14-3249-80

antibody from Invitrogen Antibodies

Targeting: CDH1 CD324, UVO, uvomorulin

Provider product page for 14-3249-80

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References [46]
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Immunocytochemistry [1]
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Product number: 14-3249-80 - Provider product page
Provider: Invitrogen Antibodies
Product name: CD324 (E-Cadherin) Monoclonal Antibody (DECMA-1), eBioscience™
Antibody type: Monoclonal
Antigen: Other
Description: Description: The monoclonal antibody DECMA-1 recognizes mouse, human and canine CD324 also known as E-cadherin (Epithelial cadherin) or uvomorulin. Like the other cadherin family members P and N cadherin, E-cadherin is a transmembrane glycoprotein involved in intercellular adhesion. These proteins share a common basic structure. The extracellular portions of the proteins are largely composed of repeating domains, each with two consensus Ca2+-binding motifs. The cytoplasmic domain interacts with a-, b-, and g-catenins and actinins. These catenins connect E-cadherin with the cytoskeleton. Expression is found in most epidermal cells including melanocytes and kerotinocytes. E-cadherin is localized at the intercellular boundaries of epithelial cells in several tissues, and is thought to play a role in maintenance of tissue integrity. Loss of E-cadherin function has been implicated in the progression of a variety of cancers. E-Cadherin protein is sensitive to trypsin treatment, so exposure to trypsin should be minimized or avoided. The monoclonal antibody DECMA-1 has been shown to have functional activity by disrupting adhesion in human, mouse and dog cells. Applications Reported: This DECMA-1 antibody has been reported for use in flow cytometric analysis, immunoprecipitation, western blotting, immunohistochemical staining of frozen tissue sections, and ELISA. (Please use Functional Grade purified DECMA-1, Product # 16-3249-85 , in functional assays). Applications Tested: This DECMA-1 antibody has been tested by western blot analysis of EDTA treated MDCK cell line. This can be used at less than or equal to 2 µg/mL. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest. Purity: Greater than 90%, as determined by SDS-PAGE. Aggregation: Less than 10%, as determined by HPLC. Filtration: 0.2 µm post-manufacturing filtered.
Reactivity: Human, Mouse, Canine
Host: Rat
Isotype: IgG
Antibody clone number: DECMA-1
Vial size: 25 µg
Concentration: 0.5 mg/mL
Storage: 4° C

CDH1 protein structure - 14-3249-80 shown in red.

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Experimental details: Extended Figure 2 A E-Cadherin immunolabeling and quantification of primary KPC (n = 5 mice), KPC; Twist cKO (n = 5 mice) and KPC; Snail cKO (n = 4 mice) (scale, 100 mum). B Zeb2 immunolabeling and quantification of primary KPC (n = 6 mice), KPC; Twist cKO (n = 5 mice) and KPC; Snail cKO (n = 7 mice) (scale, 50 mum; inset scale, 20 mum). C Sox4 immunolabeling and quantification of primary KPC (n = 7 mice), KPC; Twist cKO (n = 6 mice) and KPC; Snail cKO (n = 8 mice) (scale, 50 mum; inset scale, 20 mum). D Slug immunolabeling and quantification of primary KPC (n = 4 mice), KPC; Twist cKO (n = 4 mice) and KPC; Snail cKO (n = 4 mice) tumors (scale, 50 mum; inset scale, 20 mum). E Sirius Red staining and quantification of primary KPC (n = 21 mice), KPC;Twist cKO (n = 8 mice) and KPC;Snail cKO (n = 11 mice) (scale, 200 mum; s.d.) F alphaSMA immunolabeling and quantification of primary KPC (n = 5 mice), KPC;Twist cKO (n = 5 mice) and KPC;Snail cKO (n = 5 mice) (scale, 100 mum). G CD31 immunolabeling and quantification of primary KPC (n = 4 mice), KPC;Twist cKO (n = 4 mice) and KPC;Snail cKO (n = 3 mice) (scale, 200 mum, inset scale, 100 mum). H Pimonidazole staining and quantification of primary KPC (n = 4 mice), KPC; Twist cKO (n = 4 mice) and KPC; Snail cKO (n = 4 mice) (scale, 100 mum). I CD3 immunolabeling and quantification of primary KPC (n = 5 mice), KPC;Twist cKO (n = 5 mice) and KPC;Snail cKO (n = 5 mice) (scale, 100 mum; inset scale, 25 mum). Unless otherwise indicated err

