Fig 1: The expression and clinical significance of CtBP1 in NSCLC. (A) Representative images of IHC staining of CtBP1 expression in 96 human NSCLC tissues and pair-matched normal tissues. Scale bar, 25 µm. (B) H score of IHC staining of NSCLC tissues and pair-matched normal tissues. (C) Relative mRNA level of CtBP1 in NSCLC samples and pair-matched normal tissues was analysed by real-time PCR. (D) Protein level of CtBP1 in NSCLC samples and pair-matched normal tissues was analysed by Western blotting. (E) Kaplan-Meier analysis showing the correlations between CtBP1 expression and the overall survival of patients with NSCLC, determined by log-rank test. CtBP1 low, n = 48; CtBP1 high, n = 48 (P = 0.023). Data are derived from three independent experiments and presented as mean ± SD. **P < 0.01
Fig 2: Knockdown of CtBP1 and overexpression of IRF1 affected the expression of GAS5 targets. U2OS and Saos2 cells were transfected shCtBP1 and control-shRNA to generate the U2OS-control-shRNA (U2OS), U2OS-CtBP1-KD, Saos2-control-shRNA (Saos2), Saos2-CtBP1-KD cell lines. These cell lines were then used to measure the mRNA levels of CtBP1 (A) and GAS5 and GAS5 targets including TP53, Bax, Bim, TGFB, DDB2 and ROS1 (B). U2OS and Saos2 cells were transfected with pCDNA3-Flag and pCDNA3-IRF1-Flag to generate the U2OS-pCDNA3-Flag (U2OS), U2OS-IRF1-OE, Saos2- pCDNA3-Flag (Saos2), Saos2-IRF1-OE cell lines. These cell lines were then used to measure the mRNA levels of IRF1 (C) and GAS5 and GAS5 targets (D). *P<0.05, **P<0.01 and ***P<0.001.
Fig 3: The inhibitory effect of CtBP1 knockdown on in vivo tumor growth in a lung PDTX mouse model. (A) Representative images of lung tumors from nude mice that received injections of stroke-physiology saline solution (control), control lentivirus (vector), or CtBP1-shRNA lentivector (CtBP1). (B) Tumor volumes were measured after injection every 5 days for a period of 25 days. Error bar represents SD (n=4). (C) Representative cross-sections of excised subcutaneous tumors from the control, vector and CtBP1 groups. (D) Tumor weights in the control, vector and CtBP1 groups were determined on day 25. Error bar represents SD (n=4). **P<0.01 vs. empty vector. CtBP1, carboxyl-terminal binding protein 1; PDTX, patient-derived tumor xenograft.
Fig 4: Effects of CtBP1 and SOX2 knockdown on in vivo tumor growth in a lung PDTX mouse model. (A) Representative images of lung tumor from nude mice that received injections of control lentivirus (control), or SOX2-shRNA lentivector (shSOX2), CtBP1-shRNA lentivector (shCtBP1), or SOX2-shRNA and CtBP1-shRNA in combination (shSOX2+shCtBP1). (B) Tumor volumes were measured after injection every 5 days for a period of 25 days. Error bar represents SD (n=4). (C) Representative cross-sections of excised subcutaneous tumors from the control, shSOX2, shCtBP1 and shSOX2+shCtBP1 groups. Magnification, ×400. (D) Tumor weights in the control, vector and CtBP1 groups were determined on day 25. Error bar represents SD (n=4). **P<0.01 vs. control; ##P<0.01 vs. shCtBP1+ shSOX2. PDTX, patient-derived tumor xenograft; CtBP1, carboxyl-terminal binding protein 1.
Fig 5: CtBP1 promotes macrophage recruitment and polarization in NSCLC by chemokine (C-C motif) ligand 2 (CCL2). (A) and (B) Transwell migration assay of macrophage by CM from NSCLC cells as indicated. (C) Real-time PCR analysis for the expression levels of CD68 and CD163 in THP-1 macrophages treated with CM from NSCLC cells as indicated. (D) Real-time PCR for the mRNA expression of tumour-associated macrophage (TAM) characteristic cytokines in THP-1 macrophages treated with CM from A549 cells as indicated. (E) Enzyme-linked immunosorbent assay for the secretion of tumour-associated macrophage (TAM) characteristic cytokines in THP-1 macrophages treated with CM from A549 cells as indicated. Data are derived from three independent experiments and presented as mean ± SD. *P < 0.05, **P < 0.01
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