Journal: International Journal of Molecular Sciences

Article Title: Expression of Carbonic Anhydrase I in Motor Neurons and Alterations in ALS

doi: 10.3390/ijms17111820

Figure Lengend Snippet: CA1 is expressed in human spinal cord motor neurons. Images of the normal human spinal cord immune-stained with CA1 or CA2 antibody using the DAB method (brown color) counter-stained with hematoxylin (blue color). The GαCA1 (1:500) and HRP-RbαCA2 (1:500) antibodies were used for this experiment. ( A ) A low magnification image of the ventral horn of spinal cord stained with the CA1 antibody. Two representative motor neurons are indicated by arrows. The white scale bar indicates 0.25 mm; ( B , C ) Higher magnification of spinal cord images stained with the CA1 antibody; ( D , E ) Higher magnification of spinal cord images stained with the CA2 antibody; The black scale bar indicates 50 μm for ( B – E ).

Article Snippet: Both recombinant human CA1 (#2180-CA) and CA2 (#2184-CA) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Expression of Carbonic Anhydrase I in Motor Neurons and Alterations in ALS

doi: 10.3390/ijms17111820

Figure Lengend Snippet: CA1 is differentially regulated from CA2 in ALS spinal cord. ( A ) Western blot analysis of the proteins from either the cytosolic (cyto) or microsomal (mv) fractionation extracted from the spinal cords of the control or ALS subjects probed with CA1 (HRP-GαCA1, 1:5000), CA2 (HRP-RbαCA2, 1:5000), SOD1, and PDI antibodies. An equal amount of proteins were used for each lane for either “cyto” or “mv” fraction; ( B ) Quantitative analyses of the differences in the intensities of immune-reactive signals for each protein between the control and ALS groups. All data points were indicated for each group. The dotted horizontal and solid vertical lines in each group represent “Mean ± SD” of the group value. p values are indicated for each graph, and * indicates p < 0.05.

Article Snippet: Both recombinant human CA1 (#2180-CA) and CA2 (#2184-CA) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot, Fractionation, Control

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of Carbonic Anhydrase 2 Overcomes Temozolomide Resistance in Glioblastoma Cells

doi: 10.3390/ijms23010157

Figure Lengend Snippet: Expression levels of CA2 ( A ), P-gp ( B ), and CD133 ( C ) in patient-matched initial (i) vs. recurrent (r) GBM ( n = 20 from 10 matched patients); ( D ) Representative IF staining of CA2 and stem cell marker SOX2 in three GBM tissue sections. Co-staining of CA2 (red) and SOX2 (green) confirmed CA2 in stem-like cells of GBM patients ( n = 3 from 10 recurrent GBM patients), scale bar: 25 μm. ( E – G ) mRNA expression of GBM related carbonic anhydrase genes CA2 ( E ), CA9 ( F ), and CA12 ( G ) in GBM cell lines U87 and U251 and patient-derived GSCs. Carbonic Anhydrase expression levels were detected by qPCR ( n = 3). For all genes, expression levels determined in U87 cells were set to 1. qPCR results were obtained from three independent experiments. In ( A – C , E – G ), data are presented as mean ± SD, student’s t -test was used to analyze ( A – C ), One-way ANOVA was used to analyze ( E – G ), * p < 0.05; ** p < 0.01; *** p < 0.001, ns: not significant.

Article Snippet: U87 and U251 CA2 expressing cells were generated by transfection with CA2 plasmid (RC201974, ORIGENE, Rockville, MD, USA) using 2.5 μg of plasmid DNA mix Lipofectamine LTX Reagent (15338100, Invitrogen, Waltham, MA, USA).

Techniques: Expressing, Staining, Marker, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of Carbonic Anhydrase 2 Overcomes Temozolomide Resistance in Glioblastoma Cells

doi: 10.3390/ijms23010157

Figure Lengend Snippet: ( A , C ) Generation of stable CA2 overexpressing U87 and U251 cell lines as confirmed by Western Blot and qPCR. ( B , D ) The proliferation of control cells (U87_Ctrl, U251_Ctrl) and CA2 overexpressing (U87_CA2, U251_CA2) cells was measured by CellTiter Glo ( n = 3 independent replicates). ( E ) Invasion of U251_Ctrl (Left panel) and U251_CA2 (Right panel) cells stained with DAPI (scale bar: 75 μm). ( F ) Relative invasion of U251_Ctrl and U251_CA2 cells ( n = 6). ( G , I ) CA2 overexpressing cells have increased oxidative mitochondrial metabolism. The oxygen consumption rate (OCR) was measured by a seahorse XFe96 metabolic-flux analyzer. U87_CA2 cells ( G ) and U251_CA2 cells ( I ) significantly increased mitochondrial basal respiration, maximal respiration, and ATP production ( n = 7–8). ( H , J ) CA2 overexpressing cells have elevated levels of glycolysis rate. The extracellular acidification rates (ECAR) were measured by a seahorse XFe96 metabolic-flux analyzer. U87_CA2 cells ( H ) and U251_CA2 cells ( J ) significantly increased glycolytic activity ( n = 7–8). Results were obtained from three independent experiments. Data are presented as mean ± SD, student’s t -test was used to analyze the data ** p < 0.01; *** p < 0.001, ns: not significant.

Article Snippet: U87 and U251 CA2 expressing cells were generated by transfection with CA2 plasmid (RC201974, ORIGENE, Rockville, MD, USA) using 2.5 μg of plasmid DNA mix Lipofectamine LTX Reagent (15338100, Invitrogen, Waltham, MA, USA).

Techniques: Western Blot, Staining, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of Carbonic Anhydrase 2 Overcomes Temozolomide Resistance in Glioblastoma Cells

doi: 10.3390/ijms23010157

Figure Lengend Snippet: Effect of the pan-CA inhibitor acetazolamide (ACZ) and a potent CA2 inhibitor brinzolamide (BRZ) on invasion and energy metabolism in GBM cell lines. ( A , B ) Molecular structures of ACZ and BRZ. ( C , D ) U251_CA2 cells’ overall metabolism after ACZ and BRZ stimulation was measured by a seahorse XFe96 metabolic-flux analyzer. ACZ and BRZ significantly decreased oxidative metabolism ( C ) and also reduced the level of glycolysis rate ( D ) in U251_CA2 cells ( n = 5–6). U251_CA2 cells significantly decreased mitochondrial basal respiration, maximal respiration, and ATP production compared to U251_Ctrl cells after a 24 h stimulation with either 400 μM ACZ ( E ) or 400 μM BRZ ( G ). U251_CA2 cells also significantly reduced glycolytic activity compared to U251_Ctrl cells after 400 μM ACZ ( F ) and BRZ ( H ) stimulation for 24 h. ( I ) Invasion assay of U251_CA2 cells stained with DAPI after ACZ and BRZ stimulation for 24 h with concentrations indicated (scale bar: 75 μm). ( J ) Quantification of the proportion of invasive cells. Both 100 μM and 400 μM BRZ significantly decreased cell invasion of U251_CA2 cells, whereas only 400 μM ACZ reduced the invasiveness of cells ( n = 6). ( K , L ) U251_CA2 cell significantly reduced cell invasiveness compared to U251_Ctrl cells after treatment with 400 μM BRZ. However, 400 μM ACZ did not change the invasiveness of U251_CA2 cells compared to the control group. Results were obtained from three independent experiments. Data are presented as mean ± SD, student’s t -test was used to analyze ( E – H , K , L ) One-way ANOVA was used to analyze ( J ), * p < 0.05; ** p < 0.01; *** p < 0.001, ns: not significant.

Article Snippet: U87 and U251 CA2 expressing cells were generated by transfection with CA2 plasmid (RC201974, ORIGENE, Rockville, MD, USA) using 2.5 μg of plasmid DNA mix Lipofectamine LTX Reagent (15338100, Invitrogen, Waltham, MA, USA).

