Journal: Frontiers in Veterinary Science

Article Title: Detection of bimodal survivin expressions in canine cancer types by flow cytometry compared to immunohistochemistry

doi: 10.3389/fvets.2025.1552415

Figure Lengend Snippet: Analysis of survivin expression sites by IHC and WB. (A) Images of HE staining and survivin expression via IHC in canine melanoma cell lines (CMM2, CMeC, and LMeC) are shown. (B) Images of HE staining and survivin expression via IHC in murine cell lines (p815, CT26, and B16F10) are shown. (C) The cytosol and total survivin expression patterns via WB are indicated. (D) The expression levels of total survivin (blue bar) and cytosolic survivin (orange bar) corrected on the basis of β-actin expression are shown via density of plot analysis via ImageJ software.

Article Snippet: A total of six cell lines were used: canine malignant melanoma lines [CMM2, CMeC2, LMeC; provided by Dr. Takayuki Nakagawa, Department of Veterinary Surgery, University of Tokyo; ( )], the murine malignant melanoma line B16F10, the murine mast cell tumor line p815, and the murine colon cancer line CT26 (distributed for a fee by the JCRB Cell Bank).

Techniques: Expressing, Staining, Software

Journal:

Article Title: Vascular endothelial growth factor overproduced by tumour cells acts predominantly as a potent angiogenic factor contributing to malignant progression

doi: 10.1046/j.1365-2613.1999.00122.x

Figure Lengend Snippet: (a) VEGF and (b) bFGF productivity of cancer cells transfected with VEGF cDNA. ▪ parental cells; □ transfected clones. The levels of VEGF and bFGF contained in conditioned medium and cell extract, respectively, were determined by sandwich ELISA. Sample values were plotted against a recombinant VEGF or bFGF standard curve (mean ± SE, n = 3). ND: not detectable. QG90, RPMI4788, MCF-7 and their transfected clones were cultured in RPMI1640 containing 10% FCS (normal). Hormones-stripped FCS with (E2+) or without (E2−) 10−8 M 17 β-oestradiol was used for MCF-7 and its transfected clone, M2–24-G10.

Article Snippet: The human colon cancer cell line, RPMI4788 ( Moore & Koike 1964 ; Naomoto et al . 1987 ), was a generous gift from Dr Akio Hizuta (First Department of Surgery, Okayama University Medical School, Okayama, Japan).

Techniques: Transfection, Clone Assay, Sandwich ELISA, Recombinant, Cell Culture

Journal:

Article Title: Vascular endothelial growth factor overproduced by tumour cells acts predominantly as a potent angiogenic factor contributing to malignant progression

doi: 10.1046/j.1365-2613.1999.00122.x

Figure Lengend Snippet: HUVE cell growth stimulatory activities in conditioned media of VEGF-transfected clones of (a) ○ QG90; □ Q1-1-C1; • Q1-24-E6; ▪ Q1-24-F6; ▴ Q1-24-H2, (b) ○ RPMI4788; • S0A12; ▪ S2G2 and (c) ○ MCF-7; • M2-24-D10; ▪ M2-24-E11; □ M2-24-G10 cells. HUVE cells were cultured in the presence of various concentrations of conditioned medium from each clone, indicated in Figure 1. After 4 days, HUVE cell growth was determined by MTT assay. The HUVE cell growth induced by 2.5 ng/ml of human recombinant VEGF165 was used as a positive control (♦). Results are presented as the percentage of the control without any growth factor. Each point indicates the mean of duplicate determinations.

Article Snippet: The human colon cancer cell line, RPMI4788 ( Moore & Koike 1964 ; Naomoto et al . 1987 ), was a generous gift from Dr Akio Hizuta (First Department of Surgery, Okayama University Medical School, Okayama, Japan).

Techniques: Transfection, Clone Assay, Cell Culture, MTT Assay, Recombinant, Positive Control, Control

Journal:

Article Title: Vascular endothelial growth factor overproduced by tumour cells acts predominantly as a potent angiogenic factor contributing to malignant progression

doi: 10.1046/j.1365-2613.1999.00122.x

Figure Lengend Snippet: Growth curves of VEGF-transfected clones of (a) ○ QG90; □ Q1-1-C1; • Q1-24-E6; ▪ Q1-24-F6; ▴ Q1-24-H2, (b) ○ RPMI4788; • S0A12; ▪ S2G2 and (c) ○ MCF-7; • M2-24-D10; ▪ M2-24-E11; □ M2-24-G10 cells. The number of cells was counted with a Coulter Counter (mean ± SE, n = 3).

