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: Oncogene

Article Title: The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion

doi: 10.1038/onc.2017.219

Figure Lengend Snippet: Identification of the CAIX Interactome using the BioID proximal ligation strategy and validation of high confidence proximal CAIX-associating proteins by co-immunoprecipitation. ( a ) Schematic showing the domain structure of the CAIX-BirA* fusion protein used for the BioID studies. SP, signal peptide; PG, proteoglycan-like domain; CA, catalytic domain; TM, transmembrane domain; IC, intracellular domain. ( b ) FACS analysis showing levels of constitutive expression of the CAIX-BirA* fusion protein expressed by MDA-MB-231 cells (MDA-MB-231-CAIX-BirA*) cultured in normoxia compared to levels of endogenous CAIX expression by WT MDA-MB-231 cells cultured in either normoxia or hypoxia. The filled (gray) histograms indicate specific CAIX staining whereas the dotted lined histograms indicate staining by isotype-specific control IgG. ( c ) Image showing exogenous CAIX (red) localized at the cell surface of MDA-MB-231-CAIX-BirA* cells grown in normoxia. Scale bar, 10 μ M . ( d – g ) Co-IP of high confidence proximal CAIX-associating proteins ( d ) ITGB1, ( e ) ITGA2 ( f ) CD98hc and ( g ) MMP14 with exogenous CAIX expressed by MDA-MB-231-CAIX-BirA* cells cultured in normoxia. ( h – j ) Co-IP of ( h ) ITGB1, ( i ) ITGA2 and ( j ) MMP14 with endogenous CAIX expressed by WT MDA-MB-231 cells cultured in either normoxia (N) or hypoxia (H). Isotype-specific IgG was used as a control for co-IPs and data in ( d ) to ( j ) are representative of at least two independent experiments. IgG, immunoglobulin G.

Article Snippet: Indicated concentrations of recombinant CAIX (Cat no. 2188-CA, R&D systems) were pre-incubated with activated MMP14 (Recombinant pro-MMP14, Cat.no.

Techniques: Ligation, Biomarker Discovery, Immunoprecipitation, Expressing, Cell Culture, Staining, Control, Co-Immunoprecipitation Assay

Journal: Oncogene

Article Title: The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion

doi: 10.1038/onc.2017.219

Figure Lengend Snippet: Selected CAIX-BirA*FLAG high-confidence proximal associating proteins identified by BioID in MDA-MB-231 cells

Article Snippet: Indicated concentrations of recombinant CAIX (Cat no. 2188-CA, R&D systems) were pre-incubated with activated MMP14 (Recombinant pro-MMP14, Cat.no.

Techniques:

Journal: Oncogene

Article Title: The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion

doi: 10.1038/onc.2017.219

Figure Lengend Snippet: CAIX colocalizes with integrins and MMP14 in pseudopodia-like protrusions resembling lamellipodia. ( a ) Images of MDA-MB-231-CAIX-BirA* cells cultured on type 1 collagen in normoxia, showing colocalization (yellow, arrows in merge) of exogenously expressed CAIX (red) with ITGB1, ITGA2, MMP14 and cofilin (each marker in green), but not with FAK (green; arrowheads). Scale bar, 10 μm. ( b ) Images of WT MDA-MB-231 cells grown in either normoxia or hypoxia, showing colocalization of hypoxia-induced CAIX (red) with F-actin (green) in pseudopodia-like protrusions (arrows, merge). Scale bar, 10 μm. ( c ) Images of WT MDA-MB-231 cells cultured on type 1 collagen in hypoxia, showing colocalization of hypoxia-induced CAIX (red) with F-actin, ITGA2 and MMP14 (green) at leading edges of cells (arrows, merge), but not at focal adhesions. Scale bar, 10 μm. ( d and e ) 3D maximum projections of 2D images of MDA-MB-231-CAIX-BirA* cells transfected with GFP and cultured on type 1 collagen in normoxia, showing robust colocalization of CAIX (red) with ( d ) ITGA2 and ( e ) MMP14 (blue) in pseudopodia-like protrusions and depletion of cytoplasmic GFP (green) from these regions. Images for each channel are shown in the upper panels and merged images are shown in the lower panels. Scale bar, 10 μm.

Article Snippet: Indicated concentrations of recombinant CAIX (Cat no. 2188-CA, R&D systems) were pre-incubated with activated MMP14 (Recombinant pro-MMP14, Cat.no.

Techniques: Cell Culture, Marker, Transfection

Journal: Oncogene

Article Title: The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion

doi: 10.1038/onc.2017.219

Figure Lengend Snippet: CAIX regulates migration of breast cancer cells. ( a ) Images of wound-induced cell migration by the indicated MDA-MB-231 cells cultured in normoxia. Scale bar, 300 μm. Dashed lines demarcate the wound boundary at t =0 h and yellow shading indicates the cell-free area. ( b ) Quantification of migration by the cells described in a . *** P <0.001. ( c ) Images of wound-induced migration of MDA-MB-231 CAIX-BirA* cells cultured in normoxia and treated with increasing concentrations of U-104. Scale bar, 300 μm. Wound boundaries and cell-free area are demarcated as described in a . ( d ) Quantification of migration by the cells described in c . * P <0.05, *** P <0.001. Data in panels ( b ) and ( d ) show the mean±s.e.m. of technical replicates ( n =16) and are representative of two independent experiments. ( e ) Wound-induced migration of WT MDA-MB-231 cells cultured in normoxia (black bars) or hypoxia (gray bars) and treated with U-104. Data show the mean±s.e.m. of technical replicates ( n =12) and are representative of three independent experiments. ** P <0.01, *** P <0.001. Statistical analysis was performed using Student’s t -test ( b ) or ANOVA ( d , e ).

Article Snippet: Indicated concentrations of recombinant CAIX (Cat no. 2188-CA, R&D systems) were pre-incubated with activated MMP14 (Recombinant pro-MMP14, Cat.no.

Techniques: Migration, Cell Culture

Journal: Oncogene

Article Title: The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion

doi: 10.1038/onc.2017.219

Figure Lengend Snippet: CAIX colocalizes with MMP14 at invadopodia and regulates type I collagen degradation. ( a ) Images of a MDA-MB-231-CAIX-BirA* cell (white dashed line shows boundary) cultured on type I collagen in normoxia, identifying a ROI (yellow box) containing invadopodia (arrows) labeled for Cortactin (cyan), CAIX (red) and MMP14 (green). Profile plots showing colocalization of CAIX and MMP14 at cortactin-positive invadopodia (1 and 2) along the indicated lines (insets) are shown at the right. Scale bar, 10 μm. ( b ) Images of an MDA-MB-231-CAIX-BirA* cell (white dashed line shows cell boundary) cultured on DQ type I collagen in normoxia, identifying an ROI (yellow box) containing regions of collagen degradation (1 and 2, arrows) that are labeled for DQ collagen (cyan), CAIX (red) and MMP14 (green). Profile plots showing colocalization of the signals along the indicated lines (insets) are shown to the right. Scale bar, 10 μm. ( c ) Images showing WT MDA-MB-231 cells cultured in hypoxia on fluorescently labeled gelatin (blue, with black areas denoting regions of degradation) and stained for CAIX (green) and markers of invadopodia (red), including cortactin, Tks5 and MMP14 to demonstrate colocalization at mature invadopodia (white arrows; yellow foci). Scale bars, 10 μm (left), 2 μm (right). ( d ) Quantification of DQ collagen degradation by the indicated MDA-MB-231 cell lines grown in normoxia. *** P <0.001. ( e ) Quantitation of degradation of DQ collagen by MDA-MB-231 CAIX KO and CAIX rescue cells cultured in normoxia. * P <0.05. Data in ( d ) and ( e ) show the mean±s.e.m. of technical replicates ( n =5) and are representative of three independent experiments. ( f ) Dose-dependent inhibition of DQ collagen degradation by CAIX rescue cells treated with U-104. Data show the mean±s.e.m. of technical replicates ( n =4) and are representative of two independent experiments. * P <0.05, ** P <0.01. ( g ) Quantification of DQ collagen degradation by WT MDA-MB-231 and CAIX KO cells cultured in hypoxia. Data show the mean±s.e.m. of technical replicates ( n =8) and are representative of three independent experiments. ** P <0.01. ( h and i ) Invasion through Matrigel by highly metastatic MDA-MB-231 LM2-4 breast cancer cells cultured in hypoxia and ( h ) expressing CAIX-targeted (shCAIX) shRNA or ( i ) treated with U-104. Data show the mean±s.e.m. of three independent experiments. *** P <0.001. Western blots of hypoxia-induced CAIX expression are shown as insets.

Article Snippet: Indicated concentrations of recombinant CAIX (Cat no. 2188-CA, R&D systems) were pre-incubated with activated MMP14 (Recombinant pro-MMP14, Cat.no.

Techniques: Cell Culture, Labeling, Staining, Quantitation Assay, Inhibition, Expressing, shRNA, Western Blot

