Fig 2: Expression of RasGRP4 in lymphoma and normal tissues. (a) RasGRP4 was the most abundantly expressed protein among the RasGRP family members in lymphoma. (b) Representative images of the expression of RasGRP4 in DLBCL, FL, and Burkitt lymphoma, which was higher than in reactive lymph nodes. DLBCL showed the highest expression level. Scale bars: 1000 μm (above), 125 μm (below). (c) Representative WB images showing that the expression of RasGRP4 protein was significantly higher in DLBCL patients than in controls. (d and e) Representative images showing that RasGRP4 expression was higher in SUDHL-4 and Raji cell lines compared with normal/activated B cells, as determined by flow cytometry and WB. N indicates the number of independent experiments. **P < 0.01

Fig 3: MAPK-associated signaling pathways downstream of RasGRP4 are involved in DLBCL. (a) Representative WB images showing the differences in expression and phosphorylation levels of the downstream signaling proteins between SUDHL-4 cells and normal/activated B cells. (b and c) Quantification of the levels (in a) and the phosphorylation ratio (in a). Compared with normal/activated B cells, the ratios of P-ERK1/2:ERK1/2 and P-ERK5:ERK5 were increased in DLBCL cells, while the ratio of P-JNK:JNK was decreased, and no significant change in the P-P38:P38 ratio was detected. (d) Representative WB images showing the differences in expression and phosphorylation levels of the downstream signaling proteins in RasGRP4-knockdown SUDHL-4 cells and the control group. (e and f) Quantification of the levels (in d) and the phosphorylation ratio (in d). Knockdown of RasGRP4 decreased the ERK expression and increased the JNK expression in SUDHL-4 cells; densitometry was analyzed using ImageJ software. N indicates the number of independent experiments. **P < 0.01, *P < 0.05

Fig 4: Knockdown of RasGRP4 led to injured mitochondria and upregulated caspase-3 activity and oxidative stress in DLBCL. (a) Representative WB images of the cleaved caspase-3 expression in the NC and shRNA groups. (b) Quantification of the ratio of cleaved caspase-3/procaspase-3 (in a), which showed that the ratio was significantly higher in RasGRP4-knockdown groups than NC groups after analysis by ImageJ. (c) Representative histograms of mitochondrial membrane potential measured by flow cytometry with JC-1 probe. (d) Quantification of the red/green fluorescence ratio (in c), which was significantly lower when RasGRP4 was downregulated. The levels of oxidative stress markers ROS (e), MDA (f), and SOD (g) were determined using commercial assay kits. RasGRP4 knockdown significantly increased the oxidative stress in lymphoma cells. (h) RasGRP4 knockdown resulted in lymphoma cell injury. Intracellular LDH content was significantly higher in the knockdown groups than in the NC groups. N indicates the number of independent experiments. **P < 0.01, *P < 0.05

Fig 5: Effective knockdown of RasGRP4 by lentiviral shRNA plasmids and result in decreased DLBCL cell proliferation. (a) Representative images of WB analysis, which show the different efficiencies of the three shRNAs in decreasing RasGRP4 expression in DLBCL. (b) Quantification of the densitometric assessment of protein bands (in a) using ImageJ software. The expression levels of RasGRP4 after transfection with shRNA-1, − 2, and − 3 were 72, 29, and 44%, respectively. (c) RasGRP4 mRNA levels were evaluated by qRT-PCR. N indicates the number of independent experiments. (d) Quantification of the cell number in the two groups (in A). The number of DLBCL cells was significantly lower in the RasGRP4-knockdown group compared with control. (e) Growth of RasGRP4-knockdown SUDHL-4 cells was significantly slower than the controls. (f) RasGRP4 knockdown disrupted the SUDHL-4 cell cycle. (g) Representative histograms of the apoptosis rate measured by flow cytometry in the RasGRP4-knockdown and control groups. (h) Quantification of apoptosis rate (in g), which showed that the proportion of cells in early and late apoptosis was significantly increased by RasGRP4 knockdown. N indicates the number of independent experiments. **P < 0.01