applied mouse genome survey microarray Search Results


4106 rs mouse wnt3 atcc l wnt3  (ATCC)
97
ATCC 4106 rs mouse wnt3 atcc l wnt3
4106 Rs Mouse Wnt3 Atcc L Wnt3, supplied by ATCC, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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a32955 rnase inhibitor  (New England Biolabs)
99
New England Biolabs a32955 rnase inhibitor
A32955 Rnase Inhibitor, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mouse anti gsk 3β antibody  (Cell Signaling Technology Inc)
96
Cell Signaling Technology Inc mouse anti gsk 3β antibody
Figure 6. AEG-1 activates Wnt/β-catenin signaling via targeting <t>GSK-3β</t> in glioma cells. (A) Enriched top 10 KEGG pathways for Affymetrix microarray of AEG-1 NC (N = 3) and KD (N = 3) glioma cells. 22 DEGs were enriched in KEGG_WNT_SIGNALING PATHWAY. P-value = 1.02E−06. [drawn by R 3.6.0 (https://cran.r- project.org/doc/FAQ/R-FAQ.html#Citing-R)]. (B) Enrichment plot of the Wnt signaling pathway from GSEA; ‘h’ and ‘l’ represented AEG-1 high and low expression, respectively. NES = 1.523, NOM P-value = 0.008, FDR q-value = 0.216 [drawn by GSEA tool (version 4.1.0)]. (C) Western blot bands of AEG-1, β-catenin, GSK-3β, cyclin D1, and CD44 in NC and shAEG-1 glioma cell lines. (D) Relative protein abundance was calculated by ImageJ and GraphPad Prism 8.2.1 software. (E, F) In U251 and U87 cells, Co-IP assays showed the direct interaction of AEG-1 and GSK-3β. (G) Immunofluorescence assays were used to detect the localization of AEG-1 (red) and GSK-3β (green) in U251 and U87 cells. All data were presented as the mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Mouse Anti Gsk 3β Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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murine neb  (New England Biolabs)
96
New England Biolabs murine neb
Figure 6. AEG-1 activates Wnt/β-catenin signaling via targeting <t>GSK-3β</t> in glioma cells. (A) Enriched top 10 KEGG pathways for Affymetrix microarray of AEG-1 NC (N = 3) and KD (N = 3) glioma cells. 22 DEGs were enriched in KEGG_WNT_SIGNALING PATHWAY. P-value = 1.02E−06. [drawn by R 3.6.0 (https://cran.r- project.org/doc/FAQ/R-FAQ.html#Citing-R)]. (B) Enrichment plot of the Wnt signaling pathway from GSEA; ‘h’ and ‘l’ represented AEG-1 high and low expression, respectively. NES = 1.523, NOM P-value = 0.008, FDR q-value = 0.216 [drawn by GSEA tool (version 4.1.0)]. (C) Western blot bands of AEG-1, β-catenin, GSK-3β, cyclin D1, and CD44 in NC and shAEG-1 glioma cell lines. (D) Relative protein abundance was calculated by ImageJ and GraphPad Prism 8.2.1 software. (E, F) In U251 and U87 cells, Co-IP assays showed the direct interaction of AEG-1 and GSK-3β. (G) Immunofluorescence assays were used to detect the localization of AEG-1 (red) and GSK-3β (green) in U251 and U87 cells. All data were presented as the mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Murine Neb, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mouse gene 1.0 st array  (Thermo Fisher)
90
Thermo Fisher mouse gene 1.0 st array
Figure 6. AEG-1 activates Wnt/β-catenin signaling via targeting <t>GSK-3β</t> in glioma cells. (A) Enriched top 10 KEGG pathways for Affymetrix microarray of AEG-1 NC (N = 3) and KD (N = 3) glioma cells. 22 DEGs were enriched in KEGG_WNT_SIGNALING PATHWAY. P-value = 1.02E−06. [drawn by R 3.6.0 (https://cran.r- project.org/doc/FAQ/R-FAQ.html#Citing-R)]. (B) Enrichment plot of the Wnt signaling pathway from GSEA; ‘h’ and ‘l’ represented AEG-1 high and low expression, respectively. NES = 1.523, NOM P-value = 0.008, FDR q-value = 0.216 [drawn by GSEA tool (version 4.1.0)]. (C) Western blot bands of AEG-1, β-catenin, GSK-3β, cyclin D1, and CD44 in NC and shAEG-1 glioma cell lines. (D) Relative protein abundance was calculated by ImageJ and GraphPad Prism 8.2.1 software. (E, F) In U251 and U87 cells, Co-IP assays showed the direct interaction of AEG-1 and GSK-3β. (G) Immunofluorescence assays were used to detect the localization of AEG-1 (red) and GSK-3β (green) in U251 and U87 cells. All data were presented as the mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Mouse Gene 1.0 St Array, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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integrin a5 subunit  (Cell Signaling Technology Inc)
95
Cell Signaling Technology Inc integrin a5 subunit
Figure 4. Identification of Tinagl1-Interacting Proteins (A–C) LM2 cells expressing the C-terminal HA-tagged Tinagl1 (Tinagl1-HA) were lysed and immunoprecipitated (IP) with immunoglobulin G (IgG) (control) or anti- HA antibody. The IP samples were subjected to silver staining and WB (A) before mass spectrometry analysis. Tinagl1-interacting partners were clustered with KEGG pathway analysis, and the three top pathways are shown in (B). The members of the top three pathways have overlaps. The EGFR and <t>integrin</t> b1 subunits are the core members of each pathways (C). (D and E) LM2 cells stably expressing Tinagl1-HA were lysed and IP with IgG or anti-HA antibodies. The IP samples were subjected to WB analysis with the indicated antibodies to detect the interaction with EGFR and the integrin b1 subunit (D), and with integrins av, or <t>a5</t> subunits (E). See also Figure S4.
Integrin A5 Subunit, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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5246s  (Cell Signaling Technology Inc)
98
Cell Signaling Technology Inc 5246s
Figure 4. Identification of Tinagl1-Interacting Proteins (A–C) LM2 cells expressing the C-terminal HA-tagged Tinagl1 (Tinagl1-HA) were lysed and immunoprecipitated (IP) with immunoglobulin G (IgG) (control) or anti- HA antibody. The IP samples were subjected to silver staining and WB (A) before mass spectrometry analysis. Tinagl1-interacting partners were clustered with KEGG pathway analysis, and the three top pathways are shown in (B). The members of the top three pathways have overlaps. The EGFR and <t>integrin</t> b1 subunits are the core members of each pathways (C). (D and E) LM2 cells stably expressing Tinagl1-HA were lysed and IP with IgG or anti-HA antibodies. The IP samples were subjected to WB analysis with the indicated antibodies to detect the interaction with EGFR and the integrin b1 subunit (D), and with integrins av, or <t>a5</t> subunits (E). See also Figure S4.
5246s, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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atcc crl  (ATCC)
99
ATCC atcc crl
Figure 4. Identification of Tinagl1-Interacting Proteins (A–C) LM2 cells expressing the C-terminal HA-tagged Tinagl1 (Tinagl1-HA) were lysed and immunoprecipitated (IP) with immunoglobulin G (IgG) (control) or anti- HA antibody. The IP samples were subjected to silver staining and WB (A) before mass spectrometry analysis. Tinagl1-interacting partners were clustered with KEGG pathway analysis, and the three top pathways are shown in (B). The members of the top three pathways have overlaps. The EGFR and <t>integrin</t> b1 subunits are the core members of each pathways (C). (D and E) LM2 cells stably expressing Tinagl1-HA were lysed and IP with IgG or anti-HA antibodies. The IP samples were subjected to WB analysis with the indicated antibodies to detect the interaction with EGFR and the integrin b1 subunit (D), and with integrins av, or <t>a5</t> subunits (E). See also Figure S4.
Atcc Crl, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/atcc crl/product/ATCC
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smart seq v4 ultra low input rna kit  (TaKaRa)
99
TaKaRa smart seq v4 ultra low input rna kit
Figure 4. Identification of Tinagl1-Interacting Proteins (A–C) LM2 cells expressing the C-terminal HA-tagged Tinagl1 (Tinagl1-HA) were lysed and immunoprecipitated (IP) with immunoglobulin G (IgG) (control) or anti- HA antibody. The IP samples were subjected to silver staining and WB (A) before mass spectrometry analysis. Tinagl1-interacting partners were clustered with KEGG pathway analysis, and the three top pathways are shown in (B). The members of the top three pathways have overlaps. The EGFR and <t>integrin</t> b1 subunits are the core members of each pathways (C). (D and E) LM2 cells stably expressing Tinagl1-HA were lysed and IP with IgG or anti-HA antibodies. The IP samples were subjected to WB analysis with the indicated antibodies to detect the interaction with EGFR and the integrin b1 subunit (D), and with integrins av, or <t>a5</t> subunits (E). See also Figure S4.
Smart Seq V4 Ultra Low Input Rna Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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resource source identifier label it nucleic acid labeling kit mirus cat  (Mirus Bio)
98
Mirus Bio resource source identifier label it nucleic acid labeling kit mirus cat
Figure 4. Identification of Tinagl1-Interacting Proteins (A–C) LM2 cells expressing the C-terminal HA-tagged Tinagl1 (Tinagl1-HA) were lysed and immunoprecipitated (IP) with immunoglobulin G (IgG) (control) or anti- HA antibody. The IP samples were subjected to silver staining and WB (A) before mass spectrometry analysis. Tinagl1-interacting partners were clustered with KEGG pathway analysis, and the three top pathways are shown in (B). The members of the top three pathways have overlaps. The EGFR and <t>integrin</t> b1 subunits are the core members of each pathways (C). (D and E) LM2 cells stably expressing Tinagl1-HA were lysed and IP with IgG or anti-HA antibodies. The IP samples were subjected to WB analysis with the indicated antibodies to detect the interaction with EGFR and the integrin b1 subunit (D), and with integrins av, or <t>a5</t> subunits (E). See also Figure S4.
Resource Source Identifier Label It Nucleic Acid Labeling Kit Mirus Cat, supplied by Mirus Bio, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rabbit foxa2  (Cell Signaling Technology Inc)
96
Cell Signaling Technology Inc rabbit foxa2
Fig. 1 | Identification of CD177+ and CD275+ ADE subpopulations. a, Schematic representation of hESC differentiation toward DE. b,c, Representative FACS plots of apparently homogeneous <t>FOXA2+/SOX17+</t> DE (b) showing a heterogenous population marked by CXCR4+/CD117+ cells (c) (n = 3 (b), n = 6 (c) biologically independent experiments). d–g, Gene expression profiles of CXCR4+/CD117−, CXCR4high/CD117high, CXCR4mid/CD117mid and CXCR4low/CD117low cells for FOXA2 (d), SOX17 (e), CER1 (f) and HHEX (g) (ANOVA, n = 3 biologically independent experiments). Data are represented as mean ± s.e.m.; P < 0.05 and P < 0.01. Statistically nonsignificant results are not indicated in the figure. h, Summary of the antibody screen identifying and isolating CD177 and CD275 as markers of ADE subpopulations. CXCR4 and FOXA2 are used as controls to identify the whole DE. i, hPSCs and hPSC-derived DE stained for CXCR4, CD177 and CD275 as shown by live-cell FACS (n = 10 biologically independent experiments). AA, activin A; D, day.
Rabbit Foxa2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rpn2232 ethylenediaminetetraacetic acid edta thermo fisher  (Thermo Fisher)
99
Thermo Fisher rpn2232 ethylenediaminetetraacetic acid edta thermo fisher
Fig. 1 | Identification of CD177+ and CD275+ ADE subpopulations. a, Schematic representation of hESC differentiation toward DE. b,c, Representative FACS plots of apparently homogeneous <t>FOXA2+/SOX17+</t> DE (b) showing a heterogenous population marked by CXCR4+/CD117+ cells (c) (n = 3 (b), n = 6 (c) biologically independent experiments). d–g, Gene expression profiles of CXCR4+/CD117−, CXCR4high/CD117high, CXCR4mid/CD117mid and CXCR4low/CD117low cells for FOXA2 (d), SOX17 (e), CER1 (f) and HHEX (g) (ANOVA, n = 3 biologically independent experiments). Data are represented as mean ± s.e.m.; P < 0.05 and P < 0.01. Statistically nonsignificant results are not indicated in the figure. h, Summary of the antibody screen identifying and isolating CD177 and CD275 as markers of ADE subpopulations. CXCR4 and FOXA2 are used as controls to identify the whole DE. i, hPSCs and hPSC-derived DE stained for CXCR4, CD177 and CD275 as shown by live-cell FACS (n = 10 biologically independent experiments). AA, activin A; D, day.
Rpn2232 Ethylenediaminetetraacetic Acid Edta Thermo Fisher, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


