Journal: British Journal of Cancer

Article Title: An intrinsic purine metabolite AICAR blocks lung tumour growth by targeting oncoprotein mucin 1

doi: 10.1038/s41416-023-02196-z

Figure Lengend Snippet: a Diagram showing the strategy employed to shortlist AICAR-binding proteins. The three steps include in silico screening using FINDSITE comb2.0 , protein expression assay, and thermal stability assay. b Time-dependent western blotting and relative quantification of protein expression for AICAR-binding proteins. The treatment responses on H1975 cells treated with 1 mM AICAR for 1 and 2 h were grouped by strong inactivation (type 1) and weak response (type 2). GAPDH was used as a loading control. N = 2–3 replicates. c Dose-dependent western blotting and relative quantification of protein expression for MUC1-CT and TMEM70. H1975 cells were treated with increasing doses of AICAR (0, 0.4, 1.3, and 4.4 mM) for 22 h, followed by a western blot assay. β-actin was used as a loading control. N = 3 replicates. d Thermal stability assay for MUC1-CT and TMEM70. H1975 cells were treated with 1 mM AICAR for 15 min. The cell pellets were heated for 3 min at their respective temperature (37–55 °C), followed by a western blot assay. N = 2 replicates. e Immunofluorescence staining for MUC1-CT in H441 cells. The cells were treated with 0.3 mM AICAR for 4 h and then incubated with rabbit anti-MUC1-CT primary antibody followed by goat anti-rabbit IgG conjugated with Alexa Fluor 555. The nucleus was counterstained with DAPI. The images were taken using a Keyence fluorescent microscope. Scale bar, 50 μm. f qRT-PCR analysis for MUC1-CT targeting genes. Expression levels for CTGF , PGM2 , and ENO1 were analysed by qRT-PCR in H1975 cells treated with 0.3 mM AICAR for 4 h. GAPDH was used as an endogenous control. N = 3 replicates. g qRT-PCR analysis for MUC1 expression in H1975 cells with MUC1 overexpression (OE). The cells were transfected with a lentiviral vector containing MUC1 or scrambled control, followed by 0.5 µg/ml puromycin selection. The relative MUC1 expression level in scrambled control cells was calibrated as 1. GAPDH was used as an endogenous control. N = 3 replicates. h Organoid formation assay for AICAR treatment response in H1975 cells overexpressing MUC1 . The cells with MUC1 overexpression or a scrambled control vector were plated at 2000 cells per well and treated with vehicle or 0.3 mM AICAR continuously for nine days. Images were taken with an EVOS microscope, and the treatment responses were quantified using a 3D Celltiter-Glo assay. N = 4–5 replicates. Scale bar, 300 µm. Data are mean ± s.e.m. and were analysed with Brown-Forsythe and Welch ANOVA ( b , c , f , h ); Welch’s t -test ( d , g ). * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant.

Article Snippet: Taqman gene expression probes included MUC1 (Hs00159357_m1), Egfr (Mm01187858_m1), Muc1 (Mm00449604_m1), CTGF (Hs00170014_m1), ENO1 (Hs00361415_m1), and PGM2 (Hs01055491_m1).

Techniques: Binding Assay, In Silico, Expressing, Stability Assay, Western Blot, Quantitative Proteomics, Control, Immunofluorescence, Staining, Incubation, Microscopy, Quantitative RT-PCR, Over Expression, Transfection, Plasmid Preparation, Selection, Tube Formation Assay, Glo Assay

Journal: British Journal of Cancer

Article Title: An intrinsic purine metabolite AICAR blocks lung tumour growth by targeting oncoprotein mucin 1

doi: 10.1038/s41416-023-02196-z

Figure Lengend Snippet: a Top KEGG signalling pathways differentially expressed in H1975 cells treated with AICAR. The cells were treated with 1 mM AICAR for 4 h, followed by whole transcriptomic analysis. N = 3 replicates. b Enrichment plot by gene set enrichment analysis for the JAK-STAT signalling pathway in H1975 cells treated with AICAR. Profile of the running enrichment score (ES) (top) and positions of gene set members on the rank-ordered list (bottom) were shown. N = 3 replicates. c A heat map showing top enriched genes of the JAK-STAT signalling pathway in H1975 cells treated with AICAR compared with vehicle-treated cells. d Longitudinal analysis of p-JAK1, p-TYK2, and MUC1-CT expression. H1975 cells were treated with 1 mM AICAR for 0, 1, and 2 h followed by a western blot assay. β-actin was used as a loading control. N = 3 replicates. e Confocal images for co-localisation of MUC1-CT and p-JAK1 in lung cancer cells. H1975 cells were co-incubated with Armenian hamster anti-MUC1-CT and rabbit anti-p-JAK1 primary antibodies for 2 h. Then the cells were incubated with secondary antibodies conjugated with Alexa Fluor 488 or 555. The nucleus was counterstained with DAPI. The images (top: lower magnification; bottom: higher magnification) were taken using a Zeiss confocal fluorescent microscope. Scale Bar, 50 µm (top) and 20 µm (bottom). f Duolink ligation assay and confocal imaging for physical MUC1-JAK1 interactions. H441 cells were treated with vehicle or 1 mM AICAR for 1 h. After treatment, the cells were incubated with mouse anti-ZO-1 primary antibody overnight, followed by anti-mouse IgG conjugated with Alexa Fluor 488. Then the cells were co-incubated with anti-MUC1 and anti-JAK1 primary antibodies, followed by incubation with proximity ligation assay probes conjugated with Cy3, ligation, and amplification steps. The nucleus was counterstained with DAPI. The images were taken using a Zeiss confocal fluorescent microscope, and the Duolink dots were quantified using Image J. Scale Bar, 20 µm. g Cell viability assay of H1975 cells treated with AICAR and VX-509. Cells were plated in a 96-well plate and treated with AICAR (1 mM) with or without VX-509 (10 μM). The cell viability was measured three days after treatment. Values were normalised to a vehicle-treated control group. N = 3–4 replicates. Data are mean ± s.e.m. and were analysed with Welch’s t -test ( a , b , f ); Brown-Forsythe and Welch one-way ANOVA ( d , g ). * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant.

Article Snippet: Taqman gene expression probes included MUC1 (Hs00159357_m1), Egfr (Mm01187858_m1), Muc1 (Mm00449604_m1), CTGF (Hs00170014_m1), ENO1 (Hs00361415_m1), and PGM2 (Hs01055491_m1).

Techniques: Expressing, Western Blot, Control, Incubation, Microscopy, Ligation, Imaging, Proximity Ligation Assay, Amplification, Viability Assay

Journal: British Journal of Cancer

Article Title: An intrinsic purine metabolite AICAR blocks lung tumour growth by targeting oncoprotein mucin 1

doi: 10.1038/s41416-023-02196-z

Figure Lengend Snippet: a Western blotting and quantitative analysis for p-EGFR (Y1068), EGFR, and MUC1-CT in EGFR TL ( T790M; L858R )-induced lung tissues from transgenic mice. The mice were fed doxycycline (Dox)-impregnated food pellets for 0, 1, and 2 weeks followed by whole lung-tissue extraction. N = 2 replicates. b qRT-PCR analysis of gene expression for EGFR and MUC1 in EGFR TL -induced lung tissues. Gapdh was used as an endogenous control. EG0, EG1, and EG2 represent tissues from the mice fed with dox-impregnated food pellets for 0, 1, and 2 weeks, respectively. N = 3 replicates. c Western blotting and quantitative analysis for p-EGFR (Y1068), EGFR, and MUC1-CT in EGFR TL -induced lung tissues after EGFR inactivation. The mice fed with Dox-impregnated food pellets for 8 weeks were given either the same Dox diet for an additional 2 weeks (EG10) or a regular diet for 2 weeks (EG8off2). Then the whole lung tissues were extracted for protein expression assay. N = 2 replicates. d qRT-PCR analysis of gene expression for EGFR and MUC1 in mouse EGFR TL -induced lung tissues after EGFR inactivation. The lung tissues from EG14 and EG8OFF2 mice were extracted for RNA analysis. GAPDH was used as an endogenous control. N = 3 replicates. e Western blotting and quantitative analysis for p-EGFR (Y1068) and EGFR expression in H1975 cells with MUC1 overexpression (OE). β-actin was used as a loading control. N = 3 replicates. f Western blotting and quantitative analysis for p-EGFR (Y1068), EGFR, and MUC1-CT expression in H1975 cells treated with 1 mM AICAR for one and 2 h. β-actin was used as a loading control. N = 3 replicates. g qRT-PCR analysis for MUC1 gene expression in H1975 cells with MUC1 knockdown. The cells were transfected with a lentiviral vector containing shRNA against MUC1 (shMUC1) or a scrambled control vector (sh-Control), followed by a 0.5 µg/ml puromycin selection. GAPDH was used as an endogenous control. N = 3 replicates. h Cell viability assay of H1975 cells treated with osimertinib and VX-509. 3000 cells with MUC1 knockdown (sh-MUC1) and a negative control vector (sh-control) were plated in a 96-well plate and treated with VX-509 (10 μM), osimertinib (0.5 μM), or both. The cell viability was measured three days after treatment. Values were normalised to a vehicle-treated sh-control group. N = 4 replicates. Data are mean ± s.e.m. and were analysed with unpaired two-tailed t -test ( c , d , e , g ); one-way ANOVA ( a , b ); Brown-Forsythe and Welch ANOVA ( f , h ). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant.

Article Snippet: Taqman gene expression probes included MUC1 (Hs00159357_m1), Egfr (Mm01187858_m1), Muc1 (Mm00449604_m1), CTGF (Hs00170014_m1), ENO1 (Hs00361415_m1), and PGM2 (Hs01055491_m1).

