Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Limited Association Between Ascorbate Concentrations and Vitamin C Transporters in Renal Cell Carcinoma Cells and Clinical Samples.

doi: 10.33594/000000435

Figure Lengend Snippet: Fig. 1. Protein levels of SVCT1 and SVCT2 in ccRCC cell lines in vitro. (A) SVCT antibodies were validated using blocking peptides. Antibodies for SVCT1 and SVCT2 were pre-absorbed with blocking peptides before incubation of the membranes. Western blots show Caki-1 cell lysates probed with the respective antibodies with (+) or without (-) blocking peptides. The specific band for SVCT1 was confirmed at ~65 kDa and SVCT2 was identified as a double band at ~72 and 100 kDa (indicated by arrows). (B) Protein levels of SVCT1 and SVCT2 in 786-O, Caki-1 and Caki- 2 cells was analysed by Western blotting. β-actin was used as a loading control. (C) Quantification of re- lative protein levels of SVCT1 and SVCT2 (both size bands) in ccRCC cell lines. Mean ± SD, n = 3.

Article Snippet: The following primary antibodies were used: anti-human SVCT1 (polyclonal rabbit, Aviva Systems, San Diego, CA, USA, OAAB09000), anti-human SVCT2 (polyclonal rabbit, Atlas Antibodies, Stockholm, Sweden, HPA052825) and anti-β-actin (monoclonal mouse, Sigma-Aldrich, A5316).

Techniques: In Vitro, Blocking Assay, Incubation, Western Blot, Control

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Limited Association Between Ascorbate Concentrations and Vitamin C Transporters in Renal Cell Carcinoma Cells and Clinical Samples.

doi: 10.33594/000000435

Figure Lengend Snippet: Fig. 2. Levels of ascorbate transporters in response to ascorbate over time in ccRCC cell lines. The ccRCC cells 786-0 (A-C), Caki-1 (D-F) and Caki-2 (G-I) were analysed for ascorbate, SVCT1 and SVCT2 (both bands). Cells were treated with 0 μM (A, D, G), 50 μM (B, E, H) or 500 μM ascorbate (C, F, I) for 16 h and then cultured without ascorbate for 24 h. Ascorbate and protein levels of SVCT1 and SVCT2 was measured at indicated time points. Protein levels are relative to untreated cells at 0 h, and did not change significantly by ascorbate addition or removal. Data are from three independent experiments and are presented as mean ± SD. Stati- stical significance was determined by One-way ANOVA with Dunnett’s Multiple Comparison Test; * p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: The following primary antibodies were used: anti-human SVCT1 (polyclonal rabbit, Aviva Systems, San Diego, CA, USA, OAAB09000), anti-human SVCT2 (polyclonal rabbit, Atlas Antibodies, Stockholm, Sweden, HPA052825) and anti-β-actin (monoclonal mouse, Sigma-Aldrich, A5316).

Techniques: Cell Culture, Comparison

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Limited Association Between Ascorbate Concentrations and Vitamin C Transporters in Renal Cell Carcinoma Cells and Clinical Samples.

doi: 10.33594/000000435

Figure Lengend Snippet: Fig. 3. Localisation of SVCT2 in 786-0, Caki-1 and Caki-2 cells during exposure to ascorbate. Cellular loca- lisation of SVCT2 in response to extracellular ascorbate (500 μM) was monitored for 5-60 min. Immunoflu- orescence images for SVCT2 (red) and Phalloidin (green to stain F-actin) of fixed cells were taken at indica- ted time points and are shown as merged images. Nuclei were visualised with DAPI (blue). Red arrows show examples of cytoplasmic spots, purple arrows illustrate nuclear localisation of SVCT2. 63x objective (oil), scale bar = 20 μm. Note that photos are enlarged and cropped to show individual cells of interest.

Article Snippet: The following primary antibodies were used: anti-human SVCT1 (polyclonal rabbit, Aviva Systems, San Diego, CA, USA, OAAB09000), anti-human SVCT2 (polyclonal rabbit, Atlas Antibodies, Stockholm, Sweden, HPA052825) and anti-β-actin (monoclonal mouse, Sigma-Aldrich, A5316).