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Experimental details: Extended Figure 3 A Immunolabeling of primary tumors (n = 3 mice) for alphaSMA (red), CK8 (green), Ki-67 (white) and DAPI (blue); yellow arrows point to EMT + cells (scale, 20 mum). B Representative dot plots of circulating YFP + cells. C Images of serial sections of KPC; LSL-YFP lung and liver metastasis stained for H&E or immunolabeled for CK19 or YFP. Yellow dashed box represents magnified areas in panel below (scale, 200 mum; magnification scale, 100 mum). D KPC metastatic tumors stained for Twist and Snail (n = 3 mice; scale, 50 mum; inset scale, 20 mum). E Zeb1 immunolabeling and quantification of metastatic KPC (n = 4 mice), KPC; Twist cKO (n = 3 mice) and KPC; Snail cKO (n = 4 mice) (scale, 50 mum; inset scale, 20 mum). F alphaSMA immunolabeling and quantification of metastatic KPC (n = 3 mice), KPC; Twist cKO (n = 3 mice) and KPC; Snail cKO (n = 3 mice) (scale, 50 mum; inset scale, 20 mum). G E-Cadherin staining on serial sections of alphaSMA immunolabeling and quantification of metastatic KPC (n = 4 mice), KPC; Twist cKO (n = 3 mice) and KPC; Snail cKO (n = 4 mice) (scale, 50 mum; inset scale, 20 mum). H Ki-67 immunolabeling and quantification of metastatic KPC (n = 7 mice), KPC; Twist cKO (n = 3 mice) and KPC; Snail cKO (n = 3 mice) (scale, 50 mum). Unless otherwise indicated error bars represent s.e.m, percentages indicated represent percent decrease from control, and significance determined by One-way ANOVA. * P

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Experimental details: Figure 2 Chemoresistant NSCLC cells exhibit an activated EMT pathway. ( a ) Morphological images of chemoresistant A549_R and parental A549 cells. Scale bar, 100 mu m. ( b ) A549_R and A549 cells were immunostained for Vimentin, beta -catenin and E-cadherin (green). Nuclei were counterstained with DAPI (blue). Scale bar, 25 mu m ( c-e ) mRNA levels of EMT-TFs and CDH2/N-cadherin in A549_R, H358_R and H460_R cells and the corresponding parental cells (A549, H358 and H460) were analyzed by qPCR. Data are shown as mean+-S.D. of three independent experiments ( n =3). ( f ) Different NSCLC cells are at distinct stages along the EMT. mRNA levels of EMT-TFs and Vimentin in PC9, H358, A549 and H460 cells were analyzed by qPCR and results are shown in mean+-S.D. of three independent experiments ( n =3). ( g ) NSCLC cells at distinct EMT stages exhibit differential sensitivity to MTA. PC9, H358, A549 and H460 cells treated with the indicated MTA doses for 5 days were analyzed by XTT assay. The results are shown as mean+-S.D. of triplicate experiments. * P

