Journal: Nature

Article Title: Intratumoral heterogeneity generated by Notch signaling promotes small cell lung cancer

doi: 10.1038/nature22323

Figure Lengend Snippet: a , Quantification of the frequency (Freq.) of Hes1 pos cells in TKO tumors at different stages of tumor development. Scores: 0 (0%), 1 (1-20%), 2 (20-60%), 3 (>60%). Hyperplasias ( n =26, from 5 mice, defined by area < 16,600 μm 2 ) and early tumors ( n =69, from 5 mice) were analyzed 3 months after Ad-CMV-Cre); late tumors ( n =83, from 5 mice) and liver metastases ( n =54, from 5 mice) were analyzed 6-7 months after Ad-CMV-Cre (data for the late tumors are shown for comparison and are the same as in ). b , As in a, for human SCLC tumor microarray sections and segregated by clinical stage. H-scores: stage I (123.9; n =71 sections), stage II (135.3; n =68 sections), stages III+IV (148.5; n =33 sections). c , Representative immunofluorescence (IF) for GFP in TKO Hes1 GFP/+ SCLC tumors. d , Representative flow cytometry plots of cells isolated from pooled tumors from a TKO Hes1 GFP/+ mouse. Arrows depict the sequential gating strategy for enriching for single, live (by exclusion of 7-aminoactinomycin D (7-AAD)) and lineage (CD45, CD31, TER-119)-negative cells. CD24 labels >98% of Cre-recombined cells and thus further enriches for tumor cells . e , IF of a TKO Rosa26 lox-stop-lox-tdTomato ;Hes1 GFP/+ tumor section showing co-localization of GFP and Tomato signals. f , Flow cytometry shows that GFP high cells in pooled tumors from a TKO Rosa26 lox-stop-lox-tdTomato ;Hes1 GFP/+ mouse infected with Ad-CMV-Cre are positive for Tomato expression (representative of n =2 mice). g , Genotyping PCR analysis for recombination (Δ) or the unrecombined (floxed) alleles at the Rb , p53 and p130 loci in GFP neg and GFP high tumor cells sorted from 3 TKO Hes1 GFP/+ mice. DNA from fl/fl and/or Δ/Δ cells serve as controls. Scale bars, 50 μm.

Article Snippet: TKO Hes1 GFP/+ mice were injected intraperitoneally with 100mg/kg EdU (5-ethynyl-2’-deoxyuridine; Life Technologies) 8 hours before euthanasia.

Techniques: Comparison, Microarray, Immunofluorescence, Flow Cytometry, Isolation, Infection, Expressing

Journal: Nature

Article Title: Intratumoral heterogeneity generated by Notch signaling promotes small cell lung cancer

doi: 10.1038/nature22323

Figure Lengend Snippet: a , Schematic for the generation of TKO allografts. A TKO tumor was isolated, digested to form single cells and implanted subcutaneously in NSG mice. b , Representative hematoxylin and eosin (H&E) staining and IHC for Ascl1 in allograft tumor sections. c , Tumor volumes of TKO allografts treated with the indicated drugs ( n =16 tumors per group, 1 experiment). The experiment was stopped at day 18 and samples were collected for analysis. d,e , Representative IHC for Hes1 (d) or double IHC for Hes1 (brown) and Ascl1 (pink) (e) in TKO allograft tumors collected 18 days after the start of treatment. f,g , IHC staining quantification for the frequency of Hes1 pos CC3 pos (f) and Hes1 pos Ki67 pos (g) cells in murine TKO allografts after 18 days of treatment ( n =8 tumors per group, except for C/I and tarex in (f) ( n =7 tumors each). h,i , IHC staining quantification for the frequency of HES1 pos CC3 pos (h) and HES1 pos KI67 pos (i) cells in xenograft tumors after 31 days of treatment (for h, control/tarex groups: n =4 tumors each; C/I: n =3 tumors; tarex+C/I: n =2 tumors; for i, control/tarex groups: n =4 tumors each; C/I and tarex+C/I groups: n =5 tumors each). j,k , IHC staining quantification for the frequency of Ascl1 pos CC3 pos (j) and Ascl1 pos Ki67 pos (k) cells in murine TKO allografts after 18 days of treatment ( n =8 tumors per group, except for the C/I group in (j) ( n =7 tumors). l,m , IHC staining quantification for the frequency (%) of ASCL1 pos CC3 pos (l) and ASCL1 pos KI67 pos (m) cells in xenograft tumors after 31 days of treatment ( n =4 tumors per group, except for control and tarex groups in (l) ( n =2 tumors) and the tarex+C/I group in (m) ( n =3 tumors). n,o , Representative IHC (n) and quantification of HES1 pos (o) cells in LU66 PDX tumors collected 56 days after the start of treatment (control: n =3 tumors; tarex: n =4 tumors; C/I and tarex+C/I: n =5 tumors per group). *P < 0.05; **P < 0.01; ***P < 0.001; **** P < 0.0001. Statistical significance was determined by two-tailed unpaired Student’s t -test. Data are represented as mean ± SEM. Scale bars, 50 μm.

Article Snippet: TKO Hes1 GFP/+ mice were injected intraperitoneally with 100mg/kg EdU (5-ethynyl-2’-deoxyuridine; Life Technologies) 8 hours before euthanasia.

Techniques: Isolation, Staining, Immunohistochemistry, Control, Two Tailed Test

Journal: Nature

Article Title: Intratumoral heterogeneity generated by Notch signaling promotes small cell lung cancer

doi: 10.1038/nature22323

Figure Lengend Snippet: a,b , Representative Hes1 IHC (a) and frequency of Hes1 pos cells (b) in mouse SCLC ( n =5 mice, 83 tumors). c,d , As in a,b, for human SCLC ( n =172 sections). e , Flow cytometry of %GFP high cells from pooled TKO Hes1 GFP/+ tumors ( n =18 mice). f , qRT-PCR of Notch pathway genes in GFP high relative to GFP neg tumor cells ( n =3 mice). g , As in e, with mice treated with DMSO ( n =5 mice) or DBZ ( n =4 mice). h , Immunoblots of GFP high cell lines grown with Dll4 or 72 hours after removal from Dll4. i , GFP and Uchl1 (NE marker) immunofluorescence in TKO Hes1 GFP/+ tumors (representative of n =3 biological replicas). j , Images of cell cultures established from GFP neg and GFP high tumor cells (representative of n =5 biological replicas). Inset: NE KP1 SCLC cell line. k , EdU incorporation in cells sorted from TKO Hes1 GFP/+ tumors ( n =3 mice). *P < 0.05; **P < 0.01; ***P < 0.001. Two-tailed paired (f,k) or unpaired (g) Student’s t -test. Data are mean ± s.d. Scale bars, 50 μm.

Article Snippet: TKO Hes1 GFP/+ mice were injected intraperitoneally with 100mg/kg EdU (5-ethynyl-2’-deoxyuridine; Life Technologies) 8 hours before euthanasia.

