There was a significant difference

Techniques Used: Expressing

34) Product Images from "Uev1A facilitates osteosarcoma differentiation by promoting Smurf1-mediated Smad1 ubiquitination and degradation"

Article Title: Uev1A facilitates osteosarcoma differentiation by promoting Smurf1-mediated Smad1 ubiquitination and degradation

Journal: Cell Death & Disease

doi: 10.1038/cddis.2017.366

UEV1A overexpression inhibits OS-related proto-oncogene and stem cell marker gene expression and cell proliferation. ( a ) Effects of ectopic UEV gene expression on the expression of OS-related proto-oncogenes. Total RNA was extracted from control and UEV -overexpressed cells followed by qRT-PCR analysis. Data are presented as the mean±S.D. ( b ) WB analysis of c-Myc and Aldh1 levels. ( c ) UEV1A overexpression reduces U2OS cell proliferation. After stably transfected U2OS cells were treated with Dox for 4 weeks, collected cells were seeded in the 6D plate with fresh Dox-containing medium and incubated for the indicated period before cell counting. The Dox- population followed the same treatment except adding Dox. ( d ) Ectopic UEV1A expression reduces cellular Sox2 and Oct4 levels as revealed by WB. ( e ) Synergistic effects of ectopic UEV1A expression and ADM treatment on CDK4 expression. Total RNAs were extracted for the qRT-PCR analysis and results are presented as mean±S.D. ( f ) UEV1A overexpression sensitizes U2OS cells to the anticancer agent ADM. The UEV1A- transfected cells were untreated or treated with 0.8 μ g/ml ADM for 48 h. UEV1A -transfected U2OS cells without Dox induction were used as a control
Figure Legend Snippet: UEV1A overexpression inhibits OS-related proto-oncogene and stem cell marker gene expression and cell proliferation. ( a ) Effects of ectopic UEV gene expression on the expression of OS-related proto-oncogenes. Total RNA was extracted from control and UEV -overexpressed cells followed by qRT-PCR analysis. Data are presented as the mean±S.D. ( b ) WB analysis of c-Myc and Aldh1 levels. ( c ) UEV1A overexpression reduces U2OS cell proliferation. After stably transfected U2OS cells were treated with Dox for 4 weeks, collected cells were seeded in the 6D plate with fresh Dox-containing medium and incubated for the indicated period before cell counting. The Dox- population followed the same treatment except adding Dox. ( d ) Ectopic UEV1A expression reduces cellular Sox2 and Oct4 levels as revealed by WB. ( e ) Synergistic effects of ectopic UEV1A expression and ADM treatment on CDK4 expression. Total RNAs were extracted for the qRT-PCR analysis and results are presented as mean±S.D. ( f ) UEV1A overexpression sensitizes U2OS cells to the anticancer agent ADM. The UEV1A- transfected cells were untreated or treated with 0.8 μ g/ml ADM for 48 h. UEV1A -transfected U2OS cells without Dox induction were used as a control

Techniques Used: Over Expression, Marker, Expressing, Quantitative RT-PCR, Western Blot, Stable Transfection, Transfection, Incubation, Cell Counting

35) Product Images from "c-Myb knockdown increases the neomycin-induced damage to hair-cell-like HEI-OC1 cells in vitro"

Article Title: c-Myb knockdown increases the neomycin-induced damage to hair-cell-like HEI-OC1 cells in vitro

Journal: Scientific Reports

doi: 10.1038/srep41094

c-Myb expression in the cochlear hair cells. Immunofluorescence staining showed the expression pattern of c-Myb (red) in the postnatal cochlea. Myosin 7a (green) and Sox2 (grey) were used as HC and SC markers, respectively. Scale bars = 30 μm.
Figure Legend Snippet: c-Myb expression in the cochlear hair cells. Immunofluorescence staining showed the expression pattern of c-Myb (red) in the postnatal cochlea. Myosin 7a (green) and Sox2 (grey) were used as HC and SC markers, respectively. Scale bars = 30 μm.

Techniques Used: Expressing, Immunofluorescence, Staining

36) Product Images from "Genes Involved in the Transcriptional Regulation of Pluripotency Are Expressed in Malignant Tumors of the Uterine Cervix and Can Induce Tumorigenic Capacity in a Nontumorigenic Cell Line"

Article Title: Genes Involved in the Transcriptional Regulation of Pluripotency Are Expressed in Malignant Tumors of the Uterine Cervix and Can Induce Tumorigenic Capacity in a Nontumorigenic Cell Line

Journal: Stem Cells International

doi: 10.1155/2019/7683817

HaCaT cell line increases the efficiency of sphere formation in the presence of the OSKM-N factors. OSKM-N gene overexpression enhances cancer stem cell properties in HaCaT cells. HaCaT cells were cotransfected with OSKM-N gene plasmids and cultured in sphere-forming media. (a) Spheres formed by HaCaT cells compared with HaCaT transfected with OCT4, SOX2, KLF4, C-MYC, NANOG, and a combination of SOX2 plus NANOG (SN). (b) Efficiency of sphere formation of OSKM-N gene-transfected HaCaT cells and (c) length of sphere. The scale bar is 50 μ m. Experiments were performed in triplicate, and the values are shown as the mean ± standard deviation. ×40 magnification.
Figure Legend Snippet: HaCaT cell line increases the efficiency of sphere formation in the presence of the OSKM-N factors. OSKM-N gene overexpression enhances cancer stem cell properties in HaCaT cells. HaCaT cells were cotransfected with OSKM-N gene plasmids and cultured in sphere-forming media. (a) Spheres formed by HaCaT cells compared with HaCaT transfected with OCT4, SOX2, KLF4, C-MYC, NANOG, and a combination of SOX2 plus NANOG (SN). (b) Efficiency of sphere formation of OSKM-N gene-transfected HaCaT cells and (c) length of sphere. The scale bar is 50 μ m. Experiments were performed in triplicate, and the values are shown as the mean ± standard deviation. ×40 magnification.

Techniques Used: Over Expression, Cell Culture, Transfection, Standard Deviation

Differential expression of stemness- and pluripotency-related genes in tumor biopsies and normal tissues. The scatter plots illustrate data from 85 cervical cancer and 6 normal samples grouped by tumor and normal tissue. The gene expression intensity values were obtained by microarray analyses for OCT4 , SOX2 , KLF4 , C-MYC , and NANOG . Patients were grouped by cervical cancer (CC) biopsies and cervical normal tissue (CNT).
Figure Legend Snippet: Differential expression of stemness- and pluripotency-related genes in tumor biopsies and normal tissues. The scatter plots illustrate data from 85 cervical cancer and 6 normal samples grouped by tumor and normal tissue. The gene expression intensity values were obtained by microarray analyses for OCT4 , SOX2 , KLF4 , C-MYC , and NANOG . Patients were grouped by cervical cancer (CC) biopsies and cervical normal tissue (CNT).

