EMBO/SourceData huggingface dataset:
Figure 6(b) Confirmation of the expression of GFP-tagged human SAMHD1 (WT, and DN, DA, TA, and RE mutants). Histone H3 expression is shown as a loading control.(c) CSR complementation assay in Samhd1 KO cells. Data are shown as the % IgA switching relative to the IgA switching in WT cells transfected with empty vector, and are derived from three independent experiments (mean ± sd; two-tailed unpaired Student's t-test).(e) Effect of deoxyribonucleoside supplementation individually or in combination on the rate of IgM to IgA switching in CH12F3-2A cells. Data are presented as % IgA switching relative to the IgA switching in cells treated with an equivalent volume of the solvent in which the deoxyribonucleosides were dissolved (n=3; mean ± sd; two-tailed unpaired Student's t-test; ** = p ≤ 0.01; *** = p ≤ 0.001).(f) Effect of deoxyribonucleoside supplementation individually or in combination on the frequency of IgH/c-Myc translocations in CH12F3-2A cells (n = 2; mean ± sd; Fisher's exact test; ns = not significant; *** = p ≤ 0.001). The concentration dNTPs used for supplementation either individually or in combination were as follows: 2′-deoxyguanosine (dG), 0.15 mM; 2′-deoxyadenosine (dA), 1.0 mM; 2′-deoxycytidine (dC), 12 mM; and 2′-deoxythymidine (dT), 1.6 mM.
Figure 7(b) FACS profile of the percentage of WT and Samhd1 KO CH12F3-2A cells undergoing IgA switching following transfection with the indicated plasmids. The number in the FACS plot represents the percentage of cells expressing IgA on their surface. (c) Summary of the FACS data obtained from two independent experiments. The data are shown as percentage of IgA expressing cells upon CSR induced by blunt (left) or staggered (right) DSBs (n = 2; mean + sd; two-tailed unpaired Student's t-test); ns = not significant.(d) Length of resected DNA at Sμ-Sα junctions obtained from the joining of DSBs with blunt ends. Statistical significance as evaluated by two-tailed unpaired Student's t-test is shown. (e) Summary of the characteristics of Sμ-Sα recombination junctions resulting from the joining of DSBs with blunt ends.(f) Length of resected DNA at Sμ-Sα junctions obtained from the joining of staggered DSBs. Left: Length of Sμ and Sα overhangs were 35 nt and 98 nt, respectively. Right: Length of Sμ and Sα overhangs were 35 nt and 187 nt, respectively. (g) Summary of the characteristics of Sμ-Sα recombination junctions resulting from the joining of staggered DSBs. Statistical significance as evaluated by Fisher's exact test is shown.(h) Summary of the average length of insertions at Sμ-Sα recombination junctions. Statistical significance as evaluated by two-tailed unpaired Student's t-test is shown.Analysis of the effect of TdT expression on the frequency of CSR as well as characteristics of Sμ-Sα recombination junctions resulting from the joining of DSBs with blunt ends. (j) Top: Schematic representation of the experimental design to analyze the effect of TdT expression on CSR in WT and Samhd1 KO cells. Bottom: Western blot analysis of the expression of TdT in WT and Samhd1 KO cells. Tubulin was used as a loading control.(k) FACS profile of the percentage IgA switching in WT or Samhd1 KO CH12F3-2A cells with or without TdT expression. (l) Summary of the FACS data obtained from two independent experiments (n = 2; mean + sd; two-tailed unpaired Student's t-test; * = p ≤ 0.05).(m) Summary of the characteristics of Sμ-Sα recombination junctions. Statistical significance as evaluated by Fisher's exact test is shown. ns = not significant.