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Experimental details: Figure 2 Growth and androgen-responsiveness of prostate organoids from normal prostate epithelium. ( a ) Flow-sorting strategy to eliminate EpCAM - E-cadherin - cells from dissociated prostate tissue for organoid culture. ( b ) Low-power view of organoids at 20 days after plating, showing heterogeneity of phenotype. ( c ) Higher-power view showing hollow and filled budding organoid (arrow). ( d,e ) H&E staining of sections from a hollow organoid ( d ) and a multi-layered organoid ( e ). ( f ) Many proliferating cells are detectable by Ki67 immunostaining (arrows). ( g ) Organoids have an outer layer that expresses the basal marker CK5 (arrowheads). ( h ) Outer cells express the basal marker p63 (arrowheads), while interior cells are positive for the luminal marker CK18. ( i ) Nuclear immunostaining of AR (arrows) in organoids cultured in standard conditions with DHT. ( j ) Nuclear immunostaining for Foxa1 (arrows). ( k ) Tissue recombination of normal organoids with rat embryonic urogenital mesenchyme followed by renal grafting results in reconstitution of prostate tissue. ( l,m ) Organoids at passage 4 were passaged as single-cell suspensions and plated in the presence of DHT ( l ) or absence of DHT ( m ). ( n,o ) Strong nuclear AR immunostaining in the presence of DHT ( n ) and weak cytoplasmic AR immunostaining in the absence of DHT ( o ). ( p ) qPCR analysis of expression of AR downstream genes in organoids cultured in the presence or absence of DHT. Results are from a si

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Experimental details: Figure 1 Genetic targeting of EMT reduces renal fibrosis and improves tubular health. ( a-c ) Representative images (8 visual fields for each tissue analyzed) of H&E ( a ), MTS ( b ) and Sirius Red ( c ) staining of kidneys from the indicated experimental groups. Scale bar, 100 ~m; insert, 25 ~m. ( d ) Number of healthy tubules. WT contralat., n = 4; Twist cKO contralat., n = 3; WT UUO, n = 9; Twist cKO UUO, n = 9. ( e-f ) Interstitial fibrosis, based on MTS ( e ) and Sirius Red ( f ) staining. WT contralat., n = 4; Twist cKO contralat., n = 4; WT UUO, n = 9; Twist cKO UUO, n = 9. ( g ) Blood urea nitrogen (BUN) levels. Healthy, n = 3; WT NTN, n = 7; Twist cKO NTN; n = 7. WT vehicle, n = 4; WT FA, n = 4; Twist cKO FA, n = 4. ( h ) Representative images (8 visual fields for each tissue analyzed) of immunolabeling for alphaSMA and YFP (left) and quantification of the percent YFP + alphaSMA + cells per total number of YFP + tubular epithelial cells (right). WT UUO, n = 3; Twist cKO UUO, n = 4. Scale bar, 20 ~m. White arrowheads, YFP + /alphaSMA + cells. ( i ) Representative images (3 visual fields for each tissue analyzed) of YFP + proximal tubules and alphaSMA-RFP + myofibroblasts in UUO-treated mice (left) and quantification of the percent of YFP + RFP + cells (right). gammaGT-Cre; LSL -EYFP LoxP/+ ; alphaSMA-RFP, n = 3; gammaGT-Cre; LSL -EYFP LoxP/+ ; alphaSMA-RFP; Twist cKO , n = 4. Scale bar, 20 ~m. ( j ) Percent E-cadherin + alphaSMA + cells (as measured by flow cytometry)

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Experimental details: Fig. 3 miR-384 mimic inhibits EMT, migration and invasion of NPC cells. a levels of mesenchyme marker protein Vimentin and epithelium-marker protein E-cadherin in cells measured by western blot analysis; b , c number of migrated ( b ) and invaded ( c ) cells determined by Transwell assays. Data are exhibited as mean +- SD from three independent experiments; in panel ( a ), data were analyzed using two-way ANOVA, while data in panels ( b ) and ( c ) were analyzed by one-way ANOVA, and Tukey''s multiple comparison test was used for the post-hoc test after ANOVA; * p < 0.05; ** p < 0.01 vs. the Mock group; # p < 0.05 vs. the InC group

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Experimental details: Fig. 4 Caspase-11 mediates STAT1 activation during CAC. a Western blot analysis of phosho-/total STAT1, STAT3, IkappaBalpha, and actin (loading control) in colon homogenates from AOM/DSS-treated WT and Casp11 -/- mice on day 105 post initial AOM IP injection. Each lane represents an individual mouse. b Total STAT1 and c pSTAT1 expression densitometry (relative to actin) in colon homogenates from AOM/DSS-treated WT and Casp11 -/- mice. ** p < 0.01 (two-tailed unpaired t-test). Representative immunofluorescent images of d pSTAT1; and e total STAT1; co-stained for the epithelial marker E-cadherin (red) and DAPI (blue) in distal colon sections from AOM/DSS-treated WT and Casp11 -/- mice at day 105 (scale bar = 20 mum)

