Journal: Cell metabolism

Article Title: Cancer Lipid Metabolism Confers Antiangiogenic Drug Resistance.

doi: 10.1016/j.cmet.2018.05.005

Figure Lengend Snippet: Figure 1. Tumor and Angiogenesis Responses to AAD in Adipose and Non-adipose Tissues (A–H) CRC (A–D) and PDAC (E–H) tumors implanted in subcutaneous (non-adipose) and inguinal WAT were treated with a NIIgG or an anti-VEGF neutralizing antibody (n = 8–10 mice per group). Tumor growth (A–C and E–G) was measured as volumes (A, B, E, and F) and weight (C and G). Percentages of tumor inhibition were calculated (D and H). (I and K) Micrographs of CD31+ microvessels (red) in association with NG2+ pericytes (green in upper panels), leakiness of 70-kDa dextran (green in middle panels), and perfusion of 2000-kDa dextran (green in lower panels) in NIIgG- and anti-VEGF-treated non-adipose and adipose CRC (I) and PDAC (K) cancers. Arrows in upper panels point to NG2+ pericytes in association with tumor vessels. Arrowheads in middle panels indicate leaked dextran signals. Arrows in lower panels indicate perfused tumor vessels. Bar represents 100 mm. (J and L) Quantification of CD31+ tumor vessels (n = 5–10 random fields per group), pericyte-associated vessels (n = 5–10 random fields per group), extravasated 70-kDa dextran signals (n = 4–7 random fields per group), and perfusion of 2,000-kDa dextran (n = 4–7 random fields per group) in CRC (J) and PDAC (L) cancers. *p < 0.05; **p < 0.01; ***p < 0.001. NS, not significant. Data presented as means ± SEM. See also Figures S1 and S2.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, Peptides, and Recombinant Proteins Etomoxir Dr. Wolf-BioScience N/A MTT Sigma-Aldrich Cat# M5655 Clodronate liposomes FormuMax Cat# F70101C-N Control Liposomes FormuMax Cat# F70101-N Proteinase inhibitor cocktail Sigma-Aldrich Cat# P8340 Phosphatase inhibitor cocktail Cell Signaling Cat# 5870 Bodipy 558/568 C12 Invitrogen Cat# D-3835 D-luciferin PerkinElmer Cat# 122799 Critical Commercial Assays Cpt1 shRNA lentiviral particles Santa Cruz Biotechnology Cat# sc-40377-V Mycoplasma detection kit Lonza Cat# LT07-318 Pimonidazole Hypoxyprobe Cat# HP6-x cDNA Synthesis Kit Thermo Scientific Cat# K1632 Fast SYBR Green Master Mix Applied Biosystems Cat# 4385612 ATP assay kit Abnova Cat# KA0806 NEFA-HR(2) Assay Reagent 1 Wako Chemicals Cat# 434-91795 NEFA-HR(2) Assay Reagent 2 Wako Chemicals Cat# 434-91995 NEFA-HR(2) Assay Standard Wako Chemicals Cat# 270-77000 2000-kDa-lysinated fluorescein-labeled dextran Invitrogen Cat# D7137 2000-kDa-lysinated tetramethylrhodamine-labeled dextran Invitrogen Cat# D7139 70-kDa-lysinated fluorescein-labeled dextran Invitrogen Cat# D1822 70-kDa-lysinated tetramethylrhodamine-labeled dextran Invitrogen Cat# D1818 GeneJET RNA Purification Kit Thermo Scientific Cat# K0732 Deposited Data Affymetrix microarray data, mouse colorectal cancer grown in non-adipose or white adipose environment This paper GEO:GSE113507 Affymetrix microarray data, mouse colorectal cancer grown in healthy liver or fatty liver with or without antiangiogenic treatment This paper GEO:GSE113508 Experimental Models: Cell Lines MC38 murine colon adenocarcinoma Dr. Rubén Hernández, Center for Applied Medical Research, University of Navarra, Spain N/A Hepa1-6 murine hepatocellular carcinoma ATCC CRL-1830 PancO2 murine pancreatic ductal adenocarcinoma Dr. Maximilian Schnurr, Munich University, Germany N/A Experimental Models: Organisms/Strains Mouse: C57BL/6NJ Jackson Laboratory RRID:IMSR_JAX:005304 Mouse: CB17/Icr-Prkdcscid/IcrCrl Charles River RRID:IMSR_CRL:236 Oligonucleotides qRT-PCR primers.

