Journal: bioRxiv

Article Title: Cachd1 is a novel Frizzled- and LRP6-interacting protein required for neurons to acquire left-right asymmetric character

doi: 10.1101/2022.05.16.492129

Figure Lengend Snippet: (A, left) Representative scatter plot of flow cytometry testing binding of FLAG-tagged CACHD1 prey protein to human FZD7-eGFP transiently transfected HEK293E cells (detected by phycoerythrin (PE)-conjugated secondary antibody; A, right) without (blue) or with (red) pre-incubation with anti-Frizzled antibody OMP-18R5; secondary only negative control (grey). n = 3; one-tailed paired t -test (D. F. = 2, t = 9.53, *** p = 0.0054). (B) Dot plot of human (blue diamonds) or zebrafish (blue circles) CACHD1, or negative control CD200R (grey) prey protein binding (∆M PE ) to cells transiently transfected with eGFP fusion protein constructs indicated (transfections verified by antibody labelling in bold). Each dot represents a single experiment; horizontal bars denote the mean and error bars represent 95% confidence intervals. One way Welch test of means (Cachd1 prey v. CD200R prey, not assuming equal variances; F = 132.32, D. F num = 30.00, D. F denom = 34.67, p = 5.09 × 10 −28 ), post hoc pairwise t -tests with non-pooled standard deviations, Benjamini & Hochberg correction for multiple testing; only significant differences between Cachd1 and CD200R prey for individual transfections are presented here for clarity, ** 0.05 > p > 0.01, *** 0.01 > p > 0.005, **** p < 0.005. (C) SPR-based determination of K D for mouse CACHD1 ECD analyte binding to immobilised FZD5 CRD , LRP6 P3E3P4E4 (3-4, left panel), and normalised response curves for different CACHD1 ECD :FZD CRD interactions. RU, response units.

Article Snippet: Mouse Fzd5 cysteine rich domain (UniProt: Q9EQD0, residues A27-T157), human FZD7 CRD (UniProt: O75084, residues Q33-G170), human FZD8 CRD (UniProt: Q9H461, residues A28-T158) were synthesized (Genscript).

Techniques: Flow Cytometry, Binding Assay, Transfection, Incubation, Negative Control, One-tailed Test, Protein Binding, Construct

Journal: Frontiers in Endocrinology

Article Title: Human immature testicular tissue organ culture: a step towards fertility preservation and restoration

doi: 10.3389/fendo.2023.1242263

Figure Lengend Snippet: Spermatogonial differentiation. (A, B) Hematoxylin and eosin staining of cultured immature testicular tissue showing the presence of primary spermatocytes. Scale bars: 50 µm (C, D) Immunofluorescence of SYCP3+ primary spermatocytes and VASA+ germ cells of post thaw ITT (C) and post cultured ITT (D) . Dashed white line: basal lamina. Scale bars: 50 µm. (E–H) Percentage of SYCP3+ primary spermatocytes per total number of VASA+ germ cells in prepubertal and peripubertal patients on day 7, 16 and 32 in four in vitro conditions, none (E) , FSH (F) , hCG (G) and FSH + hCG (H) . Raw data VASA+/SYCP3+ germ cell numbers for post-thaw day 0 as well as days 7, 16 and 32 of culture are shown in Supplementary Table 3 .

Article Snippet: Goat anti-human DDX4 (VASA, 1:100, AF2030 Thermo Scientific) was used as a broad germ cell marker, goat or mouse anti-human UCHL1 (1:200, LS-B16043-50 or 7863-1004 Bio-Rad) for undifferentiated spermatogonia, rabbit anti-human SYCP3 (1:300, NB300-232SS Novus Biologicals) for primary spermatocytes, rabbit anti-human CREM (1:100, LS-B13702-50) for early spermatids, mouse anti-human KI67 (1:50 550609 BD Biosciences) for proliferation, rabbit anti-human cleaved CASPASE 3 (1:300, 96615 Cell Signaling Technology) for apoptosis, rabbit or goat anti-human SOX9 (1:400 Ab5535, Fisher Scientific or 1:100 AF3075 R&D) for Sertoli cells, rabbit anti-human AR (1:100 Ab271891 Abcam) for mature Sertoli cell, rabbit anti-human CLAUDIN 11 (1:100 36-4500 Invitrogen) for tight junctions of blood-testis barrier and rabbit anti-human INSL3 (1:1500, HPA028615 Sigma) for mature Leydig cells.

Techniques: Staining, Cell Culture, Immunofluorescence, In Vitro

Journal: Oncotarget

Article Title: The proteoglycan-like domain of carbonic anhydrase IX mediates non-catalytic facilitation of lactate transport in cancer cells

doi: 10.18632/oncotarget.25371

Figure Lengend Snippet: (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% CO 2 / 10 mM HCO 3 - . (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% CO 2 / 10 mM HCO 3 - (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.