Techniques Used: Expressing, Microarray

The mRNA levels of OCT4 , SOX2 , and NANOG are high in cancer stem cell-enriched cultures. The messenger RNA levels of OCT4 , SOX2 , and NANOG are higher in the cancer stem cell-enriched cultures grown as spheres from HeLa (HeLa SP) and SiHa (SiHa SP) cells than in their monolayer cultures (HeLa and SiHa, respectively) grown conventionally. Experiments were performed in triplicate, and the values are expressed as mean ± standard deviation. Beta2-microglobulin ( β 2M) was used as the housekeeping gene. ∗ p value
Figure Legend Snippet: The mRNA levels of OCT4 , SOX2 , and NANOG are high in cancer stem cell-enriched cultures. The messenger RNA levels of OCT4 , SOX2 , and NANOG are higher in the cancer stem cell-enriched cultures grown as spheres from HeLa (HeLa SP) and SiHa (SiHa SP) cells than in their monolayer cultures (HeLa and SiHa, respectively) grown conventionally. Experiments were performed in triplicate, and the values are expressed as mean ± standard deviation. Beta2-microglobulin ( β 2M) was used as the housekeeping gene. ∗ p value

Techniques Used: Standard Deviation

OSKM-N proteins are expressed in cervical cancer. (a) Expression levels of OCT4 (i), SOX2 (ii), KLF4 (iii), C-MYC (iv), and NANOG (v) proteins in cervical cancer tumors were measured by western blot (WB). Tumor samples (T1-T10) were compared with a nontumor sample (NT1). WB experiments were performed in triplicate; the values are expressed as mean ± standard deviation (normalized to β -actin) using densitometric analysis. (b) The expression of OSKM-N in tumor cells from T1 and T2 samples was observed by immunohistochemistry (arrows). ×20 magnification. ∗ p
Figure Legend Snippet: OSKM-N proteins are expressed in cervical cancer. (a) Expression levels of OCT4 (i), SOX2 (ii), KLF4 (iii), C-MYC (iv), and NANOG (v) proteins in cervical cancer tumors were measured by western blot (WB). Tumor samples (T1-T10) were compared with a nontumor sample (NT1). WB experiments were performed in triplicate; the values are expressed as mean ± standard deviation (normalized to β -actin) using densitometric analysis. (b) The expression of OSKM-N in tumor cells from T1 and T2 samples was observed by immunohistochemistry (arrows). ×20 magnification. ∗ p

Techniques Used: Expressing, Western Blot, Standard Deviation, Immunohistochemistry

Pluripotency factors are expressed in CC with different clinical outcomes. OSKM-N factors are overexpressed in cervical cancer tumor cells (arrows). P16 INK4A was used as an indirect indicator of the presence of HPV and the degree of epithelial injury. The expression of OCT4, SOX2, and KLF4 was examined in 44 CC tissues from which the clinical response to treatment was known. Representative images of four samples are shown. ×40 magnification.
Figure Legend Snippet: Pluripotency factors are expressed in CC with different clinical outcomes. OSKM-N factors are overexpressed in cervical cancer tumor cells (arrows). P16 INK4A was used as an indirect indicator of the presence of HPV and the degree of epithelial injury. The expression of OCT4, SOX2, and KLF4 was examined in 44 CC tissues from which the clinical response to treatment was known. Representative images of four samples are shown. ×40 magnification.

Techniques Used: Expressing

37) Product Images from "Characterization of Lgr6+ Cells as an Enriched Population of Hair Cell Progenitors Compared to Lgr5+ Cells for Hair Cell Generation in the Neonatal Mouse Cochlea"

Article Title: Characterization of Lgr6+ Cells as an Enriched Population of Hair Cell Progenitors Compared to Lgr5+ Cells for Hair Cell Generation in the Neonatal Mouse Cochlea

Journal: Frontiers in Molecular Neuroscience

doi: 10.3389/fnmol.2018.00147

Re-sort analysis, immunostaining, and q-PCR of flow-sorted Lgr5+ and Lgr6+ cells from the postnatal cochlea. (A) At P3, Lgr5 was expressed in the third row of Deiters’ cells (DC3), the inner pillar cells (IPs), the inner phalangeal cells (IPCs), and the lateral GER, while Lgr6 was only expressed in the IPs. (B) Cryosection showed that Lgr5 was expressed in DC3s, IPs, IPCs and the GER, and Lgr6 was only expressed in a subset of IPs in the P3 organ of Corti. (C) GFP+ cells and GFP– cells were isolated using flow cytometry. Re-sort analysis of GFP+ cells demonstrated > 90% purity. (D) Immunostaining of Lgr5+ cells and Lgr6+ cells from the cochlea showed a high percentage of Sox2+ (95.4% and 95.2%, respectively) and GFP+ (95.8% and 96.6%, respectively) cells, and no Myo7a+ cells, among the sorted cells. (E,F) q-PCR showed that isolated Lgr5+ cells and Lgr6+ cells had significantly higher Lgr5 and Lgr6 expression, slightly higher Sox2 expression, and significantly lower Brn3.1 expression compared to the Lgr5- cells and Lgr6– cells, respectively. Scale bars are 20 μm. ∗∗ p
Figure Legend Snippet: Re-sort analysis, immunostaining, and q-PCR of flow-sorted Lgr5+ and Lgr6+ cells from the postnatal cochlea. (A) At P3, Lgr5 was expressed in the third row of Deiters’ cells (DC3), the inner pillar cells (IPs), the inner phalangeal cells (IPCs), and the lateral GER, while Lgr6 was only expressed in the IPs. (B) Cryosection showed that Lgr5 was expressed in DC3s, IPs, IPCs and the GER, and Lgr6 was only expressed in a subset of IPs in the P3 organ of Corti. (C) GFP+ cells and GFP– cells were isolated using flow cytometry. Re-sort analysis of GFP+ cells demonstrated > 90% purity. (D) Immunostaining of Lgr5+ cells and Lgr6+ cells from the cochlea showed a high percentage of Sox2+ (95.4% and 95.2%, respectively) and GFP+ (95.8% and 96.6%, respectively) cells, and no Myo7a+ cells, among the sorted cells. (E,F) q-PCR showed that isolated Lgr5+ cells and Lgr6+ cells had significantly higher Lgr5 and Lgr6 expression, slightly higher Sox2 expression, and significantly lower Brn3.1 expression compared to the Lgr5- cells and Lgr6– cells, respectively. Scale bars are 20 μm. ∗∗ p

Techniques Used: Immunostaining, Polymerase Chain Reaction, Flow Cytometry, Isolation, Cytometry, Expressing

38) Product Images from "Cancer-associated fibroblasts release exosomal microRNAs that dictate an aggressive phenotype in breast cancer"

Article Title: Cancer-associated fibroblasts release exosomal microRNAs that dictate an aggressive phenotype in breast cancer

Journal: Oncotarget

doi: 10.18632/oncotarget.14752

Effects of miRs-21, -143, and -378e on stemness and EMT markers in triple-negative breast cancer cells BT549 cells were transfected with miRs: scr, -21, -143, and -378e, for 24h. Real Time PCR was performed to analyze oct3/4, sox2, and snail mRNA levels a . In addition, BT549 cells were transfected with these miRs alone or in combination for 48h. Western blot analysis was performed to evaluate nanog, oct3/4, sox2, and zeb protein levels b . MDA-MB-231 cells were transfected with miRs: scr, -21, -143, and -378e, for 24h. Real Time PCR was performed to analyze oct3/4, nanog, sox2, and snail mRNA levels (a). In addition, MDA-MB-231 cells were transfected with these miRs alone or in combination for 48h. Western blot analysis was performed to evaluate zeb, nanog, and oct3/4 protein levels (b). T47D cells were transfected with anti-miRs: -scr (control), -21, -143, and -378e, for 48h. Real Time PCR was performed to analyze sox2 mRNA levels c . In addition, T47D cells were transfected with anti-miRs (alone or in combination; final concentration: 200nM) for 72h. Western blot analysis was performed to evaluate e-cadherin, zeb, and slug protein levels d . Western blot analysis from representative experiments. Actin or α-tubulin were used as loading controls. The experiments were repeated at least three times (b, d). In a and c, data were obtained from three independent experiments and are presented as mean value ± SD. P-value calculated using Student's t test. * p
Figure Legend Snippet: Effects of miRs-21, -143, and -378e on stemness and EMT markers in triple-negative breast cancer cells BT549 cells were transfected with miRs: scr, -21, -143, and -378e, for 24h. Real Time PCR was performed to analyze oct3/4, sox2, and snail mRNA levels a . In addition, BT549 cells were transfected with these miRs alone or in combination for 48h. Western blot analysis was performed to evaluate nanog, oct3/4, sox2, and zeb protein levels b . MDA-MB-231 cells were transfected with miRs: scr, -21, -143, and -378e, for 24h. Real Time PCR was performed to analyze oct3/4, nanog, sox2, and snail mRNA levels (a). In addition, MDA-MB-231 cells were transfected with these miRs alone or in combination for 48h. Western blot analysis was performed to evaluate zeb, nanog, and oct3/4 protein levels (b). T47D cells were transfected with anti-miRs: -scr (control), -21, -143, and -378e, for 48h. Real Time PCR was performed to analyze sox2 mRNA levels c . In addition, T47D cells were transfected with anti-miRs (alone or in combination; final concentration: 200nM) for 72h. Western blot analysis was performed to evaluate e-cadherin, zeb, and slug protein levels d . Western blot analysis from representative experiments. Actin or α-tubulin were used as loading controls. The experiments were repeated at least three times (b, d). In a and c, data were obtained from three independent experiments and are presented as mean value ± SD. P-value calculated using Student's t test. * p

