Journal: The Journal of Experimental Medicine

Article Title: Suppression of CCL2 angiocrine function by adrenomedullin promotes tumor growth

doi: 10.1084/jem.20211628

Figure Lengend Snippet: The G s -coupled adrenomedullin receptor CALCRL is required for endothelial and tumor cell proliferation in vitro and in vivo. (a and b) HUVECs were transfected with control siRNA or siRNA directed against GNAS or the indicated GPCR RNAs, and HUVEC proliferation in the absence and presence of GFP-MeWo tumor cells was determined by staining for Ki67. Shown are the ranked average ratios of Ki67-positive HUVECs co-cultured with MeWo cells and Ki67-positive HUVECs grown alone (a) and representative immunofluorescence staining (b; n = 4 independently evaluated dates). (c–f) HUVECs transfected with control siRNA or siRNA directed against CALCRL (e and f) or pretreated without or with 1 µM of the adrenomedullin receptor antagonist AM22-52 (c and d) were cultured with GFP-MeWo for the indicated time periods. Thereafter, Ki67-positive HUVECs were determined by staining with anti-Ki67 antibody (red) with DAPI (blue; c, n = 3 independent experiments), the number of GFP-expressing tumor cells was determined by either immunofluorescence (d and e, n = 3 independent experiments), or the expression level of GFP was determined by Western blot analysis (f). The bar diagrams show the statistical evaluation or represent the relative densitometric values based on Fiji software (f; n = 3 independent experiments). (g) B16-F10 melanoma cells were injected subcutaneously in control or EC-Calcrl-KO mice, and tumor growth was determined ( n = 7 mice for each genotype). (h) E0771 breast cancer cells were injected into mammary fat pad of control or EC-Calcrl-KO mice, and tumor growth was determined ( n = 5 mice for each genotype). (i) Immunohistochemistry of B16-F10 tumors grown in control or EC-Calcrl-KO mice. Sections were analyzed for markers of endothelial cells (PECAM1; green), perivascular cells (α-SMA; red) or proliferating cells (Ki67; red), and were stained with DAPI (blue). Bar diagrams show the statistical evaluation ( n = 6 mice for each genotype). Bar length: 100 μm (b–e and i). Data represent mean values ± SEM; **, P ≤ 0.01; ***, P ≤ 0.001 (two-way ANOVA and Bonferroni’s post hoc test [f–h], two-tailed Student’s t test [c–e and i]).

Article Snippet: MeWo cells were from CLS, and E0771 were from tebu-bio.

Techniques: In Vitro, In Vivo, Transfection, Control, Staining, Cell Culture, Immunofluorescence, Expressing, Western Blot, Software, Injection, Immunohistochemistry, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Suppression of CCL2 angiocrine function by adrenomedullin promotes tumor growth

doi: 10.1084/jem.20211628

Figure Lengend Snippet: Tumor cell–derived adrenomedullin promotes endothelial and tumor cell proliferation in vitro. (a) HUVECs transfected with control siRNA or siRNA directed against GNAS or ADM were cultured without (basal) or with 10 nM adrenomedullin for 24 h and were then stained with an anti-Ki67 antibody. The bar diagram shows the statistical evaluation. (b) GFP-MeWo cells were transfected with control siRNA or siRNA directed against CALCRL or ADM and were cultured without (basal) or with 2 nM adrenomedullin for 24 h. Thereafter, the number of GFP-expressing tumor cells was determined. The bar diagram shows the statistical evaluation (a and b; n = 2 independent experiments, two evaluated areas per experiments). (c and d) HUVECs were transfected with control siRNA or siRNA directed against ADM and were cultured with GFP-MeWo for the indicated time periods. Endothelial proliferation was determined by staining with anti-Ki67 antibody (c), or the expression level of GFP was determined by Western blot analysis as an indicator of tumor cell growth (d). (e and f) HUVECs were cultured with GFP-MeWo transfected with control siRNA or siRNA directed against ADM for the indicated time periods. Endothelial proliferation was determined by staining with anti-Ki67 antibody (e), or the tumor cell proliferation was determined by quantifying expression level of GFP by Western blot analysis (f). The bar diagrams show the statistical evaluation (c and e; n = 3 independent experiments) or represent the relative densitometric values based on Fiji software (d and f; n = 3 independent experiments). Bar length: 100 μm (a–c and e). Data represent mean values ± SEM; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (one-way ANOVA and Tukey’s post hoc test [a and b], two-way ANOVA and Bonferroni’s post hoc test [d and f], and two-tailed Students t test [c and e]).

Article Snippet: MeWo cells were from CLS, and E0771 were from tebu-bio.