Article Snippet: The human colon cancer cell line, RPMI4788 ( Moore & Koike 1964 ; Naomoto et al . 1987 ), was a generous gift from Dr Akio Hizuta (First Department of Surgery, Okayama University Medical School, Okayama, Japan).

Techniques: Transfection, Clone Assay

Journal:

Article Title: Vascular endothelial growth factor overproduced by tumour cells acts predominantly as a potent angiogenic factor contributing to malignant progression

doi: 10.1046/j.1365-2613.1999.00122.x

Figure Lengend Snippet: Tumorigenic potentials of transfected clones of QG90, RPMI4788 and MCF-7 cells

Article Snippet: The human colon cancer cell line, RPMI4788 ( Moore & Koike 1964 ; Naomoto et al . 1987 ), was a generous gift from Dr Akio Hizuta (First Department of Surgery, Okayama University Medical School, Okayama, Japan).

Techniques: Transfection, Clone Assay

Journal:

Article Title: Vascular endothelial growth factor overproduced by tumour cells acts predominantly as a potent angiogenic factor contributing to malignant progression

doi: 10.1046/j.1365-2613.1999.00122.x

Figure Lengend Snippet: In vivo angiogenic activity of VEGF-transfected clones of (a) QG90 and (b) RPMI4788 cells. A Millipore chamber containing each clone or HBSS was implanted s.c. into a dorsal air sac of a BALB/c mouse (three to six mice/group). After 4 days, the area of new capillarization induced by each clone was determined with an image analyser. Results (mean ± SE) are presented as percentages of the mean area of the control group treated with a HBSS-containing chamber. * P < 0.01 vs. the control group; ns, not significant (by Student's t-test).

Article Snippet: The human colon cancer cell line, RPMI4788 ( Moore & Koike 1964 ; Naomoto et al . 1987 ), was a generous gift from Dr Akio Hizuta (First Department of Surgery, Okayama University Medical School, Okayama, Japan).

Techniques: In Vivo, Activity Assay, Transfection, Clone Assay, Control

Journal:

Article Title: Vascular endothelial growth factor overproduced by tumour cells acts predominantly as a potent angiogenic factor contributing to malignant progression

doi: 10.1046/j.1365-2613.1999.00122.x

Figure Lengend Snippet: Hematoxylin and eosin-stained sections of tumours 32 days after s.c. injection of (a) RPMI4788 cells and the transfected clones, (b) S0A12 and (c) S2G2 cells into nude mice. RPMI4788 cells yielded small, poorly vascularlized tumours (a), whereas both clones yielded well-vascularlized ones (b, c). Arrow heads indicate blood vessels. Scale bar = 400μm.

Article Snippet: The human colon cancer cell line, RPMI4788 ( Moore & Koike 1964 ; Naomoto et al . 1987 ), was a generous gift from Dr Akio Hizuta (First Department of Surgery, Okayama University Medical School, Okayama, Japan).

Techniques: Staining, Injection, Transfection, Clone Assay

Journal: Nature Communications

Article Title: Tumor evolution selectively inactivates the core microRNA machinery for immune evasion

doi: 10.1038/s41467-021-27331-3

Figure Lengend Snippet: a Schematic overview of clonal mutation profiling of MC38 cell line-derived tumors grown in host mice harboring different levels of T cell immunity. b Heatmap showing allele frequency (AF) of hotspot mutations enriched (AF > 0.1) in at least two different tumors from the immunocompetent group ( n = 10 mice for immunodeficient, n = 13 for immunocompetent, and n = 8 for immunotherapy group). A total of 59 mutations in 53 genes were categorized to PD-1–dependent and –independent groups as indicated. Tumor-infiltrating lymphocytes were calculated by mMCP counter. See also Supplementary Fig. . Source data are provided as a source data file.

Article Snippet: MC38 cell line was provided by WuXi AppTec and cultured using DMEM (Gibco) with 10% fetal bovine serum (FBS), respectively.

