Jun Lu

DEGs in whole transcriptome sequencing between HGC-27 cells treated with 5-HT and control.

Supplementary Figure 9. HTR2B overexpression correlates with poor prognosis. (A) Kaplan–Meier survival analysis comparing disease-specific survival rates based on low vs. high expression of HTR2B mRNA in TCGA cohorts (n = 370). (B) Forest plot showing the hazard ratio for 5-year overall survival (OS) in our local dataset. (C) Kaplan–Meier survival analysis comparing overall survival in patients stratified by high versus low IHC scores for HTR2B, further classified by TNM stage of gastric cancer in the local cohort (n = 188). (D) Representative IHC staining of HIF1α in human gastric tumors. (E and F) Kaplan–Meier survival analysis comparing overall survival (E) and disease-specific survival (F) rates based on low vs. high expression of HIF1α mRNA in TCGA cohorts (n = 370).

Supplementary Data 1-Whole-transcriptome sequencing of 49 human gastric tumor tissues and adjacent non-tumor tissues in our center

Supplementary Figure 8. ABCD1 modulates lipid metabolism to attenuate lipid peroxidation. (A-C) AGS and HGC-27 cells from both si-control and si-ABCD1 groups were treated with vehicle or 5-HT (10 μM) for 48 h. Representative confocal images show fluorescence staining in AGS (A) and HGC-27 cells (C) using C11-BODIPY dye. (B) The rate of intracellular lipid ROS in AGS cells was measured by C11-BODIPY fluorescence (n = 3). **p < 0.01; ns, not significant. (D) Expression levels of HIF1α, ABCD1, ACSl4, and GPX4 were evaluated by immunoblot analysis in AGS and HGC-27 cells after 24h treatment with vehicle or 5-HT (10 μM) under normoxic (20% O2) or hypoxic (1% O2) conditions. (E) Representative IHC images showing HIF1α expression in subcutaneous xenograft tumor tissues from SB204741-treated vs. control groups and sh-HTR2B vs. sh-vector groups. (F) After treatment with PP2 (50 nM), AGS and HGC-27 cells were cultured with 5-HT (10 μM) for another 24 h. Levels of Fyn, HIF1α, ABCD1, and phosphorylated and total forms of Akt and mTOR were assessed by immunoblot. (G and H) Representative IHC images of p85 (G) and Fyn (H) from SB204741-treated vs. control or sh-HTR2B vs. sh-vector groups in subcutaneous xenograft tumor tissues.

Supplementary Figure 7. Nuclear translocation of HIF1α upon 5-HT stimulation. (A) Volcano plots of significant DEGs show downregulation of SIRT6 and EGLN2, genes known to negatively regulate HIF1α transcription. These are based on the DEG profiles of HGC-27 cells treated with vehicle versus those treated with 5-HT at 10 μM (n = 3). (B) The proteins Fyn, HIF1α, ABCD1, ACSL4, GPX4, phosphorylated and total Akt, and mTOR were measured by immunoblot in both sh-vector and sh-HTR2B cells. (C and D) Immunofluorescence staining was performed to analyze the subcellular localization of HIF1α in HGC-27 (C) and AGS (D) cells, with or without 5-HT (10 μM) treatment for 24 h. (E-H) AGS and HGC-27 cells from both si-control and si-ABCD1 groups were treated with vehicle or 5-HT (10 μM) for 48 h. Representative confocal images show fluorescence staining in AGS (E) and HGC-27 cells (G) via DCFH-DA dye. Rates of intracellular ROS were measured by DCFH-DA fluorescence in AGS (F) and HGC-27 cells (H) at the indicated time points (n = 3). *p < 0.05, ***p < 0.001; ns, not significant.

Clinicopathological characteristics of 188 patients with gastric cancer in Fujian Medical University Union Hospital (FJMUUH)

Supplementary Figure 2. HTR2B plays a protective role in gastric cells. (A and B) Cell viability of HGC-27 (A) and AGS (B) cells at different concentrations of 5-HT in the absence of serum for 24 h (n = 3). (C-H) Cell viability was measured in five gastric cell lines (AGS, HGC-27, BGC-823, MKN-45, and NCI-N87) and the non-malignant cell line (GES-1) in the presence of vehicle or 5-HT (10 μM) (n = 3). *p < 0.05, ** p < 0.01. (I) Cell viability of HGC-27 cells at different concentrations of α-Me-HTP (a specific agonist of HTR2B) and SB204741 (a selective HTR2B antagonist) in the absence of serum for 24 h (n = 3). *p < 0.05, **** p < 0.0001. (J) Representative confocal images showing fluorescence staining in AGS cells treated with vehicle, 5-HT (10 μM), α-Me-HTP (100 μM), or SB204741 (100 μM) for 48 h using the EdU-488 dye. (K) The efficiency of HTR2B knockdown by shRNA interference in AGS and HGC-27 cells was quantified using western blot analysis. (L) Viability of cells exposed to 10 μM 5-HT under standard culture conditions (10% (v/v) FBS) was assessed (n = 3). (M) Cell viability was evaluated in the presence of 100 μM SB204741 under standard culture conditions (10% (v/v) FBS) (n = 3).

Supplementary Figure 3. HTR2B gene silencing or treatment with antagonist reduced the gastric tumor burden in vivo. (A) HE and IHC staining (CK-pan, CD45) were performed to verify the nature of tumor tissues harvested from patient-derived xenograft (PDX) mice as gastric cancer. (B and C) Subcutaneous xenograft tumors harvested after 3 weeks of SB204741 treatment. *p < 0.05. (D) 5-HT levels in the culture supernatants of AGS, HGC-27 and GES-1 cells measured with an enzyme-linked immunosorbent assay (ELISA) kit (n = 3). (E) Plasma concentrations of 5-HT in nu/nu nude mice quantified using an ELISA kit (n = 10). (F and G) Representative IHC images of proliferation markers (Ki-67, PCNA) in subcutaneous xenograft tumors. (H-K) Annexin V-PE/7-AAD apoptosis flow assay in HGC-27 (H and I) and AGS (J and K) cells in the presence of vehicle or 5-HT (10 μM) for 48 h. PE Annexin V and 7-AAD negative (viable or no measurable apoptosis); PE Annexin V positive and 7-AAD negative (early apoptosis, membrane integrity present); and PE Annexin V and 7-AAD positive (end apoptosis and death).