Journal: Exploration

Article Title: STIP1 drives Metabolic Reprogramming in Esophageal Squamous Cell Carcinoma via AHCY‐LDHA Axis

doi: 10.1002/exp.20240198

Figure Lengend Snippet: FIGURE 1 Heat-stimulation promotes glycolysis and induces STIP1 overexpression. A. WB experiment was performed to measure the glycolysis enzymes level. B. Glucose uptake was detected by kit after 65◦C water stimulation. C. STIP1 expression was heightened in esophageal tissues of mice treated with hot water compared to normal controls. D. Across a panel of human esophageal cell lines, STIP1 protein levels were elevated in multiple ESCC lines versus normal esophageal epithelial cells (upper panel). The bands intensities were subjected to quantitative analysis (lower panel). E. In analysis of 5 paired patient ESCC tumor and adjacent normal tissues, STIP1 protein was increased in tumor samples compared to matched normal epithelium (upper panel). The bands intensities were subjected to quantitative analysis (lower panel). TCGA database was used to evaluate STIP1 expression across normal and cancerous tissues (F), examining its distribution across tumor stages (G) and investigating correlations between STIP1 expression and patient prognostic outcomes (H). I. By immunohistochemistry of ESCC tissue microarrays, STIP1 staining was more intense in tumor areas versus paired normal epithelium (40x and 100x magnifications shown). Quantitative analysis confirmed significantly higher STIP1 protein levels in ESCC tumors compared to paired normal tissues(J) and in unpaired ESCC versus normal esophagus (K). Stratifying by clinicopathologic characteristics, STIP1 expression was incrementally increased with higher lymph node involvement (L), poorer tumor grade (M), and advanced tumor stage (N). O. Relationship between STIP1 expression level and overall survival for tissue microarray. P. Schematic of the 4NQO-induced esophageal cancer model.

Article Snippet: Cells were then incubated overnight at 4◦C with primary antibodies targeting STIP1 (Proteintech, 68155-1-Ig, 1:200 dilution) and AHCY (Proteintech, 10757-2-AP, 1:100 dilution).

Techniques: Over Expression, Expressing, Immunohistochemistry, Staining, Microarray

Journal: Exploration

Article Title: STIP1 drives Metabolic Reprogramming in Esophageal Squamous Cell Carcinoma via AHCY‐LDHA Axis

doi: 10.1002/exp.20240198

Figure Lengend Snippet: FIGURE 2 STIP1 promotes cell growth and PDXO development. A. Stable STIP1 knockdown ESCC cell lines were generated, with efficient STIP1 protein reduction confirmed by western blotting. B. STIP1 knockdown significantly decreased ESCC cell proliferation based on MTT assays. C. D Anchorage independent growth and plate colony formation assay from different cells with STIP1 knockdown. Colonies were counted using Image Pro- Plus (Scale bar: 200 µm). E. STIP1 was overexpressed in KYSE410 cells and the expression of STIP1 was determined by western blot. F. After overexpressed STIP1, cell proliferation was measured by MTT assay. G, H. Anchorage independent growth and plate colony formation assay from KYSE410 with STIP1 overexpression. Colonies were counted using Image J-Plus (Scale bar: 200 µm). I-J Esophageal squamous cell carcinoma cells with stable knockdown of STIP1 expression via short hairpin RNAs (shRNAs) or non-targeting control shRNA were subcutaneously injected into athymic nude mice (n = 8 per group) to evaluate effects on tumorigenicity. Tumor weights were calculated in the right panel. K-L. Intertumoral injection of STIP1 shRNA in PDXs suppressed tumor growth compared to control shRNA. Tumor weights were calculated in the right panel. M-N. STIP1 knockdown reduced the number and size of PDXOs. Organoids absorbance was measured in the right panel. All data statistical differences were evaluated using Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Cells were then incubated overnight at 4◦C with primary antibodies targeting STIP1 (Proteintech, 68155-1-Ig, 1:200 dilution) and AHCY (Proteintech, 10757-2-AP, 1:100 dilution).