Techniques: Flow Cytometry, Quantitative RT-PCR, Western Blot, Marker, Immunofluorescence, Two Tailed Test

Journal: Nature

Article Title: Intratumoral heterogeneity generated by Notch signaling promotes small cell lung cancer

doi: 10.1038/nature22323

Figure Lengend Snippet: a , Unsupervised clustering of qRT-PCR ( n =46 cells) from one TKO Hes1 GFP/+ tumor (representative of n =2 experiments). Dark blue: undetectable expression. Arrowheads: NE cells that express at least one Notch receptor but have undetectable Hes1 . b , Flow cytometry of freshly isolated GFP neg cells grown on Dll4 or PBS control (representative of n =3 biological replicas). c , Immunoblot of (b). GFP neg cells grown on Dll4 were re-sorted for GFP neg and GFP high populations. d , Relative number of GFP high cells generated from freshly-isolated GFP neg cells grown on Dll4 after DBZ treatment ( n =3 biological replicas). e , Flow cytometry of a GFP high cell line treated with DMSO or DBZ while grown with or without Dll4 or co-cultured with 3 NE cell lines without Dll4 ( n =3 biological replicas). Median GFP intensity normalized to DMSO; -Dll4. f , Freshly isolated GFP neg cells that became GFP high after Dll4 exposure were replated with or without Dll4. Flow cytometry and images are shown (representative of n =2 biological replicas). *P < 0.05; **P < 0.01; ***P < 0.001. Two-tailed paired Student’s t -test. Data are mean ± s.d. Scale bars, 50 μm.

Article Snippet: TKO Hes1 GFP/+ mice were injected intraperitoneally with 100mg/kg EdU (5-ethynyl-2’-deoxyuridine; Life Technologies) 8 hours before euthanasia.

Techniques: Quantitative RT-PCR, Expressing, Flow Cytometry, Isolation, Control, Western Blot, Generated, Cell Culture, Two Tailed Test

Journal: Nature

Article Title: Intratumoral heterogeneity generated by Notch signaling promotes small cell lung cancer

doi: 10.1038/nature22323

Figure Lengend Snippet: a , Cell viability assay of GFP neg , GFP high or a mixture of GFP neg and GFP high cells (bulk) in Matrigel ( n =3 biological replicas). b,c , Relative luciferase activity (b) and EdU incorporation (c) of luciferase-labeled KP1 cells in 2% serum with or without GFP high cells ( n =3 biological replicas, with n =3 technical replicas each for (b)). d,e , As in b,c, with conditioned media from KP1 or from GFP high cell lines ( n =3 biological replicas, with n =3 technical replicas each for (d)). f , Quantification of %CC3 pos cells in GFP neg or GFP high tumor cells in TKO Hes1 GFP/+ mice treated acutely with cisplatin/etoposide ( n =15 tumors per group, from 3 mice). g , Tumor volumes of TKO SCLC allografts ( n =5 tumors per group, one experiment). h , Quantification of Hes1 pos cells ( n =8 tumors except tarex+C/I ( n =6 tumors), one experiment) in TKO allografts after 18 days of treatment. i , Tumor volumes of PDX model OMP-LU66 treated with control antibody ( n =10 tumors), tarex ( n =9), C/I ( n =20), or tarex + C/I ( n =24) (one experiment). j , Model. In SCLC tumors, Notch signaling is activated in a subset of NE cells, leading to a loss of NE features. Non-NE SCLC cells promote the growth of NE cells. Combining chemotherapy and Notch inhibition can target both the fast cycling NE cells and the non-NE cells, respectively. *P < 0.05; **P < 0.01; *** P < 0.001; **** P < 0.0001. For a-f, statistical significance determined by two-tailed paired (a-e) or unpaired (f) Student’s t -test; data are mean ± s.d. For g-i, statistical significance determined by two-tailed unpaired t-test against the control group when applicable or between the C/I and Tarex+C/I groups; data are mean ± SEM. Scale bars, 50 μm.

Article Snippet: TKO Hes1 GFP/+ mice were injected intraperitoneally with 100mg/kg EdU (5-ethynyl-2’-deoxyuridine; Life Technologies) 8 hours before euthanasia.

Techniques: Viability Assay, Luciferase, Activity Assay, Labeling, Control, Inhibition, Two Tailed Test

Journal: Nature

Article Title: Intratumoral heterogeneity generated by Notch signaling promotes small cell lung cancer

doi: 10.1038/nature22323

Figure Lengend Snippet: a,b , Co-immunofluorescence for GFP and EdU (a) or phospho-histone H3 (b) in TKO Hes1 GFP/+ tumors. c , Weight of tumors formed from freshly sorted GFP neg and GFP high tumor cells implanted subcutaneously in immunocompromised NSG mice ( n =6 tumors each; two-tailed unpaired Student’s t -test). d , Flow cytometry of GFP in a tumor formed from freshly sorted GFP neg cells implanted subcutaneously in immunocompromised NSG mice (representative of n =6 biological replicas). *P < 0.05. Data are represented as mean ± s.d. Scale bars, 50 μm.

Article Snippet: TKO Hes1 GFP/+ mice were injected intraperitoneally with 100mg/kg EdU (5-ethynyl-2’-deoxyuridine; Life Technologies) 8 hours before euthanasia.

Techniques: Immunofluorescence, Two Tailed Test, Flow Cytometry

Journal: Nature

Article Title: Intratumoral heterogeneity generated by Notch signaling promotes small cell lung cancer

doi: 10.1038/nature22323

Figure Lengend Snippet: a , Representative IHC for Hes1 and cleaved caspase-3 (CC3) in serial TKO tumor sections. Inset: higher magnification of a positive control for CC3 (tumor from a mouse treated with chemotherapy). b , Predicted numbers and ratio of GFP neg and GFP high tumor cells if the two populations divide independently of each other, and GFP neg cells cycle approximately 3 times faster than GFP high cells. c , Representative IHC in serial sections from TKO Hes1 GFP/+ tumors initiated by Adeno-CGRP-Cre. d , Quantification of the frequency (Freq.) of Hes1 pos cells in TKO hyperplasias ( n =23, from 5 mice) and tumors ( n =50, from 7 mice) induced by Adeno-CGRP-Cre. Scores: 0 (0%), 1 (1-20%), 2 (20-60%) 3 (>60%). e , Flow cytometry of %GFP high cells in pooled tumors from TKO Hes1 GFP/+ mice ( n =4) infected with Ad-CGRP-Cre. f , Images of freshly isolated GFP neg cells grown on dishes coated with Dll4 or PBS control (representative of n =3 biological replicas). g,h , Freshly isolated GFP neg cells that remained GFP neg after culture on Dll4-coated dishes were replated on dishes coated with Dll4 ligand (+Dll4) or PBS control (-Dll4). g , Flow cytometry and images (representative of n =2 biological replicas). h , GFP neg and GFP high cells that formed after this second round of Dll4 stimulation were sorted and analyzed by immunoblot. Control: GFP high cell line. i , Relative number of GFP high cells formed from freshly isolated GFP neg cells grown on Dll4 after two weeks of tarextumab treatment ( n =3 biological replicas each). j , Single cell qRT-PCR ( n =45 each) of H29, H82 and H889 human SCLC cell lines. Heatmap was generated by unsupervised clustering of each cell line. Dark blue regions indicate undetectable expression. k , qRT-PCR for HES1 after 72 hours of culture with or without Dll4. Data is normalized to GAPDH ( n =3 biological replicas with n =3 technical replicas each). l,m , GFP high cell lines were treated with DMSO or DBZ while grown with or without Dll4 or co-cultured with 3 individual NE cell lines in the absence of Dll4. GFP expression was analyzed by flow cytometry after 72 hours. l , Flow cytometry of GFP high cell line #1 (representative of n =3 biological replicas; GFP intensity quantified in ). m , Quantification of GFP intensity in GFP high cell line #2; relative median GFP intensity normalized to the DMSO -Dll4 condition ( n =3 biological replicas). n,o , Representative images (n) and qRT-PCR (o) of GFP high cell lines cultured in the presence of absence of Dll4 for more than a month ( n =3 biological replicas with n =3 technical replicas). p , Freshly isolated GFP neg cells that became GFP high after culture on Dll4 were replated on dishes coated with Dll4 ligand or PBS control and analyzed by immunoblot after a month. GFP neg cell lysate: positive control for Ascl1. *P < 0.05; **P < 0.01. Statistical significance was determined by two-tailed paired Student’s t -test. Data are represented as mean ± s.d. Scale bars, 50 μm.

