Gsk2656157

Lyophilized lipids, 1-deoxysphinganine (Avanti; cat 860493) and sphinganine (Avanti; cat 860498), were resuspended to stock concentrations at 5 mM in EtOH and subsequently added to retinal organoid culture media at a concentration of 1 μM. For control conditions, equivalent amounts of EtOH were added to the media. ISRIB (Sigma; cat SML0843) was administered at 200 nM. GSK2656157 (Bio Vision; cat 9466) was administered at 500 nM. AA147 was obtained from the Kelly Lab at Scripps Research and resuspended in dimethyl sulfoxide (DMSO); organoids were administered at 10 μM daily. PF429242 (Sigma-Aldrich; cat SML0667) was resuspended in water and administered at 10 μM. CP7 was obtained from the Walter Lab at UCSF, resuspended in DMSO, and administered at 7 μM. 4μ8 C (EMD Millipore; cat 412512) and STF-083010 (Sigma; cat 412510) were resuspended in DMSO and administered at 32 µM. For drug experiments, 1-dSA and drugs were added to organoid culture media at concurrent times. Organoids were cultured in experimental conditions for 4 days unless otherwise stated with a condition-specific media change on day 2. For GSK2656157 and ISRIB experiments, drugs were added on day 0 and day 2. For all other drug experiments, drugs were added daily.

HA‐LIPIN1^WT, HA‐LIPIN^Mut, and mitoPLD‐GFP overexpression constructs were kind gifts from Hiromi Sesaki (Johns Hopkins) and described previously (Adachi et al, 2016 (link)). The PA‐PLA1‐GFP overexpression construct was purchased from Addgene (#162880). Perk^WT and Perk^PSP overexpression plasmids were kind gifts from Jonathan Lin (Stanford; Yuan et al, 2018 (link)). Plasmids containing shRNA were purchased from Sigma in the pLKO.1 vector: mouse Prelid1 shRNA (TRCN0000345802), human PRELID1 shRNA (TRCN0000130829). All compounds used in this study were purchased: thapsigargin (Tg; Fisher Scientific), GSK2656157 (BioVision Inc.), ISRIB (Sigma), CCCP (Sigma), rapamycin (Selleckchem), and ionomycin (Sigma).

Antibodies including HT-7 (catalog no.: MN1000; Invitrogen), AT8 (catalog no.: MN1020; Invitrogen), YFP (catalog no.: ab6556; Abcam), HSP90 (catalog no.: ab13492; Abcam), lamin A/C (catalog no.: 2032; Cell Signaling Technology), GAPDH (catalog no.: ab8245; Abcam), T-PERK (catalog no.: 3192; Cell Signaling Technology), phosphorylated PERK (catalog no.: 3179; Cell Signaling Technology), eIF2α (catalog no.: 5324; Cell Signaling Technology), p-eIF2α (catalog no.: 5324; Cell Signaling Technology) were pretested to detect the targeted proteins. Small molecules included GSK2656157 (catalog no.: 9466-5; BioVision), GSK2606414 (catalog no.: S7307; Selleckchem), salubrinal (catalog no.: S2923; Selleckchem), ISRIB (catalog no.: S7400; Selleckchem); Ceapin-A7 (catalog no.: SML2330; Sigma–Aldrich), 4u8C (catalog no.: CAS14003-96-4; Calbiochem), and AA147 (product no.: 6538059; ChemBridge) were prepared in dimethyl sulfoxide following the manufacturer’s instruction and stored at −80 °C as stock solution. ER stress–inducing chemical, thapsigargin, was dissolved in dimethyl sulfoxide and added to the cell culture media at a concentration of 300 nM. The working solutions were freshly prepared with −80 °C stock solution.