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. 2021 Aug 30;12(9):823. doi: 10.1038/s41419-021-04108-6

Fig. 2. The cytotoxic effect of transfected tRFs and piRNAs in neuronal culture.

Fig. 2

Synthetic tRFs and piRNAs were transfected in primary neuron culture. For controls, a scrambled sequence of tRF was synthesized (tRF-random), or the FAM tag was added at the 5′ of the control (5′-FAM-tRF-random). Then, their toxicity was assessed by the ATP content and LDH release assays. A After 4 h of tRFs transfection, typical morphological change of neurons under confocal. White arrows pointed to the swollen neurons. Trial n = 3. B The swollen neuron morphology was also observed after glutamate treatment for 3 h. Trial n = 3. C After 3 h transfection, the total cellular ATP level was determined. Three synthesized piRNAs were tested, including piR-rno-318194 (piR-1), piR-rno-888357 (piR-2), and piR-rno-2898628 (piR-3). Because of variations in each culture condition (different embryo age and cell number), the ATP levels of the controls were set as 1, and relative ATP level changes of the other conditions (tRFs and piRNAs) were plotted. Trial n = 2−4. One-way ANONA analysis was performed for comparison of more than three groups in all of the following figures, with post-hoc test Tukey for comparison between two groups. * P < 0.05, ** P < 0.01, and *** P < 0.001. D For the LDH assay to quantify cell death, the cell medium and neurons were collected separately at 12 h after transfection. The cell death index = LDHmedium/ (LDHmedium + LDHcell) × 100. The conditions of treatment were listed on the dot graph. Trial n = 3.