Journal: STAR Protocols

Article Title: Protocol for quantifying drug sensitivity in 3D patient-derived ovarian cancer models

doi: 10.1016/j.xpro.2024.103274

Figure Lengend Snippet:

Article Snippet: CD326 (EpCAM) antibody, anti-human, PE, REAfinity (flow cytometry 1:200) , Miltenyi Biotec , Cat# 130-110-999; RRID: AB_2657495.

Techniques: Immunofluorescence, Flow Cytometry, Control, Recombinant, Red Blood Cell Lysis, Saline, Software, Cell Culture, Plasmid Preparation, Microscopy

Journal: Frontiers in Immunology

Article Title: Impact of an Immune Modulator Mycobacterium-w on Adaptive Natural Killer Cells and Protection Against COVID-19

doi: 10.3389/fimmu.2022.887230

Figure Lengend Snippet: Relationship between NKG2C + ANK cells and COVID-19: Scatter dot with bar plot showing expression of NKG2C + ANK cells at baseline in the overall cohort (no infection, n=64 and infection, n=16), control group (no infection, n=20 and infection, n=10) and Mw group (no infection, n=44 and infection, n=6) with respect to SARS-CoV-2 infection. Black and red upside shaded triangles represent NKG2C + ANK for Control group and Mw group respectively and unshaded upside triangles represent NKG2C + ANK of both groups combined. ***p < 0.001, **p < 0.01 and *P < 0.05.

Article Snippet: For surface staining, 0.5 × 10 6 cells were washed with phosphate-buffered saline and stained with the following antibodies that were used for phenotypic analysis: CD3(APC-H7, SK-7) CD16 (PE-Cy7, B73.1), CD56 (APC R700, NCAM16.2), CD57 (BV605, NK-1), NKG2A (PE-Cy7, Z199), CD4 (APC-H7), CD8 (Per-CP Cy), CD45RA (FITC), and CD45RO (BV605), from BD Biosciences, (San Jose, CA, United States) and NKG2C (PE, REA205) from Miltenyi Biotec (Bergisch Gladbach, Germany).

Techniques: Expressing, Infection, Control

Journal: Frontiers in Immunology

Article Title: Impact of an Immune Modulator Mycobacterium-w on Adaptive Natural Killer Cells and Protection Against COVID-19

doi: 10.3389/fimmu.2022.887230

Figure Lengend Snippet: Impact of Mw on both ANK cells and iNK) cells: (A) Scatter dot with bar plot showing the expression of NKG2C + ANK cells (Mw group -n=30 and control group- n=15) and (B) NKG2A + iNK cells (Mw group-n=30 and control group- n=15) with and without of Mw vaccine at baseline and day 60. (C) Scatter dot with bar plot showing kinetics of NKG2C + ANK cells, and (D) NKG2A + iNK cells expression in Mw group (n=30) at baseline, day 30, day 60, and day 100. (E) log2FC expression of NKG2C + and NKG2A + in both Mw group (n=30) and vontrol group (n=15) at day 60 after normalization with baseline. Red upside and downside shaded triangles represent NKG2C + ANK and NKG2A + iNK, respectively, for Mw group. Black upside and downside shaded triangles represent NKG2C + ANK and NKG2A + iNK, respectively, for control group. ****p < 0.0001, **p <0.01, ***p <0.001, and ns, p-value not significant.

Article Snippet: For surface staining, 0.5 × 10 6 cells were washed with phosphate-buffered saline and stained with the following antibodies that were used for phenotypic analysis: CD3(APC-H7, SK-7) CD16 (PE-Cy7, B73.1), CD56 (APC R700, NCAM16.2), CD57 (BV605, NK-1), NKG2A (PE-Cy7, Z199), CD4 (APC-H7), CD8 (Per-CP Cy), CD45RA (FITC), and CD45RO (BV605), from BD Biosciences, (San Jose, CA, United States) and NKG2C (PE, REA205) from Miltenyi Biotec (Bergisch Gladbach, Germany).

Techniques: Expressing, Control

Journal: Frontiers in Immunology

Article Title: Impact of an Immune Modulator Mycobacterium-w on Adaptive Natural Killer Cells and Protection Against COVID-19

doi: 10.3389/fimmu.2022.887230

Figure Lengend Snippet: Impact of Mw on upregulation of NKG2C + ANK cells with respect to NKG2C expression at baseline: Scatter dot with bar plot showing the kinetics of (A) NKG2C + ANK cells (n=15) and (C) NKG2A + iNK (n=15) cells expression in Mw group at baseline, days 30 and 60 in respect to <15% NKG2C at baseline. (B) NKG2C + ANK (n=15) cells and (D) NKG2A + iNK (n=15) cell expression in Mw group at baseline, days 30 and 60 in respect to >15% NKG2C at baseline. Log2FC expression of (E) NKG2C + ANK and (F) NKG2A + iNK cells at day 60 after normalization with baseline value in Mw group (>/<15%, n=15) and control group. Red upside and downside shaded triangles represent NKG2C + ANK and NKG2A + iNK respectively for Mw group. Black upside and downside shaded triangles represent NKG2C + ANK and NKG2A + iNK, respectively, for control group. ****p < 0.0001, **p < 0.01, *p < 0.05, and ns, p-value not significant.

Article Snippet: For surface staining, 0.5 × 10 6 cells were washed with phosphate-buffered saline and stained with the following antibodies that were used for phenotypic analysis: CD3(APC-H7, SK-7) CD16 (PE-Cy7, B73.1), CD56 (APC R700, NCAM16.2), CD57 (BV605, NK-1), NKG2A (PE-Cy7, Z199), CD4 (APC-H7), CD8 (Per-CP Cy), CD45RA (FITC), and CD45RO (BV605), from BD Biosciences, (San Jose, CA, United States) and NKG2C (PE, REA205) from Miltenyi Biotec (Bergisch Gladbach, Germany).

Techniques: Expressing, Control

Journal: Frontiers in Immunology

Article Title: Impact of an Immune Modulator Mycobacterium-w on Adaptive Natural Killer Cells and Protection Against COVID-19

doi: 10.3389/fimmu.2022.887230

Figure Lengend Snippet: Mw showed sustained effect on NKG2C/NKG2A ratios until 100 days and Impact of Mw intracellular cytokine (IFN-γ) release: Scatter dot with bar plot showing (A) NKG2C/NKG2A ratio of control group (n=15) and Mw group (n=30) at baseline and day 60. (B) Kinetics of NKG2C/NKG2A ratio at baseline, days 30, 60, and 100 in Mw group. (C, D) Kinetics of NKG2C/NKG2A ratio at baseline and day 60 on the basis of >/< 15% NKG2C at baseline in Mw group and control group, respectively. (E) intracellular IFN-γ release in both control (n=5) and Mw (n=5) groups at baseline and day 60. Red and black shaded circles represent Mw group and control group, respectively. ****p < 0.0001, *p < 0.05 and ns, p-value not significant.

Article Snippet: For surface staining, 0.5 × 10 6 cells were washed with phosphate-buffered saline and stained with the following antibodies that were used for phenotypic analysis: CD3(APC-H7, SK-7) CD16 (PE-Cy7, B73.1), CD56 (APC R700, NCAM16.2), CD57 (BV605, NK-1), NKG2A (PE-Cy7, Z199), CD4 (APC-H7), CD8 (Per-CP Cy), CD45RA (FITC), and CD45RO (BV605), from BD Biosciences, (San Jose, CA, United States) and NKG2C (PE, REA205) from Miltenyi Biotec (Bergisch Gladbach, Germany).

Techniques: Control

Journal: Frontiers in Immunology

Article Title: Impact of an Immune Modulator Mycobacterium-w on Adaptive Natural Killer Cells and Protection Against COVID-19

doi: 10.3389/fimmu.2022.887230

Figure Lengend Snippet: Impact of the second dose of Mw on ANK and INK cells: Scatter dot with bar plot showing expression of (A) NKG2C + ANK (day 60, single dose; n=17, double dose; n=12 and day 100, single dose; n=17, double dose; n=12), (B) NKG2A + iNK cells (day 60, single dose; n=12, double dose; n=12 and day 100, single dose; n=17, double dose; n=12) and (C) NKG2C/NKG2A (day 60, single dose; n=17, double dose; n=12 and day 100, single dose; n=17, double dose; n=12) ratio with respect to single and double dose of Mw vaccine at day 60 and 100. Red upside and downside shaded triangles represent NKG2C + ANK and NKG2A + iNK, respectively, for Mw group. Red-shaded circle represents Mw group. ***p < 0.001, *p < 0.05 and ns, p value not significant.

Article Snippet: For surface staining, 0.5 × 10 6 cells were washed with phosphate-buffered saline and stained with the following antibodies that were used for phenotypic analysis: CD3(APC-H7, SK-7) CD16 (PE-Cy7, B73.1), CD56 (APC R700, NCAM16.2), CD57 (BV605, NK-1), NKG2A (PE-Cy7, Z199), CD4 (APC-H7), CD8 (Per-CP Cy), CD45RA (FITC), and CD45RO (BV605), from BD Biosciences, (San Jose, CA, United States) and NKG2C (PE, REA205) from Miltenyi Biotec (Bergisch Gladbach, Germany).