Article Snippet: TKO Hes1 GFP/+ mice were injected intraperitoneally with 100mg/kg EdU (5-ethynyl-2’-deoxyuridine; Life Technologies) 8 hours before euthanasia.

Techniques: Positive Control, Flow Cytometry, Infection, Isolation, Control, Western Blot, Quantitative RT-PCR, Generated, Expressing, Cell Culture, Two Tailed Test

Journal: Nature

Article Title: Intratumoral heterogeneity generated by Notch signaling promotes small cell lung cancer

doi: 10.1038/nature22323

Figure Lengend Snippet: a , Flow cytometry showing gates used to sort for tumor cells with negative (neg), intermediate (int), and high levels of GFP from a TKO Hes1 GFP/+ mouse (representative of n =4 biological replicas). b , qRT-PCR for Notch pathway genes and NE genes in sorted cells ( n =4 biological replicas with n =3 technical replicas each). c-e , Single cell qRT-PCR of GFP neg , GFP int and GFP high cells sorted from one TKO Hes1 GFP/+ tumor ( n =32 cells sorted per group; 20 cells with low expression of housekeeping genes were excluded from the heatmaps). (c) Unsupervised clustering segregates the cells into two main groups. (d) Supervised clustering and (e) analysis of Hes1 mRNA (normalized to Gapdh ) shows that the number of GFP int cells with detectable Hes1 levels is intermediate between GFP neg and GFP high cells. f , Representative images of freshly isolated cells in culture (without Dll4). Scale bars, 50 μm. *P < 0.05; **P < 0.01; ***P < 0.001; **** P < 0.0001. Statistical significance was determined by two-tailed paired Student’s t -test. Data are represented as mean ± s.d.

Article Snippet: TKO Hes1 GFP/+ mice were injected intraperitoneally with 100mg/kg EdU (5-ethynyl-2’-deoxyuridine; Life Technologies) 8 hours before euthanasia.

Techniques: Flow Cytometry, Quantitative RT-PCR, Expressing, Isolation, Two Tailed Test

Journal: Nature

Article Title: Intratumoral heterogeneity generated by Notch signaling promotes small cell lung cancer

doi: 10.1038/nature22323

Figure Lengend Snippet: a , GSEA signatures enriched in GFP high (astroglial signature) and GFP neg (neuronal signature) tumor cells based on microarray data of tumors from n =3 mice. b , Images of GFP neg , GFP high or bulk tumor cells (mixture of GFP neg and GFP high ) 7 days after isolation from TKO Hes1 GFP/+ mice and seeded at equal numbers in 50% Matrigel (representative of n =3 biological replicas). c , Immunofluorescence for Uchl1 (NE marker) and GFP 10 days after the cells were sorted from TKO Hes1 GFP/+ tumors and in 50% Matrigel. Note: Most of the spheroids in the bulk culture were composed of only one type of cell (Uchl1 pos GFP neg or Uchl1 neg GFP pos ; the mixed spheroid shown here are the minority. d , Quantification of the number of each type of spheroids in each culture condition from (c) ( n =3 mice, 2 sections each for immunostaining). e-g , Relative luciferase activity of a luciferase-labeled mouse SCLC NE cell line seeded alone or co-cultured with GFP high cell lines ( n =2 (e) or n =3 (f,g) biological replicas with n =3 technical replicas each). e , 2% serum, KP2 NE cell line. f,g , 10% serum, KP1 (f) and KP2 (g) cell lines. h-j , As in e-g, but the cells were fixed and collected for EdU analysis by flow cytometry ( n =2 (h) or n =3 (i,j) biological replicas). k , Representative images of KP1 cells seeded in conditioned media (CM) (2% serum) from KP1 cells or from GFP high cell lines. l , Relative luciferase activity of luciferase-labeled KP2 cells 72 hours after seeding in CM (2% serum) from KP2 cells or from GFP high cell lines ( n =3 biological replicas with n =3 technical replicas each). m , As in l, but the cells were fixed and collected for EdU analysis by flow cytometry ( n =3 biological replicas). n,o , As in l, but with KP1 (n) or KP2 cells (o) in 10% serum ( n =3 biological replicas with n =3 technical replicas each). p,q , AlamarBlue cell viability assay for NE SCLC cell lines 72 hours after culture with the indicated recombinant proteins ( n =3 biological replicas with n =3 technical replicas each). r , ELISA assay for midkine in supernatant from NE ( n =4) and GFP high ( n =4) cell lines ( n =2 biological replicas each). s , Luminex assay for midkine in serum plasma from normal (control; n =11) and SCLC ( n =15) patients. t , Relative luciferase activity of luciferase-labeled mouse SCLC NE cell lines ( n =4) 72 hours after seeding alone or co-cultured with GFP high cell lines and treated with 10 μM DBZ or DMSO control. Data are normalized to NE-monoculture with DMSO ( n =3 biological replicas with n =3 technical replicas each). *P < 0.05; **P < 0.01; ***P < 0.001. Statistical significance was determined by two-tailed paired Student’s t -test, except for (r,s: two-tailed unpaired Student’s t -test). Data are represented as mean ± s.d. Scale bars, 50 μm.

Article Snippet: TKO Hes1 GFP/+ mice were injected intraperitoneally with 100mg/kg EdU (5-ethynyl-2’-deoxyuridine; Life Technologies) 8 hours before euthanasia.

Techniques: Microarray, Isolation, Immunofluorescence, Marker, Immunostaining, Luciferase, Activity Assay, Labeling, Cell Culture, Flow Cytometry, Viability Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Luminex, Clinical Proteomics, Control, Two Tailed Test

Journal: Nature

Article Title: Intratumoral heterogeneity generated by Notch signaling promotes small cell lung cancer

doi: 10.1038/nature22323

Figure Lengend Snippet: a,b , Average cell viability (MTT assay) of NE ( n =4) and GFP high ( n =3) cell lines 48 hours after cisplatin (a) or etoposide (b) treatment ( n =3 biological replicas with n =3 technical replicas each). c,d , Quantification of cells expressing cleaved caspase-3 (CC3) (c) and Hes1 (d) in tumors in TKO mice acutely treated with cisplatin and etoposide (CC3: n =49; Hes1: n =41 tumors) or a vehicle control (CC3: n =48; Hes1: n =67 tumors) (data from 2 groups of 3 mice previously described in in but analyzed as number of CC3 pos per tumor, not per mouse). e , Representative immunofluorescence of tumors from TKO Hes1 GFP/+ mice acutely treated with acutely treated with cisplatin and etoposide. Scale bars, 50 μm. f , Quantification of Hes1 pos cells in the tumors of TKO Rosa26 lox-stop-lox-Luciferase mice after 3 weeks of weekly saline ( n =190 tumors, from 7 mice) or cisplatin ( n =217 tumors, from 8 mice) treatments. g-i , Overall survival of all ( g ; n =65, 49 HES1 neg , 16 HES1 pos ), stages I-III ( h ; n =26, 19 HES1 neg , 7 HES1 pos ) or stage IV ( i ; n =39, 30 HES1 neg , 9 HES1 pos ) SCLC patients with HES1-negative or HES1-positive tumors. j-l , Progression-free survival of all ( j ; n =67, 51 HES1 neg , 16 HES1 pos ), stages I-III ( k ; n =28, 21 HES1 neg , 7 HES1 pos ) or stage IV ( l ; n =39, 30 HES1 neg , 9 HES1 pos ) SCLC patients with HES1-negative or HES1-positive tumors. *P < 0.05; **P < 0.01; ***P < 0.001; **** P < 0.0001. Statistical significance was determined by two-tailed paired (a,b) or unpaired (c,d,f) Student’s t -test. For g-l, probability was calculated using a weighted log-rank test with emphasis on later time points (see methods). Data are represented as mean ± s.d.

