2.5 Validation of siRNA-mediated knockdown of COPI-specific target genes

To validate the knockdown effect of COPI observed in high-throughput screening results, primarily verified the silencing effect of siRNA, specific siRNAs targeting COPA and COPB1 (each target gene is represented by three different siRNAs, Table 1) were synthesized from GenePharma and transfected into HeLa cells using Lipofectamine RNAiMAX. After 48 h of transfection, cell lysates were harvested, and the protein expression levels of the target proteins as well as the mRNA transcription levels (primers shown in Table 2) were assessed using reverse transcription-quantitative PCR (RT-qPCR) or western blotting.

Sequences of siRNA for COPA and COPB1 silence.

*Negative control: non-specific siRNA.

Primers for reverse transcription-quantitative PCR (RT-qPCR).

A total of 24 h post-transfection, GFP-expressing C. psittaci was utilized to infect the transfected cells at an MOI of 1. The cell supernatant and pellets were collected at various time points and the intracellular growth and proliferation levels of C. psittaci were quantitatively analyzed.