Journal: EBioMedicine

Article Title: Antibody-drug conjugate T-DM1 treatment for HER2+ breast cancer induces ROR1 and confers resistance through activation of Hippo transcriptional coactivator YAP1

doi: 10.1016/j.ebiom.2019.04.061

Figure Lengend Snippet: Overexpression of ROR1/YAP1 in breast cancer is associated with poor overall survival outcomes. (a) Graphical representation of cancer types in which ROR1 is most frequently altered in the TCGA dataset. (b) ROR1 and YAP1 mutation analysis of 960 breast cancer samples in the TCGA dataset. (c) Significant co-occurrences of ROR1 and YAP1 in the TCGA dataset. (d) Boxplot comparing ROR1 and YAP1 expression in ER + PR+/HER2-; ER + PR+/HER2+; ER-PR-/HER2+; subtypes samples in the TCGA dataset (one-way ANOVA). (e) Immunohistochemical detection of ROR1 and YAP1 in disease free, ER + PR+/HER2-; ER + PR+/HER2+; ER-PR-/HER2+; breast cancer subtypes from our own data cohort (one-way ANOVA). The boxes show the median ±1 quartile, with whiskers extending to the most extreme data point within 1.5 interquartile ranges from the box boundaries. (f) Survival analysis of ROR1 and YAP1 high and low expressing breast cancer patients based on expression intensity (Log-rank test; p < .0001, scale bar: 50 μM). (g) Bar graph showing the ROR1 and YAP1 expression intensity (%) in treatment naïve, sensitive, partially sensitive and resistant patient tumors (**p < .0, two-tailed Student's t -test).

Article Snippet: Human ROR1 (AF2000, R&D System, USA), HER2 (MAB1129, R & D System, USA) and YAP1(MAB8094, R & D System, USA) antibodies were purchased from R & D System, USA.

Techniques: Over Expression, Mutagenesis, Expressing, Immunohistochemical staining, Two Tailed Test

Journal: EBioMedicine

Article Title: Antibody-drug conjugate T-DM1 treatment for HER2+ breast cancer induces ROR1 and confers resistance through activation of Hippo transcriptional coactivator YAP1

doi: 10.1016/j.ebiom.2019.04.061

Figure Lengend Snippet: Treatment resistant BC cells overexpress ROR1 have a higher sphere forming efficiency: (a) Administration of therapy leads to the increase of ROR1 expression intensity in HER2 + treatment naïve and treatment exposed BC patient ( p = 3.021e-02. unpaired two-sided t -test). (b) Tumor cells were dissociated from treatment naïve, sensitive and resistant HER2 + BC patients and equal numbers of cells were cultured in the supplemented CSC medium and stained for ROR1 expression in the spheres. Matched HER2 + BC patient's tissues were stained for ROR1 expression by immunofluorescence (Scale bar: 50 μM). (c) FACS analysis of ROR1 + and ROR1 − population in treatment naïve, sensitive and resistant cells from primary HER2 + BC patient tumors. (d) Equal number of sorted ROR1 + and ROR1 − cells from primary BC patients ( n = 2) were cultured in the supplemented CSC medium and analyzed for sphere forming efficiency ( p < .0002, paired two-sided t -test). (e) Comparison of epithelial-to-mesenchymal transition related genes showing mRNA expression from treatment naïve, sensitive and resistant ROR1 + and ROR1 − primary HER2 + BC patients cell fractions (results are mean fold change +/− SE, n = 3). (f) Comparison of ROR1 mRNA expression in primary HER2 + treatment naïve, sensitive and resistant BC patient tumors (results are mean fold change +/− SE, n = 3). (g) In vitro treatment of HER2 + primary tumors cells treated with T-DM1 (5 nM) and assessed for sphere forming efficiency (black arrowhead). Below is shown immunoblotting of ROR1 from each group of patients' protein samples (results are mean +/− SE, n = 3 run in triplicate; Scale bar: 100 μM).

Article Snippet: Human ROR1 (AF2000, R&D System, USA), HER2 (MAB1129, R & D System, USA) and YAP1(MAB8094, R & D System, USA) antibodies were purchased from R & D System, USA.

Techniques: Expressing, Cell Culture, Staining, Immunofluorescence, Comparison, In Vitro, Western Blot

Journal: EBioMedicine

Article Title: Antibody-drug conjugate T-DM1 treatment for HER2+ breast cancer induces ROR1 and confers resistance through activation of Hippo transcriptional coactivator YAP1

doi: 10.1016/j.ebiom.2019.04.061

Figure Lengend Snippet: T-DM1 treatment alters ROR1 expression without significant changes in HER2 expression: (a) Immunostaining of HER2 expression from treatment naïve ( n = 5), sensitive (n = 5) and resistant (n = 5) HER2 + BC patient's tumor samples (Scale bar: 50 μM). (b) HCC1954 and MCF-7-HER2+ cells were cultured on a glass cover slip in 24-well plates and treated either with vehicle (DMSO) or 5 nM of T-DM1 for 5 days. Cells were fixed and immunofluorescence stained images captured for HER2 (red) and nucleus DAPI (blue) (Scale bar: 50 μM). (c) HCC1954 and MCF-7-HER2+ cells were cultured, treated and fixed as (b), and immunofluorescently stained for ROR1 (red) and nuclear DAPI (blue) expression (Scale bar: 50 μM). (d) Treatment naïve BC patient tumor cells, (e) HCC1954(HER2+), and (f) MCF-7-HER2+ cells were treated with T-DM1 (5 nM) for 5 days, trypsinized and cultured in supplemented CSC medium in sphere culture for 10 days (10 days is denoted as first generation). After 10 days, spheres were dissociated and re-cultured in CSC medium for an additional 10 days (20 days denoted as second generation) followed by dissociation of spheres and then re-cultured again in CSC medium (30 days denoted as third generation). In every 10-day culture cycle, sphere forming efficiency was examined in ROR1 + and ROR1 − populations by counting the number and size of spheres. (Bar graph represents the mean +/− SE, n = 3; run in triplicate; p = ns [not significant]; p = .05; p = .01, p = .001; one-way ANOVA). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Human ROR1 (AF2000, R&D System, USA), HER2 (MAB1129, R & D System, USA) and YAP1(MAB8094, R & D System, USA) antibodies were purchased from R & D System, USA.

Techniques: Expressing, Immunostaining, Cell Culture, Immunofluorescence, Staining