Techniques Used: Transfection, Real-time Polymerase Chain Reaction, Western Blot, Multiple Displacement Amplification, Concentration Assay

NF exosomes transfected with miRs -21, -143, -378e, similarly to CAF exosomes, promote stemness and epithelial-mesenchymal transition phenotype NF (patients #5, and #6) were cultured for 48h. Then, exosomes were isolated from NF-conditioned media with ExoQuick-TC™ solution, and transfected using Exo-Fect™ solution with scrambled miRs (scr, control), or miRs -21, -143, -378e, alone or in combination (final concentration: 130nM). T47D cells were cultured in the presence of NF exosomes (patient #5) transfected with either scrambled miRs (control) or miRs for 48h. Cells were harvested and grown under non-adherent conditions in stem medium. After four days, the capacity of cells to form spheres was assessed a . Sphere diameter distribution for 10 representative fields b . Scale bar: 100μm c . In addition, T47D cells were cultured in the presence of NF exosomes (patients #5, and #6) transfected with either scrambled miRs (control) or miRs for 24h d or 48h e . Real Time PCR was performed to analyze nanog, sox2, and snail mRNA (d). Western blot analysis was performed to evaluate zeb, nanog, and oct3/4 protein levels. Western blot analysis from representative experiments. Actin was used as loading control. The experiments were repeated at least three times (e). In a, b, d, data were obtained from three independent experiments and are presented as mean value ± SD. P-value calculated using Student's t test. * p
Figure Legend Snippet: NF exosomes transfected with miRs -21, -143, -378e, similarly to CAF exosomes, promote stemness and epithelial-mesenchymal transition phenotype NF (patients #5, and #6) were cultured for 48h. Then, exosomes were isolated from NF-conditioned media with ExoQuick-TC™ solution, and transfected using Exo-Fect™ solution with scrambled miRs (scr, control), or miRs -21, -143, -378e, alone or in combination (final concentration: 130nM). T47D cells were cultured in the presence of NF exosomes (patient #5) transfected with either scrambled miRs (control) or miRs for 48h. Cells were harvested and grown under non-adherent conditions in stem medium. After four days, the capacity of cells to form spheres was assessed a . Sphere diameter distribution for 10 representative fields b . Scale bar: 100μm c . In addition, T47D cells were cultured in the presence of NF exosomes (patients #5, and #6) transfected with either scrambled miRs (control) or miRs for 24h d or 48h e . Real Time PCR was performed to analyze nanog, sox2, and snail mRNA (d). Western blot analysis was performed to evaluate zeb, nanog, and oct3/4 protein levels. Western blot analysis from representative experiments. Actin was used as loading control. The experiments were repeated at least three times (e). In a, b, d, data were obtained from three independent experiments and are presented as mean value ± SD. P-value calculated using Student's t test. * p

Techniques Used: Transfection, Cell Culture, Isolation, Concentration Assay, Real-time Polymerase Chain Reaction, Western Blot

CAF exosomes promote stemness, epithelial–mesenchymal transition, and anchorage-independent cell growth T47D cells were cultured under non-adherent conditions in stem medium in the absence (control) or presence of either NF exosomes (patients #5, #6, and #10) or CAF exosomes (patients #3, #7, and #9). After four days, the capacity of cells to form spheres was assessed a . Sphere diameter distribution (control cells and T47D cells treated with either NF#6 ex or CAF#7 ex) for 10 representative fields. Scale bar: 100μm b . T47D cells were cultured in the absence (control) or presence of either NF exosomes (patients #5, #6, and #10) or CAF exosomes (patients #7, and #12). After 24h, cells were harvested and cultured in soft agar c . Scale bar: 100μm d . T47D cells were cultured in the absence (control) or presence of either NF ex (patients #5, #6, and #10) or CAF ex (patients #3, #7, #9, #12 and #13) for 24h e . or 48h f . Real Time PCR was performed to analyze oct3/4, nanog, sox2, snail and zeb mRNA levels (e). Western blot analysis was performed to evaluate nanog, sox2, oct3/4, zeb, e-cadherin and snail protein levels (f). BT549 cells were cultured in the absence (control) or presence of either NF exosomes (patient #6) or CAF exosomes (patient #7) for 48h. Western blot analysis was performed to evaluate e-cadherin and oct3/4 protein levels. MDA-MB-231 cells were cultured in the absence (control) or presence of either NF exosomes (patient #5) o CAF exosomes (patient #12) for 48h. Western blot analysis was performed to evaluate oct3/4 protein level g . Western blot from representative experiments is shown. Actin or vinculin were used as loading controls. The experiments were repeated at least three times (f, g). In a, b, c, e, data were obtained from three independent experiments and are presented as mean value ± SD. P-value was calculated using one-way ANOVA followed by Bonferroni's post hoc testing. * p
Figure Legend Snippet: CAF exosomes promote stemness, epithelial–mesenchymal transition, and anchorage-independent cell growth T47D cells were cultured under non-adherent conditions in stem medium in the absence (control) or presence of either NF exosomes (patients #5, #6, and #10) or CAF exosomes (patients #3, #7, and #9). After four days, the capacity of cells to form spheres was assessed a . Sphere diameter distribution (control cells and T47D cells treated with either NF#6 ex or CAF#7 ex) for 10 representative fields. Scale bar: 100μm b . T47D cells were cultured in the absence (control) or presence of either NF exosomes (patients #5, #6, and #10) or CAF exosomes (patients #7, and #12). After 24h, cells were harvested and cultured in soft agar c . Scale bar: 100μm d . T47D cells were cultured in the absence (control) or presence of either NF ex (patients #5, #6, and #10) or CAF ex (patients #3, #7, #9, #12 and #13) for 24h e . or 48h f . Real Time PCR was performed to analyze oct3/4, nanog, sox2, snail and zeb mRNA levels (e). Western blot analysis was performed to evaluate nanog, sox2, oct3/4, zeb, e-cadherin and snail protein levels (f). BT549 cells were cultured in the absence (control) or presence of either NF exosomes (patient #6) or CAF exosomes (patient #7) for 48h. Western blot analysis was performed to evaluate e-cadherin and oct3/4 protein levels. MDA-MB-231 cells were cultured in the absence (control) or presence of either NF exosomes (patient #5) o CAF exosomes (patient #12) for 48h. Western blot analysis was performed to evaluate oct3/4 protein level g . Western blot from representative experiments is shown. Actin or vinculin were used as loading controls. The experiments were repeated at least three times (f, g). In a, b, c, e, data were obtained from three independent experiments and are presented as mean value ± SD. P-value was calculated using one-way ANOVA followed by Bonferroni's post hoc testing. * p

