Journal: bioRxiv

Article Title: Loss of STARD7 Impairs Mitochondrial Phospholipid Homeostasis and Contributes to Mitochondrial Myopathy

doi: 10.1101/2025.11.27.691062

Figure Lengend Snippet: Loss of STARD7 disrupts fast-twitch–associated gene expression and fiber-type composition in soleus muscle (A) PCA of RNA-seq data from control (cont) and mKO soleus muscles. Each dot represents an individual animal (n = 3 per group). (B) Volcano plot of differentially expressed genes (DEGs). Differential expression was analyzed using the edgeR package after applying filterByExpr to remove low-expression genes. Genes with |log fold change| > 1 and FDR < 0.05 were defined as DEGs. Fast-twitch–related genes, including Mybpc2 , Actn3 , Pvalb , Mct4 , and Myh4 , were significantly downregulated in mKO. (C) Heatmap of DEGs (FDR < 0.05, |log fold change| > 1) in the soleus. (D) Functional enrichment analysis of DEGs using the clusterProfiler package for Gene Ontology (GO) and WikiPathways. GO terms and pathways with adjusted p < 0.05 were considered significantly enriched. (E) Immunoblot analysis of fast-twitch markers. Each lane represents an independent animal (n = 3). CBB staining confirms equal loading. (F) Representative image of soleus cross-sections stained with isoform-specific myosin heavy chain antibodies. Blue, type I (MyHC-I); red, type IIa (MyHC-IIa); green, type IIb (MyHC-IIb). Fibers lacking these isoforms were classified as type IIx. Scale bar, 200 μm.

Article Snippet: Antibodies used were anti-STARD7 (15191-1-AP), UQCRC2 (14742-1-AP), PVALB (29312-1-AP), MYBPC2 (25257-1-AP), MCT4 (22787-1-AP), MYH4 (20140-1-AP), MYH7 (22280-1-AP), CRLS1 (14845-1-AP), and PGS1 (17149-1-AP) (Proteintech, Rosemont, IL, USA); anti-TAZ (sc-365810) (Santa Cruz Biotechnology, Dallas, TX, USA); OXPHOS complexes (ab110413) (Abcam, Cambridge, UK); anti-OPA1 (612606) (BD Biosciences, San Jose, CA, USA); and 4-HNE (MHN-100P) (JaICA, Shizuoka, Japan).

Techniques: Gene Expression, RNA Sequencing, Control, Muscles, Quantitative Proteomics, Expressing, Functional Assay, Western Blot, Staining

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Monocarboxylate transporter 4, associated with the acidification of synovial fluid, is a novel therapeutic target for inflammatory arthritis.

doi: 10.1002/art.39270

Figure Lengend Snippet: Figure 2. Monocarboxylate transporter 4 (MCT4) mRNA and protein are up-regulated in rheumatoid arthritis synovial fibroblasts (RASFs). A, Lev- els of mRNA transcripts of Na1/H1 exchanger 1 (NHE-1), vacuolar-type H1ATPase (V-ATPase), MCT1, and MCT4 relative to that of b-actin in nor- mal SFs, 4 osteoarthritis SF (OASF) lines, and 4 RASF lines. B, Western blot analysis of MCT4 and b-actin protein in normal SFs, OASF lines, and RASF lines. C, Quantification depiction of the findings shown in B. Shown is MCT4 protein expression relative to b-actin protein levels in the 4 OASF lines and the 4 RASF lines. Values in A and C are the mean 6 SD. * 5 P , 0.05; ** 5 P , 0.01 by Student’s t-test. D, RA synovial tissue specimens incubated with rabbit anti-MCT4 antibodies (left) and with control normal rabbit serum (middle), and OA synovial tissue specimens incubated with anti-MCT4 antibodies (right). Boxed areas at the top are shown at higher magnification at the bottom. Results are representative of 8 RA synovial tis- sue specimens and 5 OA synovial tissue specimens. Bars 5 100 mm (top); 10 mm (bottom). Original magnification 3 100 (top); 3 400 (bottom).