Techniques: Western Blot, Transgenic Assay, Extraction, Quantitative RT-PCR, Gene Expression, Control, Expressing, Over Expression, Knockdown, Transfection, Plasmid Preparation, shRNA, Selection, Viability Assay, Negative Control, Two Tailed Test

Journal: British Journal of Cancer

Article Title: An intrinsic purine metabolite AICAR blocks lung tumour growth by targeting oncoprotein mucin 1

doi: 10.1038/s41416-023-02196-z

Figure Lengend Snippet: a qRT-PCR analysis of gene expression for EGFR and MUC1 in EGFR TL -induced lung tissues from transgenic mice. The far normal and tumour tissues from transgenic mice 14 weeks after Dox induction were extracted for RNA analysis. Gapdh was used as an endogenous control. N = 3 replicates. b Differential MUC1 gene expression in lung adenocarcinoma (LUAD) compared with tumour-adjacent tissues by analysing the TCGA_LUAD dataset. N = 59 (normal), and N = 517 (tumour). c Patients’ overall survival in lung adenocarcinoma patients at stages II–IV. The median expression levels of MUC1 were used for a cut-off of high and low expression for MUC1 . N = 135. d Patients’ disease-free survival in lung adenocarcinoma patients at stages II–IV. The median expression levels of MUC1 were used for a cut-off of high and low expression for MUC1 . N = 105. e Xenograft tumour growth in mice treated with AICAR. The xenograft tumour was pre-established by implanting 1 million H1975 cells subcutaneously. When the tumour reached 45 mm 3 , the mice were treated with 300 mg/kg/day AICAR in PBS or a vehicle subcutaneously for ten days. The tumour was measured with a digital caliper, and the tumour size was calculated. N = 7 replicates. f Xenograft tumour images and relative weight quantification from mice treated with AICAR or PBS. The average tumour weight from the PBS-treated group is normalised as 1. N = 7 replicates. g Mouse body weight after treatment with AICAR or PBS for ten days. N = 7 replicates. h , i H&E staining of subcutaneous tumours ( h ) and liver tissues ( I ) from H1975 cell line-derived xenograft (CDX) treated with PBS or AICAR. Scale bar, 125 μm. j – l Immunofluorescence staining for Ki-67 ( j ), γ-H 2 AX ( k ), and p21 Cip1 ( l ) in subcutaneous tumours from H1975 CDX treated with PBS or AICAR. Scale bar, 100 μm. Data are mean ± s.e.m. and were analysed with unpaired two-tailed t -test ( a , b ); log-rank test ( c , d ); Welch’s t -test ( e – g ). * p < 0.05; ** p < 0.01; **** p < 0.0001; ns, not significant.

Article Snippet: Taqman gene expression probes included MUC1 (Hs00159357_m1), Egfr (Mm01187858_m1), Muc1 (Mm00449604_m1), CTGF (Hs00170014_m1), ENO1 (Hs00361415_m1), and PGM2 (Hs01055491_m1).

Techniques: Quantitative RT-PCR, Gene Expression, Transgenic Assay, Control, Expressing, Staining, Derivative Assay, Immunofluorescence, Two Tailed Test

Journal: British Journal of Cancer

Article Title: An intrinsic purine metabolite AICAR blocks lung tumour growth by targeting oncoprotein mucin 1

doi: 10.1038/s41416-023-02196-z

Figure Lengend Snippet: a A diagram showing mechanisms of AICAR’s anticancer roles. In MUC1-dependent tumours, AICAR treatment directly binds and degrades MUC1-CT, increasing DNA damage in tumour cells. The degraded MUC1-CT de-stabilises p-EGFR and p-JAK1, further inactivating tumour-supportive signals. Created with BioRender.com. b Treatment response to VX-509 and osimertinib and AICAR in H1975 cells. 3000 cells were plated in a 96-well plate and treated with VX-509 (10 μM), osimertinib (0.5 μM), AICAR (1 mM), or a combination. The cell viability was measured 3 days after treatment. Values were normalised to a vehicle-treated group. N = 4 replicates. c , d Growth of PDX ( c ) and transgenic mouse EGFR TL -induced lung tumour ( d )-derived organoids treated with AICAR, osimertinib, and VX-509. 2000 cells were plated in organoid-culture media followed by treatments with AICAR (1 mM), osimertinib (0.5 μM), VX-509 (10 μM), or combinations for 10 days. The media were replenished every three days. The 3D cultures’ size was measured on day ten by ImageJ. The organoid tumour area in the vehicle-treated group was normalised as 100%. Scale bar, 50 μm. N = 6–12 replicates. Data are mean ± s.e.m. and were analysed with Brown-Forsythe and Welch ANOVA ( b , c , d ). * p < 0.05; ** p < 0.01; **** p < 0.0001.

Article Snippet: Taqman gene expression probes included MUC1 (Hs00159357_m1), Egfr (Mm01187858_m1), Muc1 (Mm00449604_m1), CTGF (Hs00170014_m1), ENO1 (Hs00361415_m1), and PGM2 (Hs01055491_m1).

Techniques: Transgenic Assay, Derivative Assay

Journal: Cell reports

Article Title: Molecular Stressors Engender Protein Connectivity Dysfunction through Aberrant N -Glycosylation of a Chaperone

doi: 10.1016/j.celrep.2020.107840

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-Flotillin-1 , Cell Signaling Technology , Cat# 3253, RRID:AB_2106734.

Techniques: Recombinant, Clinical Proteomics, Membrane, Isolation, Cell Fractionation, Extraction, Viability Assay, Transfection, Software, Western Blot, Staining

Journal: Cancers

Article Title: Niclosamide Revitalizes Sorafenib through Insulin-like Growth Factor 1 Receptor (IGF-1R)/Stemness and Metabolic Changes in Hepatocellular Carcinoma

doi: 10.3390/cancers15030931

Figure Lengend Snippet: High IGF-1R expression and malignant properties in sorafenib-resistant HCC cell lines. ( A ) Cell morphology of sorafenib-naïve HCC cells (HepG2215 and Hep3B) and sorafenib-resistant HCC cells (HepG2215_R and Hep3B_R). ( B ) Cell viability and IC50 values of sorafenib-naïve HCC cells (HepG2215 and Hep3B) and sorafenib-resistant HCC cells (HepG2215_R and Hep3B_R) under sorafenib treatment (0, 5, 10, 15, 20 and 50 μM). ( C ) Expression of IGF-1R protein in sorafenib-naïve HCC cells (HepG2215 and Hep3B) and sorafenib-resistant HCC cells (HepG2215_R and Hep3B_R). The results of three replicates were shown in . ( D ) Gene expression profile of sorafenib-naïve HCC and sorafenib-resistant HCC obtained using real-time RT-qPCR analyses. ( E ) Secondary sphere formation assay. Bar = 100 μm. ( F ) Migration ability assay. Bar = 100 μm. For all quantification, data are the mean ± SD of at least three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, as obtained from Student’s t test.

Article Snippet: Hep3B cells (HBV + /HBsAg + human HCC, HB-8064) were purchased from the American Type Culture Collection (Manassas, VA, USA) and HepG2215 cells (HBV + /HBsAg + human hepatoblastoma) were kindly provided by Dr. Jun-Jen Liu (Institute of Medical Biotechnology, Taipei Medical University, Taipei, Taiwan).

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Tube Formation Assay, Migration

Journal: Cancers

Article Title: Niclosamide Revitalizes Sorafenib through Insulin-like Growth Factor 1 Receptor (IGF-1R)/Stemness and Metabolic Changes in Hepatocellular Carcinoma

doi: 10.3390/cancers15030931

Figure Lengend Snippet: The combination of niclosamide and sorafenib enhances cell death and reduces the activity of stemness-related properties in sorafenib-resistant HCC cells and their organoids. ( A ) Cell viability assay of the sorafenib-resistant HCC cells under different experimental conditions for 24 and 48 h. ( B ) Protein expression in the sorafenib-resistant HCC cells under different experimental conditions for 24 h, including stemness-related markers (IGF-1R, OCT4) and drug resistance markers (ABCG2). The results of three replicates were shown in . ( C ) Secondary sphere formation assay. Bar = 100 μm. ( D ) Migration ability assay. Bar = 100 μm. ( E ) Generation and identification of tumor organoids derived from the sorafenib-resistant HepG2215_R and Hep3B_R cells. The red circle shows the pseudoglandular pattern structure of HCC. Bar = 100 μm. ( F ) Cell viability assay of the sorafenib-resistant HCC organoids under different experimental conditions for 72 h. ( G ) Gene expression of the sorafenib-resistant HCC cells under different experimental conditions for 24 h, including stemness-related markers ( OCT4 ), drug resistance ( ABCG2 ), and EMT ( IGF-1R and VIMENTIN ). Control group (Ctrl), sorafenib alone (S, 10 µM), niclosamide alone (N, 3 µM), and niclosamide plus sorafenib (S + N). For all quantification, data are the mean ± SD of at least three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, as obtained from one-way ANOVA.

Article Snippet: Hep3B cells (HBV + /HBsAg + human HCC, HB-8064) were purchased from the American Type Culture Collection (Manassas, VA, USA) and HepG2215 cells (HBV + /HBsAg + human hepatoblastoma) were kindly provided by Dr. Jun-Jen Liu (Institute of Medical Biotechnology, Taipei Medical University, Taipei, Taiwan).