Journal: EBioMedicine

Article Title: Antibody-drug conjugate T-DM1 treatment for HER2+ breast cancer induces ROR1 and confers resistance through activation of Hippo transcriptional coactivator YAP1

doi: 10.1016/j.ebiom.2019.04.061

Figure Lengend Snippet: T-DM1 treatment-induced ROR1 + cells enriched with CSC features show higher self-renewal efficiency and increased resistance with enriched CSC features. (a) Treatment naïve HER2 + BC patients tumor cells were enzymatically dissociated and cultured in supplemented CSC medium, treated with either vehicle (DMSO) and 5 nM T-DM1 and incubated for 10 days (follow the schematic diagram). Spheres were dissociated for single cells and subjected to FACS sorting for ROR1. Extreme limiting dilution assay (ELDA) was performed by plating sorted cells with increasing cell numbers and analyzed for stemness frequencies by ‘R' statistical package “statmod” ( p < .0427; one-way ANOVA). (right) showed the confidence interval for 1/stemness frequency. (b) Bulk cells were isolated and treated as described in (a) and analyzed for sphere forming efficiency (Bar graph represents the mean +/− SE, n = 3, run in triplicate). (c) HER2 + patient tumor cells treated with either vehicle or T-DM1 for 5 days, harvested and FACS sorted for ROR1 + and ROR1 − populations, and then sorted cells were cultured in supplemented CSC medium [follow the schematic diagram] for 10 days and analyzed for the sphere forming efficiency from each cell population (black arrowhead; p = .001 for T-DM1 treatment; p = .05 for vehicle, unpaired two-sided t -test, Scale bar: 100 μM). (d) HCC1954 cells were treated and analyzed as described in (c) [black arrowhead; p = .01 for T-DM1 treatment; p = ns (not significant) for vehicle, unpaired two-sided t -test; Scale bar: 100 μM]. (e) HCC1954 cells were either treated with vehicle or T-DM1 and sorted as described in (c) and quantitatively analyzed for Bmi1, Nanog, Oct3/4. Sox2 mRNA in ROR1 + and ROR1 − populations. (Bar graph represents the mean +/− SE, n = 3; run in triplicate; p = .05; p = .01; p = .001; one-way ANOVA). (f) ROR1 + cells were more significantly resistant than ROR1 − cells at individual T-DM1 treatment and showed higher EC50 in response to T-DM1 as compared to ROR1 − cells. (Bar graph represents the mean +/− SE, n = 3; run in triplicate; p = .05, p = .01).

Article Snippet: Human ROR1 (AF2000, R&D System, USA), HER2 (MAB1129, R & D System, USA) and YAP1(MAB8094, R & D System, USA) antibodies were purchased from R & D System, USA.

Techniques: Cell Culture, Incubation, Limiting Dilution Assay, Isolation

Journal: EBioMedicine

Article Title: Antibody-drug conjugate T-DM1 treatment for HER2+ breast cancer induces ROR1 and confers resistance through activation of Hippo transcriptional coactivator YAP1

doi: 10.1016/j.ebiom.2019.04.061

Figure Lengend Snippet: T-DM1 treatment induced ROR1 + cells are enriched for CD44, ALDH and YAP1 target genes: (a-b) Freshly dissociated treatment naïve and treatment resistant HER2 + BC patients tumor cells were FACS sorted and analyzed for ROR1 subpopulation stained with ALDH and CD44 antibodies (Results are median +/− interquartile range (IQR) n = 5; unpaired two-sided t -test). (c-d. Patients tumor cells were either treated with vehicle (DMSO) and/or T-DM1 for 5 days, and sorted as (a) Analysis of ALDH and CD44 expression by FACS and immunofluorescence staining in ROR1 + and ROR1 − subpopulation (Scale bar: 50 μM). (e) HCC1954 cells treated, maintained, sorted and analyzed as (d). (g) Cells were treated and sorted as (d), extracted total RNA followed by qRT-PCR for YAP1, TEAD1, CTGF and CCND1 in ROR1 + and ROR1 − populations (Bar graph represents the mean +/− SE, n = 3; run in triplicate; p = .001, 0.01, 0.05, 0.001 and 0.001). (h) Detection of CTGF from treatment naïve and treatment exposed patients' blood serum by ELISA assay (Bar graph represents the mean +/− SE, n = 3; run in triplicate; ** p < .01; Students t -test).

Article Snippet: Human ROR1 (AF2000, R&D System, USA), HER2 (MAB1129, R & D System, USA) and YAP1(MAB8094, R & D System, USA) antibodies were purchased from R & D System, USA.

Techniques: Staining, Expressing, Immunofluorescence, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: EBioMedicine

Article Title: Antibody-drug conjugate T-DM1 treatment for HER2+ breast cancer induces ROR1 and confers resistance through activation of Hippo transcriptional coactivator YAP1

doi: 10.1016/j.ebiom.2019.04.061

Figure Lengend Snippet: Silencing ROR1 sensitizes T-DM1 treatment-induced ROR1 + spheroids forming efficiency and tumor initiation: (a) Patients tumor cells, (b) HCC1954, and (c) MCF-7 HER2+ cells. T-DM1 and ROR1-shRNA co-treatment resulted in eradication of sphere forming efficiency. (d-f) Loss of ROR1 expression in the spheres was analyzed by immunofluorescence staining (Scale bar: 50 μM) (red-ROR1, blue-DAPI) in HER2 + BC patients tumor cells, HCC1954 and MCF-7-HER2+ cells (Bar graph represents the mean +/− SE, n = 3; run in triplicate; p = .05; p = .01; one-way ANOVA). (g) Average number of spheres assessed for HCC1954 and MCF-7-HER2+ cells after transfecting with control shRNA and ROR1-shRNA. (h) Representation of tumors grown in mice after transfecting HCC1954 cells with either control-shRNA or ROR-shRNA. Table below indicates the number of tumors initiated in each group with percentage. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Human ROR1 (AF2000, R&D System, USA), HER2 (MAB1129, R & D System, USA) and YAP1(MAB8094, R & D System, USA) antibodies were purchased from R & D System, USA.

Techniques: shRNA, Expressing, Immunofluorescence, Staining, Control

Journal: EBioMedicine

Article Title: Antibody-drug conjugate T-DM1 treatment for HER2+ breast cancer induces ROR1 and confers resistance through activation of Hippo transcriptional coactivator YAP1

doi: 10.1016/j.ebiom.2019.04.061

Figure Lengend Snippet: T-DM1 treatment- induced ROR1 + cells have high tumor forming capacity in vivo : (a) Schematic representation of cell sorting and tumor initiation assay. (b) Fresh HER2 + breast tumor cells (n = 5 patient) were dissociated and FACS sorted ROR1 + and ROR1 − cells as described previously. Equal number of sorted and unsorted bulk cells from each patient tumor were injected subcutaneously in Nu/J mice (n = 5) and tumor growth and incidence were monitored for 8 weeks. ROR1 + cells were capable of generating larger tumors (5 out of 5 tumors), ROR1 − cells (2 out of 5 tumors) and unsorted bulk cells (3 out of 5 tumors) generated tumors but smaller in size (Scale bar 5 mm). (c) The weight distributions of tumors generated from ROR + , ROR1 − and unsorted bulk tumor cells are shown in all patient's tumors. Results are median +/− IQR (Inter quartile range), p = .01 and 0.05; repeated measure ANOVA. (d) HCC1954 cells were either treated with vehicle (DMSO) and/or T-DM1 for 5 days and sorted based on T-DM1 induced cell surface ROR1 overexpression and injected to mice. Tumor growth and incidence were monitored for 8 weeks. On the basis of tumor incidence in the indicated time, a Kaplan-Meier survival curve was generated using Log-rank test (Log-rank P = .03). “R” statistical software “survival” and “survminer” packages were used to analyze the tumor-free survival curve. (e) In vivo limiting dilution assay was performed using HCC1954 cells and treatment and sorting performed as described in (a). Sorted 100, 1000, 10,000 and 100,000 cells were mixed with Matrigel and injected subcutaneously in Nu/J mice (n = 5, Scale bar 5 mm). (f) Tumor initiating capacity was monitored over 8 weeks and CSC frequency was calculated using “R” statistical software “statmod” package. The frequency of tumor-initiating cells for 3 mice is graphed. Results are median +/− IQR (repeated measure ANOVA).

Article Snippet: Human ROR1 (AF2000, R&D System, USA), HER2 (MAB1129, R & D System, USA) and YAP1(MAB8094, R & D System, USA) antibodies were purchased from R & D System, USA.

Techniques: In Vivo, FACS, Injection, Generated, Over Expression, Software, Limiting Dilution Assay

Journal: EBioMedicine

Article Title: Antibody-drug conjugate T-DM1 treatment for HER2+ breast cancer induces ROR1 and confers resistance through activation of Hippo transcriptional coactivator YAP1

doi: 10.1016/j.ebiom.2019.04.061

Figure Lengend Snippet: YAP1 regulates T-DM1 treatment-induced ROR1 overexpression and CSC enrichment. (a) HCC1954 cells were transduced with full length YAP1 cDNA (PIN20YYAP1). Doxycycline (1 μg/ml) was added in the cell culture medium to induce the YAP1 expression. Western blot was performed using antibodies to YAP1, ROR1 and HER2. (b) Immunofluorescence staining for YAP1 (green; white arrow head) and ROR1 (red; white arrowhead) and DAPI (blue) confirming the transduction of YAP1 in the presence and absence of doxycycline (Scale bar: 50 μM). (c) YAP1 was knockdown by two independent YAP1 siRNA and Western Blot was performed to confirm the expression of YAP1 and ROR1 (red label box siRNA was used in our experiments). (d) HCC1954 cells were transfected with non-targeting and YAP1 specific siRNA for 48 h. Cells were then treated with T-DM1 for 48 h, followed by treatment with ROR1-shRNA for 48 h. Treated cells were trypsinized, cultured in the supplemented CSC medium, and assessed for sphere forming efficiency (black arrowhead) Bar graph represents the mean +/− SE, n = 3; run in triplicate; p = .05; p = .01; one-way ANOVA. [Scale bar: 100 μM]. (e) Immunoblots showing the expression of YAP1, ROR1 and HER2 after treatment of HCC1954 cells with 2 μg/mL verteporfin. (f) Quantification of sphere forming efficiency (black arrowhead) after verteporfin treatment at 2 μg/ml and vehicle (DMSO) in HCC1954 cells. Bar graph represents the mean +/− SE, n = 3; run in triplicate; p = .05; p = .01; Student's t -test; Scale bar: 100 μM). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Human ROR1 (AF2000, R&D System, USA), HER2 (MAB1129, R & D System, USA) and YAP1(MAB8094, R & D System, USA) antibodies were purchased from R & D System, USA.

Techniques: Over Expression, Transduction, Cell Culture, Expressing, Western Blot, Immunofluorescence, Staining, Knockdown, Transfection, shRNA

Journal: Life

Article Title: A Histological Analysis and Detection of Complement Regulatory Protein CD55 in SARS-CoV-2 Infected Lungs

doi: 10.3390/life14091058

Figure Lengend Snippet: Representative overlay immunofluorescence images of lung tissues derived from COVID–/ARDS–, COVID+/ARDS+, and COVID–/ARDS+ patients in 200× (upper row: ( A , C , E , G ). Scalebar: 100 µm) and 630× (lower row: ( B , D , F ). Scalebar: 20 µm) magnification. Red (Cy3) = CD55, blue (DAPI) = cell nuclei. The primary antibody against CD55 was omitted in the negative control. ( A ) The blood vessel inside the lung tissues has been indicated by the oval lining. The yellow arrow indicates a staining artifact. The white arrows in image ( B ) depict alveolar macrophages with positive CD55 staining. ( H ) Graphic representation of relative CD55 protein fluorescence intensity measured in [COVID–/ARDS–, n = 4], [COVID+/ARDS+, n = 5] and [COVID–/ARDS+, n = 3], lung tissues. Mean with standard deviation (SD). The COVID–/ARDS– group has been normalized to 100. Repeated Measures one-way ANOVA using Dunnett’s multiple comparisons (*). * = p ≤ 0.05.

Article Snippet: For antigen retrieval, the slides were treated in a pressure cooker (Rommelbacher ElektroHausgeräte GmbH, Dinkelsbühl, Germany) at 97 °C, pH 6, and steam pressure of 1 bar for 10 min. After the antigen retrieval procedure and cooling of the slides upto RT, the slides were first rinsed in PBS for 5 min before the primary antibody, CD55 (Antigen Affinity-purified Polyclonal Goat IgG, Catalog Number: AF2009, R&D systems, Minneapolis, MN, USA) [ ] diluted in blocking solution was pipetted onto the slides and incubated at 4 °C overnight.

Techniques: Immunofluorescence, Derivative Assay, Negative Control, Staining, Fluorescence, Standard Deviation

Journal: Life

Article Title: A Histological Analysis and Detection of Complement Regulatory Protein CD55 in SARS-CoV-2 Infected Lungs

doi: 10.3390/life14091058

Figure Lengend Snippet: ( A ) A representative overlay immunofluorescence image of COVID–/ARDS– lung tissue containing a vessel with circa 200 µm thick muscle layer (double-headed red arrow). Red (Cy3) = CD55, blue (DAPI) = cell nuclei. ( B ) Image A in the processing sequence of the LASX software analysis tool (Version 3.5.7.23225) containing blue as well as red channels, ( C ) single red channel extraction from image B, ( D ) a binary image derived from image C. Scalebar 100 µm.

Article Snippet: For antigen retrieval, the slides were treated in a pressure cooker (Rommelbacher ElektroHausgeräte GmbH, Dinkelsbühl, Germany) at 97 °C, pH 6, and steam pressure of 1 bar for 10 min. After the antigen retrieval procedure and cooling of the slides upto RT, the slides were first rinsed in PBS for 5 min before the primary antibody, CD55 (Antigen Affinity-purified Polyclonal Goat IgG, Catalog Number: AF2009, R&D systems, Minneapolis, MN, USA) [ ] diluted in blocking solution was pipetted onto the slides and incubated at 4 °C overnight.

Techniques: Immunofluorescence, Sequencing, Software, Extraction, Derivative Assay

Journal: Infection and Immunity

Article Title: Role of Src Kinases in Mobilization of Glycosylphosphatidylinositol-Anchored Decay-Accelerating Factor by Dr Fimbria-Positive Adhering Bacteria ^▿ †

doi: 10.1128/IAI.01052-10

Figure Lengend Snippet: Mobilization of hDAF around adhering Dr-positive E. coli requires activated Src kinases. (A) Immunoblot analysis of hDAF, activated Src kinases (Src-Y418P), Src kinases (Src), and actin in the Triton X-100-extracted fraction (E) and the Triton X-100-resistant fraction (R) isolated from HeLa cells infected with IH11128 strain (wt), Dr-positive (Dr+) or Dr-negative (Dr−) E. coli, or not infected (NI). (Left panel) the different fractions were analyzed by SDS-PAGE, followed by Western blotting with appropriate antibodies. (Right panel) Quantification of activated Src kinases (Src-Y418P). (B) Knockdown expression of hDAF by specific siRNA in HeLa cells. The HeLa cells were transfected or not transfected (NT) with a control or with a specific hDAF siRNA. hDAF expression was analyzed by Western blotting using anti-hDAF AF2009 antibody (left panel) and quantified (right panel). (C) Knockdown expression of hDAF results in a decrease in Dr-positive E. coli-induced activation of Src kinase. Western blotting (left panel) and quantification (right panel) of activated Src kinases (Src-Y418P). (D) The Src kinase inhibitor PP2 inhibited the Dr-positive E. coli-induced activation of Src kinase, whereas its inactive analog, PP3, did not. Western blotting (top panel) and quantification (lower panel) of activated Src kinases (Src-Y418P). Each immunoblot (A to D) is representative of three independent experiments. Blotting with antiactin pAb was used to confirm gel loading. Blots were quantified using Image J software. The value reported represents the mean ± SEM. *, P < 0.05 (Student's t test) versus the appropriate control cells; **, P < 0.01 (Student's t test) versus the appropriate control cells. (E) Immunofluorescence analysis of hDAF mobilization in infected HeLa cells treated with PP2 (+PP2) or PP3 (+PP3). hDAF (red) was labeled with monoclonal antibody 8D11, and the Dr fimbriae (green) were labeled with the J63 antibody. 3D analysis was performed on a cross-section of the immunofluorescence images (white square). (F) Immunofluorescence analysis of hDAF mobilization in HEK293 cells infected with Dr+ E. coli and treated with PP3 (+PP3) or PP2 (+PP2). hDAF (red) was labeled with polyclonal AF2009 antibody, and Dr+ E. coli bacteria were visualized by differential interference contrast (Dic). All micrographs are representative of three independent experiments.