Figure 1C, D. Oxidative stress biochemical markers (lipid peroxidation, ROS production, GSH levels) (C) and p-ERK expression (D) in PD and control (CNTR) fibroblasts (n=6). E, F. Oxidative stress biochemical markers (lipid peroxidation, ROS production, GSH levels) (E) and p-ERK expression (F) in PD and control (CNTR) myoblasts (n=4). In all instances indicators of stress were increased in PD, compared to the respective control samples. Data information: In each experiment at least biological triplicates were analyzed for each cell line or tissue sample; each assay was performed at least in duplicate. Data are presented as mean ± SD. Student's t-test was applied. Statistically significant comparison p-values are indicated. Fi
Figure 2A, B. Western Blot analysis of the autophagy marker LC3 (A) and quantitative analyses (B) in untreated control fibroblasts (CNTR) and in 2 PD fibroblasts cell lines (PD1, PD2). PD cells were untreated (NT) or subjected to different treatments to modulate autophagy (starvation, STAR; rapamycin, RAPA; MK6-83; bafilomycin, BAFI). Data information: Starvation of cells was performed for 4 hours; treatments with 20 µM rapamycin or 30 µM MK6-83 or 100nM bafilomycin was performed for 24 hours. Data presented as mean ± SD. P values were calculated with ANOVA Šídák's multiple comparisons test. Statistically significant comparison p-values are indicated.C. ROS production, lipid peroxidation, and GSH levels in PD fibroblasts (n=3). CNTR (white bars), CNTR after treatment (dark grey bars), PD samples (black bars) and PD after treatment (light grey bars). Physiological or pharmacological enhancement of autophagy resulted in correction of oxidative stress in PD cells, while bafilomycin treatment further increased stress in CNTR and PD cells. Data information: Starvation of cells was performed for 4 hours; treatments with 20 µM rapamycin or 30 µM MK6-83 or 100nM bafilomycin was performed for 24 hours. Data presented as mean ± SD. P values were calculated with ANOVA Šídák's multiple comparisons test. Statistically significant comparison p-values are indicated.
Figure 3A, B, C. Correlations between the levels of single stress indicators and correction of GAA activity by rhGAA in 6 different PD fibroblasts. ROS production and lipid peroxidation inversely correlate with the GAA levels attained in cells after incubation with rhGAA for 4 hours. The analysis of correlation was calculated, and the coefficient of Pearson is indicated. D
Figure 4A. Cell viability in control and PD fibroblasts (3 different cell lines) measured by MTT assay in the presence of increased concentrations (0-100 µM) of either sodium arsenite (ARS) or tert-butyl-peroxide (TBP) at different time points. Data presented as a mean ± SD. B. ROS production, lipid peroxidation, and GSH levels in fibroblasts (3 different cell lines) after 6 hours of treatment with 100 µM ARS and or 10 µM TBP. Both oxidative agents induced increases in oxidative stress in control (CNTR) and in PD cells. Data presented as a mean ± SD. Significance was calculated by one-way ANOVA followed by Šídák's multiple comparisons test. C Data information: statistically significant p-values are indicated.
Figure 5A. Effect of antioxidants on ROS production, lipid peroxidation, and GSH levels in PD fibroblasts (3 different cell lines for each treatment, each cell line assayed at least in ducplicate). Cells were incubated for 24 hours with: idebenone (IDE) 0.5 µM; edaravone (EDA) 50 µM; N-acetylcysteine (NAC) 0.5 mM; resveratrol (RESV) 30 µM. Mean of control (CNTR) values are taken as equal to 100. The results are shown as mean ± SD. Data information: To calculate statistical significance one-way ANOVA was applied for all experiments followed by Dunnett's test. Statistically significant p-values are indicated.E. Confocal immunofluorescence analysis of GAA and LAMP2; representative fibroblast cell lines (PD1). Confocal 63x images; Scale bar 50 µm; Brightness +20%. F. Percent of GAA/LAMP2 colocalization in PD1. The results are expressed as means ± SD of 5 images for each condition. G. Percent of GAA/LAMP2 colocalization; mean of the analyses performed in 3 PD fibroblast cell lines. For each PD patient, 5 images for each condition were quantified. The results are expressed as means ± SD of 5 images for each condition. Data information: To calculate statistical significance one-way ANOVA was applied for all experiments followed by Dunnett's test. Statistically significant p-values are indicated.
Figure 6D. Mean of results obtained in control (two cell lines in duplicate) and PD fibroblast (3 different cell lines, each cell line tested in duplicate in three different experiments). For each cell line, the amount of M6PR positive cells was normalized, taking that observed in not-treated fibroblasts as 1. Data presented as mean ± SD. Data information: For statistical analysis a one-way ANOVA followed by Šídák's post hoc test was applied.