Techniques: Activity Assay, Invasion Assay, Staining

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of Carbonic Anhydrase 2 Overcomes Temozolomide Resistance in Glioblastoma Cells

doi: 10.3390/ijms23010157

Figure Lengend Snippet: Effect of combined treatment with TMZ and either ACZ or BRZ on CA2 overexpressing GBM cell lines U87 and U251. ( A ) Co-treatment with 500 μM TMZ and ACZ/BRZ on U87_Ctrl cells and U87_CA2 cells for 5 d. U87_CA2 cells significantly decreased cell viability compared to U87_Ctrl cells after BRZ + TMZ treatment. However, only 400 μM ACZ + TMZ decreased cell viability compared to the U87_Ctrl group ( n = 3). ( B ) In U87_CA2 cells treated with TMZ, ACZ/BRZ, or TMZ + ACZ/BRZ, TMZ plus 400 μM ACZ decreased the viability compared to TMZ alone. However, 100 μM ACZ in combination with TMZ did not change the cell viability. Both 100 μM and 400 μM BRZ in combination with 500 μM TMZ decreased the viability significantly compared to TMZ treatment alone ( n = 3). ( C ) Co-treatment with 30 μM TMZ and ACZ/BRZ on U251_Ctrl cells and U251_CA2 cells for 5 d. U251_CA2 cells decreased cell viability compared to U251_Ctrl cells after 400 μM BRZ + TMZ treatment. However, ACZ + TMZ did not change cell viability compared to the U251_Ctrl group ( n = 3). ( D ) In U251_CA2 cells, ACZ/BRZ has no cytotoxic effect, TMZ plus 400 μM ACZ decreased the viability compared to TMZ alone. Both 100 μM and 400 μM BRZ in combination with 500 μM TMZ decreased the viability significantly compared to TMZ treatment alone ( n = 3). * (Red): compared to Control group; # (Purple): compared to TMZ group; & (Orange): compared to ACZ 100 μM + TMZ group; % (Black): compared to BRZ 100 μM + TMZ group; $ (Green): compared to ACZ 400 μM + TMZ group. Results were obtained from 3 independent experiments. Data are presented as mean ± SD, Two-way ANOVA was used to analyze ( A , C ), One-way ANOVA was used to analyze ( B , D ), ***, $$$, &&&, ###, p < 0.001, ns: not significant.

Article Snippet: U87 and U251 CA2 expressing cells were generated by transfection with CA2 plasmid (RC201974, ORIGENE, Rockville, MD, USA) using 2.5 μg of plasmid DNA mix Lipofectamine LTX Reagent (15338100, Invitrogen, Waltham, MA, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of Carbonic Anhydrase 2 Overcomes Temozolomide Resistance in Glioblastoma Cells

doi: 10.3390/ijms23010157

Figure Lengend Snippet: Combined treatment of BRZ and TMZ increases cell death in GBM stem-like cells. ( A – C ) mRNA expression of CA2 after TMZ treatment at 3 d and 5 d were detected by RT-PCR, the mRNA level of CA2 increased after TMZ stimulation ( n = 3). ( D ) Visualization of the morphology of GSCs (Nr. 2016/175) using light microscopy after TMZ and CA inhibitor stimulation for 10 d. ( E – G ) Cell viability of co-treatment measured by CellTiter-Glo assay. Co-treatment with 500 μM TMZ and 400 μM ACZ decreased the viability compared to TMZ alone. However, 100 μM ACZ in combination with TMZ did not change the cell viability. Both 100 μM and 400 μM BRZ in combination with 500 μM TMZ decreased the viability significantly compared to TMZ treatment alone ( n = 3). * (Red): compared to Control group; # (Purple): compared to TMZ group; & (Orange): compared to ACZ 100 μM + TMZ group; % (Black): compared to BRZ 100 μM + TMZ group; $ (Green): compared to ACZ 400 μM + TMZ group. Results were obtained from 3 independent experiments. In ( A – C , E – G ), data are presented as mean ± SD, student’s t -test was used to analyze ( A – C ), One-way ANOVA was used to analyze ( E – G ), *, & p < 0.05; ## p < 0.01; ***, ###, &&&, $$$ p < 0.001, ns: not significant.