Article Snippet: TKO Hes1 GFP/+ mice were injected intraperitoneally with 100mg/kg EdU (5-ethynyl-2’-deoxyuridine; Life Technologies) 8 hours before euthanasia.

Techniques: MTT Assay, Expressing, Control, Immunofluorescence, Luciferase, Saline, Two Tailed Test

Journal: Nature

Article Title: Thermal stress induces glycolytic beige fat formation via a myogenic state.

doi: 10.1038/s41586-018-0801-z

Figure Lengend Snippet: Fig. 2 | MyoD+ progenitors in inguinal WAT give rise to glycolytic beige fat. a, Schematic of the experiment. b, Immunofluorescent staining of GFP and UCP in the inguinal WAT of mice in a. Scale bar, 100 µm. c, Quantification of GFP+ beige adipocytes among total UCP1+ adipocytes in inguinal WAT. n = 11 biologically independent samples. ND, not detected. Data are mean ± s.e.m. d, PCA of transcriptome from indicated tissues. n = 3 biologically independent samples. e, GO analysis of enriched genes in GFP+ beige fat in d. f, Expression of glucose metabolism genes. n = 3 biologically independent samples. P < 0.05 by unpaired Student’s two-sided t-test. g, Expression of glucose metabolism genes in inguinal WAT of wild-type and β-less mice. n = 3 biologically independent samples. P < 0.05 by one-way ANOVA followed by Tukey’s test.

Article Snippet: The following antibodies were used in this study: GFP antibody (GFP-1020, Aves), UCP1 antibody (ab-10983, Abcam), ENO1 antibody (ab-155102, Abcam), myosin (skeletal) antibody (M7523-.2ML, Sigma-Aldrich), myoD antibody (sc-760, Santa Cruz Biotech); Alexa Fluor 488 goat anti-chicken (A-11039, Life Technologies), Alexa Fluor 546 goat anti-rabbit (A-11035, Life Technologies), Alexa Fluor 647 goat anti-rabbit (A-21244, Life Technologies), and biotinylated goat anti-rabbit (BA-1000, Vector), CD31-PE/Cy7 antibody (BD Biosciences), CD45-PE/Cy7 antibody (BD Biosciences), CD34-APC antibody (Biolegend), and CD29-APC/Cy7 antibody (Biolegend).

Techniques: Staining, Expressing

Journal: Nature

Article Title: Thermal stress induces glycolytic beige fat formation via a myogenic state.

doi: 10.1038/s41586-018-0801-z

Figure Lengend Snippet: Fig. 4 | GABPα promotes g-beige fat differentiation in myoblasts. a, HOMER motif analysis based on transcriptomics from β-less mice (left) and g-beige fat (right). P values represent enrichment of indicated binding motifs. b, Oil red O and BODIPY staining of differentiated C2C12 cells expressing empty vector or indicated factors under pro-adipogenic conditions. Scale bar, 100 µm. c, mRNA expression of indicated genes in differentiated C2C12 cells. n = 3–4. d, mRNA expression of Ucp1 and Pgc1a in differentiated C2C12 cells treated with or without forskolin (cAMP activation). n = 6–8. e, Immunofluorescent staining of MHC and ENO1 in differentiated C2C12 cells. DAPI counter stain. Scale bar, 100 µm. f, ECAR in differentiated C2C12 cells. n = 10 biologically independent samples. Data are mean ± s.e.m., and analysed by two-way ANOVA followed by Bonferroni’s test. g, Glucose oxidation in indicated cells. n = 6. h, Fatty acid oxidation in indicated cells. n = 5. i, Immunofluorescent staining of GFP and UCP1 in the inguinal WAT of control and GabpaMyod1-KO mice. Mice were pre-treated with β-blocker and acclimated to 15 °C. tdTomato or DAPI counter stain. Scale bar, 100 µm. j, Quantification of GFP+UCP1+ adipocytes in i. n = 6. Data are expressed as mean ± s.e.m. b, e, i, Images represent three independent experiments. c, d, g, h, Data are mean ± s.e.m. of biologically independent samples; ANOVA followed by Tukey’s test.

Article Snippet: The following antibodies were used in this study: GFP antibody (GFP-1020, Aves), UCP1 antibody (ab-10983, Abcam), ENO1 antibody (ab-155102, Abcam), myosin (skeletal) antibody (M7523-.2ML, Sigma-Aldrich), myoD antibody (sc-760, Santa Cruz Biotech); Alexa Fluor 488 goat anti-chicken (A-11039, Life Technologies), Alexa Fluor 546 goat anti-rabbit (A-11035, Life Technologies), Alexa Fluor 647 goat anti-rabbit (A-21244, Life Technologies), and biotinylated goat anti-rabbit (BA-1000, Vector), CD31-PE/Cy7 antibody (BD Biosciences), CD45-PE/Cy7 antibody (BD Biosciences), CD34-APC antibody (Biolegend), and CD29-APC/Cy7 antibody (Biolegend).

Techniques: Binding Assay, Staining, Expressing, Plasmid Preparation, Activation Assay, Control

Journal: PLoS ONE

Article Title: miR-7977 inhibits the Hippo-YAP signaling pathway in bone marrow mesenchymal stromal cells

doi: 10.1371/journal.pone.0213220

Figure Lengend Snippet: Summary of results of GSEA showing p-value <0.00001 and q-value <0.25.

Article Snippet: Expression plasmid of human miR-7977 (MIR7977-MiRNA) and empty vector control (pCMV-MIR) were purchased from OriGene Inc. MSCs or HTS-5 cells were transfected with 2 μg of the 8xGTIIC-luciferase, YAP1 (GFP-tagged)-pCMV6-AC-GFP, control pMirTarget plasmid (pCMV-MIR) or pCMV-MIR-7977 using Lipofectamine LTX transfection reagent (Life Technologies).

Techniques:

Journal: PLoS ONE

Article Title: miR-7977 inhibits the Hippo-YAP signaling pathway in bone marrow mesenchymal stromal cells

doi: 10.1371/journal.pone.0213220

Figure Lengend Snippet: (A) Enrichment plots from GSEA are shown. The panel was obtained by GSEA using the gene set database, c6all.v6.0.symbols.gmt [Oncogenic signature]. (B) The core enrichment of genes was visualized as heatmap. Left side showed control MSCs and Right side showed miR-7977-transduced MSCs. (C) The expression of each gene associated with YAP1 and tight junction was visualized as a molecular interaction network made by Cytoscape software. Gene symbols are overlaid on each node and interactions between genes are shown as dotted lines with an arrow (edge). The fold change of gene expression [Log 2 (miR-7977 transduced MSCs/ Control transduced MSCs)] was superimposed on the molecular interaction network.