Techniques: Mutagenesis, Derivative Assay

Journal: Nature Communications

Article Title: Tumor evolution selectively inactivates the core microRNA machinery for immune evasion

doi: 10.1038/s41467-021-27331-3

Figure Lengend Snippet: a Schematic overview of in vivo CRISPR screen to validate candidates from immune-selected mutations. b Tumor growth curves of MC38 tumors in nude mice ( n = 8), and WT mice treated with rat IgG2a and IgG2b isotype ( n = 8), PD-1 antibody ( n = 10), PD-L1 antibody ( n = 10), or CD4 and CD8 antibodies ( n = 7). Data are represented as mean ± s.e.m., **** P < 0.0001, significance was determined using two-way analysis of variance (ANOVA). c – e Distribution histograms of log Fold-change (FC) for all 10 sgRNAs targeting Cd274 ( c ), Pdcd1 ( d ) or Ankrd52 ( e ) as indicated in red lines, overlaid on gray gradient depicting the overall distribution (Cut-off: |FC | > 1.5, P < 0.05 for enrichment or depletion, analyzed by edgeR). f , Volcano plot for selected top guides for Cd274 and Ankrd52 (Cut-off: |FC | > 1.5, P < 0.05, analyzed by MAGeCK). g In vivo competition assay with equal number mixture of MC38 cells infected with sgRNA for non-targeting control (NT) or Ankrd52 ( An ) in WT mice ( n = 5) and nude mice ( n = 5). Data are represented as mean ± s.e.m., *** P = 0.0004, **** P < 0.0001, significance was determined using two-tailed unpaired Student’s t -test to compare sg Ankrd52 vs sgNT #2. h , i , Flow cytometry analysis of CD4 + ( h ) and CD8 + ( i ) T cell populations from NT and Ankrd52 knockout ( An KO) tumors ( n = 5 per group). Data are representative of two independent experiments and represented as mean ± s.e.m., significance was determined using two-tailed unpaired Student’s t -test. See also Supplementary Fig. – . Source data are provided as a source data file.

Article Snippet: MC38 cell line was provided by WuXi AppTec and cultured using DMEM (Gibco) with 10% fetal bovine serum (FBS), respectively.

Techniques: In Vivo, CRISPR, Competitive Binding Assay, Infection, Control, Two Tailed Test, Flow Cytometry, Knock-Out

Journal: Nature Communications

Article Title: Tumor evolution selectively inactivates the core microRNA machinery for immune evasion

doi: 10.1038/s41467-021-27331-3

Figure Lengend Snippet: a Schematic overview of CRISPR screen using co-culture of OT-I T cell with MC38-OVA cells. b Cell viability of MC38-OVA cells co-cultured with OT-I T cells during 2.5 days ( n = 3 per group per timepoint). Data are represented as mean ± s.d., **** P < 0.0001, significance is determined using multiple two-tailed Student’s t -test. c – e Distribution histograms of log 2 FC values for all ten sgRNAs targeting Jak1 ( c ), B2m ( d ), or Ankrd52 ( e ) (Cut-off: FC > 1.4, P < 0.05 for enrichment, analyzed by edgeR). f Killing of MC38-OVA cells with indicated sgRNAs by OT-I T cells at indicated ratio in co-culture. Data are representative of three independent experiments and represented as mean ± s.e.m., ** P < 0.01, *** P < 0.001, significance was determined using multiple two-tailed Student’s t -test (sgNT #1 vs sg Ankrd52 #1, P = 3.72×10 −3 for 1:3, P = 1.74×10 −3 for 1:5; sgNT #2 vs sg Ankrd52 #1, P = 3.64×10 −3 for 1:3, P = 1.67×10 −3 for 1:5; sgNT #1 vs sg Ankrd52 #2, P = 2.28 × 10 −4 for 1:3, P = 4.43 × 10 −3 for 1:5; sgNT #2 vs sg Ankrd52 #2, P = 2.7 × 10 −4 for 1:3, P = 4.57 × 10 −3 for 1:5). See also Supplementary Figs. and . Source data are provided as a source data file.

Article Snippet: MC38 cell line was provided by WuXi AppTec and cultured using DMEM (Gibco) with 10% fetal bovine serum (FBS), respectively.

