Journal: Neuro-Oncology

Article Title: Dissecting inherent intratumor heterogeneity in patient-derived glioblastoma culture models

doi: 10.1093/neuonc/now253

Figure Lengend Snippet: Floating cells display enhanced mesenchymal properties. (A) MGG6 NS and FC mRNA were analyzed by qRT-PCR for different mesenchymal markers including CD44, vimentin, YKL-40, N-cad, and MMP2. (B) Cell lysates from different cultures were analyzed by western blotting for pSTAT3, STAT3, CD44, C/EBPβ, and β-actin. (C) FACS analysis showing proportion of CD44 positive cells in FC and their corresponding NS from different GSC cultures. (D) MGG29 NS were fractionated into CD44high and CD44low subpopulation by FACS and predominant cells were established and labeled with GFP and mCherry, respectively. Left: fluorescence analysis of a mixture of these 2 subpopulations cultured in serum; middle: FC collected and analyzed for both GFP and mCherry; right: flow cytometry of CD44high cells in both GFP and mCherry populations. (E) NS and FC were differentiated using StemPro Chondrogenesis Differentiation Kit and stained with fast green and safranin O (positive, #; negative, +). (F) NS and FC were cultured as a monolayer and their ability to invade/migrate was evaluated using scratch healing assay. Bright field micrographs are showing for different groups. Scale bar, 50 μm.

Article Snippet: CD44 expression was measured in 500000 cells dissociated with 2 mM EDTA and incubated in 80 μL PBS/0.1% bovine serum albumin at 4°C for 30 min in the dark with mouse monoclonal anti-human CD44 antibody conjugated to fluorescein isothiocyanate (FITC, 1:10; Miltenyi Biotec) or respective mouse immunoglobulin G1 control in the presence of FcR blocking reagent (1:5).

Techniques: Quantitative RT-PCR, Western Blot, Labeling, Fluorescence, Cell Culture, Flow Cytometry, Staining

Journal: Nature Communications

Article Title: N 1 -methyladenosine methylation in tRNA drives liver tumourigenesis by regulating cholesterol metabolism

doi: 10.1038/s41467-021-26718-6

Figure Lengend Snippet: a Overview of immunohistochemistry analysis of endogenous m 1 A levels in a panel of 191 clinically defined human HCC samples. Samples are arranged by grades and then sorted by microvascular invasion (MVI) (gray: “no”; red: “yes”). Paired samples are connected with gray lines. b Representative immunohistochemistry images of endogenous m 1 A levels (brown) in grade III HCC tumor tissue. Scale bar, 100 μm. n = 4 independent experiments. c LC-MS/MS quantification of m 1 A/A and m 6 A/A ratios in total RNA purified from HCC samples. Data are means ± SD. n = 4 biologically independent experiments. Exact P values from left to right: 0.052, 0.00082, 0.00043, 0.026, 0.011, 0.043, 0.0075, 0.13, 0.084, 0.055. d Kaplan–Meier plots of overall survival based on m 1 A levels in the tissue microarray assay (TMA) cohort. The median expression value is used as a cut-off. P values for Kaplan–Meier curves were calculated using a log-rank test. e LC-MS/MS quantification of m 1 A levels in total RNA, large RNA (≥200 nucleotide, nt, mostly composed of rRNA) and small RNA (<200 nt, mostly composed of tRNA) purified from HCC primary CSCs and non-CSCs, presented as percentage of unmodified A. Data are means ± SD. n = 5 biologically independent experiments. Exact P values from left to right: 0.0052, 0.0063, 0.0065, 0.067, 0.11, 0.075, 0.0017, 0.0089, and 0.0084. f Global m 1 A levels were detected in liver CSCs (C) and non-CSCs (N) from HCC primary tumors using dot blot assay. MB (methyl blue) was used as loading controls. n = 4 biologically independent samples. g Representative immunofluorescence staining of m 1 A and CD13 in HCC primary tumor cells and respective oncosphere cells (S). DAPI, 4,6-diamidino-2-phenylindole. Scale bar, 10 μm. n = 4 biologically independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001, and NS, not significant ( P > 0.05) by two-tailed Student’s t -test.

Article Snippet: PE-anti-human IgG (12-4998-82), fluorescein isothiocyanate (FITC)-anti-human CD13 (clone no. WM-15, 11-0138-42), FITC-anti-human IgG (31529) were purchased from eBioscience (San Diego, USA).

