Cell Culture and Genetic Manipulation

Cells were cultured in DMEM (Life Tech.) supplemented with 8% fetal bovine serum (FBS), penicillin and streptomycin. FLCN-HA, EGFP-PAT1 or LAMP1-EGFP plasmid was introduced into HEK293 cells using the TurboFect transfection reagent (Life Tech.). Stable cell lines were selected on the bases of the neomycin resistance. For RNAi experiments, RNAiMax diluted in OptiMEM (Life Tech.) was used to deliver siRNA according to the manufacturer's instructions; shRNA sequences were cloned into the expressing plasmid (pCD513B-U6) and co-transfected with the helper plasmids (GAG, REV and VSV-G) into HEK293T cells using TurboFect transfection reagent (Life Tech.). Purified and concentrated virus was used to infect cells with puromycin selection. To confirm the data of the knockdown experiments, we tested at least two RNAi of each target gene following published works and obtained similar results, including two siRNAs of FLCN (siFLCN-1 and 2 in ref. 21 (link)), two shRNAs of FLCN (shFLCN-1 and 2 in ref. 29 (link)), two siRNAs of PAT1 (si158 and si160 in ref. 30 (link)) and two siRNAs of PAT4 (si435 and si437 in ref. 30 (link)). After transfection, cells were allowed to recover for at least 36 hrs before the following treatment.

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Wu X., Zhao L., Chen Z., Ji X., Qiao X., Jin Y, & Liu W. (2016). FLCN Maintains the Leucine Level in Lysosome to Stimulate mTORC1. PLoS ONE, 11(6), e0157100.

Publication 2016

DMEM TurboFect transfection reagent RNAiMax OptiMEM

Cell lines Cells Fetal bovine serum Flcn Gene Hek293 cells Lamp1 Neomycin Penicillin Plasmids Puromycin Rnai Shrnas Sirnas Streptomycin Transfection Virus

Corresponding Organization : Northwest A&F University

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Variable analysis

independent variables

FLCN-HA plasmid introduction
EGFP-PAT1 plasmid introduction
LAMP1-EGFP plasmid introduction
SiRNA delivery targeting FLCN, PAT1, and PAT4
ShRNA sequences targeting FLCN

dependent variables

Stable cell line selection based on neomycin resistance
Evaluation of knockdown experiments

control variables

DMEM culture medium supplemented with 8% FBS, penicillin, and streptomycin
TurboFect transfection reagent used for plasmid introduction
RNAiMax and OptiMEM used for siRNA delivery
Puromycin selection for shRNA-expressing cells
Minimum 36-hour recovery period after transfection

controls

Positive control: Knockdown experiments using at least two RNAi (siRNA or shRNA) targeting each gene (FLCN, PAT1, and PAT4) based on published works
Negative control: Not explicitly mentioned

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