Techniques: CRISPR, Co-Culture Assay, Cell Culture, Two Tailed Test

Journal: Nature Communications

Article Title: Tumor evolution selectively inactivates the core microRNA machinery for immune evasion

doi: 10.1038/s41467-021-27331-3

Figure Lengend Snippet: a Hallmark gene sets enriched for commonly down-regulated genes in Ankrd52 -null (both An KO1 and An KO2, 1.5-fold-change cut-off, P < 0.05, analyzed by edgeR) MC38 cells compared to control cells after IFNγ treatment. b , c Enrichment of genes associated with IFNγ response in Ankrd52 -null cells exposed to IFNγ. d Heatmap showing down-regulated IFNγ responsive gene expression in Ankrd52 -null cells by RNA-seq analysis ( n = 2 per group per condition). e , f Cxcl9 ( e ) and Cxcl10 ( f ) mRNA level in control and ANKRD52 -null MC38 cells treated with IFNγ ( n = 3 per group). Data are representative of two independent experiments and represented as mean ± s.e.m., significance was determined using two-tailed unpaired Student’s t -test. g Abundance of IFNγ signaling proteins in control and Ankrd52 -null MC38 cells treated with IFNγ. Data are representative of five independent experiments. h Abundance of membrane MHC-I expression in control and Ankrd52 -null MC38 cells after treatment with IFNγ. MFI (mean fluorescence intensity) of H2-K b was normalized by the responding group without IFNγ treatment ( n = 3 per group per condition). Data are representative of four independent experiments and represented as mean ± s.d., significance was determined using two-tailed unpaired Student’s t -test. i Presentation of OVA-derived peptide (SIINFEKL) in OVA-treated control and Ankrd52 -null MC38 cells. MFI of SIINFEKL-H2K b was normalized by the responding group without IFNγ treatment ( n = 3 per group per condition). Data are representative of three independent experiments and represented as mean ± s.d., significance was determined using two-tailed unpaired Student’s t -test. See also Supplementary Figs. and . Source data are provided as a source data file.

Article Snippet: MC38 cell line was provided by WuXi AppTec and cultured using DMEM (Gibco) with 10% fetal bovine serum (FBS), respectively.

Techniques: Control, Gene Expression, RNA Sequencing, Two Tailed Test, Membrane, Expressing, Fluorescence, Derivative Assay

Journal: Nature Communications

Article Title: Tumor evolution selectively inactivates the core microRNA machinery for immune evasion

doi: 10.1038/s41467-021-27331-3

Figure Lengend Snippet: a Localization of clinical hotspot ANKRD52 mutations from combined COSMIC and OncoWuXi database. b Protein abundance of p-STAT1 and p-STAT3 in Ankrd52 -null MC38 cells expressing WT or indicated mutant ANKRD52 after IFNγ treatment. Data are representative of two independent experiments. c Membrane MHC-I level in Ankrd52 -null MC38 cells expressing WT or indicated mutant ANKRD52 after IFNγ treatment. MFI of H2-K b was normalized by the responding group without IFNγ treatment ( n = 5 per group per condition). Data are representative of two independent experiments and represented as mean ± s.d., significance was determined using two-tailed unpaired Student’s t -test. d Killing of OVA-treated Ankrd52 -null MC38 cells expressing WT or mutant ANKRD52 by OT-I T cells ( n = 3 per group per condition). Data are representative of two independent experiments and represented as mean ± s.d., significance was determined using two-tailed unpaired Student’s t -test. e Bar plot showing the percentage of patients with mutations in ANKRD52 across cancer patients receiving ICB therapies (anti-CTLA-4 or anti-PD-1) reported by Van Allen et al., Science 2015 and Riaz et al., Cell 2017. The non-response group is composed of SD and PD. f Bar plot showing the percentage of patients with mutations in ANKRD52 across multiple cancer types. Source data are provided as a source data file.

Article Snippet: MC38 cell line was provided by WuXi AppTec and cultured using DMEM (Gibco) with 10% fetal bovine serum (FBS), respectively.

Techniques: Quantitative Proteomics, Expressing, Mutagenesis, Membrane, Two Tailed Test

Journal: Nature Communications

Article Title: Tumor evolution selectively inactivates the core microRNA machinery for immune evasion

