Fig 2: Treatment with GA inhibited the growth of A549 cells in vivo. (A) and (B) The GA treatment suppressed the formation of tumours by A549 cells in vivo. Mice were treated with 0.9% NaCl (model), GA (high dose, 100 mg/kg; low dose, 50 mg/kg) or Kobe (80 mg/kg). (A) Tumour weights were monitored. (B) Growth curves of the xenograft tumours. Values are presented as means ± S.D. (n = 6). (C) Images of H&E (original magnification × 100 and × 400), and IHC staining of the tumour tissues show the levels of P-ERK1/2, scale bar: 50 µm. (D) and (E) Levels of the phosphorylated c-RAF-S259, B-RAF-S729, B-RAF-T401 and ERK1/2 proteins in the tumour tissues were analysed by Western blotting. The data are reported as means ± S.D. (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 compared to the model.

Fig 3: Raf1 is overexpressed in T-cell lymphoma tissues. (A) Raf1 expression in lymphoid neoplasm DLBC and normal tissues was analyzed from mRNA sequencing data from TCGA database. Red denotes cancer tissue, and gray denotes normal tissue. (B) mRNA expression levels of Raf1 in cancerous lymph nodes vs. adjacent normal tissues from T-cell lymphoma patients, and in stage III/IV vs. stage I/II tumor tissues. (C) Immunohistochemistry staining of Raf1 in cancerous lymph nodes or adjacent normal tissues from T-cell lymphoma patients. Magnification, ×400. (D) Western blot analysis of protein expression of Raf1 in cancerous lymph nodes or adjacent normal tissues from T-cell lymphoma patients. ***P<0.001. DLBC, diffuse large B-cell lymphoma; TCGA, The Cancer Genome Atlas.

Fig 4: Effect of BPTF or Raf1 on the proliferation of T-cell lymphoma. (A) mRNA and (B) protein expression levels of BPTF and Raf1 in H9 cells and Hut-102 cells. ***P<0.001 vs. H9 cells. (C) Confirmation of successful silencing of Raf1 in Hut-102 cells by shRaf1. ***P<0.001 vs. NC. (D) Cell viability of Hut-102 cells transfected with shBPTF, shRaf1 or NC. MTT assays were performed 24 h after transfection. ***P<0.001 vs. NC. (E) Representative micrographs (left) and relative quantification (right) from colony formation assays. *P<0.05 and **P<0.01 vs. NC. (F) Flow cytometry plots and quantitative analysis of Annexin V/propidium iodide staining assay. ***P<0.001 vs. NC. (G) Flow cytometric analysis of cell cycle phase distribution assay. Histograms depict the proportion of Hut-102 cells in G1, S and G2 phases. **P<0.01 and ***P<0.001 vs. NC. (H) Western blot analysis of Bax, Bcl-2, and β-actin. The histogram depicts the signal ration of Bax/Bcl-2. ***P<0.001 vs. NC. BPTF, bromodomain PHD finger transcription factor; NC, negative control; sh, short hairpin.

Fig 5: GA inhibits GTP binding and the activation of downstream effectors. (A) GA dose-dependently inhibits GTP binding and the activation of downstream effectors in vitro. (B) and (C) Western blots showing the levels of activated downstream effectors, c-RAF phosphorylated at S338 and S259 and phosphorylated ERK1/2 levels, in A549 cells and HepG2 cells after GA treatment. GAPDH expression was used as an internal control for normalization. The assay was performed three times, Error bars indicate means ± S.D. (*P < 0.05, **P < 0.01, ***P < 0.001 compared to the control). (D) and (E) A549 cells were cultured with 20 or 40 µmol/L GA. Migration (upper panel) and invasion (lower panel) were investigated using Transwell and Matrigel assays (n = 5), scale bar: 50 µm. (F) and (G) PI staining was performed to investigate the effect of GA on the cell cycle. Cells were treated with GA (20 or 40 µmol/L) for 24 h and then stained with PI. GA induced S phase arrest in A549 cells. Histogram shows the percentage of cells in G0/G1, G2/M and S Phase (n = 3).