Techniques: Staining

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Limited Association Between Ascorbate Concentrations and Vitamin C Transporters in Renal Cell Carcinoma Cells and Clinical Samples.

doi: 10.33594/000000435

Figure Lengend Snippet: Fig. 4. Localisation of SVCT2 in 786-0, Caki-1 and Caki-2 cells during exposure to ascorbate. Cellular loca- lisation of SVCT2 in response to extracellular ascorbate (500 μM) was monitored for 2-8 h. Immunofluore- scence images for SVCT2 (red) and Phalloidin (green to stain F-actin) of fixed cells were taken at indicated time points and are shown as merged images. Nuclei were visualised with DAPI (blue). Red arrows show examples of cytoplasmic spots, purple arrows illustrate nuclear localisation of SVCT2. 63x objective (oil), scale bar = 20 μm.

Article Snippet: The following primary antibodies were used: anti-human SVCT1 (polyclonal rabbit, Aviva Systems, San Diego, CA, USA, OAAB09000), anti-human SVCT2 (polyclonal rabbit, Atlas Antibodies, Stockholm, Sweden, HPA052825) and anti-β-actin (monoclonal mouse, Sigma-Aldrich, A5316).

Techniques: Staining

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Limited Association Between Ascorbate Concentrations and Vitamin C Transporters in Renal Cell Carcinoma Cells and Clinical Samples.

doi: 10.33594/000000435

Figure Lengend Snippet: Fig. 6. Immunohistochemical detection of ascorbate transporters in renal cortex and RCC tumour tissue. Tissue sections of human renal cortex, pRCC and ccRCC tumours were analysed for ascorbate transpor- ter distribution by immunohistochemistry. In normal renal cortex tissue, SVCT1 was localised to the apical membrane of proximal tubular epithelial cells (P; indicated with arrowheads) and was absent in distal tu- bules (D). In pRCC and ccRCC tissue, staining of SVCT1 was cytoplasmic. Immunoreactivity of SVCT2 was seen in the cytoplasm of both cortex and tumour tissue. The negative control (no primary antibody) of renal cortex showed marked unspecific background staining that was especially strong in proximal tubular cells. No staining was seen in the negative controls of tumour tissue. Representative photos for SVCT1, SVCT2 and control are taken from serial sections of the same location; 20x objective, scale bar = 50 μm.

Article Snippet: The following primary antibodies were used: anti-human SVCT1 (polyclonal rabbit, Aviva Systems, San Diego, CA, USA, OAAB09000), anti-human SVCT2 (polyclonal rabbit, Atlas Antibodies, Stockholm, Sweden, HPA052825) and anti-β-actin (monoclonal mouse, Sigma-Aldrich, A5316).

Techniques: Immunohistochemical staining, Immunohistochemistry, Membrane, Staining, Negative Control, Control

Journal: Free radical biology & medicine

Article Title: Mitochondrial dysfunction in the APP/PSEN1 mouse model of Alzheimer’s disease and a novel protective role for ascorbate

doi: 10.1016/j.freeradbiomed.2017.08.021

Figure Lengend Snippet: A) The oxygen consumed by mitochondrial isolated from WT cortex was significantly increased with the addition of 100μM ASC (p = 0.0017) compared to vehicle following establishment of baseline. Oxygen consumption within groups increased with the addition of each new substrate compared to the prior condition (p < 0.0001). Asterices (*) indicate a main effect of ASC following addition of ASC and of each substrate B) SVCT2 xpression is only slightly increased, (not significant), in mitochondria isolated from SVCT2Tg mice compared to WT mitochondria (p = 0.55, n= 5 per group). C) ASC content is significantly elevated,, in SVCT2Tg mitochondria at 4 months (p = 0.05, n = 5–9 per group). D) Mitochondrial isolates from SVCT2Tg mice show no difference in mitochondrial membrane potential measured by relative fluorescence from accumulated TMRE (p = 0.3409, n = 5–7 per group). E) Mitochondrial isolates from SVCT2Tg mice show a significant decrease in levels of ROS as measured by relative fluorescence from oxidized dihydrofluorescein (p = 0.0008, n = 6–8).