Article Snippet: U87 and U251 CA2 expressing cells were generated by transfection with CA2 plasmid (RC201974, ORIGENE, Rockville, MD, USA) using 2.5 μg of plasmid DNA mix Lipofectamine LTX Reagent (15338100, Invitrogen, Waltham, MA, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Light Microscopy, Glo Assay

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of Carbonic Anhydrase 2 Overcomes Temozolomide Resistance in Glioblastoma Cells

doi: 10.3390/ijms23010157

Figure Lengend Snippet: BRZ is more effective than ACZ in causing re-sensitization of long-term TMZ-resistant GSCs. ( A – G ) qPCR measurement for three GBM-related CA genes ( CA2 , CA9 , and CA12 ), three monocarboxylate transporters ( MCT-1 , MCT-4 , and SLC4A4 ), and drug-resistance gene ( P-gp ) which showed differential expression between TMZ resistant cell and its paired control/DMSO cells ( n = 3). ( H ) Cell morphology changes after TMZ and CA inhibitor stimulation for 14 d. ( I ) Co-treatment with C50 dose (3 μM) of TMZ and 100 μM/400 μM ACZ or BRZ did not decrease the cell viability compared to single treatment with TMZ on DMSO control cells ( n = 3). ( J ) Both 100 μM and 400 μM BRZ in combination with IC50 dose (250 μM) TMZ decreased the viability significantly compared to TMZ treatment alone on TMZ-resistant cells, however, ACZ in combination with TMZ did not change the cell viability ( n = 3). * (Red): compared to Control group; # (Purple): compared to TMZ group; & (Orange): compared to ACZ 100 μM + TMZ group; % (Black): compared to BRZ100 μM + TMZ group; $ (Green): compared to ACZ 400 μM + TMZ group. Results were obtained from 3 independent experiments. Data are presented as mean ± SD, One-way ANOVA was used to analyze the data, * p < 0.05; **, %% p < 0.01; ***, ###, $$$ p < 0.001, ns: not significant.

Article Snippet: U87 and U251 CA2 expressing cells were generated by transfection with CA2 plasmid (RC201974, ORIGENE, Rockville, MD, USA) using 2.5 μg of plasmid DNA mix Lipofectamine LTX Reagent (15338100, Invitrogen, Waltham, MA, USA).

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of Carbonic Anhydrase 2 Overcomes Temozolomide Resistance in Glioblastoma Cells

doi: 10.3390/ijms23010157

Figure Lengend Snippet: The combination of BRZ and TMZ increased cell death in GBM stem-like cells by activating autophagy ( A , B ) Autophagy marker LC3 immunostaining of U251_Ctrl and U251_CA2 cells after TMZ and ACZ/BRZ stimulation for 24 h. Co-treatment with TMZ and BRZ induced the expression of LC3 puncta in U251_CA2 cells (scale bar: 50 μm). ( C , D ) Western Blotting of autophagy-related proteins and CA2 protein in U251_Ctrl and U251_CA2 cells with the same treatment as in ( A ). TMZ plus BRZ did not increase the protein expression of LC3II in U251_Ctrl cells compared to TMZ treatment alone ( C ) but increased in U251_CA2 cells ( D ) ( n = 3). ( E – G ) Western Blotting of autophagy-related proteins and CA2 protein in GBM stem cells with the same treatment as in E–G for 24 h stimulation. Compared with the control group, TMZ monotherapy has a tendency to increase the protein level of LC3II, but the expression of it is significantly increased in BRZ alone and TMZ combined with BRZ treatment ( n = 3). Results were obtained from three independent experiments. Data are presented as mean ± SEM, One-way ANOVA was used to analyze the data, * p < 0.05; ** p < 0.01; *** p < 0.001, ns: not significant.

Article Snippet: U87 and U251 CA2 expressing cells were generated by transfection with CA2 plasmid (RC201974, ORIGENE, Rockville, MD, USA) using 2.5 μg of plasmid DNA mix Lipofectamine LTX Reagent (15338100, Invitrogen, Waltham, MA, USA).

Techniques: Marker, Immunostaining, Expressing, Western Blot