PMC

Antibody staining of fixed and permeabilized oocytes, expressing MCT1+CAIX-WT (A), MCT4+CAIX-WT (B), MCT1+CAIX-ΔPG (C), MCT4+CAIX-ΔPG (D), and a native oocyte as control (E). CAIX was labeled with an antibody, mapping against the C-terminal region of CAIX. Pictures were taken with a confocal laser scanning microscope.

(A) Western blot of CAIX, in normoxic and hypoxic MCF-7 and MDA-MB-231 cells with Anti-PG (Ab-M75, upper blots). β-tubulin was used as loading control (lower blots). (B) Quantification of the protein level of CAIX in normoxic and hypoxic MDA-MB-231 and MCF-7 cells, respectively, relative to the protein level of β-tubulin. Data are represented as mean + SEM. Significance in differences was tested with Student's t-test, following a Shapiro-Wilk test. The significance indicators above the bars for normoxic and hypoxic MCF-7 cells refer to the corresponding values for normoxic and hypoxic MDA-MB-231 cells (gray bars).

(A) Original recordings of the relative change in intracellular lactate concentration during application of 0.3, 1, 3, 10, and 30 mM of lactate, in MDA-MB-231 breast cancer cells, incubated under normoxia (black traces) or hypoxia (blue traces). (B) Relative change in intracellular lactate concentration, as induced by application of lactate in MDA-MB-231 cells, incubated under normoxia (gray) or hypoxia (blue), plotted against the extracellular lactate concentration. (C, D) Rate of change in intracellular pH, as induced by application (C) and removal (D) of lactate in MDA-MB-231 cells, incubated under normoxia (gray) or hypoxia (blue), plotted against the extracellular lactate concentration. Data are represented as mean ± SEM. Significance in differences was tested with Student's t-test, following a Shapiro-Wilk test. The significance indicators above each pair of dots refer to the difference between normoxic and hypoxic cells at the respective lactate concentration.

(A) Staining of nuclei with Hoechst (blue) in hypoxic MDA-MB-231 cells before (A₁) and 3 days after production of a scratch through the culture (A₂-A₄). Cells remained either untreated (A₁, A₂) or where incubated with Anti-PG (A₃) or Anti-CA (A₄). (B-E) Size of the gap (%) in MDA-MB-231 (B, C) and MCF-7 (D, E) cell cultures, 0–3 days after scratching. Cells were incubated under normoxic (B, D) or hypoxic (C, E) conditions in the presence of Anti-PG (red) and Anti-CA (green), respectively, or without antibody (gray). Data are represented as mean ± SEM. Significance in differences was tested with ANOVA, followed by means comparison. The red significance indicators depict differences between cells treated with Anti-PG and control cells, the green significance indicators depict differences between cells treated with Anti-CA and control cells at the respective time points.

(A, B) Staining of nuclei with Hoechst (blue) in MCF-7 (A) and MDA-MB-231 cells (B) after 3 days in culture at 1% O₂. Hypoxic cells remained either untreated (A₁, B₁) or were incubated with 5 μg/ml Anti-PG (A₂, B₂) or 5 μg/ml Anti-CA (A₃, B₃). (C, D) Total number of nuclei/mm² in MCF-7 (C) and MDA-MB-231 (D) cell cultures, kept for 0–3 days under the conditions as described in (A and B). Data are represented as mean ± SEM. Significance in differences was tested with ANOVA, followed by means comparison. The red significance indicators depict differences between cells treated with Anti-PG and control cells, the green significance indicators depict differences between cells treated with Anti-CA and control cells at the respective time points.

(A) Amino acid sequence of the human CAIX proteoglycan-like domain. Negatively charged amino acids are labelled in red, positively charged amino acids are labelled in blue. (B, C) Original recordings of the change in intracellular H⁺ concentration ([H⁺]_i) in oocytes expressing (B) MCT1 or (C) MCT4 (black trace), MCT1/4 + CAIX-WT (blue trace), and MCT1/4 + CAIX-ΔPG (red trace), respectively, during application of 3 and 10 mM of lactate and 5% CO₂ / 10 mM HCO₃^-. (D-I) Rate of change in intracellular H⁺ concentration (Δ[H⁺]/Δt) as induced by application (D, G) and removal (E, H) of 3 and 10 mM lactate, and application of 5% CO₂ / 10 mM HCO₃^- (F, I), respectively, in oocytes expressing MCT1/4 (gray), MCT1/4+CAIX-WT (blue), and MCT1+CAII-ΔPG (red), respectively. Data are represented as mean + SEM. Significance in differences was tested with ANOVA, followed by means comparison. The black significance indicators above the bars for MCT1/4+CAIX/CAIX-ΔPG-coexpressing oocytes refer to the values from oocytes expressing MCT1/4 alone. The blue significance indicators above the bars for MCT1/4+CAIX-ΔPG-coexpressing oocytes refer to the values from oocytes coexpressing MCT1/4 + CAIX.

