Structured Review

Abcam rabbit mab rack1
<t>Rack1</t> deficiency induces proliferation of a population of intestinal crypt cells enriched for stem and progenitor cells. A and B : immunofluorescent costaining for Rack1 (green, panels at left ) and Sox9 (red, panels at right ) in the small intestine ( A ) or colon ( B ) of a Rack1 −i/−i mouse (panels at bottom ) and a Rack1 fl/fl littermate (panels at top ). C : costaining for Rack1 (panel at left ) and CD44 (panel at right ) in the small intestine of a Rack1 −i/−i mouse. Scale bar: 100 μm. Data are representative of up to 50 fields examined for each of 3–8 mice.
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1) Product Images from "Rack1 function in intestinal epithelia: regulating crypt cell proliferation and regeneration and promoting differentiation and apoptosis"

Article Title: Rack1 function in intestinal epithelia: regulating crypt cell proliferation and regeneration and promoting differentiation and apoptosis

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

doi: 10.1152/ajpgi.00240.2017

Rack1 deficiency induces proliferation of a population of intestinal crypt cells enriched for stem and progenitor cells. A and B : immunofluorescent costaining for Rack1 (green, panels at left ) and Sox9 (red, panels at right ) in the small intestine ( A ) or colon ( B ) of a Rack1 −i/−i mouse (panels at bottom ) and a Rack1 fl/fl littermate (panels at top ). C : costaining for Rack1 (panel at left ) and CD44 (panel at right ) in the small intestine of a Rack1 −i/−i mouse. Scale bar: 100 μm. Data are representative of up to 50 fields examined for each of 3–8 mice.
Figure Legend Snippet: Rack1 deficiency induces proliferation of a population of intestinal crypt cells enriched for stem and progenitor cells. A and B : immunofluorescent costaining for Rack1 (green, panels at left ) and Sox9 (red, panels at right ) in the small intestine ( A ) or colon ( B ) of a Rack1 −i/−i mouse (panels at bottom ) and a Rack1 fl/fl littermate (panels at top ). C : costaining for Rack1 (panel at left ) and CD44 (panel at right ) in the small intestine of a Rack1 −i/−i mouse. Scale bar: 100 μm. Data are representative of up to 50 fields examined for each of 3–8 mice.

Techniques Used: Mouse Assay

Rack1 deficiency reduces apoptotic activity as measured by cleavage of caspase-3 following radiation injury. A : Rack1 +/−i and Rack1 −i/−i mice received whole body X-ray radiation and were euthanized 72 h later. B : CAG-Cre-ER : Rack1 +/fl and CAG-Cre-ER:Rack1 fl/fl mice treated with tamoxifen (0.2 g/kg) for 5 days ( Rack1 +/− and Rack1 −/− mice, respectively) were irradiated and euthanized 96 h later. Immunofluorescent costaining for Rack1 (green, panels at left ) and cleaved caspase-3 (red, panels in center ) was performed in small intestinal sections. Merged images are shown in the panels at right . Scale bar: 100 μm. Data are representative of up to 50 fields examined from 3 or more mice ( A ) or 2 mice ( B ).
Figure Legend Snippet: Rack1 deficiency reduces apoptotic activity as measured by cleavage of caspase-3 following radiation injury. A : Rack1 +/−i and Rack1 −i/−i mice received whole body X-ray radiation and were euthanized 72 h later. B : CAG-Cre-ER : Rack1 +/fl and CAG-Cre-ER:Rack1 fl/fl mice treated with tamoxifen (0.2 g/kg) for 5 days ( Rack1 +/− and Rack1 −/− mice, respectively) were irradiated and euthanized 96 h later. Immunofluorescent costaining for Rack1 (green, panels at left ) and cleaved caspase-3 (red, panels in center ) was performed in small intestinal sections. Merged images are shown in the panels at right . Scale bar: 100 μm. Data are representative of up to 50 fields examined from 3 or more mice ( A ) or 2 mice ( B ).

Techniques Used: Activity Assay, Mouse Assay, Irradiation

Rack1-deleted epithelia of irradiated (IR) Rack1 −i/−i mice contain areas of high-grade dysplasia. Mice received whole body X-ray radiation, as indicated. Immunofluorescent staining for Rack1 (green, panels at left ) or hematoxylin and eosin analysis of small intestinal sections from a nonirradiated (NR) Rack1 fl/fl mouse ( A ) or an irradiated (IR) Rack1 −i/−i mouse ( C ) and its irradiated Rack1 +/−i littermate control ( B ). Scale bar: 100 μm. Data are representative of up to 50 fields examined from 5 mice.
Figure Legend Snippet: Rack1-deleted epithelia of irradiated (IR) Rack1 −i/−i mice contain areas of high-grade dysplasia. Mice received whole body X-ray radiation, as indicated. Immunofluorescent staining for Rack1 (green, panels at left ) or hematoxylin and eosin analysis of small intestinal sections from a nonirradiated (NR) Rack1 fl/fl mouse ( A ) or an irradiated (IR) Rack1 −i/−i mouse ( C ) and its irradiated Rack1 +/−i littermate control ( B ). Scale bar: 100 μm. Data are representative of up to 50 fields examined from 5 mice.

Techniques Used: Irradiation, Mouse Assay, Staining

Generation of a mouse line with receptor for activated C kinase 1 (Rack1) deleted in intestinal epithelia. A : generation of floxed-Rack1 mice. First panel: Rack1 ( Rk1 ) targeting vector. PL452 vector containing mouse genomic Rack1 fragments (amplified from mouse 129/Sv genomic DNA) with a loxP sequence inserted into intron 1 and a neo gene (flanked by loxP ) inserted into intron 2. 5′-Arm (2.15 kb): Rack1 5′-untranslated region (UTR), exon 1, most of intron 1, and a loxP sequence added to the 3′-end. 3′-Arm (4.29 kb): Rack1 sequence extending from intron 2 to 7. Second panel: the chromosomal Rack1 gene. Third panel: the floxed-Rack1 allele containing the neo gene. Following electroporation of the targeting vector into embryonic stem (ES) cells, ES clones were selected by neo + (positive selection) and tk − (negative selection), and homologous recombinants were identified by PCR and Southern blot analyses. Fourth panel: the floxed-Rack1 allele. To generate a chromosomal copy of floxed-Rack1 in the ES cell line, neo was deleted by transient expression of Cre recombinase. B : Rack1 deletion in the intestine of a mouse carrying vil-Cre and floxed-Rack1 ( vil-Cre : Rack1 +/fl ). Intestinal DNA from a vil-Cre : Rack1 +/fl mouse was examined for Rack1 deletion by PCR analyses. Tail DNA analysis was included as a control. The 2.6-kb fragment corresponds to the floxed-Rack1 allele (exon 2 present), and the 2.2-kb fragment corresponds to the mutant Rack1 allele (exon 2 deleted). p., proximal; d., distal.
Figure Legend Snippet: Generation of a mouse line with receptor for activated C kinase 1 (Rack1) deleted in intestinal epithelia. A : generation of floxed-Rack1 mice. First panel: Rack1 ( Rk1 ) targeting vector. PL452 vector containing mouse genomic Rack1 fragments (amplified from mouse 129/Sv genomic DNA) with a loxP sequence inserted into intron 1 and a neo gene (flanked by loxP ) inserted into intron 2. 5′-Arm (2.15 kb): Rack1 5′-untranslated region (UTR), exon 1, most of intron 1, and a loxP sequence added to the 3′-end. 3′-Arm (4.29 kb): Rack1 sequence extending from intron 2 to 7. Second panel: the chromosomal Rack1 gene. Third panel: the floxed-Rack1 allele containing the neo gene. Following electroporation of the targeting vector into embryonic stem (ES) cells, ES clones were selected by neo + (positive selection) and tk − (negative selection), and homologous recombinants were identified by PCR and Southern blot analyses. Fourth panel: the floxed-Rack1 allele. To generate a chromosomal copy of floxed-Rack1 in the ES cell line, neo was deleted by transient expression of Cre recombinase. B : Rack1 deletion in the intestine of a mouse carrying vil-Cre and floxed-Rack1 ( vil-Cre : Rack1 +/fl ). Intestinal DNA from a vil-Cre : Rack1 +/fl mouse was examined for Rack1 deletion by PCR analyses. Tail DNA analysis was included as a control. The 2.6-kb fragment corresponds to the floxed-Rack1 allele (exon 2 present), and the 2.2-kb fragment corresponds to the mutant Rack1 allele (exon 2 deleted). p., proximal; d., distal.

Techniques Used: Mouse Assay, Plasmid Preparation, Amplification, Sequencing, Electroporation, Clone Assay, Selection, Polymerase Chain Reaction, Southern Blot, Expressing, Mutagenesis

Rack1 deficiency decreases differentiation of crypt cells into enterocyte, goblet, and enteroendocrine cell lineages and promotes differentiation into Paneth cells. Immunofluorescent costaining for Rack1 (Rk1; green, panels at left ) and the differentiation markers (red, panels at right ) sucrase isomaltase (SI; A ), synaptophysin (Syn; B ), lysozyme (Lyso; C ), or mucin-2 (Muc2; D and E ) in the small intestine ( A – D ) or colon ( E ) of Rack1 −i/−i mice. DNA is labeled by Hoechst 33342 fluorescent staining (blue). Scale bar: 100 μm. Data are representative of up to 50 fields examined from 3 or more mice.
Figure Legend Snippet: Rack1 deficiency decreases differentiation of crypt cells into enterocyte, goblet, and enteroendocrine cell lineages and promotes differentiation into Paneth cells. Immunofluorescent costaining for Rack1 (Rk1; green, panels at left ) and the differentiation markers (red, panels at right ) sucrase isomaltase (SI; A ), synaptophysin (Syn; B ), lysozyme (Lyso; C ), or mucin-2 (Muc2; D and E ) in the small intestine ( A – D ) or colon ( E ) of Rack1 −i/−i mice. DNA is labeled by Hoechst 33342 fluorescent staining (blue). Scale bar: 100 μm. Data are representative of up to 50 fields examined from 3 or more mice.

Techniques Used: Mouse Assay, Labeling, Staining

2) Product Images from "Wogonoside inhibits IL-1β induced catabolism and hypertrophy in mouse chondrocyte and ameliorates murine osteoarthritis"

Article Title: Wogonoside inhibits IL-1β induced catabolism and hypertrophy in mouse chondrocyte and ameliorates murine osteoarthritis

Journal: Oncotarget

doi: 10.18632/oncotarget.18374

Wogonoside inhibit extracellular matrix degradation from IL-1β induced mice chondrocyte (A-B) The protein expression of MMP-3, MMP-9, MMP-13 and ADAMTS-5 in chondrocytes treated as above. (C-D) The representative MMP-13 was detected by the immunofluorescence combined with DAPI staining for nuclei (original magnification × 200, scale bar: 50 μm). The data in the figures represent the averages ± S.D. Significant differences between the treatment and control groups are indicated as **P
Figure Legend Snippet: Wogonoside inhibit extracellular matrix degradation from IL-1β induced mice chondrocyte (A-B) The protein expression of MMP-3, MMP-9, MMP-13 and ADAMTS-5 in chondrocytes treated as above. (C-D) The representative MMP-13 was detected by the immunofluorescence combined with DAPI staining for nuclei (original magnification × 200, scale bar: 50 μm). The data in the figures represent the averages ± S.D. Significant differences between the treatment and control groups are indicated as **P

Techniques Used: Mouse Assay, Expressing, Immunofluorescence, Staining

3) Product Images from "Biologic Properties of Mesenchymal Stem Cells Derived From Bone Marrow and Adipose Tissue"

Article Title: Biologic Properties of Mesenchymal Stem Cells Derived From Bone Marrow and Adipose Tissue

Journal:

doi: 10.1002/jcb.20904

Transcription factor mRNA and expression in MSCs. Total cellular RNA analyzed by RT-PCR for Oct-4, Sox-2, and Rex-1 mRNA expression ( A ). The cell lysate from MSC types at different passages were subjected to Western blot analysis using Oct-4 and Sox-2
Figure Legend Snippet: Transcription factor mRNA and expression in MSCs. Total cellular RNA analyzed by RT-PCR for Oct-4, Sox-2, and Rex-1 mRNA expression ( A ). The cell lysate from MSC types at different passages were subjected to Western blot analysis using Oct-4 and Sox-2

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

Oct-4 and Sox-2 localization in MSCs. Immunohistochemical staining of rBMSCs ( A ), hBMSCs ( B ), rASCs ( C ), and hASCs ( D ). Cells were fixed, permeabilized, and stained to visualize Oct-4 and Sox-2. Distribution and localization of these transcription factors
Figure Legend Snippet: Oct-4 and Sox-2 localization in MSCs. Immunohistochemical staining of rBMSCs ( A ), hBMSCs ( B ), rASCs ( C ), and hASCs ( D ). Cells were fixed, permeabilized, and stained to visualize Oct-4 and Sox-2. Distribution and localization of these transcription factors

Techniques Used: Immunohistochemistry, Staining

4) Product Images from "Diabetic Vascular Calcification Mediated by the Collagen Receptor Discoidin Domain Receptor 1 via the Phosphoinositide 3-Kinase/Akt/Runt-Related Transcription Factor 2 Signaling Axis"

Article Title: Diabetic Vascular Calcification Mediated by the Collagen Receptor Discoidin Domain Receptor 1 via the Phosphoinositide 3-Kinase/Akt/Runt-Related Transcription Factor 2 Signaling Axis

Journal: Arteriosclerosis, thrombosis, and vascular biology

doi: 10.1161/ATVBAHA.118.311238

Discoidin domain receptor 1 (DDR1) mediates vascular smooth muscle cell (VSMC) calcification through the phosphoinositide 3-kinase (PI3K)/Akt signaling axis. Ddr1 +/+ and Ddr1 −/− VSMCs were cultured in calcifying media for 12 d and treated daily with dimethyl sulfoxide (Con.), insulin (Ins.), or wortmannin (Wort.) ( A ). Insulin stimulated VSMC calcification while wortmannin attenuated calcification. DDR1 immunoprecipitation was performed on cell lysates from Ddr1 +/+ VSMCs treated with insulin, wortmannin, or type-I collagen (Col-1) ( B ). Treatment with Col-1 led to increased DDR1 activation (pY), and Col-1 or insulin treatment resulted in increased association of DDR1 with PI3K subunits (p110β and p85). Wortmannin attenuated this interaction.
Figure Legend Snippet: Discoidin domain receptor 1 (DDR1) mediates vascular smooth muscle cell (VSMC) calcification through the phosphoinositide 3-kinase (PI3K)/Akt signaling axis. Ddr1 +/+ and Ddr1 −/− VSMCs were cultured in calcifying media for 12 d and treated daily with dimethyl sulfoxide (Con.), insulin (Ins.), or wortmannin (Wort.) ( A ). Insulin stimulated VSMC calcification while wortmannin attenuated calcification. DDR1 immunoprecipitation was performed on cell lysates from Ddr1 +/+ VSMCs treated with insulin, wortmannin, or type-I collagen (Col-1) ( B ). Treatment with Col-1 led to increased DDR1 activation (pY), and Col-1 or insulin treatment resulted in increased association of DDR1 with PI3K subunits (p110β and p85). Wortmannin attenuated this interaction.

Techniques Used: Cell Culture, Immunoprecipitation, Activation Assay

5) Product Images from "Cell viability assessed in a reproducible model of human osteoblasts derived from human adipose-derived stem cells"

Article Title: Cell viability assessed in a reproducible model of human osteoblasts derived from human adipose-derived stem cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0194847

Analysis of hASC markers. (A) CD90. (B) CD105. (C) Nanog. (D) Sox-2. (E) STRO-1. (F) CD45RO. (G) CD117. (H) Quantification of expression of positive and negative markers. Graphs show the expression of different markers in hASCs. The expression of the markers was assessed using flow cytometry by means of the intensity of the emitted fluorescence (FL1 and FL2). The expression of the positive markers, CD90, CD105, and STRO-1 (surface markers mesenchymal) and Nanog and Sox-2 (pluripotency markers) is compared to the decreased expression of negative markers (CD45RO and CD117). Data are represented as mean ± standard deviation. The statistical analyses were obtained using an ANOVA followed by Dunnett’s test with P
Figure Legend Snippet: Analysis of hASC markers. (A) CD90. (B) CD105. (C) Nanog. (D) Sox-2. (E) STRO-1. (F) CD45RO. (G) CD117. (H) Quantification of expression of positive and negative markers. Graphs show the expression of different markers in hASCs. The expression of the markers was assessed using flow cytometry by means of the intensity of the emitted fluorescence (FL1 and FL2). The expression of the positive markers, CD90, CD105, and STRO-1 (surface markers mesenchymal) and Nanog and Sox-2 (pluripotency markers) is compared to the decreased expression of negative markers (CD45RO and CD117). Data are represented as mean ± standard deviation. The statistical analyses were obtained using an ANOVA followed by Dunnett’s test with P

Techniques Used: Expressing, Flow Cytometry, Cytometry, Fluorescence, Standard Deviation

6) Product Images from "B7-H1 is correlated with malignancy-grade gliomas but is not expressed exclusively on tumor stem-like cells"

Article Title: B7-H1 is correlated with malignancy-grade gliomas but is not expressed exclusively on tumor stem-like cells

Journal: Neuro-Oncology

doi: 10.1215/15228517-2009-014

CD133 + and CD133 − glioma xenografts (BT1) both showed histological features of primary glioblastoma and expressed similar levels of B7-H1 protein, but CD133 + cells had more tumor growth capability and a higher proliferation index. (A) Sagittal contrast-enhanced MR image revealed temporal space-occupying tumor with heterogeneous enhancement. Hematoxylin and eosin (H E) staining showed histological features of glioblastoma multiforme (GBM). Glial fibrillary acidic protein (GFAP), Ki67, doublecortin (DCX), and nestin were expressed by the primary tumor. (B) Tumors formed in severe combined immunodeficiency mice after subcutaneously inoculation of 1 × 10 7 CD133 + or CD133 − cells in 27 days (left three panels). The adjacent pleura and lung tissue were infiltrated with CD133 + cells (arrow). CD133 + cells had more tumor growth capability based on the secondary tumor size. B7-H1 was expressed at the similar degree in secondary glioma cells (right). Red histogram, B7-H1 expression in CD133 − xenograft cells (25.65%); green histogram, B7-H1 expression in CD133 + xenograft cells (24.62%); black histogram, isotype control. (C) CD133 − or CD133 + cells could also initiate gliomas in 30 days when implanted into the right striatum. However, the latter had enhanced tumor growth and vascularity (left). CD133 + cell–induced glioblastoma had a higher proliferation index compared to the CD133 − xenografts (middle and right, p
Figure Legend Snippet: CD133 + and CD133 − glioma xenografts (BT1) both showed histological features of primary glioblastoma and expressed similar levels of B7-H1 protein, but CD133 + cells had more tumor growth capability and a higher proliferation index. (A) Sagittal contrast-enhanced MR image revealed temporal space-occupying tumor with heterogeneous enhancement. Hematoxylin and eosin (H E) staining showed histological features of glioblastoma multiforme (GBM). Glial fibrillary acidic protein (GFAP), Ki67, doublecortin (DCX), and nestin were expressed by the primary tumor. (B) Tumors formed in severe combined immunodeficiency mice after subcutaneously inoculation of 1 × 10 7 CD133 + or CD133 − cells in 27 days (left three panels). The adjacent pleura and lung tissue were infiltrated with CD133 + cells (arrow). CD133 + cells had more tumor growth capability based on the secondary tumor size. B7-H1 was expressed at the similar degree in secondary glioma cells (right). Red histogram, B7-H1 expression in CD133 − xenograft cells (25.65%); green histogram, B7-H1 expression in CD133 + xenograft cells (24.62%); black histogram, isotype control. (C) CD133 − or CD133 + cells could also initiate gliomas in 30 days when implanted into the right striatum. However, the latter had enhanced tumor growth and vascularity (left). CD133 + cell–induced glioblastoma had a higher proliferation index compared to the CD133 − xenografts (middle and right, p

Techniques Used: Staining, Mouse Assay, Expressing

The effect of interferon-γ (IFN-γ) on B7-H1 expression in CD133 + and CD133 − cells. (A) Representative example of flow cytometric (FCM) analysis for B7-H1 and CD133 expression in tumor BT1. Time points shown are before (day 0) or after (day 2) incubation with 100 ng/ml IFN-γ. (B) Percentage of CD133 + cells (left) and CD133 − cells (right) expressing B7-H1 were determined by FCM analysis before and after IFN-γ treatment. (C) B7-H1 expression in either CD133 + or CD133 − tumor subpopulations was upregulated by IFN-γ treatment after 48 h (left, mean ± SD, n = 8; * p
Figure Legend Snippet: The effect of interferon-γ (IFN-γ) on B7-H1 expression in CD133 + and CD133 − cells. (A) Representative example of flow cytometric (FCM) analysis for B7-H1 and CD133 expression in tumor BT1. Time points shown are before (day 0) or after (day 2) incubation with 100 ng/ml IFN-γ. (B) Percentage of CD133 + cells (left) and CD133 − cells (right) expressing B7-H1 were determined by FCM analysis before and after IFN-γ treatment. (C) B7-H1 expression in either CD133 + or CD133 − tumor subpopulations was upregulated by IFN-γ treatment after 48 h (left, mean ± SD, n = 8; * p

Techniques Used: Expressing, Flow Cytometry, Incubation

B7-H1 can be expressed by brain tumor stem-like cells and predominantly by nondividing tumor cells. (A) Double immunolabeling performed with antibodies (Abs) against B7-H1 and against neural stem/progenitor cell markers nestin (left, red), CD133 (top right, red), TUC-4 (middle right, red), and doublecortin (DCX; bottom right, red). Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI; blue). Arrow shows large B7-H1 + tumor cell with marked nuclear atypia. (B) Double immunostaining with anti-B7-H1 and anti-Ki67 antibodies reveal predominant B7-H1 expression on Ki67 − cells (left). Flow cytometric analysis confirmed this finding (right top), and B7-H1 levels were significantly different between Ki67 + cells and Ki67 − cells (bottom right; mean ± SD, n = 8; * p
Figure Legend Snippet: B7-H1 can be expressed by brain tumor stem-like cells and predominantly by nondividing tumor cells. (A) Double immunolabeling performed with antibodies (Abs) against B7-H1 and against neural stem/progenitor cell markers nestin (left, red), CD133 (top right, red), TUC-4 (middle right, red), and doublecortin (DCX; bottom right, red). Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI; blue). Arrow shows large B7-H1 + tumor cell with marked nuclear atypia. (B) Double immunostaining with anti-B7-H1 and anti-Ki67 antibodies reveal predominant B7-H1 expression on Ki67 − cells (left). Flow cytometric analysis confirmed this finding (right top), and B7-H1 levels were significantly different between Ki67 + cells and Ki67 − cells (bottom right; mean ± SD, n = 8; * p

Techniques Used: Immunolabeling, Double Immunostaining, Expressing, Flow Cytometry

7) Product Images from "Stemness and inducing differentiation of small cell lung cancer NCI-H446 cells"

Article Title: Stemness and inducing differentiation of small cell lung cancer NCI-H446 cells

