Additional file 1 of Tunneling nanotubes induced by pseudorabies virus facilitate viral transmission in neuronal cells to evade the immune system

Supplementary Material 1: S1 Fig. Confirmation of PRV-US3 eukaryotic expression plasmids and kinase-inactivated US3 expression plasmids. a. Plasmid identification by PCR. The amplification products were of the expected size for the fragment of interest (1164 bp). b. Plasmid identification by double enzyme digestion. After double enzyme digestion, Ea-US3 and hSD-US3 plasmids showed vector fragment of 6249 bp and the target fragment of 1164 bp. c. Plasmid identification by sequencing. The fragment sequences of the US3 gene inserted in the plasmid were 100% homologous to those of the template. d. Identification of viral US3 expression by Western blot. Both Ea-US3 and hSD-US3 plasmids could express US3 protein in correct size. e. Kinase-inactivated US3 expression plasmids identification by sequencing. The fragment sequences of the kinase-inactivated US3 gene have undergone the correct mutation at the predetermined nucleotide loci. Compared with AAG sequence at positions 406–408 of the US3 gene, the nucleotide sequence at the same positions of the US3-136 gene has been replaced with GGT as designed. Compared with GAC sequence at positions 661-663 of the US3 gene, the nucleotide sequence at the same positions of the US3-221 gene has been replaced with GCT as designed. f. Identification of kinase-inactivated US3 protein expression by Western blot. Both kinase-inactive US3-136 and US3-221 plasmids could express US3 protein in correct size, with the US3-136 plasmid exhibiting relatively low expression levels. S2 Fig. TNTs fomation in neuronal-differentiated SK-N-SH cells following PRV infection. a. The differentiated SK-N-SH cells exhibited neuronal morphological characteristics, with small, round cell bodies and long axons. b. and c. The differentiated SK-N-SH cells expressed the neuronal markers TUBB3 (b) and MAP2 (c). d. hSD-1/2019 infection induced actin-rich filamentous structures that connect the dfferentiated neurons. Arrows indicate TNTs. S3 Fig. PRV hSD-1/2019 infection induced TNTs in SH-SY5Y and mouse primary neurons. a and b. hSD-1/2019 induced TNTs in SH-SY5Y cells. Arrows indicated TNTs. c and d. hSD-mCherry induced TNTs in mouse primary neurons. Arrows indicate TNTs. S4 Fig. Comparison of PRV US3 amino acid sequences between Ea and hSD-1/2019 strains.