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Experimental details: Fig. 1 Reprogramming trajectories of cocktails containing Oct4, Oct6, Oct4 defSox2 , or GFP in chemically defined medium. a The cartoons represent the preferences of the POU proteins for the DNA-dependent homodimerization on MORE versus heterodimerization with Sox2 on SoxOct elements determined using quantitative biochemical assays . MORE DNA is shown in orange and SoxOct DNA in blue; m1 is monomer 1 and m2 is monomer2. The thickness of the arrows illustrates the DNA binding preferences. b Experimental design of reprogramming and RNA-seq, ATAC-seq, and ChIP-seq experiments. c Whole-well scans from a 6-well plate using GFP channel ( Oct4 -GFP), Nanog immunofluorescence and merged panels; for OG2-MEF cells reprogrammed with Oct4-SK at day 8; scale 5 mm. d Whole-well scans (upper panel) of wells from 12-well plate using GFP channel for three POU factors (Oct4, Oct4 defSox2 , and Oct6); scale 5 mm. Representative phase contrast (middle panel) and corresponding Oct4 -GFP fluorescence (lower panel) images of reprogramming experiments; time-point: day 8 post transduction; scale 200 mum. e Hierarchically clustered heatmap based on r2 correlation coefficients using RNA-seq reads as input. iPSCs and ESCs expression data are from GSE93029 . f Mean gene expression trajectories for indicated categories (upper panel) or a representative gene from each category (lower panel) in four reprogramming conditions. See Supplementary Fig. 1E for a larger panel of genes for each category. g Fraction

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Experimental details: Figure 3 Leptin stimulated epithelial-mesenchymal transition (EMT) in CHMp cells. ( a ) Matrigel invasion assay and calculated invasion index of CHMp and siRNA-transfected cells. Invaded cells were counted under light microscopy (40x) using ImageJ software. ( b ) Comparison of EMT-related gene ( E-cadherin and vimentin ) expression using real-time PCR analysis among experimental groups. ( c ) Western blot analysis of tumor invasion-related genes. All graphs are visualized as mean +- SEM with at least three replicates. The column bars with different alphabetical letters indicate significant difference among groups ( p < 0.05). Combi: combination of leptin and Allo-aca; NC: negative control siRNA.

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Experimental details: Figure 4 Fluorescence staining of epithelial markers in the lungs. Notes: Lung sections from SCID-beige mice administered intravenously with PKH26-labeled hADSCs were observed under aconfocal laser microscope. ( A ) Fluorescence of PKH-26 (red) was observed with nuclear counterstaining by DAPI (blue) (x200). ( B ) Expression of epithelial cells markers TTF-1, Pro SP-C, beta-Catenin, and CDH1 visualized by Alexa Fluor 488 (green) in PKH26-labeled hADSCs (red) (x400). Arrow shows Alexa Flour 488 and PKH-26 merged cells. Scale bar shows 50 mum. Data are representative of more than three independent experiments. Abbreviations: hADSCs, human adipose-derived mesenchymal stem cells; DAPI, 4',6-diamidino-2-phenylindole; TTF-1, thyroid transcription factor-1; SP-C, surfactant protein C; CDH1, cadherin-1.

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Experimental details: Fig. 2. Characterization of unique 3D structures. (A) Representative bright-field image, schematic, and description for each class (prestructure, spheroid, and tubule) of organoid structures. Scale bars: 100 mum. (B) Bright-field images and corresponding immunofluorescence staining for E-cadherin (purple) in non-differentiated (E-cadherin negative; prestructures) and differentiated (E-cadherin positive; spheroids and tubules) structures. Scale bars: 50 mum. (C) Representative bright-field images of tubule development in the culture model. Tubules are often part of more complex 3D structures, including the globular, supportive prestructures. Scale bars: 25 mum. (D) Differentiated tubule structures demonstrate basolateral localization of Na + /K + -ATPase (green). Nuclei are stained with DAPI (blue). Scale bar: 50 mum. (E) Spheroids and tubules additionally demonstrate primary cilia, as indicated by acetyl-alpha-tubulin (red) puncta on the apical membrane. Basolateral Na + /K + -ATPase (green) and nuclear DAPI staining (blue) are also shown. Arrows indicate cilia, positively stained by acetyl-alpha-tubulin. Scale bars: 25 mum.

