Shrna construct

We used lentivirus-mediated transduction for shRNA-dependent down-regulation of SLC38A5 in MB231 cells. The shRNA constructs specific for human SLC38A5 were obtained from Sigma–Aldrich, which lists four constructs that had been validated for a significant silencing of the transporter (>65%) in the fibroblast cell line MCH58. We performed a preliminary study to screen the efficacy of all these four constructs for their ability to down-regulate SLC38A5 in MB231 cells. Based on this experiments, we selected two of these constructs (Cat. no. TRCN44005 and TRCN44007) that we found to down-regulate SLC38A5 in MB231 cells by 80%. The experimental protocol followed the manufacturer's directions for transduction and for the selection of stable clones. The extent of down-regulation in MB231 cells was monitored by measuring the SLC38A5 mRNA levels by quantitative RT-PCR and also by functional assays involving serine uptake.

Rat pituitary GH3 cells were cultured at 37 °C in a humidified atmosphere containing 95 % air and 5 % CO₂. The culture medium was DMEM supplemented with 10 % fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin. GH3 cells were transfected with small hairpin RNA (shRNA) constructs (Sigma-Aldrich) or the VSVG-mEmerald plasmid (modified from Addgene plasmid #31947) with Lipofectamine 2000 (Invitrogen). For growth hormone secretion assay, cells were transferred to serum free medium 48 h after transfection and incubated for 2 h before ELISA. Live cell confocal images were acquired 48 h after transfection, using spinning disk confocal scan head (CSU-X/M2 N, Yokogawa) attached to an inverted microscope (IX-81, Olympus) and an EMCCD camera (DU897BV, Andor) controlled by Micro-Manager software. Images (512 × 512 pixels, voxel size 0.0946 μm/pixel) were taken every 0.5 s for 400 frames. Live images were analyzed in NIH ImageJ with the MTrackJ plugin. VSVG containing vesicles (10/cell) were randomly selected in 11 Otg1 knockdown cells and 11 scramble shRNAs treated cells. Directionality of each vesicle was defined as its real transport distance divided by linear distance between the start and end positions.

ShRNA constructs were purchased from Sigma-Aldrich (St. Louis, MO, USA). The specific constructs are listed in Table S3.
Control shRNA or targeted shRNA-expressing pseudoviruses were generated by cotransfecting 293T cells with the shRNA vectors (4–5 shRNAs per gene) and pHCMV-G [40] (link). The shRNA was delivered into target cells following the manufacturer's recommendations, using the pLKO.1-puro backbone vector. For transduction, HeLa, Jurkat and Cos-1 cells were incubated with the shRNA lentiviral stock overnight. 24 hours post-transduction, the medium was aspirated and replaced with fresh medium containing puromycin at 2 ug/ml for HeLa or 4 ug/ml for Jurkat and Cos-1. After 1 week of puromycin selection, the cells were ready for experiments.

HT1080 cells were transfected by the same procedure previously outlined in [29 (link)]. shRNA constructs targeting the MMP 1, MMP 9, MMP 7, MMP 11 genes were purchased from Sigma Aldrich. PCR studies were performed after lentiviral mediated transduction and only shRNAs showing more than 85% knockdown were used for subsequent studies (Supplementary Figure 2H–2K). Genomic sequences for the knockdowns are listed in Table 1.

The following primers were used for qPCR experiments: 36B4 5′-CAGCAAGTGGGAAGGTGTAATCC-3′ (fwd), 5′-CCATTCTATCATCAACGGGTACAA-3′(rev), TNFR1 5′-TGCAGGAAGAACCAGTACCG-3′ (fwd), 5′-TTCGTGCACTCCAGGCTTTT-3′ (rev), TNFR2 5′-CATGCCGGCTCAGAGAATACT-3′ (fwd), 5′-CACCTGGTCAGAGCTACAGC-3′ (rev), DR4 5′-GGTCGTACCTAGCTCAGCTG-3′ (fwd), 5′-CTGTACATGGGAGGCAAGCA-3′ (rev), DR5 5′-GTGGAGCTAAGTCCCTGCAC-3′ (fwd), 5′-TCCCCACTGTGCTTTGTACC-3′ (rev), CD95 5′-CCATAAGCCCTGTCCTCCAG-3′ (fwd), 5′- TGGTATTCTGGGTCCGGGT-3′ (rev), CHOP 5′-CATCACCACACCTGAAAGCA-3′ (fwd), 5′-TCAGCTGCCATCTCTGCA-3′ (rev), CFLAR 5′-GAACAGCTTGGCGCTCAAC-3′ (fwd), 5′-GCCAAGAATCTGGGATATACCATG-3′ (rev). The results were normalized to 36B4 expression using the 2^−ΔΔCt method.^{41 (link)} Primers were designed with Geneious 10.0.5 software (Biomatters Ltd, Auckland, New Zealand) and synthesized by Eurofins Genomics (Ebersberg, Germany).
The following shRNA constructs were obtained from Sigma for specific knockdown of the indicated target genes: