The off-target effects of dynamin inhibitor Dyngo-4a during the internalization of dextran by bone marrow mesenchymal stem cells in rat

by Selleckchem | May 26 2023

Objective To investigate the possible off-target effects of dynamin (DNM) inhibitor Dyngo-4a in dynamin-dependent endocytic pathways. Methods Bone marrow mesenchymal stem cells (BMSCs) obtained from SD rats were isolated and cultured, and identified by flow cytometry. The cells were divided into inhibitor control group, Dyngo-4a-treated group, negative control siRNA (si-NC) transfection group, DNM2 siRNA transfection (si-DNM2) group, si-DNM2 and Dyngo-4a co-treated group. Real time quantitative PCR and Western blot analysis were used to verify the silencing efficiencies of DNM2 gene and CCK-8 assay were used to detect the cell viability after Dyngo-4a treatment. Confocal microscopy was used to detect the number and mean fluorescence intensity (MFI) of transferrin-Dylight649-positive and dextran-TMR-positive vesicles. Results The mRNA and protein expression levels of DNM2 were down-regulated using small interfering RNA. The number of transferrin-Dylight649-positive vesicles significantly decreased in si-DNM2 group compared with si-NC group. For the number and MFI of dextran-TMR-positive vesicles, no significant change was observed between the si-DNM2 group and the si-NC group, but there was a significant reduction in the si-DNM2 and Dyngo-4a co-treated group compared with the si-DNM2 group. A significant decrease was also found in the Dyngo-4a-treated group compared with the inhibitor control group. Conclusion The off-target effects of dynamin inhibitor Dyngo-4a presents in the internalization of dextran by BMSCs.

Comments：

Based on the study, the researchers aimed to investigate the possible off-target effects of the dynamin inhibitor Dyngo-4a in dynamin-dependent endocytic pathways. To achieve this, they isolated and cultured bone marrow mesenchymal stem cells (BMSCs) from SD rats and identified them using flow cytometry. The cells were then divided into different groups: inhibitor control group, Dyngo-4a-treated group, negative control siRNA (si-NC) transfection group, DNM2 siRNA transfection (si-DNM2) group, and si-DNM2 and Dyngo-4a co-treated group.

Real-time quantitative PCR and Western blot analysis were used to confirm the silencing efficiencies of the DNM2 gene, and the cell viability after Dyngo-4a treatment was assessed using a CCK-8 assay. Confocal microscopy was employed to detect the number and mean fluorescence intensity (MFI) of transferrin-Dylight649-positive and dextran-TMR-positive vesicles.

The results of the study showed that the mRNA and protein expression levels of DNM2 were down-regulated using small interfering RNA. Moreover, the number of transferrin-Dylight649-positive vesicles significantly decreased in the si-DNM2 group compared with the si-NC group. However, no significant change was observed between the si-DNM2 group and the si-NC group concerning the number and MFI of dextran-TMR-positive vesicles.

Furthermore, there was a significant reduction in the si-DNM2 and Dyngo-4a co-treated group compared with the si-DNM2 group regarding the number and MFI of dextran-TMR-positive vesicles. Similarly, a significant decrease was also found in the Dyngo-4a-treated group compared with the inhibitor control group.

Based on these findings, the study concludes that the off-target effects of dynamin inhibitor Dyngo-4a present in the internalization of dextran by BMSCs.