Article Snippet: Detection of CAIX was performed either with rabbit anti-human CAIX polyclonal antibody (2 μg/ml; NB100-417, Novus Biologicals), for samples of cells treated with anti-CAIX monoclonal antibodies, or with mouse anti-human CAIX monoclonal antibody clone Anti-PG (M75; 0.4 μg/ml).

Techniques: Sequencing, Concentration Assay, Expressing, Comparison

Journal: Oncotarget

Article Title: The proteoglycan-like domain of carbonic anhydrase IX mediates non-catalytic facilitation of lactate transport in cancer cells

doi: 10.18632/oncotarget.25371

Figure Lengend Snippet: 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.

Article Snippet: Detection of CAIX was performed either with rabbit anti-human CAIX polyclonal antibody (2 μg/ml; NB100-417, Novus Biologicals), for samples of cells treated with anti-CAIX monoclonal antibodies, or with mouse anti-human CAIX monoclonal antibody clone Anti-PG (M75; 0.4 μg/ml).

Techniques: Staining, Expressing, Control, Labeling, Laser-Scanning Microscopy

Journal: Oncotarget

Article Title: The proteoglycan-like domain of carbonic anhydrase IX mediates non-catalytic facilitation of lactate transport in cancer cells

doi: 10.18632/oncotarget.25371

Figure Lengend Snippet: (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).

Article Snippet: Detection of CAIX was performed either with rabbit anti-human CAIX polyclonal antibody (2 μg/ml; NB100-417, Novus Biologicals), for samples of cells treated with anti-CAIX monoclonal antibodies, or with mouse anti-human CAIX monoclonal antibody clone Anti-PG (M75; 0.4 μg/ml).

Techniques: Concentration Assay, Incubation, Expressing, Control, Comparison

Journal: Oncotarget

Article Title: The proteoglycan-like domain of carbonic anhydrase IX mediates non-catalytic facilitation of lactate transport in cancer cells

doi: 10.18632/oncotarget.25371

Figure Lengend Snippet: (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 18 O-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 18 O depletion of doubly labelled 13 C 18 O 2 . 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 18 O depletion of doubly labelled 13 C 18 O 2 . 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).

Article Snippet: Detection of CAIX was performed either with rabbit anti-human CAIX polyclonal antibody (2 μg/ml; NB100-417, Novus Biologicals), for samples of cells treated with anti-CAIX monoclonal antibodies, or with mouse anti-human CAIX monoclonal antibody clone Anti-PG (M75; 0.4 μg/ml).

Techniques: Mass Spectrometry, Expressing, Labeling, Activity Assay, Comparison, Incubation

Journal: Oncotarget

Article Title: The proteoglycan-like domain of carbonic anhydrase IX mediates non-catalytic facilitation of lactate transport in cancer cells

doi: 10.18632/oncotarget.25371

Figure Lengend Snippet: (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).

Article Snippet: Detection of CAIX was performed either with rabbit anti-human CAIX polyclonal antibody (2 μg/ml; NB100-417, Novus Biologicals), for samples of cells treated with anti-CAIX monoclonal antibodies, or with mouse anti-human CAIX monoclonal antibody clone Anti-PG (M75; 0.4 μg/ml).

Techniques: Incubation, Concentration Assay, Comparison, Control, Activity Assay, Mass Spectrometry, Western Blot

Journal: Oncotarget

Article Title: The proteoglycan-like domain of carbonic anhydrase IX mediates non-catalytic facilitation of lactate transport in cancer cells

doi: 10.18632/oncotarget.25371

Figure Lengend Snippet: (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).

Article Snippet: Detection of CAIX was performed either with rabbit anti-human CAIX polyclonal antibody (2 μg/ml; NB100-417, Novus Biologicals), for samples of cells treated with anti-CAIX monoclonal antibodies, or with mouse anti-human CAIX monoclonal antibody clone Anti-PG (M75; 0.4 μg/ml).

Techniques: Incubation, Concentration Assay, Comparison, Control, Activity Assay, Mass Spectrometry, Western Blot

Journal: Oncotarget

Article Title: The proteoglycan-like domain of carbonic anhydrase IX mediates non-catalytic facilitation of lactate transport in cancer cells

doi: 10.18632/oncotarget.25371

Figure Lengend Snippet: (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).

Article Snippet: Detection of CAIX was performed either with rabbit anti-human CAIX polyclonal antibody (2 μg/ml; NB100-417, Novus Biologicals), for samples of cells treated with anti-CAIX monoclonal antibodies, or with mouse anti-human CAIX monoclonal antibody clone Anti-PG (M75; 0.4 μg/ml).

Techniques: Western Blot, Control