Journal: Cell Death & Disease

doi: 10.1038/cddis.2013.152

Induced neurogenic differentiation of NCI-H446 cells. After treatment with TSA, NCI-H446 cells changed into neuron-like appearance with many neurites interconnected as a network ( a1 , control; a2 – a4 , TSA treated for 2, 5, and 7 days, respectively). The differentiated cancer cells were strongly positive for immunofluorescence staining of neuron markers BM88 ( b1 , control; b2 – b4 , TSA treated for 2, 5, and 7 days, respectively) and NF-200 ( c1 , control; c2 – c4 , TSA treated for 2, 5, and 7 days, respectively). Treatment with TSA could inhibit proliferating of the cancer cells, so that the number of these cells was decreased gradually due to death of differentiated cells ( d1 : in control group, most nuclei of cancer cells were positive for Ki67; d2 – d4 : after treatment with TSA for 2, 5, and 7 days, respectively, fewer and fewer nuclei of the cells were positive). After treatment with TSA, the autophagic cells stained for the autophagy marker Beclin-1 were increased gradually ( e1 , control; e2 – e4 , TSA treated for 2, 5, and 7 days, respectively). TUNEL staining showed that treatment with TSA could induce the cancer cells to undergo apoptosis ( f1 : in control group, fewer nuclei of cancer cells were positive for TUNEL staining; f2 – f4 : after treatment with TSA for 2, 5, and 7 days, respectively, more and more nuclei of the cells were positive, meanwhile, the number of total cells was decreased gradually due to apoptosis). ( g ) The proliferation index and apoptotic index indicated that the cancer cells ceased proliferation and underwent apoptosis. ( h ) Western blotting showed that TSA-treated cancer cells overexpressed NF-200 and BM88 (CEND1). The level of acetyl-H3K9 in these cells was raised due to inhibition of HDACs by TSA as well. In addition, in these differentiated cells the increasing accumulations of autophagy-related proteins, Atg7 and Beclin-1, which were cleaved, and the conversion of LC3-I to LC3-II were also detected; meanwhile, the levels of pro-apoptotic proteins, Bax, pro-caspase-3, and cleaved caspase-3, were raised. ( i ) However, the levels of Bcl-2 and Ki67 were decreased gradually. ( a1 – a4 ) The phase contrast images. ( b1 – e4 ) Immunofluorescence staining with Cy3, counterstaining nuclei with Hoechst 33342. ( f1 – f4 ) Visualizing the apotoptic nuclei with DAB by TUNEL staining. Scale bar, 100 μ m
Figure Legend Snippet: Induced neurogenic differentiation of NCI-H446 cells. After treatment with TSA, NCI-H446 cells changed into neuron-like appearance with many neurites interconnected as a network ( a1 , control; a2 – a4 , TSA treated for 2, 5, and 7 days, respectively). The differentiated cancer cells were strongly positive for immunofluorescence staining of neuron markers BM88 ( b1 , control; b2 – b4 , TSA treated for 2, 5, and 7 days, respectively) and NF-200 ( c1 , control; c2 – c4 , TSA treated for 2, 5, and 7 days, respectively). Treatment with TSA could inhibit proliferating of the cancer cells, so that the number of these cells was decreased gradually due to death of differentiated cells ( d1 : in control group, most nuclei of cancer cells were positive for Ki67; d2 – d4 : after treatment with TSA for 2, 5, and 7 days, respectively, fewer and fewer nuclei of the cells were positive). After treatment with TSA, the autophagic cells stained for the autophagy marker Beclin-1 were increased gradually ( e1 , control; e2 – e4 , TSA treated for 2, 5, and 7 days, respectively). TUNEL staining showed that treatment with TSA could induce the cancer cells to undergo apoptosis ( f1 : in control group, fewer nuclei of cancer cells were positive for TUNEL staining; f2 – f4 : after treatment with TSA for 2, 5, and 7 days, respectively, more and more nuclei of the cells were positive, meanwhile, the number of total cells was decreased gradually due to apoptosis). ( g ) The proliferation index and apoptotic index indicated that the cancer cells ceased proliferation and underwent apoptosis. ( h ) Western blotting showed that TSA-treated cancer cells overexpressed NF-200 and BM88 (CEND1). The level of acetyl-H3K9 in these cells was raised due to inhibition of HDACs by TSA as well. In addition, in these differentiated cells the increasing accumulations of autophagy-related proteins, Atg7 and Beclin-1, which were cleaved, and the conversion of LC3-I to LC3-II were also detected; meanwhile, the levels of pro-apoptotic proteins, Bax, pro-caspase-3, and cleaved caspase-3, were raised. ( i ) However, the levels of Bcl-2 and Ki67 were decreased gradually. ( a1 – a4 ) The phase contrast images. ( b1 – e4 ) Immunofluorescence staining with Cy3, counterstaining nuclei with Hoechst 33342. ( f1 – f4 ) Visualizing the apotoptic nuclei with DAB by TUNEL staining. Scale bar, 100 μ m

Techniques Used: Immunofluorescence, Staining, Marker, TUNEL Assay, Western Blot, Inhibition

Induced osteogenic differentiation of NCI-H446 cells. After cultured in osteogenic induction medium, the cancer cells changed into bigger multiform osteoblast-like cells. The osteoblast-like cancer cells showed strong activity of alkaline phosphatase ( a1 , control group; a2 , inducing for 3 days; a3 , inducing for 1 week; and a4 , inducing for 2 weeks). The Alizarin Red S staining showed the mineralized bone nodules on the surface of the osteocyte-like cancer cells ( b1 , control group; b2 , inducing for 1 week; b3 , inducing for 2 weeks; and b4 , inducing for 3 weeks). The inhibitor of Sirt1/2, cambinol, could inhibit osteogenic differentiation, however, the agonist for Sirt1, resveratrol, could promote osteogenic differentiation ( c1 , cultured in DMEM medium containing 10% FBS for 1 week; c2 , cultured in osteogenic indution medium containing 100 μ M cambinol for 2 weeks; c3 , cultured in osteogenic indution medium for 2 weeks; and c4 , cultured in osteogenic indution medium containing 100 μ M resveratrol for 2 weeks). Western blotting showed that after inducing differentiation, the levels of autophagy-related proteins (Atg7 and Beclin), were increased, and these proteins were cleaved dynamicly. LC3-I was processed into LC3-II as the indicator of autophagy, in parallel to changing levels of the apoptosis markers (caspase-3, Bax, and Bcl-2). Meanwile, the bone matrix proteins (collagen-I and osteocalcin) were increased gradually ( d ). During the differentiation process, the osteogenic regulatory proteins (Runx2 and Foxo3a) were upregulated, whereas the adipogenic regulatory protein PPAR γ was downregulated. Cambinol could inhibit expressions of the osteogenic regulatory proteins, and resveratrol could promote expressions of these proteins. ( e ) The expression of Sirtuin1 was changed gently; however, the activity of Sirtuin1/2 showed changing obviously by detection of the actylated tubulin- α . Scale bar, 50 μ m
Figure Legend Snippet: Induced osteogenic differentiation of NCI-H446 cells. After cultured in osteogenic induction medium, the cancer cells changed into bigger multiform osteoblast-like cells. The osteoblast-like cancer cells showed strong activity of alkaline phosphatase ( a1 , control group; a2 , inducing for 3 days; a3 , inducing for 1 week; and a4 , inducing for 2 weeks). The Alizarin Red S staining showed the mineralized bone nodules on the surface of the osteocyte-like cancer cells ( b1 , control group; b2 , inducing for 1 week; b3 , inducing for 2 weeks; and b4 , inducing for 3 weeks). The inhibitor of Sirt1/2, cambinol, could inhibit osteogenic differentiation, however, the agonist for Sirt1, resveratrol, could promote osteogenic differentiation ( c1 , cultured in DMEM medium containing 10% FBS for 1 week; c2 , cultured in osteogenic indution medium containing 100 μ M cambinol for 2 weeks; c3 , cultured in osteogenic indution medium for 2 weeks; and c4 , cultured in osteogenic indution medium containing 100 μ M resveratrol for 2 weeks). Western blotting showed that after inducing differentiation, the levels of autophagy-related proteins (Atg7 and Beclin), were increased, and these proteins were cleaved dynamicly. LC3-I was processed into LC3-II as the indicator of autophagy, in parallel to changing levels of the apoptosis markers (caspase-3, Bax, and Bcl-2). Meanwile, the bone matrix proteins (collagen-I and osteocalcin) were increased gradually ( d ). During the differentiation process, the osteogenic regulatory proteins (Runx2 and Foxo3a) were upregulated, whereas the adipogenic regulatory protein PPAR γ was downregulated. Cambinol could inhibit expressions of the osteogenic regulatory proteins, and resveratrol could promote expressions of these proteins. ( e ) The expression of Sirtuin1 was changed gently; however, the activity of Sirtuin1/2 showed changing obviously by detection of the actylated tubulin- α . Scale bar, 50 μ m

Techniques Used: Cell Culture, Activity Assay, Staining, Western Blot, Expressing

8) Product Images from "Inducing goat pluripotent stem cells with four transcription factor mRNAs that activate endogenous promoters"

Article Title: Inducing goat pluripotent stem cells with four transcription factor mRNAs that activate endogenous promoters

Journal: BMC Biotechnology

doi: 10.1186/s12896-017-0336-7

Expression profiles of the derived goat iPSCs.  a  the properties of goat iPS1 cell line were evaluated with ESCs markers, including OCT4, SOX2, KLF 4, NANOG, CDX2, REX, SSEA-1, TRA-1-60 and TRA-1-81. All these markers were positively stained in the goat iPS cell line. GEFs without transfections were used as the blank control, which did not express ESCs markers. Immunofluorescence staining of goat iPS1 cells without primary antibodies was used as the negative control. Scale bar: 50 μm.  b  qRT-PCR analysis confirmed that the goat iPS1 cell line express a variety of ESCs marker genes, including Oct4, Sox2, Nanog, Dax1 and Gdf3. The expression of DNA methyltranferase Dnmt3b and DNA dymethyltransferase TET1, 2, 3 were also detected with qRT-PCR.  c  Quantitative evaluation of Oct4, Sox2, and Nanog expression in different passages of goat iPS1 cell line. Up: Oct4, Middle: Sox2, Down: Nanog
Figure Legend Snippet: Expression profiles of the derived goat iPSCs. a the properties of goat iPS1 cell line were evaluated with ESCs markers, including OCT4, SOX2, KLF 4, NANOG, CDX2, REX, SSEA-1, TRA-1-60 and TRA-1-81. All these markers were positively stained in the goat iPS cell line. GEFs without transfections were used as the blank control, which did not express ESCs markers. Immunofluorescence staining of goat iPS1 cells without primary antibodies was used as the negative control. Scale bar: 50 μm. b qRT-PCR analysis confirmed that the goat iPS1 cell line express a variety of ESCs marker genes, including Oct4, Sox2, Nanog, Dax1 and Gdf3. The expression of DNA methyltranferase Dnmt3b and DNA dymethyltransferase TET1, 2, 3 were also detected with qRT-PCR. c Quantitative evaluation of Oct4, Sox2, and Nanog expression in different passages of goat iPS1 cell line. Up: Oct4, Middle: Sox2, Down: Nanog

Techniques Used: Expressing, Derivative Assay, Staining, Transfection, Immunofluorescence, Negative Control, Quantitative RT-PCR, Marker

9) Product Images from "Acid Ceramidase Maintains the Chondrogenic Phenotype of Expanded Primary Chondrocytes and Improves the Chondrogenic Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells"

Article Title: Acid Ceramidase Maintains the Chondrogenic Phenotype of Expanded Primary Chondrocytes and Improves the Chondrogenic Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0062715

Horse bone marrow cells were isolated and expanded for 3 weeks in standard culture media (without rAC) to prepare homogenous populations of MSCs. They were then placed into chondrocyte differentiation media containing TGF-beta1, but with or without rAC. A) Pellet cultures were grown for 3 weeks to prepare chondrocytes, and then fixed and analyzed by Alcian Blue (left panel) and Safranin-O (right panel) staining. Note the smaller, more diffuse pellets in the absence of rAC. Pellets were also submitted to immunostaining against B) Sox9, C) Aggrecan, or D) Collagen 2A1. Note the higher expression intensity of Sox9, Aggrecan and Collagen 2A1 in the pellets. E) Pellets were also subjected to an immunostaining against Collagen 10. Note the diminished expression of Collagen 10 in pellets exposed to rAC. DAPI (blue left panel) indicates nuclei, and the right panel (red) indicates Sox9, Aggrecan, Collagen 2A1, or Collagen 10, respectively. Merged images are to the right. Scale bars: 50 µm. * = p
Figure Legend Snippet: Horse bone marrow cells were isolated and expanded for 3 weeks in standard culture media (without rAC) to prepare homogenous populations of MSCs. They were then placed into chondrocyte differentiation media containing TGF-beta1, but with or without rAC. A) Pellet cultures were grown for 3 weeks to prepare chondrocytes, and then fixed and analyzed by Alcian Blue (left panel) and Safranin-O (right panel) staining. Note the smaller, more diffuse pellets in the absence of rAC. Pellets were also submitted to immunostaining against B) Sox9, C) Aggrecan, or D) Collagen 2A1. Note the higher expression intensity of Sox9, Aggrecan and Collagen 2A1 in the pellets. E) Pellets were also subjected to an immunostaining against Collagen 10. Note the diminished expression of Collagen 10 in pellets exposed to rAC. DAPI (blue left panel) indicates nuclei, and the right panel (red) indicates Sox9, Aggrecan, Collagen 2A1, or Collagen 10, respectively. Merged images are to the right. Scale bars: 50 µm. * = p

Techniques Used: Isolation, Staining, Immunostaining, Expressing

Rat articular chondrocytes were obtained from femurs and grown for 3 weeks in monolayer cultures using standard culture medium with or without recombinant human AC (rAC 200 U/ml). rAC was added once at the initiation of the cultures. At the end of the 3-week expansion period, the cells were harvested and analyzed. A) Total cell counts (left) revealed no differences in the presence of rAC. Western blotting for two apoptosis markers (Bax and PARP) similarly revealed no differences. B) The expanded chondrocytes were analyzed by western blots for several important chondrogenic markers, including collagens 1A2 and 2A1, aggrecan, Sox9, FGF2, and TGF-beta1. Note that in all cases these chondrocyte markers were elevated in the cells treated with rAC. In contrast to collagens 2A1 and 1A2, collagen 10 expression, a marker of hypertrophy, was lowered by rAC treatment. C) Horse articular chondrocytes were obtained surgically from femoral heads and frozen. The frozen cells were then recovered and grown in monolayer cultures for 3 weeks without rAC. At 3 weeks the cells were passaged and re-plated at a density of 1×10 6 /cm 2 , and then grown for an additional 1 week with or without rAC. At the end of this 1-week growth period the cells were analyzed by western blots, revealing that the expression of two chondrogenic markers, aggrecan and Sox9, were highly elevated in the rAC-treated horse cells. Bax expression also was reduced in the rAC cells, suggesting a reduction in apoptosis by rAC treatment. All experiments have been repeated at least 3×. Images are representative from individual experiments.
Figure Legend Snippet: Rat articular chondrocytes were obtained from femurs and grown for 3 weeks in monolayer cultures using standard culture medium with or without recombinant human AC (rAC 200 U/ml). rAC was added once at the initiation of the cultures. At the end of the 3-week expansion period, the cells were harvested and analyzed. A) Total cell counts (left) revealed no differences in the presence of rAC. Western blotting for two apoptosis markers (Bax and PARP) similarly revealed no differences. B) The expanded chondrocytes were analyzed by western blots for several important chondrogenic markers, including collagens 1A2 and 2A1, aggrecan, Sox9, FGF2, and TGF-beta1. Note that in all cases these chondrocyte markers were elevated in the cells treated with rAC. In contrast to collagens 2A1 and 1A2, collagen 10 expression, a marker of hypertrophy, was lowered by rAC treatment. C) Horse articular chondrocytes were obtained surgically from femoral heads and frozen. The frozen cells were then recovered and grown in monolayer cultures for 3 weeks without rAC. At 3 weeks the cells were passaged and re-plated at a density of 1×10 6 /cm 2 , and then grown for an additional 1 week with or without rAC. At the end of this 1-week growth period the cells were analyzed by western blots, revealing that the expression of two chondrogenic markers, aggrecan and Sox9, were highly elevated in the rAC-treated horse cells. Bax expression also was reduced in the rAC cells, suggesting a reduction in apoptosis by rAC treatment. All experiments have been repeated at least 3×. Images are representative from individual experiments.

Techniques Used: Recombinant, Western Blot, Expressing, Marker

Primary human chondrocytes obtained from the femoral head of a 74-year-old woman with OA were expanded for 3 weeks in monolayer culture with or without rAC added to the culture media (DMEM +10% FBS, rAC added once at the initiation of the culture; 170 U/ml AC). A) mRNA expression of several chondrogenic markers (collagen 2A1 [Col2a1], TGF-beta1 [TGF], Sox9 and aggrecan [ACAN]) were analyzed by quantitative RT-PCR. Note the positive influence of rAC on the expression of these markers. The expression of collagen 10 was unchanged in these cultures. B) The same experiment was repeated on a second set of cells from a patient with OA. RT-PCR analysis was performed 3×. Consistent with the results obtained with rat and equine chondrocytes ( Fig. 2 ), no significant differences in the number of cells could be found (right panels). * = p
Figure Legend Snippet: Primary human chondrocytes obtained from the femoral head of a 74-year-old woman with OA were expanded for 3 weeks in monolayer culture with or without rAC added to the culture media (DMEM +10% FBS, rAC added once at the initiation of the culture; 170 U/ml AC). A) mRNA expression of several chondrogenic markers (collagen 2A1 [Col2a1], TGF-beta1 [TGF], Sox9 and aggrecan [ACAN]) were analyzed by quantitative RT-PCR. Note the positive influence of rAC on the expression of these markers. The expression of collagen 10 was unchanged in these cultures. B) The same experiment was repeated on a second set of cells from a patient with OA. RT-PCR analysis was performed 3×. Consistent with the results obtained with rat and equine chondrocytes ( Fig. 2 ), no significant differences in the number of cells could be found (right panels). * = p

Techniques Used: Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction

10) Product Images from "Stemness and inducing differentiation of small cell lung cancer NCI-H446 cells"

Article Title: Stemness and inducing differentiation of small cell lung cancer NCI-H446 cells

Journal: Cell Death & Disease

doi: 10.1038/cddis.2013.152

The cancer cells in xenograft tumor expressed multilineage stem cell markers. These stem cell markers included cancer stem cell markers CD133 ( a ) and CD44 ( b ), neural stem cell marker Nestin ( c ), MSC marker Vimentin ( d ), metastasis-associated protein MMP-9 ( e ), stem cell transcription factors Sox-2 ( f ), Sall4 ( g ), and c-Myc ( h ), and proliferation cell nuclear antigen Ki67 ( i ). Immunofluorescence staining with Cy3, counterstaining nuclei with Hoechst 33342. Scale bar, 50 μ m
Figure Legend Snippet: The cancer cells in xenograft tumor expressed multilineage stem cell markers. These stem cell markers included cancer stem cell markers CD133 ( a ) and CD44 ( b ), neural stem cell marker Nestin ( c ), MSC marker Vimentin ( d ), metastasis-associated protein MMP-9 ( e ), stem cell transcription factors Sox-2 ( f ), Sall4 ( g ), and c-Myc ( h ), and proliferation cell nuclear antigen Ki67 ( i ). Immunofluorescence staining with Cy3, counterstaining nuclei with Hoechst 33342. Scale bar, 50 μ m

Techniques Used: Marker, Immunofluorescence, Staining

11) Product Images from "Local injection of mesenchymal stem cells protects testicular torsion-induced germ cell injury"

Article Title: Local injection of mesenchymal stem cells protects testicular torsion-induced germ cell injury

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-015-0079-0

Orbital fat-derived stem cells secreted stem cell factor and supported Leydig cells.  (A)  After orbital fat-derived stem cell (OFSC) injection, stem cell factor (SCF) was abundant in damaged testicular tissue. Human (h) immunoglobulin (IgG) and beta-2 microglobulin (β2M), two probes for human cells, were not detectable in  (B)  the sham operation group (Ctrl) and  (C)  the torsion–detorsion group (T/D).  (D)  Most hIgG-expressing and hβ2M-expressing cells could be detectable in the space between the seminiferous tubules.  (E)  Some of the human cells differentiated into P450-expressing cells and  (F)  very few human cells differentiated into sex determining region Y-box 9 (Sox-9)-positive cells. DAPI, 4,6-diamidino-2-phenylindole.
Figure Legend Snippet: Orbital fat-derived stem cells secreted stem cell factor and supported Leydig cells. (A) After orbital fat-derived stem cell (OFSC) injection, stem cell factor (SCF) was abundant in damaged testicular tissue. Human (h) immunoglobulin (IgG) and beta-2 microglobulin (β2M), two probes for human cells, were not detectable in (B) the sham operation group (Ctrl) and (C) the torsion–detorsion group (T/D). (D) Most hIgG-expressing and hβ2M-expressing cells could be detectable in the space between the seminiferous tubules. (E) Some of the human cells differentiated into P450-expressing cells and (F) very few human cells differentiated into sex determining region Y-box 9 (Sox-9)-positive cells. DAPI, 4,6-diamidino-2-phenylindole.