Article Snippet: SVCT2 expression (SVCT2 H-70 (rabbit) sc-30114, dil. 1:1000, Santa Cruz Biotechnology, Inc., USA) was normalized to heat shock protein-60 (HSP-60 (mouse) A302-844A, dil. 1:2000, Bethyl Laboratories, USA).

Techniques: Isolation, Membrane, Fluorescence

Journal: Free radical biology & medicine

Article Title: Mitochondrial dysfunction in the APP/PSEN1 mouse model of Alzheimer’s disease and a novel protective role for ascorbate

doi: 10.1016/j.freeradbiomed.2017.08.021

Figure Lengend Snippet: A) SVCT2 expression is decreased in mitochondrial isolates from mice heterozygous for SVCT2 compared to isolates with wild-type SVCT2 expression (p = 0.03, n = 5–6 per group). B) ASC content is significantly lower in SVCT2+/− mitochondria at 4 months (p = 0.024, n = 5 per group). C) Qualitative western blot analysis demonstrates detectable amyloid β expression in APP/PSEN1 and SVCT2+/−; APP/PSEN1 mice at 4 months of age, that already appears more severe in the compound mutant mice. D) SVCT2+/− mitochondrial isolates consume less oxygen (p=0.04), while mitochondrial isolates from APP/PSEN1 transgenic mice consume more oxygen when at full respiratory capacity (p = 0.005). WT n = 6, SVCT2+/− n = 5, APP/PSEN1, SVCT2+/−;APP/PSEN1 n = 6. Letters indicate a main effect of SVCT2 (a,b) and APP/PSEN1 (c,d).

Article Snippet: SVCT2 expression (SVCT2 H-70 (rabbit) sc-30114, dil. 1:1000, Santa Cruz Biotechnology, Inc., USA) was normalized to heat shock protein-60 (HSP-60 (mouse) A302-844A, dil. 1:2000, Bethyl Laboratories, USA).

Techniques: Expressing, Western Blot, Mutagenesis, Transgenic Assay

Journal: Free radical biology & medicine

Article Title: Mitochondrial dysfunction in the APP/PSEN1 mouse model of Alzheimer’s disease and a novel protective role for ascorbate

doi: 10.1016/j.freeradbiomed.2017.08.021

Figure Lengend Snippet: A) Mitochondrial isolates from SVCT2+/− mice show decreased mitochondrial membrane potential measured by relative fluorescence from accumulated TMRE (p = 0.049, n = 5–6 per group). B) Mitochondrial isolates from APP/PSEN1 transgenic mice show increased membrane potential measured by relative fluorescence from accumulated TMRE (p = 0.0085, n = 7–8 per group). C) The ATP/ADP ratio in SVCT2+/− isolates were not significantly different from WT (p= 0.195). D) The ATP/ADP ratio in APP/PSEN1 transgenic isolates was significantly lower than in WT isolates (p= 0.0299). E) Mitochondrial isolates from SVCT2+/− mice had significantly greater relative fluorescence from oxidized dihydrofluorescein (p = 0.03) than did mice that were wild-type for SVCT2. F) Mitochondrial isolates from APP/PSEN1 transgenic mice also show increased levels of ROS as measured by relative fluorescence from oxidized dihydrofluorescein (p = 0.038).

Article Snippet: SVCT2 expression (SVCT2 H-70 (rabbit) sc-30114, dil. 1:1000, Santa Cruz Biotechnology, Inc., USA) was normalized to heat shock protein-60 (HSP-60 (mouse) A302-844A, dil. 1:2000, Bethyl Laboratories, USA).

Techniques: Membrane, Fluorescence, Transgenic Assay

Journal: Free radical biology & medicine

Article Title: Mitochondrial dysfunction in the APP/PSEN1 mouse model of Alzheimer’s disease and a novel protective role for ascorbate

doi: 10.1016/j.freeradbiomed.2017.08.021

Figure Lengend Snippet: A) Amyloid β 42/40 ratio is significantly greater at 12 months compared with 5 months in APP/PSEN1 and SVCT2Tg;APP/PSEN1 mice (p< 0.0001) but does not differ according to genotype with each age group. B) Cortical ASC concentration is significantly elevated in SVCT2Tg mice compared to mice with normal SVCT2 expression (p=0.005) at 5 months, but at 12 months a significant difference is observed between SVCT2Tg and SVCT2Tg;APP/PSEN1 only (p=0.0002).