Article Snippet: A rabbit anti-human MCT4 antibody (1:100) or a rabbit anti-mouse MCT4 antibody (1:100; Santa Cruz Biotechnology) served as the primary antibody, and normal rabbit serum (1:100; Wako) served as the control.

Techniques: Western Blot, Expressing, Incubation, Control

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Monocarboxylate transporter 4, associated with the acidification of synovial fluid, is a novel therapeutic target for inflammatory arthritis.

doi: 10.1002/art.39270

Figure Lengend Snippet: Figure 3. Knockdown of monocarboxylate transporter 4 (MCT4) with small interfering RNA (siRNA). A, Rheumatoid arthritis syno- vial fibroblast (RASF) lines were transfected with MCT4 siRNA or with control siRNA (mock). MCT4 siRNA transfection caused the number of MCT4 mRNA transcripts to fall relative to the number of b-actin mRNA transcripts. Values are the mean 6 SD of 3 RASF lines. ** 5 P , 0.01 by Student’s unpaired t-test. B, Western blot analysis of MCT4 and b-actin showed that MCT4 siRNA transfec- tion abrogated MCT4 expression in RASFs. Results are representa- tive of 3 independent experiments.

Article Snippet: A rabbit anti-human MCT4 antibody (1:100) or a rabbit anti-mouse MCT4 antibody (1:100; Santa Cruz Biotechnology) served as the primary antibody, and normal rabbit serum (1:100; Wako) served as the control.

Techniques: Knockdown, Small Interfering RNA, Transfection, Control, Western Blot, Expressing

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Monocarboxylate transporter 4, associated with the acidification of synovial fluid, is a novel therapeutic target for inflammatory arthritis.

doi: 10.1002/art.39270

Figure Lengend Snippet: Figure 4. Knockdown of monocarboxylate transporter 4 (MCT4) inhibits growth of rheumatoid arthritis synovial fibroblasts (RASFs). A, Num- bers of RASFs and osteoarthritis SFs (OASFs) after transfection with MCT4 small interfering RNA (siRNA) or control siRNA (mock). B, Flow cytometry data showing dilution of 5,6-carboxyfluorescein succinimidyl ester (CFSE) in MCT4 siRNA– and control siRNA–transfected RASFs on day 3. Shown are representative histograms (left) and percentages of divided RASFs (n 5 3 cell lines) (right). C, Flow cytometric analysis of annexin V and propidium iodide (PI) staining of RASFs and OASFs transfected with MCT4 siRNA or control siRNA, and of RASFs treated with cycloheximide (CHX; final concentration 100 mg/ml) as a positive control. Shown are representative dot plots (top) and percentages of PI- negative/annexin V–positive early apoptotic cells (bottom left) and PI-positive/annexin V–positive late apoptotic cells (bottom right). D, Left, Intracellular pH and L-lactate concentrations in RASFs transfected with MCT4 siRNA or control siRNA. Right, Extracellular pH and L-lactate concentrations in medium of RASFs transfected with MCT4 siRNA or control siRNA. Values are the mean 6 SD. * 5 P , 0.05; ** 5 P , 0.01 versus control siRNA–transfected RASFs, by Student’s t-test.

Article Snippet: A rabbit anti-human MCT4 antibody (1:100) or a rabbit anti-mouse MCT4 antibody (1:100; Santa Cruz Biotechnology) served as the primary antibody, and normal rabbit serum (1:100; Wako) served as the control.