Techniques: Derivative Assay, In Vitro, Transfection, Control, Cell Culture, Staining, Expressing, Western Blot, Software, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Suppression of CCL2 angiocrine function by adrenomedullin promotes tumor growth

doi: 10.1084/jem.20211628

Figure Lengend Snippet: Role of adrenomedullin in proliferation of endothelial and tumor cells. (a and b) HUVECs transfected with control siRNA or siRNA directed against ADM were cultured with GFP-MDA 231 for the indicated time periods, then either stained with anti-Ki67 antibody (red) and with DAPI (blue; a) or lysed to analyze GFP expression as an indicator of tumor cell number (b; n = 3 independent experiments). (c) HUVECs were co-cultured for 24 h with GFP-expressing MDA-MB-231 cells transfected with control siRNA or siRNA directed against ADM. Endothelial proliferation was determined by analyzing immunofluorescence obtained with the anti-Ki67 antibody. Bar diagrams show the statistical evaluation ( n = 3 independent experiments). (d) HUVECs transfected with control siRNA or siRNA directed against Adm were cultured alone or together with MeWo cells transfected with control siRNA or siRNA directed against adrenomedullin. Thereafter, adrenomedullin levels were determined by Western blot analysis. (e) MLECs were transfected with control siRNA or siRNA directed against Gnas or Calcrl, then cultured without (basal) or with conditioned medium (CM) of B16-F10 cells transfected with control shRNA (control B16) or shRNA directed against Adm (Adm-KD-B16). Endothelial proliferation was determined by staining with anti-Ki67 antibody ( n = 2 independent experiments). (f) Expression of ADM in control B16-F10 melanoma cells and in Adm-KD-B16 was determined by Western blotting. (g) Whole mount retinae of control and EC-Adm-KO mice at P7 stained with Isolectin B4. The bar diagram shows the quantification of vasculature parameters ( n = 4 for each genotype). (h) Control B16-F10 or Adm-KD-B16 cells were cultured for 72 h, and cells were counted. The bar diagram shows the statistical evaluation ( n = 2 independent experiments). Bar length: 100 μm (a, c, e, and g); and in panel h: 50 μm. Data represent mean values ± SD; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (one-way ANOVA and Tukey’s post hoc test [e], two-way ANOVA and Bonferroni’s post hoc test [b and h] and two-tailed Student’s t test [a, c, and g]).

Article Snippet: MeWo cells were from CLS, and E0771 were from tebu-bio.

Techniques: Transfection, Control, Cell Culture, Staining, Expressing, Immunofluorescence, Western Blot, shRNA, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Suppression of CCL2 angiocrine function by adrenomedullin promotes tumor growth

doi: 10.1084/jem.20211628

Figure Lengend Snippet: Role of CCL2 in the interaction of tumor cells and endothelial cells. (a) HUVECs (ECs) transfected with control siRNA (cont) or siRNA directed against GNAS were cultured with GFP-MeWo (TCs) for 48 h (co-culture, right) or cultured alone, lysed, and then mixed with GFP-MeWo lysates (mix, left). The expression level of ADM and Gα s were analyzed by Western blotting. The bar diagram represents the relative densitometric values of the band recognized by the anti-ADM antibody based on Fiji software ( n = 3 independent experiments). (b) Western blot analysis of mammary gland lysates dissected from MMTV-PyMT mice crossed with control or EC-Gα s -KO mice showing ADM and CCL2 protein levels as well as tubulin levels as loading control. (c) Heat map showing top 50 differentially expressed genes (based on false discovery rate) that significantly have increased (red) or decreased (blue) expression following GNAS silencing (kd) in HUVECs compared to scrambled (sc) siRNA control. (d) HUVECs were incubated without or with 10 nM adrenomedullin for 3 h, and the expression of the indicated genes was determined by qRT-PCR analysis ( n = 3). (e and f) B16-F10 cells were stably transduced with control shRNA or shRNA directed against Ccr2 (e) or GFP-MeWo cells were transfected with control siRNA or siRNA directed against CCR2 (f), and were then incubated without or with 50 ng/ml CCL2 for 3 h. Thereafter, the adrenomedullin gene expression was determined by qRT-PCR analysis ( n = 4 independent experiments). (g) GFP-MeWo cells were incubated without or with 100 ng/ml pertussis toxin (PTX) overnight then incubated with 50 ng/ml CCL2 for 3 h. Thereafter, ADM expression was determined by qRT-PCR analysis. (h) HUVECs were transfected with scrambled control siRNA or siRNA directed against GNAS and were then cultured for 48 h together with GFP-MDA-MB-231 cells transfected with control siRNA or siRNA directed against CCR2 . Thereafter, the number of GFP-expressing tumor cells was determined by immunofluorescence. The bar diagram shows the statistical evaluation ( n = 2 independent experiments). Bar length: 100 μm (h). Data represent mean values ± SEM; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (two-way ANOVA and Bonferroni’s post hoc test [a and e–h] and two-tailed Student’s t test [b]).