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Experimental details: Figure 4. Immunofluorescence analysis with DAPI staining on DU145 cells. (A) Expression of E-cadherin in CMTM5-overexpressing cells and vector control cells. (B) Expression of p-EGFR in CMTM5-overexpressing cells and vector control cells. Scale bar, 100 um. ***P

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Experimental details: Figure 4 Tight junction proteins distribution in spheroids. A typical image of a spheroid. The presence of junction markers zona occludens-1 (ZO-1) and E-cadherin were confirmed via immunofluorescent staining: red, ZO-1; green, E-cadherin; blue, nuclei (DAPI).

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Experimental details: Figure 3 rLRR20 and Leptospira infection downregulated E-cadherin expression. Neutralizing anti-E-cadherin antibody (Neu-alpha-E-cad) and short interfering RNA against E-cadherin (si-E-cad) were used to verify the regulatory effects of rLRR20 on E-cadherin expression in HK2s and hRPTECs. Effect of alpha-E-cad, which inhibits the interaction between rLRR20 and E-cadherin, on the rLRR20-mediated degradation of E-cadherin in HK2s ( A ) and hRPTECs ( B ). Effect of si-E-cad on the rLRR20-mediated degradation of E-cadherin in HK2s ( C ) and hRPTECs ( D ). Effect of pathogenic ( L. santarosai ) and non-pathogenic ( L. biflexa ) Leptospira species on the E-cadherin levels in HK2s ( E ) and hRPTECs ( F ). beta-actin was used as the internal control. EMJH, Ellinghausen-McCullough-Johnson-Harris medium. *** p < 0.001; ** p < 0.01; * p < 0.05.

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Experimental details: Figure 4 ABZ treatment downregulates the snail expression in melanoma cells by increasing the accumulation of phosphorylated GSK-3 beta /Tyr216. (a) The relative transcription levels of Snail in the ABZ-treated (0.4 mu M) and control groups of A375 (left) and B16-F10 (right) melanoma cells were measured by RT-qPCR, with beta -actin as the internal control. (b) The expression of transcription factor Snail in A375 (left) and B16-F10 (right) cells was detected by western blot analysis, with beta -actin as the internal reference protein. (c-d) The expression levels of cytoplasmic proteins AKT, pAKT, GSK-3 beta , pGSK-3 beta (Ser9/Tyr216) and Snail, and nuclear protein pSnail in A375 and B16-F10 cells were also determined by western blotting, with beta -actin and PCNA as the internal controls for the cytoplasmic and nuclear proteins, respectively. The histograms show the relative density of AKT/pAKT, GSK-3 beta /pGSK-3 beta (Ser9/Tyr216), and Snail/p-Snail. (e) A375 cells were cotreated with or without MG132 and 0.4 mu M ABZ for 24 h western blot (up) was used to detect the expression levels of AKT, pGSK-3 beta /Tyr216, Snail, N-cadherin, and E-cadherin in the cytoplasm of A375 cells. The histogram (bottom) shows the relative density of AKT, pGSK-3 beta /Tyr216, Snail, E-cadherin, and N-cadherin. (f) Histogram showing the relative expression intensity of pGSK-3 beta (Ser9/Tyr216) and pAKT after immunohistochemical staining of mouse metastatic lung cancer tissues. Scale bars = 100

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Experimental details: Figure 3 Inefficient Reprogramming Progression of OKMS Reprogramming Intermediates (A) E-CAD and Nanog -GFP expression changes during TNG MKOS/OKMS reprogramming. Red indicates E-CAD - Nanog -GFP - , white indicates E-CAD + Nanog -GFP - , and green indicates E-CAD + Nanog -GFP + . (B) CD44 and ICAM1 expression changes during TNG MKOS/OKMS reprogramming with E-CAD, Nanog -GFP expression color codes in (A). (C) Flow cytometry analysis of sorted day-10 E-CAD -/+ 2NG- ( Nanog -GFP - CD44 - ICAM1 - ), 3NG - ( Nanog -GFP - CD44 - ICAM1 + ), and 3NG + ( Nanog -GFP + CD44 - ICAM1 + ) cells after a 24-hr culture. dox, doxycycline. (D) E-CAD -/+ 2NG - , 3NG - , and 3NG + ( Nanog -GFP + CD44 - ICAM1 + ) cells on day 10 were seeded at clonal density, and Nanog -GFP + iPSC colonies were counted after 10 days of further culture. The graph depicts the relative Nanog -GFP + CFA compared to that of MKOS 3NG + cells. Error bars represent SD; n = 3 independent experiments.

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Experimental details: Figure 1 Epithelial-to-Mesenchymal Transition Reported by Historical Lineage-Tracing Reporter and Current E-Cadherin Status in Murine Metastatic Mammary Carcinoma Model (A) Schematic of fluorescent transgenic mouse model, carrying polyoma middle-T (PyMT) oncogene driven by the mammary gland specific MMTV promoter, Cre recombinase under the control of the fsp1 promoter, endogenous E-cad labeled with mCFP, and ubiquitous expression from the R26 locus of membranous RFP-STOP, which is flanked by lox P sites, leading to excision and subsequent expression of membranous GFP upon Cre expression. (B) Top panel: representative multi-photon images of the fluorescent PyMT mammary tumors from recipient mice. Scale bar, 100 mum. Bottom panel: high-magnification images of the E-cad-mCFP and RFP channel of the boxed area in the top left image. Scale bar, 50 mum. (C) Representative scatterplot from flow cytometry analysis of primary tumor of recipient mice for either absence of endogenous E-cad-mCFP expression and antibody staining (E-cad LO ) or presence of high expression of E-cad and staining for Ab (E-cad HI tumor cells) or subdivision of RFP + or GFP + cells. (D) Relative mRNA expression of classical EMT marker genes (E-cad, Vimentin [Vim], fibronectin [FN], Zeb1, N-cad [N-cadherin], Slug) determined using qPCR of sorted RFP + E-cad Hi , RFP + E-cad LO , RFP + GFP + , and GFP + cells from primary tumors. Plotted mean and SD of n = 3 mice. (E) Quantification of lung metastases of all tran

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Experimental details: Figure 2 Mesenchymal E-cad LO Population Exceeds fsp1 -Lineage-Traced Population in Primary Tumor and Circulation and Has Metastatic Potential (A) Percentage of GFP + (green), GFP + RFP + (yellow), and RFP + (red) tumor cells in primary tumors of recipient mice. n = 16 mice. (B) Percentage of E-cad HI (blue), E-cad LO (light blue) tumor cells in primary tumors of recipient mice. n = 16 mice. (C) Comparison of percentage of E-cad LO (light blue) with GFP + (green) and GFP + RFP + (yellow) tumor cells in primary tumors of recipient mice. n = 16 mice. (D) Percentage of GFP + (green), GFP + RFP + (yellow), and RFP + (red) circulating tumor cells collected from the right cardiac chamber of recipient mice. n = 16 mice. (E) Percentage of E-cad HI (blue), E-cad LO (light blue) circulating tumor cells collected from the right cardiac chamber of recipient mice. n = 16 mice. (F) Comparison of percentage of E-cad LO (light blue) with GFP + (green) and GFP + RFP + (yellow) circulating tumor cells collected from the right cardiac chamber of recipient mice. n = 16 mice. (G) Top panel: schematic overview of experimental setup: fluorescence-activated cell sorting (FACS)-sorted E-cad HI or E-cad LO cells of orthotopically transplanted mice were injected in the tail vein of acceptor mice. Bottom panel: quantification of lung metastases of all transplanted mice. n = 3 mice per condition. (H) Representative multi-photon image of lung metastasis from (G). Scale bar, 500 mum. Right panels: zoom of

