Journal: BMC biology

Article Title: Proton export upregulates aerobic glycolysis.

doi: 10.1186/s12915-022-01340-0

Figure Lengend Snippet: Fig. 1 Over-expression of CA-IX in MCF-7 breast cancer cells increases glycolytic metabolism in vitro. A Overall survival Kaplan–Meier curve in ER-positive luminal B breast cancer comparing low and high CA9 gene expression n = 226 (kmplot.com). Statistical analysis using Log-rank P test p = 0.0433. B Immunoblotting of protein lysates from MCF-7 cells transfected with empty vector (MOCK-2) or Ca9 vector (M1 and M6). Proteins from total cell extracts were immunoblotted for CA-IX, CA-II, CA-XII, and B-actin (loading control). C Representative immunocytochemistry images of CA-IX protein expression in MCF-7, MOCK-2, and CA-IX clones M1 and M6. CA-IX clones (M1 & M6) exhibit CA-IX membrane staining, whereas MOCK-2 and parental MCF-7 cells do not. DAPI nuclear stain (blue), wheat germ agglutinin membrane stain (green), and CA-IX stain (red). D Glucose induced proton production rate (PPR) using the Seahorse extracellular flux analyzer, measured post glucose injection (N = 8 biological replicates per group). E Glucose uptake of cells in each group (n = 3 biological replicates) over 24 h, measured as luminescence generated using Glucose Uptake-Glo assay (Promega). F Lactate measured in extracellular media after 24 h using Sigma kit (n = 3 biological replicates per group). G Basal oxygen consumption rate (OCR) measured using the Seahorse extracellular flux analyzer in 5.8 mM glucose concentration (N = 8 biological replicates per group). D–G Data are shown as mean ± SD, statistical analysis using ordinary one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Article Snippet: Carbonic anhydrase 9 (CA9) construct was designed by Origene based on the sequence(Accession Number: NM_001216; Homo sapiens) and cloned into a pCMV6 vector (PS10001, Origene, MD) to form pCMV6/CA-IX vectors (CQ10630, RC204839 subclone, Origene, MD) in which CA9 gene expression was driven under the CMV promoter.

Techniques: Over Expression, In Vitro, Gene Expression, Western Blot, Transfection, Plasmid Preparation, Control, Immunocytochemistry, Expressing, Clone Assay, Membrane, Staining, Injection, Generated, Glo Assay, Concentration 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