Article Snippet: MeWo cells were from CLS, and E0771 were from tebu-bio.

Techniques: Transfection, Control, Cell Culture, Co-Culture Assay, Expressing, Western Blot, Software, Incubation, Quantitative RT-PCR, Stable Transfection, Transduction, shRNA, Gene Expression, Immunofluorescence, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Suppression of CCL2 angiocrine function by adrenomedullin promotes tumor growth

doi: 10.1084/jem.20211628

Figure Lengend Snippet: Endothelial CCL2 regulates adrenomedullin expression and release in tumor cells in vitro. (a–d) HUVECs were transfected with scrambled control siRNA or siRNA directed against GNAS and/or CCL2 and were cultured with GFP-MeWo for the indicated time periods. Endothelial cell proliferation was determined by staining for Ki67 (a), and the number of GFP-expressing tumor cells was determined by immunofluorescence (b) or determined as the protein level of GFP by Western blot analysis (c). In addition, expression of ADM was analyzed by immunoblotting (d). The bar diagrams show the statistical evaluation (a and b; n = 3 independent experiments) or the relative densitometric values based on Fiji software (c and d; n = 3 independent experiments). (e) HUVECs were transfected with control siRNA or siRNA directed against GNAS and cells were co-cultured for 24 h with MeWo cells transfected with control siRNA or siRNA directed against CCR2 . Thereafter, the expression level of ADM was determined by Western blot analysis. The bar diagrams represent the relative densitometric values of the band recognized by the anti-ADM antibody based on Fiji software ( n = 3 independent experiments). Bar length: 100 μm (a and b). Data represent mean values ± SEM; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (two-way ANOVA and Bonferroni’s post hoc test [c and e], and one-way ANOVA and Tukey’s post hoc test [a, b, and d]).

Article Snippet: MeWo cells were from CLS, and E0771 were from tebu-bio.

Techniques: Expressing, In Vitro, Transfection, Control, Cell Culture, Staining, Immunofluorescence, Western Blot, Software

Journal: Frontiers in Microbiology

Article Title: Adenovirus Isolated From a Cat Is Related to Human Adenovirus 1

doi: 10.3389/fmicb.2019.01430

Figure Lengend Snippet: Replication of the feline adenovirus in different human ( Homo sapiens ) cell lines.

Article Snippet: , Melanoma , MEWO (ATCC HTB-65) , ∗ CPE-/+ , ∗ IFA-/+.

Techniques: Transformation Assay

Journal: International Journal of Molecular Sciences

Article Title: Human Microfibrillar-Associated Protein 4 (MFAP4) Gene Promoter: A TATA-Less Promoter That Is Regulated by Retinol and Coenzyme Q10 in Human Fibroblast Cells

doi: 10.3390/ijms21218392

Figure Lengend Snippet: The expression of pEGFP- pMFAP4 plasmids in mouse fibroblast NIH/3T3 cells, human melanoma MeWo cells, and mouse melanoma B16-F10 cells. ( A ) Fluorescence images. Scale bar = 100 μm. ( B ) Relative fluorescence intensity. Data are presented as means ± SE.

Article Snippet: Detroit 551 cells (BCRC 60118, human normal fibroblast cells), MeWo cells (BCRC 60540, human melanoma cells), NIH/3T3 cells (BCRC 60008, mouse fibroblast cells), and B16-F10 cells (BCRC 60031, mouse melanoma cells) were all obtained from the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan, ROC).

Techniques: Expressing, Fluorescence

Journal: Bioengineering & Translational Medicine

Article Title: Polymeric nanoparticle‐based delivery of TRAIL DNA for cancer‐specific killing

doi: 10.1002/btm2.10019

Figure Lengend Snippet: Cancer cell types, sources, and culture conditions

Article Snippet: MeWo , Melanoma (metastatic to lymph node) , Human , Dr. Martin Pomper, Department of Radiology, Johns Hopkins University , High‐glucose DMEM with pyruvate and l ‐glutamine + 10% FBS, 1% anti‐anti.

Techniques: Cell Culture

Journal: Bioengineering & Translational Medicine

Article Title: Polymeric nanoparticle‐based delivery of TRAIL DNA for cancer‐specific killing

doi: 10.1002/btm2.10019

Figure Lengend Snippet: Leading nanoparticle formulations for cancer cell transfection

Article Snippet: MeWo , Melanoma (metastatic to lymph node) , Human , Dr. Martin Pomper, Department of Radiology, Johns Hopkins University , High‐glucose DMEM with pyruvate and l ‐glutamine + 10% FBS, 1% anti‐anti.

Techniques: Polymer, Transfection