Techniques: Expressing

Journal: Oncology letters

Article Title: 7-difluoromethoxyl-5,4'-di-n-octyl genistein inhibits ovarian cancer stem cell characteristics through the downregulation of FOXM1.

doi: 10.3892/ol.2014.2080

Figure Lengend Snippet: Figure 4. Overexpression of FOXM1 attenuates the inhibitory effect of DFOG on the self‑renewal of OCSLCs derived from the SKOV3 cell line. (A) Western blot analysis showing the overexpression of FOXM1 following transfection with plasmid pcDNA3.1‑FOXM1 in the SFCs derived from the SKOV3 cells. The overexpression of FOXM1 reduced the DFOG‑induced downregulation of FOXM1, CD133, CD44 and ALDH1 expression. (B) The sphere‑forming ability of the SFCs derived from the SKOV3 cells exposed to DFOG was partly rescued following overexpression of FOXM1. Data are presented as the mean ± standard deviation (n=3). *P<0.05 vs. 0.1% DMSO. #P<0.05 vs. cells treated with the same concentration of DFOG using pcDNA3.1 transfection. DFOG, 7‑difluorome thoxyl‑5,4'‑di‑n‑octylgenistein; FOXM1, forkhead box M1; OCSLCs, ovarian cancer stem‑like cells; GEN, genistein; SFCs, sphere‑forming cells.

Article Snippet: The FOXM1 cDNA plasmid was purchased from OriGene Technologies Inc. (Rockville, MD, USA).

Techniques: Over Expression, Derivative Assay, Western Blot, Transfection, Plasmid Preparation, Expressing, Standard Deviation, Concentration Assay

Journal: Oncology letters

Article Title: 7-difluoromethoxyl-5,4'-di-n-octyl genistein inhibits ovarian cancer stem cell characteristics through the downregulation of FOXM1.

doi: 10.3892/ol.2014.2080

Figure Lengend Snippet: Figure 3. DOFG downregulates FOXM1 and CSC marker expression in OCSLCs derived from SKOV3 cells. (A) FOXM1 is highly expressed in SFCs from the SKOV3 cell line compared with the corresponding parental cells. (B) DFOG downregulated the expression of FOXM1 and CSC markers (CD133, CD44, and ALDH1) in the SFCs derived from the SKOV3 cells. DFOG, 7‑difluoromethoxyl‑5,4'‑di‑n‑octylgenistein; FOXM1, forkhead box M1; CSCs, cancer stem cells; OCSLCs, ovarian cancer stem‑like cells; GEN, genistein; SFCs, sphere‑forming cells.

Article Snippet: The FOXM1 cDNA plasmid was purchased from OriGene Technologies Inc. (Rockville, MD, USA).

Techniques: Marker, Expressing, Derivative Assay

Journal: Nature genetics

Article Title: Meningioma DNA methylation groups identify biological drivers and therapeutic vulnerabilities

doi: 10.1038/s41588-022-01061-8

Figure Lengend Snippet: a, Confocal microscopy and quantification of Annexin V in IOMM-Lee cells from Extended data figure 4e treated with actinomycin D or vehicle control for 24 hours. DNA is marked with DAPI. Scale bar 10 μM. From left to right, 96, 101, 95, 90, 98, or 75 cells are shown (ANOVA, one-sided). b, Immunoblot for Merlin, Caspase-7, cleaved Caspase-7 (cCaspase-7), or GAPDH in IOMM-Lee cells from a. c, Quantification of Annexin V confocal microscopy in MSC1 cells stably expressing sgNTC or sgNF2-2. Cells were treated as in a. From left to right, 29, 19, 40, or 30 cells are shown (ANOVA, one-sided). d, Representative images of cleaved Caspase-3 (cCaspase-3) immunohistochemistry from CH-157MN xenografts stably expressing doxycycline-inducible Merlin encoding a FLAG tag (NF2-FLAG) in NU/NU mice after 7 days of doxycycline (n=6) or vehicle treatment (n=6), and 24 hours after 4 Gy ionizing radiation (n=6) or control treatment (n=6). Scale bar 100 μM. e, Immunoblot for Merlin, IRF8, Tubulin, or Histone H3 (HH3) in cytoplasmic or nuclear fractions of M10GdCas9-KRAB cells from Extended data figure 4b. f, Normalized proteomic proximity-labeling mass spectrometry from M10G cells stably expressing Merlin constructs with APEX tags. From left to right, 2 or 3 biological replicates are shown. g, Immunoblot for IRF8 or FLAG after FLAG immunoprecipitation from M10G cells stably expressing Merlin encoding a FLAG tag (NF2FLAG). EV, empty vector. h, QPCR for the glucocorticoid receptor (NR3C1) in IOMM-Lee cells expressing a non-targeting control siRNA (siNTC) or siRNAs suppressing NR3C1 (siNR3C1). 3 biological replicates per condition (Student’s t test, one-sided). Lines represent means, and error bars represent standard error of the means.