Figure 6. AEG-1 activates Wnt/β-catenin signaling via targeting GSK-3β in glioma cells. (A) Enriched top 10 KEGG pathways for Affymetrix microarray of AEG-1 NC (N = 3) and KD (N = 3) glioma cells. 22 DEGs were enriched in KEGG_WNT_SIGNALING PATHWAY. P-value = 1.02E−06. [drawn by R 3.6.0 (https://cran.r- project.org/doc/FAQ/R-FAQ.html#Citing-R)]. (B) Enrichment plot of the Wnt signaling pathway from GSEA; ‘h’ and ‘l’ represented AEG-1 high and low expression, respectively. NES = 1.523, NOM P-value = 0.008, FDR q-value = 0.216 [drawn by GSEA tool (version 4.1.0)]. (C) Western blot bands of AEG-1, β-catenin, GSK-3β, cyclin D1, and CD44 in NC and shAEG-1 glioma cell lines. (D) Relative protein abundance was calculated by ImageJ and GraphPad Prism 8.2.1 software. (E, F) In U251 and U87 cells, Co-IP assays showed the direct interaction of AEG-1 and GSK-3β. (G) Immunofluorescence assays were used to detect the localization of AEG-1 (red) and GSK-3β (green) in U251 and U87 cells. All data were presented as the mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Journal: Scientific reports

Article Title: AEG-1 silencing attenuates M2-polarization of glioma-associated microglia/macrophages and sensitizes glioma cells to temozolomide.

doi: 10.1038/s41598-021-96647-3

Figure Lengend Snippet: Figure 6. AEG-1 activates Wnt/β-catenin signaling via targeting GSK-3β in glioma cells. (A) Enriched top 10 KEGG pathways for Affymetrix microarray of AEG-1 NC (N = 3) and KD (N = 3) glioma cells. 22 DEGs were enriched in KEGG_WNT_SIGNALING PATHWAY. P-value = 1.02E−06. [drawn by R 3.6.0 (https://cran.r- project.org/doc/FAQ/R-FAQ.html#Citing-R)]. (B) Enrichment plot of the Wnt signaling pathway from GSEA; ‘h’ and ‘l’ represented AEG-1 high and low expression, respectively. NES = 1.523, NOM P-value = 0.008, FDR q-value = 0.216 [drawn by GSEA tool (version 4.1.0)]. (C) Western blot bands of AEG-1, β-catenin, GSK-3β, cyclin D1, and CD44 in NC and shAEG-1 glioma cell lines. (D) Relative protein abundance was calculated by ImageJ and GraphPad Prism 8.2.1 software. (E, F) In U251 and U87 cells, Co-IP assays showed the direct interaction of AEG-1 and GSK-3β. (G) Immunofluorescence assays were used to detect the localization of AEG-1 (red) and GSK-3β (green) in U251 and U87 cells. All data were presented as the mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Cells were incubated with rabbit anti-AEG-1 antibody (Proteintech, 1:1000), mouse anti-GSK-3β antibody (Cell Signaling Technology, 1:1000), and rabbit anti-γH2AX (Cell Signaling Technology, 1:400), diluted using 1% FBS, at 4 °C overnight.

Techniques: Microarray, Expressing, Western Blot, Quantitative Proteomics, Software, Co-Immunoprecipitation Assay, Immunofluorescence

Figure 4. Identification of Tinagl1-Interacting Proteins (A–C) LM2 cells expressing the C-terminal HA-tagged Tinagl1 (Tinagl1-HA) were lysed and immunoprecipitated (IP) with immunoglobulin G (IgG) (control) or anti- HA antibody. The IP samples were subjected to silver staining and WB (A) before mass spectrometry analysis. Tinagl1-interacting partners were clustered with KEGG pathway analysis, and the three top pathways are shown in (B). The members of the top three pathways have overlaps. The EGFR and integrin b1 subunits are the core members of each pathways (C). (D and E) LM2 cells stably expressing Tinagl1-HA were lysed and IP with IgG or anti-HA antibodies. The IP samples were subjected to WB analysis with the indicated antibodies to detect the interaction with EGFR and the integrin b1 subunit (D), and with integrins av, or a5 subunits (E). See also Figure S4.

Journal: Cancer cell

Article Title: Tinagl1 Suppresses Triple-Negative Breast Cancer Progression and Metastasis by Simultaneously Inhibiting Integrin/FAK and EGFR Signaling.