Article Snippet: Expression plasmid of human miR-7977 (MIR7977-MiRNA) and empty vector control (pCMV-MIR) were purchased from OriGene Inc. MSCs or HTS-5 cells were transfected with 2 μg of the 8xGTIIC-luciferase, YAP1 (GFP-tagged)-pCMV6-AC-GFP, control pMirTarget plasmid (pCMV-MIR) or pCMV-MIR-7977 using Lipofectamine LTX transfection reagent (Life Technologies).

Techniques: Expressing, Software

Journal: PLoS ONE

Article Title: miR-7977 inhibits the Hippo-YAP signaling pathway in bone marrow mesenchymal stromal cells

doi: 10.1371/journal.pone.0213220

Figure Lengend Snippet: (A) qRT-PCR for STK4 was conducted after transduction of control or miR-7977 for primary MSCs (5 nM) and HTS-5 (20 nM). The left panel shows the results in MSC (n = 5), and the right panel shows the results in the HTS-5 mesenchymal cell line (n = 3). (B) Immunoblot analysis of Hippo signaling pathway after transfer of control siRNA or miR-7977 into HTS-5. Anti-STK4 Ab, anti-YAP1 and anti- phospho-YAP1 Ser127 were used. As internal standard, anti-β-actin was used.

Article Snippet: Expression plasmid of human miR-7977 (MIR7977-MiRNA) and empty vector control (pCMV-MIR) were purchased from OriGene Inc. MSCs or HTS-5 cells were transfected with 2 μg of the 8xGTIIC-luciferase, YAP1 (GFP-tagged)-pCMV6-AC-GFP, control pMirTarget plasmid (pCMV-MIR) or pCMV-MIR-7977 using Lipofectamine LTX transfection reagent (Life Technologies).

Techniques: Quantitative RT-PCR, Transduction, Western Blot

Journal: PLoS ONE

Article Title: miR-7977 inhibits the Hippo-YAP signaling pathway in bone marrow mesenchymal stromal cells

doi: 10.1371/journal.pone.0213220

Figure Lengend Snippet: (A) Nuclear localization of GFP-tagged YAP1 after transduction of YAP1 (GFP-tagged)—pCMV6-AC-GFP into HTS-5. Subsequently, control or 20 nM miR-7977 mimic was transfected, and the cells were visualized. Left panel: control siRNA transfer. Right panel: miR-7977 transfer. Scale = 10 μm. (B) Schematic diagram of luciferase assay to evaluate the interaction of TEAD and YAP1. (C) Transduction of pCMV-MIR-7977 and 8xGTIIC-luciferase into the HTS-5 stromal cell line and analysis of luciferase activity 2 days after transduction. Luciferase activity was normalized to the activity of an internal control (renilla luciferase). * P < 0.05, pCMV-MIR (control, n = 8) vs. pCMV-MIR-7977 (n = 8) (Student’s t -test). Data represent three independent experiments, each done in octuplicate.

Article Snippet: Expression plasmid of human miR-7977 (MIR7977-MiRNA) and empty vector control (pCMV-MIR) were purchased from OriGene Inc. MSCs or HTS-5 cells were transfected with 2 μg of the 8xGTIIC-luciferase, YAP1 (GFP-tagged)-pCMV6-AC-GFP, control pMirTarget plasmid (pCMV-MIR) or pCMV-MIR-7977 using Lipofectamine LTX transfection reagent (Life Technologies).

Techniques: Transduction, Transfection, Luciferase, Activity Assay

Journal: Oncogene

Article Title: Autophagy suppresses Ras-driven epithelial tumourigenesis by limiting the accumulation of reactive oxygen species

doi: 10.1038/onc.2017.175

Figure Lengend Snippet: Autophagy inhibition in Ras-activated tissues results in cell- and non-cell-autonomous effects on tissue growth. ( a ) Three-dimensional (3D) reconstruction of Ras V12 Atg RNAi and control eye-antennal mosaic discs from L3 wandering larvae. Mutant clones are GFP+ and tissues were counterstained with DAPI (blue). ( b ) In discs expressing both Ras V12 and Atg1 RNAi in clones, the proportion of GFP tissue was significantly higher than in the control Ras V12 mRFP samples (49.4±2.4%, compared to 38.9±1.2%, P =0.00028). This trend was also observed in Ras V12 Atg8a RNAi , although the difference was not significantly different (42.9±2.3%, P =0.068). ( c ) Non-cell-autonomous proliferation in wild-type tissue (non-GFP) surrounding Ras V12 GFP , Ras V12 Atg1 RNAi or Ras V12 Atg8a RNAi clones (GFP+), as seen by EdU incorporation. ( d ) Non-cell-autonomous caspase activation in wild-type tissue (non-GFP) surrounding Ras V12 Atg1 RNAi or Ras V12 Atg8a RNAi clones (GFP+), as seen by Dcp1 staining. Arrows indicate the apoptotic wave before the MF. ( e ) Apoptosis is confirmed by TUNEL assay around clones expressing Ras V12 and Atg8a -RNAi. ( f ') Quantification of data in ( d ). ( g ) Quantification of data in ( e ). Error bars=s.e.m. Statistics: one-way ANOVA with Tukey's multiple correction test. Scale bars: 50 μm.

Article Snippet: Tissue was fixed in 4% paraformaldehyde for 20 min at RT, washed 3x times with PBS+0.5% Triton X-100, incubated with antibody against GFP and washed and incubated with secondary antibodies, before EdU revelation using Click-iT protocol (Life Technologies, Thermo Fisher).

Techniques: Inhibition, Control, Mutagenesis, Clone Assay, Expressing, Activation Assay, Staining, TUNEL Assay

Journal: Nature communications

Article Title: Beta 1-integrin-c-Met cooperation reveals an inside-in survival signalling on autophagy-related endomembranes.

doi: 10.1038/ncomms11942

Figure Lengend Snippet: Figure 2 | b1-integrin is required for sustained c-Met-dependent ERK1/2 phosphorylation in detached cells, c-Met-dependent in vivo tumorigenesis and invasion. (a–d) Western blots for: (a) tubulin and phospho-ERK1/2 in b1A and GD25 (b1 / ) cells, stimulated with HGF for 0, 15 and 120 min; (b) Phospho-c-Met (Y1234-355), c-Met, phospho-ERK1/2, ERK 1/2 and tubulin in M1268T c-Met-expressing NIH3T3; (c) phospho-c-Met (Y1234-355), GFP (c-Met-GFP: p195, precursor; p170, mature b chain), b1-integrin, phospho-ERK1/2 and tubulin in c-Met-GFP cells incubated with tetracycline (Tet) for 0 or 16 h; (d) b1-integrin, phospho-ERK1/2, and tubulin in A549 cells, stimulated without ( ) or with ( þ ) HGF for 120 min in suspension; (b–d) All cells were transfected with control (Cont) or b1-integrin (b1) (human cells: oligo 1, Qiagen; mouse cells: oligo 3, Dharmacon) siRNA. Graphs represent phospho- ERK1/2/tubulin ratios (means±s.e.m.), normalized to appropriate controls: (a,d) no HGF; (b,c) siRNA control (Cont), obtained by densitometric analysis (n ¼ 3 to 6). (e) Tumour growth curves, over time, of M1268T c-Met-expressing NIH3T3 cells, transfected with control (Cont) or b1-integrin (b1) siRNA. Data are mean tumour volume (mm3)±s.e.m. of n ¼ 5 mice per group. (f) Pictures of the lungs of mice dissected 21 days after injection into the tail vein with either control (Cont) (n ¼ 4) or b1-integrin (b1) (n ¼ 5) siRNA-transfected M1268T c-Met-expressing NIH3T3 cells. Graph represents the mean number of macroscopic tumours per mouse±s.e.m. (g) Mean number±s.e.m. of disseminated WT and M1268T c-Met-expressing NIH3T3 cells per zebrafish embryo 24 h after injection. Cells were transfected with control (Cont) or b1-integrin (b1) siRNA (n ¼ 3, average of 25 embryos per condition per experiment). t-Test, * Po0.05; **Po0.01; ***Po0.001.

