Journal: Scientific reports

Article Title: MCT4 is induced by metastasis-enhancing pathogenic mitochondrial NADH dehydrogenase gene mutations and can be a therapeutic target.

doi: 10.1038/s41598-021-92772-1

Figure Lengend Snippet: Figure 1. Lactate efflux and the expression of lactate transporter MCTs. (a) Lactate efflux by P29 cybrids. Error bar: SD. (b) RT-qPCR analysis of CD147 mRNA expression in P29 cybrids. Error bar: SD. (c) RT-qPCR analysis of MCT1 mRNA expression in P29 cybrids. Error bar: SD. (d) Western blot analysis of the expression of CD147 and MCT1 in P29 cybrids. β-Actin was used as a loading control. (e) RT-qPCR analysis of the expression of MCT2, MCT3 and MCT4 mRNA in P29 cybrids. nd: not detected. Error bar: SD. (f) Western blot analysis of the expression of MCT4 in P29 cybrids. β-Actin was used as a loading control. (g) Immunofluorescent staining of MCT4 in P29mtP29 and P29mtB82M cells. Uncropped Western blots images are shown in Supplementary Fig. S12.

Article Snippet: MCT4 was detected with a mouse monoclonal anti-MCT4 antibody (SC-376140, 1:200 dilution, Santa Cruz Biotechnology, Dalla, TX, USA) and a VECTASTAIN Elite ABC HRP kit (Vector Laboratories, Burlingame, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Staining

Journal: Scientific reports

Article Title: MCT4 is induced by metastasis-enhancing pathogenic mitochondrial NADH dehydrogenase gene mutations and can be a therapeutic target.

doi: 10.1038/s41598-021-92772-1

Figure Lengend Snippet: Figure 2. Effect of MCT4 knockdown on the invasiveness of P29mtB82M cells. (a) Western blot analysis of the expression of MCT4 in P29mtB82M cells transfected with a control siRNA (siCont) or an MCT4 siRNA (siMCT4 #1 and #2). β-Actin was used as a loading control. Uncropped Western blot images are shown in Supplementary Fig. S12. (b) Proliferation of P29mtB82M cells transfected with a control siRNA (siCont) or an MCT4 siRNA (siMCT4 #1 and #2), as measured by WST assay. The cells were cultured for 2 days. Error bar: SD. (c) Gelatine invadopodia assay. Images showing the degradation of FITC-labelled gelatine at the invadopodia of P29mtB82M cells transfected with a control siRNA (siCont) or an MCT4 siRNA (siMCT4 #1 and #2). Bar. 20 μm. (d) Quantitation of the degraded area/ cell in the invadopodia assay. Error bar: SD. (e) Matrigel invasion assay. Images showing invasion of P29mtB82M cells transfected with a control siRNA (siCont) or an MCT4 siRNA (siMCT4 #1 and #2). (f) Quantitation of the number of invaded cells. Error bar: SD. (g) RT-qPCR analysis of the expression of MCT4 and matrix degradation-related genes in P29mtB82M cells transfected with a control siRNA (siCont) or MCT4 siRNAs (siMCT4 #1 and #2). Error bar: SD.

Article Snippet: MCT4 was detected with a mouse monoclonal anti-MCT4 antibody (SC-376140, 1:200 dilution, Santa Cruz Biotechnology, Dalla, TX, USA) and a VECTASTAIN Elite ABC HRP kit (Vector Laboratories, Burlingame, CA, USA).

Techniques: Knockdown, Western Blot, Expressing, Transfection, Control, WST Assay, Cell Culture, Invadopodia Assay, Quantitation Assay, Invasion Assay, Quantitative RT-PCR

Journal: Scientific reports

Article Title: MCT4 is induced by metastasis-enhancing pathogenic mitochondrial NADH dehydrogenase gene mutations and can be a therapeutic target.

doi: 10.1038/s41598-021-92772-1

Figure Lengend Snippet: Figure 4. MCT expression in NSCLC cell lines. (a–e) RT-qPCR analysis of the expression of CD147 and MCT1-4. nd: not detected. (f) Western blot analysis of MCT4 expression. β-Actin was used as a loading control. Uncropped Western blot images are shown in Supplementary Fig. S12.

Article Snippet: MCT4 was detected with a mouse monoclonal anti-MCT4 antibody (SC-376140, 1:200 dilution, Santa Cruz Biotechnology, Dalla, TX, USA) and a VECTASTAIN Elite ABC HRP kit (Vector Laboratories, Burlingame, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control

Journal: Scientific reports

Article Title: MCT4 is induced by metastasis-enhancing pathogenic mitochondrial NADH dehydrogenase gene mutations and can be a therapeutic target.

doi: 10.1038/s41598-021-92772-1

Figure Lengend Snippet: Figure 5. IHC analysis of MCT4 expression in NSCLC tissues. NSCLC tissues harbouring predicted pathogenic ND mutations such as homoplasmy (LuAdBrM5) or heteroplasmy (or heterogeneous state) (LuAdBrM1 and LgCa169) and NSCLC tissues having no pathogenic ND mutation (LgCa183, LgCa188 and LgCa193) were immunostained for MCT4 using a monoclonal MCT4 antibody. LuAdBrM5 and LuAdBrM1 tissues were strongly positive for MCT4 on cell membranes. LgCa169 tissues contain both MCT4-positive and MCT4- negative regions, which might represent cancer cells with and without the mutation, respectively. Cancer cells in LgCa183, LgC188 and LgCa193 tissues were negative for MCT4, but stromal cells were positive. Bar: 100 μm.

Article Snippet: MCT4 was detected with a mouse monoclonal anti-MCT4 antibody (SC-376140, 1:200 dilution, Santa Cruz Biotechnology, Dalla, TX, USA) and a VECTASTAIN Elite ABC HRP kit (Vector Laboratories, Burlingame, CA, USA).

Techniques: Expressing, Mutagenesis

Journal: Scientific reports

Article Title: MCT4 is induced by metastasis-enhancing pathogenic mitochondrial NADH dehydrogenase gene mutations and can be a therapeutic target.

doi: 10.1038/s41598-021-92772-1

Figure Lengend Snippet: Figure 6. NAC suppresses MCT4 expression. (a) Effect of NAC on ROS generation in P29mtB82 cells by FACS analysis. Cells were treated with 20 mM NAC for 24 h, and levels of mtROS and cellular ROS were detected by MitoSOX Red and DCF-DA, respectively. (b, c) RT-qPCR and Western blot analyses of MCT4 and MCT1 expressions in P29mtB82M (b) and H358 cells (c), both of which were treated with 20 mM NAC for the indicated times. (d) FACS analysis of the effect of MitoPQ on mtROS generation in P29mtP29 and P29mtA11 cells. The cells were treated with 10 µM MitoPQ for 24 h. mtROS were detected by MitoSOX Red. (e) Effect of MitoTEMPO on MCT4 expression in P29mtB82M cells by Western blotting. The cells were treated with 20 µM MitoTEMPO for the indicated times. (f) Effect of H2O2 and MitoPQ. P29mtP29 and P29mtA11 cells were treated with H2O2 or MitoPQ at various concentrations for 3 and 2 days, respectively. Lysates from P29mtB82M cells were used as a positive control. β-Actin was used as a loading control. Uncropped Western blot images are available as Supplementary Fig. S12.

Article Snippet: MCT4 was detected with a mouse monoclonal anti-MCT4 antibody (SC-376140, 1:200 dilution, Santa Cruz Biotechnology, Dalla, TX, USA) and a VECTASTAIN Elite ABC HRP kit (Vector Laboratories, Burlingame, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Positive Control, Control

Journal: Scientific reports

Article Title: MCT4 is induced by metastasis-enhancing pathogenic mitochondrial NADH dehydrogenase gene mutations and can be a therapeutic target.

doi: 10.1038/s41598-021-92772-1

Figure Lengend Snippet: Figure 7. Analyses of signalling pathways involved in MCT4 expression in P29mtB82M and H358 cells by signalling pathway inhibitors. (a) Effects of various inhibitors on MCT4 expression in P29mtB82M cells. The cells were treated with the inhibitors at the indicated concentrations for 2 days. (b–e) Effect of LY294002, rapamycin, BML-275 and PF-4709671 on the expressions of MCT4 and MCT1. P29mtB82M cells were treated with LY294002 (b), rapamycin (c), BML-275 (d) or PF-4709671 (e) at the indicated concentrations for 2 days. The results of RT-qPCR and Western blotting are shown. (f) Comparison of phosphorylation levels of various signalling elements in P29mtP29 and P29mtB82M cells. Ratios of phosphorylated/total protein (p/t) are shown. (g) Effects of various inhibitors on MCT4 expression in H358 cells. Cells were treated with the inhibitors at the indicated concentrations for 2 days. For Western blotting, β-Actin was used as a loading control. Ratios of MCT4/β-actin are shown. Uncropped Western blot images are provided as Supplementary Fig. S12.

Article Snippet: MCT4 was detected with a mouse monoclonal anti-MCT4 antibody (SC-376140, 1:200 dilution, Santa Cruz Biotechnology, Dalla, TX, USA) and a VECTASTAIN Elite ABC HRP kit (Vector Laboratories, Burlingame, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Comparison, Phospho-proteomics, Control

Journal: International Journal of Molecular Sciences

Article Title: Lactate Transporter Monocarboxylate Transporter 4 Induces Bone Pain in Head and Neck Squamous Cell Carcinoma

doi: 10.3390/ijms19113317

Figure Lengend Snippet: The expression of monocarboxylate transporter 4 (MCT4) in head and neck normal tissues, head and neck cancers, and human oral squamous cell lines. ( A ) Immunohistochemistry analysis of MCT4 in head and neck normal tissue ( a ) and head and neck cancer (HNC) ( b ). Scale Bar = 200 μm. ( B ) Scatterplot of the MCT4-positive areas in the head and neck normal tissues ( n = 11) and head and neck squamous cell carcinoma (HNSCC) ( n = 70). Error bars: mean ± standard deviation (SD). There was a significantly increased expression of MCT4 in the HNSCC samples ( p < 0.0001). ( C ) Expression of MCT4 in human oral squamous cell carcinoma cell lines (HSC-2, -3, -4; SAS, OSC-19) and a human breast cancer cell line (MCF7) analyzed by western blotting.

Article Snippet: Puromycin dihydrochloride (cat. No. #sc-108071), control shRNA plasmid-A (#sc108060), MCT4 shRNA plasmid (h2) (#sc-45892-SH) and anti-MCT4 antibody (anti-rabbit, monoclonal, #sc-50329) were purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Expressing, Immunohistochemistry, Standard Deviation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Lactate Transporter Monocarboxylate Transporter 4 Induces Bone Pain in Head and Neck Squamous Cell Carcinoma

doi: 10.3390/ijms19113317

Figure Lengend Snippet: Reduction of MCT4 protein in HNSCC SAS cells. ( A ) The expression of MCT4 in SAS cells. After the stable transfection of SAS cells with control shRNA (sh-control) and MCT4 shRNA (sh-MCT4), the transfected and non-transfected cells (parental) were analyzed by western blotting. The expression of MCT4 in sh-MCT4 analyzed with image blot density was approximately one-half that of the parental cells. ( B ) Immunofluorescence analysis of MCT4 and 4′,6-diamidino-2-phenylindole (DAPI) for nuclear staining in the cells. Left, MCT4 (green); middle, DAPI (blue); right, merge. Sections were incubated with rabbit anti-MCT4 (1:100), then with Alexa Fluor 488 anti-rabbit IgG (1:1000) and encapsulated with DAPI. Scale Bar = 100 μm. ( C ) Proliferation assay. We cultured sh-control and sh-MCT4 cells for seven days and then counted the number of each ( n = 3). There was no significant difference between the cells. Error bars: mean ± SD. ( D ) Each group of SAS cells was cultured for 24 h. The conditioned medium was then collected and the concentration of lactic acid was measured ( n = 3). Error bars: mean ± SD; * p < 0.01 vs. sh-MCT4. ( E ) Each group of SAS cells was cultured for 48 h. The conditioned medium was then collected and the extra cellular pH (pH e ) was measured ( n = 3). Error bars: mean ± SD; * p < 0.01 vs. sh-MCT4.

Article Snippet: Puromycin dihydrochloride (cat. No. #sc-108071), control shRNA plasmid-A (#sc108060), MCT4 shRNA plasmid (h2) (#sc-45892-SH) and anti-MCT4 antibody (anti-rabbit, monoclonal, #sc-50329) were purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Expressing, Stable Transfection, Control, shRNA, Transfection, Western Blot, Immunofluorescence, Staining, Incubation, Proliferation Assay, Cell Culture, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Lactate Transporter Monocarboxylate Transporter 4 Induces Bone Pain in Head and Neck Squamous Cell Carcinoma

doi: 10.3390/ijms19113317

Figure Lengend Snippet: Neurite outgrowth from rat primary dorsal root ganglia (DRG) cells co-cultured with each group of SAS cells. ( A ) Neurite outgrowth from primary neuron cells in co-culture with control medium ( a ), parental SAS ( b ), sh-control ( c ), and sh-MCT4 ( d ) SAS cells for five days, labeled with calcein acetoxymethyl (AM). Scale Bar=50μm. ( B ) Quantitative data of ( A) . Error bars: mean ± SD; * = p < 0.05 vs. control; # = p < 0.05 vs. sh-MCT4.

Article Snippet: Puromycin dihydrochloride (cat. No. #sc-108071), control shRNA plasmid-A (#sc108060), MCT4 shRNA plasmid (h2) (#sc-45892-SH) and anti-MCT4 antibody (anti-rabbit, monoclonal, #sc-50329) were purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Cell Culture, Co-Culture Assay, Control, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Lactate Transporter Monocarboxylate Transporter 4 Induces Bone Pain in Head and Neck Squamous Cell Carcinoma

doi: 10.3390/ijms19113317

Figure Lengend Snippet: Bone resorption of the tibia in SAS cell-injected mice. ( A ) Normal bone marrow tissue and SAS cells colonized in bone marrow (hematoxylin and eosin) at 100× magnification. ( B ) X-ray photographs showing bone resorptions of the tibia at post-operative day (POD) seven in SAS cell-injected mice. The lower panel compares the bone resorption areas. There was no significant difference between the parental and sh-MCT4 groups ( n = 5). Error bars: mean ± SD.

Article Snippet: Puromycin dihydrochloride (cat. No. #sc-108071), control shRNA plasmid-A (#sc108060), MCT4 shRNA plasmid (h2) (#sc-45892-SH) and anti-MCT4 antibody (anti-rabbit, monoclonal, #sc-50329) were purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Injection

Journal: International Journal of Molecular Sciences

Article Title: Lactate Transporter Monocarboxylate Transporter 4 Induces Bone Pain in Head and Neck Squamous Cell Carcinoma

doi: 10.3390/ijms19113317

Figure Lengend Snippet: Bone pain and lactate concentration in sham mice and SAS cell-injected mice. ( A ) Lactate concentration in cardiac blood sampling or tibiae marrow ( n = 5). ( a ) Lactic acid concentration in cardiac blood. ( b ) Lactic acid concentration in tibial bone marrow. Error bars: mean ± SD. * = p < 0.05 vs. sh-control. ( B ) Thermal sensitivity results of each group. The test was performed every other day from POD one to seven ( n = 5). Error bars: mean ± SD. * = p < 0.05 vs. sham; ** = p < 0.01 vs. sham; # = p < 0.05 vs. sh-MCT4.

Article Snippet: Puromycin dihydrochloride (cat. No. #sc-108071), control shRNA plasmid-A (#sc108060), MCT4 shRNA plasmid (h2) (#sc-45892-SH) and anti-MCT4 antibody (anti-rabbit, monoclonal, #sc-50329) were purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Concentration Assay, Injection, Sampling, Control

Journal: International Journal of Molecular Sciences

Article Title: Lactate Transporter Monocarboxylate Transporter 4 Induces Bone Pain in Head and Neck Squamous Cell Carcinoma

doi: 10.3390/ijms19113317

Figure Lengend Snippet: Excitation of sensory nerves determined by p-ERK expression in DRGs in sham, parental, sh-control, and sh-MCT4-injected mice shown by TRPV1 expression. ( A ) Western blotting results. ( B ) Immunofluorescence analysis of p-ERK and TRPV1. ( left ) p-ERK or TRPV1 (green); ( right ) CGRP (red). Scale Bar = 50 μm. Sections were processed as described in the Materials and Methods section.

Article Snippet: Puromycin dihydrochloride (cat. No. #sc-108071), control shRNA plasmid-A (#sc108060), MCT4 shRNA plasmid (h2) (#sc-45892-SH) and anti-MCT4 antibody (anti-rabbit, monoclonal, #sc-50329) were purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Expressing, Control, Injection, Western Blot, Immunofluorescence

Journal: Cell Death Discovery

Article Title: MCT4-driven CAF-mediated metabolic reprogramming in breast cancer microenvironment is a vulnerability targetable by miR-425-5p

doi: 10.1038/s41420-024-01910-x

Figure Lengend Snippet: Representative Western blot analysis of MCT4 performed on plasmamembrane fractions of Cancer-Associated Fibroblasts (CAFs) ( n = 4) and Normal Fibroblasts (NFs) ( n = 3). Na + /K+ ATPase was used for data normalization of the fractionation in the sample preparation. Densitometry data are expressed after normalization on Na + /K+ ATPase band intensity A . A representative confocal microscopy image of MCT4 expression in CAFs ( n = 3) (DAPI blue, MCT4 red) B . Three different subpopulations of two primary fibroblasts were collected at different growing distances from the tumor (intratumoral, IT; peritumoral, PT; normal, NF) and MCT4 gene expression was evaluated by quantitative real-time PCR. Values are expressed as a mean of two biological replicates of technical duplicates C . Culture media from NFs ( n = 2) and CAFs ( n = 2) were collected and assessed by a colorimetric L-lactate assay. Values are expressed as mean of two technical replicates D . Graph shows mean ± SD. * P values < 0.05; *** p < 0.001. Bar = 100 μm.

Article Snippet: Plasmid transfection in MS-5 (2500 ng of SLC16A3 tGFP-tagged supplied by Origene CAT#: RG211987) was carried out using Lipofectamine 3000 reagent (Invitrogen).

Techniques: Western Blot, Fractionation, Sample Prep, Confocal Microscopy, Expressing, Real-time Polymerase Chain Reaction, Lactate Assay

Journal: Cell Death Discovery

Article Title: MCT4-driven CAF-mediated metabolic reprogramming in breast cancer microenvironment is a vulnerability targetable by miR-425-5p

doi: 10.1038/s41420-024-01910-x

Figure Lengend Snippet: Bioinformatics predictions (TargetScan and miRANDA) indicated miR-425-5p as a putative miRNA targeting MCT4 mRNA A . Representative Real Time PCR for miR-425-5p upon miR-425-5p and anti miR-425-5p transfection in Cancer-Associated Fibroblasts (CAF n = 1) and Normal Fibroblasts (NF n = 1) respectively. Data are expressed as mean of two technical replicates B . Representative Western Blot analysis of MCT4 upon miR-425-5p and anti-miR-425 transfection in CAFs ( n = 4) and NFs ( n = 2) respectively C . Dual luciferase assay of the predicted binding site for miR-425-5p on the 3’UTR region of SLC16A3. Data are expressed as mean of two technical replicates D . Quantitative Real Time PCR for miR-425-5p performed on CAFs ( n = 3) and NFs ( n = 2), to evaluate miR-425-5p levels. Values are expressed as mean of two technical replicates E . Graph shows mean ± SD over control. * P values < 0.05.