doi: 10.1016/j.ccell.2018.11.016

Figure Lengend Snippet: Figure 4. Identification of Tinagl1-Interacting Proteins (A–C) LM2 cells expressing the C-terminal HA-tagged Tinagl1 (Tinagl1-HA) were lysed and immunoprecipitated (IP) with immunoglobulin G (IgG) (control) or anti- HA antibody. The IP samples were subjected to silver staining and WB (A) before mass spectrometry analysis. Tinagl1-interacting partners were clustered with KEGG pathway analysis, and the three top pathways are shown in (B). The members of the top three pathways have overlaps. The EGFR and integrin b1 subunits are the core members of each pathways (C). (D and E) LM2 cells stably expressing Tinagl1-HA were lysed and IP with IgG or anti-HA antibodies. The IP samples were subjected to WB analysis with the indicated antibodies to detect the interaction with EGFR and the integrin b1 subunit (D), and with integrins av, or a5 subunits (E). See also Figure S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Tinagl1, in 1:1000 (WB), 2 mg (IP), Rabbit ProteinTech Cat#12077-1-AP; RRID: AB_2058942 Tinagl1, in 1:100 (IHC), Rabbit Sigma-Aldrich Cat#HPA048695; RRID: AB_2680497 b-actin, in 1:10,000 (WB), mouse Abcam Cat#ab6276; RRID: AB_2223210 EGFR, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4267; RRID: AB_2246311 p-EGFR (Try1068), in 1:1000 (WB), 1:100 (IHC), Rabbit Cell Signaling Technology Cat#3777; RRID: AB_2096270 FAK, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#3285; RRID: AB_10694068 p-FAK (Try397), in 1:1000 (WB), 1:100 (IHC), Rabbit Cell Signaling Technology Cat#8556; RRID: AB_10891442 p-FAK (Try925), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#3284; RRID: AB_2253227 AKT, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4691; RRID: AB_915783 p-ATK (S473), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4060; RRID: AB_2315049 ERK1/2, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4695; RRID: AB_390779 p-ERK1/2 (Thr202/Tyr204), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4370; RRID: AB_2315112 Integrin b1 subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Cell Signaling Technology Cat#34971 Integrin a5 subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Cell Signaling Technology Cat#4705; RRID: AB_10827978 Integrin av subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Abcam Cat#ab179475; RRID: AB_2716738 Integrin a3 subunit, in 1:1000 (WB), Rabbit Abcam Cat#ab190731 Integrin a4 subunit, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#8440P Integrin aM subunit, in 1:1000 (WB), Rabbit Abcam Cat#ab8878; RRID: AB_306831 HA, in 2 mg (IP), Mouse Santa Cruz Biotechnology Cat#sc-7392; RRID: AB_627809 HA, in 1:1000 (WB), Rat Roche, 11867423001 Cat#11867423001; RRID: AB_10094468 MYC, in 1:1000 (WB), 2 mg (IP), Mouse Santa Cruz Biotechnology Cat#sc-40; RRID: AB_627268 FLAG, in 1:1000 (WB), 2 mg (IP), Mouse Sigma-Aldrich Cat#F7425; RRID: AB_439687 His, in 1:1000 (WB), Mouse Sigma-Aldrich Cat#H1029; RRID: AB_260015 GFP, in 1:1000 (WB), Mouse Santa Cruz Biotechnology Cat#sc-9996; RRID: AB_627695 Fibronectin, in 1:1000 (WB), Rabbit ProteinTech Cat#15613-1-AP; RRID: AB_2105691 EGF, in 1:1000 (WB), Mouse Santa Cruz Biotechnology Cat#sc-275; RRID: AB_631417 (Continued on next page) e1 Cancer Cell 35, 1–17.e1–e7, January 14, 2019

Techniques: Expressing, Immunoprecipitation, Control, Silver Staining, Mass Spectrometry, Stable Transfection

Figure 5. Tinagl1 Inhibits integrin/FAK and EGFR Signaling Pathways (A) Gene set enrichment analysis of lung metastatic nodules formed by LM2 cells stably expressing the vector control or Tinagl1 were dissected and digested. n = 3 per group. (B) Heatmap representation of microarray data displaying the expression of EGFR or integrin/FAK regulated genes in the control versus Tinagl1-expressing LM2 cells. (C) Heatmap representation of microarray data displaying the expression of genes compensated by integrin/FAK (left) or EGFR (right) in control versus Tinagl1- expressing LM2 cells.

Journal: Cancer cell

Article Title: Tinagl1 Suppresses Triple-Negative Breast Cancer Progression and Metastasis by Simultaneously Inhibiting Integrin/FAK and EGFR Signaling.

doi: 10.1016/j.ccell.2018.11.016

Figure Lengend Snippet: Figure 5. Tinagl1 Inhibits integrin/FAK and EGFR Signaling Pathways (A) Gene set enrichment analysis of lung metastatic nodules formed by LM2 cells stably expressing the vector control or Tinagl1 were dissected and digested. n = 3 per group. (B) Heatmap representation of microarray data displaying the expression of EGFR or integrin/FAK regulated genes in the control versus Tinagl1-expressing LM2 cells. (C) Heatmap representation of microarray data displaying the expression of genes compensated by integrin/FAK (left) or EGFR (right) in control versus Tinagl1- expressing LM2 cells.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Tinagl1, in 1:1000 (WB), 2 mg (IP), Rabbit ProteinTech Cat#12077-1-AP; RRID: AB_2058942 Tinagl1, in 1:100 (IHC), Rabbit Sigma-Aldrich Cat#HPA048695; RRID: AB_2680497 b-actin, in 1:10,000 (WB), mouse Abcam Cat#ab6276; RRID: AB_2223210 EGFR, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4267; RRID: AB_2246311 p-EGFR (Try1068), in 1:1000 (WB), 1:100 (IHC), Rabbit Cell Signaling Technology Cat#3777; RRID: AB_2096270 FAK, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#3285; RRID: AB_10694068 p-FAK (Try397), in 1:1000 (WB), 1:100 (IHC), Rabbit Cell Signaling Technology Cat#8556; RRID: AB_10891442 p-FAK (Try925), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#3284; RRID: AB_2253227 AKT, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4691; RRID: AB_915783 p-ATK (S473), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4060; RRID: AB_2315049 ERK1/2, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4695; RRID: AB_390779 p-ERK1/2 (Thr202/Tyr204), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4370; RRID: AB_2315112 Integrin b1 subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Cell Signaling Technology Cat#34971 Integrin a5 subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Cell Signaling Technology Cat#4705; RRID: AB_10827978 Integrin av subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Abcam Cat#ab179475; RRID: AB_2716738 Integrin a3 subunit, in 1:1000 (WB), Rabbit Abcam Cat#ab190731 Integrin a4 subunit, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#8440P Integrin aM subunit, in 1:1000 (WB), Rabbit Abcam Cat#ab8878; RRID: AB_306831 HA, in 2 mg (IP), Mouse Santa Cruz Biotechnology Cat#sc-7392; RRID: AB_627809 HA, in 1:1000 (WB), Rat Roche, 11867423001 Cat#11867423001; RRID: AB_10094468 MYC, in 1:1000 (WB), 2 mg (IP), Mouse Santa Cruz Biotechnology Cat#sc-40; RRID: AB_627268 FLAG, in 1:1000 (WB), 2 mg (IP), Mouse Sigma-Aldrich Cat#F7425; RRID: AB_439687 His, in 1:1000 (WB), Mouse Sigma-Aldrich Cat#H1029; RRID: AB_260015 GFP, in 1:1000 (WB), Mouse Santa Cruz Biotechnology Cat#sc-9996; RRID: AB_627695 Fibronectin, in 1:1000 (WB), Rabbit ProteinTech Cat#15613-1-AP; RRID: AB_2105691 EGF, in 1:1000 (WB), Mouse Santa Cruz Biotechnology Cat#sc-275; RRID: AB_631417 (Continued on next page) e1 Cancer Cell 35, 1–17.e1–e7, January 14, 2019