Techniques: Knockdown, Generated, Western Blot, Colony Assay, Expressing, MTT Assay, Over Expression, Control, shRNA, Injection

Journal: Exploration

Article Title: STIP1 drives Metabolic Reprogramming in Esophageal Squamous Cell Carcinoma via AHCY‐LDHA Axis

doi: 10.1002/exp.20240198

Figure Lengend Snippet: FIGURE 3 STIP1 plays a crucial role in glycolysis. A. Western blot shows STIP1 knockdown in KYSE450 and KYSE30 cells reduce protein levels of key glycolytic enzymes including PKM2, PKM, LDHA, ENO1 and ALDOA in ESCC cells. β-actin is shown as a loading control. B. STIP1 knockdown decreases glucose consumption in ESCC cells. Values are normalized to total protein content. C. STIP1 knockdown reduces lactate secretion in ESCC cells. Lactate levels in conditioned media were normalized to cell number. D. STIP1 knockdown lowers LDHA catalytic activity in ESCC cell lysates. E. Overexpression STIP1 enhance LDHA enzyme activity. LDHA activity was determined by measuring NADH levels spectrophotometrically. F,G. ECAR reflecting glycolytic flux, is decreased or increased after STIP1 knockdown in KYSE450 or overexpression in KYSE410 cells. ECAR was measured using a Seahorse XFe96 analyzer under basal conditions and in response to glucose, oligomycin, and 2-DG. Data are mean ± SD of triplicate experiments. *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t-test.

Article Snippet: Cells were then incubated overnight at 4◦C with primary antibodies targeting STIP1 (Proteintech, 68155-1-Ig, 1:200 dilution) and AHCY (Proteintech, 10757-2-AP, 1:100 dilution).

Techniques: Western Blot, Knockdown, Control, Activity Assay, Over Expression

Journal: Exploration

Article Title: STIP1 drives Metabolic Reprogramming in Esophageal Squamous Cell Carcinoma via AHCY‐LDHA Axis

doi: 10.1002/exp.20240198

Figure Lengend Snippet: FIGURE 4 STIP1 interacts with AHCY to enhance binding to LDHA, thereby promoting glycolytic flux. A. Mass spectrometry analysis identifies glycolytic enzymes as STIP1-interacting proteins. B, C. Endogenous and exogenous co-immunoprecipitation verifies binding between STIP1 and AHCY D. Immunofluorescence staining shows co-localization of STIP1 (green) and AHCY (red) in the cytoplasm of ESCC cells (DAPI stain in blue). E. The AHCY-STIP1 complex was modeled using Alphafold3. Then the AHCY in the model was aligned to the experimental structure (PDB 1LI4), and colored by C-alpha RMSD using ChimeraX. Red indicates high RMSD and larger conformational change. F. Knockdown or overexpression of STIP1 reduces or enhances AHCY enzyme activity, respectively. G,H. Co-immunoprecipitation confirms endogenous and exogenous interaction between AHCY and LDHA. I. Endogenous IP assay to check the binding of STIP1 and LDHA. J. HEK293T cells were transfected with Myc-tagged AHCY and truncated LDHA including 1–274, 1–160, 161–274. Immunoprecipitation was performed using anti-HA affinity gel followed by western blots using anti-Myc antibody. K. Immunofluorescence shows cytoplasmic co-localization of AHCY (green) and LDHA (red) in ESCC cells. L,M. STIP1 knockdown or overexpression reduces or enhances the AHCY-LDHA interaction. N,O. Knockdown or overexpression of STIP1 or AHCY decreases or increases the SAM:SAH ratio. Data are mean ± SD of 3 experiments. *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t-test.