Techniques: Immunohistochemistry, Liquid Chromatography with Mass Spectroscopy, Purification, Microarray, Expressing, Dot Blot, Immunofluorescence, Staining, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Insight into immune profile associated with vitiligo onset and anti-tumoral response in melanoma patients receiving anti-PD-1 immunotherapy

doi: 10.3389/fimmu.2023.1197630

Figure Lengend Snippet: IL-17-expressing cells are differently modulated in the skin lesion and in the circulation of melanoma patients developing vitiligo during anti-PD-1 therapy. (A) Representative images of the immunohistochemistry (panels a to d) and immunofluorescence (panels e, f) analysis performed using antibodies directed against CD56 (panels a–c), IL-17 (panels d–f) and TCR alpha V 7.2 (panels g–i) on normal skin biopsies and on biopsies from patients affected by vitiligo without melanoma (vitiligo) and who developed vitiligo during immunotherapy (vitiligo DI). Magnification 200x. (B) Quantitative analyses of immunohistochemical staining. The mean value + standard error of the mean (SEM) of the cell count obtained for five different fields is shown, ***p<0.05 as assessed by Kruskal-Wallis test.

Article Snippet: For immunofluorescence, 4-µm sections were dewaxed, rehydrated, and after quenching endogenous peroxidase with 3% bovine serum albumin in 1x phosphate buffer, achieving antigen retrieval and blocking non-specific binding sites, sections were incubated with the monoclonal antibody anti-TCR V alpha 7.2 FITC conjugated (cod.130-123-929, Miltenyi Biotech; 1:50) and incubated for 1 hour at 37°C in a humid chamber.

Techniques: Expressing, Immunohistochemistry, Immunofluorescence, Staining, Cell Counting

Journal: Frontiers in Immunology

Article Title: Insight into immune profile associated with vitiligo onset and anti-tumoral response in melanoma patients receiving anti-PD-1 immunotherapy

doi: 10.3389/fimmu.2023.1197630

Figure Lengend Snippet: Expression of the immune cell subsets in the primary melanoma and metastases of patients developing or not vitiligo during anti-PD-1 therapy. (A) Representative images of immunohistochemistry (panels a to i) and immunofluorescence (l–n) obtained using anti-CD25 (a–c), anti-CD56 (d–f), anti-IL-17A (g–i), and anti-TCR alpha V 7.2 (j–l) in primary melanoma and metastasis from patients who developed (vitiligo) or not vitiligo (not vitiligo) during therapy. A–I 100x magnification, panel L-N 200X magnification. Magnification 200x. (B) Quantitative analyses of immunohistochemical staining. The mean value + standard error of the mean (SEM) of the cell count obtained for five different fields is shown, *p<0.05 as assessed by Kruskal-Wallis test.

Article Snippet: For immunofluorescence, 4-µm sections were dewaxed, rehydrated, and after quenching endogenous peroxidase with 3% bovine serum albumin in 1x phosphate buffer, achieving antigen retrieval and blocking non-specific binding sites, sections were incubated with the monoclonal antibody anti-TCR V alpha 7.2 FITC conjugated (cod.130-123-929, Miltenyi Biotech; 1:50) and incubated for 1 hour at 37°C in a humid chamber.

Techniques: Expressing, Immunohistochemistry, Immunofluorescence, Staining, Cell Counting

Journal: PLoS ONE

Article Title: The Use of a New CellCollector to Isolate Circulating Tumor Cells from the Blood of Patients with Different Stages of Prostate Cancer and Clinical Outcomes - A Proof-of-Concept Study

doi: 10.1371/journal.pone.0158354

Figure Lengend Snippet: The CTCs were identified and enumerated via a) positive nuclear staining (Hoechst), b) positive cytokeratin staining, and c) negative CD45 staining. d) Overlay of all images showing size and morphological characteristics.

Article Snippet: Subsequently, the cells were blocked with 3% bovine serum albumin (BSA) (PAA) in PBS for 1 h. Primary antibodies, including anti-pan-cytokeratin-fluorescein isothiocyanate (FITC) (CK8, CK18 and CK19, Abcam) and anti-CD45- allophycocyanin (APC, Invitrogen), were added for 1 h. The wire was then rinsed 3 times with 3 ml of PBS, and the nuclei were counterstained with Hoechst 33258 (Invitrogen).

