Fig 1: SPL overexpression reduces ER stress–induced UPRmt. A: HEK293 cells were transfected with either empty vector (EV) or SPL_WT plasmid, and the cell lysates were used to perform the SPL fluorogenic substrate assay according to manufacturer’s protocol (n = 5 biological replicates). B–F: WT MEFs were transfected with either EV or SPL_WT and treated with TG (100 nM; 12 h). Total RNA was analyzed by qRT-PCR for Atf5, Atf4, Ddit3, Lonp1, Hspa9, and Gapdh mRNA (n = 4 biological replicates). G: WT MEFs were transfected with the indicated plasmids and treated with TG (100 nM; 4 h). MF protein lysates were analyzed by Western blotting using specific antibodies for LONP1, mtHSP70, and CLPP1 and CE protein lysates with antibodies for p-IRE1, IRE1, FLAG, p-eif2a, and ß-actin. The membrane was stained with Ponceau S. (n = 3 biological replicates; a representative blot is shown). Data information: All data are mean ± SEM; (n = 4) Unpaired t test with Welch’s correction. * P = 0.05, ** P = 0.01, *** P =0.001. All data are mean ± SEM; (n = 3) Unpaired t test. * P = 0.05, ** P = 0.01. ATF, activating transcription factor; CE, total cellular extract; CLPP1, caseinolytic protease-1; ER, endoplasmic reticulum; HEK293, Human embryonic kidney 293; IRE1, inositol-requiring enzyme-1; MEFs, mouse embryonic fibroblasts; MF, mitochondrial fraction; mtHSP70, mitochondrial heat shock protein 70; LONP1, Lon protease-1; SPL, sphingosine 1-phosphate lyase; mt, mitochondria; TG, thapsigargin; UPR, unfolded protein response.
Fig 2: Stability of POL?AA449T mutant protein in vitro and in vivo. (A) Schematic representation of a typical thermofluor stability assay. This assay uses a fluorescent dye, SYPRO Orange, to monitor the temperature-induced unfolding of proteins. When the temperature starts to rise and unfold the protein, the SYPRO Orange dye fluoresces by binding to exposed hydrophobic patches. (B) Thermofluor stability assay to evaluate thermostability of mPOL?AWT (black) and mPOL?AA449T (blue), in absence (solid line) or presence (dashed line) of mPOL?B. (C) Size-exclusion chromatogram of mPOL?AWT (black line) and mPOL?AA449T (blue line) in presence of mPOL?B, to evaluate interaction between POL?A and POL?B. (D) SDS-PAGE of the selected peak fractions from (C) of mPOL?AWT and mPOL?B. (E) SDS-PAGE of the selected peak fractions from (C) of mPOL?AA449T and mPOL?B. Note the brackets highlighting unbound POL?B (free from POL?AA449T). (F) Western blot analysis of steady-state levels of POL?A, LONP1 and POL?B upon siRNA-mediated knockdown of LONP1, POL?B and POL?A, in HeLa cells. ß-actin was used as loading control. (G) Quantification of POL?A levels upon siRNA-mediated knockdown of LONP1 (F). POL?A levels were normalized to ß-actin and presented as FOLD change from cells treated with control siRNA. Data are presented as mean ± SEM. Two tailed unpaired Student's t-test: ***P < 0.001. (n = 3). (H) Quantification of POL?A levels upon siRNA-mediated knockdown of POL?B (F). POL?A levels were normalized to ß-actin and presented as fold change from cells treated with control siRNA. Data are presented as mean ± SEM. Two tailed unpaired Student's t-test: ***P < 0.001. (n = 3). (I) Western blot analysis of steady-state levels of POL?A in heart of Lonp1+/+ and Lonp1-/- animals. An anti-LONP1 antibody was used to confirm gene knockout and HSC70 was used as loading control. (J) Quantification of POL?A levels in heart of Lonp1+/+ and Lonp1-/- animals (I). POL?A levels were normalized to HSC70 and presented as fold change from Lonp1+/+. Data are presented as mean ± SEM. Two tailed unpaired Student's t-test: *P < 0.05. (n = 6).