(A, B) Original recordings of the change in intracellular H⁺ concentration ([H⁺]_i) in oocytes coexpressing MCT1+CAIX-WT (A) and MCT4+CAIX (B), respectively, during application of 3 and 10 mM lactate. Cells were pre-incubated for 24 h with 0.4 μg/ml Anti-PG (red traces), 0.4 μg/ml Anti-CA (green traces) or without antibodies (black traces) before the measurements were carried out. (C, D) Rate of change in intracellular H⁺ concentration (Δ[H⁺]/Δt) as induced by application (C, E) and removal (D, F) of 3 and 10 mM lactate, in oocytes expressing MCT1/4 (gray) and MCT1/4+CAIX-WT (blue), respectively, in the presence of Anti-PG, Anti-CA or in the absence of antibodies (control). Data are represented as mean + SEM. Significance in differences was tested with ANOVA, followed by means comparison. The black significance indicators above the bars for MCT1/4+CAIX-coexpressing oocytes refer to the corresponding values from oocytes expressing MCT1/4 alone (gray bars). The blue significance indicators above the bars for MCT1/4+CAIX-coexpressing oocytes, incubated with Anti-PG or Anti-CA refer to the values from MCT1/4+CAIX-coexpressing oocytes not incubated with antibody (blue bars). No significant changes were found between MCT1/4-expressing oocytes incubated with or without antibodies (gray bars).

(A) Original recordings of the log enrichment (LE), as measured by gas-analysis mass spectrometry, of a pool of 20 lysed, native oocytes (black trace) and pools of 20 lysed oocytes, expressing either CAIX-WT (blue trace) or CAIX-ΔPG (red trace). The beginning of the traces shows the rate of degradation of the ¹⁸O-labeled substrate in the non-catalyzed reaction. The black arrowhead indicates addition of oocyte lysate. (B) Enzymatic activity of lysates from 20 native oocytes (gray) and 20 oocytes expressing either CAIX-WT (blue) or CAIX-ΔPG (red), respectively. One unit is defined as 100% stimulation of the non-catalyzed ¹⁸O depletion of doubly labelled ¹³C¹⁸O₂. Data are represented as mean + SEM. Significance in differences was tested with ANOVA, followed by means comparison. The black significance indicators above the bars from CAIX-expressing oocytes refer to the values from native cells (gray bar). The blue asterisks above the bar from CAIX-ΔPG-expressing oocytes refer to the values from CAIX-WT-expressing oocytes (blue bar). (C) Enzymatic activity of cell lysate from 20 native (gray) and CAIX-expressing oocytes (blue, red and green), respectively. Cell lysates were incubated for 2h with 5 μg/ml of Anti-PG (red), 5 μg/ml of Anti-CA (green), or without antibody (blue). One unit is defined as 100% stimulation of the non-catalyzed ¹⁸O depletion of doubly labelled ¹³C¹⁸O₂. Data are represented as mean + SEM. Significance in differences was tested with ANOVA, followed by means comparison. The black significance indicators above the bars from CAIX-expressing oocytes refer to the values from native cells (gray bar). The blue asterisks above the bar from CAIX-expressing oocytes, incubated with antibodies refer to the values from CAIX-WT-expressing oocytes without antibody (blue bar). The red asterisks above the bar from CAIX-expressing oocytes, incubated with Anti-CA refer to the values from CAIX-WT-expressing oocytes, incubated with Anti-PG (red bar).

