Journal: Cell Reports Methods

Article Title: MPA PASS software enables stitched multiplex, multidimensional EV repertoire analysis and a standard framework for reporting bead-based assays

doi: 10.1016/j.crmeth.2021.100136

Figure Lengend Snippet: Detection antibodies used

Article Snippet: CA9 , Miltenyi Biotec , Cat# 130-110-058; RRID: AB_2651327.

Techniques: Labeling, Recombinant

Journal: Cell Reports Methods

Article Title: MPA PASS software enables stitched multiplex, multidimensional EV repertoire analysis and a standard framework for reporting bead-based assays

doi: 10.1016/j.crmeth.2021.100136

Figure Lengend Snippet:

Article Snippet: CA9 , Miltenyi Biotec , Cat# 130-110-058; RRID: AB_2651327.

Techniques: Clinical Proteomics, Recombinant, Saline, Modification, Software

Journal: Cell Death & Disease

Article Title: Sanguinarine inhibits epithelial–mesenchymal transition via targeting HIF-1α/TGF-β feed-forward loop in hepatocellular carcinoma

doi: 10.1038/s41419-019-2173-1

Figure Lengend Snippet: a MHCC-97H and SMMC-7721 cells were treated with 100 μM CoCl 2 or incubated in 1% O 2 for 24 h. HIF1A, CA9, VEGF, and TGFB1 mRNA expression values were assessed by RT-qPCR. Gene expression is normalized to ACTB. MHCC-97H and SMMC-7721 cells were treated with 100 μM CoCl 2 or incubated in 1% O 2 for 24 or 48 h. b TGF-β secretion was determined by ELISA. Mean + SEM ( n = 5), * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, one-way ANOVA followed by Bonferroni posttest in comparison with control. c HIF-1α, ARNT, PHD2, CA9, and TGF-β (SN, supernatant) protein levels were determined by western blotting. Quantification plots are shown below. d MHCC-97H and SMMC-7721 cells were stimulate d with 10 ng/mL TGF-β for 24 h. HIF1A, CA9, and VEGF mRNA expression values were assessed by RT-qPCR. Gene expression is normalized to ACTB. e MHCC-97H and SMMC-7721 cells were stimulat e d with 0, 5, and 10 ng/mL TGF-β for 24 h. HIF-1α, ARNT, PHD2 and VEGF, and CA9 protein levels were examined by western blotting. Quantification plots are shown on the right. f MHCC-97H and SMMC-7721 cells were treated with 100 μM CoCl 2 in the presence of absence of 0.5 μM TGF-β receptor inhibitor LY2157299. HIF1A expression was assessed by RT-qPCR. Gene expression is normalized to ACTB and control group was assigned as 1. g MHCC-97H and SMMC-7721 cells were treated with 100 μM CoCl 2 in the presence of absence of 0.5 μM TGF-β receptor inhibitor LY2157299. HIF-1α and CA9 were determined by western blotting. Quantification plots are shown below. h MHCC-97H and SMMC-7721 cells were treated with 100 μM CoCl 2 in the presence of absence of 0.5 μM LY2157 2 99 for 48 h. LY2157299 was removed and treated with 5 ng/mL TGF-β for additional 24 h. HIF1A expression was assessed by RT-qPCR. Gene expression is normalized to ACTB and shown as fold change. Data were expressed as mean ± SEM ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 compared with untreated control cells or as indicated. Data are representative of three independent experiments.

Article Snippet: E-cadherin, N-cadherin, Vimentin, Snail, VEGF, ARNT, CA9, PHD2, TGF-β rabbit mAb, Phospho-AKT, HIF-1α, GAPDH, and β-actin mouse mAb were obtained from Protein Technology Group (Chicago, Illinois, USA), and PI3K-p110α, PI3K-p110β, Phospho-p85, Phospho-Smad2/3, and α-SMA rabbit mAb were purchased from Cell Signaling (Boston, Massachusetts, USA).

Techniques: Incubation, Expressing, Quantitative RT-PCR, Gene Expression, Enzyme-linked Immunosorbent Assay, Comparison, Control, Western Blot

Journal: Cell Death & Disease

Article Title: Sanguinarine inhibits epithelial–mesenchymal transition via targeting HIF-1α/TGF-β feed-forward loop in hepatocellular carcinoma

doi: 10.1038/s41419-019-2173-1

Figure Lengend Snippet: a MHCC-97H cells were treated with sanguinarine in the absence or presence of 1% O 2 for 12 h. HIF-1α (red), ARNT (green), DAPI (blue) staining, and 3-channel merged images indicated the nuclear translocation of HIF-1α. Scale bars, 200 μm. MHCC-97H and SMMC-7721 cells were treated with different concentrations of sanguinarine in the absence or presence of 1% O 2 or 100 μM CoCl 2 for 24 h. b HIF-1α, CA9, VEGF, N-cadherin, and Vimentin expression levels were assessed by western blotting. Quantification plots are shown on the right. Data were expressed as mean ± SEM ( n = 3). The results shown were representative of three independent experiments. c VEGF and d TGF-β production was analyzed by ELISA. Data are represented as mean + SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, one-way ANOVA followed by Bonferroni posttest in comparison with control. # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001 one-way ANOVA followed by Bonferroni posttest in comparison with CoCl 2 or 1% O 2 samples.