Techniques: Knockdown, Transfection, Small Interfering RNA, Control, Flow Cytometry, Staining, Concentration Assay, Positive Control

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Monocarboxylate transporter 4, associated with the acidification of synovial fluid, is a novel therapeutic target for inflammatory arthritis.

doi: 10.1002/art.39270

Figure Lengend Snippet: Figure 5. Knockdown of monocarboxylate transporter 4 (MCT4) induces intrinsic apoptosis of rheumatoid arthritis synovial fibroblasts (RASFs). A, Western blot analysis of RASF lines transfected with MCT4 small interfering RNA (siRNA) or control siRNA (mock) to determine caspase 3, caspase 9, and caspase 8 protein expression. B, Levels of cleaved caspase 3 and caspase 9 expression relative to pro- caspase 3 and procaspase 9 expression levels, respectively, for all 3 RASF lines. Values are the mean 6 SD. * 5 P , 0.05 versus control siRNA–transfected RASFs, by Student’s t-test.

Article Snippet: A rabbit anti-human MCT4 antibody (1:100) or a rabbit anti-mouse MCT4 antibody (1:100; Santa Cruz Biotechnology) served as the primary antibody, and normal rabbit serum (1:100; Wako) served as the control.

Techniques: Knockdown, Western Blot, Transfection, Small Interfering RNA, Control, Expressing

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Monocarboxylate transporter 4, associated with the acidification of synovial fluid, is a novel therapeutic target for inflammatory arthritis.

doi: 10.1002/art.39270

Figure Lengend Snippet: Figure 6. Knockdown of monocarboxylate transporter 4 (MCT4) reduces severity of collagen-induced arthritis (CIA) in mice. A, Synovial tissue from the knee joint of a mouse with CIA and a control naive mouse after incubation with a rabbit anti-MCT4 antibody. Bars 5 100 mm. B, Left, Expression of 6-FAM in the knee joint 24 hours after electroporation, as observed under a fluorescence stereomicroscope. Right, Brightfield image of the same joint. C, Synovial tissue from the left knee joints of mice with CIA after electrotransfer of MCT4-specific small interfering RNA (siRNA) or control siRNA (mock) and incubation with a rabbit anti-MCT4 antibody. Bars 5 100 mm. D, Top, Hematoxylin and eosin (H&E)–stained synovial tissue from the knee joints of mice with CIA 72 hours after electrotransfer of MCT4-specific siRNA or control siRNA. Bars 5 100 mm. Middle, Histologic scores (n 5 10 mice per group). Values are the mean 6 SD. * 5 P , 0.05 by Mann-Whitney U test. Bottom, TUNEL assay performed in synovial tissue from the knee joints of mice with CIA transfected with MCT4-specific siRNA or control siRNA. Bars 5 10 mm.

Article Snippet: A rabbit anti-human MCT4 antibody (1:100) or a rabbit anti-mouse MCT4 antibody (1:100; Santa Cruz Biotechnology) served as the primary antibody, and normal rabbit serum (1:100; Wako) served as the control.

Techniques: Knockdown, Control, Incubation, Expressing, Electroporation, Fluorescence, Electrotransfer, Small Interfering RNA, Staining, MANN-WHITNEY, TUNEL Assay, Transfection

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: Advanced Science

Article Title: POU2F1 Promotes Chemoresistance in Colorectal Cancer Cells via Attenuates the MDR2 Degradation Mediated by PPP1R11 Lactylation

doi: 10.1002/advs.202522316

Figure Lengend Snippet: POU2F1 restrains PPP1R11 lactylation via up‐regulating MCT4 expression. (A, B) Volcano plot (A) and Heatmap (B) showing the transcriptomic expression of genes involving the production and transport of lactate according to silencing POU2F1 cells and parental cells. The three columns of histograms on the right show the results of differential expression analysis of each gene in indicated comparisons on the top of each column. (C, D) The mRNA (C) and protein (D) levels of SMCT2, MCT3, and MCT4 in POU2F1 silencing/ overexpressing CRC cells. (E, F) The mRNA (E) and protein (F) levels of SMCT2, MCT3, and MCT4 in POU2F1 silencing CRC cells rescued with POU2F1 overexpression. (G, H) Quantification of intracellular lactate (G) and protein level of PPP1R11 lactylation (H) in the indicated cells with or without 20 mM MSC‐4381 treatment. (I, J) Quantification of intracellular lactate (I) and protein level of PPP1R11 lactylation (J) in the indicated cells with MCT4 silencing/ overexpression. Data are represented as the means ± standard deviation (SD). * p < 0.05; ** p < 0.01; *** p < 0.001; ns, no significance.

Article Snippet: Small molecular compounds, such as MG‐132 (S2619, Selleck), Bromosulfalein (Cayman), cycloheximide (C1988, Sigma), L‐lactate (L7022, Merck), Glucose (D9434, Merck) RS6212 (HY‐150753, MCE), Syrosingopine (S9907, Selleck), Rotenone (HY‐B1756, MCE), MSC‐4381 (HY‐132301, MCE), and DAPI (0100‐20, Southern Biotech) were also purchased from the indicated suppliers.

Techniques: Expressing, Quantitative Proteomics, Over Expression, Standard Deviation

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: MDA-MB-231 and EO771 BC cells were infected with 1 μ g control shRNA (cat. no. sc-108080-V; Santa Cruz Biotechnology, Inc.) or MCT4 shRNA using a lentiviral particle transduction system (multiplicity of infection, 2.0) (cat. nos. sc-45892-V and sc-40120-V; Santa Cruz Biotechnology, Inc.) in the presence of 5 μ g/ml polybrene (sc-134220; Santa Cruz Biotechnology, Inc) for 24 h. Subsequently, infected cells were cultured in DMEM with 10% FBS for 7 days in the presence of 2 μ g/ml puromycin to select cells stably expressing the shRNAs, following which subsequent experiments were performed.

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: MDA-MB-231 and EO771 BC cells were infected with 1 μ g control shRNA (cat. no. sc-108080-V; Santa Cruz Biotechnology, Inc.) or MCT4 shRNA using a lentiviral particle transduction system (multiplicity of infection, 2.0) (cat. nos. sc-45892-V and sc-40120-V; Santa Cruz Biotechnology, Inc.) in the presence of 5 μ g/ml polybrene (sc-134220; Santa Cruz Biotechnology, Inc) for 24 h. Subsequently, infected cells were cultured in DMEM with 10% FBS for 7 days in the presence of 2 μ g/ml puromycin to select cells stably expressing the shRNAs, following which subsequent experiments were performed.

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: MDA-MB-231 and EO771 BC cells were infected with 1 μ g control shRNA (cat. no. sc-108080-V; Santa Cruz Biotechnology, Inc.) or MCT4 shRNA using a lentiviral particle transduction system (multiplicity of infection, 2.0) (cat. nos. sc-45892-V and sc-40120-V; Santa Cruz Biotechnology, Inc.) in the presence of 5 μ g/ml polybrene (sc-134220; Santa Cruz Biotechnology, Inc) for 24 h. Subsequently, infected cells were cultured in DMEM with 10% FBS for 7 days in the presence of 2 μ g/ml puromycin to select cells stably expressing the shRNAs, following which subsequent experiments were performed.

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: MDA-MB-231 and EO771 BC cells were infected with 1 μ g control shRNA (cat. no. sc-108080-V; Santa Cruz Biotechnology, Inc.) or MCT4 shRNA using a lentiviral particle transduction system (multiplicity of infection, 2.0) (cat. nos. sc-45892-V and sc-40120-V; Santa Cruz Biotechnology, Inc.) in the presence of 5 μ g/ml polybrene (sc-134220; Santa Cruz Biotechnology, Inc) for 24 h. Subsequently, infected cells were cultured in DMEM with 10% FBS for 7 days in the presence of 2 μ g/ml puromycin to select cells stably expressing the shRNAs, following which subsequent experiments were performed.

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: MDA-MB-231 and EO771 BC cells were infected with 1 μ g control shRNA (cat. no. sc-108080-V; Santa Cruz Biotechnology, Inc.) or MCT4 shRNA using a lentiviral particle transduction system (multiplicity of infection, 2.0) (cat. nos. sc-45892-V and sc-40120-V; Santa Cruz Biotechnology, Inc.) in the presence of 5 μ g/ml polybrene (sc-134220; Santa Cruz Biotechnology, Inc) for 24 h. Subsequently, infected cells were cultured in DMEM with 10% FBS for 7 days in the presence of 2 μ g/ml puromycin to select cells stably expressing the shRNAs, following which subsequent experiments were performed.

Techniques: Injection, Control

Journal: Cell Communication and Signaling : CCS

Article Title: Lactate-induced MRP1 expression contributes to metabolism-based etoposide resistance in non-small cell lung cancer cells

doi: 10.1186/s12964-020-00653-3

Figure Lengend Snippet: Induction of lactate metabolism inhibits genotoxic and apoptotic effects of etoposide in NSCLC. a Treatment of cells with etoposide for 5 h resulted in an increase in lactate secretion (* p <0.05, ** P< 0.01, *** p <0.001 for difference from untreated control by ANOVA for multiple comparison, ns means no statistical difference). b The cells were treated with different concentrations of etoposide or 1% Triton X-100 as positive control for 5 h. Culture medium was collected for determination of released lactate dehydrogenase (LDH) reflecting the membrane rapture. No significant difference of LDH release was detected after etoposide treatment (difference from untreated control by ANOVA for multiple comparison, ns means no statistical difference). c The extracellular acidification rate (ECAR) was measured in cells exposed to 25 μM etoposide for 5 h. The concentrations of oligomycin, FCCP were 10 μM and the bar is representative of mean ± S.D. of three independent experiments (* p <0.05, student t-test). d Measurement of 2-DG uptake in cells after exposed etoposide for 5 h. (* p <0.05, ** P< 0.01, for difference from untreated control by ANOVA for multiple comparison, ns means no statistical difference). e Western blot analysis was performed to examine glycolysis enzymes, HK2, MCT4, LDHA, MCT1 and Glut1, in different histological lung cancer cell lines A549, H1299 and H446 treated for indicated time (upper panel, 25 μM) or for various concentrations (lower panel) of etoposide. f The cells were first stimulated either with LA (lactic acid) or NaL (sodium lactate) for 3 h followed by further treatment with 25 μM etoposide for additional 36 h and then lysed for western blot analysis using antibodies against Cleaved-PARP and γH2AX. It is lactic acid, but not its sodium salt, suppressed the levels of γH2AX in both A549 and H1299 cell lines. g Comet assay reveal that 20 mM LA (lactic acid) inhibited DNA damage induced by etoposide. The quantification was present in right panel. The bars represent the mean ± S.D. of triplicates (** p <0.01 for difference from control cells, ## p <0.01 for difference from etoposide-treated cells by ANOVA with Dunnett’s correction for multiple comparisons). h A549 and H1299 were incubated first with etoposide for 5 h and then subjected to LA (lactic acid) exposure in the presence of etoposide for additional 36 h. The results revealed that etoposide induced γ-H2AX was not inhibited by lagging treatment with LA. i A549 and H1299 cells were pre-treated with LA (20 mM) for 3 h before etoposide treatment for 36 h. Flow cytometric analysis of cell death showed that lactate significantly reduced etoposide-induced apoptosis in A549 but not in H1299 cells. The quantification was present in right panel. The bars represent the mean ± S.D. of triplicates (** p <0.01 for difference from control cells, ## p <0.01 for difference from etoposide-treated cells by ANOVA with Dunnett’s correction for multiple comparisons, ns means no statistical difference). j Cell viability assay showed that LA significantly increased A549 rather than H1299 cell viability. In A549 cells, the EC 50 (concentration for 50% of maximal effect) of etoposide was 19.87 μM, the cell survival was increased in the presence of LA with EC 50 of 28.07 μM. However, the LA stimulation had not effect of EC 50 values in H1299 cells. (* p <0.05, for difference from untreated control by ANOVA with Dunnett’s correction for multiple comparisons). k Cells were treated with different concentrations of LA for 3 h in the absence and presence of 25 μM etoposide. ATM and p-ATM expression were examined by western blot. l 24 h after transfection with wild-type p53 plasmids, cells were treated with etoposide in the absence and presence of 20 mM LA. The expression levels of Cleaved-PARP, γ-H2AX, ATM, p-ATM were analyzed by western blot

Article Snippet: Anti- SLC16A4 (MCT4) (A3016), Anti-GLUT1 (A6982) were obtained from Abclonal (Wuhan, China).

Techniques: Control, Comparison, Positive Control, Membrane, Western Blot, Single Cell Gel Electrophoresis, Incubation, Viability Assay, Concentration Assay, Expressing, Transfection

Journal: Cell Communication and Signaling : CCS

Article Title: Lactate-induced MRP1 expression contributes to metabolism-based etoposide resistance in non-small cell lung cancer cells

doi: 10.1186/s12964-020-00653-3

Figure Lengend Snippet: HIF-1α is a key regulator of drug-induced metabolic reprogramming. a and b Western blot demonstrated the time-course ( a ) and dose-dependent ( b ) induction of HIF1-α by etoposide. c A549 and H1299 cells were transfected with a HRE-luc plasmid containing five synthetic responsive elements of HIF1-α for 24 h and then treated with etoposide for 5 h. Then luciferase activity was immediately measured and normalized using the dual luciferase reporter system. The bars represent the mean ± S.D. of triplicates (* p < 0.05 for difference from untreated cells by ANOVA for multiple comparison). d A549 Cells were treated with etoposide for 5 h before determination of ROS. e A549 and H1299 cells were pretreated with increasing concentrations of N-acetylcysteine (NAC) for 30 min before 25 μM etoposide was added. Western blot was conducted 5 h later to determine levels of HIF-1α, HK2, MCT4, Glut1. f A549 cells were transfected with HIF-1α siRNA for 24 h and further treated with 25 μM etoposide for 36 h. Western blot was performed to examine the expression of HK2, MCT4, Glut1. g The cells were first stimulated either with different concentrations of LA or different degree of acidification by adding HCl into medium for 3 h followed by further treatment with 25 μM etoposide for additional 36 h and then lysed for western blot analysis of MRP1, cleaved-PARP and Snail expression. h Gradual titration of NaHCO 3 to LA-rich medium dramatically sensitize A549 cells to etoposide treatment as indicated by Cleaved-PARP levels. i Cell apoptosis assay. Addition of NaHCO 3 rescued the etoposide-induced apoptosis inhibited by lactate treatment by Annexin-PI dual staining. The quantification was presented in right panel (** p < 0.01, *** p < 0.001, for difference from the untreated cells, ## p <0.01 for difference from etoposide-transfected cells, $$ p < 0.01 for difference from the combined etoposide and LA treated cells by ANOVA with Dunnett’s correction for multiple comparisons) j Cell viability assay showed titration of lactic acid with NaNCO 3 rescued the etoposide-induced inhibition of cell viability. Corresponding EC 50 values for etoposide, Etoposide+LA and Etoposide+LA + NaNCO 3 are 20.11 μM, 27.99 μM and 19.71 μM respectively

Article Snippet: Anti- SLC16A4 (MCT4) (A3016), Anti-GLUT1 (A6982) were obtained from Abclonal (Wuhan, China).

Techniques: Western Blot, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Comparison, Expressing, Titration, Apoptosis Assay, Staining, Viability Assay, Inhibition