Article Snippet: SVCT2 expression (SVCT2 H-70 (rabbit) sc-30114, dil. 1:1000, Santa Cruz Biotechnology, Inc., USA) was normalized to heat shock protein-60 (HSP-60 (mouse) A302-844A, dil. 1:2000, Bethyl Laboratories, USA).

Techniques: Concentration Assay, Expressing

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Limited Association Between Ascorbate Concentrations and Vitamin C Transporters in Renal Cell Carcinoma Cells and Clinical Samples.

doi: 10.33594/000000435

Figure Lengend Snippet: Fig. 1. Protein levels of SVCT1 and SVCT2 in ccRCC cell lines in vitro. (A) SVCT antibodies were validated using blocking peptides. Antibodies for SVCT1 and SVCT2 were pre-absorbed with blocking peptides before incubation of the membranes. Western blots show Caki-1 cell lysates probed with the respective antibodies with (+) or without (-) blocking peptides. The specific band for SVCT1 was confirmed at ~65 kDa and SVCT2 was identified as a double band at ~72 and 100 kDa (indicated by arrows). (B) Protein levels of SVCT1 and SVCT2 in 786-O, Caki-1 and Caki- 2 cells was analysed by Western blotting. β-actin was used as a loading control. (C) Quantification of re- lative protein levels of SVCT1 and SVCT2 (both size bands) in ccRCC cell lines. Mean ± SD, n = 3.

Article Snippet: The following primary antibodies were used: anti-human SVCT1 (polyclonal rabbit, Aviva Systems, San Diego, CA, USA, OAAB09000), anti-human SVCT2 (polyclonal rabbit, Atlas Antibodies, Stockholm, Sweden, HPA052825) and anti-β-actin (monoclonal mouse, Sigma-Aldrich, A5316).

Techniques: In Vitro, Blocking Assay, Incubation, Western Blot, Control

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Limited Association Between Ascorbate Concentrations and Vitamin C Transporters in Renal Cell Carcinoma Cells and Clinical Samples.

doi: 10.33594/000000435

Figure Lengend Snippet: Fig. 2. Levels of ascorbate transporters in response to ascorbate over time in ccRCC cell lines. The ccRCC cells 786-0 (A-C), Caki-1 (D-F) and Caki-2 (G-I) were analysed for ascorbate, SVCT1 and SVCT2 (both bands). Cells were treated with 0 μM (A, D, G), 50 μM (B, E, H) or 500 μM ascorbate (C, F, I) for 16 h and then cultured without ascorbate for 24 h. Ascorbate and protein levels of SVCT1 and SVCT2 was measured at indicated time points. Protein levels are relative to untreated cells at 0 h, and did not change significantly by ascorbate addition or removal. Data are from three independent experiments and are presented as mean ± SD. Stati- stical significance was determined by One-way ANOVA with Dunnett’s Multiple Comparison Test; * p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: The following primary antibodies were used: anti-human SVCT1 (polyclonal rabbit, Aviva Systems, San Diego, CA, USA, OAAB09000), anti-human SVCT2 (polyclonal rabbit, Atlas Antibodies, Stockholm, Sweden, HPA052825) and anti-β-actin (monoclonal mouse, Sigma-Aldrich, A5316).

Techniques: Cell Culture, Comparison

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Limited Association Between Ascorbate Concentrations and Vitamin C Transporters in Renal Cell Carcinoma Cells and Clinical Samples.

doi: 10.33594/000000435

Figure Lengend Snippet: Fig. 6. Immunohistochemical detection of ascorbate transporters in renal cortex and RCC tumour tissue. Tissue sections of human renal cortex, pRCC and ccRCC tumours were analysed for ascorbate transpor- ter distribution by immunohistochemistry. In normal renal cortex tissue, SVCT1 was localised to the apical membrane of proximal tubular epithelial cells (P; indicated with arrowheads) and was absent in distal tu- bules (D). In pRCC and ccRCC tissue, staining of SVCT1 was cytoplasmic. Immunoreactivity of SVCT2 was seen in the cytoplasm of both cortex and tumour tissue. The negative control (no primary antibody) of renal cortex showed marked unspecific background staining that was especially strong in proximal tubular cells. No staining was seen in the negative controls of tumour tissue. Representative photos for SVCT1, SVCT2 and control are taken from serial sections of the same location; 20x objective, scale bar = 50 μm.