Techniques: Inhibition

Journal: Cell metabolism

Article Title: Cancer Lipid Metabolism Confers Antiangiogenic Drug Resistance.

doi: 10.1016/j.cmet.2018.05.005

Figure Lengend Snippet: Figure 2. Anti-tumor and Antiangiogenic Responses of CRC and HCC Tumors Implanted in Normal and Steatotic Livers (A and E) Morphological and bioluminescent imaging analyses of tumor signals in healthy and steatotic livers. Arrows indicate surface CRC (A) and HCC (E) tumor nodules in healthy and steatotic livers. Bar represents 1 cm. (B and F) Quantification of liver weight (n = 3–6 mice per group), liver tumor volume (n = 5–7 mice per group), surface visible nodules (n = 5–7 mice per group), and photon counts (n = 6–10 mice per group) in CRC (B) and HCC (F) cancers. (C and G) Micrographs of CD31+ microvessels (red), leakiness of 70-kDa dextran (green in middle panels), and perfusion of 2,000-kDa dextran (green in lower panels) in NIIgG- and anti-VEGF-treated non-steatotic and steatotic CRC (C) and HCC (G) cancers. Arrows in upper panels point to CD31+ tumor vessels. Arrowheads in middle panels indicate extravasated dextran signals. Arrows in lower panels indicate perfused tumor vessels. Bar represents 100 mm. (D and H) Quantification of CD31+ tumor vessels (n = 8 random fields from three to six independent tumor samples per group), extravasated 70-kDa dextran signals (n = 5–6 random fields from three to six independent tumor samples per group), and perfusion of 2,000-kDa dextran (n = 6–8 random fields from three to six independent tumor samples per group) in CRC (D) and HCC (H) cancers. *p < 0.05; **p < 0.01; ***p < 0.001. NS, not significant. Data presented as means ± SEM. See also Figure S3.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, Peptides, and Recombinant Proteins Etomoxir Dr. Wolf-BioScience N/A MTT Sigma-Aldrich Cat# M5655 Clodronate liposomes FormuMax Cat# F70101C-N Control Liposomes FormuMax Cat# F70101-N Proteinase inhibitor cocktail Sigma-Aldrich Cat# P8340 Phosphatase inhibitor cocktail Cell Signaling Cat# 5870 Bodipy 558/568 C12 Invitrogen Cat# D-3835 D-luciferin PerkinElmer Cat# 122799 Critical Commercial Assays Cpt1 shRNA lentiviral particles Santa Cruz Biotechnology Cat# sc-40377-V Mycoplasma detection kit Lonza Cat# LT07-318 Pimonidazole Hypoxyprobe Cat# HP6-x cDNA Synthesis Kit Thermo Scientific Cat# K1632 Fast SYBR Green Master Mix Applied Biosystems Cat# 4385612 ATP assay kit Abnova Cat# KA0806 NEFA-HR(2) Assay Reagent 1 Wako Chemicals Cat# 434-91795 NEFA-HR(2) Assay Reagent 2 Wako Chemicals Cat# 434-91995 NEFA-HR(2) Assay Standard Wako Chemicals Cat# 270-77000 2000-kDa-lysinated fluorescein-labeled dextran Invitrogen Cat# D7137 2000-kDa-lysinated tetramethylrhodamine-labeled dextran Invitrogen Cat# D7139 70-kDa-lysinated fluorescein-labeled dextran Invitrogen Cat# D1822 70-kDa-lysinated tetramethylrhodamine-labeled dextran Invitrogen Cat# D1818 GeneJET RNA Purification Kit Thermo Scientific Cat# K0732 Deposited Data Affymetrix microarray data, mouse colorectal cancer grown in non-adipose or white adipose environment This paper GEO:GSE113507 Affymetrix microarray data, mouse colorectal cancer grown in healthy liver or fatty liver with or without antiangiogenic treatment This paper GEO:GSE113508 Experimental Models: Cell Lines MC38 murine colon adenocarcinoma Dr. Rubén Hernández, Center for Applied Medical Research, University of Navarra, Spain N/A Hepa1-6 murine hepatocellular carcinoma ATCC CRL-1830 PancO2 murine pancreatic ductal adenocarcinoma Dr. Maximilian Schnurr, Munich University, Germany N/A Experimental Models: Organisms/Strains Mouse: C57BL/6NJ Jackson Laboratory RRID:IMSR_JAX:005304 Mouse: CB17/Icr-Prkdcscid/IcrCrl Charles River RRID:IMSR_CRL:236 Oligonucleotides qRT-PCR primers.

Techniques: Imaging

Journal: Cell metabolism

Article Title: Cancer Lipid Metabolism Confers Antiangiogenic Drug Resistance.

doi: 10.1016/j.cmet.2018.05.005

Figure Lengend Snippet: Figure 6. Blocking CPT1 Enhances AAD-Mediated Anti-tumor Effects (A and F) shRNA-Cpt1- and control-vehicle-transfected CRC (A) or HCC (F) tumor-bearing mice received NIIgG and anti-VEGF treatment. Upper panels: representative tumors. Arrows point to tumors in each group. Middle panels: extravasation of 70-kDa dextran (green). CD31+ blood vessels (red). Arrowheads indicate leaked dextran signals. Lower panels: perfusion of 2,000-kDa dextran (green). CD31+ blood vessels (red). Arrows indicate perfused tumor vessels. Bar represents 100 mm. (B and G) Immunohistochemical analysis of Ki67+ proliferating cells and activated caspase-3+ apoptotic cells in CRC (B) or HCC (G). Arrows and arrowheads point to proliferating and apoptotic cells, respectively. Bar represents 100 mm. (C and H) Quantification of tumor volumes of various CRC (C) or HCC (H) groups (n = 3–5 animals per group). (D and I) Quantification of CD31+ tumor vessels (n = 9 random fields per group), extravasated 70-kDa dextran signals (n = 9 random fields per group), and perfusion of 2,000-kDa dextran (n = 9 random fields per group) in CRC (D) or HCC (I). (E and J) Quantification of Ki67+ (n = 6 random fields per group) and cleaved caspase-3+ signals (n = 6 random fields per group) in CRC (E) or HCC (J). *p < 0.05; **p < 0.01; ***p < 0.001. NS, not significant. Data presented as means ± SEM. See also Figure S6.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, Peptides, and Recombinant Proteins Etomoxir Dr. Wolf-BioScience N/A MTT Sigma-Aldrich Cat# M5655 Clodronate liposomes FormuMax Cat# F70101C-N Control Liposomes FormuMax Cat# F70101-N Proteinase inhibitor cocktail Sigma-Aldrich Cat# P8340 Phosphatase inhibitor cocktail Cell Signaling Cat# 5870 Bodipy 558/568 C12 Invitrogen Cat# D-3835 D-luciferin PerkinElmer Cat# 122799 Critical Commercial Assays Cpt1 shRNA lentiviral particles Santa Cruz Biotechnology Cat# sc-40377-V Mycoplasma detection kit Lonza Cat# LT07-318 Pimonidazole Hypoxyprobe Cat# HP6-x cDNA Synthesis Kit Thermo Scientific Cat# K1632 Fast SYBR Green Master Mix Applied Biosystems Cat# 4385612 ATP assay kit Abnova Cat# KA0806 NEFA-HR(2) Assay Reagent 1 Wako Chemicals Cat# 434-91795 NEFA-HR(2) Assay Reagent 2 Wako Chemicals Cat# 434-91995 NEFA-HR(2) Assay Standard Wako Chemicals Cat# 270-77000 2000-kDa-lysinated fluorescein-labeled dextran Invitrogen Cat# D7137 2000-kDa-lysinated tetramethylrhodamine-labeled dextran Invitrogen Cat# D7139 70-kDa-lysinated fluorescein-labeled dextran Invitrogen Cat# D1822 70-kDa-lysinated tetramethylrhodamine-labeled dextran Invitrogen Cat# D1818 GeneJET RNA Purification Kit Thermo Scientific Cat# K0732 Deposited Data Affymetrix microarray data, mouse colorectal cancer grown in non-adipose or white adipose environment This paper GEO:GSE113507 Affymetrix microarray data, mouse colorectal cancer grown in healthy liver or fatty liver with or without antiangiogenic treatment This paper GEO:GSE113508 Experimental Models: Cell Lines MC38 murine colon adenocarcinoma Dr. Rubén Hernández, Center for Applied Medical Research, University of Navarra, Spain N/A Hepa1-6 murine hepatocellular carcinoma ATCC CRL-1830 PancO2 murine pancreatic ductal adenocarcinoma Dr. Maximilian Schnurr, Munich University, Germany N/A Experimental Models: Organisms/Strains Mouse: C57BL/6NJ Jackson Laboratory RRID:IMSR_JAX:005304 Mouse: CB17/Icr-Prkdcscid/IcrCrl Charles River RRID:IMSR_CRL:236 Oligonucleotides qRT-PCR primers.

