Journal: Developmental cell

Article Title: Myocardial polyploidization creates a barrier to heart regeneration in zebrafish

doi: 10.1016/j.devcel.2018.01.021

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: To generate the hs:(P*-S) FRT -Cre-dnEct2 transgenic line, a construct containing the following DNA elements was assembled by Gibson cloning: (1) a ~1.5-kb hsp70l promoter to drive expression in response to heat shock ( Halloran et al., 2000 ), amplified from p5E- hsp70l ( Kwan et al., 2007 ); (2) the coding sequence of a Y65A mutated version (non fluorescent) of the fluorescent protein PhiYFP amplified from pThy1-Brainbow3.2 ( Cai et al., 2013 ) flanked by FRT sites; (3) a bicistronic nls-Cre-P2A-dnEct2 cassette generated by subcloning a codon optimized, nuclear directed Cre from pAAV-pgk-Cre (a gift from Patrick Aebischer, Addgene plasmid #24593) and the Danio rerio dnEct2 amplified from cmlc2:GdnEct2 .

Techniques: Recombinant, RNAscope, HD Assay, TUNEL Assay, In Situ, Cloning, Software, Fluorsave, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: DNA-linked Inhibitor Antibody Assay (DIANA) for sensitive and selective enzyme detection and inhibitor screening

doi: 10.1093/nar/gkw853

Figure Lengend Snippet: Ultrasensitive detection of PSMA and CAIX by DIANA. ( A and B ) Plots of average C q values versus amount of human recombinant purified PSMA standard diluted in buffered solution (A), or average C q values versus amount of human CAIX present in HT-29 cell lysate diluted in buffered solution (B). Upper x-axes indicate molar concentration, while lower x-axes indicate the corresponding molar amount per well. Horizontal lines show the average background signal; dashed horizontal lines show the average background signal plus 2 s.d. Error bars show s.d. of quadruplicate measurements (A and B). ( C and D ) Plots of concentrations of PSMA (C) and CAIX (D) determined using DIANA in 36 human serum samples versus concentrations determined using ELISA in the same serum samples (CAIX) or in control citrate plasma samples (PSMA) drawn from the same individuals on the same occasion. Lines show the linear regression fit of log-transformed concentrations; dashed lines indicate values 1.25-fold higher or lower than the linear regression. Error bars show s.d. of triplicate (C) or duplicate (D) measurements.

Article Snippet: As a standard for CAIX quantification, we used lysate from cultured HT-29 cells; the CAIX concentration in the stock lysate was determined by Quantikine ELISA for human CAIX (RnD Systems).

Techniques: Recombinant, Purification, Concentration Assay, Enzyme-linked Immunosorbent Assay, Control, Clinical Proteomics, Transformation Assay

Journal: Nature Communications

Article Title: Acid-exposed and hypoxic cancer cells do not overlap but are interdependent for unsaturated fatty acid resources

doi: 10.1038/s41467-024-54435-3

Figure Lengend Snippet: a Representative pictures and quantification of Carbonic Anhydrase 9 (CA9, red) and pimonidazole (green) immunostaining in FaDu and HCT116 spheroid sections ( N = 3, n = 2); DAPI (blue) nuclear staining was used to normalize measurements. The white delimitation represents the rim of the spheroid. b Representative pictures and quantification of CA9 (red) and Hypoxia-Responsive Element-dependent GFP reporter (HRE, green) wholemount fluorescence in FaDu and HCT116 spheroids ( N = 5 and 7, respectively). c 3-dimensional modeling of CA9 (red), HRE-GFP (green), and DAPI (blue) staining in Fadu spheroids; this experiment was repeated twice with similar results. d Representative CA9 (purple) and pimonidazole (yellow) immunostaining in FaDu and HCT116 tumor sections; this experiment was repeated twice with similar results. e Representative flow cytometry analysis of CA9 staining in FaDu and HCT116 cancer cells maintained under normoxia at physiological pH 7.4 or at acidic pH 6.5; this experiment was repeated twice with similar results. The scale bar represents 100 µm for 3D tumor spheroids ( a – c ) and 200 µm for mouse tumor sections ( d ). Data are plotted as the means ± SD (** P = 0.0017, **** P < 0.0001); N indicates the number of independent experiments and n indicates the number of biological replicates (when >1). Significance was determined by two-sided Student’s t -test ( a , b ). Source data are provided as a Source Data file.

Article Snippet: The staining was performed with PE-coupled anti-CA9 antibody (130-123-340, Miltenyi, 1:200) for 20 min at 4 °C.

Techniques: Immunostaining, Staining, Fluorescence, Flow Cytometry

Journal: Nature Communications

Article Title: Acid-exposed and hypoxic cancer cells do not overlap but are interdependent for unsaturated fatty acid resources

doi: 10.1038/s41467-024-54435-3

Figure Lengend Snippet: a , b Schematic protocol of cell sorting from 3D FaDu spheroids based on CA9 immunostaining and HRE-GFP expression, created in BioRender. Feron, O. (2024) BioRender.com/i40c288 ( a ) and representative FACS plots showing gating strategy for each of the four quadrants ( b ). c Principal component analysis (PCA) discriminating the four quadrants based on RNA-seq analysis performed on 3 independent sorting experiments. d Volcano plot of differentially expressed genes between CA9+/HRE− and CA9+/HRE+ FaDu cell populations. e . KEGG pathway enrichment analysis of the differentially expressed genes between CA9+/HRE− and CA9+/HRE+ FaDu cell populations. f , g Changes in the mRNA expression of the indicated desaturases in the four distinct FaDu spheroid compartments ( f ) ( N = 3, n = 2), and in FaDu cancer cells maintained at physiological pH 7.4 or at acidic pH 6.5 ( g ) ( N = 4); results are expressed as fold-change vs . mRNA levels in CA9-/HRE- double negative cell populations and in cancer cells at pH 7.4, respectively. Data are plotted as the means ± SD ( P -values as indicated or *** P < 0.001, **** P < 0.0001); N indicates the number of independent experiments and n indicates the number of biological replicates (when >1). Significance was determined by a two-sided Student’s t -test with FDR adjustment ( d ), one-way ANOVA with Tukey’s multiple comparison test ( f ), or two-sided Student’s t -test ( g ). Source data are provided as a Source Data file.

Article Snippet: The staining was performed with PE-coupled anti-CA9 antibody (130-123-340, Miltenyi, 1:200) for 20 min at 4 °C.

Techniques: FACS, Immunostaining, Expressing, RNA Sequencing, Comparison

Journal: Nature Communications

Article Title: Acid-exposed and hypoxic cancer cells do not overlap but are interdependent for unsaturated fatty acid resources

doi: 10.1038/s41467-024-54435-3

Figure Lengend Snippet: a , b Representative contrast phase pictures ( a ) and quantification ( b ) of the effects of CRISPR-Cas9-based SCD1 gene invalidation on FaDu spheroid growth at day 7 post-formation (vs. control sgRNA); N = 3, n = 2. c – e Quantification of SCD1 (violet) ( c ) ( N = 5) and CA9 (red) immunostaining ( d ) ( N = 5), and representative pictures ( e ) of spheroids made of CRISPR-Cas9-based SCD1-silenced FaDu cells at day 10 post-formation. f , g Spheroid growth ( f ) ( N = 3, n = 3–4) and cytotoxicity (Incucyte Cytotox Green reagent) follow up ( g ) ( N = 3, n = 4) after exposure to SCD1 inhibitor (or vehicle) for 72 h in FaDu spheroids. h , i Representative pictures ( h ) and quantification of CA9 (red) ( i ) in sections of FaDu spheroids collected after 72 h exposure to SCD1 inhibitor ( N = 3, n = 2). j Flow cytometry analysis of CA9 labeling from FaDu cells isolated from spheroids after 72 h exposure to SCD1 inhibitor (or vehicle) ( N = 3). k Quantification of pimonidazole (green) in sections of FaDu spheroids collected after 72 h exposure to SCD1 inhibitor ( N = 3, n = 2). l , m Mitochondrial oxygen consumption rate (OCR) of l FaDu ( N = 3, n = 7) and HCT116 ( N = 3, n = 7–8) spheroids after 72 h SCD1 inhibition and ( m ) FaDu cancer cells transduced with the indicated SCD1 sgRNA ( N = 3, n = 3) or control sgRNA ( N = 3, n = 3–4). n Non-mitochondrial OCR of FaDu cells transduced with an SCD1-expressing vector or control plasmid and exposed to palmitate ( N = 3, n = 3) or vehicle (FA-free BSA) ( N = 3, n = 4). o – r Representative pictures ( o ) and quantification of SCD1 (violet) ( p ), CA9 (red) ( q ), and pimonidazole (green) ( r ) staining in FaDu spheroids exposed to PPAR-γ inhibitor (or vehicle) for 72 h ( N = 3); the effects of a PPAR-α inhibitor are also shown in graphs ( p – r ). All treatments ( f – l ) with SCD1 inhibitor (A939572, 32 µM) were initiated at day 7 after spheroid formation (i.e., timing 0 on graphs). All immunostaining quantifications ( c , d , i , k , p – r ) were normalized to the DAPI nuclear staining area. Data are plotted as the means ± SD ( P -values as indicated or *** P < 0.001, **** P < 0.0001); N indicates the number of independent experiments and n indicates the number of biological replicates (when >1). Significance was determined by one-way ANOVA with Tukey’s multiple comparison tests ( b – d ), Dunnet’s multiple comparison tests ( p – r ), Sidaks’s multiple comparison tests ( f , g ), two-way ANOVA with Tukey’s multiple comparison tests ( l – n ) or two-sided Student’s t -tests ( i – k ). Source data are provided as a Source Data file.

Article Snippet: The staining was performed with PE-coupled anti-CA9 antibody (130-123-340, Miltenyi, 1:200) for 20 min at 4 °C.

Techniques: CRISPR, Control, Immunostaining, Flow Cytometry, Labeling, Isolation, Inhibition, Transduction, Expressing, Plasmid Preparation, Staining, Comparison

Journal: Nature Communications

Article Title: Acid-exposed and hypoxic cancer cells do not overlap but are interdependent for unsaturated fatty acid resources

doi: 10.1038/s41467-024-54435-3

Figure Lengend Snippet: a , b Representative pictures ( a ) and quantification ( b ) of colorectal cancer patient-derived organoids after treatment with SCD1 inhibitor (A939572, 20 µM) in the presence of OA (100 µM) and/or DHA (50 µM) for 96 h ( N = 2, n = 2). c , d Effect of SCD1 inhibitor (12 µM) on the viability (96 h) ( c ) and lipid peroxidation BODIPY-C11 staining (72 h) ( d ) of 6.5/Fadu ( N = 3, n = 2) and 6.5/HCT116 ( N = 3, n = 4) cancer cells in the presence of OA (100 µM) and/or DHA (50 µM) and/or α-Tocopherol (10 µM) or vehicle(s). e Representative pictures of CA9 immunofluorescence signal (red) in equatorial sections of FaDu and HCT116 spheroids exposed for 72 h to SCD1 inhibitor (A939572, 32 µM) in the presence of OA (100 µM) or not; this experiment was repeated twice with similar results. f Representative flow chart depicting the effects of SCD1 inhibitor on the uptake of TopFluor oleate by 6.5/Fadu and 6.5/HCT116 cancer cells maintained under normoxia or hypoxia (1% O 2 ) ( N = 4, n = 4). g Schematic representation of the symbiotic relationship between hypoxic cancer cells (unable to synthesize MUFA and thus dependent on exogenous MUFA) and acidic, non-hypoxic cancer cells that may use both MUFA sources. Inhibition of SCD1 however forces the latter cell compartment to capture exogenous MUFA thereby depriving hypoxic cells from a vital source of MUFA. h Viability of FaDu and HCT116 cancer cells maintained under hypoxia (1% O 2 ) and exposed for 48 h to the conditioned medium (CM) from normoxic 6.5/FaDu and 6.5/HCT116 exposed or not to SCDi (24 h, 15 and 25 µM A939572, respectively) ( N = 3, n = 7). In some experiments, CM was supplemented by oleate (50 µM) ( N = 3, n = 4). Control conditions consist of CM + fresh addition of SCDi ( N = 3, n = 7), and non-conditioned medium (NCM) ( N = 3, n = 3). i , j MUFA amounts ( i ) and SFA/MUFA ratio ( j ) determined in the conditioned medium (CM) of normoxic 6.5/FaDu and 6.5/HCT116 cancer cells exposed or not to SCDi as above ( N = 2). Data are plotted as the means ± SD (ns: non-significant, P -values as indicated or *** P < 0.001, **** P < 0.0001); N indicates the number of independent experiments and n indicates the number of biological replicates (when >1). Significance was determined by two-way ANOVA with Tukey’s multiple comparison test ( b – h ). Source data are provided as a Source Data file.

Article Snippet: The staining was performed with PE-coupled anti-CA9 antibody (130-123-340, Miltenyi, 1:200) for 20 min at 4 °C.

Techniques: Derivative Assay, Staining, Immunofluorescence, Inhibition, Control, Comparison