Article Snippet: The following primary antibodies were used: anti-human SVCT1 (polyclonal rabbit, Aviva Systems, San Diego, CA, USA, OAAB09000), anti-human SVCT2 (polyclonal rabbit, Atlas Antibodies, Stockholm, Sweden, HPA052825) and anti-β-actin (monoclonal mouse, Sigma-Aldrich, A5316).

Techniques: Immunohistochemical staining, Immunohistochemistry, Membrane, Staining, Negative Control, Control

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Ascorbic acid uptaken by sodium-dependent vitamin C transporter 2 induces βhCG expression through Sp1 and TFAP2A transcription factors in human choriocarcinoma cells.

doi: 10.1210/jc.2012-1753

Figure Lengend Snippet: FIG. 4. AA induced hCG5 reporter gene expression through transcriptional factors, Sp1 and TFAP2A. Schematic presentation of 5-untranscriptional region and the major potentially binding sites for transcription factors on hCG5 promoter (A). After being transiently transfected with reporter constructs including WT, deletion mutants, and two Sp1- and/or three TFAP2A-binding site mutants (Sp1-mut, TFAP2A-mut, TFAP2ASp1-mut), cells were treated with indicated concentrations of AA for 48 h, and the cell lysates were harvested for dual-luciferase assay (B–D). After infection with Sp1-shRNA (Sp1-si) and/or TFAP2A-shRNA (TFAP2A-si)-expressing retroviruses, cells were transiently transfected with reporter constructs and treated with indicated concentrations of AA for 48 h, and then the cell lysates were harvested for dual-luciferase assay and Western blot assay (E–G). The luciferase activity and protein levels of Sp1 and TFAP2A derived from cells treated with 0 M AA and infected with or without control virus were defined as 1. Results are expressed as means SD of three independent cultures. **, P 0.01 vs. cells treated with 0 M AA and infected with or without control viruses; †, P 0.05, ‡, P 0.01 vs. cells transfected with 345 to 114 reporter constructs (WT) or cells infected with control viruses.

Article Snippet: The immunoblot was performed with polyclonal rabbit antimouse SVCT2 antibody (23), polyclonal rabbit antihuman Sp1 antibody (Cell Signaling Technology, Danvers, MA) and monoclonal mouse antihuman TFAP2A antibody (Sigma), respectively.

Techniques: Gene Expression, Binding Assay, Transfection, Construct, Luciferase, Infection, shRNA, Expressing, Western Blot, Activity Assay, Derivative Assay, Control, Virus

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Ascorbic acid uptaken by sodium-dependent vitamin C transporter 2 induces βhCG expression through Sp1 and TFAP2A transcription factors in human choriocarcinoma cells.

doi: 10.1210/jc.2012-1753

Figure Lengend Snippet: FIG. 5. Silence of Sp1 and TFAP2A expression abolished AA-induced hCG expression in JEG-3 cells. Twenty-four hours after plating, JEG-3 cells were infected with control or Sp1- shRNA and/or TFAP2A-shRNA-expressing retroviruses for another 24 h, and cells were treated with AA at the indicated concentrations for 48 h. Then cells were harvested for preparation of mRNA and quantitative RT-PCR for hCG mRNA expression (A), and the medium was harvested for hCG ELISA determination (B). The expression of hCG mRNA was normalized to the level of -actin mRNA, and the data are expressed relative to the mRNA levels of cells infected with control viruses and treated with 0 M AA, which were set to 1. Results are expressed as means SD of three independent cultures. **, P 0.01 vs. cells infected with control viruses and treated with 0 M AA; †, P 0.05, ‡, P 0.01 vs. cells treated with 0 M AA and infected with control viruses.

Article Snippet: The immunoblot was performed with polyclonal rabbit antimouse SVCT2 antibody (23), polyclonal rabbit antihuman Sp1 antibody (Cell Signaling Technology, Danvers, MA) and monoclonal mouse antihuman TFAP2A antibody (Sigma), respectively.

Techniques: Expressing, Infection, Control, shRNA, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay