Journal: PLOS Pathogens

Article Title: Differential carbonic anhydrase activities control EBV-induced B-cell transformation and lytic cycle reactivation

doi: 10.1371/journal.ppat.1011998

Figure Lengend Snippet: Key resources table.

Article Snippet: Chemical compound, drug , S4 , MedChemExpress , Cat No.: HY-110243 , Carbonic anhydrase inhibitor.

Techniques: In Vitro, Transformation Assay, Generated, Control, Plasmid Preparation, Sequencing, Clone Assay, Binding Assay, Mutagenesis, Polymerase Cycling Assembly, Expressing, Staining, Immunofluorescence, Transduction, Selection, Stable Transfection, Transfection, Software

Journal: Acta Pharmaceutica Sinica. B

Article Title: An immunostimulant nanomedicine enhances radioimmunotherapy by remodeling the tumor immunosuppressive landscape after radiotherapy

doi: 10.1016/j.apsb.2025.11.012

Figure Lengend Snippet: Immunosuppression after radiotherapy promotes breast cancer progression. Tumor growth curves (A) and weights (B) of mice before and after RT ( n = 6). (C) Survival curve of mice after different treatments ( n = 6). Tumor growth curves (D) and weights (E) of mice treated with RT and RT plus α -PD-L1 ( n = 6). (F) Survival curve of mice after different treatments ( n = 6). (G) Cluster analysis of differential expression genes between untreated and RT-treated tumors 48 h post-RT ( n = 3). (H) Gene Ontology (GO) analysis of tumor tissues before and after RT (select the top 10 for each item). (I) Heat map of differentially expressed genes related to apoptosis and immune suppression ( n = 3). (J) Immunofluorescence staining images of tumor tissue in saline and RT groups (scale bar = 20 μm). (K) Quantitative analysis of tumor-infiltrating CD45 + cells ( n = 6). Representative flow cytometry images (M) and quantitative analysis (L) of tumor-infiltrating MDSCs ( n = 6). (N) The TUNEL staining of tumor section (scale: 25 μm). (O) Adenosine content detection in tumor tissue ( n = 6). Data are presented as mean ± SD. ∗∗∗∗ P < 0.0001 determined by Student’s t-test.

Article Snippet: InVivo MAb anti-mouse PD-L1 was purchased from Bioxcell (USA).

Techniques: Quantitative Proteomics, Immunofluorescence, Staining, Saline, Flow Cytometry, TUNEL Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: An immunostimulant nanomedicine enhances radioimmunotherapy by remodeling the tumor immunosuppressive landscape after radiotherapy

doi: 10.1016/j.apsb.2025.11.012

Figure Lengend Snippet: FD@ATRA-enhanced radiotherapy combined with α -PD-L1 to enhance the efficacy of large-volume tumors. (A) Schematic diagram of the therapeutic process for evaluating the anti-tumor effects in a bilateral 4T1 tumor-bearing mouse model of large volume. (B) Distant tumor growth curves of mice after different treatments ( n = 5). (C) In vitro images of the distant tumors ( n = 5). (D) In vitro distant tumors mass ( n = 5). (E) Immunohistochemical staining of CD3 in tumor tissue slices after different treatments (scale bars = 100 μm). The images below were the corresponding enlarged parts (scale bars = 25 μm). (F) Immunohistochemical staining of CD8 in tumor tissue slices after various treatments (scale bars = 100 μm). The images below were the corresponding enlarged parts (scale bars = 25 μm). Data are presented as mean ± SD. ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001 determined by Student’s t -test.

Article Snippet: InVivo MAb anti-mouse PD-L1 was purchased from Bioxcell (USA).

Techniques: In Vitro, Immunohistochemical staining, Staining

Journal: Theranostics

Article Title: Hypoxia-induced TGFBI maintains glioma stem cells by stabilizing EphA2

doi: 10.7150/thno.95141

Figure Lengend Snippet: TGFBI is associated with the hypoxic microenvironment in human gliomas. (A) FISH staining of TGFBI in human GBM specimens (Ivy gap datasets). Scale bars: 2 mm; enlarged image: 100 μm. (B) Volcano map showing genes highly expressed in hypoxia region (pseudopalisade and microvascular proliferation regions). Each dot represents a gene. (C) Correlation between TGFBI and mRNA expression of hypoxia-related gene in the CCGA-GBM and TGGA-GBM datasets. CC, correlation coefficient; Dot size and color represent the correlation coefficient. (D) IF staining of TGFBI (green) and two hypoxia-associated markers, HIF1α (above, red) and CA9 (bottom, red), in human GBM specimens. Scale bars: 50 μm; enlarged image: 10 μm. (E) IHC staining demonstrating the association between TGFBI and HIF1α proteins in human gliomas. AOD, Average of density; n = 58.

Article Snippet: Primary antibodies: TGFBI (Abclonal, Cat#A11222, for IB, 1:1000; for IP, 5ug; Proteintech, Cat#10188-AP, for IHC, 1:100; for IF, 1:100), CD133 (Affinity, Cat#BF0403, for IF, 1:100), SOX2 (Proteintech, Cat#66411-1-Ig, for IB, 1:1000; Santa Cruz, Cat#365823, for IF, 1:50; for IHC, 1:50), HIF1α (Proteintech, Cat#20960-1-AP, for IB, 1:1000; for IF, 1:50; for IHC 1:50), carbonic anhydrase 9 (CA9, NOVUS, Cat#NB100-417, for IF, 1:50), OLIG2 (Proteintech, Cat#66513-1-Ig, for IB, 1:1000; for IF, 1:200), Ki67 (Proteintech, Cat#27309-1-AP, for IF, 1:200), EphA2 (Abclonal, Cat#A7183, for IB,1:1000; Santa Cruz, Cat#398832, for IF, 1:50; for IHC, 1:50; for IP, 5ug), Akt (Proteintech, Cat#60203-1-Ig, for IB, 1:5000), phospho-Akt (Ser473) (Proteintech, Cat#66444-1-Ig, for IB, 1:2000; Cell Signaling Technology, Cat#4060, for IF, 1:100; for IHC, 1:50), c-MYC (Proteintech, Cat#67447-1-Ig, for IB, 1:5000; for IF,1:100; for IHC, 1:100), phospho-EphA2 (Y588) (Proteintech, Cat#30263-1-AP, for IB, 1:1000), Integrin αV (Proteintech, Cat#27096-1-AP, for IB, 1:1000), Integrin β5 (CST, Cat#3629, for IB, 1:1000), Notch1 (Cell Signaling Technology, Cat#3608, for IB, 1:1000).

Techniques: Staining, Expressing, Immunohistochemistry

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

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activation of Carbonic Anhydrase IX by Alternatively Spliced Tissue Factor Under Late-Stage Tumor Conditions

doi: 10.1038/labinvest.2016.103

Figure Lengend Snippet: A) Cell culture conditions for modeling early stage or late stage pancreatic cancer progression. B) Expression of the hypoxia-associated proteins HIF-1α (117 kD), HIF-2α (118 kD), and CAIX (55 kD) in early (norm) or late stage (hyp) environments. β-Actin served as a loading control. HeLa cells treated with 100 μM CoCl 2 were used as a positive control for hypoxia-induced gene expression. “env,” the environment under which the cells were kept for 48 hours before lysing, with “hyp” indicating hypoxic/low glucose and “norm” indicating normoxic/high glucose. The bottom panel shows the over-expression of asTF in Pt45.P1 cells, vimentin , and the loading control β-Actin.

Article Snippet: The separated proteins were then transferred to a PVDF membrane, blocked with 5% nonfat milk, probed with antibodies to asTF (rabbit monoclonal RabMab1) , vimentin (rabbit mAb, Cell Signaling), β-actin (rabbit, Cell Signaling), HIF-1α (rabbit pAb, Bethyl Laboratories), HIF-2α (rabbit pAb, GeneTex) CAIX (mouse mAb, GeneTex), or CAXII (goat pAb, Acris), followed by probing with the corresponding HRP-conjugated secondary antibodies (Bio-Rad) and developed using ECL and H 2 O 2 .

Techniques: Cell Culture, Expressing, Control, Positive Control, Gene Expression, Over Expression