Journal: Oncogene

Article Title: The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion

doi: 10.1038/onc.2017.219

Figure Lengend Snippet: CAIX regulates breast cancer cell invasion and metastasis. ( a ) Schematic showing the domain structure of wild type (WT) huCAIX (459 aa) and constructs lacking either the intracellular domain (ΔIC), the extracellular proteoglycan-like domain (ΔPG) or the indicated point mutations in the IC domain. SP, signal peptide; CA, catalytic domain; TM, transmembrane domain. ( b ) Western blot analysis of the levels of expression of the indicated huCAIX constructs by 4T1shCAIX cells cultured in hypoxia. β-actin served as a loading control. ( c ) Analysis of CAIX catalytic activity using the in-cell carbonic anhydrase activity assay. Assays were performed in normoxia in the presence or absence of U-104 (50 μ M ) as indicated. Levels of CAIX catalytic activity were normalized by using the time required to achieve 50% of the total decrease in pH. Data were normalized to the spontaneous rate of reaction in the presence of buffer alone and the activity of cells expressing WT huCAIX was set to 100%. Data show the mean±s.e.m. of technical replicates ( n =3/group) and are representative of three independent experiments * P <0.05, *** P <0.001. ( d ) Invasion through Matrigel by the indicated 4T1 cell lines cultured in hypoxia. Data show the mean±s.e.m. of three independent experiments. * P <0.05, *** P <0.001. ( e ) Invasion through Matrigel by the 4T1shCAIX cell lines expressing WT, ΔIC and ΔPG variants of huCAIX and cultured in hypoxia. * P <0.05, *** P <0.001. ( f ) Invasion through Matrigel by 4T1 sh/WT huCAIX cells cultured in hypoxia and treated with U-104. *** P <0.001. Western blots of hypoxia-induced CAIX expression are shown as insets. For ( d )– ( f ), data show the mean±s.e.m. of three independent experiments. ( g – i ) Analysis of invasion through type 1 collagen by 4T1 sh/WT huCAIX cells cultured in hypoxia and treated with ( g ) U-104 (50 μ M ), ( h ) anti-MMP14 antibody (20 μg/ml) or ( i ) a combination of anti-MMP14 antibody (20 μg/ml) and U-104 (50 μ M ). * P <0.05, ** P <0.01. Data in ( g )–( i ) show the mean±s.e.m. of three independent experiments. ( j ) Analysis of spontaneous lung metastases formed by the indicated 4T1 cell lines following growth of orthotopic breast tumors. Data show the mean±s.e.m. n =6/group. ( k ) Analysis of experimental lung metastases formed by the indicated 4T1 cell lines. Mean±s.e.m. is shown. n =6/group. * P <0.05, ** P <0.01. Statistical analysis was performed using ANOVA ( c , d , e , k ) or Student’s t -test ( f , g , h ).

Article Snippet: Indicated concentrations of recombinant CAIX (Cat no. 2188-CA, R&D systems) were pre-incubated with activated MMP14 (Recombinant pro-MMP14, Cat.no.

Techniques: Construct, Western Blot, Expressing, Cell Culture, Control, Activity Assay

Journal: Oncogene

Article Title: The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion

doi: 10.1038/onc.2017.219

Figure Lengend Snippet: CAIX associates with MMP14 through its intracellular domain and potentiates MMP14 activity. ( a ) Co-IP of huCAIX and MMP14 from 4T1shCAIX cells cultured in hypoxia and expressing WT, ΔIC or ΔPG variants of huCAIX. ( b ) Co-IP of huCAIX and MMP14 from 4T1shCAIX cells cultured in hypoxia and expressing WT huCAIX or the T443A point mutant. ( c ) Co-IP of huCAIX and MMP14 from 4T1shCAIX cells cultured in hypoxia and expressing WT or the Y449A point mutant. ( d ) Co-IP of huCAIX and MMP14 from 4T1shCAIX cells grown in hypoxia and expressing WT huCAIX, the S448A point mutant or the ΔPG mutant. ( e ) Co-IP of huCAIX and MMP14 from 4T1shCAIX cells in hypoxia expressing WT huCAIX, the ΔIC truncation or the double point mutant S448A+Y449A. Isotype-specific IgG was used as a control for co-IPs. ( f ) Time-dependent stimulation of MMP14 activity in the presence (red bars) or absence (black bars) of equimolar concentrations of CAIX. Data show the mean±s.e.m. of technical replicates ( n =5) and are representative of two independent experiments. * P <0.05, ** P <0.01. ( g ) Dose-dependent inhibition of CAIX-mediated stimulation of MMP14 activity in the presence of increasing concentrations of U-104. Mean±s.e.m. of technical replicates ( n =5). Data are representative of two independent experiments. *** P <0.001. ( h ) Inhibition of CAIX-mediated stimulation of MMP14 activity in the presence of increasing amounts of D 2 O (heavy water). Mean±s.e.m. of technical replicates ( n =5). Data are representative of three independent experiments. *** P <0.001. Statistical analysis was performed using Student’s t -test ( f ) or ANOVA ( g , h ).

Article Snippet: Indicated concentrations of recombinant CAIX (Cat no. 2188-CA, R&D systems) were pre-incubated with activated MMP14 (Recombinant pro-MMP14, Cat.no.

Techniques: Activity Assay, Co-Immunoprecipitation Assay, Cell Culture, Expressing, Mutagenesis, Control, Inhibition

Journal: Oncogene

Article Title: The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion

doi: 10.1038/onc.2017.219

Figure Lengend Snippet: CAIX stimulates the activity of MMP14. Model for CAIX-mediated regulation of MMP14 activity. CAIX, which is upregulated in hypoxia, associates through its intracellular domain with MMP14 at functional invadopodia. At these invasive structures, CAIX functions to potentiate MMP14-mediated collagen degradation by directly contributing H + ions required for MMP14 catalytic activity.

Article Snippet: Indicated concentrations of recombinant CAIX (Cat no. 2188-CA, R&D systems) were pre-incubated with activated MMP14 (Recombinant pro-MMP14, Cat.no.

Techniques: Activity Assay, Functional Assay

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