Article Snippet: For the anoikis assay 1 106 cells were transferred to 50ml falcon tubes for 24 h (c-Met-GFP and NIH3T3 cells) or 48 h (MDA-MB-468 and A549 cells) in 10ml of serum-free (MDA-MB-468, NIH3T3 and c-Met-GFP) or full serum (A549) media þ / tetracycline (c-Met-GFP) or ±50 ngml 1 HGF (A549 and MDA-MB-468) at 37 C. Cells were put on ice and either harvested for western blot, cytospun and fixed for immunofluorescence, or stained with propidium iodide (1/100, Life Technologies) for 15min.

Techniques: Phospho-proteomics, In Vivo, Western Blot, Expressing, Incubation, Suspension, Transfection, Control, Injection

Journal: Nature communications

Article Title: Beta 1-integrin-c-Met cooperation reveals an inside-in survival signalling on autophagy-related endomembranes.

doi: 10.1038/ncomms11942

Figure Lengend Snippet: Figure 3 | The role of b1-integrin in c-Met-dependent signalling is adhesion independent though its active conformation is a positive regulator. (a) Confocal section of A549 cells stimulated with HGF-AlexaFluor-555 (HGF-555, red) for 120 min in suspension. Cells were cytospun, fixed and stained for active conformation b1-integrin (9EG7, green) and DAPI (blue). Colocalizations appear in yellow. Scale bar, 10 mm. Numbers are mean percentage colocalization±s.e.m. (n ¼ 3). (b) The mean percentage cell surface levels±s.e.m. of active conformation b1-integrin (9EG7) reported on pan-b1-integrin (DF7) assessed by flow cytometry. c-Met-GFP cells were treated with or without tetracycline (Tet) for 16 h (arbitrary units, n ¼ 4) and with or without SU11274 (2 mM) (n ¼ 3). (c,d) Confocal projections of 7 Z-sections from the base to the apex of cells. Arrows show examples of plasma membrane staining. Scale bar, 10 mm. Cells were cultured on Poly-L-lysine coated glass coverslips for 16 h with tetracycline and stained for active conformation b1-integrin (9EG7, red). c-Met-GFP is in green. (c) T-REx-293 cells, non-transfected and stably transfected with c-Met-GFP (‘c-Met-GFP cells’), at a 50/50 ratio. (d) c-Met-GFP cells in the presence of the c-Met inhibitor SU11274 (2 mM). (e) Western blots for phospho-ERK1/2, ERK1/2 and tubulin in WT and cpdm (SHARPIN null) MEFs, stimulated without ( ) or with ( þ ) HGF for 15 and 120 min in suspension and treated without ( ) or with ( þ ) 1 mM MnCl2. Graphs represent phospho-ERK1/2/ERK1/2 ratios±s.e.m. Normalized to no HGF obtained by densitometric analysis (n ¼ 3). t-Test, *Po0.05.

Article Snippet: For the anoikis assay 1 106 cells were transferred to 50ml falcon tubes for 24 h (c-Met-GFP and NIH3T3 cells) or 48 h (MDA-MB-468 and A549 cells) in 10ml of serum-free (MDA-MB-468, NIH3T3 and c-Met-GFP) or full serum (A549) media þ / tetracycline (c-Met-GFP) or ±50 ngml 1 HGF (A549 and MDA-MB-468) at 37 C. Cells were put on ice and either harvested for western blot, cytospun and fixed for immunofluorescence, or stained with propidium iodide (1/100, Life Technologies) for 15min.

Techniques: Suspension, Staining, Cytometry, Clinical Proteomics, Membrane, Cell Culture, Transfection, Stable Transfection, Western Blot

Journal: Nature communications

Article Title: Beta 1-integrin-c-Met cooperation reveals an inside-in survival signalling on autophagy-related endomembranes.

doi: 10.1038/ncomms11942

Figure Lengend Snippet: Figure 4 | c-Met and b1-integrin cooperation is endocytosis dependent and b1-integrin is required for c-Met endocytosis. (a) Western blots for phospho-ERK1/2, ERK1/2 and tubulin in b1A cells stimulated without ( ) or with ( þ ) HGF for 120 min following pre-treatment with DMSO or Dynasore (80 mM); (b) western blots for phospho-ERK1/2, clathrin and tubulin in A549 cells transfected with control (Cont) or clathrin heavy chain (CHC) siRNA and incubated in suspension for 120 min without ( ) or with ( þ ) HGF. (c) Western blots for phospho-ERK1/2, ERK1/2 and tubulin in M1268T c-Met- expressing NIH3T3 treated with DMSO or Dynasore (80 mM). (a–c) Graphs represent mean phospho-ERK1/2/tubulin ratio±s.e.m.; (a,b) upon HGF stimulation normalized to no HGF; (c) with dynasore normalized to DMSO, obtained by densitometric analysis (n ¼ 3). (d) Western blot for b1-integrin following a biotinylation internalization assay in b1A and b1A-YYFF cells incubated for 0, 5, 15 and 30 min at 37 C with HGF. Numbers represent the percentage of internalization. (e) Western blots for tubulin and phospho-ERK1/2 in b1A-YYFF cells, stimulated with HGF for 0, 15 and 120 min. Graph is mean phospho-ERK1/2/tubulin ratio±s.e.m. Normalized to HGF 0 min, obtained by densitometric analysis (n ¼ 6). (f) Western blot for c-Met following a biotinylation internalization assay in b1A and b1A-YYFF cells incubated for 0, 5, 15 and 30 min at 37 C with HGF. Numbers represent the percentage of internalization. (g) Percentage of HGF-AlexaFluor-555 (HGF-555) uptake (mean red pixels per cell/total c-Met levels)±s.e.m. after 15 min incubation in b1A and b1A-YYFF cells normalized to the uptake in GD25 cells (n ¼ 3). (h) Confocal sections of A549 cells, stimulated with HGF-AlexaFluor-555 (HGF- 555) for 120 min in suspension. Scale bar, 10 mm. Numbers are percentage of HGF-555 uptake (mean red pixels per cell)±s.e.m. in cells transfected with b1-integrin siRNA, normalized to the uptake in cells transfected with control siRNA (n ¼ 3). (i) Percentage of HGF-AlexaFluor-555 (HGF-555) uptake (mean red pixels per cell)±s.e.m. in cpdm (SHARPIN null) MEFs stimulated for 120 min in suspension, normalized to the uptake in WT MEFs (n ¼ 3). (j) Percentage of HGF-AlexaFluor-555 (HGF-555) uptake (mean red pixels per cell/total c-Met levels)±s.e.m. in b1A-YYFF cells positive for GFP-Rab21 normalized to the uptake in b1A-YYFF cells negative for GFP-Rab21 (from the same coverslips)±s.e.m. (n ¼ 3). (k) Mean phospho-ERK1/2 levels±s.e.m. in permeabilised b1A-YYFF cells, Rab21-GFP negative ( ) or positive ( þ ) (from the same population), upon stimulation with HGF for 120 min, normalized to mean phospho-ERK1/2 levels at HGF 0 min, assessed by flow cytometry (n ¼ 3). t-Test, *Po0.05; **Po0.01; NS: not significant.