Techniques: Protein-Protein interactions, Stable Transfection, Expressing, Plasmid Preparation, Control, Microarray

Figure 6. Tinagl1 Inhibits EGFR Dimerization and Blocks the Interaction between the Integrin b1 Subunit and FN (A) LM2 cells were transfected with plasmids to overexpress GFP-EGFR and EGFR-Myc. 48 hr after transfection, the cells were treated with or without 1 mg/mL of r-Tinagl1 for 1 hr, followed by 10 min of 1 ng/mL EGF treatment. The cells were then collected and immunoprecipitated with either IgG or anti-Myc antibody. IP samples were subjected to WB analysis (top), and the amount of EGFR-GFP that interacts with EGFR-Myc was quantified and normalized to the PBS treatment group (bottom). (B) LM2 cells stably expressing EGFR-Myc were pre-treated with PBS or 1 mg/mL of r-Tinagl1 for 1 hr and then treated with 1 ng/mL of EGF for another 10 min. Next, the cells were collected and the dimers were crosslinked with disuccinimidyl suberate (DSS) treatment, followed by WB analysis (top) and quantification of the ratio of dimerized EGFR in each treatment group (bottom). (C) HEK293T cells overexpressing the integrin b1 subunit were lysed. 20 mg of FN was added into the lysate, and the lysate was divided into eight groups. PBS or the indicated amount of proteins were added into each group followed by IP with IgG or anti-b1 antibody. The IP samples were then subjected to WB analysis. (D) HEK293T cells overexpressing both integrin b1 subunit and Tinagl1-HA were lysed and divided into eight groups. PBS or the indicated amount of proteins were added into the lysate, followed by IP and WB analysis.

Journal: Cancer cell

Article Title: Tinagl1 Suppresses Triple-Negative Breast Cancer Progression and Metastasis by Simultaneously Inhibiting Integrin/FAK and EGFR Signaling.

doi: 10.1016/j.ccell.2018.11.016

Figure Lengend Snippet: Figure 6. Tinagl1 Inhibits EGFR Dimerization and Blocks the Interaction between the Integrin b1 Subunit and FN (A) LM2 cells were transfected with plasmids to overexpress GFP-EGFR and EGFR-Myc. 48 hr after transfection, the cells were treated with or without 1 mg/mL of r-Tinagl1 for 1 hr, followed by 10 min of 1 ng/mL EGF treatment. The cells were then collected and immunoprecipitated with either IgG or anti-Myc antibody. IP samples were subjected to WB analysis (top), and the amount of EGFR-GFP that interacts with EGFR-Myc was quantified and normalized to the PBS treatment group (bottom). (B) LM2 cells stably expressing EGFR-Myc were pre-treated with PBS or 1 mg/mL of r-Tinagl1 for 1 hr and then treated with 1 ng/mL of EGF for another 10 min. Next, the cells were collected and the dimers were crosslinked with disuccinimidyl suberate (DSS) treatment, followed by WB analysis (top) and quantification of the ratio of dimerized EGFR in each treatment group (bottom). (C) HEK293T cells overexpressing the integrin b1 subunit were lysed. 20 mg of FN was added into the lysate, and the lysate was divided into eight groups. PBS or the indicated amount of proteins were added into each group followed by IP with IgG or anti-b1 antibody. The IP samples were then subjected to WB analysis. (D) HEK293T cells overexpressing both integrin b1 subunit and Tinagl1-HA were lysed and divided into eight groups. PBS or the indicated amount of proteins were added into the lysate, followed by IP and WB analysis.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Tinagl1, in 1:1000 (WB), 2 mg (IP), Rabbit ProteinTech Cat#12077-1-AP; RRID: AB_2058942 Tinagl1, in 1:100 (IHC), Rabbit Sigma-Aldrich Cat#HPA048695; RRID: AB_2680497 b-actin, in 1:10,000 (WB), mouse Abcam Cat#ab6276; RRID: AB_2223210 EGFR, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4267; RRID: AB_2246311 p-EGFR (Try1068), in 1:1000 (WB), 1:100 (IHC), Rabbit Cell Signaling Technology Cat#3777; RRID: AB_2096270 FAK, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#3285; RRID: AB_10694068 p-FAK (Try397), in 1:1000 (WB), 1:100 (IHC), Rabbit Cell Signaling Technology Cat#8556; RRID: AB_10891442 p-FAK (Try925), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#3284; RRID: AB_2253227 AKT, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4691; RRID: AB_915783 p-ATK (S473), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4060; RRID: AB_2315049 ERK1/2, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4695; RRID: AB_390779 p-ERK1/2 (Thr202/Tyr204), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4370; RRID: AB_2315112 Integrin b1 subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Cell Signaling Technology Cat#34971 Integrin a5 subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Cell Signaling Technology Cat#4705; RRID: AB_10827978 Integrin av subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Abcam Cat#ab179475; RRID: AB_2716738 Integrin a3 subunit, in 1:1000 (WB), Rabbit Abcam Cat#ab190731 Integrin a4 subunit, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#8440P Integrin aM subunit, in 1:1000 (WB), Rabbit Abcam Cat#ab8878; RRID: AB_306831 HA, in 2 mg (IP), Mouse Santa Cruz Biotechnology Cat#sc-7392; RRID: AB_627809 HA, in 1:1000 (WB), Rat Roche, 11867423001 Cat#11867423001; RRID: AB_10094468 MYC, in 1:1000 (WB), 2 mg (IP), Mouse Santa Cruz Biotechnology Cat#sc-40; RRID: AB_627268 FLAG, in 1:1000 (WB), 2 mg (IP), Mouse Sigma-Aldrich Cat#F7425; RRID: AB_439687 His, in 1:1000 (WB), Mouse Sigma-Aldrich Cat#H1029; RRID: AB_260015 GFP, in 1:1000 (WB), Mouse Santa Cruz Biotechnology Cat#sc-9996; RRID: AB_627695 Fibronectin, in 1:1000 (WB), Rabbit ProteinTech Cat#15613-1-AP; RRID: AB_2105691 EGF, in 1:1000 (WB), Mouse Santa Cruz Biotechnology Cat#sc-275; RRID: AB_631417 (Continued on next page) e1 Cancer Cell 35, 1–17.e1–e7, January 14, 2019