Techniques: Activity Assay, Viability Assay, Expressing, Tube Formation Assay, Migration, Derivative Assay, Gene Expression, Control

Journal: Cancers

Article Title: Niclosamide Revitalizes Sorafenib through Insulin-like Growth Factor 1 Receptor (IGF-1R)/Stemness and Metabolic Changes in Hepatocellular Carcinoma

doi: 10.3390/cancers15030931

Figure Lengend Snippet: Niclosamide enhanced the ability of sorafenib by decreasing glycolysis and mitochondrial membrane potential in sorafenib-resistant HCC cells. ( A ) Schematic of glucose metabolism in normal cells and cancer cells under normoxia. ( B ) Representative enzymes in glycolysis and oxidative phosphorylation. Glut, glucose transporter; HK2, hexokinase 2; LDH, lactic dehydrogenase; PEPPCK, phosphoenolpyruvate carboxykinase. ( C ) Gene expression levels of glycolysis-related enzymes in both sorafenib-naïve and sorafenib-resistant HCC cells ( D ) or their organoids ( E ) under different experimental conditions for 24 h were shown. Control group (Ctrl), sorafenib alone (S, 10 µM), niclosamide alone (N, 3 µM), and niclosamide plus sorafenib (S + N). Real-time RT-qPCR analyses. ( F ) JC-1 staining levels of sorafenib-resistant HCC cells (HepG2215_R and Hep3B_R) under different experimental conditions for 48 h. Control group (Ctrl), sorafenib alone (S, 10 µM), niclosamide alone (N, 3 µM), and niclosamide plus sorafenib (S + N). Flow cytometry dot plot analysis for the gating of JC1 (red)-aggregates and JC1 (green)-monomer populations. ( G ) Fluorescence intensity ratio at 590 nm/530 nm and ( H ) the cell images of ( F ). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, as obtained from one-way ANOVA.

Article Snippet: Hep3B cells (HBV + /HBsAg + human HCC, HB-8064) were purchased from the American Type Culture Collection (Manassas, VA, USA) and HepG2215 cells (HBV + /HBsAg + human hepatoblastoma) were kindly provided by Dr. Jun-Jen Liu (Institute of Medical Biotechnology, Taipei Medical University, Taipei, Taiwan).

Techniques: Membrane, Phospho-proteomics, Gene Expression, Control, Quantitative RT-PCR, Staining, Flow Cytometry, Fluorescence

Journal: Colombia Médica : CM

Article Title: Role of heat-shock proteins in infection of human adenocarcinoma cell line MCF-7 by tumor-adapted rotavirus isolates

doi: 10.25100/cm.v51i4.4196

Figure Lengend Snippet: Binding of rotavirus isolates to Hsp90, Hsp70, Hsp60, Hsp40, Hsc70, PDI and integrin β3. MCF-7 cells (A-E) or their cell membrane enriched fractions (F-J) were incubated with the indicated rotavirus isolates. After PBS-washing, the preparations were RIPA-solubilized and applied into ELISA plates coated with rabbit capture antibodies to rotavirus structural proteins. For detection, goat antibodies to HSPs, PDI, Hsc70, integrin β3 or to rotavirus were used and the reaction measured using secondary HRP-conjugated donkey anti-goat IgG and OPD substrate.

Article Snippet: The PBS-washed cells were reacted with rabbit polyclonal antibodies to rotavirus structural proteins (SP) and incubated at 37º C for 1 h. The cells were washed twice with PBS and incubated with HRP-conjugated goat anti-rabbit antibodies (0.133 μg/mL, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at 37º C for 1 h. After three washes with PBS, rotavirus antigen-positive cells were visualized using 3-amino-9-ethylcarbazole (AEC) substrate (0.64 mg/mL) in 50 mM sodium acetate buffer, pH 5.0, containing 0.04% H 2 O 2 .

Techniques: Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Colombia Médica : CM

Article Title: Role of heat-shock proteins in infection of human adenocarcinoma cell line MCF-7 by tumor-adapted rotavirus isolates

doi: 10.25100/cm.v51i4.4196

Figure Lengend Snippet: Blocking of rotavirus binding to cell surface by antibodies against Hsp90, Hsp70, Hsp60, Hsp40, and Hsc70. A-F. MCF-7 cells were incubated with F(ab’)2 antibody fragments against to Hsp90, Hsp70, Hsc70, Hsp60 or Hsp40 at the indicated dilutions, followed by incubation with the rotavirus isolates indicated in panels. After PBS- washing, the cells were solubilized with RIPA buffer before addition to ELISA wells coated with guinea pig capture antibodies to rotavirus particles. For detection, rabbit antibodies to rotavirus particles were used and the reaction measured using secondary HRP-conjugated donkey anti-rabbit IgG and OPD substrate. Data are means values ( standard deviation from three independent experiments performed in duplicate.

Article Snippet: The PBS-washed cells were reacted with rabbit polyclonal antibodies to rotavirus structural proteins (SP) and incubated at 37º C for 1 h. The cells were washed twice with PBS and incubated with HRP-conjugated goat anti-rabbit antibodies (0.133 μg/mL, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at 37º C for 1 h. After three washes with PBS, rotavirus antigen-positive cells were visualized using 3-amino-9-ethylcarbazole (AEC) substrate (0.64 mg/mL) in 50 mM sodium acetate buffer, pH 5.0, containing 0.04% H 2 O 2 .

Techniques: Blocking Assay, Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Colombia Médica : CM

Article Title: Role of heat-shock proteins in infection of human adenocarcinoma cell line MCF-7 by tumor-adapted rotavirus isolates

doi: 10.25100/cm.v51i4.4196

Figure Lengend Snippet: Binging of rotavirus particles to Hsp90, Hsp70, Hsp60 and Hsp40, and inhibition of rotavirus infection by competition for binding to these HSPs. A. MCF-7 cell membrane-enriched fractions were separately incubated with the indicated rotavirus isolates. The solubilized cell membrane fractions were immunoprecipitated with agarose bead-coupled antibodies against rotavirus particles. The immunoprecipitated proteins were analyzed by SDS-PAGE/Western blotting using antibodies against the indicated HSPs and luminol-based detection. B. MA104 cell membrane-enriched fractions were incubated with rotavirus isolate. The solubilized cell membrane fractions were immunoprecipitated with agarose bead-coupled antibodies against rotavirus particles. The immunoprecipitated proteins were analyzed by SDS-PAGE/Western blotting using antibodies against integrin β3, Hsc70, and PDI or HSPs and luminol-based detection. C. A VOBPA assay was conducted by lysing MCF-7 cells with RIPA buffer followed by non-denaturing and non-reducing PAGE and Western blotting. The membrane was incubated with rotavirus WTEW, washed quickly and treated with 2.5% glutaraldehyde. The membrane was treated with a mix of antibodies to the HSPs (lane 1) indicated above or antibodies to rotavirus TLPs (lane 2). Reactions were revealed with Western blotting Luminol Reagent. D, E. Rotavirus isolates (TRUYO, WWM, WTEW, WT-15 and ECwt-o) at MOI 0.8 were treated with the indicated concentrations of rHsp90 (D) or rHsp70 (E) before incubation with MCF-cells. Rotavirus isolates were treated with BSA (100 ng/mL) or Hsc70 (100 ng/mL) or left untreated to be used as controls. The frozen and thawed cells were treated with RIPA buffer and the lysate applied on ELISA plates coated with Guinea pig capture antibodies to rotavirus particles. Detection was performed with rabbit primary antibodies to rotavirus particles. The reaction was revealed with secondary HRP-conjugated goat anti-rabbit IgG and OPD. F. Cell viability of cells treated with recombinant proteins (rHsp90 or rHsp70) (100 (g/mL) was determined using trypan blue exclusion test. Data are presented as means ( standard deviation from three independent experiments performed in duplicate.

Article Snippet: The PBS-washed cells were reacted with rabbit polyclonal antibodies to rotavirus structural proteins (SP) and incubated at 37º C for 1 h. The cells were washed twice with PBS and incubated with HRP-conjugated goat anti-rabbit antibodies (0.133 μg/mL, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at 37º C for 1 h. After three washes with PBS, rotavirus antigen-positive cells were visualized using 3-amino-9-ethylcarbazole (AEC) substrate (0.64 mg/mL) in 50 mM sodium acetate buffer, pH 5.0, containing 0.04% H 2 O 2 .

Techniques: Inhibition, Infection, Binding Assay, Incubation, Immunoprecipitation, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, Recombinant, Standard Deviation

Journal: Colombia Médica : CM

Article Title: Role of heat-shock proteins in infection of human adenocarcinoma cell line MCF-7 by tumor-adapted rotavirus isolates

doi: 10.25100/cm.v51i4.4196

Figure Lengend Snippet: Biotinylation of cell surface proteins and immuno-precipitation. REH cells were added 1 mg/ml NHS-SS-Biotin for 30 min at 4 °C. Cells were washed with PBS containing 0.1 M glycine. Then, the cells were incubated separately with the 5 rotavirus isolates tested. Subsequently, the cells were fixed with glutaraldehyde (2.5%). Cells were immunoprecipitated using anti-rotavirus antibodies coupled to agarose beads. The precipitate was analyzed by SDS-PAGE/Western blotting and the membrane was developed with streptavidin-HRP conjugate and the Luminol kit to detect biotinylated HSPs.

Article Snippet: The PBS-washed cells were reacted with rabbit polyclonal antibodies to rotavirus structural proteins (SP) and incubated at 37º C for 1 h. The cells were washed twice with PBS and incubated with HRP-conjugated goat anti-rabbit antibodies (0.133 μg/mL, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at 37º C for 1 h. After three washes with PBS, rotavirus antigen-positive cells were visualized using 3-amino-9-ethylcarbazole (AEC) substrate (0.64 mg/mL) in 50 mM sodium acetate buffer, pH 5.0, containing 0.04% H 2 O 2 .

Techniques: Immunoprecipitation, Incubation, SDS Page, Western Blot

Journal: Colombia Médica : CM

Article Title: Role of heat-shock proteins in infection of human adenocarcinoma cell line MCF-7 by tumor-adapted rotavirus isolates

doi: 10.25100/cm.v51i4.4196

Figure Lengend Snippet: Decrease of rotavirus infection by antibodies to Hsp90, Hsp70, Hsp60 or Hsp40. A-E. MCF-7 cells in culture medium were separately treated with a serial dilution of antibodies to the indicated HSPs for 45 min at 4° C, DMEM-washed and then incubated with the rotavirus isolates indicated in panels. After 12 h.p.i., structural rotavirus antigens were detected by immunocytochemistry assay. Virus inoculated cells without antibody pre-treatment were used as a control. Data were expressed as percentage of infected cells. Data are shown as mean percentages ( standard deviation from three independent experiments performed in duplicate.