Article Snippet: For the anoikis assay 1 106 cells were transferred to 50ml falcon tubes for 24 h (c-Met-GFP and NIH3T3 cells) or 48 h (MDA-MB-468 and A549 cells) in 10ml of serum-free (MDA-MB-468, NIH3T3 and c-Met-GFP) or full serum (A549) media þ / tetracycline (c-Met-GFP) or ±50 ngml 1 HGF (A549 and MDA-MB-468) at 37 C. Cells were put on ice and either harvested for western blot, cytospun and fixed for immunofluorescence, or stained with propidium iodide (1/100, Life Technologies) for 15min.

Techniques: Western Blot, Transfection, Control, Incubation, Suspension, Expressing, Cytometry

Journal: Nature communications

Article Title: Beta 1-integrin-c-Met cooperation reveals an inside-in survival signalling on autophagy-related endomembranes.

doi: 10.1038/ncomms11942

Figure Lengend Snippet: Figure 6 | b1-integrin plays the role of an adaptor to sustain c-Met signalling on ARE. (a) Graph represents mean phospho-p52Shc/p52Shc ratios±s.e.m. upon 120 min HGF stimulation normalized to 0 min, in GD25, b1A and b1A-YYFF cells. Data were obtained by densitometric analysis of western blots (shown in Supplementary Fig. S7a) (n ¼ 3 GD25 cells, n ¼ 4 b1A and b1A-YYFF cells). (b) Western blots for phospho-p52Shc, tubulin and b1-integrin in A549 cells, transfected with control (Cont) or b1-integrin (b1) siRNA and stimulated without ( ) or with ( þ ) HGF for 120 min in suspension. Numbers are mean phospho-p52Shc/tubulin ratios±s.e.m. upon HGF stimulation normalized to no HGF, obtained by densitometric analysis (n ¼ 3). (c) WTand cpdm (Sharpin null) MEFs were incubated without ( ) or with ( þ ) HGF for 120 min in suspension. Graph represents the mean phospho-p52Shc/p52Shc

Article Snippet: For the anoikis assay 1 106 cells were transferred to 50ml falcon tubes for 24 h (c-Met-GFP and NIH3T3 cells) or 48 h (MDA-MB-468 and A549 cells) in 10ml of serum-free (MDA-MB-468, NIH3T3 and c-Met-GFP) or full serum (A549) media þ / tetracycline (c-Met-GFP) or ±50 ngml 1 HGF (A549 and MDA-MB-468) at 37 C. Cells were put on ice and either harvested for western blot, cytospun and fixed for immunofluorescence, or stained with propidium iodide (1/100, Life Technologies) for 15min.

Techniques: Western Blot, Transfection, Control, Suspension, Incubation

Journal: Nature communications

Article Title: Beta 1-integrin-c-Met cooperation reveals an inside-in survival signalling on autophagy-related endomembranes.

doi: 10.1038/ncomms11942

Figure Lengend Snippet: Figure 7 | c-Met–b1-integrin cooperation on ARE mediates c-Met-dependent anchorage-independent cell survival. (a) Confocal section of A549 cells after HGF stimulation in suspension for 48h. Cytospun and fixed cells were stained for c-Met (red), b1-integrin (green), LC3B (blue) and DAPI (magenta). Scale bar, 10mm. Numbers are mean percentage colocalization±s.e.m. between c-Met–b1-integrin and LC3B (n¼ 3). (b) Mean percentage of HGF- or tetracycline-dependent anchorage-independent survival (or protection against anoikis)±s.e.m. in cells transfected with b1-integrin (b1)(Qiagen), ATG5 (SMARTpool) or ATG13 (from one to four individual oligos used per experiment, pooled data from one or multiple individual oligos per experiment) siRNA, normalized to control (Cont) siRNA. A549 cells were stimulated with HGF for 48h in suspension. c-Met-GFP cells were stimulated with tetracycline (Tet) for 24h in suspension. The HGF- or Tet-dependent anchorage-independent survival was obtained by normalizing the data with HGF/Tet to no HGF/Tet. Cells were stained with propidium iodide and the cell viability was analysed by flow cytometry (A549: b1-integrin siRNA n¼ 6, ATG5 or ATG13 siRNA n¼ 3. c-Met-GFP: b1-integrin or ATG5 siRNA n¼ 3, ATG13 siRNA n¼ 4). (c) Western blots for phospho-ERK1/2, ERK1/2, phospho-p52Shc, tubulin and ATG5 in A549 cells, transfected with control or ATG5 siRNA and stimulated without (–) or with (þ ) HGF for 48h in suspension. Graph represents mean ratios±s.e.m. of phospho-ERK1/2/tubulin (siRNA b1-integrin: n¼ 5, siRNA ATG5: n¼ 3) and of phospho-p52Shc/tubulin (siRNA b1-integrin: n¼ 3; siRNA ATG5: n¼ 4) with HGF normalized to the mean ratios with no HGF, obtained by densitometric analysis. (d,e) Western blots for (d) phospho-ERK1/2 and tubulin or (e) phospho-ERK1/2, tubulin and ATG13 in (d) A549 cells stimulated without ( ) or with (þ ) HGF for 48h in suspension or (e) c-Met-GFP cells stimulated without ( ) or with (þ ) Tet for 24h in suspension, transfected with control or (d) one (oligo 3) or (e) two (oligo 1 and 2) individual ATG13 siRNA oligos. Graph represents mean fold increases of phospho-ERK1/2/tubulin (from one to four individual oligos used per experiment, pooled data from one or multiple individual oligos per experiment) upon HGF/Tet versus no HGF/Tet±s.e.m. (n¼ 4), obtained by densitometric analysis. (f) Western blots for phospho-c-Met (Y1234-35), c-Met, phospho-ERK1/2 and tubulin in A549 cells pre-treated with the AIIB2 b1-integrin blocking antibody (2mgml 1), an isotype control or no antibody, and stimulated without ( ) or with (þ ) HGF for 48h in suspension. Graph represents mean phospho- ERK1/2/tubulin ratios±s.e.m. with HGF normalized to the ratios with no HGF obtained by densitometric analysis (n¼ 3). t-Test, *Po0.05; **Po0.01; ***Po0.001.

Article Snippet: For the anoikis assay 1 106 cells were transferred to 50ml falcon tubes for 24 h (c-Met-GFP and NIH3T3 cells) or 48 h (MDA-MB-468 and A549 cells) in 10ml of serum-free (MDA-MB-468, NIH3T3 and c-Met-GFP) or full serum (A549) media þ / tetracycline (c-Met-GFP) or ±50 ngml 1 HGF (A549 and MDA-MB-468) at 37 C. Cells were put on ice and either harvested for western blot, cytospun and fixed for immunofluorescence, or stained with propidium iodide (1/100, Life Technologies) for 15min.

Techniques: Suspension, Staining, Transfection, Control, Cytometry, Western Blot, Blocking Assay

Journal: Nature communications

Article Title: Beta 1-integrin-c-Met cooperation reveals an inside-in survival signalling on autophagy-related endomembranes.

doi: 10.1038/ncomms11942

Figure Lengend Snippet: Figure 8 | c-Met–b1-integrin intracellular cooperation mediates c-Met-dependent anchorage-independent growth, in vivo tumorigenesis and invasion. (a) Mean area of b1A, GD25 and b1A-YYFF colonies treated with ( þ ) HGF in soft agar normalized to the mean area without HGF ( )±s.e.m. (n ¼ 3, each experiment performed in duplicate). (b) Tumour growth curves, over time, of GD25, b1A and b1A-YYFF cells mixed with MRC5 fibroblasts and treated daily with DMSO or PHA-665752 (PHA, 100 nM) by topical application onto the surface of the skin where cells had been injected from day 1 after injection. Graphs represent the mean tumour volumes (mm3)±s.e.m. of n ¼ 5 mice per group measured daily. (c) Mean number of disseminated b1A and b1A-YYFF cells per zebrafish embryo 24 h after injection in the yolk sac±s.e.m. Cells were incubated with HGF and treated with DMSO or PHA-665752 (PHA, 100 nM) (n ¼ 3, average of 26 embryos per condition per experiment). (d) Mean number of disseminated A549 cells per embryo 24 h after injection in the yolk sac±s.e.m. Cells were transfected with control or ATG5 siRNA and incubated without ( ) or with ( þ ) HGF (n ¼ 3, average of 39 embryos per condition per experiment). (e) Model of b1-integrin–c-Met ‘inside-in’ signalling, promoting survival of cancer cells during metastasis and in the establishment of tumours: In unstimulated cells, c-Met and b1-integrin form a complex at the plasma membrane. c-Met activation in either a ligand-dependent (A549/ MDA-MB-468/MEF cells) or -independent (c-Met-GFP/M1268T c-Met-expressing NIH3T3 cells) manner results in internalization and activation of b1-integrin, which is the major form to internalize with c-Met. b1-integrin and its active conformation are in turn required for an optimal endocytosis of c-Met. Thus, they both need each other for their optimal endocytosis. They co-internalize in a clathrin-dependent manner. Internalized c-Met-b1-integrin complex progressively accumulates on ‘autophagy-related endomembranes’ (ARE), which are LC3B-positive endomembranes. b1-integrin promotes sustained c-Met signalling from ARE likely through acting as an adaptor that links c-Met to p52Shc, which in turn activates the downstream signalling pathway ERK1/2. Altogether, c-Met-b1-integrin cooperation, that we named ‘inside-in signalling’ is required for anchorage-independent survival that may help cancer cells to survive as they invade from the primary tumour, travel from the primary tumour to the secondary site during metastasis, leading to tumour growth and metastasis. t-Test for a and c; ANOVA test for b; Mann–Whitney U-test for d; *Po0.05; **Po0.01; ***Po0.001; NS: not significant.

Article Snippet: For the anoikis assay 1 106 cells were transferred to 50ml falcon tubes for 24 h (c-Met-GFP and NIH3T3 cells) or 48 h (MDA-MB-468 and A549 cells) in 10ml of serum-free (MDA-MB-468, NIH3T3 and c-Met-GFP) or full serum (A549) media þ / tetracycline (c-Met-GFP) or ±50 ngml 1 HGF (A549 and MDA-MB-468) at 37 C. Cells were put on ice and either harvested for western blot, cytospun and fixed for immunofluorescence, or stained with propidium iodide (1/100, Life Technologies) for 15min.

Techniques: In Vivo, Injection, Incubation, Transfection, Control, Clinical Proteomics, Membrane, Activation Assay, Expressing, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: Mesenchymal Stromal Cell-derived Extracellular Vesicles Promote Myeloid-biased Multipotent Hematopoietic Progenitor Expansion via Toll-Like Receptor Engagement *

doi: 10.1074/jbc.M116.745653

Figure Lengend Snippet: MSC-derived EVs activate NF-κB and Notch signaling to promote HSPC differentiation. A, diagrammatic representation of the lentiviral vector design used to generate the NF-κB enhancer-luciferase reporter cell line. Stably transduced and sorted SIM-A9 microglial cells were used for EV exposure followed by luciferase assay. The SIM-A9-NF-κB-Luc cells were exposed with MSC EVs and incubated for 48 h followed by luciferase assay. Non-treated SIM-A9-NF-κB-Luc and LPS-treated SIM-A9-NF-κB-Luc cells were used as negative and positive controls, respectively. Luciferase activity was measured as relative florescence units (RFU). B, quantitative mRNA expression of NF-κB and Notch-1 signaling and downstream targets involved in cell proliferation in MSC EV-exposed HSPCs. C, cytokines released by HSPCs after 48 h co-culture with MSC EVs when compared with S100 were measured using Luminex. D, quantitative mRNA expression of targets relevant for hematopoiesis and TLR4-mediated activation was analyzed in MSC EV-exposed WT and MyD88−/− HSPCs. E, TLR4 signaling was specifically inhibited in WT MSC EV-exposed HSPCs using TAK-242. Error bars indicate means ± S.E.

Article Snippet: Generation of NF-κB Luc GFP Reporter Cells Human embryonic kidney (HEK-293T) cells were seeded at a density of 1.5 × 107 cells per 15-cm tissue culture dish (Corning), precoated with 0.01% poly- l -lysine (Sigma), and transfected by using the transducing plasmid pHAGE NF-κB-TA-luc-UBC-GFP-W and helper constructs pLP1, pLP2, and pLP/VSVG (Gibco-Invitrogen) ( 55 , 56 ).

Techniques: Derivative Assay, Plasmid Preparation, Luciferase, Stable Transfection, Incubation, Activity Assay, Expressing, Co-Culture Assay, Luminex, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: Mesenchymal Stromal Cell-derived Extracellular Vesicles Promote Myeloid-biased Multipotent Hematopoietic Progenitor Expansion via Toll-Like Receptor Engagement *

doi: 10.1074/jbc.M116.745653

Figure Lengend Snippet: MSC EV exposure induces myelosuppression and HSC exhaustion via TLR4. A, to examine HSPC repopulation potential, EV- or S100-exposed HSPCs were competitively transplanted into irradiation-conditioned CD45-mismatched recipients. B, donor chimerism and lymphoid versus myeloid contribution were examined at 4 weeks (short-term HSC) and 16 weeks (long-term HSC) after engraftment. C, competitively transplanted WT and Tlr4−/− donor chimerism and myeloid contribution were examined at 4 and 8 weeks after engraftment. D, model of BM-MSC-derived EVs effect on HSPCs. The HSPC transcriptome toward differentiation and proliferation undergoes activation after MSC EV exposure. The NF-κB signaling pathway up-regulation by MSC EVs results the entry into G1 cell cycle phase followed by MPP2 and MPP3/4 expansion.

Article Snippet: Generation of NF-κB Luc GFP Reporter Cells Human embryonic kidney (HEK-293T) cells were seeded at a density of 1.5 × 107 cells per 15-cm tissue culture dish (Corning), precoated with 0.01% poly- l -lysine (Sigma), and transfected by using the transducing plasmid pHAGE NF-κB-TA-luc-UBC-GFP-W and helper constructs pLP1, pLP2, and pLP/VSVG (Gibco-Invitrogen) ( 55 , 56 ).

Techniques: Irradiation, Derivative Assay, Activation Assay