Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2022 May 13;219(6):e20201405. doi: 10.1084/jem.20201405

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).

PMC Copyright notice

Figure S2. — Defective autophagy in Gimap6^−/− lymphocytes. (A) Quantitative PCR analysis of Gimap6 mRNA expression in T cells isolated from WT (Ctrl) and Gimap6^−/− (KO) mice. ND, not detectable. Data represent three experiments. (B and C) Whole splenocytes from WT (Ctrl) and Gimap6^−/− (KO) mice were treated with 100 nM Baf or an equal volume of vehicle (100% ethanol, Veh) for 2 h and then intracytoplasmically stained with antibody against LC3. (B) Representative flow plots of splenocytes gating on B cells (B220⁺), CD4 T cells (CD3⁺CD4⁺), CD8 T cells (CD3⁺CD8⁺), NK cells (NK.1⁺CD3⁻), and NKT cells (NK1.1⁺CD3⁺). (C) Quantification of autophagic flux (gMFI LC3_Baf − gMFI LC3_Veh)/gMFI LC3_Veh. n_KO = 7; n_Ctrl = 9. Data represent three experiments. (D) Representative flow plots showing LC3-II levels in naive (T_N; CD44⁻) or memory (T_MEM; CD44⁺) CD4 or CD8 T cells. (E) Autophagic flux as calculated in D. n_KO = 7; n_Ctrl = 9. Data represent three experiments. (F) WB of enriched CD4 T cells activated for the indicated number of days (D) with 1 µg/ml of plate bound anti-CD3 and anti-CD28. On the day of harvest, cells were incubated for 2 h with vehicle (100% ethanol) or 10 nM Baf and then lysed in radioimmunoprecipitation assay buffer. Lysates were run on Bis-Tris gels and transferred onto PVDF membranes before immunoblotting with antibodies against mouse GIMAP6, LC3, and β-actin. Cells from one to three mice were pooled for each experiment. Shown is one representative experiment of three. (G and H) Whole mouse splenocytes were activated for 3 or 5 d with 1 µg/ml plate bound anti-CD3 and soluble anti-CD28 before staining with MitoSox Red for 15 min. (G) Representative flow plots showing MitoSox Red staining of CD4 and CD8 T cells. (H) Quantification of G. n_KO = 4; n_Ctrl = 11. Shown is one of two experiments. (I) Representative flow plot showing LC3-II staining in proliferating Pt 1 and control (Ctrl) T cells activated for 3 d. The number of divisions each population has undergone is shown above, as indicated by dilution of CTV. Data represent three experiments. (J) Purified T cells from controls (Ctrl, including two NC and family members) and Pt 1 were activated for 15 d with Dynabeads Human T-Activator CD3/CD28 before staining with MitoSox Red for 15 min. Flow plots showing MitoSox Red staining (left) and quantification (right). Data represent three experiments. An unpaired t-test was used for A, C, E, and H (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Bars (A, C, E, H, and J) represent mean ± SEM.