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Experimental details: Figure 3. Reprogramming-Specific Gene Expression Patterns Are Important for Conversion to iPSCs (A) t-SNE plots based on Figure 2A highlighting the expression of MEF-associated mesenchymal genes that are downregulated as cells transition from MEFs to pluripotency. Top schematic indicates the pattern of expression. (B) Percentage of Cdh1+ cells that also co-express the indicated MEF genes on the x axis. The percentage of MEF gene-expressing cells that express Cdh1 is presented in brackets on the x axis. Note that because of the limit of detection of single-cell transcriptional analysis, co-expression may be underestimated. (C) (i) t-SNE plots based on Figure 2A illustrating co-expression of Cdh1 with Twist1. Note that because of the limit of detection of single-cell transcriptional analysis, co-expression may be underestimated. (ii) Immunofluorescent staining for CDH1 and TWIST1. Percentage of CDH1+/TWIST1+ colonies on A2S day 4 shown below image. Scale bar, 10 mum. (D) t-SNE plots based on Figure 2A highlighting the expression of DNA replication and cell-cycle-associated genes. Top schematic indicates the pattern of expression. (E) Left: percentage of cells that are Ki67+ at each indicated reprogramming time point in FBS or A2S systems. Right: immunofluorescent staining of Ki67 during FBS and A2S reprogramming (day 9 and day 4, respectively). Scale bar, 50 mum. (F) t-SNE plot based on Figure 2A for the anti-proliferation gene Cdkn1c. Top schematic indicates the patter

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Experimental details: Figure 1 Nanog+ Colonies from Neural Stem Cell and Astrocyte Reprogramming Can Emerge Independent of E-Cadherin or SSEA1 (A) Immunofluorescence (IF) images of NANOG colonies on day 10 of reprogramming NSC with E-cadherin and/or SSEA1. Scale bar, 50 mum. Insets, magnification of field. (B) Scheme of experiment presented in (C) and (D). Dox was added to cells on day 0 (d0) and reprogramming cultures were fixed on indicated days. (C) Counts of Nanog+ (N+), E-cadherin+ (E+) and Nanog+/E-cadherin+ (N+E+) colonies from (i) NSC, (ii) astrocyte, and (iii) MEF reprogramming cultures, fixed on days indicated on X axis. Counts from three independent experiments are stacked. Statistical significance of difference between N+ and N+E+ colonies by paired two-tailed t test; * p < 0.05, ** p < 0.01. Non-significant differences are not marked. (D) As in (C) above except for SSEA1+. SSEA1 = S+, Nanog+/SSEA1 = N+S+. t test; * p < 0.05, ** p < 0.01. (E) Combined counts of NANOG+/SSEA1+/E-cadherin. Counts from two independent experiments are stacked. Note that the number of S+E+ colonies and E+S+ colonies differ because of scoring between large fields of SSEA containing multiple E-cadherin+ colonies.

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Experimental details: Figure 3 Late Reprogramming Markers Are Enriched in the N+E+ Population (A) Flow cytometry plot of reprogramming culture stained with Nanog-647 and E-cadherin-488. (B) RNA-Seq read counts (transcripts per million [TPM]) for E-cadherin, labeled Cdh1 in figure, (left, Y axis) and Nanog (right, Y axis) for N+E+ and N+E- populations from three independent experiments. (C) Heatmap of genes 1.5-fold differentially expressed between N+E+ and N+E- populations (set to 1) ranked by average TPM from three independent experiments. (D) Functional annotation of gene expression in N+E+ and N+E- populations. (E) Left: Immunofluorescence images of NANOG, E-cadherin, and DPPA4 on d11 of NSC reprogramming. Right: Counts from two independent experiments of Dppa4+ colonies in N+E+ and N+E- colonies are stacked. Scale bar, 50 mum. Insets, magnification of field. (F) Comparison of d14 N+ and N+E+ colonies following withdrawal of dox on the day indicated on the X axis. Counts from four independent experiments are stacked. No significant difference between N+ and N+E+ colonies by paired two-tailed t test.