Journal: The Journal of Clinical Investigation

Article Title: TYRO3 induces anti–PD-1/PD-L1 therapy resistance by limiting innate immunity and tumoral ferroptosis

doi: 10.1172/JCI139434

Figure Lengend Snippet: (A) Volcano plot of 4T1-P and Tyro3-OE differentially expressed genes. adj, adjusted. (B) SLC3A2 expression in patients with melanoma with high (n = 52) and low (n = 19) TYRO3 expression levels who received anti–PD-1 therapy. ****P < 0.0001, by 2-tailed, unpaired Student’s t test. (C) Relative lipid ROS in CD45− tumor cells. 4T1-P plus IgG versus 4T1-P plus anti–PD-1, *P = 0.037; Tyro3-OE plus IgG versus Tyro3-OE plus anti–PD-1, NS P = 0.92; and 4T1-P plus anti–PD-1 versus Tyro3-OE plus anti–PD-1, ***P = 0.0004, by 2-way ANOVA. (D) MFI of IFN-γ expression in CD8+ T cells from anti–PD-1–treated 4T1-P and Tyro3-OE tumors. NS P = 0.626, by 2-tailed, unpaired Student’s t test. (E) Percentage of 7-AAD+ cells in 4T1-P and Tyro3-OE cells treated with 2 μM erastin and/or 5 μM Fer-1 for 48 hours (n = 3). £P = 0.013, by 2-tailed, unpaired Student’s t test. (F) Relative lipid ROS in 4T1-P and Tyro3-OE cells treated with 10 μM erastin and/or 10 μM Fer-1 for 8 hours (n = 3). **P = 0.0013, by 2-tailed, unpaired Student’s t test. (G) Percentage of 7-AAD+ cells in 4T1-R and Tyro3−/− cells treated with 2 μM erastin and/or 5 μM Fer-1 for 24 hours (n = 3). ****P < 0.0001, by 2-tailed, unpaired Student’s t test. (H) Relative lipid ROS in 4T1-R and Tyro3−/− cells treated with 10 μM erastin and/or 10 μM Fer-1 for 8 hours (n = 3). ****P < 0.0001, †††P = 0.000124, and ††P = 0.00125, by 2-tailed, unpaired Student’s t test. (I) A dual-luciferase reporter assay was performed by cotransfecting ARE-reporter-luciferase and pRL-TK with a TYRO3-OE plasmid, and cells were primed with 2 μM MK2206 for 24 hours (n = 3). ##P = 0.002 and NS P = 0.115, by 2-tailed, unpaired Student’s t test. (J) Relative lipid ROS in 4T1-P and Tyro3-OE cells primed with 2 μM MK2206 for 24 hours, and then treated with 10 μM erastin for 8 hours (n = 3). §P = 0.02, §§P = 0.003, NS P = 0.052, and NS P = 0.79, by 2-tailed, unpaired Student’s t test. (K) Relative lipid ROS in 4T1 cells primed with or without 200 nM Pros1 for 24 hours and then treated with 10 μM erastin and/or 10 μM Fer-1 for 8 hours (n = 3). ¶P = 0.013 and ****P < 0.0001, by 2-tailed, unpaired Student’s t test. (L) Relative lipid ROS in 4T1 Tyro3−/− cells primed with or without 200 nM Pros1 for 24 hours and then treated with 10 μM erastin and/or 10 μM Fer-1 for 8 hours (n = 3). NS P = 0.059, NS P = 0.53, and NS P = 0.58, by 2-tailed, unpaired Student’s t test. Data are presented as the mean ± SD.

Article Snippet: Paraffin-embedded tissue array slides containing melanoma sections (ME804b, US Biomax) were pretreated using a Melanin Bleach Kit (24883-1, Polysciences) and stained with anti-SLC3A2 antibody (1:100; 47213S, Cell Signaling Technology) and anti-TYRO3 (1:100; OM223993, Omnimabs) as described above.

Techniques: Expressing, Luciferase, Reporter Assay, Plasmid Preparation

Journal: EMBO Reports

Article Title: APEX2‐mediated RAB proximity labeling identifies a role for RAB21 in clathrin‐independent cargo sorting

doi: 10.15252/embr.201847192

Figure Lengend Snippet: RAB21 is required for endosomal sorting of direct clathrin‐independent cargos. RAB21 associates with WASH/retromer subcomplexes at endosomes. RAB21 could either (i) recruit WASH/retromer or (ii) be recruited by WASH/retromer or (iii) be part of a positive feedback loop that would allow WASH/retromer and RAB21 recruitment at endosomes. Endosomal RAB21 would be required for WASH‐mediated F‐actin polymerization. Although the data do not directly demonstrate a direct link between F‐actin generation and cargo sorting, we propose that RAB21‐dependent F‐actin generation would be required for sorting of a CIE cargo subclass (MCT1, SLC3A2, Basigin, and CD44), while it would not be required for other cargos (CI‐MPR or Glut1). In RAB21 knockout cells, decreased WASH/retromer endosomal localization is observed, which results in reduced endosomal F‐actin and direct CIE cargo misrouting. This ultimately leads to the lysosomal degradation of these misrouted cargos (demonstrated for SLC3A2). Dotted line between VARP and RAB21 indicates that their coregulation with the retromer is speculative.

Article Snippet: Antibodies used for immunoblotting were anti‐GFP (1:500, Roche #11814460001), anti‐RAB21 (1:1,000, Sigma #R4405 or 1:1,000, Invitrogen #PA5‐34404), anti‐RAB4 (1:1,000, Cell Signaling #2167), anti‐RAB5 (1:1,000, Cell Signaling #3547), anti‐RAB7 (1:1,000, Cell Signaling #9367), anti‐GAPDH‐HRP (1:1,000, Cell Signaling #8884), anti‐Myc (1:1,000, Cell Signaling #2278), anti‐HA (1:1,000, Cell Signaling #3724), anti‐SLC3A2 (1:800, Cell Signaling #13180), anti‐Strumpellin (1:500, Santa Cruz #377146), anti‐VPS35 (1:500, Santa Cruz #374372), anti‐VPS26 (1:500, Santa Cruz #390304), anti‐VPS29 (1:500, Santa Cruz #398874), anti‐FAM21 and anti‐WASH (1:10,000, gift from D. Billadeau), anti‐CAPZα (1:500, Santa Cruz #374302), anti‐APPL1 (1:1,000, Cell Signaling #3858), anti‐VARP (1:500, Bethyl Laboratories #A302‐997A), anti‐tubulin (1:2,500, Sigma #T9026), streptavidin‐HRP (1:1,000, Thermo Fisher #N100), and anti‐rabbit and mouse HRP (1:10,000, Jackson Laboratories #115‐035‐144 and #115‐035‐146, respectively).

Techniques: Knock-Out