Article Snippet: The PBS-washed cells were reacted with rabbit polyclonal antibodies to rotavirus structural proteins (SP) and incubated at 37º C for 1 h. The cells were washed twice with PBS and incubated with HRP-conjugated goat anti-rabbit antibodies (0.133 μg/mL, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at 37º C for 1 h. After three washes with PBS, rotavirus antigen-positive cells were visualized using 3-amino-9-ethylcarbazole (AEC) substrate (0.64 mg/mL) in 50 mM sodium acetate buffer, pH 5.0, containing 0.04% H 2 O 2 .

Techniques: Infection, Serial Dilution, Incubation, Immunocytochemistry, Standard Deviation

Journal: Colombia Médica : CM

Article Title: Role of heat-shock proteins in infection of human adenocarcinoma cell line MCF-7 by tumor-adapted rotavirus isolates

doi: 10.25100/cm.v51i4.4196

Figure Lengend Snippet: Effect of inhibition of Hsp90 and Hsp70 on rotavirus infection. A-E. MCF-7cells were seeded in 96-well plates with different concentrations of Hsp90 inhibitors (17AAG, NvP Auy or Radicicol) or Hsp70 inhibitors (Quercetin (QC) or VER155008) for 1 h at 37° C. Cells were DMEM-washed and inoculated with the rotavirus isolates indicated in panels and incubated for 45 min at 4° C.Cells were DMEM-washed and inoculated with the rotavirus isolates indicated in panels and incubated for 12 h at 37( C. Viral structural antigens were detected by immunocytochemistry assay and infection was expressed as percentage of infected cells. F. Cells were incubated with 300 (M QC for 30 min at 43( C or 37( C and the percentage of infected cells determined by immunocytochemistry assay using rabbit antibodies to rotavirus structural antigens. G. Trypan blue-cell viability assay of cells treated with 600 (M of the inhibitors indicated in the bottom of the panel. Data are shown as mean percentages ( standard deviation from three independent experiments performed in duplicate.

Article Snippet: The PBS-washed cells were reacted with rabbit polyclonal antibodies to rotavirus structural proteins (SP) and incubated at 37º C for 1 h. The cells were washed twice with PBS and incubated with HRP-conjugated goat anti-rabbit antibodies (0.133 μg/mL, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at 37º C for 1 h. After three washes with PBS, rotavirus antigen-positive cells were visualized using 3-amino-9-ethylcarbazole (AEC) substrate (0.64 mg/mL) in 50 mM sodium acetate buffer, pH 5.0, containing 0.04% H 2 O 2 .

Techniques: Inhibition, Infection, Incubation, Immunocytochemistry, Viability Assay, Standard Deviation

Journal: Colombia Médica : CM

Article Title: Role of heat-shock proteins in infection of human adenocarcinoma cell line MCF-7 by tumor-adapted rotavirus isolates

doi: 10.25100/cm.v51i4.4196

Figure Lengend Snippet: Increase of rotavirus infection by overexpression of rHsp90 and rHsp70. MCF-7 cells were transfected with the indicated plasmids expressing rHsp90 or rHsp70. After 24 h of transfection, the cells were infected with the indicated rotavirus isolates for 12 h at 37( C. A-E. Expression of recombinant proteins was detected by immunofluorescence using specific antibodies to them Representative photographs are shown. Non-transfected cells were used as a control. F. Transfected or non-transfected cells with Hsp90- or Hsp70-containing plasmids were separately infected or left uninfected with the indicated rotavirus isolates. Aliquots of transfected cells expressing rHsp90 were treated the inhibitor 17AAG before infection with rotavirus isolates. Cells transfected with the empty plasmid or non-transfected were used as a control. Infection was expressed as percentage of cells being positive to rotavirus structural antigens. G. Representative photographs of transfected cells infected or left uninfected with the rotavirus isolates. Rotavirus antigens were detected with secondary HRP-conjugated antibodies (reddish-brown), and recombinant HSPs (rHsp90 or rHsp70) were detected with secondary FITC-conjugated antibodies (green). 17AAG was used as an inhibitor of Hsp90. Control cells consisted of non-infected cells transfected with HSP-plasmid (either rHsp90 or rHsp70) and rotavirus infected or left uninfected cells transfected with the empty plasmid (-HSP-plasmid). Data are shown as mean percentages ( standard deviation from three independent experiments performed in duplicate.

Article Snippet: The PBS-washed cells were reacted with rabbit polyclonal antibodies to rotavirus structural proteins (SP) and incubated at 37º C for 1 h. The cells were washed twice with PBS and incubated with HRP-conjugated goat anti-rabbit antibodies (0.133 μg/mL, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at 37º C for 1 h. After three washes with PBS, rotavirus antigen-positive cells were visualized using 3-amino-9-ethylcarbazole (AEC) substrate (0.64 mg/mL) in 50 mM sodium acetate buffer, pH 5.0, containing 0.04% H 2 O 2 .

Techniques: Infection, Over Expression, Transfection, Expressing, Recombinant, Immunofluorescence, Plasmid Preparation, Standard Deviation

Journal: Colombia Médica : CM

Article Title: Role of heat-shock proteins in infection of human adenocarcinoma cell line MCF-7 by tumor-adapted rotavirus isolates

doi: 10.25100/cm.v51i4.4196

Figure Lengend Snippet: Unión de aislamientos de rotavirus a Hsp90, Hsp70, Hsp60, Hsp40, Hsc70, PDI e integrina β3. Las células MCF-7 (A-E) o sus fracciones enriquecidas de membrana celular (F-J) se incubaron con los aislamientos de rotavirus indicados. Después del lavado con PBS, las preparaciones se solubilizaron con RIPA y se aplicaron en placas de ELISA recubiertas con anticuerpos de captura de conejo contra proteínas estructurales de rotavirus. Para la detección, se usaron anticuerpos de cabra contra HSP, PDI, Hsc70, integrina β3 o contra rotavirus y se midió la reacción usando anticuerpo secundario de burro anti-IgG de cabra conjugado con HRP y se reveló usando sustrato de OPD.

Article Snippet: The PBS-washed cells were reacted with rabbit polyclonal antibodies to rotavirus structural proteins (SP) and incubated at 37º C for 1 h. The cells were washed twice with PBS and incubated with HRP-conjugated goat anti-rabbit antibodies (0.133 μg/mL, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at 37º C for 1 h. After three washes with PBS, rotavirus antigen-positive cells were visualized using 3-amino-9-ethylcarbazole (AEC) substrate (0.64 mg/mL) in 50 mM sodium acetate buffer, pH 5.0, containing 0.04% H 2 O 2 .

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Colombia Médica : CM

Article Title: Role of heat-shock proteins in infection of human adenocarcinoma cell line MCF-7 by tumor-adapted rotavirus isolates

doi: 10.25100/cm.v51i4.4196

Figure Lengend Snippet: Bloqueo de la unión de rotavirus a la superficie celular con anticuerpos contra Hsp90, Hsp70, Hsp60, Hsp40 y Hsc70. A-F. Las células MCF-7 se incubaron con fragmentos de anticuerpo F(ab')2 contra Hsp90, Hsp70, Hsc70, Hsp60 o Hsp40 en las diluciones indicadas, seguido de incubación con los aislados de rotavirus indicados en los paneles. Después del lavado con PBS, las células se solubilizaron con tampón RIPA antes de la adición a los pozos de ELISA recubiertos con anticuerpos de captura de cobayo contra proteínas estructurales de rotavirus. Para la detección, se usaron anticuerpos de conejo contra partículas de rotavirus y se midió la reacción usando IgG secundario de burro anti-conejo conjugado con HRP y se reveló utilizando sustrato de OPD. Los datos son valores medios ± desviación estándar de tres experimentos independientes realizados por duplicado.

Article Snippet: The PBS-washed cells were reacted with rabbit polyclonal antibodies to rotavirus structural proteins (SP) and incubated at 37º C for 1 h. The cells were washed twice with PBS and incubated with HRP-conjugated goat anti-rabbit antibodies (0.133 μg/mL, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at 37º C for 1 h. After three washes with PBS, rotavirus antigen-positive cells were visualized using 3-amino-9-ethylcarbazole (AEC) substrate (0.64 mg/mL) in 50 mM sodium acetate buffer, pH 5.0, containing 0.04% H 2 O 2 .

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Colombia Médica : CM

Article Title: Role of heat-shock proteins in infection of human adenocarcinoma cell line MCF-7 by tumor-adapted rotavirus isolates

doi: 10.25100/cm.v51i4.4196

Figure Lengend Snippet: Unión de partículas de rotavirus a Hsp90, Hsp70, Hsp60 y Hsp40, e inhibición de la infección por rotavirus al competir con HSPs de la célula. A. Las fracciones enriquecidas en la membrana celular de MCF-7 se incubaron por separado con los aislados de rotavirus indicados. Las fracciones de membrana celular solubilizadas se inmunoprecipitaron con anticuerpos acoplados a perlas de agarosa contra partículas de rotavirus. Las proteínas inmunoprecipitadas se analizaron mediante SDS-PAGE/Western blotting usando anticuerpos contra las HSPs indicadas y detección basada en luminol. B. Se incubaron fracciones enriquecidas en la membrana celular MA104 con aislado de rotavirus. Las fracciones de membrana celular solubilizadas se inmunoprecipitaron con anticuerpos acoplados a perlas de agarosa contra partículas de rotavirus. Las proteínas inmunoprecipitadas se analizaron mediante SDS-PAGE/Western blotting usando anticuerpos contra la integrina β3, Hsc70, PDI o HSP y detección basada en luminol. C. Se realizó un ensayo de VOBPA lisando células MCF-7 con tampón RIPA seguido de PAGE no desnaturalizante y no reductor y transferencia Western. La membrana se incubó con rotavirus WTEW, se lavó rápidamente y se trató con glutaraldehído al 2,5%. La membrana se trató con una mezcla de anticuerpos contra las HSPs (carril 1) indicadas anteriormente o anticuerpos contra las TLPs de rotavirus (carril 2). Las reacciones se revelaron con reactivo Western Luminol de transferencia. D, E. Los aislamientos de rotavirus (TRUYO, WWM, WTEW, WT-15 y ECwt-o) a un MOI 0.8 se trataron con las concentraciones indicadas de rHsp90 (D) o rHsp70 (E) antes de la incubación con células MCF-7. Los aislamientos de rotavirus se trataron con BSA (100 ng/mL) o Hsc70 (100 ng/ml) o se dejaron sin tratar para utilizarlos como controles. Las células congeladas y descongeladas se trataron con tampón RIPA y el lisado se aplicó sobre placas ELISA recubiertas con anticuerpos de captura de cobayo contra partículas de rotavirus. La detección se realizó con anticuerpos primarios de conejo contra partículas de rotavirus. La reacción se reveló con anticuerpos secundarios IgG de cabra anti-conejo conjugados con HRP y se revelo con sustrato de OPD. F. Se determinó la viabilidad celular de las células tratadas con proteínas recombinantes (rHsp90 o rHsp70) (100 µg/mL) usando una prueba de exclusión con azul de tripano. Los datos se presentan como promedios ± desviación estándar (DS) de tres experimentos independientes realizados por duplicado.