Techniques: Blocking Assay, shRNA, Control, Transfection, Immunohistochemical staining

Journal: Nature biotechnology

Article Title: Generation of pancreatic β cells from CD177 + anterior definitive endoderm.

doi: 10.1038/s41587-020-0492-5

Figure Lengend Snippet: Fig. 1 | Identification of CD177+ and CD275+ ADE subpopulations. a, Schematic representation of hESC differentiation toward DE. b,c, Representative FACS plots of apparently homogeneous FOXA2+/SOX17+ DE (b) showing a heterogenous population marked by CXCR4+/CD117+ cells (c) (n = 3 (b), n = 6 (c) biologically independent experiments). d–g, Gene expression profiles of CXCR4+/CD117−, CXCR4high/CD117high, CXCR4mid/CD117mid and CXCR4low/CD117low cells for FOXA2 (d), SOX17 (e), CER1 (f) and HHEX (g) (ANOVA, n = 3 biologically independent experiments). Data are represented as mean ± s.e.m.; P < 0.05 and P < 0.01. Statistically nonsignificant results are not indicated in the figure. h, Summary of the antibody screen identifying and isolating CD177 and CD275 as markers of ADE subpopulations. CXCR4 and FOXA2 are used as controls to identify the whole DE. i, hPSCs and hPSC-derived DE stained for CXCR4, CD177 and CD275 as shown by live-cell FACS (n = 10 biologically independent experiments). AA, activin A; D, day.

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology Animals and other organisms Human research participants Clinical data Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Human CXCR4-PE,Miltenyi Biotech,130-098-354, dilution 1:40; Human CXCR4-APC,Miltenyi Biotech, 120-010-802, dilution 1:40; Human CD117-APC, Miltenyi Biotech, 130-091-733, dilution 1:40; Human CD117-PE, Miltenyi Biotech, 130-091-734, dilution 1:40; FOXA2-Alexa Fluor® 488, R and D, IC2400G; dilution 1:10 SOX17-APC, R and D, IC1924A; dilution 1:10 Human CD177-APC, Miltenyi Biotech, 120-017-498; dilution 1:20 Human CD275-APC, Miltenyi Biotech, 120-012-112; dilution 1:20 PE Mouse anti-PDX1, BD PharmingenTM, 562161; dilution 1:40 4 nature research | reporting sum m ary O ctober 2018 Alexa Fluor® 647 Mouse anti-Nkx6.1, BD PharmingenTM, 563338; dilution 1:40 Alexa Fluor® 647 Mouse IgG1 κ Isotype Control, BD PharmingenTM, 563023; dilution 1:40 Rabbit FOXA2, Cell signalling, 8186; dilution 1:1000 Goat SOX17 Acris/Novus GT15094, dilution 1:1000 Goat CER1 R&D Systems AF1075, dilution 1:1000 Mouse β-catenin BD 610154, dilution 1:1000 Guinea pig INSULIN Thermo Schientific PA1-26938, dilution 1:100 Guinea pig C-Peptide Abcam ab30477, dilution 1:300 Rabbit MAFA Betalogics LP9872, dilution 1:100 Rabbit MAFA ,Novus Biologicals, NB400-137, dilution 1:100 Rabbit GLUT1 Thermo Fisher PA1-37782, dilution 1:100 Goat GATA6 R&D Systems AF1700, dilution 1:1000 Mouse SOX2 Abgent / Bio Cat AM2048, dilution 1:1000 Rabbit CDX2 Santa Cruz sc-134468, dilution 1:1000 Mouse GCG Sigma G2654-.2ML, dilution 1:300 Goat PDX1 R&D Systems AF2419, dilution 1:500 Rabbit NKX6.1Novus biologicalsNBP1-49672, dilution 1:500 Goat NKX6.1R&D systemsAF5857, dilution 1:300 Rabbit p-JNK Cell signalling 4668, dilution 1:1000 Rabbit DVL2 Cell signalling 3216, dilution 1:1000 Mouse GAPDH Merck Biosciences CB1001, dilution 1:6000 Validation All primary antibodies were validated for their expression on undifferentiated cells and/or pancreatic human sections/islets.