Techniques: Staining

Journal: British Journal of Cancer

Article Title: Newly characterised ex vivo colospheres as a three-dimensional colon cancer cell model of tumour aggressiveness

doi: 10.1038/sj.bjc.6605173

Figure Lengend Snippet: Colospheres derived from colon carcinoma patient tissue. ( A ) Photomicrograph of a representative colosphere generated on tissue culture plastic; ( B ) HES staining; ( C ) anti-Ki67 staining. Magnification: × 40. ( D–F ) Confocal immunofluorescence of colospheres. ( D ) TOPRO-3 staining; ( E ) EpCAM staining; ( F ) overlay of ( D ) and ( E ). Scale bar: 50 μ m.

Article Snippet: Disaggregated colospheres, spheroids and xenografts were assessed using an Epics XL cytometre (Coulter, Villepinte, France) with the following antibodies: anti-human CD133/2 (clone 293C3) phycoerythrin, anti-human EpCAM (clone HEA-125) fluorescein isothiocyanate (Miltenyi-Biotec SAS, Paris, France) and anti-human CD44-FITC (clone G44-26; BD Biosciences), at appropriate dilutions.

Techniques: Derivative Assay, Generated, Staining, Immunofluorescence

Journal: Nature Communications

Article Title: Apolipoprotein A-IV binds αIIbβ3 integrin and inhibits thrombosis

doi: 10.1038/s41467-018-05806-0

Figure Lengend Snippet: ApoA-IV competes with Fg binding to αIIbβ3 integrin. a Effective 2D affinity of apoA-IV-αIIbβ3 versus Fg-αIIbβ3 within contact area. The adhesion frequency ( P a ) depends on the densities of receptor and ligand, therefore the weaker ligand apoA-IV required four-fold higher density (40 µm −2 ) than Fg (8 µm −2 ) yet only generated approximately one-fold higher P a . Affinity to αIIbβ3 for apoA-IV (2.13 × 10 −4 μm 4 ) is 43% of that for Fg (4.99 × 10 −4 μm 4 ) (Grey, Fg-αIIbβ3; dark grey, apoA-IV-αIIbβ3). b Dose-dependent inhibition of Fg-αIIbβ3 by recombinant apoA-IV in Biomembrane Force Probe assay. c , d Dose-dependent inhibition of Fg-αIIbβ3 by recombinant human apoA-IV in ELISA ( c ) and a flow cytometry assay ( d ). The inhibitory effect of varying apoA-IV concentrations (0–10 µM) on the interaction between immobilized αIIbβ3 (1 µg) and Fg (1 µM) ( c , left). The concentration–response curves were fit to the half maximal inhibitory model (methods) to determine the IC50 for apoA-IV. The apoA-IV IC50 for the inhibition of Fg-αIIbβ3 was found to be 1.32 ± 0.42 µM. Control experiments confirm that the ELISA signal observed is indeed from specific binding interactions and not from non-specific or off-target binding ( c , right). FITC-conjugated human Fg binding to the human gel-filtered platelets that were incubated with increasing doses of human recombinant apoA-IV or BSA control was analyzed by flow cytometry ( d ). Platelets were activated by TRAP (250 µM). P a adhesion frequency, A c contact area, m r αIIbβ3 density, m l Fg or apoA-IV density, k off 2D off-rate, A c k on 2D effective on-rate, rH-ApoA-IV recombinant human apoA-IV. n = 4. Unpaired, two-tailed Student’s t -test. Mean ± SEM. NS not significant, * P < 0.05, ** P < 0.01 and *** P < 0.001

Article Snippet: Goat anti-human apoA-IV antibody (N-20, sc-19036; C-20, sc-19038), mouse anti-human apoA-IV antibody (G-8; sc-374543), mouse IgG kappa binding protein conjugated to HRP (sc-516102), fluorescein isothiocyanate (FITC)-conjugated donkey anti-goat IgG, and FITC-conjugated human Fg were purchased from Santa Cruz (Dallas, Texas, USA).

Techniques: Binding Assay, Generated, Inhibition, Recombinant, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Concentration Assay, Control, Incubation, Two Tailed Test