(A) Original recordings of the change in intracellular pH (pH_i) during application of 3 and 10 mM of lactate, in MDA-MB-231 breast cancer cells, incubated under normoxia (left traces) or hypoxia (right traces), in the presence of 5 μg/ml Anti-PG (red traces), 5 μg/ml Anti-CA (green traces) or in the absence of antibodies (black traces). (B, C) Rate of change in intracellular pH, as induced by application (B) and removal (C) of lactate in MDA-MB-231 cells, incubated under normoxia (gray) or hypoxia (blue), in the presence of Anti-PG, Anti-CA or in the absence of antibodies (Ctrl). (D) Relative change in intracellular lactate concentration, as induced by application of lactate in MDA-MB-231 cells, incubated under normoxia (gray) or hypoxia (blue) in the presence of Anti-PG, Anti-CA or in the absence of antibodies (Ctrl). Data are represented as mean ± SEM. Significance in differences was tested with ANOVA, followed by means comparison. The significance indicators above the bars from cells, incubated with antibody refer to the corresponding values from control cells (Ctrl). (E) CA catalytic activity of MDA-MB-231 cells, as measured by gas-analysis mass spectrometry, in the presence 5 μg/ml Anti-PG (red), 5 μg/ml Anti-CA (green), 30 μM of the CA inhibitor EZA (yellow) and in the absence of antibodies or inhibitors (Ctrl). Data are represented as mean + SEM. Significance in differences was tested with ANOVA, followed by means comparison. The black asterisks above the bars from cells treated with antibodies or EZA refer to the values from control cells (gray bar). The red asterisks above the bars from cells treated with Anti-CA or EZA refer to the values from cells treated with Anti-PG (red bar). The green significance indicator above the bar from cells treated with EZA refers to the values from cells treated with Anti-CA (green bar). (F) Western blot against CAIX and β-tubulin as loading control, in MDA-MB-231 cells, incubated in the presence Anti-PG, Anti-CA or in the absence of antibodies (Ctrl). (G) Quantification of the protein level of CAIX in MDA-MB-231 cells, incubated in the presence Anti-PG (red), Anti-CA (green) or in the absence of antibodies (gray) relative to the protein level of β-tubulin. Data are represented as mean + SEM. Significance in differences was tested with ANOVA, followed by means comparison. The black significance indicators above the bars from cells, incubated with antibody refer to the corresponding values from control cells (gray bar). The red significance indicator above the bar from cells treated with Anti-CA refers to the values from cells treated with Anti-PG (red bar).

(A) Original recordings of the change in intracellular pH (pH_i) during application of 3 and 10 mM of lactate in MCF-7 breast cancer cells, incubated under normoxia (left traces) or hypoxia (right traces), in the presence of 5 μg/ml Anti-PG (red traces), 5 μg/ml Anti-CA (green traces) or in the absence of antibodies (black traces). (B, C) Rate of change in intracellular pH, as induced by application (B) and removal (C) of lactate in MCF-7 cells, incubated under normoxia (gray) or hypoxia (blue), in the presence of Anti-PG, Anti-CA or in the absence of antibodies (Ctrl). (D) Relative change in intracellular lactate concentration, as induced by application of lactate in MCF-7 cells, incubated under normoxia (gray) or hypoxia (blue) in the presence of Anti-PG, Anti-CA or in the absence of antibodies (Ctrl). Data are represented as mean ± SEM. Significance in differences was tested with ANOVA, followed by means comparison. The significance indicators above the bars from cells, incubated with antibody refer to the corresponding values from control cells (Ctrl). (E) CA catalytic activity of MCF-7 cells, as measured by gas-analysis mass spectrometry, in the presence 5 μg/ml Anti-PG (red), 5 μg/ml Anti-CA (green), 30 μM of the CA inhibitor EZA (yellow) and in the absence of antibodies or inhibitors (Ctrl). Data are represented as mean + SEM. Significance in differences was tested with ANOVA, followed by means comparison. The black asterisks above the bars from cells treated with antibodies or EZA refer to the values from control cells (gray bar). The red asterisks above the bars from cells treated with Anti-CA or EZA refer to the values from cells treated with Anti-PG (red bar). The green significance indicator above the bar from cells treated with EZA refers to the values from cells treated with Anti-CA (green bar). (F) Western blot against CAIX and β-tubulin as loading control, in MCF-7 cells, incubated in the presence Anti-PG, Anti-CA or in the absence of antibodies (Ctrl). (G) Quantification of the protein level of CAIX in MCF-7 cells, incubated in the presence Anti-PG (red), Anti-CA (green) or in the absence of antibodies (gray) relative to the protein level of β-tubulin. Data are represented as mean + SEM. Significance in differences was tested with ANOVA, followed by means comparison. The black significance indicators above the bars from cells, incubated with antibody refer to the corresponding values from control cells (gray bar). The red significance indicator above the bar from cells treated with Anti-CA refers to the values from cells treated with Anti-PG (red bar).