Article Snippet: Gene over-expression For transient over-expression of FOXM1 or USF1, M10G cells were transfected with pCMV6-FOXM1 (#RC202246, Origene) or pCMV6-USF1 (#RC204915, Origene) plasmids at a ratio of 1 μg of DNA to 2 μl of FuGENE transfection reagent (#E2311, Promega).

Techniques: Confocal Microscopy, Control, Western Blot, Stable Transfection, Expressing, Immunohistochemistry, FLAG-tag, Labeling, Mass Spectrometry, Construct, Immunoprecipitation, Plasmid Preparation

Journal: Nature genetics

Article Title: Meningioma DNA methylation groups identify biological drivers and therapeutic vulnerabilities

doi: 10.1038/s41588-022-01061-8

Figure Lengend Snippet: a, Ki-67 labeling index from meningioma clinical pathology reports (n=206) across DNA methylation groups (ANOVA, one-sided). b, Representative images of meningioma Ki-67 and FOXM1 immunohistochemistry (n=92) across meningioma DNA methylation groups. Scale bar 10 μM. c, Meningioma DNA methylation analysis of chromosome 9p segment copy number deletions of any size containing the entire CDKN2A/B locus across Merlin-intact (n=8 of 192 meningiomas, 4%), Immune-enriched (n=5 of 216 meningiomas, 2%), and Hypermitotic (n=24 of 157 meningiomas, 15%) DNA methylation groups (n=565, Chi-squared test, two-sided). d, Meningioma DNA methylation (n=565) of CDKN2A (cg26349275) or CDKN2B (cg08390209) across DNA methylation groups (ANOVA, one-sided). e, tSNE plot of meningioma and meningioma cell line DNA methylation profiles. Four representative meningiomas from each DNA methylation group are shown. Triplicate meningioma M10GdCas9-KRAB cultures stably expressing a non-targeting control single-guide RNA (sgNTC) or single-guide RNAs suppressing NF2 (sgNF2), CDKN2A (sgCDKN2A), or CDKN2B (sgCDKN2B) are shown. Differences in DNA methylation groups are captured in tSNE1, and a positive shift from Immune-enriched meningiomas to Hypermitotic meningiomas mimics the shift from M10GdCas9-KRAB-sgNTC and M10GdCas9-KRAB-sgNF2 cells to M10GdCas9-KRAB-sgCDKN2A and M10GdCas9-KRAB-sgCDKN2B cells. Differences between tumors and cell lines, such as the tumor microenvironment, are captured in tSNE2. f, Meningioma DNA methylation analysis of chromosome 1p segment copy number amplifications of any size containing the entire USF1 locus across Merlin-intact (n=0 of 192 meningiomas, 0%), Immune-enriched (n=2 of 216 meningiomas, 4%), and Hypermitotic (n=38 of 157 meningiomas, 24%) DNA methylation groups (n=565, Chi-squared test, two-sided). g, USF1 ChIP-QPCR in DI98 meningioma cells for the CDK6 promoter compared to negative control primers targeting a gene desert (NC1) or a gene not predicted to be bound by USF1 (NC2) from ChIP sequencing. **p=0.001 (Student’s t test, one-sided, no adjustment for multiple comparisons). h, QPCR for CDK6 in M10GdCas9-KRAB cells expressing sgNTC or a single-guide RNA suppressing USF1 (sgUSF1), or M10G cells over-expressing USF1 or empty vector (EV). *p=0.003, **p=0.001 (Student’s t test, one-sided, no adjustment for multiple comparisons). i, Relative colony area of CH-157MN cells stably over-expressing USF1 or EV after 10 days of clonogenic growth. **p=0.001 (Student’s t test, one-sided, no adjustment for multiple comparisons). Lines represent means, and error bars represent standard error of the means.