Techniques Used: Cell Culture, Real-time Polymerase Chain Reaction, Western Blot, Multiple Displacement Amplification

miR-21, miR-143, and miR-378e promote stemness and epithelial–mesenchymal transition T47D cells were transfected with scrambled (scr, control) or miRs -21, -143, and -378e, alone or in combination (final concentration: 100nM). After 48h, cells were harvested and cultured under non-adherent conditions in stem medium. After four days, the capacity of cells to form spheres was assessed a . Sphere diameter distribution for 10 representative fields. Scale bar: 100μm b . T47D cells were transfected with anti-miRs (alone or in combination, final concentration: 200nM) or scrambled anti-miR (anti-miR-scr, control). After 24h, cells were harvested and cultured under non-adherent conditions in stem medium. After four days, the capacity of cells to form spheres was assessed c . Sphere diameter distribution for 10 representative fields. Scale bar: 50μm d . ALDEFLUOR assays were performed in T47D cells transfected with scrambled or miRs -21, -143, and -378e for 48h e . T47D cells were transfected with scrambled, miRs -21, -143, or -378e for 48h f . or 72h g . Real Time PCR was performed to analyze oct3/4, nanog, sox2, and snail mRNA levels (f). In addition, T47D cells were transfected with the miRs alone or in combination for 72h. Western blot analysis was performed to evaluate nanog, sox2, oct3/4, zeb, and e-cadherin protein levels (g). In a, b, c, d, e, f, data were obtained from three independent experiments and are presented as mean value ± SD. P-value was calculated using Student's t test. * p
Figure Legend Snippet: miR-21, miR-143, and miR-378e promote stemness and epithelial–mesenchymal transition T47D cells were transfected with scrambled (scr, control) or miRs -21, -143, and -378e, alone or in combination (final concentration: 100nM). After 48h, cells were harvested and cultured under non-adherent conditions in stem medium. After four days, the capacity of cells to form spheres was assessed a . Sphere diameter distribution for 10 representative fields. Scale bar: 100μm b . T47D cells were transfected with anti-miRs (alone or in combination, final concentration: 200nM) or scrambled anti-miR (anti-miR-scr, control). After 24h, cells were harvested and cultured under non-adherent conditions in stem medium. After four days, the capacity of cells to form spheres was assessed c . Sphere diameter distribution for 10 representative fields. Scale bar: 50μm d . ALDEFLUOR assays were performed in T47D cells transfected with scrambled or miRs -21, -143, and -378e for 48h e . T47D cells were transfected with scrambled, miRs -21, -143, or -378e for 48h f . or 72h g . Real Time PCR was performed to analyze oct3/4, nanog, sox2, and snail mRNA levels (f). In addition, T47D cells were transfected with the miRs alone or in combination for 72h. Western blot analysis was performed to evaluate nanog, sox2, oct3/4, zeb, and e-cadherin protein levels (g). In a, b, c, d, e, f, data were obtained from three independent experiments and are presented as mean value ± SD. P-value was calculated using Student's t test. * p

Techniques Used: Transfection, Concentration Assay, Cell Culture, Real-time Polymerase Chain Reaction, Western Blot

39) Product Images from "Model for long QT syndrome type 2 using human iPS cells demonstrates arrhythmogenic characteristics in cell culture"

Article Title: Model for long QT syndrome type 2 using human iPS cells demonstrates arrhythmogenic characteristics in cell culture

Journal: Disease Models & Mechanisms

doi: 10.1242/dmm.008409

Characterization of iPSCs. (A) Morphology of the iPSC colonies is similar to those of hESCs. The colonies are rather roundish and the edges are well defined and sharp, which is typical for a stem cell colony. (B) Expression of pluripotency markers in LQT2-specific iPSCs is shown by RT-PCR. All the endogenous pluripotency genes studied were turned on in iPSCs by passage 6 (top panel). As a positive control, they were also expressed in hESCs (H7). Expression of Sox2 and very modest expression of Rex1 and Myc was found also in EBs, which were used as a negative control. β-actin served as a loading control. None of the exogenous genes were expressed in iPSCs at passage 11. As a positive control, PCR was also done using the transfected plasmids as templates (bottom panel). RT-PCR results were similar for all the iPSC lines. (C) Immunocytochemical staining of the cells shows that pluripotency markers are expressed also at the protein level. The expression of Nanog, Oct3/4, Sox2, SSEA-4, TRA1-60 and TRA1-81 was similar in all iPSC lines and there were no differences between LQT2-specific and control lines. (D) Karyotypes of all the iPSC lines were analyzed and proved to be normal. (E) Teratomas were made from one LQT2-specific line and two control lines to further confirm the pluripotency of the lines. Tissues from all three germ layers were found in teratomas from every line. (F) EBs were also formed from all the lines to show the pluripotent differentiation capacity. The EB-derived cells from both LQT2-iPSC and all control iPSC lines expressed markers from the three embryonic germ layers.
Figure Legend Snippet: Characterization of iPSCs. (A) Morphology of the iPSC colonies is similar to those of hESCs. The colonies are rather roundish and the edges are well defined and sharp, which is typical for a stem cell colony. (B) Expression of pluripotency markers in LQT2-specific iPSCs is shown by RT-PCR. All the endogenous pluripotency genes studied were turned on in iPSCs by passage 6 (top panel). As a positive control, they were also expressed in hESCs (H7). Expression of Sox2 and very modest expression of Rex1 and Myc was found also in EBs, which were used as a negative control. β-actin served as a loading control. None of the exogenous genes were expressed in iPSCs at passage 11. As a positive control, PCR was also done using the transfected plasmids as templates (bottom panel). RT-PCR results were similar for all the iPSC lines. (C) Immunocytochemical staining of the cells shows that pluripotency markers are expressed also at the protein level. The expression of Nanog, Oct3/4, Sox2, SSEA-4, TRA1-60 and TRA1-81 was similar in all iPSC lines and there were no differences between LQT2-specific and control lines. (D) Karyotypes of all the iPSC lines were analyzed and proved to be normal. (E) Teratomas were made from one LQT2-specific line and two control lines to further confirm the pluripotency of the lines. Tissues from all three germ layers were found in teratomas from every line. (F) EBs were also formed from all the lines to show the pluripotent differentiation capacity. The EB-derived cells from both LQT2-iPSC and all control iPSC lines expressed markers from the three embryonic germ layers.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control, Negative Control, Polymerase Chain Reaction, Transfection, Staining, Derivative Assay

40) Product Images from "Cux2 Activity Defines a Subpopulation of Perinatal Neurogenic Progenitors in the Hippocampus"

Article Title: Cux2 Activity Defines a Subpopulation of Perinatal Neurogenic Progenitors in the Hippocampus