Article Snippet: E-cadherin, N-cadherin, Vimentin, Snail, VEGF, ARNT, CA9, PHD2, TGF-β rabbit mAb, Phospho-AKT, HIF-1α, GAPDH, and β-actin mouse mAb were obtained from Protein Technology Group (Chicago, Illinois, USA), and PI3K-p110α, PI3K-p110β, Phospho-p85, Phospho-Smad2/3, and α-SMA rabbit mAb were purchased from Cell Signaling (Boston, Massachusetts, USA).

Techniques: Staining, Translocation Assay, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Comparison, Control

Journal: Cell Death & Disease

Article Title: Sanguinarine inhibits epithelial–mesenchymal transition via targeting HIF-1α/TGF-β feed-forward loop in hepatocellular carcinoma

doi: 10.1038/s41419-019-2173-1

Figure Lengend Snippet: a HepG2 cells were treated with different concentrations of sanguinarine in the absence or presence of 10 ng/mL TGF-β for 24 h. Snail (green), α-SMA (green), p-Smad2/3 (green), DAPI (blue) staining and 2-channel merged images indicated the nuclear translocation of Snail and p-Smad2/3, and expression of α-SMA. The results shown were representative of three independent experiments. Scale bars, 200 μm. b SMMC-7721 cells were treated with different concentrations of sanguinarine in the absence or presence of 10 ng/mL TGF-β. Protein levels of N-cadherin, Vimentin, and Snail were analyzed by western blotting. Quantification plots are shown below. c MHCC-97H cells were treated with different concentrations of sanguinarine in the absence or presence of 10 ng/mL TGF-β. Protein levels of N-cadherin, Snail, HIF-1α, ARNT, PHD2, and CA9 were analyzed by western blotting. GAPDH or β-actin served as controls. Quantification plots are shown below. Western blot analysis of p100α, p110β, p-p85, and p-AKT expression were demonstrated in d HepG2 and e SMMC-7721 cells treated with indicated concentrations of sanguinarine in the absence or presence of 10 ng/mL TGF-β for 48 h. Quantification plots are shown below. Data were expressed as mean ± SEM ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 compared with untreated control cells. The results shown were representative of three independent experiments. # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001 one-way ANOVA followed by Bonferroni posttest in comparison with TGF-β-treated samples.

Article Snippet: E-cadherin, N-cadherin, Vimentin, Snail, VEGF, ARNT, CA9, PHD2, TGF-β rabbit mAb, Phospho-AKT, HIF-1α, GAPDH, and β-actin mouse mAb were obtained from Protein Technology Group (Chicago, Illinois, USA), and PI3K-p110α, PI3K-p110β, Phospho-p85, Phospho-Smad2/3, and α-SMA rabbit mAb were purchased from Cell Signaling (Boston, Massachusetts, USA).

Techniques: Staining, Translocation Assay, Expressing, Western Blot, Control, Comparison

Journal: bioRxiv

Article Title: Chromosome-Specific Aneuploidy Engineering via dCas9-Induced Centromeric Chromatin Relaxation

doi: 10.1101/2025.04.25.650684

Figure Lengend Snippet: a , Schematics showing the experiment setup of Chr3 aneuploidy engineering using CRISPRt with Chr3-sg2 (Chr3-Ct) or NT-sg1 (CTRL), and VHL allele-specific KO ( VHL -ASK) in K1088N PTECs. Cells were cultured for 7 days (D7) or 11 days (D7+4) after treatments. Cells with Chr3-Ct or VHL -ASK on D7+4 were selected for CA9-positive (CA9+) cells with flow sorting. The six samples fixed for scRNA-seq are marked in red. b, Heatmaps showing single-cell chromosome copy number profiles inferred from scRNA-seq data of the indicated samples. The number of cells analysed (n) is shown. Rows represent individual cells and columns represent chromosomes. Copy number status is shown in blue (loss), red (gain) or white (neutral). c, Violin plots showing InferCNV residual expression for Chr3p and Chr3q of the indicated samples. The dots represent individual cells. The blue and red dashed lines represent the thresholds (see Methods) for copy number loss and gain, respectively. The cell proportions with the indicated copy number status are shown in the tables.

Article Snippet: Cells were stained with 1:50 diluted anti-CA9-PE antibody (Miltenyi, 130-131-321) in flow sorting buffer (1% bovine serum albumin (BSA) and 1μM EDTA in PBS) for 30 min at 4 °C in the dark.