Techniques: Transfection, Immunoprecipitation, Stable Transfection, Expressing

Figure 7. Tinagl1 Inhibits TNBC Progression by Simultaneously Targeting the Integrin/FAK and EGFR Signaling Pathways (A) 104 LM2 cells was injected into the MFP of NSG mice. Mice were intravenously treated with the indicated reagents twice per week when tumors reached 2 mm in diameter. n = 6 mice per group. (B) WB analysis for the activation of EGFR and FAK in primary tumor of each group after 5 weeks of the treatments as in (A). (C) Quantification of tumor volumes of each treatment group of (A). n = 12 tumors per group. (D and E) Lungs were collected and spontaneous metastasis was examined by ex vivo BLI at the endpoint. n = 6 lungs per group. Representative lungs (D) and quantitative data (E) is shown. Data represent means ± SEM. n.s., not significant; p > 0.05, **p < 0.001, ***p < 0.0001. Significance determined by one-way ANOVA analysis with Dunnett’s test for multiple comparisons. See also Figure S7.

Journal: Cancer cell

Article Title: Tinagl1 Suppresses Triple-Negative Breast Cancer Progression and Metastasis by Simultaneously Inhibiting Integrin/FAK and EGFR Signaling.

doi: 10.1016/j.ccell.2018.11.016

Figure Lengend Snippet: Figure 7. Tinagl1 Inhibits TNBC Progression by Simultaneously Targeting the Integrin/FAK and EGFR Signaling Pathways (A) 104 LM2 cells was injected into the MFP of NSG mice. Mice were intravenously treated with the indicated reagents twice per week when tumors reached 2 mm in diameter. n = 6 mice per group. (B) WB analysis for the activation of EGFR and FAK in primary tumor of each group after 5 weeks of the treatments as in (A). (C) Quantification of tumor volumes of each treatment group of (A). n = 12 tumors per group. (D and E) Lungs were collected and spontaneous metastasis was examined by ex vivo BLI at the endpoint. n = 6 lungs per group. Representative lungs (D) and quantitative data (E) is shown. Data represent means ± SEM. n.s., not significant; p > 0.05, **p < 0.001, ***p < 0.0001. Significance determined by one-way ANOVA analysis with Dunnett’s test for multiple comparisons. See also Figure S7.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Tinagl1, in 1:1000 (WB), 2 mg (IP), Rabbit ProteinTech Cat#12077-1-AP; RRID: AB_2058942 Tinagl1, in 1:100 (IHC), Rabbit Sigma-Aldrich Cat#HPA048695; RRID: AB_2680497 b-actin, in 1:10,000 (WB), mouse Abcam Cat#ab6276; RRID: AB_2223210 EGFR, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4267; RRID: AB_2246311 p-EGFR (Try1068), in 1:1000 (WB), 1:100 (IHC), Rabbit Cell Signaling Technology Cat#3777; RRID: AB_2096270 FAK, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#3285; RRID: AB_10694068 p-FAK (Try397), in 1:1000 (WB), 1:100 (IHC), Rabbit Cell Signaling Technology Cat#8556; RRID: AB_10891442 p-FAK (Try925), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#3284; RRID: AB_2253227 AKT, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4691; RRID: AB_915783 p-ATK (S473), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4060; RRID: AB_2315049 ERK1/2, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4695; RRID: AB_390779 p-ERK1/2 (Thr202/Tyr204), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4370; RRID: AB_2315112 Integrin b1 subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Cell Signaling Technology Cat#34971 Integrin a5 subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Cell Signaling Technology Cat#4705; RRID: AB_10827978 Integrin av subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Abcam Cat#ab179475; RRID: AB_2716738 Integrin a3 subunit, in 1:1000 (WB), Rabbit Abcam Cat#ab190731 Integrin a4 subunit, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#8440P Integrin aM subunit, in 1:1000 (WB), Rabbit Abcam Cat#ab8878; RRID: AB_306831 HA, in 2 mg (IP), Mouse Santa Cruz Biotechnology Cat#sc-7392; RRID: AB_627809 HA, in 1:1000 (WB), Rat Roche, 11867423001 Cat#11867423001; RRID: AB_10094468 MYC, in 1:1000 (WB), 2 mg (IP), Mouse Santa Cruz Biotechnology Cat#sc-40; RRID: AB_627268 FLAG, in 1:1000 (WB), 2 mg (IP), Mouse Sigma-Aldrich Cat#F7425; RRID: AB_439687 His, in 1:1000 (WB), Mouse Sigma-Aldrich Cat#H1029; RRID: AB_260015 GFP, in 1:1000 (WB), Mouse Santa Cruz Biotechnology Cat#sc-9996; RRID: AB_627695 Fibronectin, in 1:1000 (WB), Rabbit ProteinTech Cat#15613-1-AP; RRID: AB_2105691 EGF, in 1:1000 (WB), Mouse Santa Cruz Biotechnology Cat#sc-275; RRID: AB_631417 (Continued on next page) e1 Cancer Cell 35, 1–17.e1–e7, January 14, 2019

Techniques: Protein-Protein interactions, Injection, Activation Assay, Ex Vivo

Fig. 1 | Identification of CD177+ and CD275+ ADE subpopulations. a, Schematic representation of hESC differentiation toward DE. b,c, Representative FACS plots of apparently homogeneous FOXA2+/SOX17+ DE (b) showing a heterogenous population marked by CXCR4+/CD117+ cells (c) (n = 3 (b), n = 6 (c) biologically independent experiments). d–g, Gene expression profiles of CXCR4+/CD117−, CXCR4high/CD117high, CXCR4mid/CD117mid and CXCR4low/CD117low cells for FOXA2 (d), SOX17 (e), CER1 (f) and HHEX (g) (ANOVA, n = 3 biologically independent experiments). Data are represented as mean ± s.e.m.; P < 0.05 and P < 0.01. Statistically nonsignificant results are not indicated in the figure. h, Summary of the antibody screen identifying and isolating CD177 and CD275 as markers of ADE subpopulations. CXCR4 and FOXA2 are used as controls to identify the whole DE. i, hPSCs and hPSC-derived DE stained for CXCR4, CD177 and CD275 as shown by live-cell FACS (n = 10 biologically independent experiments). AA, activin A; D, day.

Journal: Nature biotechnology

Article Title: Generation of pancreatic β cells from CD177 + anterior definitive endoderm.

doi: 10.1038/s41587-020-0492-5

Figure Lengend Snippet: Fig. 1 | Identification of CD177+ and CD275+ ADE subpopulations. a, Schematic representation of hESC differentiation toward DE. b,c, Representative FACS plots of apparently homogeneous FOXA2+/SOX17+ DE (b) showing a heterogenous population marked by CXCR4+/CD117+ cells (c) (n = 3 (b), n = 6 (c) biologically independent experiments). d–g, Gene expression profiles of CXCR4+/CD117−, CXCR4high/CD117high, CXCR4mid/CD117mid and CXCR4low/CD117low cells for FOXA2 (d), SOX17 (e), CER1 (f) and HHEX (g) (ANOVA, n = 3 biologically independent experiments). Data are represented as mean ± s.e.m.; P < 0.05 and P < 0.01. Statistically nonsignificant results are not indicated in the figure. h, Summary of the antibody screen identifying and isolating CD177 and CD275 as markers of ADE subpopulations. CXCR4 and FOXA2 are used as controls to identify the whole DE. i, hPSCs and hPSC-derived DE stained for CXCR4, CD177 and CD275 as shown by live-cell FACS (n = 10 biologically independent experiments). AA, activin A; D, day.

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology Animals and other organisms Human research participants Clinical data Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Human CXCR4-PE,Miltenyi Biotech,130-098-354, dilution 1:40; Human CXCR4-APC,Miltenyi Biotech, 120-010-802, dilution 1:40; Human CD117-APC, Miltenyi Biotech, 130-091-733, dilution 1:40; Human CD117-PE, Miltenyi Biotech, 130-091-734, dilution 1:40; FOXA2-Alexa Fluor® 488, R and D, IC2400G; dilution 1:10 SOX17-APC, R and D, IC1924A; dilution 1:10 Human CD177-APC, Miltenyi Biotech, 120-017-498; dilution 1:20 Human CD275-APC, Miltenyi Biotech, 120-012-112; dilution 1:20 PE Mouse anti-PDX1, BD PharmingenTM, 562161; dilution 1:40 4 nature research | reporting sum m ary O ctober 2018 Alexa Fluor® 647 Mouse anti-Nkx6.1, BD PharmingenTM, 563338; dilution 1:40 Alexa Fluor® 647 Mouse IgG1 κ Isotype Control, BD PharmingenTM, 563023; dilution 1:40 Rabbit FOXA2, Cell signalling, 8186; dilution 1:1000 Goat SOX17 Acris/Novus GT15094, dilution 1:1000 Goat CER1 R&D Systems AF1075, dilution 1:1000 Mouse β-catenin BD 610154, dilution 1:1000 Guinea pig INSULIN Thermo Schientific PA1-26938, dilution 1:100 Guinea pig C-Peptide Abcam ab30477, dilution 1:300 Rabbit MAFA Betalogics LP9872, dilution 1:100 Rabbit MAFA ,Novus Biologicals, NB400-137, dilution 1:100 Rabbit GLUT1 Thermo Fisher PA1-37782, dilution 1:100 Goat GATA6 R&D Systems AF1700, dilution 1:1000 Mouse SOX2 Abgent / Bio Cat AM2048, dilution 1:1000 Rabbit CDX2 Santa Cruz sc-134468, dilution 1:1000 Mouse GCG Sigma G2654-.2ML, dilution 1:300 Goat PDX1 R&D Systems AF2419, dilution 1:500 Rabbit NKX6.1Novus biologicalsNBP1-49672, dilution 1:500 Goat NKX6.1R&D systemsAF5857, dilution 1:300 Rabbit p-JNK Cell signalling 4668, dilution 1:1000 Rabbit DVL2 Cell signalling 3216, dilution 1:1000 Mouse GAPDH Merck Biosciences CB1001, dilution 1:6000 Validation All primary antibodies were validated for their expression on undifferentiated cells and/or pancreatic human sections/islets.

Techniques: Gene Expression, Derivative Assay, Staining

Fig. 2 | Molecular profiling of CD177+, CD275+ and CXCR4+ DE subpopulations reveals distinct signatures. a, Summary of differentiation protocol toward DE/ADE followed by MACS to enrich for CD177, CD275 and CXCR4 populations. b, Principal component analysis showing that mRNA-derived transcriptome profiles are characteristic of different DE/ADE subpopulations (n = 3 biologically independent experiments). c–e, Bar graphs of selected and significantly enriched gene ontology terms in CD275+ versus CXCR4+ (c), CD177+ versus CD275+ (d) and CD177+ versus CXCR4+ (e) DE populations (n = 3 biologically independent experiments). Enrichment P values are calculated by HOMER findGO.pl based on the cumulative hypergeometric distribution. f,g, Validation of the microarray analysis by qPCR for noncanonical WNT/PCP components and ligands (f) and canonical WNT components and ligands (g). Data were normalized to 18S (ANOVA, n = 3 biologically independent experiments). Data are represented as mean ± s.e.m.; P < 0.05 and P < 0.01. Statistically nonsignificant results are not indicated in the figure. h,i, Western blot analysis (h) and quantification (i) of WNT/PCP components such as p-JNK and DVL2 in ADE subpopulations (n = 3 biologically independent experiments). GAPDH is used as a loading control. Data are represented as mean ± s.e.m. j, Immunofluorescence analysis validated the exclusive localization of β-catenin in the membrane in CD177+ ADE cells and in the cytoplasm and nucleus in CD275+ ADE and CXCR4+ DE cells (n = 3 biologically independent experiments). FOXA2 is used as a nuclear marker. Scale bars, 20 µm and 10 µm in inset. PC1/2, principal component 1/2.

Journal: Nature biotechnology

Article Title: Generation of pancreatic β cells from CD177 + anterior definitive endoderm.

doi: 10.1038/s41587-020-0492-5

Figure Lengend Snippet: Fig. 2 | Molecular profiling of CD177+, CD275+ and CXCR4+ DE subpopulations reveals distinct signatures. a, Summary of differentiation protocol toward DE/ADE followed by MACS to enrich for CD177, CD275 and CXCR4 populations. b, Principal component analysis showing that mRNA-derived transcriptome profiles are characteristic of different DE/ADE subpopulations (n = 3 biologically independent experiments). c–e, Bar graphs of selected and significantly enriched gene ontology terms in CD275+ versus CXCR4+ (c), CD177+ versus CD275+ (d) and CD177+ versus CXCR4+ (e) DE populations (n = 3 biologically independent experiments). Enrichment P values are calculated by HOMER findGO.pl based on the cumulative hypergeometric distribution. f,g, Validation of the microarray analysis by qPCR for noncanonical WNT/PCP components and ligands (f) and canonical WNT components and ligands (g). Data were normalized to 18S (ANOVA, n = 3 biologically independent experiments). Data are represented as mean ± s.e.m.; P < 0.05 and P < 0.01. Statistically nonsignificant results are not indicated in the figure. h,i, Western blot analysis (h) and quantification (i) of WNT/PCP components such as p-JNK and DVL2 in ADE subpopulations (n = 3 biologically independent experiments). GAPDH is used as a loading control. Data are represented as mean ± s.e.m. j, Immunofluorescence analysis validated the exclusive localization of β-catenin in the membrane in CD177+ ADE cells and in the cytoplasm and nucleus in CD275+ ADE and CXCR4+ DE cells (n = 3 biologically independent experiments). FOXA2 is used as a nuclear marker. Scale bars, 20 µm and 10 µm in inset. PC1/2, principal component 1/2.

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology Animals and other organisms Human research participants Clinical data Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Human CXCR4-PE,Miltenyi Biotech,130-098-354, dilution 1:40; Human CXCR4-APC,Miltenyi Biotech, 120-010-802, dilution 1:40; Human CD117-APC, Miltenyi Biotech, 130-091-733, dilution 1:40; Human CD117-PE, Miltenyi Biotech, 130-091-734, dilution 1:40; FOXA2-Alexa Fluor® 488, R and D, IC2400G; dilution 1:10 SOX17-APC, R and D, IC1924A; dilution 1:10 Human CD177-APC, Miltenyi Biotech, 120-017-498; dilution 1:20 Human CD275-APC, Miltenyi Biotech, 120-012-112; dilution 1:20 PE Mouse anti-PDX1, BD PharmingenTM, 562161; dilution 1:40 4 nature research | reporting sum m ary O ctober 2018 Alexa Fluor® 647 Mouse anti-Nkx6.1, BD PharmingenTM, 563338; dilution 1:40 Alexa Fluor® 647 Mouse IgG1 κ Isotype Control, BD PharmingenTM, 563023; dilution 1:40 Rabbit FOXA2, Cell signalling, 8186; dilution 1:1000 Goat SOX17 Acris/Novus GT15094, dilution 1:1000 Goat CER1 R&D Systems AF1075, dilution 1:1000 Mouse β-catenin BD 610154, dilution 1:1000 Guinea pig INSULIN Thermo Schientific PA1-26938, dilution 1:100 Guinea pig C-Peptide Abcam ab30477, dilution 1:300 Rabbit MAFA Betalogics LP9872, dilution 1:100 Rabbit MAFA ,Novus Biologicals, NB400-137, dilution 1:100 Rabbit GLUT1 Thermo Fisher PA1-37782, dilution 1:100 Goat GATA6 R&D Systems AF1700, dilution 1:1000 Mouse SOX2 Abgent / Bio Cat AM2048, dilution 1:1000 Rabbit CDX2 Santa Cruz sc-134468, dilution 1:1000 Mouse GCG Sigma G2654-.2ML, dilution 1:300 Goat PDX1 R&D Systems AF2419, dilution 1:500 Rabbit NKX6.1Novus biologicalsNBP1-49672, dilution 1:500 Goat NKX6.1R&D systemsAF5857, dilution 1:300 Rabbit p-JNK Cell signalling 4668, dilution 1:1000 Rabbit DVL2 Cell signalling 3216, dilution 1:1000 Mouse GAPDH Merck Biosciences CB1001, dilution 1:6000 Validation All primary antibodies were validated for their expression on undifferentiated cells and/or pancreatic human sections/islets.

Techniques: Derivative Assay, Biomarker Discovery, Microarray, Western Blot, Control, Immunofluorescence, Membrane, Marker