Journal: Hippocampus

doi: 10.1002/hipo.22370

Birth dating analysis in Cux2 neo/neo mutants. A–E: EdU was injected at E17.5 and the embryos ( n = 5 for each group) were analyzed at P14 by immunohistochemistry using anti-Calb antibody (green) and EdU staining (red). A–C: EdU pulsing at E17.5 revealed labeling of mature Calb + neurons in the outer layer of the DG. E: The percentage of cells double positive for Calb and EdU (Calb + EdU + ) in the total EdU-positive cells (EdU + ) were not significantly different between Cux2 neo/+ controls (C) and Cux2 neo/neo mutants (D) ( P = 0.09). F–I and K–N: EdU was injected at P5 and the analyses were done at P21 ( n = 4 for each group). Pulsing at P5 labeled deeper regions of the dentate blade and resulted in EdU co-staining with Calb (F–I) and Sox2 (K–N). J: The percentage of Calb + /EdU + cells over total EdU + was comparable between controls and mutants ( P = 0.61). O: The percentage of Sox2 + /EdU + in the total EdU + pool (retention index) was similar between controls and mutants ( P = 0.83). Scale bars: 20 µm. Abbreviations as in Figure 3 ; Calb, Calbindin. [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com .]
Figure Legend Snippet: Birth dating analysis in Cux2 neo/neo mutants. A–E: EdU was injected at E17.5 and the embryos ( n = 5 for each group) were analyzed at P14 by immunohistochemistry using anti-Calb antibody (green) and EdU staining (red). A–C: EdU pulsing at E17.5 revealed labeling of mature Calb + neurons in the outer layer of the DG. E: The percentage of cells double positive for Calb and EdU (Calb + EdU + ) in the total EdU-positive cells (EdU + ) were not significantly different between Cux2 neo/+ controls (C) and Cux2 neo/neo mutants (D) ( P = 0.09). F–I and K–N: EdU was injected at P5 and the analyses were done at P21 ( n = 4 for each group). Pulsing at P5 labeled deeper regions of the dentate blade and resulted in EdU co-staining with Calb (F–I) and Sox2 (K–N). J: The percentage of Calb + /EdU + cells over total EdU + was comparable between controls and mutants ( P = 0.61). O: The percentage of Sox2 + /EdU + in the total EdU + pool (retention index) was similar between controls and mutants ( P = 0.83). Scale bars: 20 µm. Abbreviations as in Figure 3 ; Calb, Calbindin. [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com .]

Techniques Used: Injection, Immunohistochemistry, Staining, Labeling

Model of DG morphogenesis and Cux2 activity in perinatal hippocampus progenitors. A: Schematic of DG morphogenesis in the rodent brain. The first wave of granule cell precursors arises at fetal stages (E15.5 in the mouse) and migrates away from the 3rd ventricle (3rdV) region to populate the growing subventicular (SVZ) dentate matrix in the medial region of the telencephalon. These cells are fated to give rise to the outer-most granule cell neurons. As DG development continues within the first few weeks of life, a second germinative matrix forms deep within in the SVZ of the medial telencephalon. This is the dentate knot. Cux2 activity is high at these early stages of DG morphogenesis. As the animal progresses to adulthood, the tertiary germinative matrix forms and becomes populated with long-lived multipotent NPs. This region is the SGZ that houses Type 1 cells capable of giving rise to nascent neurons well into adult life. B: Model of Cux2 localization is a subset of Type 1 progenitors that originate in the dentate germinal regions at fetal stages and persists to the first weeks of life. This is a transitory progenitor that is transiting from a Type 1-like Nestin+/GFAP+/Sox2+ radial glia to a Sox2+ Type 2 cell, which lack a radial process and astroglial markers. Cux2 was also detected in DCX + neuroblasts (Type 3 cells) and immature newly formed granule cells identified by Calretinin expression. Fate mapping of Cux2-expressing progenitors revealed labeling of Calbindin-positive DG cells, and not SGZ progenitors, suggesting that the Cux2 + progenitor does not self-renew and directly generates granule cells. Abbreviations as in Figure 3 . [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com .]
Figure Legend Snippet: Model of DG morphogenesis and Cux2 activity in perinatal hippocampus progenitors. A: Schematic of DG morphogenesis in the rodent brain. The first wave of granule cell precursors arises at fetal stages (E15.5 in the mouse) and migrates away from the 3rd ventricle (3rdV) region to populate the growing subventicular (SVZ) dentate matrix in the medial region of the telencephalon. These cells are fated to give rise to the outer-most granule cell neurons. As DG development continues within the first few weeks of life, a second germinative matrix forms deep within in the SVZ of the medial telencephalon. This is the dentate knot. Cux2 activity is high at these early stages of DG morphogenesis. As the animal progresses to adulthood, the tertiary germinative matrix forms and becomes populated with long-lived multipotent NPs. This region is the SGZ that houses Type 1 cells capable of giving rise to nascent neurons well into adult life. B: Model of Cux2 localization is a subset of Type 1 progenitors that originate in the dentate germinal regions at fetal stages and persists to the first weeks of life. This is a transitory progenitor that is transiting from a Type 1-like Nestin+/GFAP+/Sox2+ radial glia to a Sox2+ Type 2 cell, which lack a radial process and astroglial markers. Cux2 was also detected in DCX + neuroblasts (Type 3 cells) and immature newly formed granule cells identified by Calretinin expression. Fate mapping of Cux2-expressing progenitors revealed labeling of Calbindin-positive DG cells, and not SGZ progenitors, suggesting that the Cux2 + progenitor does not self-renew and directly generates granule cells. Abbreviations as in Figure 3 . [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com .]

Techniques Used: Activity Assay, Expressing, Labeling

Cux2 is expressed in Nestin, GFAP, and Sox2 positive radial glia. A–E: Cux2 (A) co-localization in Sox2 (B) and Nestin (C) positive cells in the SGZ at P21. D: Cux2 (green), Sox2 (red), and Nestin (white) co-localization in Type 1 radial glia (red arrowhead). Cux2 was also expressed in Sox2 + but Nestin-negative Type 2 cells (white arrowhead). E: High magnification (63×) Z-stack image of Cux2 (green) co-localization in a Sox2 (red) and Nestin (white) double-positive radial glia Type 1 progenitor (red arrowhead). Cux2 was also co-expressed in Sox2 + /Nestin − Type 2 cells (white arrowhead). F–K: Cux2 (F) co-localization in Sox2 (G) and GFAP (H) positive cells in the SGZ. I: Cux2 (green), Sox2 (red), and GFAP (white) co-localization in Type 1 radial glia (red arrowhead). K: High magnification (63×) Z-stack image of Cux2 (green) co-localization in a Sox2 (red) and GFAP (white) double-positive Type 1 progenitor possessing a radial glia (red arrowhead). Cux2 was also co-expressed in Sox2 + /GFAP − Type 2 cells (white arrowhead). K–M: EdU (red) co-staining with Nestin (green, arrowhead, K), and Sox2 (green, arrowhead, L). Cux2 (green) was not detected in dividing EdU + SGZ cells (red, arrowhead, M). N: Chart summarizing Cux2 expression in Type 1 (Nestin + /Sox2 + ) and Type 2 (Sox2 + /Nestin − ) cells at P21. A greater proportion of Cux2 expression was detected in Type 2 (15.7%) cells vs. Type 1 cells (5.5%). Cux2 was also co-localized in Type 3 cells (neuroblasts) and nascent neurons (see Fig. 4 ). Scale bars: A and K, 25 µm; E and J, 8 µm. Abbreviations: h, hilus; gcl, granule cell layer; SGZ, subgranular zone. [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com .]
Figure Legend Snippet: Cux2 is expressed in Nestin, GFAP, and Sox2 positive radial glia. A–E: Cux2 (A) co-localization in Sox2 (B) and Nestin (C) positive cells in the SGZ at P21. D: Cux2 (green), Sox2 (red), and Nestin (white) co-localization in Type 1 radial glia (red arrowhead). Cux2 was also expressed in Sox2 + but Nestin-negative Type 2 cells (white arrowhead). E: High magnification (63×) Z-stack image of Cux2 (green) co-localization in a Sox2 (red) and Nestin (white) double-positive radial glia Type 1 progenitor (red arrowhead). Cux2 was also co-expressed in Sox2 + /Nestin − Type 2 cells (white arrowhead). F–K: Cux2 (F) co-localization in Sox2 (G) and GFAP (H) positive cells in the SGZ. I: Cux2 (green), Sox2 (red), and GFAP (white) co-localization in Type 1 radial glia (red arrowhead). K: High magnification (63×) Z-stack image of Cux2 (green) co-localization in a Sox2 (red) and GFAP (white) double-positive Type 1 progenitor possessing a radial glia (red arrowhead). Cux2 was also co-expressed in Sox2 + /GFAP − Type 2 cells (white arrowhead). K–M: EdU (red) co-staining with Nestin (green, arrowhead, K), and Sox2 (green, arrowhead, L). Cux2 (green) was not detected in dividing EdU + SGZ cells (red, arrowhead, M). N: Chart summarizing Cux2 expression in Type 1 (Nestin + /Sox2 + ) and Type 2 (Sox2 + /Nestin − ) cells at P21. A greater proportion of Cux2 expression was detected in Type 2 (15.7%) cells vs. Type 1 cells (5.5%). Cux2 was also co-localized in Type 3 cells (neuroblasts) and nascent neurons (see Fig. 4 ). Scale bars: A and K, 25 µm; E and J, 8 µm. Abbreviations: h, hilus; gcl, granule cell layer; SGZ, subgranular zone. [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com .]