Techniques Used: Derivative Assay, Injection, Expressing

12) Product Images from "Cortical Endogenic Neural Regeneration of Adult Rat after Traumatic Brain Injury"

Article Title: Cortical Endogenic Neural Regeneration of Adult Rat after Traumatic Brain Injury

Journal: PLoS ONE

doi: 10.1371/journal.pone.0070306

The percentage of neural progenitor cells (NPCs), newborn neurons and astrocytes in BrdU + Cells. (A) A statistic diagram for the number of BrdU + cells in the peri-injured cortex at 1, 3, 7, 14 and 28 dpi. The number of BrdU + cells peaked at 7 dpi then decreased. (B) A statistic diagram for the percentage of NPCs, newborn neurons and astrocytes in BrdU + Cells. The percentage of astrocytes (GFAP + /BrdU + ) in newborn cells (BrdU + ) was more than that of newborn neurons (DCX + /BrdU + + MAP-2 + /BrdU + +NeuN + /BrdU + ) or NPCs (nestin + /sox-2 + ) in the corresponding time point. At 28 dpi, almost all newborn cells were astrocytes. * p
Figure Legend Snippet: The percentage of neural progenitor cells (NPCs), newborn neurons and astrocytes in BrdU + Cells. (A) A statistic diagram for the number of BrdU + cells in the peri-injured cortex at 1, 3, 7, 14 and 28 dpi. The number of BrdU + cells peaked at 7 dpi then decreased. (B) A statistic diagram for the percentage of NPCs, newborn neurons and astrocytes in BrdU + Cells. The percentage of astrocytes (GFAP + /BrdU + ) in newborn cells (BrdU + ) was more than that of newborn neurons (DCX + /BrdU + + MAP-2 + /BrdU + +NeuN + /BrdU + ) or NPCs (nestin + /sox-2 + ) in the corresponding time point. At 28 dpi, almost all newborn cells were astrocytes. * p

Techniques Used:

Activated GFAP + /sox-2 + radial glial (RG)-like cells in peri-injured cortex. RG-like cells were detected by GFAP (green) and sox-2 (red) antibody. Arrows denote the GFAP + /sox-2 + cells. (A, C, E, G, I) GFAP + /sox-2 + RG-like cells were observed in the peri-injured cortex at 1, 3, 7, 14 and 28 dpi. (B, D, F, H, J) GFAP + /sox-2 + RG-like cells were not found in the sham group. (K) A statistic diagram for the number of GFAP + /sox-2 + RG-like cells in the peri-injured cortex at 1, 3, 7, 14 and 28 dpi. The number of GFAP + /sox-2 + RG-like cells peaked at 3 dpi then decreased. * p
Figure Legend Snippet: Activated GFAP + /sox-2 + radial glial (RG)-like cells in peri-injured cortex. RG-like cells were detected by GFAP (green) and sox-2 (red) antibody. Arrows denote the GFAP + /sox-2 + cells. (A, C, E, G, I) GFAP + /sox-2 + RG-like cells were observed in the peri-injured cortex at 1, 3, 7, 14 and 28 dpi. (B, D, F, H, J) GFAP + /sox-2 + RG-like cells were not found in the sham group. (K) A statistic diagram for the number of GFAP + /sox-2 + RG-like cells in the peri-injured cortex at 1, 3, 7, 14 and 28 dpi. The number of GFAP + /sox-2 + RG-like cells peaked at 3 dpi then decreased. * p

Techniques Used:

Endogenic nestin + /sox-2 + neural progenitor cells (NPCs) in the peri-injured cortex. NPCs were detected by nestin (green) and sox-2 (red) antibody. Arrows denote the nestin + /sox-2 + cells. (A, C, E, G, I) Nestin + /sox-2 + NPCs were observed in the peri-injured cortex at 1, 3, 7, 14 and 28 dpi. (B, D, F, H, J) No Nestin + /sox-2 + NPCs were found in the cortex of rats in the sham group. (K) A statistic diagram for the number of nestin + /sox-2 + NPCs in the peri-injured cortex at 1, 3, 7, 14 and 28 dpi. The number of Nestin + /sox-2 + NPCs peaked at 3 dpi then decreased. * p
Figure Legend Snippet: Endogenic nestin + /sox-2 + neural progenitor cells (NPCs) in the peri-injured cortex. NPCs were detected by nestin (green) and sox-2 (red) antibody. Arrows denote the nestin + /sox-2 + cells. (A, C, E, G, I) Nestin + /sox-2 + NPCs were observed in the peri-injured cortex at 1, 3, 7, 14 and 28 dpi. (B, D, F, H, J) No Nestin + /sox-2 + NPCs were found in the cortex of rats in the sham group. (K) A statistic diagram for the number of nestin + /sox-2 + NPCs in the peri-injured cortex at 1, 3, 7, 14 and 28 dpi. The number of Nestin + /sox-2 + NPCs peaked at 3 dpi then decreased. * p

Techniques Used:

Endogenic nestin + /sox-2 + neural progenitor cells (NPCs) in the corpus callosum (CC) and posterior periventricle (pPV). NPCs were detected by nestin (green) and sox-2 (red) antibody. Arrows denote the nestin + /sox-2 + cells. (A) At 1 dpi many nestin + /sox-2 + NPCs were observed in the pPV and MCC where was adjacent to pPV. (C, E) At 3 and 7 dpi many nestin + /sox-2 + NPCs emerged in the ICC and LCC, especially in the latter where was adjacent to the peri-injured cortex. (G, I) Nestin + /sox-2 + NPCs were scarcely found in the pPV and CC at 14 and 28 dpi. (B, D, F, H, J) Nestin + /sox-2 + NPCs were not found in the sham group. (K) A statistic diagram for the number of nestin + /sox-2 + NPCs in the MCC, ICC and LCC at 1, 3, 7, 14 and 28 dpi. * p
Figure Legend Snippet: Endogenic nestin + /sox-2 + neural progenitor cells (NPCs) in the corpus callosum (CC) and posterior periventricle (pPV). NPCs were detected by nestin (green) and sox-2 (red) antibody. Arrows denote the nestin + /sox-2 + cells. (A) At 1 dpi many nestin + /sox-2 + NPCs were observed in the pPV and MCC where was adjacent to pPV. (C, E) At 3 and 7 dpi many nestin + /sox-2 + NPCs emerged in the ICC and LCC, especially in the latter where was adjacent to the peri-injured cortex. (G, I) Nestin + /sox-2 + NPCs were scarcely found in the pPV and CC at 14 and 28 dpi. (B, D, F, H, J) Nestin + /sox-2 + NPCs were not found in the sham group. (K) A statistic diagram for the number of nestin + /sox-2 + NPCs in the MCC, ICC and LCC at 1, 3, 7, 14 and 28 dpi. * p

Techniques Used: Immunocytochemistry

13) Product Images from "Isolation and Characterization of Neural Stem Cells from the Rat Inferior Colliculus"

Article Title: Isolation and Characterization of Neural Stem Cells from the Rat Inferior Colliculus

Journal: Stem Cells International

doi: 10.1155/2019/5831240

Neurosphere of the IC after 4 days of growth on a glass coverslip. After two days on PDL- and laminin-coated coverslips, these neurospheres showed an outgrowth of sphere branches with an increase in length over the course of time. Nuclei were stained with DAPI (blue). The neural progenitor cell marker Nestin (green) was positive in cells inside the sphere and in the branches. Progenitor cells within the neurospheres were stained positive for the neuronal migration protein doublecortin (DCX) (red).
Figure Legend Snippet: Neurosphere of the IC after 4 days of growth on a glass coverslip. After two days on PDL- and laminin-coated coverslips, these neurospheres showed an outgrowth of sphere branches with an increase in length over the course of time. Nuclei were stained with DAPI (blue). The neural progenitor cell marker Nestin (green) was positive in cells inside the sphere and in the branches. Progenitor cells within the neurospheres were stained positive for the neuronal migration protein doublecortin (DCX) (red).

Techniques Used: Staining, Marker, Migration

Tissue sections (20 μ m) of IC of PND6 rats with immunohistochemical staining of the progenitor cell markers Nestin (a) and doublecortin (DCX) (b).
Figure Legend Snippet: Tissue sections (20 μ m) of IC of PND6 rats with immunohistochemical staining of the progenitor cell markers Nestin (a) and doublecortin (DCX) (b).

Techniques Used: Immunohistochemistry, Staining

Generation of neural progenitor cells and stem cell division within neurospheres incubated with BrdU (10 μ M) in NSC medium for 24 h on glass coverslips. Mitotic cells were indicated by BrdU incorporation (green). Cell nuclei were stained blue by DAPI. Neural progenitor cell markers could be identified and were stained red. Atoh1 was detected in the nucleus, whereas Sox-2, Nestin, and doublecortin (DCX) showed staining in the somata of the cells.
Figure Legend Snippet: Generation of neural progenitor cells and stem cell division within neurospheres incubated with BrdU (10 μ M) in NSC medium for 24 h on glass coverslips. Mitotic cells were indicated by BrdU incorporation (green). Cell nuclei were stained blue by DAPI. Neural progenitor cell markers could be identified and were stained red. Atoh1 was detected in the nucleus, whereas Sox-2, Nestin, and doublecortin (DCX) showed staining in the somata of the cells.

Techniques Used: Incubation, BrdU Incorporation Assay, Staining

14) Product Images from "Stemness and inducing differentiation of small cell lung cancer NCI-H446 cells"

Article Title: Stemness and inducing differentiation of small cell lung cancer NCI-H446 cells

Journal: Cell Death & Disease

doi: 10.1038/cddis.2013.152

Induced osteogenic differentiation of NCI-H446 cells. After cultured in osteogenic induction medium, the cancer cells changed into bigger multiform osteoblast-like cells. The osteoblast-like cancer cells showed strong activity of alkaline phosphatase ( a1 , control group; a2 , inducing for 3 days; a3 , inducing for 1 week; and a4 , inducing for 2 weeks). The Alizarin Red S staining showed the mineralized bone nodules on the surface of the osteocyte-like cancer cells ( b1 , control group; b2 , inducing for 1 week; b3 , inducing for 2 weeks; and b4 , inducing for 3 weeks). The inhibitor of Sirt1/2, cambinol, could inhibit osteogenic differentiation, however, the agonist for Sirt1, resveratrol, could promote osteogenic differentiation ( c1 , cultured in DMEM medium containing 10% FBS for 1 week; c2 , cultured in osteogenic indution medium containing 100 μ M cambinol for 2 weeks; c3 , cultured in osteogenic indution medium for 2 weeks; and c4 , cultured in osteogenic indution medium containing 100 μ M resveratrol for 2 weeks). Western blotting showed that after inducing differentiation, the levels of autophagy-related proteins (Atg7 and Beclin), were increased, and these proteins were cleaved dynamicly. LC3-I was processed into LC3-II as the indicator of autophagy, in parallel to changing levels of the apoptosis markers (caspase-3, Bax, and Bcl-2). Meanwile, the bone matrix proteins (collagen-I and osteocalcin) were increased gradually ( d ). During the differentiation process, the osteogenic regulatory proteins (Runx2 and Foxo3a) were upregulated, whereas the adipogenic regulatory protein PPAR γ was downregulated. Cambinol could inhibit expressions of the osteogenic regulatory proteins, and resveratrol could promote expressions of these proteins. ( e ) The expression of Sirtuin1 was changed gently; however, the activity of Sirtuin1/2 showed changing obviously by detection of the actylated tubulin- α . Scale bar, 50 μ m
Figure Legend Snippet: Induced osteogenic differentiation of NCI-H446 cells. After cultured in osteogenic induction medium, the cancer cells changed into bigger multiform osteoblast-like cells. The osteoblast-like cancer cells showed strong activity of alkaline phosphatase ( a1 , control group; a2 , inducing for 3 days; a3 , inducing for 1 week; and a4 , inducing for 2 weeks). The Alizarin Red S staining showed the mineralized bone nodules on the surface of the osteocyte-like cancer cells ( b1 , control group; b2 , inducing for 1 week; b3 , inducing for 2 weeks; and b4 , inducing for 3 weeks). The inhibitor of Sirt1/2, cambinol, could inhibit osteogenic differentiation, however, the agonist for Sirt1, resveratrol, could promote osteogenic differentiation ( c1 , cultured in DMEM medium containing 10% FBS for 1 week; c2 , cultured in osteogenic indution medium containing 100 μ M cambinol for 2 weeks; c3 , cultured in osteogenic indution medium for 2 weeks; and c4 , cultured in osteogenic indution medium containing 100 μ M resveratrol for 2 weeks). Western blotting showed that after inducing differentiation, the levels of autophagy-related proteins (Atg7 and Beclin), were increased, and these proteins were cleaved dynamicly. LC3-I was processed into LC3-II as the indicator of autophagy, in parallel to changing levels of the apoptosis markers (caspase-3, Bax, and Bcl-2). Meanwile, the bone matrix proteins (collagen-I and osteocalcin) were increased gradually ( d ). During the differentiation process, the osteogenic regulatory proteins (Runx2 and Foxo3a) were upregulated, whereas the adipogenic regulatory protein PPAR γ was downregulated. Cambinol could inhibit expressions of the osteogenic regulatory proteins, and resveratrol could promote expressions of these proteins. ( e ) The expression of Sirtuin1 was changed gently; however, the activity of Sirtuin1/2 showed changing obviously by detection of the actylated tubulin- α . Scale bar, 50 μ m

Techniques Used: Cell Culture, Activity Assay, Staining, Western Blot, Expressing

15) Product Images from "Regulation of stem-like cancer cells by glutamine through β-catenin pathway mediated by redox signaling"

Article Title: Regulation of stem-like cancer cells by glutamine through β-catenin pathway mediated by redox signaling

Journal: Molecular Cancer

doi: 10.1186/s12943-017-0623-x

Decrease of SP subpopulation in A549 cells treated with hydrogen peroxide. a A549 cells were cultured without or with H 2 O 2 (50 μM) for 48 h, cells were harvested and stained with Hoechst33342 to determine SP cells. b A549 cells were treated without or with H 2 O 2 (50 μM) for 48 h, then RNA was isolated for real-time RT-PCR for analysis of expression of ALDH-1 and SOX-2. β-actin was used as an internal control. c A549 cells was treated with 5 mM aminotriazole (ATZ) for 72 h with or without pretreatment with NAC, cell lysates were subjected to western blotting to measure the expression of ABCG2 and SOX-2. d A549 cells were treated with 5 mM aminotriazole (ATZ) for 48 h, cells were harvested and stained with Hoechst 33342 to determine SP cells. e A549 cells was treated with glutamine-free medium for 72 h with or without pre-treatment of 2 mM NAC, the cells were collected for SP detection. **, p
Figure Legend Snippet: Decrease of SP subpopulation in A549 cells treated with hydrogen peroxide. a A549 cells were cultured without or with H 2 O 2 (50 μM) for 48 h, cells were harvested and stained with Hoechst33342 to determine SP cells. b A549 cells were treated without or with H 2 O 2 (50 μM) for 48 h, then RNA was isolated for real-time RT-PCR for analysis of expression of ALDH-1 and SOX-2. β-actin was used as an internal control. c A549 cells was treated with 5 mM aminotriazole (ATZ) for 72 h with or without pretreatment with NAC, cell lysates were subjected to western blotting to measure the expression of ABCG2 and SOX-2. d A549 cells were treated with 5 mM aminotriazole (ATZ) for 48 h, cells were harvested and stained with Hoechst 33342 to determine SP cells. e A549 cells was treated with glutamine-free medium for 72 h with or without pre-treatment of 2 mM NAC, the cells were collected for SP detection. **, p

Techniques Used: Cell Culture, Staining, Isolation, Quantitative RT-PCR, Expressing, Western Blot

Impact of glutamine on A549 cells clonogenic capacity and the expression of stem-cell associated molecules. a - b A549 cells were cultured in RPMI 1640 medium with or without glutamine or incubated without or with 1 U/ml L-Asparaginase. Colony numbers were counted after 2 weeks of culturing. The quantitative results of 3 independent experiments are shown in bar graphs showing mean ± SD. Images of representative colonies formed were shown in the lower panels. c Representative photographs of A549 cells cultured in medium with or without glutamine for 72 h, original magnification is 400 ×. d Effect of glutamine on expression of genes ABCG2 and SOX-2. A549 cells were cultured in RPMI 1640 with or without glutamine (300 mg/L) for 48 h, and RNA was isolated for real-time RT-PCR for detection of SOX-2 and ABCG2 expression. β-actin was used as an internal control for normalization. e Western blot analysis of ABCG2 and SOX-2. A549 cells were cultured in RPMI 1640 medium with or without glutamine (300 mg/l) for 72 h, and cell lysates were subjected to western blotting to measure the expression of ABCG2, SOX-2 and β-actin. f Effect of L-Asparaginase on expression of genes ABCG2 and SOX-2. A549 cells were cultured in the absence or presence of 1 U/ml L-Asparaginase for 72 h, and RNA was isolated for real-time RT-PCR analysis of SOX-2 and ABCG2. β-actin was used as an internal control. g A549 cells were cultured in RPMI 1640 medium without or with L-Asparaginase (1 U/ml) for indicated time, and cell lysates were subjected to western blotting to measure the expression of ABCG2 and SOX-2. (H, I) Effect of glutamine and L-Asparaginase on ABCG2 expression. A549 cells were cultured in RPMI 1640 medium with or without glutamine ( h ) or incubated without or with 1 U/ml L-Asparaginase ( i ) for the indicated time, and cells were collected and incubated with anti-ABCG2 antibody, detection of membrane protein ABCG2 was measured by flow cytometry analysis. Each Bar represents the mean ± SD of the relative fluorescence intensity from 3 independent experiments. *, p
Figure Legend Snippet: Impact of glutamine on A549 cells clonogenic capacity and the expression of stem-cell associated molecules. a - b A549 cells were cultured in RPMI 1640 medium with or without glutamine or incubated without or with 1 U/ml L-Asparaginase. Colony numbers were counted after 2 weeks of culturing. The quantitative results of 3 independent experiments are shown in bar graphs showing mean ± SD. Images of representative colonies formed were shown in the lower panels. c Representative photographs of A549 cells cultured in medium with or without glutamine for 72 h, original magnification is 400 ×. d Effect of glutamine on expression of genes ABCG2 and SOX-2. A549 cells were cultured in RPMI 1640 with or without glutamine (300 mg/L) for 48 h, and RNA was isolated for real-time RT-PCR for detection of SOX-2 and ABCG2 expression. β-actin was used as an internal control for normalization. e Western blot analysis of ABCG2 and SOX-2. A549 cells were cultured in RPMI 1640 medium with or without glutamine (300 mg/l) for 72 h, and cell lysates were subjected to western blotting to measure the expression of ABCG2, SOX-2 and β-actin. f Effect of L-Asparaginase on expression of genes ABCG2 and SOX-2. A549 cells were cultured in the absence or presence of 1 U/ml L-Asparaginase for 72 h, and RNA was isolated for real-time RT-PCR analysis of SOX-2 and ABCG2. β-actin was used as an internal control. g A549 cells were cultured in RPMI 1640 medium without or with L-Asparaginase (1 U/ml) for indicated time, and cell lysates were subjected to western blotting to measure the expression of ABCG2 and SOX-2. (H, I) Effect of glutamine and L-Asparaginase on ABCG2 expression. A549 cells were cultured in RPMI 1640 medium with or without glutamine ( h ) or incubated without or with 1 U/ml L-Asparaginase ( i ) for the indicated time, and cells were collected and incubated with anti-ABCG2 antibody, detection of membrane protein ABCG2 was measured by flow cytometry analysis. Each Bar represents the mean ± SD of the relative fluorescence intensity from 3 independent experiments. *, p

Techniques Used: Expressing, Cell Culture, Incubation, Isolation, Quantitative RT-PCR, Western Blot, Flow Cytometry, Cytometry, Fluorescence

16) Product Images from "Stemness and inducing differentiation of small cell lung cancer NCI-H446 cells"

Article Title: Stemness and inducing differentiation of small cell lung cancer NCI-H446 cells

Journal: Cell Death & Disease

doi: 10.1038/cddis.2013.152

Induced neurogenic differentiation of NCI-H446 cells. After treatment with TSA, NCI-H446 cells changed into neuron-like appearance with many neurites interconnected as a network ( a1 , control; a2 – a4 , TSA treated for 2, 5, and 7 days, respectively). The differentiated cancer cells were strongly positive for immunofluorescence staining of neuron markers BM88 ( b1 , control; b2 – b4 , TSA treated for 2, 5, and 7 days, respectively) and NF-200 ( c1 , control; c2 – c4 , TSA treated for 2, 5, and 7 days, respectively). Treatment with TSA could inhibit proliferating of the cancer cells, so that the number of these cells was decreased gradually due to death of differentiated cells ( d1 : in control group, most nuclei of cancer cells were positive for Ki67; d2 – d4 : after treatment with TSA for 2, 5, and 7 days, respectively, fewer and fewer nuclei of the cells were positive). After treatment with TSA, the autophagic cells stained for the autophagy marker Beclin-1 were increased gradually ( e1 , control; e2 – e4 , TSA treated for 2, 5, and 7 days, respectively). TUNEL staining showed that treatment with TSA could induce the cancer cells to undergo apoptosis ( f1 : in control group, fewer nuclei of cancer cells were positive for TUNEL staining; f2 – f4 : after treatment with TSA for 2, 5, and 7 days, respectively, more and more nuclei of the cells were positive, meanwhile, the number of total cells was decreased gradually due to apoptosis). ( g ) The proliferation index and apoptotic index indicated that the cancer cells ceased proliferation and underwent apoptosis. ( h ) Western blotting showed that TSA-treated cancer cells overexpressed NF-200 and BM88 (CEND1). The level of acetyl-H3K9 in these cells was raised due to inhibition of HDACs by TSA as well. In addition, in these differentiated cells the increasing accumulations of autophagy-related proteins, Atg7 and Beclin-1, which were cleaved, and the conversion of LC3-I to LC3-II were also detected; meanwhile, the levels of pro-apoptotic proteins, Bax, pro-caspase-3, and cleaved caspase-3, were raised. ( i ) However, the levels of Bcl-2 and Ki67 were decreased gradually. ( a1 – a4 ) The phase contrast images. ( b1 – e4 ) Immunofluorescence staining with Cy3, counterstaining nuclei with Hoechst 33342. ( f1 – f4 ) Visualizing the apotoptic nuclei with DAB by TUNEL staining. Scale bar, 100 μ m
Figure Legend Snippet: Induced neurogenic differentiation of NCI-H446 cells. After treatment with TSA, NCI-H446 cells changed into neuron-like appearance with many neurites interconnected as a network ( a1 , control; a2 – a4 , TSA treated for 2, 5, and 7 days, respectively). The differentiated cancer cells were strongly positive for immunofluorescence staining of neuron markers BM88 ( b1 , control; b2 – b4 , TSA treated for 2, 5, and 7 days, respectively) and NF-200 ( c1 , control; c2 – c4 , TSA treated for 2, 5, and 7 days, respectively). Treatment with TSA could inhibit proliferating of the cancer cells, so that the number of these cells was decreased gradually due to death of differentiated cells ( d1 : in control group, most nuclei of cancer cells were positive for Ki67; d2 – d4 : after treatment with TSA for 2, 5, and 7 days, respectively, fewer and fewer nuclei of the cells were positive). After treatment with TSA, the autophagic cells stained for the autophagy marker Beclin-1 were increased gradually ( e1 , control; e2 – e4 , TSA treated for 2, 5, and 7 days, respectively). TUNEL staining showed that treatment with TSA could induce the cancer cells to undergo apoptosis ( f1 : in control group, fewer nuclei of cancer cells were positive for TUNEL staining; f2 – f4 : after treatment with TSA for 2, 5, and 7 days, respectively, more and more nuclei of the cells were positive, meanwhile, the number of total cells was decreased gradually due to apoptosis). ( g ) The proliferation index and apoptotic index indicated that the cancer cells ceased proliferation and underwent apoptosis. ( h ) Western blotting showed that TSA-treated cancer cells overexpressed NF-200 and BM88 (CEND1). The level of acetyl-H3K9 in these cells was raised due to inhibition of HDACs by TSA as well. In addition, in these differentiated cells the increasing accumulations of autophagy-related proteins, Atg7 and Beclin-1, which were cleaved, and the conversion of LC3-I to LC3-II were also detected; meanwhile, the levels of pro-apoptotic proteins, Bax, pro-caspase-3, and cleaved caspase-3, were raised. ( i ) However, the levels of Bcl-2 and Ki67 were decreased gradually. ( a1 – a4 ) The phase contrast images. ( b1 – e4 ) Immunofluorescence staining with Cy3, counterstaining nuclei with Hoechst 33342. ( f1 – f4 ) Visualizing the apotoptic nuclei with DAB by TUNEL staining. Scale bar, 100 μ m

Techniques Used: Immunofluorescence, Staining, Marker, TUNEL Assay, Western Blot, Inhibition

The generation of clones derived from NCI-H446 cells in anchorage-dependent or -independent conditions. ( A ) The generation of clones in anchorage-dependent condition. The outline of the clones was sharp; the cancer cells in these clones grew in confluent condition: (a) primary clone and (b) subclone. The cells of these clones at different growth stages expressed NCAM: (c) primary clones and (d) subclones. The undifferentiated subclone cells showed very weak immunofluorescence signal for neuron marker NF-200 (e). When treated with ATRA (for 2, 3, and 5 days, respectively), the subclones ceased expansion, and the cells in these clones spread out and differentiated to neuron-like cells with very strong immunofluorescence staining for NF-200 (f). ( B ) The generation of colonies in anchorage-independent condition. Most cells seeded in agarose could generate colonies in anchorage-independent conditions: (a) the colonies grew for 2 weeks and (b) the colonies grew for 3 weeks. The cancer cells in these colonies were positive for NCAM by immunofluorescence staining: (c) one colony grew for 2 weeks and (d) two colonies grew for 3 weeks at different depth, which were fusing. As isolated from agarose and cultured in adherent condition, the spherical colony attached and expanded fast to form adherent monolayer cells, which expressed NCAM as well: (e) an intact colony and expanded cells from it and (f) magnified image of e. Immunofluorescence staining with Cy3, counterstaining nuclei with Hoechst 33342. Scale bar, 20 μ m (a and b); 50 μ m (c–f)
Figure Legend Snippet: The generation of clones derived from NCI-H446 cells in anchorage-dependent or -independent conditions. ( A ) The generation of clones in anchorage-dependent condition. The outline of the clones was sharp; the cancer cells in these clones grew in confluent condition: (a) primary clone and (b) subclone. The cells of these clones at different growth stages expressed NCAM: (c) primary clones and (d) subclones. The undifferentiated subclone cells showed very weak immunofluorescence signal for neuron marker NF-200 (e). When treated with ATRA (for 2, 3, and 5 days, respectively), the subclones ceased expansion, and the cells in these clones spread out and differentiated to neuron-like cells with very strong immunofluorescence staining for NF-200 (f). ( B ) The generation of colonies in anchorage-independent condition. Most cells seeded in agarose could generate colonies in anchorage-independent conditions: (a) the colonies grew for 2 weeks and (b) the colonies grew for 3 weeks. The cancer cells in these colonies were positive for NCAM by immunofluorescence staining: (c) one colony grew for 2 weeks and (d) two colonies grew for 3 weeks at different depth, which were fusing. As isolated from agarose and cultured in adherent condition, the spherical colony attached and expanded fast to form adherent monolayer cells, which expressed NCAM as well: (e) an intact colony and expanded cells from it and (f) magnified image of e. Immunofluorescence staining with Cy3, counterstaining nuclei with Hoechst 33342. Scale bar, 20 μ m (a and b); 50 μ m (c–f)