Article Snippet: Gene over-expression For transient over-expression of FOXM1 or USF1, M10G cells were transfected with pCMV6-FOXM1 (#RC202246, Origene) or pCMV6-USF1 (#RC204915, Origene) plasmids at a ratio of 1 μg of DNA to 2 μl of FuGENE transfection reagent (#E2311, Promega).

Techniques: Labeling, DNA Methylation Assay, Immunohistochemistry, Stable Transfection, Expressing, Control, ChIP-qPCR, Negative Control, ChIP-sequencing, Plasmid Preparation

Journal: Nature genetics

Article Title: Meningioma DNA methylation groups identify biological drivers and therapeutic vulnerabilities

doi: 10.1038/s41588-022-01061-8

Figure Lengend Snippet: a, Predicted network of FOXM1-regulated pathways in Hypermitotic meningiomas based on H3K27ac ChIP sequencing of 25 meningiomas with matched RNA sequencing and DNA methylation profiling (15 Hypermitotic, 10 non-Hypermitotic). b, Immunoblot for Merlin, FOXM1, or GAPDH in IOMM-Lee meningioma cells stably expressing a non-targeting ontrol shRNA (shNTC) or shRNAs suppressing NF2 (shNF2-1 or shNF2-2), after treatment with actinomycin D or vehicle control for 24 hours. c, QPCR for FOXM1 in M10G meningioma cells over-expressing FOXM1 or empty vector (EV). 3 biological replicates per condition. ***p≤0.0001 (Student’s t test, one-sided). d, Quantification of Annexin V confocal microscopy in M10G cells over-expressing FOXM1 or EV after treatment with actinomycin D or vehicle control for 24 hours. From left to right, 57, 58, 65, or 60 cells are shown (ANOVA, one-sided). Lines represent means, and error bars represent standard error of the means.

Article Snippet: Gene over-expression For transient over-expression of FOXM1 or USF1, M10G cells were transfected with pCMV6-FOXM1 (#RC202246, Origene) or pCMV6-USF1 (#RC204915, Origene) plasmids at a ratio of 1 μg of DNA to 2 μl of FuGENE transfection reagent (#E2311, Promega).

Techniques: ChIP-sequencing, RNA Sequencing, DNA Methylation Assay, Western Blot, Stable Transfection, Expressing, shRNA, Control, Plasmid Preparation, Confocal Microscopy

Journal: Nature genetics

Article Title: Meningioma DNA methylation groups identify biological drivers and therapeutic vulnerabilities

doi: 10.1038/s41588-022-01061-8

Figure Lengend Snippet: a, Relative colony area of M10G, BenMen, CH-157MN, or IOMM-Lee meningioma cells after 10 days of clonogenic growth and treatment with abemaciclib, ribociclib, or palbociclib. 3 biological replicates per condition per timepoint. b, Relative colony area of M10GdCas9-KRAB cells expressing sgNTC, sgCDKN2A, or sgCDKN2B after 10 days of clonogenic growth and treatment with abemaciclib. 3 biological replicates per condition. *p=0.002, **p=0.001 (Student’s t test, one-sided). Data are normalized to growth with vehicle treatment of each cell lines. c, Relative colony area of CH-157MN cells stably over-expressing USF or empty vector (EV) after 10 days of clonogenic growth and treatment with abemaciclib. 3 biological replicates per condition. **p=0.001 (Student’s t test, one-sided). Data are normalized to growth with vehicle treatment of each cell lines. d, Quantification of BenMen peri-organoid intensity after 10 days of growth and treatment with abemaciclib or vehicle control Representative images of meningioma (red) and organoid (green) cells are shown. Scale bar 100 μM. 5 biological replicates per condition (ANOVA, one-sided). e, Representative immunoblots from CH-157MN xenografts in NU/NU mice (left) harvested at intervals after a single treatment of abemaciclib (100 μg/g) via oral gavage (right). f, Representative images of CH-157MN xenograft Ki-67 immunohistochemistry after a daily treatment of abemaciclib or control. Scale bar 1 mm. Lines represent means, and error bars represent standard error of the means.

Article Snippet: Gene over-expression For transient over-expression of FOXM1 or USF1, M10G cells were transfected with pCMV6-FOXM1 (#RC202246, Origene) or pCMV6-USF1 (#RC204915, Origene) plasmids at a ratio of 1 μg of DNA to 2 μl of FuGENE transfection reagent (#E2311, Promega).

Techniques: Inhibition, Expressing, Stable Transfection, Plasmid Preparation, Control, Western Blot, Immunohistochemistry