Techniques Used: Staining, Expressing

Cux2 fate-mapped cells were Calbindin-positive mature gcl neurons. A–C: Cux2-cre; tdTomato fate mapped cells in the DG of P21 mice. C: Tomato + fate-mapped cells showed limited or no localization with high Cux2 + expressors, in the SGZ and instead localized to outer layer DG cells expressing low Cux2 levels (arrow in B, C). D–F: Tomato fate-mapped cells localized to Calbindin-expressing gcl neurons. G–I: Tomato fate-mapped cells of the P21 DG did not localize with DCX + neuroblasts. H, I: Arrow identifies a tomato + cell in the SGZ that did not express DCX (arrowhead). J–L: Tomato fate-mapped cells of the P21 DG did not localize with GFAP + astroglia. K, L: Arrow identifies a GFAP + astrocyte in the outer layer of the DG that was negative for tomato. M–O: Tomato + cells did not co-localize with Sox2 in the SGZ or occasional Sox2 + cell in the outer layer of the DG (arrow). Scale bar: A, 50 µm. Abbreviations: Calb, Calbindin; ol, outer layer of the DG; SGZ, subgranular zone. [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com .]
Figure Legend Snippet: Cux2 fate-mapped cells were Calbindin-positive mature gcl neurons. A–C: Cux2-cre; tdTomato fate mapped cells in the DG of P21 mice. C: Tomato + fate-mapped cells showed limited or no localization with high Cux2 + expressors, in the SGZ and instead localized to outer layer DG cells expressing low Cux2 levels (arrow in B, C). D–F: Tomato fate-mapped cells localized to Calbindin-expressing gcl neurons. G–I: Tomato fate-mapped cells of the P21 DG did not localize with DCX + neuroblasts. H, I: Arrow identifies a tomato + cell in the SGZ that did not express DCX (arrowhead). J–L: Tomato fate-mapped cells of the P21 DG did not localize with GFAP + astroglia. K, L: Arrow identifies a GFAP + astrocyte in the outer layer of the DG that was negative for tomato. M–O: Tomato + cells did not co-localize with Sox2 in the SGZ or occasional Sox2 + cell in the outer layer of the DG (arrow). Scale bar: A, 50 µm. Abbreviations: Calb, Calbindin; ol, outer layer of the DG; SGZ, subgranular zone. [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com .]

Techniques Used: Mouse Assay, Expressing

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    Santa Cruz Biotechnology rabbit anti sox2
    Morphologies, culture characteristics, and cell proliferation rates of cryopreserved human dental pulp tissues harvested from extracted wisdom teeth (hDPSCs-cryo). ( A ) Cell morphologies of hDPSCs-cryo at passage 0 (P0) and passage 3 (P3) showing homogeneous plate adherent fibroblast-like morphology (scale bar = 100 µm); ( B ) Cell proliferation rate by population doubling time (PDT) of hDPSCs-cryo produced normal cell proliferation curvatures. Data represent the mean ± SD of three independent experiments; ( C ) Cell cycle analysis of hDPSCs-cryo showed normal DNA content in the gap 0/1 (G0/G1), synthesis (S), or gap 2/mitotic (G2/M) phases of the cell cycle; ( D ) Western blot analysis revealed positive protein expression of the pluripotent markers, octamer-binding transcription factor 4 (Oct4), Nanog, and sex determining region Y-box 2 <t>(Sox2);</t> and ( E ) The relative quantification of pluripotent marker expressions ( D ) to the control protein (β-actin) revealed relatively higher expression of Nanog over Oct4 or Sox2. Data represent the mean ± SD of three independent experiments.
    Rabbit Anti Sox2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti sox2/product/Santa Cruz Biotechnology
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    91
    Santa Cruz Biotechnology antisera goat anti sox2
    Chondrocyte-secreted factors do not affect supporting-cell proliferation. ( A ) In a brightfield image of a utricular bubble culture established at E17.5, the utricle and ampullae semicircular canals are labeled. A piece of cartilage was dissected from the same E17.5 inner ear and positioned near the edge of the utricle, indicated by the magenta line. ( B ) In a utricular bubble preparation after 24 hr in culture, immunolabeling for <t>Sox2</t> (green) reveals the size and position of the sensory epithelia of the utricle; EdU (white) demonstrated the distribution of proliferating cells adjacent to (pink line) and distant from the cartilage. The scale bar represents 100 μm. DOI: http://dx.doi.org/10.7554/eLife.25681.016
    Antisera Goat Anti Sox2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 162 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antisera goat anti sox2/product/Santa Cruz Biotechnology
    Average 91 stars, based on 162 article reviews
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    Morphologies, culture characteristics, and cell proliferation rates of cryopreserved human dental pulp tissues harvested from extracted wisdom teeth (hDPSCs-cryo). ( A ) Cell morphologies of hDPSCs-cryo at passage 0 (P0) and passage 3 (P3) showing homogeneous plate adherent fibroblast-like morphology (scale bar = 100 µm); ( B ) Cell proliferation rate by population doubling time (PDT) of hDPSCs-cryo produced normal cell proliferation curvatures. Data represent the mean ± SD of three independent experiments; ( C ) Cell cycle analysis of hDPSCs-cryo showed normal DNA content in the gap 0/1 (G0/G1), synthesis (S), or gap 2/mitotic (G2/M) phases of the cell cycle; ( D ) Western blot analysis revealed positive protein expression of the pluripotent markers, octamer-binding transcription factor 4 (Oct4), Nanog, and sex determining region Y-box 2 (Sox2); and ( E ) The relative quantification of pluripotent marker expressions ( D ) to the control protein (β-actin) revealed relatively higher expression of Nanog over Oct4 or Sox2. Data represent the mean ± SD of three independent experiments.

    Journal: International Journal of Molecular Sciences

    Article Title: Cholinergic Nerve Differentiation of Mesenchymal Stem Cells Derived from Long-Term Cryopreserved Human Dental Pulp In Vitro and Analysis of Their Motor Nerve Regeneration Potential In Vivo

    doi: 10.3390/ijms19082434

    Figure Lengend Snippet: Morphologies, culture characteristics, and cell proliferation rates of cryopreserved human dental pulp tissues harvested from extracted wisdom teeth (hDPSCs-cryo). ( A ) Cell morphologies of hDPSCs-cryo at passage 0 (P0) and passage 3 (P3) showing homogeneous plate adherent fibroblast-like morphology (scale bar = 100 µm); ( B ) Cell proliferation rate by population doubling time (PDT) of hDPSCs-cryo produced normal cell proliferation curvatures. Data represent the mean ± SD of three independent experiments; ( C ) Cell cycle analysis of hDPSCs-cryo showed normal DNA content in the gap 0/1 (G0/G1), synthesis (S), or gap 2/mitotic (G2/M) phases of the cell cycle; ( D ) Western blot analysis revealed positive protein expression of the pluripotent markers, octamer-binding transcription factor 4 (Oct4), Nanog, and sex determining region Y-box 2 (Sox2); and ( E ) The relative quantification of pluripotent marker expressions ( D ) to the control protein (β-actin) revealed relatively higher expression of Nanog over Oct4 or Sox2. Data represent the mean ± SD of three independent experiments.

    Article Snippet: The membranes were then incubated with primary antibodies, including goat anti-Oct4 (1:200, 43–50 kDa, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), mouse anti-Nanog (1:200, 35 kDa, Santa Cruz), rabbit anti-Sox2 (1:200, 34 kDa, Santa Cruz), and rabbit anti-β-actin (1:1000, Cell signaling, Danvers, MA, USA) overnight at 4 °C.

    Techniques: Produced, Cell Cycle Assay, Western Blot, Expressing, Binding Assay, Marker

    Chondrocyte-secreted factors do not affect supporting-cell proliferation. ( A ) In a brightfield image of a utricular bubble culture established at E17.5, the utricle and ampullae semicircular canals are labeled. A piece of cartilage was dissected from the same E17.5 inner ear and positioned near the edge of the utricle, indicated by the magenta line. ( B ) In a utricular bubble preparation after 24 hr in culture, immunolabeling for Sox2 (green) reveals the size and position of the sensory epithelia of the utricle; EdU (white) demonstrated the distribution of proliferating cells adjacent to (pink line) and distant from the cartilage. The scale bar represents 100 μm. DOI: http://dx.doi.org/10.7554/eLife.25681.016

    Journal: eLife

    Article Title: Elastic force restricts growth of the murine utricle

    doi: 10.7554/eLife.25681

    Figure Lengend Snippet: Chondrocyte-secreted factors do not affect supporting-cell proliferation. ( A ) In a brightfield image of a utricular bubble culture established at E17.5, the utricle and ampullae semicircular canals are labeled. A piece of cartilage was dissected from the same E17.5 inner ear and positioned near the edge of the utricle, indicated by the magenta line. ( B ) In a utricular bubble preparation after 24 hr in culture, immunolabeling for Sox2 (green) reveals the size and position of the sensory epithelia of the utricle; EdU (white) demonstrated the distribution of proliferating cells adjacent to (pink line) and distant from the cartilage. The scale bar represents 100 μm. DOI: http://dx.doi.org/10.7554/eLife.25681.016

    Article Snippet: The primary antisera—goat anti-Sox2 (Santa Cruz), rabbit anti-Myo7A (Proteus Bioscience), rabbit anti-GFP (Torrey Pines Biolabs), mouse anti-Yap (Santa Cruz), and rabbit anti-Yap (Cell Signaling)—were reconstituted in blocking solution and applied overnight at 4˚C.

    Techniques: Labeling, Immunolabeling

    Yap controls utricular growth in organotypic bubble cultures. ( A ) A schematic representation of an experiment on an organotypic bubble culture shows the time at which the utricles are transfected with virus expressing GFP or GFP-YTIP and the duration of EdU labeling. ( B ) Sox2 antiserum (red) labels the sensory epithelia of whole-mount preparations of utricular bubbles infected with virus expressing GFP or GFP-YTIP. As demonstrated by the incorporation of EdU (white), supporting cells in E17.5 utricles in GFP virus-infected cultures, but not in GFP-YTIP virus-infected cultures, re-enter the cell cycle. GFP, which demonstrates infected cells, is shown in green. The scale bar represents 100 μm. ( C ) The area of the sensory epithelia and ( D ) the number of EdU-positive cells in GFP virus-infected controls are increased significantly as compared to GFP-YTIP virus-infected utricles (means ± SEMs; area p

    Journal: eLife

    Article Title: Elastic force restricts growth of the murine utricle

    doi: 10.7554/eLife.25681

    Figure Lengend Snippet: Yap controls utricular growth in organotypic bubble cultures. ( A ) A schematic representation of an experiment on an organotypic bubble culture shows the time at which the utricles are transfected with virus expressing GFP or GFP-YTIP and the duration of EdU labeling. ( B ) Sox2 antiserum (red) labels the sensory epithelia of whole-mount preparations of utricular bubbles infected with virus expressing GFP or GFP-YTIP. As demonstrated by the incorporation of EdU (white), supporting cells in E17.5 utricles in GFP virus-infected cultures, but not in GFP-YTIP virus-infected cultures, re-enter the cell cycle. GFP, which demonstrates infected cells, is shown in green. The scale bar represents 100 μm. ( C ) The area of the sensory epithelia and ( D ) the number of EdU-positive cells in GFP virus-infected controls are increased significantly as compared to GFP-YTIP virus-infected utricles (means ± SEMs; area p

    Article Snippet: The primary antisera—goat anti-Sox2 (Santa Cruz), rabbit anti-Myo7A (Proteus Bioscience), rabbit anti-GFP (Torrey Pines Biolabs), mouse anti-Yap (Santa Cruz), and rabbit anti-Yap (Cell Signaling)—were reconstituted in blocking solution and applied overnight at 4˚C.

    Techniques: Transfection, Expressing, Labeling, Infection

    Geometry of the utricle and simplification to a mathematical model. ( A ) In a transverse section of a utricle at E17.5, Sox2-positive cells (green) form the sensory epithelium that sits at the bottom of the fluid-filled organ. Myo7A (red) labels hair cells. DAPI (blue) stains the nuclei of all the cells in the utricle and demonstrates the presence of Sox2-neganive non-sensory cells that are contingent with the sensory epithelium and are lining the rest of the utricle. The mesenchyme surrounding the utricle is also visualized by DAPI staining. ( B ) In a schematic representation, the utricle is surrounded by mesenchymal cells (light blue) that attach it to the surrounding cartilage (purple). We term both these tissues the elastic matrix. This matrix exerts compressive elastic forces that oppose the expansion of the utricle (red arrows), These forces are transmitted tangentially to the inner layer of the ellipsoidal chamber and act on the transitional non-sensory epithelium (blue), which then opposes the expansion of the sensory epithelium (green and magenta). The effective elastic force is the sum of that owing to the transitional epithelium and the tangential force applied by the elastic matrix (orange arrows). ( C ) We simplify the treatment of the model by considering the utricle as a two-dimensional structure. The sensory epithelium (green) forms a surface that is surrounded by a band of translational epithelium of inner radius R 1 and outer radius R 2 and with a Young’s modulus E 1 (blue). This epithelium is in turn bounded by an elastic matrix of inner radius R 2 and Young’s modulus E 2 (purple). The transitional epithelium and elastic matrix can be treated as one effective material of Young’s modulus E , which we term the surrounding elastic band (orange fringes). DOI: http://dx.doi.org/10.7554/eLife.25681.008

    Journal: eLife

    Article Title: Elastic force restricts growth of the murine utricle

    doi: 10.7554/eLife.25681

    Figure Lengend Snippet: Geometry of the utricle and simplification to a mathematical model. ( A ) In a transverse section of a utricle at E17.5, Sox2-positive cells (green) form the sensory epithelium that sits at the bottom of the fluid-filled organ. Myo7A (red) labels hair cells. DAPI (blue) stains the nuclei of all the cells in the utricle and demonstrates the presence of Sox2-neganive non-sensory cells that are contingent with the sensory epithelium and are lining the rest of the utricle. The mesenchyme surrounding the utricle is also visualized by DAPI staining. ( B ) In a schematic representation, the utricle is surrounded by mesenchymal cells (light blue) that attach it to the surrounding cartilage (purple). We term both these tissues the elastic matrix. This matrix exerts compressive elastic forces that oppose the expansion of the utricle (red arrows), These forces are transmitted tangentially to the inner layer of the ellipsoidal chamber and act on the transitional non-sensory epithelium (blue), which then opposes the expansion of the sensory epithelium (green and magenta). The effective elastic force is the sum of that owing to the transitional epithelium and the tangential force applied by the elastic matrix (orange arrows). ( C ) We simplify the treatment of the model by considering the utricle as a two-dimensional structure. The sensory epithelium (green) forms a surface that is surrounded by a band of translational epithelium of inner radius R 1 and outer radius R 2 and with a Young’s modulus E 1 (blue). This epithelium is in turn bounded by an elastic matrix of inner radius R 2 and Young’s modulus E 2 (purple). The transitional epithelium and elastic matrix can be treated as one effective material of Young’s modulus E , which we term the surrounding elastic band (orange fringes). DOI: http://dx.doi.org/10.7554/eLife.25681.008

    Article Snippet: The primary antisera—goat anti-Sox2 (Santa Cruz), rabbit anti-Myo7A (Proteus Bioscience), rabbit anti-GFP (Torrey Pines Biolabs), mouse anti-Yap (Santa Cruz), and rabbit anti-Yap (Cell Signaling)—were reconstituted in blocking solution and applied overnight at 4˚C.

    Techniques: Staining, Activated Clotting Time Assay

    Subcellular Yap localization in organotypic bubble cultures. In utricles maintained for 4 d as organotypic bubble cultures in 640 Pa collagen gels (top panels), nuclear Yap labeling (green) occurs in Sox2-positive supporting cells (red) at the periphery of the macula. EdU incorporation (white) demonstrates cell proliferation. In similar preparations of the utricles maintained in 40 Pa gels (lower panels), nuclear Yap labeling and EdU incorporation occur in the Sox2-positive supporting cells throughout the sensory epithelium. The scale bar represents 25 μm. DOI: http://dx.doi.org/10.7554/eLife.25681.032

    Journal: eLife

    Article Title: Elastic force restricts growth of the murine utricle

    doi: 10.7554/eLife.25681

    Figure Lengend Snippet: Subcellular Yap localization in organotypic bubble cultures. In utricles maintained for 4 d as organotypic bubble cultures in 640 Pa collagen gels (top panels), nuclear Yap labeling (green) occurs in Sox2-positive supporting cells (red) at the periphery of the macula. EdU incorporation (white) demonstrates cell proliferation. In similar preparations of the utricles maintained in 40 Pa gels (lower panels), nuclear Yap labeling and EdU incorporation occur in the Sox2-positive supporting cells throughout the sensory epithelium. The scale bar represents 25 μm. DOI: http://dx.doi.org/10.7554/eLife.25681.032

    Article Snippet: The primary antisera—goat anti-Sox2 (Santa Cruz), rabbit anti-Myo7A (Proteus Bioscience), rabbit anti-GFP (Torrey Pines Biolabs), mouse anti-Yap (Santa Cruz), and rabbit anti-Yap (Cell Signaling)—were reconstituted in blocking solution and applied overnight at 4˚C.

    Techniques: Labeling

    NSC culture and characterization. Morphological analysis of NSCs from mouse cortex. Phase-contrast photomicrographs of suspension neurospheres ( a ) and monolayer culture cells ( b ). Scale bar = 100 μm. Identification of cultured NSCs. Fluorescent photomicrographs of NSCs for Nestin ( c ), SOX2 ( d ), GFAP ( e ), and DCX ( f ). Nuclei were stained with DAPI. Scale bar = 100 μm. Quantifications for GFAP + , Nestin + , SOX2 + , and DCX + cells ( g )

    Journal: Stem Cell Research & Therapy

    Article Title: Adjudin-preconditioned neural stem cells enhance neuroprotection after ischemia reperfusion in mice

    doi: 10.1186/s13287-017-0677-0

    Figure Lengend Snippet: NSC culture and characterization. Morphological analysis of NSCs from mouse cortex. Phase-contrast photomicrographs of suspension neurospheres ( a ) and monolayer culture cells ( b ). Scale bar = 100 μm. Identification of cultured NSCs. Fluorescent photomicrographs of NSCs for Nestin ( c ), SOX2 ( d ), GFAP ( e ), and DCX ( f ). Nuclei were stained with DAPI. Scale bar = 100 μm. Quantifications for GFAP + , Nestin + , SOX2 + , and DCX + cells ( g )

    Article Snippet: Then 3 days later, cells were immunostained with mouse anti-Nestin (Millipore), goat anti-Sox2 (Santa Cruz Technology), rabbit anti-glial fibrillary acidic protein (GFAP) (Millipore), mouse anti-Doublecortin (Santa Cruz Technology), and rabbit anti-Ki67 (1:200; Abcam, Cambridge, MA, USA).

    Techniques: Cell Culture, Staining

    Organ-of-Corti Cytoarchitecture of Wild-Type and Mpzl2- Mutant ( Mpzl2 ko/ko ) Mice Close-ups of the organ of Corti from representative frozen sections (10 μm) prepared from one representative wild-type mouse (A, E, and I) and three Mpzl2 -mutant mice (B–D, F–H, and J–L). Hair cells and supporting cells were immunolabeled for Myosin VIIa (green) and SOX2 (red), respectively, in the apical, middle, and basal turns of the cochlea. Actin in the organ of Corti was stained with phalloidin (purple). Arrowheads point to inner hair cells (IHCs), whitehead arrows to outer hair cells (OHCs), and arrows to Deiters cells (DCs). Asterisks mark abnormalities. The scale bar represents 10 μm.

    Journal: American Journal of Human Genetics

    Article Title: MPZL2, Encoding the Epithelial Junctional Protein Myelin Protein Zero-like 2, Is Essential for Hearing in Man and Mouse

    doi: 10.1016/j.ajhg.2018.05.011

    Figure Lengend Snippet: Organ-of-Corti Cytoarchitecture of Wild-Type and Mpzl2- Mutant ( Mpzl2 ko/ko ) Mice Close-ups of the organ of Corti from representative frozen sections (10 μm) prepared from one representative wild-type mouse (A, E, and I) and three Mpzl2 -mutant mice (B–D, F–H, and J–L). Hair cells and supporting cells were immunolabeled for Myosin VIIa (green) and SOX2 (red), respectively, in the apical, middle, and basal turns of the cochlea. Actin in the organ of Corti was stained with phalloidin (purple). Arrowheads point to inner hair cells (IHCs), whitehead arrows to outer hair cells (OHCs), and arrows to Deiters cells (DCs). Asterisks mark abnormalities. The scale bar represents 10 μm.

    Article Snippet: Antibodies used were the following: primary antibodies, rabbit anti-Kir4.1 (AB5818 Chemicon), rat anti-ZO-1 (sc-33725, Santa Cruz); rabbit anti-KCNQ1 (sc-20816, Santa Cruz), rabbit anti-Myosin VIIa (25-6790, Proteus), and goat anti-SOX2 (sc-17320, Santa Cruz).

    Techniques: Mutagenesis, Mouse Assay, Immunolabeling, Staining