Techniques Used: Clone Assay, Derivative Assay, Immunofluorescence, Marker, Staining, Isolation, Cell Culture

17) Product Images from "Stemness and inducing differentiation of small cell lung cancer NCI-H446 cells"

Article Title: Stemness and inducing differentiation of small cell lung cancer NCI-H446 cells

Journal: Cell Death & Disease

doi: 10.1038/cddis.2013.152

The cancer cells in xenograft tumor expressed multilineage stem cell markers. These stem cell markers included cancer stem cell markers CD133 ( a ) and CD44 ( b ), neural stem cell marker Nestin ( c ), MSC marker Vimentin ( d ), metastasis-associated protein MMP-9 ( e ), stem cell transcription factors Sox-2 ( f ), Sall4 ( g ), and c-Myc ( h ), and proliferation cell nuclear antigen Ki67 ( i ). Immunofluorescence staining with Cy3, counterstaining nuclei with Hoechst 33342. Scale bar, 50 μ m
Figure Legend Snippet: The cancer cells in xenograft tumor expressed multilineage stem cell markers. These stem cell markers included cancer stem cell markers CD133 ( a ) and CD44 ( b ), neural stem cell marker Nestin ( c ), MSC marker Vimentin ( d ), metastasis-associated protein MMP-9 ( e ), stem cell transcription factors Sox-2 ( f ), Sall4 ( g ), and c-Myc ( h ), and proliferation cell nuclear antigen Ki67 ( i ). Immunofluorescence staining with Cy3, counterstaining nuclei with Hoechst 33342. Scale bar, 50 μ m

Techniques Used: Marker, Immunofluorescence, Staining

The NCI-H446 cells expressed stem cell markers. When cultured on laminin-coated glass coverslips, the cancer cells could attach well and expanded in adherent monolayer on the substrate ( a1 – i1 ). However, as cultured at high cell density, the cancer cells could generate tumorspheres semiattached to culture plates ( a2 – i2 ). These cells could express stem cell markers: ( a ) CD133; ( b ) Sall4; ( c ) Oct4; ( d ) Nestin; ( e ) NCAM; ( f ) S100 β ; ( g ) Vimentin; ( h ) CD44; and ( i ) CD105. Immunofluorescence staining with Cy3, counterstaining nuclei with Hoechst 33342. Scale bar, 50 μ m
Figure Legend Snippet: The NCI-H446 cells expressed stem cell markers. When cultured on laminin-coated glass coverslips, the cancer cells could attach well and expanded in adherent monolayer on the substrate ( a1 – i1 ). However, as cultured at high cell density, the cancer cells could generate tumorspheres semiattached to culture plates ( a2 – i2 ). These cells could express stem cell markers: ( a ) CD133; ( b ) Sall4; ( c ) Oct4; ( d ) Nestin; ( e ) NCAM; ( f ) S100 β ; ( g ) Vimentin; ( h ) CD44; and ( i ) CD105. Immunofluorescence staining with Cy3, counterstaining nuclei with Hoechst 33342. Scale bar, 50 μ m

Techniques Used: Cell Culture, Immunofluorescence, Staining

18) Product Images from "Stemness and inducing differentiation of small cell lung cancer NCI-H446 cells"

Article Title: Stemness and inducing differentiation of small cell lung cancer NCI-H446 cells

Journal: Cell Death & Disease

doi: 10.1038/cddis.2013.152

Induced neurogenic differentiation of NCI-H446 cells. After treatment with TSA, NCI-H446 cells changed into neuron-like appearance with many neurites interconnected as a network ( a1 , control; a2 – a4 , TSA treated for 2, 5, and 7 days, respectively). The differentiated cancer cells were strongly positive for immunofluorescence staining of neuron markers BM88 ( b1 , control; b2 – b4 , TSA treated for 2, 5, and 7 days, respectively) and NF-200 ( c1 , control; c2 – c4 , TSA treated for 2, 5, and 7 days, respectively). Treatment with TSA could inhibit proliferating of the cancer cells, so that the number of these cells was decreased gradually due to death of differentiated cells ( d1 : in control group, most nuclei of cancer cells were positive for Ki67; d2 – d4 : after treatment with TSA for 2, 5, and 7 days, respectively, fewer and fewer nuclei of the cells were positive). After treatment with TSA, the autophagic cells stained for the autophagy marker Beclin-1 were increased gradually ( e1 , control; e2 – e4 , TSA treated for 2, 5, and 7 days, respectively). TUNEL staining showed that treatment with TSA could induce the cancer cells to undergo apoptosis ( f1 : in control group, fewer nuclei of cancer cells were positive for TUNEL staining; f2 – f4 : after treatment with TSA for 2, 5, and 7 days, respectively, more and more nuclei of the cells were positive, meanwhile, the number of total cells was decreased gradually due to apoptosis). ( g ) The proliferation index and apoptotic index indicated that the cancer cells ceased proliferation and underwent apoptosis. ( h ) Western blotting showed that TSA-treated cancer cells overexpressed NF-200 and BM88 (CEND1). The level of acetyl-H3K9 in these cells was raised due to inhibition of HDACs by TSA as well. In addition, in these differentiated cells the increasing accumulations of autophagy-related proteins, Atg7 and Beclin-1, which were cleaved, and the conversion of LC3-I to LC3-II were also detected; meanwhile, the levels of pro-apoptotic proteins, Bax, pro-caspase-3, and cleaved caspase-3, were raised. ( i ) However, the levels of Bcl-2 and Ki67 were decreased gradually. ( a1 – a4 ) The phase contrast images. ( b1 – e4 ) Immunofluorescence staining with Cy3, counterstaining nuclei with Hoechst 33342. ( f1 – f4 ) Visualizing the apotoptic nuclei with DAB by TUNEL staining. Scale bar, 100 μ m
Figure Legend Snippet: Induced neurogenic differentiation of NCI-H446 cells. After treatment with TSA, NCI-H446 cells changed into neuron-like appearance with many neurites interconnected as a network ( a1 , control; a2 – a4 , TSA treated for 2, 5, and 7 days, respectively). The differentiated cancer cells were strongly positive for immunofluorescence staining of neuron markers BM88 ( b1 , control; b2 – b4 , TSA treated for 2, 5, and 7 days, respectively) and NF-200 ( c1 , control; c2 – c4 , TSA treated for 2, 5, and 7 days, respectively). Treatment with TSA could inhibit proliferating of the cancer cells, so that the number of these cells was decreased gradually due to death of differentiated cells ( d1 : in control group, most nuclei of cancer cells were positive for Ki67; d2 – d4 : after treatment with TSA for 2, 5, and 7 days, respectively, fewer and fewer nuclei of the cells were positive). After treatment with TSA, the autophagic cells stained for the autophagy marker Beclin-1 were increased gradually ( e1 , control; e2 – e4 , TSA treated for 2, 5, and 7 days, respectively). TUNEL staining showed that treatment with TSA could induce the cancer cells to undergo apoptosis ( f1 : in control group, fewer nuclei of cancer cells were positive for TUNEL staining; f2 – f4 : after treatment with TSA for 2, 5, and 7 days, respectively, more and more nuclei of the cells were positive, meanwhile, the number of total cells was decreased gradually due to apoptosis). ( g ) The proliferation index and apoptotic index indicated that the cancer cells ceased proliferation and underwent apoptosis. ( h ) Western blotting showed that TSA-treated cancer cells overexpressed NF-200 and BM88 (CEND1). The level of acetyl-H3K9 in these cells was raised due to inhibition of HDACs by TSA as well. In addition, in these differentiated cells the increasing accumulations of autophagy-related proteins, Atg7 and Beclin-1, which were cleaved, and the conversion of LC3-I to LC3-II were also detected; meanwhile, the levels of pro-apoptotic proteins, Bax, pro-caspase-3, and cleaved caspase-3, were raised. ( i ) However, the levels of Bcl-2 and Ki67 were decreased gradually. ( a1 – a4 ) The phase contrast images. ( b1 – e4 ) Immunofluorescence staining with Cy3, counterstaining nuclei with Hoechst 33342. ( f1 – f4 ) Visualizing the apotoptic nuclei with DAB by TUNEL staining. Scale bar, 100 μ m

Techniques Used: Immunofluorescence, Staining, Marker, TUNEL Assay, Western Blot, Inhibition

19) Product Images from "Diabetic Vascular Calcification Mediated by the Collagen Receptor Discoidin Domain Receptor 1 via the Phosphoinositide 3-Kinase/Akt/Runt-Related Transcription Factor 2 Signaling Axis"

Article Title: Diabetic Vascular Calcification Mediated by the Collagen Receptor Discoidin Domain Receptor 1 via the Phosphoinositide 3-Kinase/Akt/Runt-Related Transcription Factor 2 Signaling Axis

Journal: Arteriosclerosis, thrombosis, and vascular biology

doi: 10.1161/ATVBAHA.118.311238

Discoidin domain receptor 1 (DDR1) deletion results in attenuated vascular smooth muscle cell (VSMC) calcification and is accompanied by reduced RUNX2 (runt-related transcription factor 2) nuclear localization and activity in vitro. Ddr1 +/+ and Ddr1 −/− VSMCs were cultured in normal or calcifying media for 2 or 12 d. Alizarin red stain ( A ) and o-cresolphthalein assay ( B ) showed reduced calcification in Ddr1 −/− VSMCs after 12 d in calcifying media. RUNX2 immunostaining revealed reduced nuclear localization of RUNX2 in Ddr1 −/− VSMCs ( C ), which was significant after 2 or 12 d in calcifying media ( D ). Subcellular fractionation of VSMCs showed reduced nuclear RUNX2 levels in Ddr1 −/− VSMCs ( E ) cultured in normal ( F ) and calcifying ( G ) conditions for 2 d. RUNX2 transcriptional activity was reduced in Ddr1 −/− VSMCs after 2 d in calcifying media ( H ). Osteocalcin mRNA was reduced at 2 and 12 d in calcifying media ( I ). α-SMA (smooth muscle α-actin) was increased while cleaved caspase-3 was reduced in Ddr1 −/− compared with Ddr1 +/+ VSMCs after 2-d culture in normal or calcifying media ( J ). B and F – H , Student t test (* P
Figure Legend Snippet: Discoidin domain receptor 1 (DDR1) deletion results in attenuated vascular smooth muscle cell (VSMC) calcification and is accompanied by reduced RUNX2 (runt-related transcription factor 2) nuclear localization and activity in vitro. Ddr1 +/+ and Ddr1 −/− VSMCs were cultured in normal or calcifying media for 2 or 12 d. Alizarin red stain ( A ) and o-cresolphthalein assay ( B ) showed reduced calcification in Ddr1 −/− VSMCs after 12 d in calcifying media. RUNX2 immunostaining revealed reduced nuclear localization of RUNX2 in Ddr1 −/− VSMCs ( C ), which was significant after 2 or 12 d in calcifying media ( D ). Subcellular fractionation of VSMCs showed reduced nuclear RUNX2 levels in Ddr1 −/− VSMCs ( E ) cultured in normal ( F ) and calcifying ( G ) conditions for 2 d. RUNX2 transcriptional activity was reduced in Ddr1 −/− VSMCs after 2 d in calcifying media ( H ). Osteocalcin mRNA was reduced at 2 and 12 d in calcifying media ( I ). α-SMA (smooth muscle α-actin) was increased while cleaved caspase-3 was reduced in Ddr1 −/− compared with Ddr1 +/+ VSMCs after 2-d culture in normal or calcifying media ( J ). B and F – H , Student t test (* P

Techniques Used: Activity Assay, In Vitro, Cell Culture, Staining, Immunostaining, Fractionation

20) Product Images from "The DNA methyl-transferase protein DNMT1 enhances tumor-promoting properties of breast stromal fibroblasts"

Article Title: The DNA methyl-transferase protein DNMT1 enhances tumor-promoting properties of breast stromal fibroblasts

Journal: Oncotarget

doi: 10.18632/oncotarget.23411

DNMT1 downregulation suppresses myofibroblasts CAF-64 cells were transfected with 3 different DNMT1-siRNA sequences (A), (B) and (C) (CAF64-si) and a scrambled sequence was used as control (CAF64-c). (A) Total RNA was extracted and used for qRT-PCR. Error bars represent mean ± S.D. (B) and (E) Whole-cell lysates were prepared from CAF64si and CAF64-c, and then were used for immunoblotting analysis using specific antibodies against the indicated proteins. (C) SFCM from the indicated cells were collected after 24 h and the levels of the indicated proteins were determined by ELISA and were presented in the respective histograms. Error bars indicate mean ± S.D (n=3). * , P
Figure Legend Snippet: DNMT1 downregulation suppresses myofibroblasts CAF-64 cells were transfected with 3 different DNMT1-siRNA sequences (A), (B) and (C) (CAF64-si) and a scrambled sequence was used as control (CAF64-c). (A) Total RNA was extracted and used for qRT-PCR. Error bars represent mean ± S.D. (B) and (E) Whole-cell lysates were prepared from CAF64si and CAF64-c, and then were used for immunoblotting analysis using specific antibodies against the indicated proteins. (C) SFCM from the indicated cells were collected after 24 h and the levels of the indicated proteins were determined by ELISA and were presented in the respective histograms. Error bars indicate mean ± S.D (n=3). * , P

Techniques Used: Transfection, Sequencing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Schematic representation of the IL-6/STAT3/NF-κB feedback loop, and the implication of HuR and DNMT1 See text for more details.
Figure Legend Snippet: Schematic representation of the IL-6/STAT3/NF-κB feedback loop, and the implication of HuR and DNMT1 See text for more details.

Techniques Used:

DNMTl upregulation in CAFs is mediated through HuR-dependent stabilization (A) and (D) Exponentially growing cells were treated with actinomycin D (5 μg/ml) for various periods of time, and total RNA was extracted and the level of the DNMT1 mRNA was assessed using qRT-PCR. The values were determined and normalized against GAPDH. Graphs show the proportion of the DNMT1 mRNA remaining post-treatment, and the dotted lines indicate the DNMT1 mRNA half life, the corresponding values are in brackets. Error bars indicate mean ± S.D (n=3). (B) Whole-cell lysates were prepared from the indicated cells, and used for imrnunoblotting analysis. (C) Total RNA was extracted from CAF-64 cells expressing either HuR-siRNA (CAF64-Hsi) or control siRNA (CAF64-Hc), and the levels of the indicated genes were assessed by qRT-PCR and normalized against GAPDH. Error bars represent mean ± S.D. * , P
Figure Legend Snippet: DNMTl upregulation in CAFs is mediated through HuR-dependent stabilization (A) and (D) Exponentially growing cells were treated with actinomycin D (5 μg/ml) for various periods of time, and total RNA was extracted and the level of the DNMT1 mRNA was assessed using qRT-PCR. The values were determined and normalized against GAPDH. Graphs show the proportion of the DNMT1 mRNA remaining post-treatment, and the dotted lines indicate the DNMT1 mRNA half life, the corresponding values are in brackets. Error bars indicate mean ± S.D (n=3). (B) Whole-cell lysates were prepared from the indicated cells, and used for imrnunoblotting analysis. (C) Total RNA was extracted from CAF-64 cells expressing either HuR-siRNA (CAF64-Hsi) or control siRNA (CAF64-Hc), and the levels of the indicated genes were assessed by qRT-PCR and normalized against GAPDH. Error bars represent mean ± S.D. * , P

Techniques Used: Quantitative RT-PCR, Expressing

Ectopic expression of DNMT1 activates breast stromal fibroblasts TCF-64 cells were transfected with a plasmid bearing the DNMT1 ORF (TCF64-orf) or an empty vector (TCF64-c). (A) Phase contrast images of cells several days post-transfection. Fluid Cell Imaging Station was utilized, scale bar = 50 μM. (B) Whole-cell lysates were prepared from the indicated cells, and were used for immunoblotting analysis using antibodies against the indicated proteins. (C) Total RNA was extracted and the mRNA levels of the indicated genes were assessed using qRT-PCR. Error bars represent mean ± S.D (n=3). * , P
Figure Legend Snippet: Ectopic expression of DNMT1 activates breast stromal fibroblasts TCF-64 cells were transfected with a plasmid bearing the DNMT1 ORF (TCF64-orf) or an empty vector (TCF64-c). (A) Phase contrast images of cells several days post-transfection. Fluid Cell Imaging Station was utilized, scale bar = 50 μM. (B) Whole-cell lysates were prepared from the indicated cells, and were used for immunoblotting analysis using antibodies against the indicated proteins. (C) Total RNA was extracted and the mRNA levels of the indicated genes were assessed using qRT-PCR. Error bars represent mean ± S.D (n=3). * , P

Techniques Used: Expressing, Transfection, Plasmid Preparation, Imaging, Quantitative RT-PCR

Ectopic expression of DNMT1 enhances the paracrine procarcinogenic effects of breast stromal fibroblasts in vitro and in vivo (A) SFCM from the indicated cells were collected after 24 h and the levels of the indicated proteins were determined by ELISA and were presented in the respective histograms. Error bars indicate mean±S.D. ** , P
Figure Legend Snippet: Ectopic expression of DNMT1 enhances the paracrine procarcinogenic effects of breast stromal fibroblasts in vitro and in vivo (A) SFCM from the indicated cells were collected after 24 h and the levels of the indicated proteins were determined by ELISA and were presented in the respective histograms. Error bars indicate mean±S.D. ** , P

Techniques Used: Expressing, In Vitro, In Vivo, Enzyme-linked Immunosorbent Assay

DNMTl is upregulated in CAFs versus TCFs (A) Formalin-fixed paraffin-embedded sections of breast resection specimens from 10 patients encompassing both invasive ductal carcinoma and their corresponding histologically normal adjacent tissues were utilized for immunohistochemical analysis using anti-DNMTI antibody. Zeiss Axio microscope was utilized, scale bar = 50 μM. (B) Whole-cell lysates were prepared from the indicated cells and were used for immunoblotting analysis, CAFs (T), TCFs (N). GAPDH was used as internal control. (C) . Total RNA was extracted and the level of the DNMT1 mRNA was assessed by qRT-PCR. Error bars represent mean ± S.D (n=3). * , P
Figure Legend Snippet: DNMTl is upregulated in CAFs versus TCFs (A) Formalin-fixed paraffin-embedded sections of breast resection specimens from 10 patients encompassing both invasive ductal carcinoma and their corresponding histologically normal adjacent tissues were utilized for immunohistochemical analysis using anti-DNMTI antibody. Zeiss Axio microscope was utilized, scale bar = 50 μM. (B) Whole-cell lysates were prepared from the indicated cells and were used for immunoblotting analysis, CAFs (T), TCFs (N). GAPDH was used as internal control. (C) . Total RNA was extracted and the level of the DNMT1 mRNA was assessed by qRT-PCR. Error bars represent mean ± S.D (n=3). * , P

Techniques Used: Formalin-fixed Paraffin-Embedded, Immunohistochemistry, Microscopy, Quantitative RT-PCR

21) Product Images from "Preclinical Evidence for Combined Use of Aromatase Inhibitors and NSAIDs as Preventive Agents of Tobacco-Induced Lung Cancer"

Article Title: Preclinical Evidence for Combined Use of Aromatase Inhibitors and NSAIDs as Preventive Agents of Tobacco-Induced Lung Cancer

Journal: Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer

doi: 10.1016/j.jtho.2017.11.126

A, Pulmonary macrophages from either control or E2 treated mice were measured by F4/80 immunostaining and quantitated by the number of F4/80 positive cells per field. Results are the mean ± S.E. B, P-NFκB, C, VEGF and D , IL-17A and were measured in lung preneoplasias by immunostaining and each field quantitated for degree of staining. P-values as analyzed by Chi-square test. *P
Figure Legend Snippet: A, Pulmonary macrophages from either control or E2 treated mice were measured by F4/80 immunostaining and quantitated by the number of F4/80 positive cells per field. Results are the mean ± S.E. B, P-NFκB, C, VEGF and D , IL-17A and were measured in lung preneoplasias by immunostaining and each field quantitated for degree of staining. P-values as analyzed by Chi-square test. *P

Techniques Used: Mouse Assay, Immunostaining, Staining

22) Product Images from "Regulation of stem-like cancer cells by glutamine through β-catenin pathway mediated by redox signaling"

Article Title: Regulation of stem-like cancer cells by glutamine through β-catenin pathway mediated by redox signaling

Journal: Molecular Cancer

doi: 10.1186/s12943-017-0623-x

Decrease of SP subpopulation in A549 cells treated with hydrogen peroxide. a A549 cells were cultured without or with H 2 O 2 (50 μM) for 48 h, cells were harvested and stained with Hoechst33342 to determine SP cells. b A549 cells were treated without or with H 2 O 2 (50 μM) for 48 h, then RNA was isolated for real-time RT-PCR for analysis of expression of ALDH-1 and SOX-2. β-actin was used as an internal control. c A549 cells was treated with 5 mM aminotriazole (ATZ) for 72 h with or without pretreatment with NAC, cell lysates were subjected to western blotting to measure the expression of ABCG2 and SOX-2. d A549 cells were treated with 5 mM aminotriazole (ATZ) for 48 h, cells were harvested and stained with Hoechst 33342 to determine SP cells. e A549 cells was treated with glutamine-free medium for 72 h with or without pre-treatment of 2 mM NAC, the cells were collected for SP detection. **, p
Figure Legend Snippet: Decrease of SP subpopulation in A549 cells treated with hydrogen peroxide. a A549 cells were cultured without or with H 2 O 2 (50 μM) for 48 h, cells were harvested and stained with Hoechst33342 to determine SP cells. b A549 cells were treated without or with H 2 O 2 (50 μM) for 48 h, then RNA was isolated for real-time RT-PCR for analysis of expression of ALDH-1 and SOX-2. β-actin was used as an internal control. c A549 cells was treated with 5 mM aminotriazole (ATZ) for 72 h with or without pretreatment with NAC, cell lysates were subjected to western blotting to measure the expression of ABCG2 and SOX-2. d A549 cells were treated with 5 mM aminotriazole (ATZ) for 48 h, cells were harvested and stained with Hoechst 33342 to determine SP cells. e A549 cells was treated with glutamine-free medium for 72 h with or without pre-treatment of 2 mM NAC, the cells were collected for SP detection. **, p

Techniques Used: Cell Culture, Staining, Isolation, Quantitative RT-PCR, Expressing, Western Blot