Article Snippet: Plasmid transfection in MS-5 (2500 ng of SLC16A3 tGFP-tagged supplied by Origene CAT#: RG211987) was carried out using Lipofectamine 3000 reagent (Invitrogen).

Techniques: Real-time Polymerase Chain Reaction, Transfection, Western Blot, Luciferase, Binding Assay, Control

Journal: Cell Death Discovery

Article Title: MCT4-driven CAF-mediated metabolic reprogramming in breast cancer microenvironment is a vulnerability targetable by miR-425-5p

doi: 10.1038/s41420-024-01910-x

Figure Lengend Snippet: Metabolic changes in MCT4-transfected MS-5 cells with or without miR-425-5p overexpression, as assessed by glycolytic proton efflux rate (glycoPER) kinetics A , proton efflux rate (PER) B , extracellular acidification rate (ECAR) C , oxygen consumption rate OCR D , and ATP production E . Data are presented as a mean of biological duplicates of eight technical replicates ± SD over control.

Article Snippet: Plasmid transfection in MS-5 (2500 ng of SLC16A3 tGFP-tagged supplied by Origene CAT#: RG211987) was carried out using Lipofectamine 3000 reagent (Invitrogen).

Techniques: Transfection, Over Expression, Control

Journal: Cell Death Discovery

Article Title: MCT4-driven CAF-mediated metabolic reprogramming in breast cancer microenvironment is a vulnerability targetable by miR-425-5p

doi: 10.1038/s41420-024-01910-x

Figure Lengend Snippet: MiR-425-5p alters the metabolism of breast Cancer-associated Fibroblasts (CAFs) by downregulating MCT4 and indirectly reducing GLUT1, leading to decreased lactate extrusion. The interaction between miR-425-5p reprogrammed CAFs and breast cancer epithelial cells has a significant impact on breast cancer cell viability, proliferation, and migration. Angiogenesis is also influenced. This graphical abstract was drawn by using and adapting pictures from Servier Medical Art (Smart.Servier.com), provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license ( http://creativecommons.org/licenses/by/3.0/ ).

Article Snippet: Plasmid transfection in MS-5 (2500 ng of SLC16A3 tGFP-tagged supplied by Origene CAT#: RG211987) was carried out using Lipofectamine 3000 reagent (Invitrogen).

Techniques: Migration

Journal: Cell Reports

Article Title: Targeting Metabolic Symbiosis to Overcome Resistance to Anti-angiogenic Therapy

doi: 10.1016/j.celrep.2016.04.028

Figure Lengend Snippet: Tumors Establish Metabolic Symbiosis to Overcome Nintedanib Treatment (A) Representative pictures of combinatorial immunofluorescence staining for MCT1, MCT4, and CD31 on histological sections of tumors from mice treated with either vehicle or nintedanib (50 mg/kg/day) are shown, as indicated. DAPI was used to visualize cell nuclei. The scale bars represent 100 μm. (B) Quantification of the closest distance separating blood vessels from MCT4+ areas by immunofluorescence co-staining for MCT4 and CD31 on Py2T tumors from ST and LT vehicle or nintedanib-treated mice. Note that, in ST vehicle-treated tumors, MCT4 was not significantly expressed and thus the distance to blood vessels could not be determined. (C and D) Quantification of the Glut1+ area fraction (C) and the MCT4+ area fraction within Glut1+ areas (D) by immunofluorescence co-staining for MCT4 and Glut1 on Py2T tumors from ST and LT vehicle or nintedanib-treated mice. (E) Representative microphotographs of immunofluorescence co-staining for MCT4 and Glut1 on histological sections of tumors from ST and LT vehicle or nintedanib-treated mice. DAPI is used to visualize cell nuclei. The scale bars represent 100 μm. (F) Quantification of the hypoxic (pimonidazole+) area fraction within Glut1+ areas by immunofluorescence co-staining for pimonidazole and Glut1 on Py2T tumors from ST and LT vehicle or nintedanib-treated mice. (G) Representative microphotographs of immunofluorescence co-staining for pimonidazole and Glut1 on histological sections of tumors from ST and LT vehicle or nintedanib-treated mice. DAPI was used to visualize cell nuclei. The scale bars represent 100 μm. (H) Quantification of the MCT4+ area fraction within pimonidazole+ areas by immunofluorescence co-staining for MCT4 and pimonidazole on Py2T tumors from ST and LT vehicle or nintedanib-treated mice. (I) Representative microphotographs of immunofluorescence co-staining for MCT4 and pimonidazole on histological sections of tumors from ST and LT vehicle or nintedanib-treated mice. DAPI is used to visualize cell nuclei. The scale bars represent 100 μm. (J) Representative microphotographs of immunofluorescence co-staining for PGC1α and CD31 on histological sections of tumors from ST and LT vehicle or nintedanib-treated mice. DAPI is used to visualize cell nuclei. The scale bars represent 50 μm. n = 4 mice per group. Mann - Whitney U test. ∗ p < 0.05; ∗∗ p < 0.01. See also Figure S4 .

Article Snippet: Subconfluent Py2T cells were transfected with 2 μg of MCT4 CRISPR/Cas9 KO plasmid and 2 μg of MCT4 HDR plasmid (Santa Cruz Biotechnology; sc-429828 and sc-429828HDR, respectively).

Techniques: Immunofluorescence, Staining, MANN-WHITNEY

Journal: Cell Reports

Article Title: Targeting Metabolic Symbiosis to Overcome Resistance to Anti-angiogenic Therapy

doi: 10.1016/j.celrep.2016.04.028

Figure Lengend Snippet: Targeting Glycolysis or Metabolic Symbiosis in Combination with Nintedanib Treatment Delays Tumor Growth (A and B) Primary tumor growth over time (A) and tumor weights at the experimental endpoint (B) of mice treated with either vehicle or nintedanib (50 mg/kg/day) in combination with 3PO (70 mg/kg/day) or solvent are shown. 3PO treatment was initiated 8 days after the initiation of nintedanib treatment and then continued as combinatorial treatment (LT treatment). In (A), data are displayed as mean tumor volumes ± SEM. (C) Quantification of microvessel densities by immunofluorescence staining for CD31 on histological tumor sections from LT nintedanib and 3PO-treated mice. Values represent the number of counts per each area of microscopic field of view, and means are displayed. n = 6–8 mice per group. (D) Pericyte coverage was assessed by immunofluorescence staining for CD31 and NG2 on histological tumor sections from LT nintedanib and 3PO-treated mice. Values represent the percentage of NG2+ blood vessels, and means are displayed. n = 4–5 mice per group. (E and F) Primary tumor growth over time (E) and tumor weights at the experimental endpoint (F) of mice injected with Py2T wild-type (WT) or Py2T CRISPR MCT4 no. 1 and no. 2 cells and treated with either vehicle control or nintedanib (50 mg/kg/day) are shown. Nintedanib treatment was initiated 19 days after tumor cell injection, once the tumors were palpable. Mice injected with CRISPR MCT4 no. 1 cells presented a delayed tumor onset and were therefore treated once the tumors became palpable (days 27–38). In (E), data are displayed as mean tumor volumes ± SEM. n = 4–7 mice per group. Mann - Whitney U test. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. See also Figure S5 .

Article Snippet: Subconfluent Py2T cells were transfected with 2 μg of MCT4 CRISPR/Cas9 KO plasmid and 2 μg of MCT4 HDR plasmid (Santa Cruz Biotechnology; sc-429828 and sc-429828HDR, respectively).

Techniques: Solvent, Immunofluorescence, Staining, Injection, CRISPR, Control, MANN-WHITNEY

Journal: Cell Reports

Article Title: Targeting Metabolic Symbiosis to Overcome Resistance to Anti-angiogenic Therapy

doi: 10.1016/j.celrep.2016.04.028

Figure Lengend Snippet: Glycolysis Induced by Hypoxia Can Be Reverted by Treatment with 3PO or the Loss of MCT4 (A and B) Shown are the measurements of representative oxygen consumption rates (A; OCRs) and extracellular acidification rates (B; ECARs) of Py2T cells cultured in normoxic or hypoxic conditions. n = 5. (C–F) Quantification of ATP-coupled respiration (C), glycolysis (D), glycolytic capacity (E), and glycolytic reserve (F) of Py2T cells cultured under normoxic or hypoxic conditions. See for details. Data are displayed as mean ± SD. n = 5 (glycolytic reserve: n = 4). Statistical significance was calculated using two-way ANOVA test. (G and H) ECAR/OCR ratio of Py2T cells cultured under normoxic or hypoxic conditions and treated with DMSO, 0.5 μM or 1 μM nintedanib (G), or 5 μM 3PO (H). Data are displayed as mean ± SD. n = 4. Two-way ANOVA test. (I) ECAR/OCR ratio of Py2T WT cells or Py2T CRISPR MCT4 no. 1 and no. 2 cells cultured under normoxic or hypoxic conditions. Data are displayed as mean ± SD. n = 4. Two-way ANOVA test. (J) The percentages of apoptotic Py2T WT cells or Py2T CRISPR MCT4 no. 1 and no. 2 cells cultured under normoxic or hypoxic conditions were assessed using flow cytometry analysis of annexin-V-expressing cells. Data are displayed as mean ± SD. n = 3. One-way ANOVA test. (K) Cell-cycle analysis for Py2T WT cells or Py2T CRISPR MCT4 clones no. 1 and no. 2 cultured under normoxic or hypoxic conditions was performed using EdU staining. Data are displayed as mean ± SD. n = 3. Two-way ANOVA test. ∗ p < 0.05; ∗∗ p < 0.01.

Article Snippet: Subconfluent Py2T cells were transfected with 2 μg of MCT4 CRISPR/Cas9 KO plasmid and 2 μg of MCT4 HDR plasmid (Santa Cruz Biotechnology; sc-429828 and sc-429828HDR, respectively).

Techniques: Cell Culture, CRISPR, Flow Cytometry, Expressing, Cell Cycle Assay, Clone Assay, Staining

Journal: Cell Reports

Article Title: Targeting Metabolic Symbiosis to Overcome Resistance to Anti-angiogenic Therapy

doi: 10.1016/j.celrep.2016.04.028

Figure Lengend Snippet: Targeting Metabolic Symbiosis Overcomes Resistance to Anti-angiogenic Therapy Anti-angiogenic therapy induces hypoxia and reduces the supply of nutrients. As a result, tumor cells shift their metabolism toward a hyperglycolytic state and establish metabolic symbiosis: tumor cells in hypoxic areas upregulate glycolysis, increase lactate production, and export lactate via MCT4. On the other hand, lactate is taken up by tumor cells in more-oxygenated regions of the tumor and is directly fueling the citric acid cycle and thus oxidative phosphorylation. As a consequence, tumor cells in normoxic tumor regions reduce glucose consumption, which increases its diffusion distance. Ablating MCT4 expression (MCT4 KO or shMCT4) or inhibition of glycolysis (3PO) disrupts this homeostatic interplay and decreases tumor growth.

Article Snippet: Subconfluent Py2T cells were transfected with 2 μg of MCT4 CRISPR/Cas9 KO plasmid and 2 μg of MCT4 HDR plasmid (Santa Cruz Biotechnology; sc-429828 and sc-429828HDR, respectively).

Techniques: Phospho-proteomics, Diffusion-based Assay, Expressing, Inhibition

Journal: International Journal of Oncology

Article Title: Lactate secreted via MCT4 from bone-colonizing breast cancer excites sensory neurons via GPR81

doi: 10.3892/ijo.2023.5487

Figure Lengend Snippet: Expression of MCT4 in normal human breast tissues, human BC tissues and BC cell lines. (A) Immunohistochemical analysis of MCT4 expression in (a) normal breast tissue and (b) BC tissue. High power images are also shown. Scale bar, 100 µ m. (B) Scatterplot of the MCT4-positive areas in the normal human breast tissues (n=10) and human BC tissues (n=40). Immunohistochemical staining positive area (%) is shown. Data are presented as the mean ± SD. * P<0.0001 vs. normal tissue. (C) Expression of MCT4 and MCT1 in BC cell lines (human MDA-MB-231 and MCF-7 and mouse 4T1 and EO771) analyzed by western blotting; (a) representative blot images and (b) relative ratio (MDA-MB-231 is indicated as 1.0) (n=3 per group). (D) Lactate concentration in the culture supernatants harvested from the four BC cell cultures. The lactate concentration in the culture supernatants was then determined. Data are presented as the mean ± SD (n=4 per group). * P<0.05 vs. MCF-7. BC, breast cancer; MCT, monocarboxylate transporter.

Article Snippet: Puromycin dihydrochloride (cat. no. sc-108071), control short hairpin (sh)RNA plasmid-A (cat. no. sc-108060), MCT4 shRNA lentivirus particles (mouse, cat. no. sc-40120-V; human, cat. no. sc-45892-V), anti-MCT4 antibody (rabbit polyclonal; cat. no. sc-50329) and anti-MCT1 antibody (mouse monoclonal; cat. no. sc-365501) were purchased from Santa Cruz Biotechnology, Inc. Anti-phosphorylated (p)-p44/42 MAPK antibody (pERK1/2; rabbit monoclonal; cat. no. 4370), anti-p44/42 MAPK antibody (ERK1/2; rabbit monoclonal; cat. no. 4695), anti-p-cAMP response element binding protein (pCREB) antibody (rabbit monoclonal; cat. no. 9198), anti-CREB antibody (rabbit monoclonal; cat. no. 9197), HRP-conjugated IgG antibody (goat anti-rabbit; cat. no. 7074), HRP-conjugated IgG antibody (goat anti-mouse; cat. no. 7076) and Alexa Fluor 488-conjugated IgG (H+L) F(ab') 2 fragment (goat anti-rabbit; cat. no. 4412) were purchased from Cell Signaling Technology, Inc. Anti-calcitonin gene-related peptide (CGRP) antibody (goat polyclonal; cat. no. ab36001) and Alexa Fluor 647-conjugated IgG H&L (donkey anti-goat; cat. no. ab150135) were purchased from Abcam.

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot, Concentration Assay

Journal: International Journal of Oncology

Article Title: Lactate secreted via MCT4 from bone-colonizing breast cancer excites sensory neurons via GPR81

doi: 10.3892/ijo.2023.5487

Figure Lengend Snippet: DRG sensory neuron excitation and mechanical allodynia determined in mice intratibially injected with EO771 mouse BC cells. (A) Expression of MCT4 in parental, sh-control and sh-MCT4 EO771 mouse BC cells examined by western analysis; (a) representative blot images and (b) relative ratio (parental is indicated as 1.0). * P<0.05 vs. parental. (B) Radiograph of tumor growth showing bone destruction in the tibia of mice injected with sh-control and sh-MCT4 mouse EO771 BC cells at day 15 (arrows). (C) Expression of pERK1/2 (green, upper panel) and CGRP (red, middle panel) in DRGs harvested from mice intratibially injected with parental, sh-control and sh-MCT4 EO771 cells at day 15 by immunofluorescence. Scale bar, 50 µ m. (D) Excitation of the F11 DRG sensory neuron cells as determined by pERK1/2 and pCREB expression. The F11 DRG neuron cells were cultured in neuron basal medium in the absence or presence of conditioned medium of the sh-control and sh-MCT4 EO771 cell cultures (30%, v/v) for 15 min, lysed and the expression of pERK1/2 and pCREB was determined by western blot analysis; (a) representative blot images (control is indicated as 1.0 normalized to β-actin) and (b) relative ratio of pERK1/2 and pCREB expression (pERK1/2/ERK1/2 and pCREB/CREB) * P<0.05 vs. sh-MCT4 CM. (E) Progression of the hind-paw mechanical allodynia in tibias of mice that received sham, parental, sh-control and sh-MCT4 EO771 cells (n=5 per group). The pain behavior test was performed every 3 days after cell injection. Mechanical allodynia seen in all mice at day 3 is due to the surgical trauma at day 0. Data are presented as the mean ± SD. * P<0.05 vs. sham; † P<0.05 vs. parental; ‡ P<0.05 vs. sh-control. DRG, dorsal root ganglion; MCT4, monocarboxylate transporter 4; CGRP, calcitonin gene-related peptide; CREB, cAMP response element binding protein; sh, short hairpin; p, phosphorylated; CM, conditioned medium.

Article Snippet: Puromycin dihydrochloride (cat. no. sc-108071), control short hairpin (sh)RNA plasmid-A (cat. no. sc-108060), MCT4 shRNA lentivirus particles (mouse, cat. no. sc-40120-V; human, cat. no. sc-45892-V), anti-MCT4 antibody (rabbit polyclonal; cat. no. sc-50329) and anti-MCT1 antibody (mouse monoclonal; cat. no. sc-365501) were purchased from Santa Cruz Biotechnology, Inc. Anti-phosphorylated (p)-p44/42 MAPK antibody (pERK1/2; rabbit monoclonal; cat. no. 4370), anti-p44/42 MAPK antibody (ERK1/2; rabbit monoclonal; cat. no. 4695), anti-p-cAMP response element binding protein (pCREB) antibody (rabbit monoclonal; cat. no. 9198), anti-CREB antibody (rabbit monoclonal; cat. no. 9197), HRP-conjugated IgG antibody (goat anti-rabbit; cat. no. 7074), HRP-conjugated IgG antibody (goat anti-mouse; cat. no. 7076) and Alexa Fluor 488-conjugated IgG (H+L) F(ab') 2 fragment (goat anti-rabbit; cat. no. 4412) were purchased from Cell Signaling Technology, Inc. Anti-calcitonin gene-related peptide (CGRP) antibody (goat polyclonal; cat. no. ab36001) and Alexa Fluor 647-conjugated IgG H&L (donkey anti-goat; cat. no. ab150135) were purchased from Abcam.

Techniques: Injection, Expressing, Control, Western Blot, Immunofluorescence, Cell Culture, Binding Assay

Journal: International Journal of Oncology

Article Title: Lactate secreted via MCT4 from bone-colonizing breast cancer excites sensory neurons via GPR81

doi: 10.3892/ijo.2023.5487

Figure Lengend Snippet: Role of MCT4 of EO771 mouse breast cancer cells in sensory neuron excitation. (A) Secretion of lactate by parental, sh-control and sh-MCT4 EO771 cells. Cells were cultured for 24 h, the culture supernatants were harvested, and lactate concentration was determined. Data are presented as the mean ± SD (n=3). * P<0.01 vs. parental and sh-control. (B) pHe of the supernatants of parental, sh-control and sh-MCT4 EO771 cells. pHe of the same supernatants shown in A was determined. Data are presented as the mean ± SD (n=3). * P<0.01 vs. parental and sh-control. (C) Intracellular Ca 2+ mobilization in the F11 DRG neuron cells. MCT4, monocarboxylate transporter 4; pHe, extracellular pH; sh, short hairpin; CM, conditioned medium.

Article Snippet: Puromycin dihydrochloride (cat. no. sc-108071), control short hairpin (sh)RNA plasmid-A (cat. no. sc-108060), MCT4 shRNA lentivirus particles (mouse, cat. no. sc-40120-V; human, cat. no. sc-45892-V), anti-MCT4 antibody (rabbit polyclonal; cat. no. sc-50329) and anti-MCT1 antibody (mouse monoclonal; cat. no. sc-365501) were purchased from Santa Cruz Biotechnology, Inc. Anti-phosphorylated (p)-p44/42 MAPK antibody (pERK1/2; rabbit monoclonal; cat. no. 4370), anti-p44/42 MAPK antibody (ERK1/2; rabbit monoclonal; cat. no. 4695), anti-p-cAMP response element binding protein (pCREB) antibody (rabbit monoclonal; cat. no. 9198), anti-CREB antibody (rabbit monoclonal; cat. no. 9197), HRP-conjugated IgG antibody (goat anti-rabbit; cat. no. 7074), HRP-conjugated IgG antibody (goat anti-mouse; cat. no. 7076) and Alexa Fluor 488-conjugated IgG (H+L) F(ab') 2 fragment (goat anti-rabbit; cat. no. 4412) were purchased from Cell Signaling Technology, Inc. Anti-calcitonin gene-related peptide (CGRP) antibody (goat polyclonal; cat. no. ab36001) and Alexa Fluor 647-conjugated IgG H&L (donkey anti-goat; cat. no. ab150135) were purchased from Abcam.

Techniques: Control, Cell Culture, Concentration Assay

Journal: International Journal of Oncology

Article Title: Lactate secreted via MCT4 from bone-colonizing breast cancer excites sensory neurons via GPR81

doi: 10.3892/ijo.2023.5487

Figure Lengend Snippet: Role of MCT4 in lactate metabolism in MDA-MB-231 human BC cells. (A) MCT4 expression in parental, sh-control and sh-MCT4 MDA-MB-231 human BC cells analyzed by western blotting; (a) representative blot images and (b) relative ratio (parental is indicated as 1.0). * P<0.01 vs. parental and sh-control (n=3). (B) Cell viability of parental, sh-control and sh-MCT4 MDA-MB-231 human BC cells. Cells were cultured for 48 h and the cell number was counted. There was no significant difference in the numbers of cells. Data are presented as the mean ± SD (n=3). (C) Intracellular levels of lactate in parental, sh-control and sh-MCT4 MDA-MB-231 human BC cells. Data are presented as the mean ± SD (n=3). * P<0.01 vs. parental and sh-control (n=3). (D) Extracellular levels of lactate in parental, sh-control and sh-MCT4 MDA-MB-231 human BC cells. Data are presented as the mean ± SD (n=3). * P<0.01 vs. parental and sh-control (n=3). BC, breast cancer; MCT4, monocarboxylate transporter 4; sh, short hairpin.

Article Snippet: Puromycin dihydrochloride (cat. no. sc-108071), control short hairpin (sh)RNA plasmid-A (cat. no. sc-108060), MCT4 shRNA lentivirus particles (mouse, cat. no. sc-40120-V; human, cat. no. sc-45892-V), anti-MCT4 antibody (rabbit polyclonal; cat. no. sc-50329) and anti-MCT1 antibody (mouse monoclonal; cat. no. sc-365501) were purchased from Santa Cruz Biotechnology, Inc. Anti-phosphorylated (p)-p44/42 MAPK antibody (pERK1/2; rabbit monoclonal; cat. no. 4370), anti-p44/42 MAPK antibody (ERK1/2; rabbit monoclonal; cat. no. 4695), anti-p-cAMP response element binding protein (pCREB) antibody (rabbit monoclonal; cat. no. 9198), anti-CREB antibody (rabbit monoclonal; cat. no. 9197), HRP-conjugated IgG antibody (goat anti-rabbit; cat. no. 7074), HRP-conjugated IgG antibody (goat anti-mouse; cat. no. 7076) and Alexa Fluor 488-conjugated IgG (H+L) F(ab') 2 fragment (goat anti-rabbit; cat. no. 4412) were purchased from Cell Signaling Technology, Inc. Anti-calcitonin gene-related peptide (CGRP) antibody (goat polyclonal; cat. no. ab36001) and Alexa Fluor 647-conjugated IgG H&L (donkey anti-goat; cat. no. ab150135) were purchased from Abcam.

Techniques: Expressing, Control, Western Blot, Cell Culture

Journal: International Journal of Oncology

Article Title: Lactate secreted via MCT4 from bone-colonizing breast cancer excites sensory neurons via GPR81

doi: 10.3892/ijo.2023.5487

Figure Lengend Snippet: Role of MCT4 in BC-BP induction in mice intratibially injected with MDA-MB-231 human BC cells. (A) Radiograph of tumor growth showing bone destruction in the tibia (arrows) of mice injected with the MDA-MB-231 sh-control and sh-MCT4 human BC cells at day 7 after cell injection. (B) Quantitative analysis of the area of bone destruction seen in A. Data are presented as the mean ± SD (n=5). (C) Hind-paw mechanical allodynia in tibiae of mice intratibially injected with parental, sh-control and sh-MCT4 MDA-MB-231 human BC cells. The behavior test was performed at day 7. Data are presented as the mean ± SD (n=5). * P<0.05 vs. sham and ** P<0.05 vs. parental and sh-control. (D) Lactate concentrations in the bone marrow fluids harvested from tibiae injected with sh-control or sh-MCT4 MDA-MB-231 BC cells or PBS. Data are presented as the mean ± SD (n=4). * P<0.05 vs. PBS and ** P<0.05 vs. sh-control. BC, breast cancer; BP, bone pain; MCT4, monocarboxylate transporter 4; sh, short hairpin.

Article Snippet: Puromycin dihydrochloride (cat. no. sc-108071), control short hairpin (sh)RNA plasmid-A (cat. no. sc-108060), MCT4 shRNA lentivirus particles (mouse, cat. no. sc-40120-V; human, cat. no. sc-45892-V), anti-MCT4 antibody (rabbit polyclonal; cat. no. sc-50329) and anti-MCT1 antibody (mouse monoclonal; cat. no. sc-365501) were purchased from Santa Cruz Biotechnology, Inc. Anti-phosphorylated (p)-p44/42 MAPK antibody (pERK1/2; rabbit monoclonal; cat. no. 4370), anti-p44/42 MAPK antibody (ERK1/2; rabbit monoclonal; cat. no. 4695), anti-p-cAMP response element binding protein (pCREB) antibody (rabbit monoclonal; cat. no. 9198), anti-CREB antibody (rabbit monoclonal; cat. no. 9197), HRP-conjugated IgG antibody (goat anti-rabbit; cat. no. 7074), HRP-conjugated IgG antibody (goat anti-mouse; cat. no. 7076) and Alexa Fluor 488-conjugated IgG (H+L) F(ab') 2 fragment (goat anti-rabbit; cat. no. 4412) were purchased from Cell Signaling Technology, Inc. Anti-calcitonin gene-related peptide (CGRP) antibody (goat polyclonal; cat. no. ab36001) and Alexa Fluor 647-conjugated IgG H&L (donkey anti-goat; cat. no. ab150135) were purchased from Abcam.

Techniques: Injection, Control

Journal: Arquivos Brasileiros de Cardiologia

Article Title: SLC26A4-AS1 Agrava a Hipertrofia Cardíaca Induzida por AngII Aumentando a Expressão de SLC26A4

doi: 10.36660/abc.20210933

Figure Lengend Snippet: – Sequência de iniciadores para SLC26A4-AS1 e GAPDH

Article Snippet: Subsequently, the membrane was cultured with primary antibodies against ANP (1μg/ml, ab237632, Abcam, Cambridge, MA, USA), BNP (1μg/ml, ab92500, Abcam), β-MHC (1/2500, ab55152, Abcam), SLC26A4 (0.25 μg/μl, 20889-1-AP, Proteintech, Chicago, USA) and GAPDH (1/2500, ab8245, Abcam) at 4°C overnight.

Techniques:

Journal: Arquivos Brasileiros de Cardiologia

Article Title: SLC26A4-AS1 Agrava a Hipertrofia Cardíaca Induzida por AngII Aumentando a Expressão de SLC26A4

doi: 10.36660/abc.20210933

Figure Lengend Snippet: – SLC26A4-AS1 é regulado para cima na hipertrofia cardíaca induzida por AngII. (A-C) Em NMVCs tratados com 150 nM AngII por 1, 6, 12 e 24 h, a expressão de biomarcadores hipertróficos (ANP, BNP e β-MHC) foi avaliada usando RT-qPCR, e o nível de proteína desses biomarcadores foi avaliada por western blot, seguida de quantificação por western blot. *P<0,05: AngII (1 h) vs AngII (0 h). *P<0,05: AngII (6 h) vs AngII (0 h). **P<0,01: AngII (12 h) vs AngII (0 h). **P<0,01: AngII (24 h) vs AngII (0 h). O número de tamanho da amostra (n) = 3. (D) A área de superfície celular em NMVCs tratadas com 150 nM AngII por 1, 6, 12 e 24 h foi avaliada usando coloração IF. Barra de escala: 10 μm. *P<0,05: AngII (1 h) vs AngII (0 h). *P<0,05: AngII (6 h) vs AngII (0 h). **P<0,01: AngII (12 h) vs AngII (0 h). **P<0,01: AngII (24 h) vs AngII (0 h). n = 3. (E) A expressão de SLC26A4-AS1 em NMVCs tratados com 150 nM AngII por 1, 6, 12 e 24 h foi detectada usando RT-qPCR. *P<0,05: AngII (1 h) vs AngII (0 h). *P<0,05: AngII (6 h) vs AngII (0 h). **P<0,01: AngII (12 h) vs AngII (0 h). **P<0,01: AngII (24 h) vs AngII (0 h). n = 3.

Article Snippet: Subsequently, the membrane was cultured with primary antibodies against ANP (1μg/ml, ab237632, Abcam, Cambridge, MA, USA), BNP (1μg/ml, ab92500, Abcam), β-MHC (1/2500, ab55152, Abcam), SLC26A4 (0.25 μg/μl, 20889-1-AP, Proteintech, Chicago, USA) and GAPDH (1/2500, ab8245, Abcam) at 4°C overnight.

Techniques: Quantitative RT-PCR, Western Blot

Journal: Arquivos Brasileiros de Cardiologia

Article Title: SLC26A4-AS1 Agrava a Hipertrofia Cardíaca Induzida por AngII Aumentando a Expressão de SLC26A4

doi: 10.36660/abc.20210933

Figure Lengend Snippet: – O silenciamento de SLC26A4-AS1 suprime a hipertrofia cardíaca induzida por AngII. (A) NMVCs foram tratados com sh-SLC26A4-AS1#1/2 ou sh-NC após o tratamento de AngII por 24 h. **P<0,01: AngII vs Controle. **P<0,01: AngII+sh-SLC26A4-AS1#1/2 vs AngII+sh-NC. n = 3. (B) A área da superfície celular foi detectada em NMVCs tratados com AngII após o silêncio de SLC26A4-AS1 usando coloração IF. Barra de escala: 10 μm. **P<0,01: AngII vs Controle. **P<0,01: AngII+sh-SLC26A4-AS1#1/2 vs AngII+sh-NC. n = 3. (C-H) Após o silenciamento de SLC26A4-AS1, a expressão de biomarcadores hipertróficos em NMVCs tratados com AngII foi medida usando RT-qPCR, e o nível desses biomarcadores foi avaliado usando western blot, seguido pela quantificação de western blot. **P<0,01: AngII vs Controle. **P<0,01: AngII+sh-SLC26A4-AS1#1/2 vs AngII+sh-NC. n = 3.

Article Snippet: Subsequently, the membrane was cultured with primary antibodies against ANP (1μg/ml, ab237632, Abcam, Cambridge, MA, USA), BNP (1μg/ml, ab92500, Abcam), β-MHC (1/2500, ab55152, Abcam), SLC26A4 (0.25 μg/μl, 20889-1-AP, Proteintech, Chicago, USA) and GAPDH (1/2500, ab8245, Abcam) at 4°C overnight.

Techniques: Quantitative RT-PCR, Western Blot

Journal: Arquivos Brasileiros de Cardiologia

Article Title: SLC26A4-AS1 Agrava a Hipertrofia Cardíaca Induzida por AngII Aumentando a Expressão de SLC26A4

doi: 10.36660/abc.20210933

Figure Lengend Snippet: – SLC26A4-AS1 funciona como um ceRNA para regular SLC26A4 na hipertrofia cardíaca induzida por AngII. (A-B) Fracionamento subcelular e ensaios de FISH foram realizados para determinar a localização de SLC26A4-AS1 em NMVCs induzidos por AngII. n = 3. (C) Ensaios RIP detectaram o enriquecimento de SLC26A4-AS1 e SLC26A4. **P<0,01: Ago2 vs IgG. n = 3. (D) A expressão de SLC26A4 em NMVCs tratados com 150 nM AngII por 1, 6, 12 e 24 h foi avaliada usando RT-qPCR. *P<0,05: AngII (1 h) vs AngII (0 h). *P<0,05: AngII (6 h) vs AngII (0 h). **P<0,01: AngII (12 h) vs AngII (0 h). **P<0,01: AngII (24 h) vs AngII (0 h). n = 3. (E-F) O nível de expressão e o nível de proteína de SLC26A4 em NMVCs tratados com AngII foram avaliados usando RT-qPCR e western blot, respectivamente, após regulação negativa de SLC26A4-AS1, seguido pela quantificação de western blot. **P<0,01: AngII vs Controle. **P<0,01: AngII+sh-SLC26A4-AS1#1/2 vs AngII+sh-NC. n = 3.

Article Snippet: Subsequently, the membrane was cultured with primary antibodies against ANP (1μg/ml, ab237632, Abcam, Cambridge, MA, USA), BNP (1μg/ml, ab92500, Abcam), β-MHC (1/2500, ab55152, Abcam), SLC26A4 (0.25 μg/μl, 20889-1-AP, Proteintech, Chicago, USA) and GAPDH (1/2500, ab8245, Abcam) at 4°C overnight.

Techniques: Quantitative RT-PCR, Western Blot

Journal: Arquivos Brasileiros de Cardiologia

Article Title: SLC26A4-AS1 Agrava a Hipertrofia Cardíaca Induzida por AngII Aumentando a Expressão de SLC26A4

doi: 10.36660/abc.20210933

Figure Lengend Snippet: – SLC26A4-AS1 promove hipertrofia cardíaca induzida por AngII via regulação para cima de SLC26A4. (A) NMVCs introduzidos com pcDNA3.1-SLC26A4, ou pcDNA3.1 foram tratados com AngII por 24 h. **P<0,01: pcDNA3.1-SLC26A4 vs pcDNA3.1. Experimentos de resgate foram conduzidos em NMVCs com a transfecção de sh-NC, sh-SLC26A4-AS1#1, sh-SLC26A4-AS1#1+pcDNA3.1 e sh-SLC26A4-AS1#1+pcDNA3.1-SLC26A4 sob tratamento com AngII . n = 3. (B) A área da superfície celular foi avaliada em NMVCs tratados com AngII usando coloração IF. **P<0,01: AngII vs Controle. **P<0,01: AngII+sh-SLC26A4-AS1#1 vs AngII+sh-NC. **P<0,01: AngII+sh-SLC26A4-AS1#1+pcDNA3.1-SLC26A4 vs AngII+sh-SLC26A4-AS1#1+pcDNA3.1. n = 3. (C-E) O nível de expressão e o nível de proteína de biomarcadores hipertróficos em NMVCs tratados com AngII foram medidos por meio de RT-qPCR e western blot, respectivamente, seguidos pela quantificação de western blot. **P<0,01: AngII vs Controle. **P<0,01: AngII+sh-SLC26A4-AS1#1 vs AngII+sh-NC. **P<0,01: AngII+sh-SLC26A4-AS1#1+pcDNA3.1-SLC26A4 vs AngII+sh-SLC26A4-AS1#1+pcDNA3.1. n = 3.

Article Snippet: Subsequently, the membrane was cultured with primary antibodies against ANP (1μg/ml, ab237632, Abcam, Cambridge, MA, USA), BNP (1μg/ml, ab92500, Abcam), β-MHC (1/2500, ab55152, Abcam), SLC26A4 (0.25 μg/μl, 20889-1-AP, Proteintech, Chicago, USA) and GAPDH (1/2500, ab8245, Abcam) at 4°C overnight.

Techniques: IF-P, Quantitative RT-PCR, Western Blot

Journal: Arquivos Brasileiros de Cardiologia

Article Title: SLC26A4-AS1 Agrava a Hipertrofia Cardíaca Induzida por AngII Aumentando a Expressão de SLC26A4

doi: 10.36660/abc.20210933

Figure Lengend Snippet: – SLC26A4-AS1 regula para cima a expressão de SLC26A4 por meio de absorção de miR-301a-3p ou miR-301b-3p. (A) Os miRNAs candidatos combinados com SLC26A4-AS1 e SLC26A4 foram obtidos no site da starBase (http://starbase.sysu.edu.cn). (B) O enriquecimento relativo de miRNAs candidatos em grupos bio-SLC26A4-AS1 foi analisado por meio do ensaio de RNA pull-down. **P<0,01: Bio-SLC26A4-AS1 vs Bio-NC. n = 3. (C) O ensaio RIP detectou o enriquecimento relativo de SLC26A4-AS1, miR-301a-3p, miR-301b-3p e SLC26A4. **P<0,01: Ago2 vs IgG. n = 3. (D-E) A expressão de miR-301a-3p ou miR-301b-3p em NMVCs tratados com 150 nM AngII por 1, 6, 12 e 24 h foi detectada usando RT-qPCR. *P<0,05: AngII (1h)/AngII (6h) vs AngII (0 h). **P<0,01: AngII (12h)/AngII (24h) vs AngII (0 h). n = 3. (F) Ensaios de luciferase repórter foram implementados para detectar a atividade de luciferase de SLC26A4-AS1 do tipo selvagem (WT) ou do tipo mutante (MUT). **P<0,01: mímicos miR-301a-3p/mímicos miR-301b-3p vs mímicos NC. n = 3. (G) Ensaios de repórter de luciferase foram realizados para detectar a atividade de luciferase de SLC26A4 3’UTR WT ou MUT. **P<0,01: mímicos miR-301a-3p/mímicos miR-301b-3p vs miméticos NC. n = 3. (H) Os efeitos da superexpressão de miR-301a-3p ou miR-301b-3p na expressão de SLC26A4 foram avaliados via RT-qPCR. **P<0,01: AngII vs Controle. **P<0,01: miméticos AngII+miR-301a-3p/miméticos miR-301b-3p vs miméticos AngII+NC. n = 3.

Article Snippet: Subsequently, the membrane was cultured with primary antibodies against ANP (1μg/ml, ab237632, Abcam, Cambridge, MA, USA), BNP (1μg/ml, ab92500, Abcam), β-MHC (1/2500, ab55152, Abcam), SLC26A4 (0.25 μg/μl, 20889-1-AP, Proteintech, Chicago, USA) and GAPDH (1/2500, ab8245, Abcam) at 4°C overnight.

Techniques: Quantitative RT-PCR, Luciferase

Journal: Arquivos Brasileiros de Cardiologia

Article Title: SLC26A4-AS1 Agrava a Hipertrofia Cardíaca Induzida por AngII Aumentando a Expressão de SLC26A4

doi: 10.36660/abc.20210933

Figure Lengend Snippet: – SLC26A4-AS1 afeta a hipertrofia cardíaca induzida por AngII via absorção de miR-301a-3p ou miR-301b-3p. (A-B) NMVCs tratados com inibidor de miR-301a-3p/inibidor de miR-301b-3p ou inibidor de NC foram tratados com AngII por 24 h. **P<0,01: inibidor de miR-301a-3p/inibidor de miR-301b-3p vs inibidor de NC. Experimentos de resgate foram conduzidos em NMVCs com a transfecção de sh-NC, sh-SLC26A4-AS1#1, sh-SLC26A4-AS1#1+inibidor de NC, sh-SLC26A4-AS1#1+miR-301a-3p inibidor e sh -SLC26A4-AS1#1+miR-301b-3p inibidor sob tratamento com AngII. n = 3. (C) A área da superfície celular foi medida em NMVCs tratados com AngII usando coloração IF. Barra de escala: 10 μm. **P<0,01: AngII vs Controle. **P<0,01: AngII+sh-SLC26A4-AS1#1 vs AngII+sh-NC. *P<0,05: AngII+sh-SLC26A4-AS1#1+inibidor de miR-301a-3p/inibidor de miR-301b-3p vs AngII+sh-SLC26A4-AS1#1+inibidor de NC. n = 3. (D-F) RT-qPCR e western blot avaliaram o nível de expressão e o nível de proteína de biomarcadores hipertróficos em NMVCs tratados com AngII, respectivamente, seguido pela quantificação de western blot. **P<0,01: AngII vs Controle. **P<0,01: AngII+sh-SLC26A4-AS1#1 vs AngII+sh-NC. *P<0,05: AngII+sh-SLC26A4-AS1#1+inibidor de miR-301a-3p/inibidor de miR-301b-3p vs AngII+sh-SLC26A4-AS1#1+inibidor de NC. n = 3.

Article Snippet: Subsequently, the membrane was cultured with primary antibodies against ANP (1μg/ml, ab237632, Abcam, Cambridge, MA, USA), BNP (1μg/ml, ab92500, Abcam), β-MHC (1/2500, ab55152, Abcam), SLC26A4 (0.25 μg/μl, 20889-1-AP, Proteintech, Chicago, USA) and GAPDH (1/2500, ab8245, Abcam) at 4°C overnight.

Techniques: Quantitative RT-PCR, Western Blot

Journal: Arquivos Brasileiros de Cardiologia

Article Title: SLC26A4-AS1 Agrava a Hipertrofia Cardíaca Induzida por AngII Aumentando a Expressão de SLC26A4

doi: 10.36660/abc.20210933

Figure Lengend Snippet: – Primer sequence for SLC26A4-AS1 and GAPDH

Article Snippet: Subsequently, the membrane was cultured with primary antibodies against ANP (1μg/ml, ab237632, Abcam, Cambridge, MA, USA), BNP (1μg/ml, ab92500, Abcam), β-MHC (1/2500, ab55152, Abcam), SLC26A4 (0.25 μg/μl, 20889-1-AP, Proteintech, Chicago, USA) and GAPDH (1/2500, ab8245, Abcam) at 4°C overnight.

Techniques: Sequencing

Journal: Arquivos Brasileiros de Cardiologia

Article Title: SLC26A4-AS1 Agrava a Hipertrofia Cardíaca Induzida por AngII Aumentando a Expressão de SLC26A4

doi: 10.36660/abc.20210933

Figure Lengend Snippet: – SLC26A4-AS1 is upregulated in AngII-induced cardiac hypertrophy. (A-C) In NMVCs treated with 150 nM AngII for 1, 6, 12, and 24 h, the expression of hypertrophic biomarkers (ANP, BNP, and β-MHC) was assessed using RT-qPCR, and the protein level of these biomarkers was evaluated through western blot, followed by western blot quantification. *P<0.05: AngII (1 h) vs AngII (0 h). *P<0.05: AngII (6 h) vs AngII (0 h). **P<0.01: AngII (12 h) vs AngII (0 h). **P<0.01: AngII (24 h) vs AngII (0 h). The number of sample size (n) = 3. (D) The cell surface area in NMVCs treated with 150 nM AngII for 1, 6, 12, and 24 h was assessed using IF staining. Scale bar: 10 μm. *P<0.05: AngII (1 h) vs AngII (0 h). *P<0.05: AngII (6 h) vs AngII (0 h). **P<0.01: AngII (12 h) vs AngII (0 h). **P<0.01: AngII (24 h) vs AngII (0 h). n = 3. (E) SLC26A4-AS1 expression in NMVCs treated with 150 nM AngII for 1, 6, 12, and 24 h was detected using RT-qPCR. *P<0.05: AngII (1 h) vs AngII (0 h). *P<0.05: AngII (6 h) vs AngII (0 h). **P<0.01: AngII (12 h) vs AngII (0 h). **P<0.01: AngII (24 h) vs AngII (0 h). n = 3.

Article Snippet: Subsequently, the membrane was cultured with primary antibodies against ANP (1μg/ml, ab237632, Abcam, Cambridge, MA, USA), BNP (1μg/ml, ab92500, Abcam), β-MHC (1/2500, ab55152, Abcam), SLC26A4 (0.25 μg/μl, 20889-1-AP, Proteintech, Chicago, USA) and GAPDH (1/2500, ab8245, Abcam) at 4°C overnight.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Staining

Journal: Arquivos Brasileiros de Cardiologia

Article Title: SLC26A4-AS1 Agrava a Hipertrofia Cardíaca Induzida por AngII Aumentando a Expressão de SLC26A4

doi: 10.36660/abc.20210933

Figure Lengend Snippet: – SLC26A4-AS1 silence suppresses AngII-induced cardiac hypertrophy. (A) NMVCs were treated with sh-SLC26A4-AS1#1/2 or sh-NC after the treatment of AngII for 24 h. **P<0.01: AngII vs Control. **P<0.01: AngII+sh-SLC26A4-AS1#1/2 vs AngII+sh-NC. n = 3. (B) Cell surface area was detected in AngII-treated NMVCs upon SLC26A4-AS1 silence using IF staining. Scale bar: 10 μm. **P<0.01: AngII vs Control. **P<0.01: AngII+sh-SLC26A4-AS1#1/2 vs AngII+sh-NC. n = 3. (C-H) After SLC26A4-AS1 silencing, the expression of hypertrophic biomarkers in AngII-treated NMVCs was measured using RT-qPCR, and the level of these biomarkers was assessed using western blot, followed by the quantification of western blot. **P<0.01: AngII vs Control. **P<0.01: AngII+sh-SLC26A4-AS1#1/2 vs AngII+sh-NC. n = 3.

Article Snippet: Subsequently, the membrane was cultured with primary antibodies against ANP (1μg/ml, ab237632, Abcam, Cambridge, MA, USA), BNP (1μg/ml, ab92500, Abcam), β-MHC (1/2500, ab55152, Abcam), SLC26A4 (0.25 μg/μl, 20889-1-AP, Proteintech, Chicago, USA) and GAPDH (1/2500, ab8245, Abcam) at 4°C overnight.

Techniques: Control, Staining, Expressing, Quantitative RT-PCR, Western Blot

Journal: Arquivos Brasileiros de Cardiologia

Article Title: SLC26A4-AS1 Agrava a Hipertrofia Cardíaca Induzida por AngII Aumentando a Expressão de SLC26A4

doi: 10.36660/abc.20210933

Figure Lengend Snippet: – SLC26A4-AS1 functions as a ceRNA to regulate SLC26A4 in cardiac hypertrophy induced by AngII. (A-B) Subcellular fractionation and FISH assays were performed to determine the location of SLC26A4-AS1 in AngII-induced NMVCs. n = 3. (C) RIP assays detected the enrichment of SLC26A4-AS1 and SLC26A4. **P<0.01: Ago2 vs IgG. n = 3. (D) The expression of SLC26A4 in NMVCs treated with 150 nM AngII for 1, 6, 12, and 24 h was assessed using RT-qPCR. *P<0.05: AngII (1 h) vs AngII (0 h). *P<0.05: AngII (6 h) vs AngII (0 h). **P<0.01: AngII (12 h) vs AngII (0 h). **P<0.01: AngII (24 h) vs AngII (0 h). n = 3. (E-F) Expression level and protein level of SLC26A4 in AngII-treated NMVCs were assessed using RT-qPCR and western blot, respectively, after SLC26A4-AS1 down-regulation, followed by the quantification of western blot. **P<0.01: AngII vs Control. **P<0.01: AngII+sh-SLC26A4-AS1#1/2 vs AngII+sh-NC. n = 3.

Article Snippet: Subsequently, the membrane was cultured with primary antibodies against ANP (1μg/ml, ab237632, Abcam, Cambridge, MA, USA), BNP (1μg/ml, ab92500, Abcam), β-MHC (1/2500, ab55152, Abcam), SLC26A4 (0.25 μg/μl, 20889-1-AP, Proteintech, Chicago, USA) and GAPDH (1/2500, ab8245, Abcam) at 4°C overnight.

Techniques: Fractionation, Expressing, Quantitative RT-PCR, Western Blot, Control

Journal: Arquivos Brasileiros de Cardiologia

Article Title: SLC26A4-AS1 Agrava a Hipertrofia Cardíaca Induzida por AngII Aumentando a Expressão de SLC26A4

doi: 10.36660/abc.20210933

Figure Lengend Snippet: – SLC26A4-AS1 promove hipertrofia cardíaca induzida por AngII via regulação para cima de SLC26A4. (A) NMVCs introduzidos com pcDNA3.1-SLC26A4, ou pcDNA3.1 foram tratados com AngII por 24 h. **P<0,01: pcDNA3.1-SLC26A4 vs pcDNA3.1. Experimentos de resgate foram conduzidos em NMVCs com a transfecção de sh-NC, sh-SLC26A4-AS1#1, sh-SLC26A4-AS1#1+pcDNA3.1 e sh-SLC26A4-AS1#1+pcDNA3.1-SLC26A4 sob tratamento com AngII . n = 3. (B) A área da superfície celular foi avaliada em NMVCs tratados com AngII usando coloração IF. **P<0,01: AngII vs Controle. **P<0,01: AngII+sh-SLC26A4-AS1#1 vs AngII+sh-NC. **P<0,01: AngII+sh-SLC26A4-AS1#1+pcDNA3.1-SLC26A4 vs AngII+sh-SLC26A4-AS1#1+pcDNA3.1. n = 3. (C-E) O nível de expressão e o nível de proteína de biomarcadores hipertróficos em NMVCs tratados com AngII foram medidos por meio de RT-qPCR e western blot, respectivamente, seguidos pela quantificação de western blot. **P<0,01: AngII vs Controle. **P<0,01: AngII+sh-SLC26A4-AS1#1 vs AngII+sh-NC. **P<0,01: AngII+sh-SLC26A4-AS1#1+pcDNA3.1-SLC26A4 vs AngII+sh-SLC26A4-AS1#1+pcDNA3.1. n = 3.

Article Snippet: Subsequently, the membrane was cultured with primary antibodies against ANP (1μg/ml, ab237632, Abcam, Cambridge, MA, USA), BNP (1μg/ml, ab92500, Abcam), β-MHC (1/2500, ab55152, Abcam), SLC26A4 (0.25 μg/μl, 20889-1-AP, Proteintech, Chicago, USA) and GAPDH (1/2500, ab8245, Abcam) at 4°C overnight.

Techniques: IF-P, Quantitative RT-PCR, Western Blot

Journal: Arquivos Brasileiros de Cardiologia

Article Title: SLC26A4-AS1 Agrava a Hipertrofia Cardíaca Induzida por AngII Aumentando a Expressão de SLC26A4

doi: 10.36660/abc.20210933

Figure Lengend Snippet: – SLC26A4-AS1 positively regulates SLC26A4 expression via sponging miR-301a-3p or miR-301b-3p. (A) Candidate miRNAs combined with SLC26A4-AS1 and SLC26A4 were obtained from the starBase website (http://starbase.sysu.edu.cn). (B) The relative enrichment of candidate miRNAs in bio-SLC26A4-AS1 groups was analyzed via RNApull-down assay. **P<0.01: Bio-SLC26A4-AS1 vs Bio-NC. n = 3. (C) The RIP assay detected the relative enrichment of SLC26A4-AS1, miR-301a-3p, miR-301b-3p, and SLC26A4. **P<0.01: Ago2 vs IgG. n = 3. (D-E) Expression of miR-301a-3p or miR-301b-3p in NMVCs treated with 150 nM AngII for 1, 6, 12, and 24 h was detected using RT-qPCR. *P<0.05: AngII (1h)/AngII (6h) vs AngII (0 h). **P<0.01: AngII (12h)/AngII (24h) vs AngII (0 h). n = 3. (F) Luciferase reporter assays were implemented to detect the luciferase activity of SLC26A4-AS1 wild-type (WT) or mutant-type (MUT). **P<0.01: miR-301a-3p mimics/miR-301b-3p mimics vs NC mimics. n = 3. (G) Luciferase reporter assays were performed to detect the luciferase activity of SLC26A4 3’UTR WT or MUT. **P<0.01: miR-301a-3p mimics/miR-301b-3p mimics vs NC mimics. n = 3. (H) The effects of miR-301a-3p or miR-301b-3p overexpression on SLC26A4 expression were assessed via RT-qPCR. **P<0.01: AngII vs Control. **P<0.01: AngII+miR-301a-3p mimics/miR-301b-3p mimics vs AngII+NC mimics. n = 3.

Article Snippet: Subsequently, the membrane was cultured with primary antibodies against ANP (1μg/ml, ab237632, Abcam, Cambridge, MA, USA), BNP (1μg/ml, ab92500, Abcam), β-MHC (1/2500, ab55152, Abcam), SLC26A4 (0.25 μg/μl, 20889-1-AP, Proteintech, Chicago, USA) and GAPDH (1/2500, ab8245, Abcam) at 4°C overnight.

Techniques: Expressing, Quantitative RT-PCR, Luciferase, Activity Assay, Mutagenesis, Over Expression, Control

Journal: Arquivos Brasileiros de Cardiologia

Article Title: SLC26A4-AS1 Agrava a Hipertrofia Cardíaca Induzida por AngII Aumentando a Expressão de SLC26A4

doi: 10.36660/abc.20210933

Figure Lengend Snippet: – SLC26A4-AS1 affects AngII-induced cardiac hypertrophy via sponging miR-301a-3p or miR-301b-3p. (A-B) NMVCs introduced with miR-301a-3p inhibitor/miR-301b-3p inhibitor, or NC inhibitor were treated with AngII for 24 h. **P<0.01: miR-301a-3p inhibitor/miR-301b-3p inhibitor vs NC inhibitor. Rescue experiments were conducted in NMVCs with the transfection of sh-NC, sh-SLC26A4-AS1#1, sh-SLC26A4-AS1#1+NC inhibitor, sh-SLC26A4-AS1#1+miR-301a-3p inhibitor, and sh-SLC26A4-AS1#1+miR-301b-3p inhibitor under AngII treatment. n = 3. (C) Cell surface area was measured in AngII-treated NMVCs using IF staining. Scale bar: 10 μm. **P<0.01: AngII vs Control. **P<0.01: AngII+sh-SLC26A4-AS1#1 vs AngII+sh-NC. *P<0.05: AngII+sh-SLC26A4-AS1#1+miR-301a-3p inhibitor/miR-301b-3p inhibitor vs AngII+sh-SLC26A4-AS1#1+NC inhibitor. n = 3. (D-F) RT-qPCR and western blot evaluated the expression level and protein level of hypertrophic biomarkers in AngII-treated NMVCs, respectively, followed by the quantification of western blot. **P<0.01: AngII vs Control. **P<0.01: AngII+sh-SLC26A4-AS1#1 vs AngII+sh-NC. *P<0.05: AngII+sh-SLC26A4-AS1#1+miR-301a-3p inhibitor/miR-301b-3p inhibitor vs AngII+sh-SLC26A4-AS1#1+NC inhibitor. n = 3.

Article Snippet: Subsequently, the membrane was cultured with primary antibodies against ANP (1μg/ml, ab237632, Abcam, Cambridge, MA, USA), BNP (1μg/ml, ab92500, Abcam), β-MHC (1/2500, ab55152, Abcam), SLC26A4 (0.25 μg/μl, 20889-1-AP, Proteintech, Chicago, USA) and GAPDH (1/2500, ab8245, Abcam) at 4°C overnight.

Techniques: Transfection, Staining, Control, Quantitative RT-PCR, Western Blot, Expressing

Journal: Cells

Article Title: Melatonin Treatment Triggers Metabolic and Intracellular pH Imbalance in Glioblastoma.

doi: 10.3390/cells11213467

Figure Lengend Snippet: Figure 3. Melatonin (aMT) disrupts the pH balance in GBM while downregulating glycolysis. (a) Representative Western blot of lactate dehydrogenase (LDH), pyruvate dehydrogenase (PDH), pyruvate kinase subtype M2 (PKM2), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) after 96 h of treatment with the vehicle or aMT. (b) Western blot quantification of lactate dehydrogenase (LDH) after 96 h of treatment with vehicle or aMT; (c) Intracellular lactate overtime after treatment with vehicle or aMT in GBM1A and QNS120; (d) Representative Western blot of Monocarboxylate transporter 4 (MCT4) after 96 h of treatment with vehicle or aMT; (e) Western blot quantification of MCT4 after 96 h of treatment with vehicle or aMT; (f) Intracellular pH after 48 or 96 h with vehicle or aMT in GBM1A; (g) Extracellular pH overtime after treatment with vehicle or aMT in GBM1A and QNS120; (h) Western blot quantification of pyruvate kinase subtype M2 (PKM2) and (i) glyceraldehyde 3-phosphate dehydrogenase (GAPDH) after 96 h of treatment with vehicle or aMT; (j) Glucose availability in media overtime in GBM1A and QNS120. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: We used the following primary antibodies: MCT4 (A304-439A; Bethyl Laboratories, Montgomery, TX, USA); LDHA (3582s; Cell Signaling Technology, Danvers, MA, USA); PDHA (3205S; Cell Signaling Technology); PKM2 (4053S; Cell Signaling Technology); and GAPDH (sc-47724; Santa Cruz Biotechnology, Inc. Dallas, TX, USA).

Techniques: Western Blot

Journal: Cells

Article Title: Melatonin Treatment Triggers Metabolic and Intracellular pH Imbalance in Glioblastoma.

doi: 10.3390/cells11213467

Figure Lengend Snippet: Figure 5. Graphical abstract. Melatonin oncostatic effects in GBM. aMT: melatonin; iPH: intracel- lular pH; ROS: reactive oxygen species; H+: proton; LDH: lactate dehydrogenase; PDH: pyruvate dehydrogenase; PKM2: pyruvate kinase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; G6PDH: glucose-6-phosphate dehydrogenase; PPP: pentose phosphate pathway, MCT4: monocar- boxylate transporter 4; GLUT: glucose transporters.

Article Snippet: We used the following primary antibodies: MCT4 (A304-439A; Bethyl Laboratories, Montgomery, TX, USA); LDHA (3582s; Cell Signaling Technology, Danvers, MA, USA); PDHA (3205S; Cell Signaling Technology); PKM2 (4053S; Cell Signaling Technology); and GAPDH (sc-47724; Santa Cruz Biotechnology, Inc. Dallas, TX, USA).

Techniques: