human cytl1 elisa kit  (Cusabio)

Journal: Signal transduction and targeted therapy

Article Title: Intracellular CYTL1, a novel tumor suppressor, stabilizes NDUFV1 to inhibit metabolic reprogramming in breast cancer.

doi: 10.1038/s41392-021-00856-1

Figure Lengend Snippet: Fig. 2 Intracellular CYTL1 inhibits glycolysis in breast cancer. a Glucose-uptake activity and b lactate production levels in MDA-MB-231 cells after treatment with rhCYTL1 at the indicated concentrations for 48 h. ns no significant. c The subcellular location of CYTL1 was analyzed by immunofluorescence in MDA-MB-231 cells. Representative data are shown from three independent experiments. Scale bar, 5 μm. d The amount of CYTL1 was determined by western blot in MDA-MB-231 cells after treatment with monensin for the indicated period. Tubulin was used as a loading control. e The OCR and f lactate production levels in MDA-MB-231 cells after treatment with 1 μM monensin for 24 h. g The supernatants and cell lysates from MDA-MB-231 cells transfected with HA-tagged CYTL1 or ΔCYTL1 expressing plasmids were determined by western blot using an anti-HA antibody. Right panel: schematic representation of the full-length CYTL1 and ΔCYTL1 plasmids. h Glucose- uptake activity and i lactate production levels in MDA-MB-231 cells overexpressing ΔCYTL1. The data are shown as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: Human CYTL1 ELISA Kit was purchased from CUSABIO Life Sciences (College Park, MD).

Techniques: Activity Assay, Western Blot, Control, Transfection, Expressing

Journal: Signal transduction and targeted therapy

Article Title: Intracellular CYTL1, a novel tumor suppressor, stabilizes NDUFV1 to inhibit metabolic reprogramming in breast cancer.

doi: 10.1038/s41392-021-00856-1

Figure Lengend Snippet: Fig. 3 Intracellular CYTL1 prevents tumor growth and metastases. a Cell numbers were counted using a Trypan exclusion assay after MDA- MB-231 cells expressing ΔCYTL1 were cultured for the indicated period. b Colony formation assay was performed after MDA-MB-231 cells expressing ΔCYTL1 were cultured for 15 days. c Cell migration of MDA-MB-231 cells expressing ΔCYTL1 was determined using transwell assay. d–h Female nude mice were subjected to orthotopic injection with MDA-MB-231 cells stably expressing ΔCYTL1. Some mice were sacrificed 28 days later, after which tumors, lungs, and sera were collected, followed by photography and measurement. Some mice were monitored until they died. d Tumor growth curves (n = 6). Upper panel: representative images of the tumors at the end of the experiments. e Tumor weight. f Survival curve of mice (n = 6). g Serum lactate levels. h Representative H&E-stained lung sections. Circles indicate metastatic foci in the lungs. Scale bar, 500 μm. Lower panel: the proportion of mice with lung metastases in each group. i–l Female C57BL/6 mice were subjected to orthotopic injection with E0771 cells stably expressing ΔCYTL1. The mice were sacrificed 36 days later, after which tumors, lungs, and sera were collected. i Tumor growth curves (n = 6). Upper panel: representative images of the tumors at the end of the experiments. j Tumor weight. k Serum lactate levels. l Representative H&E-stained lung sections. Circles indicate metastatic foci in the lungs. Scale bar, 500 μm. Lower panel: the proportion of mice with lung metastases in each group. The data are shown as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: Human CYTL1 ELISA Kit was purchased from CUSABIO Life Sciences (College Park, MD).

Techniques: Exclusion Assay, Expressing, Cell Culture, Colony Assay, Migration, Transwell Assay, Injection, Stable Transfection, Staining

Journal: Signal transduction and targeted therapy

Article Title: Intracellular CYTL1, a novel tumor suppressor, stabilizes NDUFV1 to inhibit metabolic reprogramming in breast cancer.

doi: 10.1038/s41392-021-00856-1

Figure Lengend Snippet: Fig. 4 Intracellular CYTL1 interacts with NDUFV1 and positively regulates its protein expression. a ATP and ADP levels and b NAD+ and NADH levels in the cell lysates from MDA-MB-231 cells stably expressing CYTL1 or ΔCYTL1 were determined using kit assays. c Co-IP analysis of the interaction between exogenous HA-CYTL1 or HA-ΔCYTL1 and exogenous Flag-NDUFV1 in transfected HEK293T cells. d Co-IP analysis of the interaction between endogenous NDUFV1 and exogenous HA-CYTL1 or HA-ΔCYTL1 in transfected HEK293T cells. IgG served as the negative control. Tubulin was used as a loading control. e Colocalization of HA-CYTL1 and endogenous NDUFV1 was detected by Immunofluorescence in transfected HEK293T cells. Scale bar, 5 μm. f, g NDUFV1 protein expression was determined by western blot in MDA- MB-231 cells transfected with f HA-CYTL1 or HA-ΔCYTL1 expressing plasmids or g the indicated shRNAs. Tubulin was used as a loading control. h Lactate production levels in CYTL1 or ΔCYTL1 overexpressing MDA-MB-231 cells cotransfected with the indicated shRNAs. i Glucose-uptake activity and j the OCR in MDA-MB-231 cells overexpressing NDUFV1. The data are shown as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: Human CYTL1 ELISA Kit was purchased from CUSABIO Life Sciences (College Park, MD).

Techniques: Expressing, Stable Transfection, Co-Immunoprecipitation Assay, Transfection, Negative Control, Control, Western Blot, Activity Assay

Journal: Signal transduction and targeted therapy

Article Title: Intracellular CYTL1, a novel tumor suppressor, stabilizes NDUFV1 to inhibit metabolic reprogramming in breast cancer.

doi: 10.1038/s41392-021-00856-1

Figure Lengend Snippet: Fig. 5 Intracellular CYTL1 interrupts MDM2-mediated degradation of NDUFV1 by competitive binding. a The degradation of NDUFV1 over time in the presence of cycloheximide (CHX) was monitored by western blot in MDA-MB-231 cells transfected with HA-CYTL1 or HA-ΔCYTL1 expressing plasmids. b The NDUFV1 protein level in MDA-MB-231 cells before and after treatment of 20 μM MG132 for 6 h. c Co-IP analysis of the interaction between Flag-NDUFV1 and HA-Ub in transfected HEK293T cells. d Co-IP analysis of the interaction between NDUFV1 and HA- Ub in HEK293T cells cotransfected with Myc-NC or Myc-CYTL1 after treatment of 20 μM MG132 for 6 h. e, f NDUFV1 protein expression was determined by western blot in MDA-MB-231 cells transfected with e MDM2 expressing plasmid or f siRNA specific for MDM2. Tubulin was used as a loading control. g Co-IP analysis of the interaction between MDM2 and Flag-NDUFV1 in transfected HEK293T cells. h Co-IP analysis of the interaction between MDM2 and Flag-NDUFV1 in transfected HEK293T cells cotransfected with HA-NC, HA-CYTL1, or HA-ΔCYTL1. i Schematic representation of NDUFV1 mutants that have truncated sequences. j Co-IP analysis of the interaction between HA-CYTL1 and Flag-NDUFV1 or its three mutants in transfected HEK293T cells. Stars indicate non-specific bands. k Co-IP analysis of the interaction between HA-CYTL1 and Flag-NDUFV1 or its two mutants with deletion of N-terminal sequences. l Co-IP analysis of the interaction between MDM2 and Flag-NDUFV1 or its two mutants. m Cytoplasmic and mitochondrial protein were extracted from HEK293T cells transfected with HA-NC or HA- CYTL1. Co-IP analysis was performed for the interaction between endogenous NDUFV1 and HA-CYTL1. The data are shown as the mean ± SD of three independent experiments. ***P < 0.001

Article Snippet: Human CYTL1 ELISA Kit was purchased from CUSABIO Life Sciences (College Park, MD).

Techniques: Binding Assay, Western Blot, Transfection, Expressing, Co-Immunoprecipitation Assay, Plasmid Preparation, Control

Journal: Signal transduction and targeted therapy

Article Title: Intracellular CYTL1, a novel tumor suppressor, stabilizes NDUFV1 to inhibit metabolic reprogramming in breast cancer.

doi: 10.1038/s41392-021-00856-1

Figure Lengend Snippet: Fig. 6 NDUFV1 suppresses the Y10 phosphorylation of LDHA by Src. a LDHA enzyme activity in MDA-MB-231 cells overexpressing CYTL1 was detected using kit assay. b LDHA enzyme activity in CYTL1 KO cells. c LDHA enzyme activity in MDA-MB-231 cells overexpressing NDUFV1. d, e LDHA Y10 phosphorylation level was determined by western blot in the indicated cells transfected with d Flag-NDUFV1 expressing plasmids or e the shRNAs against NDUFV1. GAPDH was used as a loading control. f MDA-MB-231 orthotopic breast cancer models were established as described in Fig. 4. The indicated proteins in tumor tissues were determined by western blot. g Co-IP analysis of the interaction between Flag-tagged NDUFV1 and endogenous Src in transfected HEK293T cells. h The levels of phosphorylated Src and total Src were determined by western blot in MDA-MB-231 cells overexpressing NDUFV1. i Co-IP analysis of the interaction between Src and LDHA in HEK293T cells transfected with Flag-NDUFV1 expressing plasmids. j A schematic model for the role of intracellular CYTL1 in regulating metabolic reprogramming in breast cancer. The data are shown as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: Human CYTL1 ELISA Kit was purchased from CUSABIO Life Sciences (College Park, MD).

Techniques: Phospho-proteomics, Activity Assay, Western Blot, Transfection, Expressing, Control, Co-Immunoprecipitation Assay