Article Snippet: The antibodies used were goat anti-hDAF polyclonal antibody (pAb) (AF2009) from R&D systems; mouse monoclonal antibody (MAb) 8D11, directed against the CCP4 domain of hDAF (Department of Pathology, Washington University School of Medicine, St. Louis, MO); rabbit anti-phospho-Src Tyr416 pAb, which recognizes phospho-Tyr416 (mammals) or phospho-Tyr418 (human), and rabbit anti-Src pAb, purchased from Cell Signaling (Denvers, MA); mouse anti-c-Src MAb (Millipore); mouse anti-Yes MAb (BD Transduction Laboratories); mouse anti-Fyn MAb and mouse anti-Lyn MAb (Exbio); rabbit antiactin pAb (Sigma); antiphosphotyrosine pY20 MAb (BD Transduction Laboratories); anti-CEACAM pAb (Dako Cytomation); anti-CEACAM MAb D14HD11 (Genovac); and rabbit anti-Dr pAb (J63), directed against the DraE adhesin subunit of Dr fimbriae ( 22 ).

Techniques: Western Blot, Isolation, Infection, SDS Page, Knockdown, Expressing, Transfection, Control, Activation Assay, Software, Immunofluorescence, Labeling, Bacteria

Journal: Infection and Immunity

Article Title: Role of Src Kinases in Mobilization of Glycosylphosphatidylinositol-Anchored Decay-Accelerating Factor by Dr Fimbria-Positive Adhering Bacteria ^▿ †

doi: 10.1128/IAI.01052-10

Figure Lengend Snippet: Activated Src kinases are mobilized around adhering Dr-positive bacteria. (A) Indirect immunofluorescence analysis of the mobilization of hDAF and activated Src kinases (Src-Y418P) around adhering Dr-positive E. coli (Dr+). The endogenous activated Src kinases (Src-Y418P) were immunolabeled using a polyclonal antibody, phospho-Src Tyr416, which recognized phosphorylated Y418 in human Src kinases (green), and hDAF (red) was stained with the monoclonal antibody 8D11. The colocalization of Src-Y418P and hDAF is visualized in the merge images as a yellow fluorescence. (B) CHO-hDAF and CHO-hDAFΔCCP4 cells transiently transfected with expression vector encoding the Src-kinase-wt-GFP (Src-wt), the constitutively activated GFP-Src kinase (Y527F), or the myristoylation-defective GFP-Src kinase (G2A) were infected with Dr-positive E. coli. Dr fimbriae (red) were labeled using the J63 antibody. GFP-Src kinase recruitment was visualized as levels of gray in the square at the top right of each image. (C) HeLa cells were transiently transfected with an expression vector encoding the Src kinase-wt-GFP (Src-wt), the constitutively activated GFP-Src kinase (Y527F), or the myristoylation-defective GFP-Src kinase (G2A) (green). Transfected HeLa cells were infected with Dr-positive E. coli or E. coli expressing the mutated Dr adhesin subunit in position 54 (Dr+D54C). Dr fimbriae (red) were labeled using the J63 antibody. GFP-Src kinase recruitment was visualized as levels of gray in the square at the top right of each image. All the micrographs are representative of three experiments.

Article Snippet: The antibodies used were goat anti-hDAF polyclonal antibody (pAb) (AF2009) from R&D systems; mouse monoclonal antibody (MAb) 8D11, directed against the CCP4 domain of hDAF (Department of Pathology, Washington University School of Medicine, St. Louis, MO); rabbit anti-phospho-Src Tyr416 pAb, which recognizes phospho-Tyr416 (mammals) or phospho-Tyr418 (human), and rabbit anti-Src pAb, purchased from Cell Signaling (Denvers, MA); mouse anti-c-Src MAb (Millipore); mouse anti-Yes MAb (BD Transduction Laboratories); mouse anti-Fyn MAb and mouse anti-Lyn MAb (Exbio); rabbit antiactin pAb (Sigma); antiphosphotyrosine pY20 MAb (BD Transduction Laboratories); anti-CEACAM pAb (Dako Cytomation); anti-CEACAM MAb D14HD11 (Genovac); and rabbit anti-Dr pAb (J63), directed against the DraE adhesin subunit of Dr fimbriae ( 22 ).

Techniques: Bacteria, Immunofluorescence, Immunolabeling, Staining, Fluorescence, Transfection, Expressing, Plasmid Preparation, Infection, Labeling

Journal: Infection and Immunity

Article Title: Role of Src Kinases in Mobilization of Glycosylphosphatidylinositol-Anchored Decay-Accelerating Factor by Dr Fimbria-Positive Adhering Bacteria ^▿ †

doi: 10.1128/IAI.01052-10

Figure Lengend Snippet: Kinase c-Src is specifically involved in the mobilization of hDAF around Dr-positive E. coli (A1 to A4). Identification of Src kinases activated and recruited around bacteria. (Top panels) HeLa cells infected with the IH11128 strain (wt) or Dr-positive E. coli (Dr+) or not infected (NI). Fyn, Lyn, c-Src, and Yes were immunoprecipitated (IP) using the appropriate antibody as described in Materials and Methods. The proteins were analyzed by SDS-PAGE, followed by Western immunoblotting. c-Src, Yes, Fyn, and Lyn were detected using specific monoclonal antibodies, and their activated state (Y418P) was revealed using the polyclonal Src-Y416P antibody. Each immunoblot is representative of three independent experiments. Blots were quantified using Image J software. The value reported represents the mean ± SEM. *, P < 0.05 (Student's t test) versus the appropriate control cells. (Lower panels) To analyze the recruitment of Fyn, Lyn, c-Src, and Yes in the vicinity of hDAF, the HeLa cells infected or not infected (NI) with Dr+ E. coli and each of the Src kinases (green) were stained using specific monoclonal antibodies. hDAF (red) was stained using polyclonal antibody AF2009. Micrographs are representative of three independent experiments. (B, top panel) HeLa cells were transfected with siRNA control or with siRNAs specific for c-Src, Yes, Fyn, or Lyn. The expression of the kinases was analyzed by SDS-PAGE and Western immunoblotted using specific antibodies. Each immunoblot is representative of three independent experiments. Blotting with anti-actin pAb was used to confirm gel loading. (B, lower panel) Consequence of knockdown of expression of specific Src kinases on mobilization of hDAF around Dr+ E. coli. The involvement of each Src kinase in the mobilization of hDAF around Dr-positive E. coli was analyzed by immunofluorescence and confocal microscopy. HeLa cells were transfected with specific siRNA of c-Src, Yes, Fyn, or Lyn and infected with Dr+ E. coli. hDAF (red) was labeled with polyclonal antibody AF2009, and the Dr fimbriae (green) were labeled with the J63 antibody. Micrographs are representative of three independent experiments.

Article Snippet: The antibodies used were goat anti-hDAF polyclonal antibody (pAb) (AF2009) from R&D systems; mouse monoclonal antibody (MAb) 8D11, directed against the CCP4 domain of hDAF (Department of Pathology, Washington University School of Medicine, St. Louis, MO); rabbit anti-phospho-Src Tyr416 pAb, which recognizes phospho-Tyr416 (mammals) or phospho-Tyr418 (human), and rabbit anti-Src pAb, purchased from Cell Signaling (Denvers, MA); mouse anti-c-Src MAb (Millipore); mouse anti-Yes MAb (BD Transduction Laboratories); mouse anti-Fyn MAb and mouse anti-Lyn MAb (Exbio); rabbit antiactin pAb (Sigma); antiphosphotyrosine pY20 MAb (BD Transduction Laboratories); anti-CEACAM pAb (Dako Cytomation); anti-CEACAM MAb D14HD11 (Genovac); and rabbit anti-Dr pAb (J63), directed against the DraE adhesin subunit of Dr fimbriae ( 22 ).

Techniques: Bacteria, Infection, Immunoprecipitation, SDS Page, Western Blot, Bioprocessing, Software, Control, Staining, Transfection, Expressing, Knockdown, Immunofluorescence, Confocal Microscopy, Labeling

Journal: Molecular cell

Article Title: Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene

doi: 10.1016/j.molcel.2017.11.004

Figure Lengend Snippet: (A) Schematic flowchart of the integrative transcriptional profiling (RNA-seq) of metastatic melanoma cell lines and NHM. (B) Log2FPKM (normalized fragments per kilobase per million mapped reads) of 824 JQ1-sensitive genes, shared between both melanoma lines. Bars represent 10–90 percentile. (C) Log2FPKM of 613 genes overexpressed in both melanoma lines over NHM2. Bars represent 10–90 percentile. (D) Heatmap of 78 genes that are the intersection of the 824 JQ1-sensitive (significantly downregulated) after 6 and/or 24 hr as in (B), and 613 genes overexpressed as in (C). Each row represents the transformed raw Z score of FPKM of an individual gene. (E) Loss-of-function proliferation mini-screen of 9 selected genes (of 78 in D) (marked in red). Data are represented as mean ± SEM. (F) AMIGO2 qRT-PCR analysis of NHM1, 501MEL, and SKmel147 cells treated with DMSO, JQ1 (JQ1[+]), or I-BET762 for 6 and 24 hr. Data are represented as mean ± SEM mRNA levels normalized to GAPDH. (G) AMIGO2 qRT-PCR analysis of SKmel147, SKmel239, A375, and SKmel2 cells treated with JQ1 (JQ1[+]) for 6 and 24 hr. Data are represented as mean ± SEM and relative to DMSO. mRNA levels are normalized to GAPDH. See also Figure S1 and primary data in Table S1.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies BRD2 Bethyl Laboratories A302-583A BRD4 Bethyl Laboratories A301-985A H3K4me1 Abcam ab8895 H3K4me3 Abcam ab1012 H3K27ac Abcam ab177178 H3K27me3 EMD 07-449 MED1 Bethyl Laboratories A300-793A macroH2A2 Bernstein Laboratory, ISMMS in house macroH2A1 Abcam ab37264 CTCF EMD 07-729 FOSL2 Santa Cruz SC-604 TEAD4 Santa Cruz SC-101184 HSP90 Santa Cruz SC-13119 GAPDH Santa Cruz SC-32233 GFP Roche 11814460001 LAMIN B1 Santa Cruz SC-6217 ALCAM Abcam ab109215 L1CAM Abcam ab20148 NRP2 Santa Cruz SC-13117 NRP1 Abcam ab81321 TUBULIN Sigma T9026 FOXM1 Cell Signaling Technology 5436 Casp3 Cell Signaling Technology 9662 PARP Cell Signaling Technology 9542 AMIGO2 (for IHC) Abnova H00347902-B01P Lot# F8271 RRID:AB_1672842 AMIGO2 (for IF and WB) R&D systems AF2080 PTK7 (N-terminal) (for WB) R&D systems AF4499 PTK7 (C-terminal) (for IF and WB) Cell Signaling 11926 Secondary rabbit anti goat (for AMIGO2 detection on WB) Sigma A5420 HRP-conjugated ACTIN Sigma A3854 Biological Samples Melanoma and normal skin tissue microarray Biomax ME208 Nevus tissue microarray IMCG, NYU MT032808 Patient derived short term cultures Osman Laboratory, NYU de Miera et al., 2012 NHM (this paper) Bernstein Laboratory, ISMMS NHM1-5 Chemicals, Peptides, and Recombinant Proteins GFP Trap-A beads Chromotek gta-10 BET inhibitor JQ1 Bradner Laboratory; Agreement #A09608 N/A BET inhibitor I-BET762 Apex Bio 501014747 IGEPAL Sigma I-8896 Micrococcal Nuclease Affymetrix 70196Y Dynabeads Protein A Immunoprécipitation kit Invitrogen 10006D Magna ChIP Protein A+G Magnetic beads Millipore 16-663 DSG Pierce (ThermoFisher) 20593 Critical Commercial Assays Click- Click-IT EdU Pacific Blue Flow Cytometry Assay Kit ThermoFisher C10636 Caspase Activity assay MBL Int.

Techniques: RNA Sequencing, Transformation Assay, Quantitative RT-PCR

Journal: Molecular cell

Article Title: Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene

doi: 10.1016/j.molcel.2017.11.004

Figure Lengend Snippet: (A) AMIGO2 expression levels detected by qRT-PCR in NHM and a panel of metastatic melanoma cell lines harboring distinct melanoma mutations. Data are normalized to GAPDH. (B and C) AMIGO2 mRNA detected by RNA-seq (B), and protein levels of NHM and patient-derived melanoma short-term cultures (STCs) (C). Data in (B) are represented in FPKM. (D) AMIGO2 mRNA levels in primary and metastatic melanoma tissue samples from TCGA (RESM-normalized values) and Xu et al., 2008 (GSE8401) (array-based expression). RNA-seq was performed by expectation-maximization. (E) AMIGO2 mRNA from TCGA samples grouped by mutational status. Data are represented in RESM. (F) Immunohistochemistry (IHC) for AMIGO2 in normal skin (n = 9, NHM are marked with arrows), nevi (n = 8), primary melanoma (n = 30), and metastatic melanoma (n = 30). Representative micrographs at 20× and 60× are shown; blow ups of representative cells are shown below. Scale bar, 100 μm. (G) Quantified IHC scores of (F) (arbitrary units). (H) Percent distribution of AMIGO2 cases by tissue type. All error bars represent mean ± SD. See also primary data in Table S2.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies BRD2 Bethyl Laboratories A302-583A BRD4 Bethyl Laboratories A301-985A H3K4me1 Abcam ab8895 H3K4me3 Abcam ab1012 H3K27ac Abcam ab177178 H3K27me3 EMD 07-449 MED1 Bethyl Laboratories A300-793A macroH2A2 Bernstein Laboratory, ISMMS in house macroH2A1 Abcam ab37264 CTCF EMD 07-729 FOSL2 Santa Cruz SC-604 TEAD4 Santa Cruz SC-101184 HSP90 Santa Cruz SC-13119 GAPDH Santa Cruz SC-32233 GFP Roche 11814460001 LAMIN B1 Santa Cruz SC-6217 ALCAM Abcam ab109215 L1CAM Abcam ab20148 NRP2 Santa Cruz SC-13117 NRP1 Abcam ab81321 TUBULIN Sigma T9026 FOXM1 Cell Signaling Technology 5436 Casp3 Cell Signaling Technology 9662 PARP Cell Signaling Technology 9542 AMIGO2 (for IHC) Abnova H00347902-B01P Lot# F8271 RRID:AB_1672842 AMIGO2 (for IF and WB) R&D systems AF2080 PTK7 (N-terminal) (for WB) R&D systems AF4499 PTK7 (C-terminal) (for IF and WB) Cell Signaling 11926 Secondary rabbit anti goat (for AMIGO2 detection on WB) Sigma A5420 HRP-conjugated ACTIN Sigma A3854 Biological Samples Melanoma and normal skin tissue microarray Biomax ME208 Nevus tissue microarray IMCG, NYU MT032808 Patient derived short term cultures Osman Laboratory, NYU de Miera et al., 2012 NHM (this paper) Bernstein Laboratory, ISMMS NHM1-5 Chemicals, Peptides, and Recombinant Proteins GFP Trap-A beads Chromotek gta-10 BET inhibitor JQ1 Bradner Laboratory; Agreement #A09608 N/A BET inhibitor I-BET762 Apex Bio 501014747 IGEPAL Sigma I-8896 Micrococcal Nuclease Affymetrix 70196Y Dynabeads Protein A Immunoprécipitation kit Invitrogen 10006D Magna ChIP Protein A+G Magnetic beads Millipore 16-663 DSG Pierce (ThermoFisher) 20593 Critical Commercial Assays Click- Click-IT EdU Pacific Blue Flow Cytometry Assay Kit ThermoFisher C10636 Caspase Activity assay MBL Int.

Techniques: Expressing, Quantitative RT-PCR, RNA Sequencing, Derivative Assay, Immunohistochemistry

Journal: Molecular cell

Article Title: Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene

doi: 10.1016/j.molcel.2017.11.004

Figure Lengend Snippet: (A) Relative growth curves of 501MEL and SKmel147 cells stably transduced with non-targeting scrambled control shRNA (shSCR) and two AMIGO2 shRNAs (shA2 #1 and #2). Values are normalized to seeding control (n = 3). (B) Colony formation assay 14 days post-infection as in (A); representative images are shown on the right. (C) Percent of cells in G1, S, and G2 for same cells as in (A), 48 hr post-transduction. (D) Percent of Annexin V-positive 501MEL and SKmel147 cells 5 days post-transduction. (E) Caspase-3 activity in whole-cell extracts of same cells as in (D). (F) PARP and cleaved CASP3 immunoblots of whole-cell lysates from 501MEL and SKmel147 cells. Actin was used as a loading control. (G) Average tumor volume in mice injected with shSCR or shA2 #2-transduced SKmel147 cells. (H) Average tumor mass and representative images of resected tumors taken 18 days post-injection (scale bar, 1 cm). Representative experiment is shown (n = 3). All values and error bars represent mean ± SD or ± SEM. See also Figures S2 and S3.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies BRD2 Bethyl Laboratories A302-583A BRD4 Bethyl Laboratories A301-985A H3K4me1 Abcam ab8895 H3K4me3 Abcam ab1012 H3K27ac Abcam ab177178 H3K27me3 EMD 07-449 MED1 Bethyl Laboratories A300-793A macroH2A2 Bernstein Laboratory, ISMMS in house macroH2A1 Abcam ab37264 CTCF EMD 07-729 FOSL2 Santa Cruz SC-604 TEAD4 Santa Cruz SC-101184 HSP90 Santa Cruz SC-13119 GAPDH Santa Cruz SC-32233 GFP Roche 11814460001 LAMIN B1 Santa Cruz SC-6217 ALCAM Abcam ab109215 L1CAM Abcam ab20148 NRP2 Santa Cruz SC-13117 NRP1 Abcam ab81321 TUBULIN Sigma T9026 FOXM1 Cell Signaling Technology 5436 Casp3 Cell Signaling Technology 9662 PARP Cell Signaling Technology 9542 AMIGO2 (for IHC) Abnova H00347902-B01P Lot# F8271 RRID:AB_1672842 AMIGO2 (for IF and WB) R&D systems AF2080 PTK7 (N-terminal) (for WB) R&D systems AF4499 PTK7 (C-terminal) (for IF and WB) Cell Signaling 11926 Secondary rabbit anti goat (for AMIGO2 detection on WB) Sigma A5420 HRP-conjugated ACTIN Sigma A3854 Biological Samples Melanoma and normal skin tissue microarray Biomax ME208 Nevus tissue microarray IMCG, NYU MT032808 Patient derived short term cultures Osman Laboratory, NYU de Miera et al., 2012 NHM (this paper) Bernstein Laboratory, ISMMS NHM1-5 Chemicals, Peptides, and Recombinant Proteins GFP Trap-A beads Chromotek gta-10 BET inhibitor JQ1 Bradner Laboratory; Agreement #A09608 N/A BET inhibitor I-BET762 Apex Bio 501014747 IGEPAL Sigma I-8896 Micrococcal Nuclease Affymetrix 70196Y Dynabeads Protein A Immunoprécipitation kit Invitrogen 10006D Magna ChIP Protein A+G Magnetic beads Millipore 16-663 DSG Pierce (ThermoFisher) 20593 Critical Commercial Assays Click- Click-IT EdU Pacific Blue Flow Cytometry Assay Kit ThermoFisher C10636 Caspase Activity assay MBL Int.

Techniques: Stable Transfection, Transduction, Control, shRNA, Colony Assay, Infection, Activity Assay, Western Blot, Injection