Article Snippet: The PBS-washed cells were reacted with rabbit polyclonal antibodies to rotavirus structural proteins (SP) and incubated at 37º C for 1 h. The cells were washed twice with PBS and incubated with HRP-conjugated goat anti-rabbit antibodies (0.133 μg/mL, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at 37º C for 1 h. After three washes with PBS, rotavirus antigen-positive cells were visualized using 3-amino-9-ethylcarbazole (AEC) substrate (0.64 mg/mL) in 50 mM sodium acetate buffer, pH 5.0, containing 0.04% H 2 O 2 .

Techniques: SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Colombia Médica : CM

Article Title: Role of heat-shock proteins in infection of human adenocarcinoma cell line MCF-7 by tumor-adapted rotavirus isolates

doi: 10.25100/cm.v51i4.4196

Figure Lengend Snippet: Biotinilación de proteínas de la superficie celular e inmunoprecipitación. Biotinilación de las proteínas de la superficie celular acoplada a inmuno-precipitación. A células de la línea REH se adicionó 1 mg/mL de NHS-SS-Biotin por 30 minutos a 4° C. Las células se lavaron con PBS que contenía 0.1 M de glicina. Luego, fueron incubadas con los 5 aislamientos de rotavirus. Posteriormente, las células se trataron con glutaraldehído (2.5% concentración final), se lisaron y fueron inmunoprecipitadas usando anticuerpos anti-rotavirus acoplados a perlas de agarosa. Posteriormente, se realizó una electroforesis (SDS-PAGE) y Western blotting y a la membrana se le aplicó estreptavidina-HRP para detectar las HSPs biotiniladas. La membrana fue revelada con el kit Luminol.

Article Snippet: The PBS-washed cells were reacted with rabbit polyclonal antibodies to rotavirus structural proteins (SP) and incubated at 37º C for 1 h. The cells were washed twice with PBS and incubated with HRP-conjugated goat anti-rabbit antibodies (0.133 μg/mL, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at 37º C for 1 h. After three washes with PBS, rotavirus antigen-positive cells were visualized using 3-amino-9-ethylcarbazole (AEC) substrate (0.64 mg/mL) in 50 mM sodium acetate buffer, pH 5.0, containing 0.04% H 2 O 2 .

Techniques: SDS Page, Western Blot

Journal: Colombia Médica : CM

Article Title: Role of heat-shock proteins in infection of human adenocarcinoma cell line MCF-7 by tumor-adapted rotavirus isolates

doi: 10.25100/cm.v51i4.4196

Figure Lengend Snippet: Disminución de la infección por rotavirus con anticuerpos contra Hsp90, Hsp70, Hsp60 o Hsp40. A-E. Las células MCF-7 en medio de cultivo se trataron por separado con una dilución en serie de anticuerpos contra las HSPs indicadas, durante 45 minutos a 4 ºC, se lavaron con DMEM y luego se incubaron con los aislados de rotavirus indicados en los paneles. Después de 12 h.p.i., se detectaron antígenos de rotavirus estructurales mediante un ensayo de inmunocitoquímica. Se utilizaron como control células inoculadas con virus sin pretratamiento con anticuerpos. Los datos se expresaron como porcentaje de células infectadas. Los datos se muestran como porcentajes promedios ± desviación estándar (DS) de tres experimentos independientes realizados por duplicado.

Article Snippet: The PBS-washed cells were reacted with rabbit polyclonal antibodies to rotavirus structural proteins (SP) and incubated at 37º C for 1 h. The cells were washed twice with PBS and incubated with HRP-conjugated goat anti-rabbit antibodies (0.133 μg/mL, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at 37º C for 1 h. After three washes with PBS, rotavirus antigen-positive cells were visualized using 3-amino-9-ethylcarbazole (AEC) substrate (0.64 mg/mL) in 50 mM sodium acetate buffer, pH 5.0, containing 0.04% H 2 O 2 .

Techniques:

Journal: Colombia Médica : CM

Article Title: Role of heat-shock proteins in infection of human adenocarcinoma cell line MCF-7 by tumor-adapted rotavirus isolates

doi: 10.25100/cm.v51i4.4196

Figure Lengend Snippet: Efecto de la inhibición de Hsp90 y Hsp70 sobre la infección por rotavirus. A-E. Se incubaron células MCF-7 en placas de 96 pozos con diferentes concentraciones de inhibidores de Hsp90 (17AAG, NvP Auy o Radicicol) o inhibidores de Hsp70 (quercetina (QC) o VER155008) durante 1h 37° C. Las células se lavaron con DMEM y se inocularon con los aislados de rotavirus indicados en los paneles durante 45 min a 4° C y luego se incubaron durante 12 ha 37º C. Los antígenos estructurales virales se detectaron mediante un ensayo de inmunocitoquímica y la infección se expresó como porcentaje de células infectadas. F. Las células se incubaron con QC 300 M durante 30 min a 43° C o 37° C y el porcentaje de células infectadas se determinó mediante un ensayo de inmunocitoquímica usando anticuerpos de conejo contra antígenos estructurales de rotavirus. G. Ensayo de viabilidad de células de azul tripán de células tratadas con 600 µM de los inhibidores indicados en la parte inferior del panel. Los datos se muestran como porcentajes medios ± desviación estándar de tres experimentos independientes realizados por duplicado.

Article Snippet: The PBS-washed cells were reacted with rabbit polyclonal antibodies to rotavirus structural proteins (SP) and incubated at 37º C for 1 h. The cells were washed twice with PBS and incubated with HRP-conjugated goat anti-rabbit antibodies (0.133 μg/mL, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at 37º C for 1 h. After three washes with PBS, rotavirus antigen-positive cells were visualized using 3-amino-9-ethylcarbazole (AEC) substrate (0.64 mg/mL) in 50 mM sodium acetate buffer, pH 5.0, containing 0.04% H 2 O 2 .

Techniques:

Journal: Colombia Médica : CM

Article Title: Role of heat-shock proteins in infection of human adenocarcinoma cell line MCF-7 by tumor-adapted rotavirus isolates

doi: 10.25100/cm.v51i4.4196

Figure Lengend Snippet: Aumento de la infección por rotavirus por sobreexpresión de rHsp90 y rHsp70. Se transfectaron células MCF-7 con los plásmidos indicados que expresan rHsp90 o rHsp70. Después de 24 h de transfección, las células se infectaron con los aislamientos de rotavirus indicados durante 12 ha 37º C. A-E. La expresión de proteínas recombinantes se detectó mediante inmunofluorescencia usando anticuerpos específicos para ellas. Se muestran fotografías representativas. Se utilizaron células no transfectadas como control. F. Las células transfectadas o no transfectadas con plásmidos que contienen Hsp90 o Hsp70 se infectaron por separado o se dejaron sin infectar con los aislamientos de rotavirus indicados. Se trataron alícuotas de células transfectadas que expresaban rHsp90 con el inhibidor 17AAG antes de la infección con aislados de rotavirus. Las células transfectadas con el plásmido vacío o no transfectadas se usaron como control. La infección se expresó como porcentaje de células positivas a antígenos estructurales de rotavirus. G. Fotografías representativas de células transfectadas infectadas o no infectadas con los aislamientos de rotavirus. Los antígenos de rotavirus se detectaron con anticuerpos secundarios conjugados con HRP (marrón rojizo), y las HSP recombinantes (rHsp90 o rHsp70) se detectaron con anticuerpos secundarios conjugados con FITC (verde). Se utilizó 17AAG como inhibidor de Hsp90. Las células de control consistían de células no infectadas transfectadas con plásmidos de HSPs (ya sea rHsp90 o rHsp70) y células infectadas con rotavirus o no infectadas, pero transfectadas con el plásmido vacío (plásmido -HSP). Los datos se muestran como porcentajes promedios ± desviación estándar (DS) de tres experimentos independientes realizados por duplicado.

Article Snippet: The PBS-washed cells were reacted with rabbit polyclonal antibodies to rotavirus structural proteins (SP) and incubated at 37º C for 1 h. The cells were washed twice with PBS and incubated with HRP-conjugated goat anti-rabbit antibodies (0.133 μg/mL, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at 37º C for 1 h. After three washes with PBS, rotavirus antigen-positive cells were visualized using 3-amino-9-ethylcarbazole (AEC) substrate (0.64 mg/mL) in 50 mM sodium acetate buffer, pH 5.0, containing 0.04% H 2 O 2 .