Article Snippet: Cells were then incubated overnight at 4◦C with primary antibodies targeting STIP1 (Proteintech, 68155-1-Ig, 1:200 dilution) and AHCY (Proteintech, 10757-2-AP, 1:100 dilution).

Techniques: Binding Assay, Mass Spectrometry, Immunoprecipitation, Immunofluorescence, Staining, Knockdown, Over Expression, Activity Assay, Transfection, Western Blot

Journal: Exploration

Article Title: STIP1 drives Metabolic Reprogramming in Esophageal Squamous Cell Carcinoma via AHCY‐LDHA Axis

doi: 10.1002/exp.20240198

Figure Lengend Snippet: FIGURE 7 LCA is a STIP1 inhibitor. A. Chemical structure of LCA. B. Computational modeling predicts binding between LCA and STIP1. C. Pull- down assay shows LCA directly interacts with STIP1 protein in vivo. D. SPR analysis verifies direct binding between LCA and STIP1. Sensorgram shows response over time as LCA passes over STIP1-conjugated chip. E. Cellular thermal shift assay demonstrates LCA binds and stabilizes STIP1 protein in ESCC cells. F. Overview of quantitative proteomics approach. KYSE450 cells were treated with LCA or DMSO control for 24 h, followed by tandem mass tag (TMT) labeling and LC-MS/MS analysis. G. After treat various doses of LCA, AHCY, LDHA, PKM2 and ALDOA protein level were measured by western blot. H,I. LCA decreases glucose consumption and LDH vitality in ESCC cells. J,K. ECAR is lowered by LCA, reflecting impaired glycolysis. Data are mean ± SD of 3 experiments. *p < 0.05, **p < 0.01, by Student’s t-test.

Article Snippet: Cells were then incubated overnight at 4◦C with primary antibodies targeting STIP1 (Proteintech, 68155-1-Ig, 1:200 dilution) and AHCY (Proteintech, 10757-2-AP, 1:100 dilution).

Techniques: Binding Assay, Pull Down Assay, In Vivo, Thermal Shift Assay, Quantitative Proteomics, Control, Labeling, Liquid Chromatography with Mass Spectroscopy, Western Blot

Journal: International journal of biological sciences

Article Title: Macrophage-derived S100A9 promotes diabetic cardiomyopathy by disturbing mitochondrial quality control via STAT3 activation.

doi: 10.7150/ijbs.111128

Figure Lengend Snippet: Figure 7. S100A9 promotes STIP1-STAT3 interaction in cardiomyocytes and diabetic heart tissues. a, Upset-Venn plot of IP-MS results in rhS100A9-treated AC16 cardiomyocytes. b-c, co-immunoprecipitation (co-IP) of MYC-STAT3 and HA-STIP1 in AC16 cardiomyocytes. d, S100A9 promoted STIP1 binding with STAT3 in AC16 cells transfected with Flag-S100A9, MYC-STAT3 and HA-STIP1. e, The predicted binding sites between STIP1 and STAT3. Optimized pose model of STIP1-STAT3 interaction by R-Dock analysis. f, Full-length STIP1 or truncated mutant STIP1 (TPR5-8 domain, Δ259-427) were co-transfected with MYC-STAT3 in AC16 cardiomyocytes. co-IP of STAT3 and STIP1 was perform with HA-tag antibody. g, co-IP of STAT3 and STIP1 was performed in STZ-induced or db/db diabetic heart tissues treated with/without paquinimod (PAQ).

Article Snippet: S100A8 (15792-1-AP), S100A9 (26992-1-AP), STIP1 (15218-1-AP), FIS1 (10956-1-AP), Tubulin (10068-1-AP), Lamin B (12987-1-AP), GAPDH (10494-1-AP), and HA-tag (51064-2-AP) were obtained from Proteintech Technology (Proteintech, China).

Techniques: Protein-Protein interactions, Immunoprecipitation, Co-Immunoprecipitation Assay, Binding Assay, Transfection, Mutagenesis