mTORC1 activity suppresses ferroptosis through a SCARB1-dependent HDL-tocopherol uptake pathway

Description

Study of the relationship between mTORC1 and ferroptosis, revealing that mTORC1 regulates SCARB1 expression and thus governs SCARB1 interaction with HDL and the uptake and delivery of HDL-derived tocopherol. Immunoblotting folder: Uncropped immunoblots from Figures 1A, 2G, 4B, 4E, 5B, S2C, S2G, and S4B. Immunoblots indicate alterations in mTOR signalling, successful CRISPRi or CRISPRa perturbation of target genes, and alterations in SCARB1 expression upon mTOR inhibition, as outlined in greater detail in the linked manuscript. Microscopy folder: Imaging of HDL uptake ± mTORC1 inhibitor RM-006 from Figure 5H - confocal images demonstrate defective uptake of Dil-HDL into lysosomes in mTORC1-inhibited cells. Display and raw images are provided in the relevant subfolders - raw image file labels are ‘condition_replicate_LAMP488_DilHDL568_image#’ and include a blue Hoechst DNA channel, a green LAMP1 AF488 channel, and a red Dil-HDL AF568 channel.

Steps to reproduce

Immunoblotting - Cells were lysed in Radioimmunoprecipitation assay (RIPA) buffer (Thermo Scientific # 89901) supplemented with Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific # 1861281), boiled, clarified, and mixed with 4x Bolt LDS samples buffer (Novex # B0007) and 10x Novex sample reducing agent (Invitrogen # B0009). Samples were run on Bolt Bis-Tris 4%–12% gels (Invitrogen # NW04125BOX) in MES or MOPS buffer (Invitrogen # B0001/2), supplemented with Bolt antioxidant (Invitrogen # BT0005). Samples were transferred to 0.45 µm LF-PVDF membranes using a Bio-Rad TransBlot Turbo semi-dry transfer system at 2.5A and 25V for 7 mins. Membranes were blocked with 5% (w/v) milk or BSA followed by primary antibody incubations for either 1 hr at room temperature or overnight at 4°C in 5% milk or BSA. Primary antibodies were as follows: SCARB1 (EP1556Y; Abcam # 52629; 1:1000), SCD1 (CD.E10; Abcam # 19862; 1:1000), pan-AKT (C67E7; Cell Signaling # 4691; 1:1000), phospho-AKT Ser473 (D9E; Cell Signaling # 4060; 1:1000), RPS6 (5G10; Cell Signaling # 2217; 1:1000), phospho-RPS6 Ser235/236 (D57.2.2E; Cell Signaling # 4858; 1:1000), 4EBP1 (53H11; Cell Signaling # 9644: 1:4000), phospho-4EBP1 Ser65 (D9G1Q; Cell Signaling # 13443; 1:1000), MTOR (7C10; Cell Signaling # 2983; 1:1000), GPX4 (Cell Signaling # 52455; 1:1000), LPCAT3 (E5G9P; Cell Signaling # 72964; 1:1000), FSP1 (F7A3L; Cell Signaling # 51676; 1:1000), GAPDH (14C10; Cell Signaling # 2118; 1:4000), and GFP (4B10; Cell Signaling # 2955; 1:1000), NQO1 (Proteintech 11451-1-AP; 1:1000), and tubulin (Sino Biological # 100109-MM05; 1:10,000). Secondary antibodies were anti-mouse 680 (Cell Signaling # 5470P; 1:4000) and/or anti-rabbit 800 (Cell Signaling # 5151P; 1:4000). Blots were imaged using a LI-COR Odyssey CLx Imaging System. Microscopy - LN229 cells treated with vehicle or 1.56 nM RM-006 for 72 hrs and 5 µg/ml Dil-HDL for 18 hrs were fixed in 4% (wt/vol) formaldehyde, permeabilised with 0.1% saponin, and labelled with LAMP1 antibody (Invitrogen; eBioH4A3; # 14-1079-80; 1:100) then AlexaFluor488 secondary antibody (Invitrogen; # A21206; 1:500) and Hoechst33342 (Invitrogen # H3570; 1:1000). Samples were imaged in Zeiss Zen Black (v14) software on an inverted Zeiss LSM780 confocal microscopy system using an oil immersion Zeiss Plan-Neofluar 40x/0.9 objective and Diode (405 nm; 3% power), Argon (488 nm; 5% power), and DPSS (561 nm: 5% power) lasers. Detector pinholes were set to give a 2.5 µm optical section and pixel dwell time was 3.15 µs with line averaging of 2. Image sizes were 1024x1024 (212.55x212.5 µm) and 12-bit depth. Cells were acquired from randomly selected fields of view and used for downstream colocalization and intensity measurements. For more information, see the methods section of the linked manuscript.

Institutions

• Icahn School of Medicine at Mount Sinai

  NY, New York
• University of California, San Francisco

  California, San Francisco
• Arc Research Institute

  California, Palo Alto

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