Fig 2: Downregulation of SETD1A decreases glycolysis in BGC-823 cells. (A–C) Glucose uptake (A), lactate production (B), and pH values (C) of SETD1A-knockdown cells were reduced under normoxic (N) and hypoxic (H) conditions for 24 h compared to control BGC-823 cells (mean ± SEM; n = 3; Student’s t-test). (D) The protein levels of HK2 and LDHA were remarkedly reduced in SETD1A-knockdown BGC-823 cells under normoxic (N) and hypoxic (H) conditions for 24 h. (E–J) The mRNA levels of GLUT1 (E), HK2 (F), PFK2 (G), PKM2 (H), LDHA (I), and MCT4 (J) were remarkedly reduced in SETD1A-knockdown BGC-823 cells under normoxic (N) and hypoxic (H) conditions for 24 h (mean ± SEM; n = 4; Student’s t-test). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 3: Expression of LDHA and PFKP proteins according to IHC. The expression of the LDHA and PFKP proteins was determined by IHC. Histological analysis included 18 CC and 6 metastatic tissues. A representative image of the experiments is shown. The detection of LDHA protein (panels A–C) and PFKP protein (panels D–F) was performed with specific antibodies. Panels A and D show the detection of LDHA and PFKP, respectively, in CC tissues of patients with DFS. Panels B and E show the expression of LDHA and PFKP, respectively, in CC tissues of patients who surviving with the disease. Panels C and F show the detection of LDHA and PFKP, respectively, in metastatic tissue from patients who surviving with the disease. The specific signal for proteins is shown in brown color and counterstained with hematoxylin in violet color. Original magnification 400x; the bars represent 20 µm. Panel G shows the quantitative analysis of LDHA and PFKP expression in CC tissues of patients with disease-free survival (n = 8) versus patients who surviving with the disease (n = 10). The average optical density and staining area of LDHA and PFKP (DOI) in the tissues were considered. The mean ± SD of three independent experiments is shown. The Mann–Whitney test was performed to assess the difference between the groups, and p < 0.05 was considered to indicate statistical significance. SD = standard deviation.

Fig 4: Validation of LDHA and PFKP expression in CC. The expression of LDHA and PFKP in CC was validated at the mRNA and protein levels by qRT–PCR and WB, respectively. Panel A shows the qRT–PCR analysis of LDHA and PFKP mRNA in 58 CCs. The expression was normalized with respect to the internal control (RPS13) and the control group by the double delta method using the final formula (2-??CT). Panels B and C show representative images of WB of LDHA and PFKP expression, respectively. OS = overall survival, DFS = disease-free survival, non-DFS = patients who died or surviving with the disease. The molecular weight of the proteins is shown in kilodaltons (kDa). The protein ß-actin was used as an internal control. All samples were derived from the same experiment, and gels and blots were processed in parallel. Panel D shows the mean expression ± SD of LDHA and PFKP proteins between patients with CC who survived (white bars, n = 47) and those who died (gray bars, n = 22). The intensity of LDHA and PFKP was normalized with respect to ß-actin. The expression is shown as optical density (OD) units. The significant differences between the groups were calculated with the Mann–Whitney U test, and p < 0.05 was considered statistically significant. Panel E shows the hazard ratio (HR) analysis in relation to LDHA and PFKP protein expression in CC. The risk of death from CC increases exponentially as protein expression (OD) increases, but it is more evident with the expression of LDHA (dark blue circles represent dead patients, while light blue circles represent surviving patients) than PFKP (dark red circles represent dead patients, while light red circles represent surviving patients). SD = standard deviation.

Fig 5: Knockdown of SETD1A reduces gastric cancer cell tumorigenesis. (A, B) Downregulation of SETD1A in BGC-823 cells inhibited xenograft tumor growth (A) in nude mice and reduced tumor weight (B) (mean ± SEM; n = 6; Student’s t-test). (C) The mRNA levels of the GLUT1, HK2, PFK2, PKM2, LDHA, and MCT4 were reduced in SETD1A-knockdown tumors (mean ± SEM; n = 6; Student’s t-test). *P < 0.05, **P < 0.01.