Techniques: Gene Expression, Derivative Assay, Staining

Journal: Nature biotechnology

Article Title: Generation of pancreatic β cells from CD177 + anterior definitive endoderm.

doi: 10.1038/s41587-020-0492-5

Figure Lengend Snippet: Fig. 2 | Molecular profiling of CD177+, CD275+ and CXCR4+ DE subpopulations reveals distinct signatures. a, Summary of differentiation protocol toward DE/ADE followed by MACS to enrich for CD177, CD275 and CXCR4 populations. b, Principal component analysis showing that mRNA-derived transcriptome profiles are characteristic of different DE/ADE subpopulations (n = 3 biologically independent experiments). c–e, Bar graphs of selected and significantly enriched gene ontology terms in CD275+ versus CXCR4+ (c), CD177+ versus CD275+ (d) and CD177+ versus CXCR4+ (e) DE populations (n = 3 biologically independent experiments). Enrichment P values are calculated by HOMER findGO.pl based on the cumulative hypergeometric distribution. f,g, Validation of the microarray analysis by qPCR for noncanonical WNT/PCP components and ligands (f) and canonical WNT components and ligands (g). Data were normalized to 18S (ANOVA, n = 3 biologically independent experiments). Data are represented as mean ± s.e.m.; P < 0.05 and P < 0.01. Statistically nonsignificant results are not indicated in the figure. h,i, Western blot analysis (h) and quantification (i) of WNT/PCP components such as p-JNK and DVL2 in ADE subpopulations (n = 3 biologically independent experiments). GAPDH is used as a loading control. Data are represented as mean ± s.e.m. j, Immunofluorescence analysis validated the exclusive localization of β-catenin in the membrane in CD177+ ADE cells and in the cytoplasm and nucleus in CD275+ ADE and CXCR4+ DE cells (n = 3 biologically independent experiments). FOXA2 is used as a nuclear marker. Scale bars, 20 µm and 10 µm in inset. PC1/2, principal component 1/2.

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology Animals and other organisms Human research participants Clinical data Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Human CXCR4-PE,Miltenyi Biotech,130-098-354, dilution 1:40; Human CXCR4-APC,Miltenyi Biotech, 120-010-802, dilution 1:40; Human CD117-APC, Miltenyi Biotech, 130-091-733, dilution 1:40; Human CD117-PE, Miltenyi Biotech, 130-091-734, dilution 1:40; FOXA2-Alexa Fluor® 488, R and D, IC2400G; dilution 1:10 SOX17-APC, R and D, IC1924A; dilution 1:10 Human CD177-APC, Miltenyi Biotech, 120-017-498; dilution 1:20 Human CD275-APC, Miltenyi Biotech, 120-012-112; dilution 1:20 PE Mouse anti-PDX1, BD PharmingenTM, 562161; dilution 1:40 4 nature research | reporting sum m ary O ctober 2018 Alexa Fluor® 647 Mouse anti-Nkx6.1, BD PharmingenTM, 563338; dilution 1:40 Alexa Fluor® 647 Mouse IgG1 κ Isotype Control, BD PharmingenTM, 563023; dilution 1:40 Rabbit FOXA2, Cell signalling, 8186; dilution 1:1000 Goat SOX17 Acris/Novus GT15094, dilution 1:1000 Goat CER1 R&D Systems AF1075, dilution 1:1000 Mouse β-catenin BD 610154, dilution 1:1000 Guinea pig INSULIN Thermo Schientific PA1-26938, dilution 1:100 Guinea pig C-Peptide Abcam ab30477, dilution 1:300 Rabbit MAFA Betalogics LP9872, dilution 1:100 Rabbit MAFA ,Novus Biologicals, NB400-137, dilution 1:100 Rabbit GLUT1 Thermo Fisher PA1-37782, dilution 1:100 Goat GATA6 R&D Systems AF1700, dilution 1:1000 Mouse SOX2 Abgent / Bio Cat AM2048, dilution 1:1000 Rabbit CDX2 Santa Cruz sc-134468, dilution 1:1000 Mouse GCG Sigma G2654-.2ML, dilution 1:300 Goat PDX1 R&D Systems AF2419, dilution 1:500 Rabbit NKX6.1Novus biologicalsNBP1-49672, dilution 1:500 Goat NKX6.1R&D systemsAF5857, dilution 1:300 Rabbit p-JNK Cell signalling 4668, dilution 1:1000 Rabbit DVL2 Cell signalling 3216, dilution 1:1000 Mouse GAPDH Merck Biosciences CB1001, dilution 1:6000 Validation All primary antibodies were validated for their expression on undifferentiated cells and/or pancreatic human sections/islets.

Techniques: Derivative Assay, Biomarker Discovery, Microarray, Western Blot, Control, Immunofluorescence, Membrane, Marker

Journal: Molecular Therapy

Article Title: Discovery of siRNA Lipid Nanoparticles to Transfect Suspension Leukemia Cells and Provide In Vivo Delivery Capability

doi: 10.1038/mt.2013.210

Figure Lengend Snippet: Expression of Caveolin 1 and 2 correlates with efficient siRNA transfection with alkylated DMA-containing SNALP-like lipid nanoparticles (SLPs). (a) Transfection efficiency of three tested leukemia cell lines, K562 (easily transfected), Molm13 (modestly transfected) and KG1 (poorly transfected), correlates with the amount of siRNAs entering into cells. Cy3-labeled control luciferase siRNAs were transfected into K562, Molm13 and KG1 cells using SLP301R. The cellular entry of siRNA was measured by quantitative fluorescent imaging (ImageStreamX, Amnis) at 2 hours after transfection. Mean Cy3 florescent intensity with SD was shown on the left. Two representative images from each cell line were shown on the right. (b) Four endocytosis-related genes were identified to be significantly underexpressed in poorly transfected KG1 and Mv4-11 cells compared with modestly transfected Molm13 and THP1 cells by comparative microarray analysis (Supplementary Table S2). (c) The expression levels of candidate genes identified in microarray were confirmed by quantitative RT-PCR in cell lines as indicated. Cav1, Cav2, and Rab13 were confirmed as underexpressed genes in poorly transfected KG1 and Mv4-11 cells compared with modestly and easily transfectd cell lines Molm13, THP1, HEL, and K562. (d) Upper panel, three groups of cell lines including easily transfected and poorly transfected adherent cell lines as well as hardest-to-transfect suspension leukemia cells, were subjected to comparative microarray analysis (Supplementary Tables S3 and S4). Lower panels, Cav1 and Cav2 were confirmed by quantitative RT-PCR as overexpressed genes in easily transfected adherent cell line HCT116, as compared with poorly transfected adherent cell lines HT29 and Colo205, and suspension leukemia cell line K562. (e) Colocalization of siRNA and Caveoloae. Cy3-labeled control luciferase siRNAs encapsulated in SLP301R were coadministered into K562 cells with Alexa647-labeled Albumins, which have been known to enter cells through Caveolae-mediated endocytosis. The cellular entry of siRNAs and Albumins was measured by quantitative fluorescent imaging (ImageStreamX, Amnis) at 30 minutes after administration. Two representative colocalization images were shown.

Article Snippet: The human adherent cancer cell lines, PC3, Colo205, HCT116, and HT-29 (ATCC, Manassas, VA); human leukemia cell lines, MV-4;11, K562, KG1, HEL, THP1 (ATCC); and MOLM13 (DSMZ, Braunschweig, Germany), were maintained in corresponding media (DMEM for adherent cell lines and RPMI for leukemia cell lines) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen).

Techniques: Expressing, Transfection, Labeling, Control, Luciferase, Imaging, Microarray, Quantitative RT-PCR, Suspension

Journal: Molecular and cellular biochemistry

Article Title: The endogenous subcellular localisations of the long chain fatty acid-activating enzymes ACSL3 and ACSL4 in sarcoma and breast cancer cells.

doi: 10.1007/s11010-018-3332-x

Figure Lengend Snippet: Fig. 1 Immunohistochemistry reveals differential expression of ACSL3 and ACSL4 in sarcomas. Multiple tissues arrays were probed using antibodies specific for either ACSL3 or ACSL4. Representa- tive images are shown for ACSL3 and ACSL4 immunohistochemical staining of matched patient samples from a and b liposarcoma, c and d fibrosarcoma, e and f leiomyosarcoma, and g and h rhabdomyo- sarcoma cores on multiple tissue arrays. Examples of negative and weak staining are presented in panels a, b and d, and high-intensity staining in the remaining panels The main image in each panel ×10 objective magnification, and each inset is ×100 objective magnifica- tion. Cytosplamic staining can be seen in higher-magnification inset images in panels c, and e–h. Note that, while ACSL3 staining was classified as weak overall in liposarcomas, it was nevertheless pos- sible to visualise ACSL3 staining on the perimeter of lipid droplets in these samples a inset

Article Snippet: Immunohistochemistry of sarcoma multiple tissue arrays Protein expression of ACSL3 and ACSL4 in tissue microarray sections of tumour samples was demonstrated using rabbit polyclonal primary antibodies raised against ACSL3 [#R2379-1 from Abiocode (Agoura Hills, California, USA)] and ACSL4 [#22401-1-AP from Proteintech Europe (Manchester, UK)], respectively.

Techniques: Immunohistochemistry, Quantitative Proteomics, Immunohistochemical staining, Staining

Journal: Molecular and cellular biochemistry

Article Title: The endogenous subcellular localisations of the long chain fatty acid-activating enzymes ACSL3 and ACSL4 in sarcoma and breast cancer cells.

doi: 10.1007/s11010-018-3332-x

Figure Lengend Snippet: Fig. 6 ACSL4 partially localises to MAM. MAM, cytoplasmic and mitochondrial fractions, were prepared from MCF-7 cells. Equal vol- umes of samples from each MCF-7 fraction were subjected to SDS- PAGE separation and immunoblotted for ACSL4, calnexin, caveolin, HMGCR and Sigma1R. Each panel represents the results of an exper- iment repeated twice with similar results

Article Snippet: Immunohistochemistry of sarcoma multiple tissue arrays Protein expression of ACSL3 and ACSL4 in tissue microarray sections of tumour samples was demonstrated using rabbit polyclonal primary antibodies raised against ACSL3 [#R2379-1 from Abiocode (Agoura Hills, California, USA)] and ACSL4 [#22401-1-AP from Proteintech Europe (Manchester, UK)], respectively.

Techniques: SDS Page

Journal: Molecular and cellular biochemistry

Article Title: The endogenous subcellular localisations of the long chain fatty acid-activating enzymes ACSL3 and ACSL4 in sarcoma and breast cancer cells.

doi: 10.1007/s11010-018-3332-x

Figure Lengend Snippet: Fig. 7 Imaging of the subcel- lular localisation of ACSL4 in MCF-7 cells. MCF-7 cells were formalin fixed and stained with antibodies to a VDAC (green) or b Sigma1R (green). ACSL4 (magenta) and chromatin counterstained with Hoechst (blue). Samples were imaged using confocal microscopy. Scale bars, 10 µm. (Color figure online)

Article Snippet: Immunohistochemistry of sarcoma multiple tissue arrays Protein expression of ACSL3 and ACSL4 in tissue microarray sections of tumour samples was demonstrated using rabbit polyclonal primary antibodies raised against ACSL3 [#R2379-1 from Abiocode (Agoura Hills, California, USA)] and ACSL4 [#22401-1-AP from Proteintech Europe (Manchester, UK)], respectively.

Techniques: Imaging, Staining, Confocal Microscopy

Journal: iScience

Article Title: Loss of physical contact in space alters the dopamine system in C. elegans

doi: 10.1016/j.isci.2022.103762

Figure Lengend Snippet:

Article Snippet: Image J software was used to measure the strongest cytoplasmic Ca 2+ intensity during flexion of the body wall muscles of an individual (n = 16 animals per condition), focused on time-lapse microscopic images with CellSens standard software (Olympus).

Techniques: Recombinant, Western Blot, Microarray, Catecholamine ELISA, Software, Imaging

Journal: Cellular signalling

Article Title: The gep proto-oncogene Gα 12 mediates LPA-stimulated activation of CREB in ovarian cancer cells

doi: 10.1016/j.cellsig.2013.08.012

Figure Lengend Snippet: A. Silencing of Gα12 in HeyA8 cells using Gα12-specific shRNA was monitored by immunoblot analysis using lysates of 25 µg protein derived from three distinct clones of Gα12-silenced cells along with cells from vector control clone. B. Gα12-shRNA-HeyA8 clones were analyzed by quantitative RT-PCR for Gα12 expression. The expression levels of Gα12 for each clone in relation to vector control cells are presented in the bar graph. C. Hey cells stably expressing shRNA against Gα12 or the vector alone (non-specific scrambled shRNA vector) were serumstarved overnight. The stably silenced Gα12 cells were treated with 20 µM of LPA for 16 hours along with one group of HeyA8 cells stably-expressing the vector alone. Additionally, one group of the vector control cells was left in serum-free media for the 16-hour treatment period. After the 16-hour treatment, nuclear lysate was obtained from each cell group and analyzed by a Protein/DNA array according to manufacturer’s protocol. Representative array data from two independent experiments are presented. Each spot on the array that corresponds to a specific transcription factor was identified according to manufacturer’s protocol. Transcription factors stimulated by LPA but absent or down-regulated in Gα12-silenced cells are scored. The arrows indicate the spots corresponding to CREB. The profiles of activated transcription factors as indicated by the binding of the respective transcription factors to the DNA-elements printed in the array were analyzed in serum-starved HeyA8 cells (Upper Panel), HeyA8 cells stimulated with LPA (Middle Panel), and LPA-stimulated HeyA8 cells in which the expression of Gα12 was silenced (Lower Panel).

Article Snippet: The nuclear lysate was then analyzed for transcription factor activation using an Affymetrix Combo Protein/DNA Array (MA1215; Santa Clara, CA) according to the manufacture’s instructions.

Techniques: shRNA, Western Blot, Derivative Assay, Clone Assay, Plasmid Preparation, Quantitative RT-PCR, Expressing, Stable Transfection, DNA Array, Binding Assay

Journal: Cellular signalling

Article Title: The gep proto-oncogene Gα 12 mediates LPA-stimulated activation of CREB in ovarian cancer cells

doi: 10.1016/j.cellsig.2013.08.012

Figure Lengend Snippet: LPA-stimulated and Gα 12 -dependent Transcription Factors in HeyA8 Cells Control HeyA8 cell expressing non-specific sh-vector or HeyA8 cells in which Gα 12 were stimulated with 20 µM LPA for 16 hrs. Nuclear extracts from these cells along with unstimulated controls were analyzed for the activation of different transcription factors using “Affymetrix Combo Protein/DNA Array” as described under Methods section. Representative array data from two independent experiments are presented here. Each spot on the array, which corresponds to a specific transcription factor, was identified using the template from the user manual. The intensities of the spots were quantified using Carestream Molecular Imaging Software version 5. Transcription factors stimulated by LPA but absent or down-regulated in Gα 12 -silenced cells were scored, quantified, and tabulated.

Article Snippet: The nuclear lysate was then analyzed for transcription factor activation using an Affymetrix Combo Protein/DNA Array (MA1215; Santa Clara, CA) according to the manufacture’s instructions.

Techniques: Expressing, Activation Assay, Imaging, Software, Inhibition, Binding Assay, Methylation