Techniques: Cell Culture, Expressing

Journal: bioRxiv

Article Title: Chromosome-Specific Aneuploidy Engineering via dCas9-Induced Centromeric Chromatin Relaxation

doi: 10.1101/2025.04.25.650684

Figure Lengend Snippet: a , Schematics showing the expected genotypes generated after Chr3-Ct or VHL -ASK in K1088N PTECs with the corresponding VHL status annotated ( VHL + for VHL proficient, and VHL- for VHL loss) and the target genotypes with VHL loss enriched by CA9-positive (CA9+) flow sorting. b, Scatter plots showing the CA9 and DAPI staining intensities of K1088N PTECs after the indicated treatment. The rectangular box shows the gating strategy to flow sort for live (DAPI-) CA9-positive (CA9+) cells. c, Violin plots showing the signature scores of a curated HIF pathway activation gene set (see Methods) in K1088N PTECs after the indicated treatment. The dots represent individual cells. The dashed line represents the calculated threshold (see Methods) for defining cells with a high (HIF-High) or a low (HIF-Low) HIF pathway signature score, used for assigning VHL proficient ( VHL +) or VHL loss ( VHL- ) status.

Article Snippet: Cells were stained with 1:50 diluted anti-CA9-PE antibody (Miltenyi, 130-131-321) in flow sorting buffer (1% bovine serum albumin (BSA) and 1μM EDTA in PBS) for 30 min at 4 °C in the dark.

Techniques: Generated, Staining, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: PIMT Binding to C-Terminal Ala459 of CAIX Is Involved in Inside-Out Signaling Necessary for Its Catalytic Activity

doi: 10.3390/ijms21228545

Figure Lengend Snippet: C -terminal amino acid Ala459 residue cooperates in the regulation of CAIX catalytic function: ( a ) Immunoblot analysis of cell lysates from C33a cells transfected with mock control, CAIX wild type (wt) or mutant (A459G) cultured 48 h in hypoxia. CAIX was detected using mouse monoclonal antibody M75 diluted 1:10 and β-actin was detected using mouse monoclonal antibody (CS3700, Cell Signaling Technology, Danvers, Massachusetts) diluted 1:5000. HRP-conjugated anti-mouse antibody (Dako Agilent, Santa Clara, California) diluted 1:5000 was used as a secondary antibody. ( b ) Fluorescence staining of CAIX in non-fixed and non-permeabilized C33a-CAIX wild type as well as A459G transfectants measured by flow cytometry. CAIX was detected using PG-domain specific mouse monoclonal antibody M75 diluted to 1 µg/mL and AlexaFluor 488-conjugated anti-mouse secondary antibody (Invitrogen, Carlsbad, California) diluted 1:1000. Results clearly demonstrate plasma membrane localization of CAIX and showed that 42.7% of C33a-CAIX-wt and 50.9% of C33a-CAIX-A459G transfectants expressed CAIX protein. C33a cells transfected with mock control plasmid were used as a negative control. The data are presented as the mean, n = 2. ( c ) Effect of Ala459 mutation on CAIX-mediated extracellular acidification. The graph shows the differences between pHe values (ΔpH) of culture media from CAIX wt or A459G-transfected and mock-transfected cells cultured 48 h in hypoxia. A459G mutant reduced acidification of extracellular pHe when compared to control wild type C33a-CAIX transfectants. The data are presented as the mean ± s.d., n = 5. Statistical significance was analyzed using the Student’s t -test and expressed as a p -value (* p < 0.05). ( d ) Effect of Ala459 mutation on migration capacity of C33a cells. The graph depicts the results of the wound healing assay given as a % of the area covered by cells migrating to close the wound at 30 h after the scratch, measured at various positions along the wounds. Area covered by C33a cells expressing CAIX-wt was set as 100%. C33a cells expressing CAIX with mutated Ala459 exhibited slower migration. The data are presented as the mean ± s.d., n = 10. Statistical significance was analyzed using the Student’s t -test and expressed as a p -value (* p < 0.05). ( e ) Accumulation of the fluorescent sulfonamide (FITC-CA-i) occurred in hypoxic MDCK cells expressing the CAIX-wt, whereas it was diminished in hypoxic MDCK cell transfected with the CAIX-A459G mutant. Images were taken using objective 10×. ( f ) In situ detection of the interaction between CAIX and AE2 using a proximity ligation assay (PLA). Analysis was performed in C33a cells transiently transfected with CAIX-wt and A459G mutant and cultured in hypoxia for an additional 48 h. Red PLA signal indicates the existence of interaction or close proximity localization also between CAIX with mutated Ala459 and AE2. C33a-mock transfectants served as a negative assay control due to a lack of one target protein. CAIX protein was post-labelled in green, nuclei are blue. Images were taken using objective 40× and zoom 3.

Article Snippet: After 48 h of hypoxic incubation, transiently transfected cells were scraped into culture medium, centrifuged at low speed, washed twice with Versene solution and incubated with the CAIX-specific mouse monoclonal M75 antibody (1 µg/mL, BMC SAS, Bratislava, Slovakia) for 30 min at 4 °C.

Techniques: Western Blot, Transfection, Mutagenesis, Cell Culture, Fluorescence, Staining, Flow Cytometry, Plasmid Preparation, Negative Control, Migration, Wound Healing Assay, Expressing, In Situ, Proximity Ligation Assay