Figure 6. TFEB/TFE3 overactivation is responsible for the abnormal mitophagy and impaired mitochondrial fitness in RagA/RagB deficient B cells.
(A) GFP+ B cells were sorted from the vector, WT TFEB, and Ca TFEB transduced B cells. Expression of the TFEB target genes was examined by qRT-PCR. (B) Representative flow plots of IgG1 expression and Celltrace violet (CTV) dilution in vector, WT TFEB, or Ca TFEB transduced GFP+ B cells. (C) Venn diagrams highlight the overlapping gene numbers in downregulated DEGs (left) and upregulated DEGs (right) between Rag KO vs WT comparison and Ca TFEB vs vector analysis. (D) Summaries of the relative MTDR (left) and TMRM (right) staining MFIs. (E) Summary of the relative MitoSox staining MFIs. (F) Summary of the relative LAMP1 staining MFIs. (G) GFP+ B cells from vector or Ca TFEB transduced B cells were sorted and subjected to transmission electron microscope (TEM) analysis. Arrows indicate mitochondria surrounded by double layer structures., scale bar = 2 μm. (H) GFP+ B cells from vector or Ca TFEB transduced B cells were sorted, and ELISA was conducted to measure the level of pS65-Ub. (I-J) B cells from WT (n = 5) and CreERRragafl/flRragbfl/fl (n = 7) mice were transduced with vector or TFEB TDN. Representative flow plots of TMRM (I) or MTDR (J) staining were presented. (K) B cells were labeled with CTV and activated with LPS/IL-4/BAFF for 3 days, expression IgG1 and CTV dilution were examined by flow cytometry. (L-N) B cells from indicated genotypes were activated with LPS/IL-4/BAFF for 3 days, TMRM or MTDR (L), MitoSox (M) or LAMP1 (N) were measured by flow cytometry. (O) Mito-QC system was used on the indicated cells to check the mitophagy. CreERRragafl/flRragbfl/fl (n = 9), CreERRragafl/flRragbfl/flTfeb fl/fl (n = 7), and WT (n = 10). (P) B cells from the indicated mice were purified and activated with LPS/IL-4/BAFF for 72 h. Mitostress assay was performed on a Seahorse XFe96 analyzer. Right, summaries of the basal respiration and maximal respiration. Data in graphs represent mean ± SEM. ns, not significant. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, one-way ANOVA (A, B, D, E, F, K, L, M, N, O and P), two-way ANOVA (I and J), two-tailed Student’s t test (H).