Techniques:

Journal: Colombia Médica : CM

Article Title: Role of heat-shock proteins in infection of human adenocarcinoma cell line MCF-7 by tumor-adapted rotavirus isolates

doi: 10.25100/cm.v51i4.4196

Figure Lengend Snippet:

Article Snippet: The PBS-washed cells were reacted with rabbit polyclonal antibodies to rotavirus structural proteins (SP) and incubated at 37º C for 1 h. The cells were washed twice with PBS and incubated with HRP-conjugated goat anti-rabbit antibodies (0.133 μg/mL, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at 37º C for 1 h. After three washes with PBS, rotavirus antigen-positive cells were visualized using 3-amino-9-ethylcarbazole (AEC) substrate (0.64 mg/mL) in 50 mM sodium acetate buffer, pH 5.0, containing 0.04% H 2 O 2 .

Techniques: Derivative Assay, In Vitro, Cell Surface Receptor Assay

Journal: Colombia Médica : CM

Article Title: Role of heat-shock proteins in infection of human adenocarcinoma cell line MCF-7 by tumor-adapted rotavirus isolates

doi: 10.25100/cm.v51i4.4196

Figure Lengend Snippet:

Article Snippet: The PBS-washed cells were reacted with rabbit polyclonal antibodies to rotavirus structural proteins (SP) and incubated at 37º C for 1 h. The cells were washed twice with PBS and incubated with HRP-conjugated goat anti-rabbit antibodies (0.133 μg/mL, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at 37º C for 1 h. After three washes with PBS, rotavirus antigen-positive cells were visualized using 3-amino-9-ethylcarbazole (AEC) substrate (0.64 mg/mL) in 50 mM sodium acetate buffer, pH 5.0, containing 0.04% H 2 O 2 .

Techniques: In Vitro

Journal: Nature cancer

Article Title: L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

doi: 10.1038/s43018-019-0006-x

Figure Lengend Snippet: a, L1CAM expression is increased in liver metastases (Met) as compared to matched primary tumors (Pri). Immunohistochemistry (IHC) for L1CAM is shown in matched normal colon, primary CRC tumor and liver metastasis sections from a representative patient. Arrows indicate L1CAM staining at the invasion front of the primary tumor. Detail of the boxed region is shown in Extended Data Fig. 1a. b, The percentage of L1CAM-expressing tumor cells in each section. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 18 paired patient samples; two-sided Wilcoxon matched-pairs signed-rank test. c, Percentage of L1CAM-expressing cells in matched pretreatment (pre-treat.) biopsies and post-treatment (post-treat.) surgically resected residual disease in patients with locally advanced rectal cancer. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 31 patients; two-sided Wilcoxon matched-pairs signed-rank test. d, Representative sections of paired pretreatment core biopsies and matched surgical resection specimens obtained after chemoradiation, from two patients with rectal adenocarcinoma, showing L1CAM expression in peripheral areas of residual adenocarcinoma after treatment. e, L1CAM immunohistochemistry in a human CRC liver metastasis resected after neoadjuvant chemotherapy, showing dense stromal infiltration and L1CAM-expressing residual tumor cell clusters. Representative of 18 samples analyzed. f, Tumor L1CAM expression is associated with greater organoid generation capacity. Median L1CAM expression is shown for freshly resected and dissociated patient CRC liver metastases measured by flow cytometry before plating of 10,000 cells in 40 μl of Matrigel using organoid medium. Organoid generation ability was assessed 14 d after plating. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 14 paired patient tumor samples; two-sided Mann-Whitney U test. g, Number of organoids (mean ± s.e.m.) grown from 10,000 L1CAMhigh or L1CAMlow cells flow-sorted from freshly resected patient CRC primary tumors (P, left) or liver metastases (Li, right), counted 14 d after surgical resection and flow sorting. From left to right, n = 3, 3, 3, 3, 3, 3, 3, 3, 3, 4, 3, 4, 4 and 11 replicates per group from each of seven patients; two-tailed Student’s t tests. h, Subcutaneous tumor volumes measured 35 d after transplantation of mice with 50,000 organoid-derived flow-sorted L1CAMhigh or L1CAMlow cells (mean ± s.e.m.); n = 5 mice per group; two-tailed Mann-Whitney U test. i, Biaxial density plot showing relative expression of L1CAM and LGR5 in 9,974 cells from four independent patient-derived metastatic CRC organoids subjected to scRNA-seq. Five clusters identified according to relative L1CAM and LGR5 expression are overlaid as colors on the density plot. j, Dual LGR5 mRNA FISH and L1CAM immunofluorescence (IF) on a patient primary CRC tissue section (top, low magnification; bottom, high magnification), showing discrete expression levels of LGR5 and L1CAM in different cell clusters, including double-positive cells. Representative field of eight tumor sections from four patients analyzed.

Article Snippet: Solid-phase L1CAM ligand binding assays used recombinant human L1CAM (human Fc tag, R&D Systems; His tag, Thermo Fisher Scientific), UltraPure BSA (Thermo Fisher Scientific), purified mouse laminin-111 (Sigma-Aldrich), purified mouse collagen IV (Cultrex, R&D Systems), purified human collagen V (Sigma-Aldrich), recombinant human tenascin C (R&D Systems) and recombinant human laminin-411, laminin-421, laminin-511 and laminin-521 (Biolamina).

Techniques: Expressing, Immunohistochemistry, Staining, Flow Cytometry, MANN-WHITNEY, Two Tailed Test, Transplantation Assay, Derivative Assay, Immunofluorescence

Journal: Nature cancer

Article Title: L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

doi: 10.1038/s43018-019-0006-x

Figure Lengend Snippet: a, L1CAM inhibition rescues the increase in organoid generation secondary to REST inhibition. MSK107Li organoids stably expressing the indicated shRNAs were grown in the presence or absence of doxycycline for 7 d before measuring cell viability (luminescence relative to day 0; in box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 6 organoid cultures per group; two-sided Mann-Whitney U tests). b, Relative mRNA levels of L1CAM and REST on day 0, normalized to GAPDH, in organoid-derived cells transduced with lentiviruses directing expression of the indicated shRNAs in the presence or absence of doxycycline. Data are shown as the mean ± s.e.m.; n = 4 organoid cultures per group; two-sided Student’s t tests. c, Left: schematic diagram showing how loss of epithelial integrity induces L1CAM expression during wound healing and tumor invasion, ultimately driving metastatic relapse. Right schematic diagram showing that loss of membrane E-cadherin in cells detached from their epithelial niche downregulates and displaces REST from the L1CAM enhancer, thus enabling L1CAM expression.

Article Snippet: Solid-phase L1CAM ligand binding assays used recombinant human L1CAM (human Fc tag, R&D Systems; His tag, Thermo Fisher Scientific), UltraPure BSA (Thermo Fisher Scientific), purified mouse laminin-111 (Sigma-Aldrich), purified mouse collagen IV (Cultrex, R&D Systems), purified human collagen V (Sigma-Aldrich), recombinant human tenascin C (R&D Systems) and recombinant human laminin-411, laminin-421, laminin-511 and laminin-521 (Biolamina).

Techniques: Inhibition, Stable Transfection, Expressing, MANN-WHITNEY, Derivative Assay, Transduction, Membrane

Journal: Nature cancer

Article Title: L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

doi: 10.1038/s43018-019-0006-x

Figure Lengend Snippet: a, Immunohistochemistry for L1CAM (top) and Ki67 (bottom) in serial sections of a representative human CRC primary tumor invasion front showing an inverse relationship between L1CAM and Ki67 expression. Representative of 16 tumors analyzed. b, Immunohistochemistry for L1CAM (top) and Ki67 (bottom) in serial sections of a representative post-treatment human CRC liver metastasis demonstrating L1CAMhighKi67low cells in organized epithelial structures and L1CAMhighKi67high cells in disrupted epithelia. Representative of 16 tumors analyzed. c, Percentage of L1CAMhigh and L1CAMlow cells that are also Ki67high in regions of intact versus disrupted glandular epithelial architecture within CRCs. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; from left to right, n = 64, 54, 28 and 34 independent fields from 16 patient tumors; two-sided Mann-Whitney U tests. d, Immunohistochemistry for L1CAM (top) and Ki67 (bottom) in serial sections of MSK107Li matched surgically resected patient CRC liver metastasis, metastasis-derived organoids and organoid-derived subcutaneous xenograft. Dashed red lines indicate the tumor-stromal boundary. Box plots indicate the percentage of Ki67+ cells among L1CAMhigh cells in the indicated sections. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; from left to right, n = 7, 9 and 9 independent fields; two-sided Mann-Whitney U tests. e,f, L1CAM is induced during normal epithelial organoid formation. Relative L1CAM mRNA levels (mean ± s.e.m.) are shown for human (e) and mouse (f) cells freshly isolated from intact colons or collected after 14 d of growth in organoid conditions. Data were normalized to GAPDH mRNA levels. n = 4 crypts or organoid cultures from each of three patients or mice; two-sided Student’s t tests. g, Relative LGR5 mRNA levels (mean ± s.e.m.) in human cells freshly isolated from intact colons or collected after 14 d of growth in organoid conditions. Data were normalized to GAPDH mRNA levels. n = 4 crypts or organoid cultures from each of three patients; two-sided Student’s t tests. h, L1CAM is induced during epithelial regeneration after colitis. C57BL/6J mice were given 3.5% DSS in their drinking water for 5 d, inducing maximal colitis by day 7, and were then maintained on water without DSS for 12 d. Mice were killed at each of the indicated time points, and their colons were collected, sectioned and either stained with Kreyberg-Jareg stain (blue, mucin; pink, collagen) or subjected to immunohistochemistry for L1CAM. Representative images of three independent experiments are shown. i, High-magnification view showing detail of L1CAM immunohistochemical staining throughout the length of the intestinal crypt, representative of three mice each from three independent experiments.

Article Snippet: Solid-phase L1CAM ligand binding assays used recombinant human L1CAM (human Fc tag, R&D Systems; His tag, Thermo Fisher Scientific), UltraPure BSA (Thermo Fisher Scientific), purified mouse laminin-111 (Sigma-Aldrich), purified mouse collagen IV (Cultrex, R&D Systems), purified human collagen V (Sigma-Aldrich), recombinant human tenascin C (R&D Systems) and recombinant human laminin-411, laminin-421, laminin-511 and laminin-521 (Biolamina).

Techniques: Immunohistochemistry, Expressing, MANN-WHITNEY, Derivative Assay, Isolation, Staining, Immunohistochemical staining

Journal: Nature cancer

Article Title: L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

doi: 10.1038/s43018-019-0006-x

Figure Lengend Snippet: a, L1CAM immunohistochemistry in a representative colon section from an L1CAMΔIEC mouse maintained on water with DSS, showing L1CAM staining restricted to submucosal neurons and no L1CAM expression in the epithelial cells of the crypt. Representative of three independent mice. b–f, L1CAM deficiency impairs epithelial healing following DSS-induced colitis. L1CAMfl/y and L1CAMΔIEC mice were given 3.5% DSS in their drinking water for 5 d, followed by 12 d of water without DSS before being killed. b, Kaplan-Meier survival curves. n = 26 mice per group from three independent experiments; two-sided Mantel-Cox test. c, Disease activity index (composite of weight loss, diarrhea and rectal bleeding) measured at the time of maximal colitis on day 7. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 26 mice per group; two-sided Mann-Whitney U test. d, Histological score (composite of inflammation, mucosal denudation and crypt dysmorphia) on day 14. L1CAMfl/y, n = 24; L1CAMΔIEC, n = 12. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; two-sided Mann-Whitney U test. e, Representative histological sections with Kreyberg-Jareg staining (blue, mucin; pink, collagen) and immunohistochemical staining for L1CAM showing denudation of mucin-producing crypts in L1CAMΔIEC mice on day 14. Panels on the right show higher magnification of less damaged areas of the colon exhibiting loss of L1CAM immunostaining in crypts of L1CAMΔIEC mice. Representative of three independent experiments. f–l, L1CAM deficiency in the progeny of LGR5-expressing cells impairs epithelial healing after DSS-induced colitis. f, Immunohistochemistry for GFP in a representative colon section from an L1CAMΔLGR5 mouse killed after 5 d of daily tamoxifen treatment. n = 3 mice. g, WTΔLGR5 and L1CAMΔLGR5 mice were treated as in f and killed, and their colon crypts were isolated and seeded for organoid generation. Representative flow cytometry assessment is shown of L1CAM expression 24 h after seeding. n = 2 mice per genotype. h, Schematic of experimental design. Mice were treated with tamoxifen for 5 d to induce Lgr5-GFP-IRES-creERT2 expression and subsequently treated with 3% DSS for 5 d, with maximal colitis by day 7. They were then maintained on water without DSS for a further 12 d before being killed. i, Representative histological sections with Kreyberg-Jareg staining (blue, mucin; pink, collagen) and immunohistochemical staining for L1CAM showing denudation of mucin-producing crypts in the distal colon in L1CAMΔLGR5 mice on day 14, while crypts are restored in L1CAMfl/y and WTΔLGR5 mice. The panels on the right show higher magnification of less damaged areas of the colon exhibiting loss of L1CAM immunostaining in crypts in L1CAMΔLGR5 mice in comparison to L1CAMfl/y and WTΔLGR5 mice. Representative of 20 evaluable mice from two independent experiments. j, Kaplan-Meier plot showing cumulative survival of L1CAMfl/y, WTΔLGR5 and L1CAMΔLGR5 mice. n = 33 mice from two independent experiments; two-sided Mantel-Cox tests. k, Disease activity index (composite of weight loss, diarrhea and rectal bleeding) measured at the time of maximal colitis on day 7. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 33 mice from two independent experiments; two-sided Mann-Whitney U tests. l, Histological scores (composite of inflammation, mucosal denudation and crypt dysmorphia) on day 14. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 20 evaluable mice from two independent experiments; two-sided Mann-Whitney U tests.

Article Snippet: Solid-phase L1CAM ligand binding assays used recombinant human L1CAM (human Fc tag, R&D Systems; His tag, Thermo Fisher Scientific), UltraPure BSA (Thermo Fisher Scientific), purified mouse laminin-111 (Sigma-Aldrich), purified mouse collagen IV (Cultrex, R&D Systems), purified human collagen V (Sigma-Aldrich), recombinant human tenascin C (R&D Systems) and recombinant human laminin-411, laminin-421, laminin-511 and laminin-521 (Biolamina).

Techniques: Immunohistochemistry, Staining, Expressing, Activity Assay, MANN-WHITNEY, Immunohistochemical staining, Immunostaining, Isolation, Flow Cytometry, Comparison

Journal: Nature cancer

Article Title: L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

doi: 10.1038/s43018-019-0006-x

Figure Lengend Snippet: a, L1CAM is required for organoid regeneration. CRC107Li organoid-derived cells were transduced with lentivirus directing the expression of either Cas9 alone or Cas9 with sgRNAs targeting L1CAM and allowed to grow under antibiotic selection for 14 d, when they were flow-sorted and seeded at a concentration of 2,000 cells per 40 μl of Matrigel in independent wells of a 96-well plate. The number of organoids (mean ± s.e.m.) established from each population 14 d after sorting and seeding is shown. From left to right, n = 10, 13 and 11 organoid cultures per group; two-tailed Mann-Whitney U test. b,c, L1CAM knockdown inhibits regrowth of multiple patient-derived organoids. Organoids derived from four patients with metastatic CRC were transduced with lentiviruses directing the expression of doxycycline (Dox)-inducible shRNA targeting L1CAM, expanded and, where indicated, treated with doxycycline for 48 h before dissociation and seeding at a concentration of 2,000 cells per 40 μl of Matrigel. Knockdown efficiencies of two independent L1CAM-targeting shRNAs in four patient-derived organoids (b) and relative cell viability on day 14 as compared to day 0 (mean ± s.e.m.) after plating of organoid-derived single cells (c) are shown. n = 6 organoid cultures per group; two-sided Student’s t tests. d, L1CAM is required for subcutaneous tumor growth in vivo. MSK107Li organoid-derived cells (50,000) expressing a doxycycline-inducible shRNA targeting L1CAM were injected subcutaneously into each flank of immunodeficient NSG mice. Where indicated, organoids were treated with doxycycline 2 d before transplantation and mice were maintained on a doxycycline diet for the duration of the experiment. Tumor volume (mean ± s.e.m.) was measured with calipers at the indicated time points after subcutaneous inoculation. n = 10 tumors from five mice per group; two-tailed Mann-Whitney U test. e, Representative image and quantification of tumor bioluminescence measured 35 d after inoculation. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 10 tumors from five mice per group; two-sided Mann-Whitney U test. f, Day 21 steady-state MSK107Li and MSK121Li organoids were incubated in medium containing 50 μM irinotecan, and L1CAM expression was measured in residual DAPI− cells 7 d later. Top- flow cytometry plots showing distribution of the data. Bottom: bars showing median fluorescence intensity of L1CAM expression in each population. From left to right, n = 6,512, 130, 8,542 and 49 cells per group, representative of three independent experiments. g, Single cells derived from CRC107Li organoids transduced with lentivirus directing expression of the indicated shRNAs were seeded at a concentration of 2,000 cells per 40 μl grown as organoids for 21 d and then treated with doxycycline and/or irinotecan (irino) as indicated. The viability assay shows the luminescence (mean ± s.e.m.) of each population relative to the luminescence at the time that drug treatment was started (day 0); n = 5 organoid cultures per group; two-sided Mann-Whitney U test. h, Solid-phase binding assay showing dose-response curves of recombinant human L1CAM-Fc binding to plates coated with equimolar concentrations of the indicated proteins. After washing, bound L1CAM-Fc was detected with horseradish peroxidase (HRP)-conjugated anti-human IgG, HRP substrate was added and OD450 was measured. Data are shown as the mean ± s.e.m; n = 5 wells per time point, representative of three independent experiments; two-tailed Mann-Whitney U test. i, L1CAM mediates the interaction of dissociated CRC cells with laminin isoforms. Single cells derived from MSK121Li organoids (3,000) cultured in the presence or absence of doxycycline to knock down L1CAM were seeded in wells coated with 30 nM of the indicated proteins. After 1 h of adhesion and extensive washing, the percentage of adherent cells (mean ± s.e.m.) was measured as the relative luminescence of each well immediately after plating. n = 10 organoid cultures per condition; two-tailed Mann-Whitney U tests.

Article Snippet: Solid-phase L1CAM ligand binding assays used recombinant human L1CAM (human Fc tag, R&D Systems; His tag, Thermo Fisher Scientific), UltraPure BSA (Thermo Fisher Scientific), purified mouse laminin-111 (Sigma-Aldrich), purified mouse collagen IV (Cultrex, R&D Systems), purified human collagen V (Sigma-Aldrich), recombinant human tenascin C (R&D Systems) and recombinant human laminin-411, laminin-421, laminin-511 and laminin-521 (Biolamina).

Techniques: Derivative Assay, Transduction, Expressing, Selection, Concentration Assay, Two Tailed Test, MANN-WHITNEY, shRNA, In Vivo, Injection, Transplantation Assay, Incubation, Flow Cytometry, Fluorescence, Viability Assay, Binding Assay, Recombinant, Cell Culture

Journal: Nature cancer

Article Title: L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

doi: 10.1038/s43018-019-0006-x

Figure Lengend Snippet: a, L1CAM expression is dynamically regulated during organoid growth. L1CAM immunohistochemistry of CRC organoids of varying size shows progressive restriction of L1CAM expression to cells at the periphery and an overall decrease in L1CAM expression with increasing organoid size. Representative of six organoid lines analyzed. b, L1CAM is induced by dissociation of normal, primary tumor and metastatic human organoids. Relative L1CAM mRNA levels, normalized to GAPDH mRNA levels (mean ± s.e.m.), were measured in intact organoids versus organoid-derived single-cell suspensions plated in Matrigel and assayed 24 h after dissociation. n = 4 replicates per group; two-sided Student’s t tests. c, Time course of regenerating organoids derived from L1CAMhigh (left) and L1CAMlow (right) cells flow-sorted from day 21 MSK107Li (top) and MSK121Li (bottom) organoids. Histograms show the distribution of L1CAM expression (measured by APC fluorescence) in each population at the indicated time points after flow sorting. Representative of three independent experiments. d,e, Dynamic induction of the L1CAMhigh phenotype by a subset of pre-existing L1CAMlow cells. CRC107Li organoids were labeled with lentivirally expressed tdTomato or GFP, and flow-sorted tdTomato+GFP−L1CAMhigh and tdTomato−GFP+L1CAMlow cells were mixed in equal proportions and allowed to regrow as organoids in the presence or absence of irinotecan. Flow plots of the distribution of tdTomato- and GFP-expressing cells (d) and the relative proportions of these cells in the population (e) are shown from monitoring by flow cytometry at the indicated time points after mixing; from left to right, n = 17, 107, 68, 251, 484 and 348 DAPI− single cells representative of three independent experiments with two organoid lines; two-sided chi-squared tests. f,g, Flow cytometry contour plots showing the distribution of L1CAM expression at the indicated time points in the presence or absence of chemotherapy (f) and median fluorescence intensity (MFI) of L1CAM expression (measured by APC) in cells derived from tdTomato+GFP−L1CAMhigh and tdTomato−GFP+L1CAMlow precursors on each day (g), representative of three independent experiments with two organoid lines.

Article Snippet: Solid-phase L1CAM ligand binding assays used recombinant human L1CAM (human Fc tag, R&D Systems; His tag, Thermo Fisher Scientific), UltraPure BSA (Thermo Fisher Scientific), purified mouse laminin-111 (Sigma-Aldrich), purified mouse collagen IV (Cultrex, R&D Systems), purified human collagen V (Sigma-Aldrich), recombinant human tenascin C (R&D Systems) and recombinant human laminin-411, laminin-421, laminin-511 and laminin-521 (Biolamina).

Techniques: Expressing, Immunohistochemistry, Derivative Assay, Fluorescence, Labeling, Flow Cytometry

Journal: Nature cancer

Article Title: L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

doi: 10.1038/s43018-019-0006-x

Figure Lengend Snippet: a,b, L1CAM is not required for intestinal adenoma formation. Male APCΔIEC and L1CAM/APCΔIEC mice were killed at 3 months of age, and their colons were collected, sectioned and examined for adenoma formation. a, Number of adenomas per mouse intestine. In box plots, boxes show the 25th–75th percentile with the median and whiskers show the minimum-maximum; n = 5 mice per group; two-sided Mann-Whitney U test. b, Mean adenoma diameter per mouse. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 5 mice per group; two-sided Mann-Whitney U test. c–e, L1CAM inhibition impairs orthotopic rectal tumor engraftment. NSG mice were given 3% DSS in their water for 5 d and then maintained on water without DSS for 2 d before intraluminal transplantation with 2 × 105 cells from dissociated MSK107Li organoids expressing doxycycline-inducible shRNA targeting L1CAM or control shRNA. Where indicated, organoids were treated in vitro with doxycycline starting 2 d before transplantation, and mice were maintained on a doxycycline diet. Mice were killed 90 d after transplantation, and their colons were collected and examined for tumor engraftment. c, Percentage of mice with an engrafted orthotopic tumor. From left to right, the stacked bar graphs show n = 7, 7, 10, 13, 9 and 7 mice per group from three independent experiments; two-sided chi-squared tests. d,e, Tumor diameter per engrafted mouse (in box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; from left to right, n = 6, 5, 6, 4, 5 and 1 mice from three independent experiments; two-sided Mann-Whitney U tests) (d) and representative H&E-stained sections (e). Arrows indicate tumour diameter. f,g, L1CAM inhibition impairs metastatic colonization of the liver. Cells (5 × 104) were derived from dissociated MSK107Li organoids with doxycycline-inducible expression of shRNA targeting L1CAM. Where indicated, organoids were treated with doxycycline starting 2 d before transplantation and mice were maintained on a doxycycline diet. Representative H&E-stained sections of liver metastases at the experimental endpoint (arrows indicate tumour diameter) (f) and quantification of ex vivo liver bioluminescence signal measured 60 d after transplantation (g) are shown. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; from left to right, n = 9, 10, 8, 9, 5 and 5 mice per group; two-sided Mann-Whitney U tests. h–l, L1CAM inhibition impairs local tumor expansion and metastasis from orthotopic cecal xenografts. h, Schematic of the experiment: cells (4 × 105) derived from MSK121Li organoids transduced with lentivirus directing the expression of tdTomato-luciferase and shRNA targeting L1CAM or control shRNA were injected into the cecal submucosa. Mice were monitored until cecal tumors were evident by ex vivo bioluminescence imaging 3 weeks after injection, randomized on the basis of bioluminescence signal and maintained on or off a doxycycline diet for 7 weeks before being killed. i–l, Quantification of whole-mouse bioluminescence signal (i) and ex vivo bioluminescence signal in the cecum (j), liver (k) and lung (l), normalized to bioluminescence at the time of randomization. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; from left to right, n = 5, 6, 12 and 11 mice per group; two-sided Mann-Whitney U tests. m–o, Combination of L1CAM inhibition with chemotherapy impairs tumor growth to a greater extent than chemotherapy alone. m, Schematic of the experiment. Cells (2 × 105) derived from MSK107Li organoids transduced with lentivirus directing the expression of tdTomato-luciferase and shRNA targeting L1CAM or control shRNA were injected subcutaneously; mice were randomized on the basis of bioluminescence intensity 5 weeks after injection and maintained on a doxycycline diet and/or treated weekly with irinotecan as indicated for 4 weeks before being killed. n,o, Ex vivo tumor volume (n) and mass (o), normalized to tumor bioluminescence at the time of randomization. From left to right, n = 10, 10, 8, 7, 10, 10, 10 and 9 tumors per group; mean ± s.e.m.; two-sided Mann-Whitney U tests.

Article Snippet: Solid-phase L1CAM ligand binding assays used recombinant human L1CAM (human Fc tag, R&D Systems; His tag, Thermo Fisher Scientific), UltraPure BSA (Thermo Fisher Scientific), purified mouse laminin-111 (Sigma-Aldrich), purified mouse collagen IV (Cultrex, R&D Systems), purified human collagen V (Sigma-Aldrich), recombinant human tenascin C (R&D Systems) and recombinant human laminin-411, laminin-421, laminin-511 and laminin-521 (Biolamina).

Techniques: MANN-WHITNEY, Inhibition, Transplantation Assay, Expressing, shRNA, In Vitro, Staining, Derivative Assay, Ex Vivo, Transduction, Luciferase, Injection, Imaging

Journal: Nature cancer

Article Title: L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

doi: 10.1038/s43018-019-0006-x

Figure Lengend Snippet: a, REST ChIP-seq analysis, showing diminution of the REST peak at the L1CAM intronic enhancer in dissociated organoid-derived cells, collected 16 h after dissociation, in comparison to intact organoids. Input control is also shown. LCA10 (gray) is not expressed in CRC organoids. Two independent organoid cultures from two patient-derived organoid lines were analyzed per condition. b, Relative mRNA levels (mean ± s.e.m.) of REST and L1CAM in intact MSK107Li organoids (day 0), cells collected 24 h after dissociation and plating as single cells (day 1), and cells collected at the indicated time points during organoid regeneration. Organoids were transduced with lentivirus constitutively expressing shRNA targeting REST or control shRNA. Gene expression was normalized to GAPDH mRNA levels. Day 1 shControl versus shREST.1: P < 0.0001 (REST), P < 0.0001 (L1CAM); day 1 shControl versus shREST.2: P = 0.007 (REST), P < 0.0001 (L1CAM); n = 4 organoid cultures per sample per time point; two-sided Student’s t tests. c, Relative expression of CDH1, REST and L1CAM (mean ± s.e.m.) in intact MSK107Li organoids transduced with lentivirus constitutively expressing shRNA targeting CDH1 or control shRNA. n = 4 organoid cultures per group; two-sided Student’s t tests. d, ChIP-PCR using antibodies against REST or isotype-control immunoglobulin in intact MSK107Li organoids transduced with lentivirus constitutively expressing shRNA targeting CDH1 or control shRNA. Fold enrichment (mean ± s.e.m.) is shown relative to the corresponding 2% input. PCR primers were selected to amplify immunoprecipitated DNA at the indicated positions relative to the L1CAM transcriptional start site. P values correspond to the comparison between shControl anti-REST and shCDH1 anti-REST; n = 3 organoid cultures per condition; two-sided Student’s t tests. e, Induction of L1CAM expression by E-cadherin knockdown can be rescued by REST but not by dominant-negative REST (dnREST). Relative mRNA levels of L1CAM, CDH1 and REST are shown in MSK107Li organoids stably expressing shRNA targeting CDH1 or control shRNA as well as cDNA expressing REST or dnREST. Gene expression was normalized to the mRNA levels of GAPDH. Data are shown as the mean ± s.e.m.; n = 4 organoid cultures per group; two-sided Student’s t tests.

Article Snippet: Solid-phase L1CAM ligand binding assays used recombinant human L1CAM (human Fc tag, R&D Systems; His tag, Thermo Fisher Scientific), UltraPure BSA (Thermo Fisher Scientific), purified mouse laminin-111 (Sigma-Aldrich), purified mouse collagen IV (Cultrex, R&D Systems), purified human collagen V (Sigma-Aldrich), recombinant human tenascin C (R&D Systems) and recombinant human laminin-411, laminin-421, laminin-511 and laminin-521 (Biolamina).

Techniques: ChIP-sequencing, Derivative Assay, Comparison, Transduction, Expressing, shRNA, Immunoprecipitation, Dominant Negative Mutation, Stable Transfection