Luciferase gl2

Pegylated human recombinant Arginase I cobalt [HuArgI (Co)-PEG5000] (Pegzilarginase) was a gift from Aeaglea BioTherapeutics (Texas, USA). The constitutively active RhoA (CA-RhoA) and the empty vector plasmid (pcDNA3.1) constructs were also gifts from Dr. Yamaguchi Hideki. Chloroquine, rapamycin and L-citrulline were purchased from Sigma-Aldrich (Darmstadt, Germany). Cyto-ID autophagy detection kit was obtained from Enzo Life Sciences (New York, USA). RhoA/Rac1/Cdc42 Activation Assay Combo Kit was from Cell BioLabs (Sand Diego, CA, USA). Actin and vinculin primary antibodies were purchased from Abcam (Cambridge, UK). Rabbit polyclonal anti-LC3 antibody was obtained from Cell Signaling (Cell Signaling Technology Inc., US). Fluorescent secondary Alexa Fluor 488-green as well as Rhodamine phalloidin stain were obtained from Invitrogen (Massachusetts, USA). Hiperfect transfection reagent, luciferase GL2 and human Flexi Tubes siRNA for RhoA were bought from Qiagen (Hilden, Germany). Lipofectamine LTX was from Waltham (Massachusetts, USA) and crystal violet was from SCP Science (Quebec, Canada).

HS5 cells were transfected with siRNA for CDCP1 (FlexiTube siRNA Hs_CDCP1__4, SI00341446, Qiagen, Hilden Germany; Stealth RNAiTM 10620312, Invitrogen), SDC1 (Hs_SDC1__1, SI00020587, Qiagen), KDM3B (Hs_JMJD1B__7, SI04270357, Qiagen), PKC-δ (Stealth RNAiTM 10620318, Invitrogen) or control siRNA (Luciferase GL2, Qiagen) using Lipofectamine RNAiMAX (Life Technologies, Grand Island NY) according to the manufacturer's protocol.
To avoid any off-target effects, four siRNAs for CDCP1 were tested, and we found that Hs_CDCP1_4 siRNA had no or minimum off-target effects compared to the other three siRNAs (Hs_CDCP1_2, SI00341432; HS_CDCP1_5, SI04170992;Hs_CDCP1_6, SI04199083, Qiagen).