Journal: Molecular cell

Article Title: Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene

doi: 10.1016/j.molcel.2017.11.004

Figure Lengend Snippet: (A) Immunofluorescence of SKmel147 cells stably expressing AMIGO2-GFP (green), stained with AMIGO2 antibody (red) and Hoechst 33342 (blue). Scale bar, 20 μm. (B) Functional annotation of AMIGO2-interacting proteins detected by GFP pull-down followed by MS in SKmel147 cells stably expressing AMIGO2-GFP (see Table S4). (C) PTK7 and GFP immunoblots following GFP pull-down from 501MEL cells stably expressing AMIGO2-GFP. (D) Full-length PTK7 (FL-PTK7), C-terminal fragments CTF1- and CTF2-PTK7, and FOXM1 immunoblots of 501MEL cells 72 hr post-infection with shSCR or shPTK7 (shP7 #1 and #2). Actin was used as a loading control. (E) Relative growth curves of 501MEL (left) and SKmel147 (right) cells stably transduced with shSCR or shPTK7 (shP7 #1 and #2). Values are normalized to seeding control (n = 3). (F) Percent Annexin V-positive cells 6 days post-transduction for same cells as in (E). (G) FL-PTK7, CTF-PTK7, and FOXM1 immunoblots of 501MEL cells 48 hr post-transduction with shSCR or shAMIGO2 (shA2 #1 and #2). Actin was used as a loading control. (H) FL-PTK7, CTF-PTK7, FOXM1, and AMIGO2 immunoblots of 501MEL cells untreated or treated with JQ1 (JQ1[+]) for 72 hr. Tubulin was used as a loading control. (I) CTF2-PTK7 immunoblot of nuclear lysates from same cell as in (G) (left). Lamin B1 was used as loading control. Signal quantification (right), normalized to Lamin B1, relative to shSCR (n = 3). All values and error bars represent mean ± SD or ± SEM. See also Figures S3 and S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies BRD2 Bethyl Laboratories A302-583A BRD4 Bethyl Laboratories A301-985A H3K4me1 Abcam ab8895 H3K4me3 Abcam ab1012 H3K27ac Abcam ab177178 H3K27me3 EMD 07-449 MED1 Bethyl Laboratories A300-793A macroH2A2 Bernstein Laboratory, ISMMS in house macroH2A1 Abcam ab37264 CTCF EMD 07-729 FOSL2 Santa Cruz SC-604 TEAD4 Santa Cruz SC-101184 HSP90 Santa Cruz SC-13119 GAPDH Santa Cruz SC-32233 GFP Roche 11814460001 LAMIN B1 Santa Cruz SC-6217 ALCAM Abcam ab109215 L1CAM Abcam ab20148 NRP2 Santa Cruz SC-13117 NRP1 Abcam ab81321 TUBULIN Sigma T9026 FOXM1 Cell Signaling Technology 5436 Casp3 Cell Signaling Technology 9662 PARP Cell Signaling Technology 9542 AMIGO2 (for IHC) Abnova H00347902-B01P Lot# F8271 RRID:AB_1672842 AMIGO2 (for IF and WB) R&D systems AF2080 PTK7 (N-terminal) (for WB) R&D systems AF4499 PTK7 (C-terminal) (for IF and WB) Cell Signaling 11926 Secondary rabbit anti goat (for AMIGO2 detection on WB) Sigma A5420 HRP-conjugated ACTIN Sigma A3854 Biological Samples Melanoma and normal skin tissue microarray Biomax ME208 Nevus tissue microarray IMCG, NYU MT032808 Patient derived short term cultures Osman Laboratory, NYU de Miera et al., 2012 NHM (this paper) Bernstein Laboratory, ISMMS NHM1-5 Chemicals, Peptides, and Recombinant Proteins GFP Trap-A beads Chromotek gta-10 BET inhibitor JQ1 Bradner Laboratory; Agreement #A09608 N/A BET inhibitor I-BET762 Apex Bio 501014747 IGEPAL Sigma I-8896 Micrococcal Nuclease Affymetrix 70196Y Dynabeads Protein A Immunoprécipitation kit Invitrogen 10006D Magna ChIP Protein A+G Magnetic beads Millipore 16-663 DSG Pierce (ThermoFisher) 20593 Critical Commercial Assays Click- Click-IT EdU Pacific Blue Flow Cytometry Assay Kit ThermoFisher C10636 Caspase Activity assay MBL Int.

Techniques: Immunofluorescence, Stable Transfection, Expressing, Staining, Functional Assay, Western Blot, Infection, Control, Transduction

Journal: Molecular cell

Article Title: Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene

doi: 10.1016/j.molcel.2017.11.004

Figure Lengend Snippet: (A) Capture of the UCSC genome browser (GRCh37/hg19) showing 800 kb region on human chromosome 12 (y axis = reads per kilobase per million reads). Ensembl genes (blue) and AMIGO2 SEs (red) are shown at the top. Putative TAD/LAD domain is shown at the bottom. (B) Zoom-in of the AMIGO2 locus showing 40 kb. Five TEs, E1–E5 (red), within the AMIGO2 SE are shown (bottom). (C) Approximately 60 kb region around the AMIGO2 SE. ChIP-seq was performed for H3K27ac for seven additional melanoma cell lines (black) and four melanoma STCs (green) (Kaufman et al., 2016; Verfaillie et al., 2015). AMIGO2 SE is shown at the bottom (red). (D) Pearson correlation of number of H3K27ac reads within the AMIGO2 SE and AMIGO2 Log2FPKM (Table S1). (E) AMIGO2 qRT-PCR in SKmel147 cells following depletion of BRD2, BRD3, or BRD4. Data are represented as mean ± SEM and normalized to PPIA. (F) AMIGO2 locus as in (B). SKmel147 RNA-seq JQ1(−)/JQ1(+) 6 hr is shown at top. (G) AMIGO2 locus as in (B). (H) FOSL2 (left), TEAD4 (right), and AMIGO2 immunoblots of SKmel147 cells 96 hr post-transduction with shSCR or shFOSL2 (shF2 #1 and #2), and shSCR or shTEAD4+TEAD1 (shT4 #1+shT1 #1 and shT4 #2+shT1 #1). HSP90 and GAPDH were used as loading controls. See also Figure S6.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies BRD2 Bethyl Laboratories A302-583A BRD4 Bethyl Laboratories A301-985A H3K4me1 Abcam ab8895 H3K4me3 Abcam ab1012 H3K27ac Abcam ab177178 H3K27me3 EMD 07-449 MED1 Bethyl Laboratories A300-793A macroH2A2 Bernstein Laboratory, ISMMS in house macroH2A1 Abcam ab37264 CTCF EMD 07-729 FOSL2 Santa Cruz SC-604 TEAD4 Santa Cruz SC-101184 HSP90 Santa Cruz SC-13119 GAPDH Santa Cruz SC-32233 GFP Roche 11814460001 LAMIN B1 Santa Cruz SC-6217 ALCAM Abcam ab109215 L1CAM Abcam ab20148 NRP2 Santa Cruz SC-13117 NRP1 Abcam ab81321 TUBULIN Sigma T9026 FOXM1 Cell Signaling Technology 5436 Casp3 Cell Signaling Technology 9662 PARP Cell Signaling Technology 9542 AMIGO2 (for IHC) Abnova H00347902-B01P Lot# F8271 RRID:AB_1672842 AMIGO2 (for IF and WB) R&D systems AF2080 PTK7 (N-terminal) (for WB) R&D systems AF4499 PTK7 (C-terminal) (for IF and WB) Cell Signaling 11926 Secondary rabbit anti goat (for AMIGO2 detection on WB) Sigma A5420 HRP-conjugated ACTIN Sigma A3854 Biological Samples Melanoma and normal skin tissue microarray Biomax ME208 Nevus tissue microarray IMCG, NYU MT032808 Patient derived short term cultures Osman Laboratory, NYU de Miera et al., 2012 NHM (this paper) Bernstein Laboratory, ISMMS NHM1-5 Chemicals, Peptides, and Recombinant Proteins GFP Trap-A beads Chromotek gta-10 BET inhibitor JQ1 Bradner Laboratory; Agreement #A09608 N/A BET inhibitor I-BET762 Apex Bio 501014747 IGEPAL Sigma I-8896 Micrococcal Nuclease Affymetrix 70196Y Dynabeads Protein A Immunoprécipitation kit Invitrogen 10006D Magna ChIP Protein A+G Magnetic beads Millipore 16-663 DSG Pierce (ThermoFisher) 20593 Critical Commercial Assays Click- Click-IT EdU Pacific Blue Flow Cytometry Assay Kit ThermoFisher C10636 Caspase Activity assay MBL Int.

Techniques: ChIP-sequencing, Quantitative RT-PCR, RNA Sequencing, Western Blot, Transduction

Journal: Molecular cell

Article Title: Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene

doi: 10.1016/j.molcel.2017.11.004

Figure Lengend Snippet: (A) Schematic of AMIGO2 SE showing enhancers E1–E5 (B), deleted sequences for CRISPR-Cas9 editing (C), and DNA segments used for E3 lucif-erase assay (D). (B) Luciferase reporter assays performed in SKmel147 cells. Negative (N1) and positive (P1) controls are shown (Figure S7A). Luciferase signal was normalized to renilla transfection control. Box-plots represent Tukey boxplots with outliers omitted. (C) CRISPR-Cas9-mediated editing of constituent enhancers E2–E3 (clones C1–C3) and E4–E5 (clones C4–C6). AMIGO2 mRNA levels by qRT-PCR (top; data represented as mean and normalized to GAPDH) and AMIGO2 immunoblots of edited SKmel147 cells (bottom) with GAPDH as loading control are shown. Non-edited clone was used as negative control. (D) Luciferase reporter assays performed in SKmel147 cells for AMIGO2 enhancer elements E3A–C and E3A deleted for TF motifs (E3A-Del) (A). Boxplots represent Tukey boxplots with outliers omitted. (E) Model of BET-regulated AMIGO2 expression in melanoma and upon BETi. NHMs express low levels of AMIGO2 due to lack of active SEs. Upon transformation, increased levels of BRD2 and BRD4 and alterations in TFs FOSL2 and TEAD4/1 trigger global epigenomic remodeling involving acquisition of SEs and activation of key melanoma survival genes, such as AMIGO2. Upon BETi treatment, BET proteins are displaced from SEs and promoters, inducing gene silencing, such as the case of AMIGO2. Since melanoma is dependent on BETs to maintain its proliferation and survival, BETi produces cytostatic and/or cytotoxic effects on melanoma cells. (F) AMIGO2 interacts with PTK7 and prevents nuclear accumulation of CTF2-PTK7. Upon BETi or AMIGO2 KD (knockdown), CTF2-PTK7 nuclear levels increase, possibly leading to changes in gene expression. See also Figure S7 and primary data in Table S7.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies BRD2 Bethyl Laboratories A302-583A BRD4 Bethyl Laboratories A301-985A H3K4me1 Abcam ab8895 H3K4me3 Abcam ab1012 H3K27ac Abcam ab177178 H3K27me3 EMD 07-449 MED1 Bethyl Laboratories A300-793A macroH2A2 Bernstein Laboratory, ISMMS in house macroH2A1 Abcam ab37264 CTCF EMD 07-729 FOSL2 Santa Cruz SC-604 TEAD4 Santa Cruz SC-101184 HSP90 Santa Cruz SC-13119 GAPDH Santa Cruz SC-32233 GFP Roche 11814460001 LAMIN B1 Santa Cruz SC-6217 ALCAM Abcam ab109215 L1CAM Abcam ab20148 NRP2 Santa Cruz SC-13117 NRP1 Abcam ab81321 TUBULIN Sigma T9026 FOXM1 Cell Signaling Technology 5436 Casp3 Cell Signaling Technology 9662 PARP Cell Signaling Technology 9542 AMIGO2 (for IHC) Abnova H00347902-B01P Lot# F8271 RRID:AB_1672842 AMIGO2 (for IF and WB) R&D systems AF2080 PTK7 (N-terminal) (for WB) R&D systems AF4499 PTK7 (C-terminal) (for IF and WB) Cell Signaling 11926 Secondary rabbit anti goat (for AMIGO2 detection on WB) Sigma A5420 HRP-conjugated ACTIN Sigma A3854 Biological Samples Melanoma and normal skin tissue microarray Biomax ME208 Nevus tissue microarray IMCG, NYU MT032808 Patient derived short term cultures Osman Laboratory, NYU de Miera et al., 2012 NHM (this paper) Bernstein Laboratory, ISMMS NHM1-5 Chemicals, Peptides, and Recombinant Proteins GFP Trap-A beads Chromotek gta-10 BET inhibitor JQ1 Bradner Laboratory; Agreement #A09608 N/A BET inhibitor I-BET762 Apex Bio 501014747 IGEPAL Sigma I-8896 Micrococcal Nuclease Affymetrix 70196Y Dynabeads Protein A Immunoprécipitation kit Invitrogen 10006D Magna ChIP Protein A+G Magnetic beads Millipore 16-663 DSG Pierce (ThermoFisher) 20593 Critical Commercial Assays Click- Click-IT EdU Pacific Blue Flow Cytometry Assay Kit ThermoFisher C10636 Caspase Activity assay MBL Int.

Techniques: CRISPR, Luciferase, Transfection, Control, Clone Assay, Quantitative RT-PCR, Western Blot, Negative Control, Expressing, Transformation Assay, Activation Assay, Knockdown, Gene Expression

Journal: Molecular cell

Article Title: Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene

doi: 10.1016/j.molcel.2017.11.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies BRD2 Bethyl Laboratories A302-583A BRD4 Bethyl Laboratories A301-985A H3K4me1 Abcam ab8895 H3K4me3 Abcam ab1012 H3K27ac Abcam ab177178 H3K27me3 EMD 07-449 MED1 Bethyl Laboratories A300-793A macroH2A2 Bernstein Laboratory, ISMMS in house macroH2A1 Abcam ab37264 CTCF EMD 07-729 FOSL2 Santa Cruz SC-604 TEAD4 Santa Cruz SC-101184 HSP90 Santa Cruz SC-13119 GAPDH Santa Cruz SC-32233 GFP Roche 11814460001 LAMIN B1 Santa Cruz SC-6217 ALCAM Abcam ab109215 L1CAM Abcam ab20148 NRP2 Santa Cruz SC-13117 NRP1 Abcam ab81321 TUBULIN Sigma T9026 FOXM1 Cell Signaling Technology 5436 Casp3 Cell Signaling Technology 9662 PARP Cell Signaling Technology 9542 AMIGO2 (for IHC) Abnova H00347902-B01P Lot# F8271 RRID:AB_1672842 AMIGO2 (for IF and WB) R&D systems AF2080 PTK7 (N-terminal) (for WB) R&D systems AF4499 PTK7 (C-terminal) (for IF and WB) Cell Signaling 11926 Secondary rabbit anti goat (for AMIGO2 detection on WB) Sigma A5420 HRP-conjugated ACTIN Sigma A3854 Biological Samples Melanoma and normal skin tissue microarray Biomax ME208 Nevus tissue microarray IMCG, NYU MT032808 Patient derived short term cultures Osman Laboratory, NYU de Miera et al., 2012 NHM (this paper) Bernstein Laboratory, ISMMS NHM1-5 Chemicals, Peptides, and Recombinant Proteins GFP Trap-A beads Chromotek gta-10 BET inhibitor JQ1 Bradner Laboratory; Agreement #A09608 N/A BET inhibitor I-BET762 Apex Bio 501014747 IGEPAL Sigma I-8896 Micrococcal Nuclease Affymetrix 70196Y Dynabeads Protein A Immunoprécipitation kit Invitrogen 10006D Magna ChIP Protein A+G Magnetic beads Millipore 16-663 DSG Pierce (ThermoFisher) 20593 Critical Commercial Assays Click- Click-IT EdU Pacific Blue Flow Cytometry Assay Kit ThermoFisher C10636 Caspase Activity assay MBL Int.

Techniques: Microarray, Derivative Assay, Recombinant, Magnetic Beads, Flow Cytometry, Caspase Activity Assay, DNA Library Preparation, Blocking Assay, Extraction, TA Cloning, Sequencing, RNA Sequencing, Western Blot, Mass Spectrometry, Expressing, Software