doi: 10.1038/s41467-021-27331-3

Figure Lengend Snippet: a p-STAT1 and p-STAT3 abundance in MC38 cells treated with IFNγ and increasing Longdaysin (0, 50, 100 μM, a CK1α inhibitor). Data are representative of three independent experiments. b Heatmap showing commonly upregulated gene expression of IFNγ signaling by RNA-seq analysis of Ankrd52 -null MC38 cells ( n = 2 replicates per group). c SOCS1 mRNA level in control and ANKRD52 -null 293 T cells overexpressing miR-155 ( n = 3 per group per condition). Data are representative of two independent experiments and represented as mean ± s.e.m., significance was determined using two-tailed unpaired Student’s t -test. d Activity of WT and mutant SOCS1 3’UTR (Rluc/Fluc) in a dual-luciferase reporter in 293 T cells overexpressing miR-155 ( n = 3 per group per condition). Data are representative of three independent experiments and represented as mean ± s.e.m., significance was determined using two-tailed unpaired Student’s t -test. e Schematic overview of qPCR test targeting mutant region to check Socs1 knockout efficiency. f Socs1 mRNA level tested by qPCR targeting mutant region in Ankrd52 -null MC38 cells with inactivated SOCS1 ( n = 5 per group). Data are represented as mean ± s.e.m., significance was determined using two-tailed unpaired Student’s t -test. g p-STAT1 and p-STAT3 abundance in Ankrd52 -null MC38 cells with inactivated SOCS1 after IFNγ treatment. Data are representative of three independent experiments. h Membrane MHC-1 expression in Ankrd52 -null MC38 cells with inactivated SOCS1 after IFNγ treatment. MFI of H2-K b was normalized by the responding group without IFNγ treatment ( n = 3 per group per condition). Data are representative of two independent experiments and represented as mean ± s.d., significance was determined using two-tailed unpaired Student’s t -test. i Killing of OVA-treated Ankrd52 -null MC38 cells with inactivated SOCS1 by OT-I T cells ( n = 3 per group per condition). Data are representative of two independent experiments and represented as mean ± s.d., significance was determined using two-tailed unpaired Student’s t -test. j Volcano plot showing the Spearman’s correlation and estimated significance of ANKRD52 with SOCS1 mRNA levels across all TCGA cancer types. Each dot represents a cancer type, blue dots indicate significant negative correlations ( P < 0.05, TIMER). See also Supplementary Figs. and . Source data are provided as a source data file.

Article Snippet: MC38 cell line was provided by WuXi AppTec and cultured using DMEM (Gibco) with 10% fetal bovine serum (FBS), respectively.

Techniques: Gene Expression, RNA Sequencing, Control, Two Tailed Test, Activity Assay, Mutagenesis, Luciferase, Knock-Out, Membrane, Expressing

Journal: Nature Communications

Article Title: Tumor evolution selectively inactivates the core microRNA machinery for immune evasion

doi: 10.1038/s41467-021-27331-3

Figure Lengend Snippet: a Diagram showing the Spearman’s correlation of top co-dependent proteins with ANKRD52 or PPP6C in CRISPR (Avana) Public 20Q3 database. Solid lines depict significant positive correlations (Correlation > 0.25, P < 0.001) and dashed lines depict weak correlation (Correlation > 0.1, P < 0.01). b , c Volcano plot showing the Spearman’s correlation and estimated significance of DICER1 ( b ) or XPO5 ( c ) with SOCS1 mRNA levels from RNA-seq data across all TCGA cancer types. Each dot represents a cancer type in TCGA; blue dots indicate significant negative correlations ( P < 0.05, TIMER). d SOCS1 mRNA level in MC38 cells with targeted sgRNAs ( n = 3 per group). Data are representative of three independent experiments and represented as mean ± s.e.m., significance was determined using two-tailed unpaired Student’s t -test. e Killing of OVA-treated MC38 cells with targeted sgRNAs by OT-I T cells ( n = 3 per group per condition). Data are representative of two independent experiments and represented as mean ± s.d., significance was determined using two-tailed unpaired Student’s t -test. f Tumor growth curves of Ago2 -null or control MC38 tumors in WT mice treated with PD-1 antibody or not ( n = 5 for NT tumor, n = 6 for NT with anti-PD-1 and n = 7 for Ago2 -null with or without anti-PD-1). Data are represented as mean ± s.e.m., significance was determined using two-tailed unpaired Student’s t -test. g Heatmap showing the Spearman’s correlation of ANKRD52 , AGO2 , DICER1 , XPO5 , DROSHA , PPP6C , or SOCS1 mRNA levels with CD4 + and CD8 + T cell abundance in tumors across all TCGA cancer types. h Model of miRNA machinery in regulation of cancer-intrinsic evasion from T cell attack. See also Supplementary Figs. – . Source data are provided as a source data file.

Article Snippet: MC38 cell line was provided by WuXi AppTec and cultured using DMEM (Gibco) with 10% fetal bovine serum (FBS), respectively.

Techniques: CRISPR, RNA Sequencing, Two Tailed Test, Control