Fig 3: Mut-H USCs had elevated UPRmt and reduced GSK3B and WNT7B.A The mRNA levels of UPRmt target genes (ATF5, HSP70, HSP60 and LONP1) were elevated in Mut-H USCs. Total RNAs were extracted from Ctr, Mut-L and Mut-H USCs, and expression of UPRmt target genes were quantified through real-time quantitative PCR (RT-qPCR). B–G The protein levels of UPRmt target genes (ATF5, HSP70, HSP60 and LONP1) were elevated in mutant-high USCs. Total protein lysates were extracted from USCs and protein levels were analyzed by western blot. ß-actin served as internal loading control. C, E, G were quantification plot for B, D, F, respectively. H mRNA levels of WNT7B were reduced in Mut-H USCs. I Protein levels of p-GSK3ß were decreased in Mut-H USCs. GADPH serves as internal control. J Quantification plot for I. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 4: Effect of LonP1 silencing on the degradation of DJ-1 unstable mutants transfected in DJ-1-null MEFs. DJ-1-null MEFs were transduced with either scrambled shRNA (scr) or specific shRNA for mouse LonP1 (sh mLonP1), as described under the Material and Methods section. (A) Panel shows a representative immunoblot developed with specific anti-LonP1 antibodies of LonP1 shRNA-mediated knockdown in DJ-1-null MEFs. Quantification is shown in the right graph. (B) Confocal fluorescence images of non-target shRNA lentiviral transduced (scr) or LonP1 shRNA lentiviral transduced (sh mLonP1) DJ-1-null MEFs growing under basal conditions and stained with Mitotracker (red), anti-LonP1 specific antibodies (green) and counterstained with DAPI (blue) for nuclei visualization. Scr DJ-1-null MEFs, panels (C) and (E) or sh mLonP1 DJ-1-null MEFs, panels (D) and (F), were transiently transfected with hDJ-1 L10P, P158? and L166P (C,D) or hDJ-1 A107P, E163K and L172Q (E,F) and 48 h after transfection were treated with 25 µg/mL cycloheximide (CHX) for the times indicated. Panels show representative immunoblots with anti-DJ-1 polyclonal antibody of the indicated hDJ-1 missense mutants transfected MEFs. Anti-tubulin antibodies were used as total protein loading control. Quantifications are shown in the graphs below as mean ± s.e.m from three different experiments. Significant differences were found between scr and sh mLonP1 DJ-1-null MEFs transfected with P158? at the time points 2 h (**p = 0.0001), 4 h (**p = 0.003), 8 h (**p = 0.0008) and 12 h (**p = 0.009), between scr and sh mLonP1 DJ-1-null MEFs transfected with L166P at the time points 2 h (**p = 0.0002), 4 h (**p = 0.0001), 8 h (**p = 0.0008) and 12 h (**p = 0.002), between scr and sh mLonP1 DJ-1-null MEFs transfected with A107P at the time points 6 h (**p = 0.0008), 12 h (*p = 0.02) and 24 h (**p = 8E-05), between scr and sh mLonP1 DJ-1-null MEFs transfected with E163K at the time points 12 h (**p = 0.009) and 24 h (*p = 0.01) and between scr and sh mLonP1 DJ-1-null MEFs transfected with L172Q at the time point 24 h (**p = 0.001).
Fig 5: IRE1-SPL signaling axis augments UPRmt. A–E: WT MEFs were treated with THI (5 mM) and TG (100 nM) for 12 h, and total RNA lysate was analyzed by qRT-PCR for Atf5, Atf4, Ddit3, Lonp1, Hspa9, and Gapdh mRNA (n = 4 biological replicates). F: WT MEFs were treated with THI (5 mM) and TG (100 nM) for 4 h, and MF protein lysates were analyzed by Western blotting using specific antibodies for LONP1, mtHSP70, and CLPP1 and CE protein lysates with antibodies for p-IRE1, IRE1, and ß-actin. Ponceau S was used as loading control. (n = 3 biological replicates; a representative blot is shown). G–J: WT MEFs were transfected with the indicated plasmids and treated with TG (100 nM: 20 h). Total RNA lysate was analyzed by qRT-PCR for Atf5, Atf4, Ddit3, Hspa9, and Gapdh mRNA (n = 4 biological replicates). Data information: All data are mean ± SEM; (n = 4) Unpaired t test with Welch’s correction. * P = 0.05, ** P = 0.01, *** P =0.001. ATF, activating transcription factor; CE, total cellular extract; CLPP1, caseinolytic protease-1; IRE1, inositol-requiring enzyme-1; MEFs, mouse embryonic fibroblasts; MF, mitochondrial fraction; mtHSP70, mitochondrial heat shock protein 70; LONP1, Lon protease-1; SPL, sphingosine 1-phosphate lyase; mt, mitochondria; TG, thapsigargin; UPR, unfolded protein response.
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