Impact of glutamine deprivation and H 2 O 2 on β-catenin pathway. a A549 cells were cultured in RPMI 1640 medium without glutamine for the indicated time, and cell lysates were subjected to western blotting to measure the expression of SOX2, p-β-catenin, β-catenin, and β-actin. b A549 cells were treated 100 μM H 2 O 2 for the indicated time, and cell lysates were subjected to western blotting to measure the expression of SOX2, p-β-catenin, β-catenin and β-actin. c Expression of β-catenin and ABCG2 mRNA in A549 cells transfected with siRNA against β-catenin or with negative control siRNA (NC). *, p
Figure Legend Snippet: Impact of glutamine deprivation and H 2 O 2 on β-catenin pathway. a A549 cells were cultured in RPMI 1640 medium without glutamine for the indicated time, and cell lysates were subjected to western blotting to measure the expression of SOX2, p-β-catenin, β-catenin, and β-actin. b A549 cells were treated 100 μM H 2 O 2 for the indicated time, and cell lysates were subjected to western blotting to measure the expression of SOX2, p-β-catenin, β-catenin and β-actin. c Expression of β-catenin and ABCG2 mRNA in A549 cells transfected with siRNA against β-catenin or with negative control siRNA (NC). *, p

Techniques Used: Cell Culture, Western Blot, Expressing, Transfection, Negative Control

Impact of glutamine on A549 cells clonogenic capacity and the expression of stem-cell associated molecules. a - b A549 cells were cultured in RPMI 1640 medium with or without glutamine or incubated without or with 1 U/ml L-Asparaginase. Colony numbers were counted after 2 weeks of culturing. The quantitative results of 3 independent experiments are shown in bar graphs showing mean ± SD. Images of representative colonies formed were shown in the lower panels. c Representative photographs of A549 cells cultured in medium with or without glutamine for 72 h, original magnification is 400 ×. d Effect of glutamine on expression of genes ABCG2 and SOX-2. A549 cells were cultured in RPMI 1640 with or without glutamine (300 mg/L) for 48 h, and RNA was isolated for real-time RT-PCR for detection of SOX-2 and ABCG2 expression. β-actin was used as an internal control for normalization. e Western blot analysis of ABCG2 and SOX-2. A549 cells were cultured in RPMI 1640 medium with or without glutamine (300 mg/l) for 72 h, and cell lysates were subjected to western blotting to measure the expression of ABCG2, SOX-2 and β-actin. f Effect of L-Asparaginase on expression of genes ABCG2 and SOX-2. A549 cells were cultured in the absence or presence of 1 U/ml L-Asparaginase for 72 h, and RNA was isolated for real-time RT-PCR analysis of SOX-2 and ABCG2. β-actin was used as an internal control. g A549 cells were cultured in RPMI 1640 medium without or with L-Asparaginase (1 U/ml) for indicated time, and cell lysates were subjected to western blotting to measure the expression of ABCG2 and SOX-2. (H, I) Effect of glutamine and L-Asparaginase on ABCG2 expression. A549 cells were cultured in RPMI 1640 medium with or without glutamine ( h ) or incubated without or with 1 U/ml L-Asparaginase ( i ) for the indicated time, and cells were collected and incubated with anti-ABCG2 antibody, detection of membrane protein ABCG2 was measured by flow cytometry analysis. Each Bar represents the mean ± SD of the relative fluorescence intensity from 3 independent experiments. *, p
Figure Legend Snippet: Impact of glutamine on A549 cells clonogenic capacity and the expression of stem-cell associated molecules. a - b A549 cells were cultured in RPMI 1640 medium with or without glutamine or incubated without or with 1 U/ml L-Asparaginase. Colony numbers were counted after 2 weeks of culturing. The quantitative results of 3 independent experiments are shown in bar graphs showing mean ± SD. Images of representative colonies formed were shown in the lower panels. c Representative photographs of A549 cells cultured in medium with or without glutamine for 72 h, original magnification is 400 ×. d Effect of glutamine on expression of genes ABCG2 and SOX-2. A549 cells were cultured in RPMI 1640 with or without glutamine (300 mg/L) for 48 h, and RNA was isolated for real-time RT-PCR for detection of SOX-2 and ABCG2 expression. β-actin was used as an internal control for normalization. e Western blot analysis of ABCG2 and SOX-2. A549 cells were cultured in RPMI 1640 medium with or without glutamine (300 mg/l) for 72 h, and cell lysates were subjected to western blotting to measure the expression of ABCG2, SOX-2 and β-actin. f Effect of L-Asparaginase on expression of genes ABCG2 and SOX-2. A549 cells were cultured in the absence or presence of 1 U/ml L-Asparaginase for 72 h, and RNA was isolated for real-time RT-PCR analysis of SOX-2 and ABCG2. β-actin was used as an internal control. g A549 cells were cultured in RPMI 1640 medium without or with L-Asparaginase (1 U/ml) for indicated time, and cell lysates were subjected to western blotting to measure the expression of ABCG2 and SOX-2. (H, I) Effect of glutamine and L-Asparaginase on ABCG2 expression. A549 cells were cultured in RPMI 1640 medium with or without glutamine ( h ) or incubated without or with 1 U/ml L-Asparaginase ( i ) for the indicated time, and cells were collected and incubated with anti-ABCG2 antibody, detection of membrane protein ABCG2 was measured by flow cytometry analysis. Each Bar represents the mean ± SD of the relative fluorescence intensity from 3 independent experiments. *, p

Techniques Used: Expressing, Cell Culture, Incubation, Isolation, Quantitative RT-PCR, Western Blot, Flow Cytometry, Cytometry, Fluorescence

23) Product Images from "Wogonoside inhibits IL-1β induced catabolism and hypertrophy in mouse chondrocyte and ameliorates murine osteoarthritis"

Article Title: Wogonoside inhibits IL-1β induced catabolism and hypertrophy in mouse chondrocyte and ameliorates murine osteoarthritis

Journal: Oncotarget

doi: 10.18632/oncotarget.18374

Effect of wogonoside on IL-1β-induced PI3K/AKT activiation (A-B) The protein expression of p-Stat3, Stat3, p-ERK1/2, ERK1/2, TRAF6 and IRAK1 in chondrocytes treated as above. (C-D) The protein expression of PI3K(p110), PI3K(p85), p-AKT and AKT in chondrocytes treated as above. The data in the figures represent the averages ± S.D. Significant differences between the treatment and control groups are indicated as **P
Figure Legend Snippet: Effect of wogonoside on IL-1β-induced PI3K/AKT activiation (A-B) The protein expression of p-Stat3, Stat3, p-ERK1/2, ERK1/2, TRAF6 and IRAK1 in chondrocytes treated as above. (C-D) The protein expression of PI3K(p110), PI3K(p85), p-AKT and AKT in chondrocytes treated as above. The data in the figures represent the averages ± S.D. Significant differences between the treatment and control groups are indicated as **P

Techniques Used: Expressing

24) Product Images from "Leptin changes differentiation fate and induces senescence in chondrogenic progenitor cells"

Article Title: Leptin changes differentiation fate and induces senescence in chondrogenic progenitor cells

Journal: Cell Death & Disease

doi: 10.1038/cddis.2016.68

Blocking the p53/p21 pathway inhibits senescence in CPCs treated with leptin. ( a ) The p38 inhibitor SB203580 (10 μ M) significantly decreased the expression of P-p38. The p53 inhibitor PFT- α (20 μ M) significantly decreased the expression of p21. ( b and c ) The percentage of SA- β -Gal-positive cells was significantly decreased after cells were treated with PFT- α and 100 ng/ml leptin but not after cells were treat with SB203580 and 100 ng/ml leptin for 48 h. Error bars represent the mean±S.D. Scale bar, 100 μ m. * P
Figure Legend Snippet: Blocking the p53/p21 pathway inhibits senescence in CPCs treated with leptin. ( a ) The p38 inhibitor SB203580 (10 μ M) significantly decreased the expression of P-p38. The p53 inhibitor PFT- α (20 μ M) significantly decreased the expression of p21. ( b and c ) The percentage of SA- β -Gal-positive cells was significantly decreased after cells were treated with PFT- α and 100 ng/ml leptin but not after cells were treat with SB203580 and 100 ng/ml leptin for 48 h. Error bars represent the mean±S.D. Scale bar, 100 μ m. * P

Techniques Used: Blocking Assay, Expressing

Activating and blocking Sirt1 affected CPCs senescence via the p53/p21 pathway induced by leptin. ( a and b ) Cells were treated with resveratrol and leptin, and the percentage of SA- β -Gal-positive cells was analysed. Cultured CPCs were treated with resveratrol (30 μ M), a Sirt1 pathway agonist, and 100 ng/ml leptin. ( c ) After 48 h of treatment, western blot analysis showed that acetylated p53 expression was inhibited. ( d ) PCR analysis of the genomic Sirt1 locus in heterozygous floxed mice with and without Cre and with or without tamoxifen. ( e and f ) Comparison of the number of p53- and p21-positive cells in wild-type and KO mice. Fluorescence microscopy images show that the p53/p21 pathway was more activated in the articular cartilage tissues of Sirt1 KO mice than in wild-type mice. Scale bar, 100 μ m. Error bars indicate the mean±S.D. * P
Figure Legend Snippet: Activating and blocking Sirt1 affected CPCs senescence via the p53/p21 pathway induced by leptin. ( a and b ) Cells were treated with resveratrol and leptin, and the percentage of SA- β -Gal-positive cells was analysed. Cultured CPCs were treated with resveratrol (30 μ M), a Sirt1 pathway agonist, and 100 ng/ml leptin. ( c ) After 48 h of treatment, western blot analysis showed that acetylated p53 expression was inhibited. ( d ) PCR analysis of the genomic Sirt1 locus in heterozygous floxed mice with and without Cre and with or without tamoxifen. ( e and f ) Comparison of the number of p53- and p21-positive cells in wild-type and KO mice. Fluorescence microscopy images show that the p53/p21 pathway was more activated in the articular cartilage tissues of Sirt1 KO mice than in wild-type mice. Scale bar, 100 μ m. Error bars indicate the mean±S.D. * P

Techniques Used: Blocking Assay, Cell Culture, Western Blot, Expressing, Polymerase Chain Reaction, Mouse Assay, Fluorescence, Microscopy

The activation of the p53/p21 pathway and the inhibition of the Sirt1 pathway promoted cell cycle arrest and senescence in CPCs that were treated with high doses of leptin. ( a ) CPCs were treated with leptin (0 ng/ml as the control or 10, 50 or 100 ng/ml) for 48 h. Compared with the control, high doses of leptin inhibited the proliferation of CPCs by CCK-8 assay. ( b ) Flow cytometry verified that the high doses of leptin induce CPCs cycle arrest by inhibiting the G 1 –S phase. ( c and d ) Staining for cell senescence. CPCs were treated with leptin (0 ng/ml as control, 10, 50 and 100 ng/ml) for 48 h, and the cells were then observed using light microscopy (× 40). Scale bar, 100 μ m. The percentage of CPCs that positively stained for SA- β -Gal was counted in five visual fields. High doses of leptin induced senescence in CPCs. ( e and f ) P-p38 and p53 expression were detected in CPCs grown in the presence of leptin (0 ng/ml as the control or 10, 50 or 100 ng/ml) for 2 days. Relative protein abundance of each blot was normalized to the grey value of β -actin. ( g and h ) Acetylp53, p21 and Sirt1 expression were detected in CPCs grown in the presence of leptin (0 ng/ml as the control or 10, 50 or 100 ng/ml) for 2 days. Relative protein abundance of each blot was normalized to the grey value of β -actin. Western blot analysis verified that the p53/p21 pathway was significantly activated and that the Sirt1 pathway was inhibited in CPCs that were treated with high doses of leptin. Error bars represent the mean±S.D. * P
Figure Legend Snippet: The activation of the p53/p21 pathway and the inhibition of the Sirt1 pathway promoted cell cycle arrest and senescence in CPCs that were treated with high doses of leptin. ( a ) CPCs were treated with leptin (0 ng/ml as the control or 10, 50 or 100 ng/ml) for 48 h. Compared with the control, high doses of leptin inhibited the proliferation of CPCs by CCK-8 assay. ( b ) Flow cytometry verified that the high doses of leptin induce CPCs cycle arrest by inhibiting the G 1 –S phase. ( c and d ) Staining for cell senescence. CPCs were treated with leptin (0 ng/ml as control, 10, 50 and 100 ng/ml) for 48 h, and the cells were then observed using light microscopy (× 40). Scale bar, 100 μ m. The percentage of CPCs that positively stained for SA- β -Gal was counted in five visual fields. High doses of leptin induced senescence in CPCs. ( e and f ) P-p38 and p53 expression were detected in CPCs grown in the presence of leptin (0 ng/ml as the control or 10, 50 or 100 ng/ml) for 2 days. Relative protein abundance of each blot was normalized to the grey value of β -actin. ( g and h ) Acetylp53, p21 and Sirt1 expression were detected in CPCs grown in the presence of leptin (0 ng/ml as the control or 10, 50 or 100 ng/ml) for 2 days. Relative protein abundance of each blot was normalized to the grey value of β -actin. Western blot analysis verified that the p53/p21 pathway was significantly activated and that the Sirt1 pathway was inhibited in CPCs that were treated with high doses of leptin. Error bars represent the mean±S.D. * P

Techniques Used: Activation Assay, Inhibition, CCK-8 Assay, Flow Cytometry, Cytometry, Staining, Light Microscopy, Expressing, Western Blot

Changes in leptin-related signalling pathways were correlated with the severity of cartilage degeneration. ( a ) p53 and Ob-Rb double-labelling (200-fold magnification) in LTP and MTP cartilage (indicated by an arrow). Scale bar, 100 μ m. ( b ) Cartilage tissue proteins were extracted from the weight-bearing areas of the LFC, MFC, LTP and MTP in OA patients. Protein western blot analysis was used to detect the protein levels of Coll-2, Sirt1, Ob-Rb, p53 and p21 in those cartilage tissue proteins. ( c ) Relative protein abundance of each blot was normalized to the grey value of GAPDH. ( d–f ) The relationships between the expression levels of Ob-Rb and Coll-2, p53 and Sirt1 were analysed in five samples. Error bars represent the mean±S.D. * P
Figure Legend Snippet: Changes in leptin-related signalling pathways were correlated with the severity of cartilage degeneration. ( a ) p53 and Ob-Rb double-labelling (200-fold magnification) in LTP and MTP cartilage (indicated by an arrow). Scale bar, 100 μ m. ( b ) Cartilage tissue proteins were extracted from the weight-bearing areas of the LFC, MFC, LTP and MTP in OA patients. Protein western blot analysis was used to detect the protein levels of Coll-2, Sirt1, Ob-Rb, p53 and p21 in those cartilage tissue proteins. ( c ) Relative protein abundance of each blot was normalized to the grey value of GAPDH. ( d–f ) The relationships between the expression levels of Ob-Rb and Coll-2, p53 and Sirt1 were analysed in five samples. Error bars represent the mean±S.D. * P

Techniques Used: Western Blot, Expressing

25) Product Images from "Cell viability assessed in a reproducible model of human osteoblasts derived from human adipose-derived stem cells"

Article Title: Cell viability assessed in a reproducible model of human osteoblasts derived from human adipose-derived stem cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0194847

Analysis of the markers present in osteoblasts. (A) CD45RO. (B) CD105. (C) STRO-1. (D) The arrows indicate the perinuclear location of Nanog. (E) The arrows indicate the location of RANKL. (F) Levels of expression of markers. Cell images showing the expression of the markers CD 45RO, RANKL, Nanog, CD105, and STRO-1 in osteoblasts cultured in osteogenic medium for 16 days. Data are expressed as mean ± standard deviation. An ANOVA followed by Tukey’s test was used to analyze the data. P
Figure Legend Snippet: Analysis of the markers present in osteoblasts. (A) CD45RO. (B) CD105. (C) STRO-1. (D) The arrows indicate the perinuclear location of Nanog. (E) The arrows indicate the location of RANKL. (F) Levels of expression of markers. Cell images showing the expression of the markers CD 45RO, RANKL, Nanog, CD105, and STRO-1 in osteoblasts cultured in osteogenic medium for 16 days. Data are expressed as mean ± standard deviation. An ANOVA followed by Tukey’s test was used to analyze the data. P

Techniques Used: Expressing, Cell Culture, Standard Deviation

Analysis of hASC markers. (A) CD90. (B) CD105. (C) Nanog. (D) Sox-2. (E) STRO-1. (F) CD45RO. (G) CD117. (H) Quantification of expression of positive and negative markers. Graphs show the expression of different markers in hASCs. The expression of the markers was assessed using flow cytometry by means of the intensity of the emitted fluorescence (FL1 and FL2). The expression of the positive markers, CD90, CD105, and STRO-1 (surface markers mesenchymal) and Nanog and Sox-2 (pluripotency markers) is compared to the decreased expression of negative markers (CD45RO and CD117). Data are represented as mean ± standard deviation. The statistical analyses were obtained using an ANOVA followed by Dunnett’s test with P
Figure Legend Snippet: Analysis of hASC markers. (A) CD90. (B) CD105. (C) Nanog. (D) Sox-2. (E) STRO-1. (F) CD45RO. (G) CD117. (H) Quantification of expression of positive and negative markers. Graphs show the expression of different markers in hASCs. The expression of the markers was assessed using flow cytometry by means of the intensity of the emitted fluorescence (FL1 and FL2). The expression of the positive markers, CD90, CD105, and STRO-1 (surface markers mesenchymal) and Nanog and Sox-2 (pluripotency markers) is compared to the decreased expression of negative markers (CD45RO and CD117). Data are represented as mean ± standard deviation. The statistical analyses were obtained using an ANOVA followed by Dunnett’s test with P

Techniques Used: Expressing, Flow Cytometry, Cytometry, Fluorescence, Standard Deviation

26) Product Images from "The alkaloids of Banisteriopsis caapi, the plant source of the Amazonian hallucinogen Ayahuasca, stimulate adult neurogenesis in vitro"

Article Title: The alkaloids of Banisteriopsis caapi, the plant source of the Amazonian hallucinogen Ayahuasca, stimulate adult neurogenesis in vitro

Journal: Scientific Reports

doi: 10.1038/s41598-017-05407-9

Effects of ayahuasca β-carboline alkaloids on adult neural stem cells proliferation. ( a ) Representative immunofluorescence images showing the expression of the cellular marker for proliferation ki67 (green) in neurospheres derived from the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the hippocampus. SVZ-derived neurospheres are shown in two panels showing the central part of the sphere (left) and the distal migration site (right). Single images from SGZ-derived neurospheres show the whole neurosphere, including the central and distal areas. DAPI was used for nuclear staining. Scale bar = 50 μm. ( b ) Representative Western blots of ki67 and the proliferating cell nuclear antigen (PCNA) levels in neurospheres treated for 7 days with each of the four alkaloids tested (1 µM). Bar graphs show the results of the quantification analyses. Each bar indicates relative protein levels expressed as mean ± SD of the quantification of at least three independent experiments corresponding to four different cellular pools. The left side of the image shows results for the subventricular zone (SVZ) of the brain. The right side of the image shows results for he subgranular zone of the hippocampus (SGZ). *p ≤ 0.05; **p ≤ 0.01 indicate significant results in the post-hoc pair-wise comparisons (Bonferroni) versus non-treated (basal) cultures.
Figure Legend Snippet: Effects of ayahuasca β-carboline alkaloids on adult neural stem cells proliferation. ( a ) Representative immunofluorescence images showing the expression of the cellular marker for proliferation ki67 (green) in neurospheres derived from the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the hippocampus. SVZ-derived neurospheres are shown in two panels showing the central part of the sphere (left) and the distal migration site (right). Single images from SGZ-derived neurospheres show the whole neurosphere, including the central and distal areas. DAPI was used for nuclear staining. Scale bar = 50 μm. ( b ) Representative Western blots of ki67 and the proliferating cell nuclear antigen (PCNA) levels in neurospheres treated for 7 days with each of the four alkaloids tested (1 µM). Bar graphs show the results of the quantification analyses. Each bar indicates relative protein levels expressed as mean ± SD of the quantification of at least three independent experiments corresponding to four different cellular pools. The left side of the image shows results for the subventricular zone (SVZ) of the brain. The right side of the image shows results for he subgranular zone of the hippocampus (SGZ). *p ≤ 0.05; **p ≤ 0.01 indicate significant results in the post-hoc pair-wise comparisons (Bonferroni) versus non-treated (basal) cultures.

Techniques Used: Immunofluorescence, Expressing, Marker, Derivative Assay, Migration, Staining, Western Blot

27) Product Images from "LSL-KrasG12D; LSL-Trp53R172H/+; Ink4flox/+; Ptf1/p48-Cre mice are an applicable model for locally invasive and metastatic pancreatic cancer"

Article Title: LSL-KrasG12D; LSL-Trp53R172H/+; Ink4flox/+; Ptf1/p48-Cre mice are an applicable model for locally invasive and metastatic pancreatic cancer

Journal: PLoS ONE

doi: 10.1371/journal.pone.0176844

The molecular profiles and EMT of neoplastic cells of KPIC tumors resemble human PC. (A) E-cadherin and CK immunostaining showed that KPIC tumors are epithelial. (B-D) Co-immuostaining p53, pMAPK and Her2 antibodies with E-cadherin antibody in KPIC tumors showed that their molecular profiles are similar to that in human PC. (E, F) Triple immunofluorescent staining of E-cadherin, vimentin and cytokeratin or Her2 staining showed that KPIC tumors harbor a significant number of EMT cells. Her2 expression in EMT cells was upregulated. (circled region, EMT region; white yellow and white arrows, EMT cells). Scale bar, 50μm.
Figure Legend Snippet: The molecular profiles and EMT of neoplastic cells of KPIC tumors resemble human PC. (A) E-cadherin and CK immunostaining showed that KPIC tumors are epithelial. (B-D) Co-immuostaining p53, pMAPK and Her2 antibodies with E-cadherin antibody in KPIC tumors showed that their molecular profiles are similar to that in human PC. (E, F) Triple immunofluorescent staining of E-cadherin, vimentin and cytokeratin or Her2 staining showed that KPIC tumors harbor a significant number of EMT cells. Her2 expression in EMT cells was upregulated. (circled region, EMT region; white yellow and white arrows, EMT cells). Scale bar, 50μm.

Techniques Used: Immunostaining, Staining, Expressing

28) Product Images from "High-mobility group box 1 released from astrocytes promotes the proliferation of cultured neural stem/progenitor cells"

Article Title: High-mobility group box 1 released from astrocytes promotes the proliferation of cultured neural stem/progenitor cells

Journal: International Journal of Molecular Medicine

doi: 10.3892/ijmm.2014.1820

Release of HMGB1 from IL-1β-stimulated astrocytes. ELISA confirmed that HMGB1 was undetectable in normal astrocyte-conditioned medium (nACM). IL-1β (0.1 ng/ml) stimulation caused astrocytes to release HMGB1 into stimulted astrocyte-conditioned medium (sACM). HMGB1 shRNA attenuated the release of HMGB1 into the ACM (HMGB1 shRNA sACM). A control shRNA (control shRNA sACM) had no effect on HMGB1 release into IL-1β-stimulated ACM. * P
Figure Legend Snippet: Release of HMGB1 from IL-1β-stimulated astrocytes. ELISA confirmed that HMGB1 was undetectable in normal astrocyte-conditioned medium (nACM). IL-1β (0.1 ng/ml) stimulation caused astrocytes to release HMGB1 into stimulted astrocyte-conditioned medium (sACM). HMGB1 shRNA attenuated the release of HMGB1 into the ACM (HMGB1 shRNA sACM). A control shRNA (control shRNA sACM) had no effect on HMGB1 release into IL-1β-stimulated ACM. * P

Techniques Used: Enzyme-linked Immunosorbent Assay, shRNA

The effects of astrocyte-derived HMGB1 on NS/PC proliferation were measured by the CCK-8 assay. Relative absorbance values of viable NS/PCs were measured by CCK-8 assay at (A) 72 h and (B) 96 h. The proliferation of NS/PCs in sACM, control shRNA sACM and HMGB1 culture media was increased following 72 and 96 h of exposure. Compared with the sACM, the proliferation of NS/PCs in HMGB1 shRNA sACM significantly decreased after 72 and 96 h. There were no intra-time point differences in NS/PC proliferation between the vehicle (NS/PC culture medium only) and control (normal ACM with IL-1β) groups at either the 72- or 96-h timepoint. * P
Figure Legend Snippet: The effects of astrocyte-derived HMGB1 on NS/PC proliferation were measured by the CCK-8 assay. Relative absorbance values of viable NS/PCs were measured by CCK-8 assay at (A) 72 h and (B) 96 h. The proliferation of NS/PCs in sACM, control shRNA sACM and HMGB1 culture media was increased following 72 and 96 h of exposure. Compared with the sACM, the proliferation of NS/PCs in HMGB1 shRNA sACM significantly decreased after 72 and 96 h. There were no intra-time point differences in NS/PC proliferation between the vehicle (NS/PC culture medium only) and control (normal ACM with IL-1β) groups at either the 72- or 96-h timepoint. * P

Techniques Used: Derivative Assay, CCK-8 Assay, shRNA

Astrocyte-derived HMGB1 on NS/PC proliferation was measured by analyzing the cell cycle. The cell cycle kinetics of NS/PCs were analyzed by flow cytometry at (A) 72 h and (B) 96 h. The proliferative capacity of NS/PCs was described as the proliferation index (PI): PI = (S + G2/M)/(G0/G1 + S + G2/M). The PIs of the sACM group, control shRNA sACM group and HMGB1 group were increased after 72 and 96 h. Compared with the sACM group, the PI of the HMGB1 shRNA sACM group was significantly decreased after 72 and 96 h. There were no intra-time point differences in PI between vehicle (NS/PC culture medium only) and control (normal ACM with IL-1β) groups at either the 72 or 96-h time point. * P
Figure Legend Snippet: Astrocyte-derived HMGB1 on NS/PC proliferation was measured by analyzing the cell cycle. The cell cycle kinetics of NS/PCs were analyzed by flow cytometry at (A) 72 h and (B) 96 h. The proliferative capacity of NS/PCs was described as the proliferation index (PI): PI = (S + G2/M)/(G0/G1 + S + G2/M). The PIs of the sACM group, control shRNA sACM group and HMGB1 group were increased after 72 and 96 h. Compared with the sACM group, the PI of the HMGB1 shRNA sACM group was significantly decreased after 72 and 96 h. There were no intra-time point differences in PI between vehicle (NS/PC culture medium only) and control (normal ACM with IL-1β) groups at either the 72 or 96-h time point. * P

Techniques Used: Derivative Assay, Flow Cytometry, Cytometry, shRNA

(A) Western blot analysis and (B) double-immunofluorescence labeling of HMGB1 expression in IL-1β-stimulated astrocytes. (A) HMGB1 protein expression was upregulated in IL-1β-stimulated astrocytes. HMGB1 shRNA specifically suppressed HMGB1 protein expression in IL-1β-stimulated astrocytes, as a control shRNA had no effect on HMGB1 protein expression in IL-1β-stimulated astrocytes. * P
Figure Legend Snippet: (A) Western blot analysis and (B) double-immunofluorescence labeling of HMGB1 expression in IL-1β-stimulated astrocytes. (A) HMGB1 protein expression was upregulated in IL-1β-stimulated astrocytes. HMGB1 shRNA specifically suppressed HMGB1 protein expression in IL-1β-stimulated astrocytes, as a control shRNA had no effect on HMGB1 protein expression in IL-1β-stimulated astrocytes. * P

Techniques Used: Western Blot, Immunofluorescence, Labeling, Expressing, shRNA

Analysis of signaling pathways involved in HMGB1-mediated NS/PC proliferation by CCK-8 assay (A and D) and western blot analysis (B and C). NS/PCs were cultured for 96 h in NS/PC culture medium containing 7 ng/ml HMGB1. (A) Compared to an IgG control, blockade of RAGE with an anti-RAGE antibody significantly inhibited HMGB1-induced NS/PC proliferation. * P
Figure Legend Snippet: Analysis of signaling pathways involved in HMGB1-mediated NS/PC proliferation by CCK-8 assay (A and D) and western blot analysis (B and C). NS/PCs were cultured for 96 h in NS/PC culture medium containing 7 ng/ml HMGB1. (A) Compared to an IgG control, blockade of RAGE with an anti-RAGE antibody significantly inhibited HMGB1-induced NS/PC proliferation. * P

Techniques Used: CCK-8 Assay, Western Blot, Cell Culture

29) Product Images from "Medullospheres from DAOY, UW228 and ONS-76 Cells: Increased Stem Cell Population and Proteomic Modifications"

Article Title: Medullospheres from DAOY, UW228 and ONS-76 Cells: Increased Stem Cell Population and Proteomic Modifications

Journal: PLoS ONE

doi: 10.1371/journal.pone.0063748

Immunostaining of TMA. Staining for Oct-4, Sox-2, beta Catenin and Nucleophosim on DAOY, ONS76 and UW228 cell lines grown as spheres are shown at 40x magnification. UW228 (second column) show smaller spheres and less intense staining for Oct-4 and Sox-2.
Figure Legend Snippet: Immunostaining of TMA. Staining for Oct-4, Sox-2, beta Catenin and Nucleophosim on DAOY, ONS76 and UW228 cell lines grown as spheres are shown at 40x magnification. UW228 (second column) show smaller spheres and less intense staining for Oct-4 and Sox-2.

Techniques Used: Immunostaining, Staining

30) Product Images from "Biologic Properties of Mesenchymal Stem Cells Derived From Bone Marrow and Adipose Tissue"

Article Title: Biologic Properties of Mesenchymal Stem Cells Derived From Bone Marrow and Adipose Tissue

Journal:

doi: 10.1002/jcb.20904

Transcription factor mRNA and expression in MSCs. Total cellular RNA analyzed by RT-PCR for Oct-4, Sox-2, and Rex-1 mRNA expression ( A ). The cell lysate from MSC types at different passages were subjected to Western blot analysis using Oct-4 and Sox-2
Figure Legend Snippet: Transcription factor mRNA and expression in MSCs. Total cellular RNA analyzed by RT-PCR for Oct-4, Sox-2, and Rex-1 mRNA expression ( A ). The cell lysate from MSC types at different passages were subjected to Western blot analysis using Oct-4 and Sox-2

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

Oct-4 and Sox-2 localization in MSCs. Immunohistochemical staining of rBMSCs ( A ), hBMSCs ( B ), rASCs ( C ), and hASCs ( D ). Cells were fixed, permeabilized, and stained to visualize Oct-4 and Sox-2. Distribution and localization of these transcription factors
Figure Legend Snippet: Oct-4 and Sox-2 localization in MSCs. Immunohistochemical staining of rBMSCs ( A ), hBMSCs ( B ), rASCs ( C ), and hASCs ( D ). Cells were fixed, permeabilized, and stained to visualize Oct-4 and Sox-2. Distribution and localization of these transcription factors

Techniques Used: Immunohistochemistry, Staining

31) Product Images from "Diabetic Vascular Calcification Mediated by the Collagen Receptor Discoidin Domain Receptor 1 via the Phosphoinositide 3-Kinase/Akt/Runt-Related Transcription Factor 2 Signaling Axis"

Article Title: Diabetic Vascular Calcification Mediated by the Collagen Receptor Discoidin Domain Receptor 1 via the Phosphoinositide 3-Kinase/Akt/Runt-Related Transcription Factor 2 Signaling Axis

Journal: Arteriosclerosis, thrombosis, and vascular biology

doi: 10.1161/ATVBAHA.118.311238

Discoidin domain receptor 1 (DDR1) mediates vascular smooth muscle cell (VSMC) calcification through the phosphoinositide 3-kinase (PI3K)/Akt signaling axis. Ddr1 +/+ and Ddr1 −/− VSMCs were cultured in calcifying media for 12 d and treated daily with dimethyl sulfoxide (Con.), insulin (Ins.), or wortmannin (Wort.) ( A ). Insulin stimulated VSMC calcification while wortmannin attenuated calcification. DDR1 immunoprecipitation was performed on cell lysates from Ddr1 +/+ VSMCs treated with insulin, wortmannin, or type-I collagen (Col-1) ( B ). Treatment with Col-1 led to increased DDR1 activation (pY), and Col-1 or insulin treatment resulted in increased association of DDR1 with PI3K subunits (p110β and p85). Wortmannin attenuated this interaction.
Figure Legend Snippet: Discoidin domain receptor 1 (DDR1) mediates vascular smooth muscle cell (VSMC) calcification through the phosphoinositide 3-kinase (PI3K)/Akt signaling axis. Ddr1 +/+ and Ddr1 −/− VSMCs were cultured in calcifying media for 12 d and treated daily with dimethyl sulfoxide (Con.), insulin (Ins.), or wortmannin (Wort.) ( A ). Insulin stimulated VSMC calcification while wortmannin attenuated calcification. DDR1 immunoprecipitation was performed on cell lysates from Ddr1 +/+ VSMCs treated with insulin, wortmannin, or type-I collagen (Col-1) ( B ). Treatment with Col-1 led to increased DDR1 activation (pY), and Col-1 or insulin treatment resulted in increased association of DDR1 with PI3K subunits (p110β and p85). Wortmannin attenuated this interaction.

Techniques Used: Cell Culture, Immunoprecipitation, Activation Assay

32) Product Images from "Leptin changes differentiation fate and induces senescence in chondrogenic progenitor cells"

Article Title: Leptin changes differentiation fate and induces senescence in chondrogenic progenitor cells

Journal: Cell Death & Disease

doi: 10.1038/cddis.2016.68

Blocking the p53/p21 pathway inhibits senescence in CPCs treated with leptin. ( a ) The p38 inhibitor SB203580 (10 μ M) significantly decreased the expression of P-p38. The p53 inhibitor PFT- α (20 μ M) significantly decreased the expression of p21. ( b and c ) The percentage of SA- β -Gal-positive cells was significantly decreased after cells were treated with PFT- α and 100 ng/ml leptin but not after cells were treat with SB203580 and 100 ng/ml leptin for 48 h. Error bars represent the mean±S.D. Scale bar, 100 μ m. * P
Figure Legend Snippet: Blocking the p53/p21 pathway inhibits senescence in CPCs treated with leptin. ( a ) The p38 inhibitor SB203580 (10 μ M) significantly decreased the expression of P-p38. The p53 inhibitor PFT- α (20 μ M) significantly decreased the expression of p21. ( b and c ) The percentage of SA- β -Gal-positive cells was significantly decreased after cells were treated with PFT- α and 100 ng/ml leptin but not after cells were treat with SB203580 and 100 ng/ml leptin for 48 h. Error bars represent the mean±S.D. Scale bar, 100 μ m. * P

Techniques Used: Blocking Assay, Expressing

Activating and blocking Sirt1 affected CPCs senescence via the p53/p21 pathway induced by leptin. ( a and b ) Cells were treated with resveratrol and leptin, and the percentage of SA- β -Gal-positive cells was analysed. Cultured CPCs were treated with resveratrol (30 μ M), a Sirt1 pathway agonist, and 100 ng/ml leptin. ( c ) After 48 h of treatment, western blot analysis showed that acetylated p53 expression was inhibited. ( d ) PCR analysis of the genomic Sirt1 locus in heterozygous floxed mice with and without Cre and with or without tamoxifen. ( e and f ) Comparison of the number of p53- and p21-positive cells in wild-type and KO mice. Fluorescence microscopy images show that the p53/p21 pathway was more activated in the articular cartilage tissues of Sirt1 KO mice than in wild-type mice. Scale bar, 100 μ m. Error bars indicate the mean±S.D. * P
Figure Legend Snippet: Activating and blocking Sirt1 affected CPCs senescence via the p53/p21 pathway induced by leptin. ( a and b ) Cells were treated with resveratrol and leptin, and the percentage of SA- β -Gal-positive cells was analysed. Cultured CPCs were treated with resveratrol (30 μ M), a Sirt1 pathway agonist, and 100 ng/ml leptin. ( c ) After 48 h of treatment, western blot analysis showed that acetylated p53 expression was inhibited. ( d ) PCR analysis of the genomic Sirt1 locus in heterozygous floxed mice with and without Cre and with or without tamoxifen. ( e and f ) Comparison of the number of p53- and p21-positive cells in wild-type and KO mice. Fluorescence microscopy images show that the p53/p21 pathway was more activated in the articular cartilage tissues of Sirt1 KO mice than in wild-type mice. Scale bar, 100 μ m. Error bars indicate the mean±S.D. * P

Techniques Used: Blocking Assay, Cell Culture, Western Blot, Expressing, Polymerase Chain Reaction, Mouse Assay, Fluorescence, Microscopy

The activation of the p53/p21 pathway and the inhibition of the Sirt1 pathway promoted cell cycle arrest and senescence in CPCs that were treated with high doses of leptin. ( a ) CPCs were treated with leptin (0 ng/ml as the control or 10, 50 or 100 ng/ml) for 48 h. Compared with the control, high doses of leptin inhibited the proliferation of CPCs by CCK-8 assay. ( b ) Flow cytometry verified that the high doses of leptin induce CPCs cycle arrest by inhibiting the G 1 –S phase. ( c and d ) Staining for cell senescence. CPCs were treated with leptin (0 ng/ml as control, 10, 50 and 100 ng/ml) for 48 h, and the cells were then observed using light microscopy (× 40). Scale bar, 100 μ m. The percentage of CPCs that positively stained for SA- β -Gal was counted in five visual fields. High doses of leptin induced senescence in CPCs. ( e and f ) P-p38 and p53 expression were detected in CPCs grown in the presence of leptin (0 ng/ml as the control or 10, 50 or 100 ng/ml) for 2 days. Relative protein abundance of each blot was normalized to the grey value of β -actin. ( g and h ) Acetylp53, p21 and Sirt1 expression were detected in CPCs grown in the presence of leptin (0 ng/ml as the control or 10, 50 or 100 ng/ml) for 2 days. Relative protein abundance of each blot was normalized to the grey value of β -actin. Western blot analysis verified that the p53/p21 pathway was significantly activated and that the Sirt1 pathway was inhibited in CPCs that were treated with high doses of leptin. Error bars represent the mean±S.D. * P
Figure Legend Snippet: The activation of the p53/p21 pathway and the inhibition of the Sirt1 pathway promoted cell cycle arrest and senescence in CPCs that were treated with high doses of leptin. ( a ) CPCs were treated with leptin (0 ng/ml as the control or 10, 50 or 100 ng/ml) for 48 h. Compared with the control, high doses of leptin inhibited the proliferation of CPCs by CCK-8 assay. ( b ) Flow cytometry verified that the high doses of leptin induce CPCs cycle arrest by inhibiting the G 1 –S phase. ( c and d ) Staining for cell senescence. CPCs were treated with leptin (0 ng/ml as control, 10, 50 and 100 ng/ml) for 48 h, and the cells were then observed using light microscopy (× 40). Scale bar, 100 μ m. The percentage of CPCs that positively stained for SA- β -Gal was counted in five visual fields. High doses of leptin induced senescence in CPCs. ( e and f ) P-p38 and p53 expression were detected in CPCs grown in the presence of leptin (0 ng/ml as the control or 10, 50 or 100 ng/ml) for 2 days. Relative protein abundance of each blot was normalized to the grey value of β -actin. ( g and h ) Acetylp53, p21 and Sirt1 expression were detected in CPCs grown in the presence of leptin (0 ng/ml as the control or 10, 50 or 100 ng/ml) for 2 days. Relative protein abundance of each blot was normalized to the grey value of β -actin. Western blot analysis verified that the p53/p21 pathway was significantly activated and that the Sirt1 pathway was inhibited in CPCs that were treated with high doses of leptin. Error bars represent the mean±S.D. * P

Techniques Used: Activation Assay, Inhibition, CCK-8 Assay, Flow Cytometry, Cytometry, Staining, Light Microscopy, Expressing, Western Blot

Changes in leptin-related signalling pathways were correlated with the severity of cartilage degeneration. ( a ) p53 and Ob-Rb double-labelling (200-fold magnification) in LTP and MTP cartilage (indicated by an arrow). Scale bar, 100 μ m. ( b ) Cartilage tissue proteins were extracted from the weight-bearing areas of the LFC, MFC, LTP and MTP in OA patients. Protein western blot analysis was used to detect the protein levels of Coll-2, Sirt1, Ob-Rb, p53 and p21 in those cartilage tissue proteins. ( c ) Relative protein abundance of each blot was normalized to the grey value of GAPDH. ( d–f ) The relationships between the expression levels of Ob-Rb and Coll-2, p53 and Sirt1 were analysed in five samples. Error bars represent the mean±S.D. * P
Figure Legend Snippet: Changes in leptin-related signalling pathways were correlated with the severity of cartilage degeneration. ( a ) p53 and Ob-Rb double-labelling (200-fold magnification) in LTP and MTP cartilage (indicated by an arrow). Scale bar, 100 μ m. ( b ) Cartilage tissue proteins were extracted from the weight-bearing areas of the LFC, MFC, LTP and MTP in OA patients. Protein western blot analysis was used to detect the protein levels of Coll-2, Sirt1, Ob-Rb, p53 and p21 in those cartilage tissue proteins. ( c ) Relative protein abundance of each blot was normalized to the grey value of GAPDH. ( d–f ) The relationships between the expression levels of Ob-Rb and Coll-2, p53 and Sirt1 were analysed in five samples. Error bars represent the mean±S.D. * P

Techniques Used: Western Blot, Expressing

33) Product Images from "Cell viability assessed in a reproducible model of human osteoblasts derived from human adipose-derived stem cells"

Article Title: Cell viability assessed in a reproducible model of human osteoblasts derived from human adipose-derived stem cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0194847

Analysis of the markers present in osteoblasts. (A) CD45RO. (B) CD105. (C) STRO-1. (D) The arrows indicate the perinuclear location of Nanog. (E) The arrows indicate the location of RANKL. (F) Levels of expression of markers. Cell images showing the expression of the markers CD 45RO, RANKL, Nanog, CD105, and STRO-1 in osteoblasts cultured in osteogenic medium for 16 days. Data are expressed as mean ± standard deviation. An ANOVA followed by Tukey’s test was used to analyze the data. P
Figure Legend Snippet: Analysis of the markers present in osteoblasts. (A) CD45RO. (B) CD105. (C) STRO-1. (D) The arrows indicate the perinuclear location of Nanog. (E) The arrows indicate the location of RANKL. (F) Levels of expression of markers. Cell images showing the expression of the markers CD 45RO, RANKL, Nanog, CD105, and STRO-1 in osteoblasts cultured in osteogenic medium for 16 days. Data are expressed as mean ± standard deviation. An ANOVA followed by Tukey’s test was used to analyze the data. P

Techniques Used: Expressing, Cell Culture, Standard Deviation

Analysis of hASC markers. (A) CD90. (B) CD105. (C) Nanog. (D) Sox-2. (E) STRO-1. (F) CD45RO. (G) CD117. (H) Quantification of expression of positive and negative markers. Graphs show the expression of different markers in hASCs. The expression of the markers was assessed using flow cytometry by means of the intensity of the emitted fluorescence (FL1 and FL2). The expression of the positive markers, CD90, CD105, and STRO-1 (surface markers mesenchymal) and Nanog and Sox-2 (pluripotency markers) is compared to the decreased expression of negative markers (CD45RO and CD117). Data are represented as mean ± standard deviation. The statistical analyses were obtained using an ANOVA followed by Dunnett’s test with P
Figure Legend Snippet: Analysis of hASC markers. (A) CD90. (B) CD105. (C) Nanog. (D) Sox-2. (E) STRO-1. (F) CD45RO. (G) CD117. (H) Quantification of expression of positive and negative markers. Graphs show the expression of different markers in hASCs. The expression of the markers was assessed using flow cytometry by means of the intensity of the emitted fluorescence (FL1 and FL2). The expression of the positive markers, CD90, CD105, and STRO-1 (surface markers mesenchymal) and Nanog and Sox-2 (pluripotency markers) is compared to the decreased expression of negative markers (CD45RO and CD117). Data are represented as mean ± standard deviation. The statistical analyses were obtained using an ANOVA followed by Dunnett’s test with P

Techniques Used: Expressing, Flow Cytometry, Cytometry, Fluorescence, Standard Deviation

34) Product Images from "The alkaloids of Banisteriopsis caapi, the plant source of the Amazonian hallucinogen Ayahuasca, stimulate adult neurogenesis in vitro"

Article Title: The alkaloids of Banisteriopsis caapi, the plant source of the Amazonian hallucinogen Ayahuasca, stimulate adult neurogenesis in vitro

Journal: Scientific Reports

doi: 10.1038/s41598-017-05407-9

Effects of ayahuasca β-carboline alkaloids on stemness of cultured adult neurospheres. Representative Western blots and bar graphs showing expression levels of the precursor cell markers musashi-1, nestin and SOX-2 after treatment with each of the four alkaloids tested (1 µM). Values in bar graphs indicate mean ± SD of the quantification of at least three independent experiments corresponding to four different cellular pools. The left side of the image shows results for the subventricular zone (SVZ) of the brain. The right side of the image shows results for the subgranular zone of the hippocampus (SGZ). *p ≤ 0.05; **P ≤ 0.01; ***p ≤ 0.001 indicate significant results in the post-hoc pair-wise comparisons (Bonferroni) versus non-treated (basal) cultures.
Figure Legend Snippet: Effects of ayahuasca β-carboline alkaloids on stemness of cultured adult neurospheres. Representative Western blots and bar graphs showing expression levels of the precursor cell markers musashi-1, nestin and SOX-2 after treatment with each of the four alkaloids tested (1 µM). Values in bar graphs indicate mean ± SD of the quantification of at least three independent experiments corresponding to four different cellular pools. The left side of the image shows results for the subventricular zone (SVZ) of the brain. The right side of the image shows results for the subgranular zone of the hippocampus (SGZ). *p ≤ 0.05; **P ≤ 0.01; ***p ≤ 0.001 indicate significant results in the post-hoc pair-wise comparisons (Bonferroni) versus non-treated (basal) cultures.

Techniques Used: Cell Culture, Western Blot, Expressing

35) Product Images from "Hyperbaric oxygen protects type II collagen in interleukin-1β-induced mandibular condylar chondrocyte via inhibiting the JNK/c-Jun signaling pathway"

Article Title: Hyperbaric oxygen protects type II collagen in interleukin-1β-induced mandibular condylar chondrocyte via inhibiting the JNK/c-Jun signaling pathway

Journal: Oncotarget

doi: 10.18632/oncotarget.19294

Immunofluorescence showed the change in expression of p-JNK, c-Jun, p-c-Jun, Sox-9 and COL2 Immunofluorescencewith six replicates showing the change in expression of p-JNK, c-Jun, p-c-Jun, Sox-9 and COL2 in the different groups.
Figure Legend Snippet: Immunofluorescence showed the change in expression of p-JNK, c-Jun, p-c-Jun, Sox-9 and COL2 Immunofluorescencewith six replicates showing the change in expression of p-JNK, c-Jun, p-c-Jun, Sox-9 and COL2 in the different groups.

Techniques Used: Immunofluorescence, Expressing

The expression of JNK, p-JNK, c-Jun, p-c-Jun, Sox-9 and COL2 in chondrocytes treated with different concentration of IL-1β (A) Western blot was used to JNK, p-JNK, c-Jun, p-c-Jun, Sox-9 and COL2 protein levels in chondrocytes treated with 0, 0.1, 1 and 10ng/ml IL-1β. Bands were quantified using Quantity One 5.0. (B) The mRNA levels of JNK, c-Jun, Sox-9 and COL2 in the chondrocytes. The 2 −ΔΔCt method was adopted with GAPDH as the reference gene. Bars represent the mean ± SD of each group (n = 6). As a control, the membrane was stripped and incubated with β-actin. Significant differences between the groups are marked with asterisks (* P
Figure Legend Snippet: The expression of JNK, p-JNK, c-Jun, p-c-Jun, Sox-9 and COL2 in chondrocytes treated with different concentration of IL-1β (A) Western blot was used to JNK, p-JNK, c-Jun, p-c-Jun, Sox-9 and COL2 protein levels in chondrocytes treated with 0, 0.1, 1 and 10ng/ml IL-1β. Bands were quantified using Quantity One 5.0. (B) The mRNA levels of JNK, c-Jun, Sox-9 and COL2 in the chondrocytes. The 2 −ΔΔCt method was adopted with GAPDH as the reference gene. Bars represent the mean ± SD of each group (n = 6). As a control, the membrane was stripped and incubated with β-actin. Significant differences between the groups are marked with asterisks (* P

Techniques Used: Expressing, Concentration Assay, Western Blot, Incubation

Inhibit JNK/c-Jun signaling pathway increase Sox-9 and COL2 expression (A) Western blot was used to evaluate the change of JNK, p-JNK, c-Jun, p-c-Jun, Sox-9 and COL2 protein levels in different groups. (B) Relative levels JNK, c-Jun, Sox-9 and COL2mRNA in the chondrocytes. The 2 −ΔΔCt method was adopted with GAPDH as the reference gene. Bars represent the mean ± SD of each group (n = 6). As a control, the membrane was stripped and incubated with β-actin. Significant differences between the groups are marked with asterisks (* P
Figure Legend Snippet: Inhibit JNK/c-Jun signaling pathway increase Sox-9 and COL2 expression (A) Western blot was used to evaluate the change of JNK, p-JNK, c-Jun, p-c-Jun, Sox-9 and COL2 protein levels in different groups. (B) Relative levels JNK, c-Jun, Sox-9 and COL2mRNA in the chondrocytes. The 2 −ΔΔCt method was adopted with GAPDH as the reference gene. Bars represent the mean ± SD of each group (n = 6). As a control, the membrane was stripped and incubated with β-actin. Significant differences between the groups are marked with asterisks (* P

Techniques Used: Expressing, Western Blot, Incubation

Western blot and RT-qRCR showed the change in expression of Sox-9 and COL2 with HBO treatment (A) Western blot was used to evaluate the change in expression of Sox-9 and COL2 protein levels in different groups. (B) Relative levels Sox-9 and COL2mRNA in the chondrocytes. The 2 −ΔΔCt method was adopted with GAPDH as the reference gene. Bars represent the mean ± SD of each group (n = 6). As a control, the membrane was stripped and incubated with β-actin. Significant differences between the groups are marked with asterisks (* P
Figure Legend Snippet: Western blot and RT-qRCR showed the change in expression of Sox-9 and COL2 with HBO treatment (A) Western blot was used to evaluate the change in expression of Sox-9 and COL2 protein levels in different groups. (B) Relative levels Sox-9 and COL2mRNA in the chondrocytes. The 2 −ΔΔCt method was adopted with GAPDH as the reference gene. Bars represent the mean ± SD of each group (n = 6). As a control, the membrane was stripped and incubated with β-actin. Significant differences between the groups are marked with asterisks (* P

Techniques Used: Western Blot, Expressing, Incubation

Western blot and RT-qRCR showed the change in expression of JNK/c-Jun signaling pathway, Sox-9 and COL2 (A) Western blot was used to evaluate the change in expression of JNK, p-JNK, c-Jun, p-c-Jun, Sox-9 and COL2 protein levels in different groups. (B) Relative levels JNK, c-Jun, Sox-9 and COL2mRNA in the chondrocytes. The 2 −ΔΔCt method was adopted with GAPDH as the reference gene. Bars represent the mean ± SD of each group (n = 6). As a control, the membrane was stripped and incubated with β-actin. Significant differences between the groups are marked with asterisks (* P
Figure Legend Snippet: Western blot and RT-qRCR showed the change in expression of JNK/c-Jun signaling pathway, Sox-9 and COL2 (A) Western blot was used to evaluate the change in expression of JNK, p-JNK, c-Jun, p-c-Jun, Sox-9 and COL2 protein levels in different groups. (B) Relative levels JNK, c-Jun, Sox-9 and COL2mRNA in the chondrocytes. The 2 −ΔΔCt method was adopted with GAPDH as the reference gene. Bars represent the mean ± SD of each group (n = 6). As a control, the membrane was stripped and incubated with β-actin. Significant differences between the groups are marked with asterisks (* P

Techniques Used: Western Blot, Expressing, Incubation

Immunohistochemistry and morphology identify of mandibular condylar chondrocytes Normal condylar chondrocytes special expressed COL2. (A) The morphology of normal chondrocytes cultured for 1, 4and 7 days was observed under a microscope. (B) The immunohistochemical staining shown that COL2 was positive in the chondrocytes.
Figure Legend Snippet: Immunohistochemistry and morphology identify of mandibular condylar chondrocytes Normal condylar chondrocytes special expressed COL2. (A) The morphology of normal chondrocytes cultured for 1, 4and 7 days was observed under a microscope. (B) The immunohistochemical staining shown that COL2 was positive in the chondrocytes.

Techniques Used: Immunohistochemistry, Cell Culture, Microscopy, Staining

36) Product Images from "Derivation of Neural Stem Cells from Human Adult Peripheral CD34+ Cells for an Autologous Model of Neuroinflammation"

Article Title: Derivation of Neural Stem Cells from Human Adult Peripheral CD34+ Cells for an Autologous Model of Neuroinflammation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0081720

Neurons with action potentials and glial cells differentiated from induced neural stem cells from adult peripheral CD34+ cells. The induced neural stem cells differentiated into βIII- tubulin positive neurons (green, Ai) in neuronal differentiation or GFAP positive astroglia (red, Aii) in astroglial differentiation medium for 1 week. Oligodendrocyte progenitor differentiation was achieved by incubating the neural stem cells with oligodendrocyte differentiation medium. O4 positive cells (green, Aiii, after 2 weeks) and a few of MBP positive cells (red, Aiv, after four weeks) were detected by immunostaining. Spontaneous neuronal differentiation was also observed in low concentration seeded neural stem cell culture (1000 cells/well in a 24 well plate) after incubation in neural stem cell medium for 1 week (Av). Action potentials were recorded from neurons differentiated for 2 weeks using whole cell patch clamp. Action potentials recorded on two separated cells are shown (Avi). Double staining with either VGLUT1 or VGAT with βIII- tubulin showed most βIII- tubulin positive neurons were either glutamatergic or gabaergic neurons while a few of doparminergic neurons were also detected by TH immunostaining after incubation in neuronal differentiation medium for 2 weeks (B). The induced neural stem cells were passaged for 17 passages and immunostained for nestin as a neural stem cell marker. The cells were also cultured in neural differentiation medium for 2 weeks and immunostained for βIII-tubulin to determine their neuronal differentiation capabilities (C).
Figure Legend Snippet: Neurons with action potentials and glial cells differentiated from induced neural stem cells from adult peripheral CD34+ cells. The induced neural stem cells differentiated into βIII- tubulin positive neurons (green, Ai) in neuronal differentiation or GFAP positive astroglia (red, Aii) in astroglial differentiation medium for 1 week. Oligodendrocyte progenitor differentiation was achieved by incubating the neural stem cells with oligodendrocyte differentiation medium. O4 positive cells (green, Aiii, after 2 weeks) and a few of MBP positive cells (red, Aiv, after four weeks) were detected by immunostaining. Spontaneous neuronal differentiation was also observed in low concentration seeded neural stem cell culture (1000 cells/well in a 24 well plate) after incubation in neural stem cell medium for 1 week (Av). Action potentials were recorded from neurons differentiated for 2 weeks using whole cell patch clamp. Action potentials recorded on two separated cells are shown (Avi). Double staining with either VGLUT1 or VGAT with βIII- tubulin showed most βIII- tubulin positive neurons were either glutamatergic or gabaergic neurons while a few of doparminergic neurons were also detected by TH immunostaining after incubation in neuronal differentiation medium for 2 weeks (B). The induced neural stem cells were passaged for 17 passages and immunostained for nestin as a neural stem cell marker. The cells were also cultured in neural differentiation medium for 2 weeks and immunostained for βIII-tubulin to determine their neuronal differentiation capabilities (C).

Techniques Used: Immunostaining, Concentration Assay, Stem Cell Culture, Incubation, Patch Clamp, Double Staining, Marker, Cell Culture

37) Product Images from "Modelling heme-mediated brain injury associated with cerebral malaria in human brain cortical organoids"

Article Title: Modelling heme-mediated brain injury associated with cerebral malaria in human brain cortical organoids

Journal: Scientific Reports

doi: 10.1038/s41598-019-55631-8

Heme treatment affects ERBB4 and NRG-1 expression in iPSCs. iPSCs were treated with heme for 18 hours. The number of cells positive for ERBB4 was assessed using flow cytometry analysis. The ERBB4 and endogenous NRG-1 (eNRG-1) protein expression levels were determined using Western blotting. ( A ) Representative flow cytometry results illustrating the percent of iPSCs that express ERBB4. ( B ) The number of cells positive for ERBB4 increased as the heme-induced injury level increased. ( C ) Cell lysates from iPSCs treated with various heme doses were immunoblotted for ERBB4 and NRG-1. Western blot densitometry analysis was performed using the ImageJ program (full blots are shown in the Supplementary Information file, Supplementary Fig. S1 ). The results show that NRG-1 expression levels increased as the heme concentration increased, while ERBB4 expression decreased in a similar manner (a: p
Figure Legend Snippet: Heme treatment affects ERBB4 and NRG-1 expression in iPSCs. iPSCs were treated with heme for 18 hours. The number of cells positive for ERBB4 was assessed using flow cytometry analysis. The ERBB4 and endogenous NRG-1 (eNRG-1) protein expression levels were determined using Western blotting. ( A ) Representative flow cytometry results illustrating the percent of iPSCs that express ERBB4. ( B ) The number of cells positive for ERBB4 increased as the heme-induced injury level increased. ( C ) Cell lysates from iPSCs treated with various heme doses were immunoblotted for ERBB4 and NRG-1. Western blot densitometry analysis was performed using the ImageJ program (full blots are shown in the Supplementary Information file, Supplementary Fig. S1 ). The results show that NRG-1 expression levels increased as the heme concentration increased, while ERBB4 expression decreased in a similar manner (a: p

Techniques Used: Expressing, Flow Cytometry, Cytometry, Western Blot, Concentration Assay

Heme-induced apoptosis of iPSCs is diminished by NRG-1. iPSCs were treated with heme for 8 hours, and NRG-1 was then added for 18 hours. Cell viability was determined using the CCK-8 assay ( A ), while the apoptotic/necrotic cell number was assessed using flow cytometry analysis ( B , C ). ( A ) Cell viability decreased significantly when iPSCs were treated with heme. NRG-1 reduced the effects of heme. ( B ) The number of apoptotic cells increased with heme compared to basal conditions. The addition of NRG-1 to heme treatment decreased not only the number of apoptotic but also the number of necrotic cells. CPT was used as a positive control for apoptosis. The data shown are the mean values + standard error (SE) (n = 3 experiments) (a: p
Figure Legend Snippet: Heme-induced apoptosis of iPSCs is diminished by NRG-1. iPSCs were treated with heme for 8 hours, and NRG-1 was then added for 18 hours. Cell viability was determined using the CCK-8 assay ( A ), while the apoptotic/necrotic cell number was assessed using flow cytometry analysis ( B , C ). ( A ) Cell viability decreased significantly when iPSCs were treated with heme. NRG-1 reduced the effects of heme. ( B ) The number of apoptotic cells increased with heme compared to basal conditions. The addition of NRG-1 to heme treatment decreased not only the number of apoptotic but also the number of necrotic cells. CPT was used as a positive control for apoptosis. The data shown are the mean values + standard error (SE) (n = 3 experiments) (a: p

Techniques Used: CCK-8 Assay, Flow Cytometry, Cytometry, Cycling Probe Technology, Positive Control

Heme induces NRG-1 and reduces ERBB4 expression in the forebrain structures of human organoids (20 days in culture). Cortical organoids were treated with heme for 8 hours, and NRG-1 was then added for 18 hours. The results showed that, compared to no treatment (a), heme induced NRG-1 expression. (b) The addition of NRG-1 to the treatment reduced its endogenous expression due to an improvement in cell injury. (c) ERBB4 expression decreased with heme treatment (e) compared to no treatment. (d) The addition of NRG-1 increased the levels of ERBB4 (f) compared to that induced by heme alone (e) but not compared to basal levels. (d) Scale bar: 20 µm.
Figure Legend Snippet: Heme induces NRG-1 and reduces ERBB4 expression in the forebrain structures of human organoids (20 days in culture). Cortical organoids were treated with heme for 8 hours, and NRG-1 was then added for 18 hours. The results showed that, compared to no treatment (a), heme induced NRG-1 expression. (b) The addition of NRG-1 to the treatment reduced its endogenous expression due to an improvement in cell injury. (c) ERBB4 expression decreased with heme treatment (e) compared to no treatment. (d) The addition of NRG-1 increased the levels of ERBB4 (f) compared to that induced by heme alone (e) but not compared to basal levels. (d) Scale bar: 20 µm.

Techniques Used: Expressing

Heme induces inflammation in cortical organoids (40 days in culture). Forebrain organoids were treated with heme for 8 hours and then NRG-1 for 18 hours. Representative images of organoids under heme and NRG-1 treatment ( A ) showing the expression of the chemokine CXCL-10, its receptor CXCR3, and BDNF in the VZ, SVZ, IZ and CP of the cortical organoids. The quantification of the staining intensity showed that heme significantly increased the expression of CXCL-10, CXCR3 and BDNF, while NRG-1 significantly reduced this effect ( B ) in all zones of the forebrain organoids.
Figure Legend Snippet: Heme induces inflammation in cortical organoids (40 days in culture). Forebrain organoids were treated with heme for 8 hours and then NRG-1 for 18 hours. Representative images of organoids under heme and NRG-1 treatment ( A ) showing the expression of the chemokine CXCL-10, its receptor CXCR3, and BDNF in the VZ, SVZ, IZ and CP of the cortical organoids. The quantification of the staining intensity showed that heme significantly increased the expression of CXCL-10, CXCR3 and BDNF, while NRG-1 significantly reduced this effect ( B ) in all zones of the forebrain organoids.

Techniques Used: Expressing, Staining

Human cortical organoids express ERBB4 and NRG-1. ( A ) The development of human cortical organoids. (a) Embryoid bodies. (b) Induction towards a neuronal fate. (c) The development of neuroepithelia. (d,e). Organoid maturation. (f) Forebrain structure within organoids (arrow) becomes evident after 40 days. ( B ) Representative sections of whole 20-day cortical organoids (a) and the inside of (b) cortical organoids stained for FOXG1 showing forebrain identity with structures displayed around the organoid core. At 40 days (c), FOXG1 staining was similar. SOX-2 (a neural stem cell marker) (red) and TUJ1 (a neuronal specific marker) (green) staining in cortical organoids at 20 (d) and 40 (e) days. ( C ) Cortical organoids expressed NRG-1 (a,b) and ERBB4 (c,d) at 20 and 40 days, respectively. Scale bar: 20 µm.
Figure Legend Snippet: Human cortical organoids express ERBB4 and NRG-1. ( A ) The development of human cortical organoids. (a) Embryoid bodies. (b) Induction towards a neuronal fate. (c) The development of neuroepithelia. (d,e). Organoid maturation. (f) Forebrain structure within organoids (arrow) becomes evident after 40 days. ( B ) Representative sections of whole 20-day cortical organoids (a) and the inside of (b) cortical organoids stained for FOXG1 showing forebrain identity with structures displayed around the organoid core. At 40 days (c), FOXG1 staining was similar. SOX-2 (a neural stem cell marker) (red) and TUJ1 (a neuronal specific marker) (green) staining in cortical organoids at 20 (d) and 40 (e) days. ( C ) Cortical organoids expressed NRG-1 (a,b) and ERBB4 (c,d) at 20 and 40 days, respectively. Scale bar: 20 µm.

Techniques Used: Staining, Marker

Heme modifies 20-day-old cortical organoid architecture by inducing early apoptosis and necrosis in its cells. Cortical organoids were treated with heme for 8 hours, and NRG-1 was then added for 18 hours. CPT treatment was used as a positive control for cell apoptosis. The results showed ( A ) that, compared to no treatment (a), heme induced the disorganization of cerebral organoid structure (b). NRG-1 improved the heme-induced lesion (c). CPT, used as a positive control for apoptosis, induced similar disorganization of cortical organoids (d). In addition, the apoptosis assay showed that heme increased cell necrosis in cortical organoids ( B ), while NRG-1 treatment reduced the injury. Representative images of cortical organoids ( C ) reinforcing the conclusion that heme had a toxic effect (b) compared to basal conditions, while NRG-1 had a protective role (d) compared to heme treatment (b). CPT also induced cell death compared to untreated condition (c) (a: p
Figure Legend Snippet: Heme modifies 20-day-old cortical organoid architecture by inducing early apoptosis and necrosis in its cells. Cortical organoids were treated with heme for 8 hours, and NRG-1 was then added for 18 hours. CPT treatment was used as a positive control for cell apoptosis. The results showed ( A ) that, compared to no treatment (a), heme induced the disorganization of cerebral organoid structure (b). NRG-1 improved the heme-induced lesion (c). CPT, used as a positive control for apoptosis, induced similar disorganization of cortical organoids (d). In addition, the apoptosis assay showed that heme increased cell necrosis in cortical organoids ( B ), while NRG-1 treatment reduced the injury. Representative images of cortical organoids ( C ) reinforcing the conclusion that heme had a toxic effect (b) compared to basal conditions, while NRG-1 had a protective role (d) compared to heme treatment (b). CPT also induced cell death compared to untreated condition (c) (a: p

Techniques Used: Cycling Probe Technology, Positive Control, Apoptosis Assay

38) Product Images from "Cochlear epithelial of dog fetuses: a new source of multipotent stem cells"

Article Title: Cochlear epithelial of dog fetuses: a new source of multipotent stem cells

Journal: Cytotechnology

doi: 10.1007/s10616-016-0049-0

Immunocytochemistry analyses of dog fetal cochlea cells with 40 days gestation. A , B Expression of mesenchymal stem cell markers: Stro-1 and CD 73, respectively. c Expression for vimentin. D – F Expression of neuronal markers: β-tubulin, myosin VIIA and nestin, respectively. H – J Expression of pluripotent stem cell markers: Oct4, Nanog and Sox 2, respectively
Figure Legend Snippet: Immunocytochemistry analyses of dog fetal cochlea cells with 40 days gestation. A , B Expression of mesenchymal stem cell markers: Stro-1 and CD 73, respectively. c Expression for vimentin. D – F Expression of neuronal markers: β-tubulin, myosin VIIA and nestin, respectively. H – J Expression of pluripotent stem cell markers: Oct4, Nanog and Sox 2, respectively

Techniques Used: Immunocytochemistry, Expressing

39) Product Images from "Klotho regulates postnatal neurogenesis and protects against age-related spatial memory loss"

Article Title: Klotho regulates postnatal neurogenesis and protects against age-related spatial memory loss

Journal: Neurobiology of aging

doi: 10.1016/j.neurobiolaging.2017.07.008

KL-deficiency reduces proliferation of neurogenic precursors A,B . WT and KO 2, 3, and 7 week stereological quantification and representative 3 week IHC of SGZ proliferating cells (Ki67). Cell nuclei labeled with DAPI. Scale bar represents 20μm. C . 3 week old WT and KO mice received 1 BrdU injection to measure cell cycle length (BrdU + +Ki67 + /BrdU + ) and cell cycle re-entry (BrdU + +Ki67 + /Ki67 + ). D . Adult hippocampus and 14 day in vitro, primary neurosphere WT qPCR normalized to the 18S ribosomal subunit and adult hippocampus (HCX). E. Choroid plexus and hippocampal representative WT KL IHC (green) and DAPI (blue) (left) with white dashed box indicating location of higher magnification images (right). Scale bar represents 100μm or 20μm. F. Representative confocal Z stack WT SGZ images of KL (red) and GFAP (green). Nuclei not shown for clarity and scale bar represents 10 μm. G. Representative confocal Z stacks WT SGZ images of KL (red) and Nestin-GFP (green) from Nestin-GFP WT mice. Nuclei not shown for clarity and scale bar represents 10 μm. H. Representative confocal Z stacks WT SGZ images of KL (red) and Sox2 (green) from WT mice. Nuclei not shown for clarity and scale bar represents 10 μm. I. Ten days after plating 500 cells/well, the number and diameter (μm) of WT/KO spheres was measured. Separate wells of KO cells received recombinant mouse KL (rKL) at plating. J. EdU was added to the media overly laying adherent cells and proliferation measured as the % of cells that were EdU+. K. Primary spheres (C) were re-plated as single cells to measure secondary sphere number and diameter (μm) 10 days later. (n=4–6 in vivo ; NSPs: n=3 independent NSP preparations; F,H are the average of all wells counted +/− S.E.M., G is the average of 4 fields per coverslip; T-test (A,C,D): **p
Figure Legend Snippet: KL-deficiency reduces proliferation of neurogenic precursors A,B . WT and KO 2, 3, and 7 week stereological quantification and representative 3 week IHC of SGZ proliferating cells (Ki67). Cell nuclei labeled with DAPI. Scale bar represents 20μm. C . 3 week old WT and KO mice received 1 BrdU injection to measure cell cycle length (BrdU + +Ki67 + /BrdU + ) and cell cycle re-entry (BrdU + +Ki67 + /Ki67 + ). D . Adult hippocampus and 14 day in vitro, primary neurosphere WT qPCR normalized to the 18S ribosomal subunit and adult hippocampus (HCX). E. Choroid plexus and hippocampal representative WT KL IHC (green) and DAPI (blue) (left) with white dashed box indicating location of higher magnification images (right). Scale bar represents 100μm or 20μm. F. Representative confocal Z stack WT SGZ images of KL (red) and GFAP (green). Nuclei not shown for clarity and scale bar represents 10 μm. G. Representative confocal Z stacks WT SGZ images of KL (red) and Nestin-GFP (green) from Nestin-GFP WT mice. Nuclei not shown for clarity and scale bar represents 10 μm. H. Representative confocal Z stacks WT SGZ images of KL (red) and Sox2 (green) from WT mice. Nuclei not shown for clarity and scale bar represents 10 μm. I. Ten days after plating 500 cells/well, the number and diameter (μm) of WT/KO spheres was measured. Separate wells of KO cells received recombinant mouse KL (rKL) at plating. J. EdU was added to the media overly laying adherent cells and proliferation measured as the % of cells that were EdU+. K. Primary spheres (C) were re-plated as single cells to measure secondary sphere number and diameter (μm) 10 days later. (n=4–6 in vivo ; NSPs: n=3 independent NSP preparations; F,H are the average of all wells counted +/− S.E.M., G is the average of 4 fields per coverslip; T-test (A,C,D): **p

Techniques Used: Immunohistochemistry, Labeling, Mouse Assay, Injection, In Vitro, Real-time Polymerase Chain Reaction, Recombinant, In Vivo

40) Product Images from "Isolation and Characterization of Neural Stem Cells from the Rat Inferior Colliculus"

Article Title: Isolation and Characterization of Neural Stem Cells from the Rat Inferior Colliculus

Journal: Stem Cells International

doi: 10.1155/2019/5831240

Results of the immunocytochemical analysis of the plated single cells of IC NSC cultures from PND6 rats. The proportions of positively stained cells in relation to the total number of cells on coverslips were evaluated. The boxplots show mean and SEM. (a) Cell proportions that are positive for the analyzed progenitor cell markers after 24 hours on glass coverslips in NSC medium (b) Differentiated cells after 6 days on glass coverslips in DIF medium. Differentiated cell types could be identified morphologically and by immunohistochemical markers of the neuroectodermal lineage. β -III-Tubulin stains cells that differentiate into the neuronal lineage, GFAP stains those that differentiate into the astroglial lineage, and MBP stains those that differentiate into the oligodendrocyte lineage.
Figure Legend Snippet: Results of the immunocytochemical analysis of the plated single cells of IC NSC cultures from PND6 rats. The proportions of positively stained cells in relation to the total number of cells on coverslips were evaluated. The boxplots show mean and SEM. (a) Cell proportions that are positive for the analyzed progenitor cell markers after 24 hours on glass coverslips in NSC medium (b) Differentiated cells after 6 days on glass coverslips in DIF medium. Differentiated cell types could be identified morphologically and by immunohistochemical markers of the neuroectodermal lineage. β -III-Tubulin stains cells that differentiate into the neuronal lineage, GFAP stains those that differentiate into the astroglial lineage, and MBP stains those that differentiate into the oligodendrocyte lineage.

Techniques Used: Staining, Immunohistochemistry

Differentiated cells from IC. NSCs after 6 days on glass coverslips in differentiation medium (DIF). (a) β -III-Tubulin (red) marked neuronal differentiated cells. The β -III-tubulin-positive cells had neuron-typical spindled, slender somata with bipolar axon configuration. (b) Astrocytes were identified by the staining of glial fibrillary acidic protein (GFAP, red). These cells showed stellate-configured branches, which is typical for astrocytes. (c) Oligodendrocytes showed positive labelling of myelin basic protein (MBP, red). Typically for oligodendrocytes, they had a peripheral-starting myelination, which is characterized by MBP staining. The proximal portions are β -tubulin-positive (green). The extensive, diversified growth of the branches is a morphological sign for oligodendrocytes. (d) Cells that were still in the undifferentiated progenitor cell stage were stained by Nestin (red). These cells had a very similar morphology to that of the GFAP-positive, astrocyte-differentiated cells. They have multipolar branches, which are labelled positively for Nestin. The cytoskeleton of all viable cells was stained by β -tubulin (green). Cell nuclei were stained with DAPI (blue).
Figure Legend Snippet: Differentiated cells from IC. NSCs after 6 days on glass coverslips in differentiation medium (DIF). (a) β -III-Tubulin (red) marked neuronal differentiated cells. The β -III-tubulin-positive cells had neuron-typical spindled, slender somata with bipolar axon configuration. (b) Astrocytes were identified by the staining of glial fibrillary acidic protein (GFAP, red). These cells showed stellate-configured branches, which is typical for astrocytes. (c) Oligodendrocytes showed positive labelling of myelin basic protein (MBP, red). Typically for oligodendrocytes, they had a peripheral-starting myelination, which is characterized by MBP staining. The proximal portions are β -tubulin-positive (green). The extensive, diversified growth of the branches is a morphological sign for oligodendrocytes. (d) Cells that were still in the undifferentiated progenitor cell stage were stained by Nestin (red). These cells had a very similar morphology to that of the GFAP-positive, astrocyte-differentiated cells. They have multipolar branches, which are labelled positively for Nestin. The cytoskeleton of all viable cells was stained by β -tubulin (green). Cell nuclei were stained with DAPI (blue).

Techniques Used: Staining

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Article Title: Silencing of heart and neural crest derivatives expressed transcript 2 attenuates transforming growth factor-β1-enhanced apoptosis of human bronchial epithelial cells
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other:

Article Title: Stemness and inducing differentiation of small cell lung cancer NCI-H446 cells
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  • 85
    Abcam anti sox 2 mouse
    Immunostaining of TMA. Staining for Oct-4, <t>Sox-2,</t> beta Catenin and Nucleophosim on DAOY, ONS76 and UW228 cell lines grown as spheres are shown at 40x magnification. UW228 (second column) show smaller spheres and less intense staining for Oct-4 and Sox-2.
    Anti Sox 2 Mouse, supplied by Abcam, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti sox 2 mouse/product/Abcam
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    Abcam mouse anti sox 2 antibody
    Relative expression of miR-34a, b and c in OSCs and U-2OS cell monolayers, detected using SYBR Green reverse transcription-quantitative polymerase chain reaction analysis. (A) Culture of OSCs in serum-free medium on days 3, 10 and 14. (B) Representative photomicrographs reveal the staining of marker antigens <t>Sox-2</t> (red) and Sca-1 (green). (C) Average expression levels of miR-34a, b and c in OSCs, compared with those in monolayer cells. *P
    Mouse Anti Sox 2 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam rabbit anti sox 2
    Nr2e1 frc/frc Müller glia misexpressed Brn3a cell-autonomously . Transverse retinal sections from P7 and P21 Nr2e1 +/+ and Nr2e1 frc/frc mice and P7 chimeras were immunostained for <t>SOX-2,</t> Brn3a, and Vimentin. a I n P7 wild-type retinas, ganglion cells labeled with Brn3a (green) appeared as a different population from Müller glia labeled with SOX-2 (red, bracket). b In Nr2e1 frc/frc retinas, the marker Brn3a (green) was misexpressed in Müller-glia somas (red, bracket), and in processes that branch in the EPL and IPL, with termini in the GCL that resemble end-feet. c - f Immunostaining for the Müller-glia markers vimentin (red) and Brn3a (green) in P7 and P21 retinas. In P7 retinas, these markers did not co-express in ( c ) Nr2e1 +/+ retinas but they did in ( d ) Nr2e1 frc/frc retinas. This also occurred in P21 retinas where the markers did not co-express in ( e ) Nr2e1 +/+ retinas but they did in ( f ) Nr2e1 frc/frc retinas. d , f Inset boxes show a magnified view of the vimentin and Brn3a co-localization in Nr2e1 frc/frc Müller-glial processes. g In P7 Wt↔ frc chimeras, misexpression of Brn3a (red) in Müller glia was seen in Nr2e1 -mutant cells (EGFP positive, green) throughout the cell soma and processes, however, this was not seen in wild-type cells (EGFP negative) where Brn3a was only expressed in ganglion cells. Representative images of a 71 % Wt↔Wt and a 58 % Wt↔ frc chimeric retina are shown. n = 3 for Nr2e1 +/+ , n = 3 for Nr2e1 frc/frc , n = 4 for Wt↔Wt, n = 4 for Wt↔ frc ; EPL, Ectopic plexiform layer; GCL, ganglion cell layer; INL, inner nuclear layer; OPL, outer plexiform layer; Hoechst, nuclear counterstain (blue); scale bar = 50 μm
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    Immunostaining of TMA. Staining for Oct-4, Sox-2, beta Catenin and Nucleophosim on DAOY, ONS76 and UW228 cell lines grown as spheres are shown at 40x magnification. UW228 (second column) show smaller spheres and less intense staining for Oct-4 and Sox-2.

    Journal: PLoS ONE

    Article Title: Medullospheres from DAOY, UW228 and ONS-76 Cells: Increased Stem Cell Population and Proteomic Modifications

    doi: 10.1371/journal.pone.0063748

    Figure Lengend Snippet: Immunostaining of TMA. Staining for Oct-4, Sox-2, beta Catenin and Nucleophosim on DAOY, ONS76 and UW228 cell lines grown as spheres are shown at 40x magnification. UW228 (second column) show smaller spheres and less intense staining for Oct-4 and Sox-2.

    Article Snippet: Anti-CD133, anti-Oct4, anti-Nanog, anti-Nestin, anti-Sox-2 mouse and goat polyclonal secondary anti-rabbit IgG H & L FITC-conjugated were purchased all from AbCam (Cambridge, UK).

    Techniques: Immunostaining, Staining

    Characterization of nestin-positive cells in the CA1 hippocampal region after transient forebrain ischemia. (A-F) Double-labeling with nestin and Ki-67 showed that some nestin-positive cells exhibited Ki-67-labeled nuclei (arrowheads) on days 3 (A-C) and 14 (D-F) after ischemia. pcl, pyramidal cell layer; sr, stratum radiatum. (G-L) Double-labeling with nestin and the transcription factors Sox-2 (G-I) or Sox-9 (J-L). Note that almost all nestin-positive cells expressed Sox-2- or Sox-9-labeled nuclei at days 3 (H, K) and 14 (I, L) after ischemia. Also note that Sox-2- or Sox-9-positive nuclei were also observed in the control hippocampus (G, J), where no nestin expression was observed. Scale bars in (C, F, I, L)=100 µm (A-L).

    Journal: Anatomy & Cell Biology

    Article Title: Characterization of nestin expression in astrocytes in the rat hippocampal CA1 region following transient forebrain ischemia

    doi: 10.5115/acb.2013.46.2.131

    Figure Lengend Snippet: Characterization of nestin-positive cells in the CA1 hippocampal region after transient forebrain ischemia. (A-F) Double-labeling with nestin and Ki-67 showed that some nestin-positive cells exhibited Ki-67-labeled nuclei (arrowheads) on days 3 (A-C) and 14 (D-F) after ischemia. pcl, pyramidal cell layer; sr, stratum radiatum. (G-L) Double-labeling with nestin and the transcription factors Sox-2 (G-I) or Sox-9 (J-L). Note that almost all nestin-positive cells expressed Sox-2- or Sox-9-labeled nuclei at days 3 (H, K) and 14 (I, L) after ischemia. Also note that Sox-2- or Sox-9-positive nuclei were also observed in the control hippocampus (G, J), where no nestin expression was observed. Scale bars in (C, F, I, L)=100 µm (A-L).

    Article Snippet: After blocking with 10% normal goat serum for 1 hour, the sections were incubated with overnight at 4℃ with a mix of monoclonal mouse anti-nestin antibody (1:500, Serotec, Oxford, UK) and one of following antibodies: polyclonal rabbit anti-GFAP (1:1,500, Millipore Corp., Temecula, CA, USA), polyclonal rabbit anti-Sox-9 (1:100, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), polyclonal rabbit anti-Ki-67 (1:1,000, Novocastra Laboratories Ltd., Newcastle-upon-Tyne, UK), polyclonal rabbit anti-S100β (1:1,000, Dako, Glostrup, Denmark) or monoclonal mouse anti-Sox-2 (1:400, Abcam, Cambridge, UK).

    Techniques: Labeling, Expressing

    Relative expression of miR-34a, b and c in OSCs and U-2OS cell monolayers, detected using SYBR Green reverse transcription-quantitative polymerase chain reaction analysis. (A) Culture of OSCs in serum-free medium on days 3, 10 and 14. (B) Representative photomicrographs reveal the staining of marker antigens Sox-2 (red) and Sca-1 (green). (C) Average expression levels of miR-34a, b and c in OSCs, compared with those in monolayer cells. *P

    Journal: Molecular Medicine Reports

    Article Title: miR-34a is downregulated in human osteosarcoma stem-like cells and promotes invasion, tumorigenic ability and self-renewal capacity

    doi: 10.3892/mmr.2017.6187

    Figure Lengend Snippet: Relative expression of miR-34a, b and c in OSCs and U-2OS cell monolayers, detected using SYBR Green reverse transcription-quantitative polymerase chain reaction analysis. (A) Culture of OSCs in serum-free medium on days 3, 10 and 14. (B) Representative photomicrographs reveal the staining of marker antigens Sox-2 (red) and Sca-1 (green). (C) Average expression levels of miR-34a, b and c in OSCs, compared with those in monolayer cells. *P

    Article Snippet: Membranes were blocked with 5% BSA (Sigma-Aldrich, Merck Millipore) in TBST (20 mM Tris, 150 mM NaCl, containing 0.3% Tween-20, pH 7.4) for 30 min. Membranes were then incubated with mouse anti-SOX-2 antibody (catalog no. ab171380; 1:1,000; Abcam) or mouse anti β-actin antibody (catalog no. ab8227; 1:1,000; Abcam) in TBST with 1% BSA (Sigma-Aldrich, Merck Millipore) at room temperature for 1 h. Following 3 washes with TBST, the membranes was incubated with anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (catalog no. ab6785; 1:5,000; Abcam) for 1 h at room temperature.

    Techniques: Expressing, SYBR Green Assay, Real-time Polymerase Chain Reaction, Staining, Marker

    Sox-2 and Sca-1 are expressed at low levels in osteosarcoma-derived form U-2OS cells. (A) mRNA (left) and protein (right) levels of Sox-2 were analyzed using RT-qPCR and semiquantitative western blot analyses. *P

    Journal: Molecular Medicine Reports

    Article Title: miR-34a is downregulated in human osteosarcoma stem-like cells and promotes invasion, tumorigenic ability and self-renewal capacity

    doi: 10.3892/mmr.2017.6187

    Figure Lengend Snippet: Sox-2 and Sca-1 are expressed at low levels in osteosarcoma-derived form U-2OS cells. (A) mRNA (left) and protein (right) levels of Sox-2 were analyzed using RT-qPCR and semiquantitative western blot analyses. *P

    Article Snippet: Membranes were blocked with 5% BSA (Sigma-Aldrich, Merck Millipore) in TBST (20 mM Tris, 150 mM NaCl, containing 0.3% Tween-20, pH 7.4) for 30 min. Membranes were then incubated with mouse anti-SOX-2 antibody (catalog no. ab171380; 1:1,000; Abcam) or mouse anti β-actin antibody (catalog no. ab8227; 1:1,000; Abcam) in TBST with 1% BSA (Sigma-Aldrich, Merck Millipore) at room temperature for 1 h. Following 3 washes with TBST, the membranes was incubated with anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (catalog no. ab6785; 1:5,000; Abcam) for 1 h at room temperature.

    Techniques: Derivative Assay, Quantitative RT-PCR, Western Blot

    Nr2e1 frc/frc Müller glia misexpressed Brn3a cell-autonomously . Transverse retinal sections from P7 and P21 Nr2e1 +/+ and Nr2e1 frc/frc mice and P7 chimeras were immunostained for SOX-2, Brn3a, and Vimentin. a I n P7 wild-type retinas, ganglion cells labeled with Brn3a (green) appeared as a different population from Müller glia labeled with SOX-2 (red, bracket). b In Nr2e1 frc/frc retinas, the marker Brn3a (green) was misexpressed in Müller-glia somas (red, bracket), and in processes that branch in the EPL and IPL, with termini in the GCL that resemble end-feet. c - f Immunostaining for the Müller-glia markers vimentin (red) and Brn3a (green) in P7 and P21 retinas. In P7 retinas, these markers did not co-express in ( c ) Nr2e1 +/+ retinas but they did in ( d ) Nr2e1 frc/frc retinas. This also occurred in P21 retinas where the markers did not co-express in ( e ) Nr2e1 +/+ retinas but they did in ( f ) Nr2e1 frc/frc retinas. d , f Inset boxes show a magnified view of the vimentin and Brn3a co-localization in Nr2e1 frc/frc Müller-glial processes. g In P7 Wt↔ frc chimeras, misexpression of Brn3a (red) in Müller glia was seen in Nr2e1 -mutant cells (EGFP positive, green) throughout the cell soma and processes, however, this was not seen in wild-type cells (EGFP negative) where Brn3a was only expressed in ganglion cells. Representative images of a 71 % Wt↔Wt and a 58 % Wt↔ frc chimeric retina are shown. n = 3 for Nr2e1 +/+ , n = 3 for Nr2e1 frc/frc , n = 4 for Wt↔Wt, n = 4 for Wt↔ frc ; EPL, Ectopic plexiform layer; GCL, ganglion cell layer; INL, inner nuclear layer; OPL, outer plexiform layer; Hoechst, nuclear counterstain (blue); scale bar = 50 μm

    Journal: Molecular Brain

    Article Title: Nr2e1 regulates retinal lamination and the development of Müller glia, S-cones, and glycineric amacrine cells during retinogenesis

    doi: 10.1186/s13041-015-0126-x

    Figure Lengend Snippet: Nr2e1 frc/frc Müller glia misexpressed Brn3a cell-autonomously . Transverse retinal sections from P7 and P21 Nr2e1 +/+ and Nr2e1 frc/frc mice and P7 chimeras were immunostained for SOX-2, Brn3a, and Vimentin. a I n P7 wild-type retinas, ganglion cells labeled with Brn3a (green) appeared as a different population from Müller glia labeled with SOX-2 (red, bracket). b In Nr2e1 frc/frc retinas, the marker Brn3a (green) was misexpressed in Müller-glia somas (red, bracket), and in processes that branch in the EPL and IPL, with termini in the GCL that resemble end-feet. c - f Immunostaining for the Müller-glia markers vimentin (red) and Brn3a (green) in P7 and P21 retinas. In P7 retinas, these markers did not co-express in ( c ) Nr2e1 +/+ retinas but they did in ( d ) Nr2e1 frc/frc retinas. This also occurred in P21 retinas where the markers did not co-express in ( e ) Nr2e1 +/+ retinas but they did in ( f ) Nr2e1 frc/frc retinas. d , f Inset boxes show a magnified view of the vimentin and Brn3a co-localization in Nr2e1 frc/frc Müller-glial processes. g In P7 Wt↔ frc chimeras, misexpression of Brn3a (red) in Müller glia was seen in Nr2e1 -mutant cells (EGFP positive, green) throughout the cell soma and processes, however, this was not seen in wild-type cells (EGFP negative) where Brn3a was only expressed in ganglion cells. Representative images of a 71 % Wt↔Wt and a 58 % Wt↔ frc chimeric retina are shown. n = 3 for Nr2e1 +/+ , n = 3 for Nr2e1 frc/frc , n = 4 for Wt↔Wt, n = 4 for Wt↔ frc ; EPL, Ectopic plexiform layer; GCL, ganglion cell layer; INL, inner nuclear layer; OPL, outer plexiform layer; Hoechst, nuclear counterstain (blue); scale bar = 50 μm

    Article Snippet: Molday, University of British Columbia (Vancouver, BC, Canada); sheep anti-Chx10 from Exalpha Biologicals, Inc. (Shirley, MA, USA); rabbit anti-cone-arrestin, rabbit anti-Sox9, rabbit anti-S-opsin, rabbit anti-mGluR1, goat anti-calretinin, chicken anti-vimentin, and guinea pig anti-GABA from Millipore (Billerica, MA, USA); mouse anti-PKCalpha, chicken anti-β-galactosidase, and rabbit anti-SOX-2 and anti-GlyT1 from Abcam (Cambridge, England); and mouse anti-GFAP from New England Biolabs (Ipswich, MA, USA).

    Techniques: Mouse Assay, Labeling, Marker, Immunostaining, Mutagenesis

    Nr2e1 frc/frc Müller glia cells were aberrantly positioned in the INL . Transverse retinal sections from P7 Nr2e1 +/+ , Nr2e1 frc/frc , and chimeric mice were immunostained for SOX-2 and Islet-1/2. a Nr2e1 +/+ SOX-2 positive Müller-glia somas (red, bracket) were localized between cholinergic amacrines in the lower INL and ON bipolars in the upper INL, the latter two cell types both labeled with Islet-1/2 (green). Müller-glia somas localized close to the middle of the INL and away from the OPL. b Nr2e1 frc/frc Müller-glia somas (red, bracket) intermingled with ON bipolars (green) and localized adjacent to the OPL. c In Wt↔ frc chimeras, the mispositioning of the Müller-glia soma was seen only in Nr2e1 -mutant cells (EGFP positive, green), which were located closer to the OPL compared to wild-type cells (EGFP negative). Representative images of a 51 % Wt↔Wt and a 58 % Wt↔ frc chimeric retina are shown. d,e Magnification of the boxes in ( c ) showing a region from the ( d ) Wt↔Wt chimera depicting Müller glia located close to the middle of the INL, and from the ( e ) Wt↔ frc chimera depicting mutant Müller glia located adjacent to the OPL. The dotted lines represent an imaginary boundary above which most wild-type cells localize. Note that in a Wt↔ frc chimera, most Nr2e1 -mutant cells (EGFP positive) localize under this line close to the OPL and most wild-type cells (EGFP negative) localize above this line and away from the OPL. n = 3 for Nr2e1 +/+ , n = 3 for Nr2e1 frc/frc , n = 4 for Wt↔Wt, n = 4 for Wt↔ frc ; EPL, Ectopic plexiform layer; GCL, ganglion cell layer; INL, inner nuclear layer; OPL, outer plexiform layer; Hoechst, nuclear counterstain (blue); scale bar = 50 μm

    Journal: Molecular Brain

    Article Title: Nr2e1 regulates retinal lamination and the development of Müller glia, S-cones, and glycineric amacrine cells during retinogenesis

    doi: 10.1186/s13041-015-0126-x

    Figure Lengend Snippet: Nr2e1 frc/frc Müller glia cells were aberrantly positioned in the INL . Transverse retinal sections from P7 Nr2e1 +/+ , Nr2e1 frc/frc , and chimeric mice were immunostained for SOX-2 and Islet-1/2. a Nr2e1 +/+ SOX-2 positive Müller-glia somas (red, bracket) were localized between cholinergic amacrines in the lower INL and ON bipolars in the upper INL, the latter two cell types both labeled with Islet-1/2 (green). Müller-glia somas localized close to the middle of the INL and away from the OPL. b Nr2e1 frc/frc Müller-glia somas (red, bracket) intermingled with ON bipolars (green) and localized adjacent to the OPL. c In Wt↔ frc chimeras, the mispositioning of the Müller-glia soma was seen only in Nr2e1 -mutant cells (EGFP positive, green), which were located closer to the OPL compared to wild-type cells (EGFP negative). Representative images of a 51 % Wt↔Wt and a 58 % Wt↔ frc chimeric retina are shown. d,e Magnification of the boxes in ( c ) showing a region from the ( d ) Wt↔Wt chimera depicting Müller glia located close to the middle of the INL, and from the ( e ) Wt↔ frc chimera depicting mutant Müller glia located adjacent to the OPL. The dotted lines represent an imaginary boundary above which most wild-type cells localize. Note that in a Wt↔ frc chimera, most Nr2e1 -mutant cells (EGFP positive) localize under this line close to the OPL and most wild-type cells (EGFP negative) localize above this line and away from the OPL. n = 3 for Nr2e1 +/+ , n = 3 for Nr2e1 frc/frc , n = 4 for Wt↔Wt, n = 4 for Wt↔ frc ; EPL, Ectopic plexiform layer; GCL, ganglion cell layer; INL, inner nuclear layer; OPL, outer plexiform layer; Hoechst, nuclear counterstain (blue); scale bar = 50 μm

    Article Snippet: Molday, University of British Columbia (Vancouver, BC, Canada); sheep anti-Chx10 from Exalpha Biologicals, Inc. (Shirley, MA, USA); rabbit anti-cone-arrestin, rabbit anti-Sox9, rabbit anti-S-opsin, rabbit anti-mGluR1, goat anti-calretinin, chicken anti-vimentin, and guinea pig anti-GABA from Millipore (Billerica, MA, USA); mouse anti-PKCalpha, chicken anti-β-galactosidase, and rabbit anti-SOX-2 and anti-GlyT1 from Abcam (Cambridge, England); and mouse anti-GFAP from New England Biolabs (Ipswich, MA, USA).

    Techniques: Mouse Assay, Labeling, Mutagenesis