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. 2021 Apr 14;13(8):1869. doi: 10.3390/cancers13081869

Figure 3.

Figure 3

TRIT1 gene amplification in primary small-cell lung cancer patients and response to arsenic trioxide. (A) MLPA assay of primary small-cell lung cancer samples. Probe mixes contained two probes for exons 4 and 9 of the TRIT1 gene (in orange). Six reference probes were also included (in grey). Values greater than 2 (two copies, corresponding to MLPA ratio of 1) were considered to indicate the presence of extra copies. Patient 1241 is shown as example of a TRIT1 two copy number case, whilst patient 391 shows TRIT1 gene amplification. (B) TRIT1 RNA expression levels derived from Affymetrix U133Plus2.0 microarray data in six primary small-cell lung cancer samples where TRIT1 copy number was determined. The only patient that exhibited TRIT1 gene amplification (#012) showed the highest TRIT1 expression level. Two copies of TRIT1 were observed in the 006, 010, 011, 014 and 018 samples. (C) IC50 determination by MTT assay. TRIT1 shRNA-depleted DMS-273 cells were significantly less sensitive to the antiproliferative effect of arsenic trioxide than were the shRNA scramble-transfected cells harboring TRIT1 gene amplification-associated overexpression. TRIT1 shRNA-mediated depletion did not affect sensitivity to cisplatin. Student’s t test, ** p < 0.01; ns, non-significant. (D) shRNA scramble (SCR, left panel) and TRIT1 shRNA-depleted (shTRIT1, right panel) DMS-273 cells were injected into the flanks of nude mice to form subcutaneous tumors. Tumor volume over time according to treatment conditions, vehicle (black lines) vs. arsenic trioxide-treated group (red lines) are shown. Black arrow indicates the time at which the mice were randomized and started to be treated with arsenic trioxide or vehicle. P values are those corresponding to Student’s t tests. Means and standard deviations (bars) are illustrated. Tumors derived from shRNA scramble-transfected DMS-273 cells were sensitive to the growth inhibition effect of arsenic trioxide (left panel), whilst TRIT1 shRNA-mediated depletion eliminates the enhanced sensitivity to arsenic trioxide, the tumor size reduction effect being similar to that obtained with the vehicle treatment (right panel). * p < 0.05; ns, non-significant.