Figure 7C. GAA activity in tissues from the Gaa KO mouse after treatment with rhGAA alone (black bars) (n of mice = 10) and with co-dosing of rhGAA and 2 gr/Kg/day NAC (n of mice =7, dark grey bars). Data presented as mean ± SD. Values obtained in tissues treated with rhGAA alone for each treatment are taken as 100. A Student's t-test was applied to compare the results in each of the tissues. D. GAA activity in tissues from the Gaa KO mouse after treatment with rhGAA alone (black bars) (n of mice =4) and with co-dosing of rhGAA and 100 mg/Kg/day IDE (n of mice =6, light grey bars). Data presented as mean ± SD. Values obtained in tissues treated with rhGAA alone for each treatment are taken as 100. A Student's t-test was applied to compare the results in each of the tissues. E E, F. Western Blot analyses of GAA and quantitative analyses of the enzyme in representative tissues from the Gaa KO mouse after treatment with rhGAA alone (n of mice =2) or in combination with NAC (n of mice =3) (E) or IDE (n of mice =3) (F). In F Data are presented as mean ± SD.G. Glycogen assay in tissues from the Gaa KO mouse after treatment with rhGAA alone (n of mice = 3) or in combination with NAC (n of mice = 3). H. Glycogen assay in tissues from the Gaa KO mouse after treatment with rhGAA alone (n of mice =4) or in combination with IDE (n of mice = 6). Data information: i data are presented as mean ± SD. Values obtained in tissues treated with rhGAA alone for each treatment are taken as 100. A Student's t-test was applied to compare the results in each tissue.
Figure 1Uncoupled (LEAK) mitochondrial respiration of soleus (SOL) and extensor digitorum longus (EDL) muscle of wild type (WT) vs. Ucp1-TG (TG) mice (WT n=9, TG n=5).Relative mRNA expression in quadriceps (Quad) of ISR components (n=8 per genotype).Representative immunoblots of ISR component eIF2α and quantification of phospho-eIF2α (p-eIF2aSer51) relative protein expression in quadriceps skeletal muscle (WT n=3, TG n=4).Multi-tissue transcriptomic profiling of Gdf15 gene expression. Heatmap is shown as raw ct expression values (n=4 per genotype). Quantification of Gdf15 mRNA expression in TG mice is shown as fold change compared to WT littermates (WT n=5, TG n=4).Skeletal muscle (Quad and Gastroc) GDF15 protein content normalized to total protein content (WT n=9, TG n=18).Ex vivo secretion of GDF15 from SOL and EDL muscle of WT vs. TG mice (n=6 per genotype) after 2h incubation normalized to muscle wet weight (mg).Mouse GDF15 plasma levels in post-absorptive state of male and female WT vs. TG mice at 20wks of age (male WT n=8, TG n=9; female WT n=5, TG n=6).Mouse GDF15 plasma levels in post-absorptive state of male WT vs. TG mice fed low-fat diet (LFD) or high-fat diet (HFD) at 24wks of age (LFD WT n=6, TG n=5; HFD WT n=5, TG n=5).Mouse GDF15 plasma levels in post-absorptive state of young (10wks) vs. old (95wks) male WT and TG mice (young WT n=10, TG n=13; old WT n=13, TG n=13).Gdf15 gene expression of differentiated C2C12 muscle cells treated with vehicle control (Ctrl) or chemical mitochondrial uncoupler (FCCP, 1µM vs. 5µM) for 5hrs (n=3 biological replicates)
Figure 2Genotyping PCR panel of Gdf15 and HSA-Ucp1 loci shown for wild type (WT), Gdf15-KO (KO), Ucp1-TG (TG) and Ucp1-TGxGdf15-KO (TGxKO) mice.Relative mRNA expression in quadriceps (Quad) of Ucp1 and Gdf15 in 20wks male WT (n=8), TG (n=7) and TGxKO (n=6) mice.Plasma mGDF15 levels at 20wks of age (WT n=9, TG n=10, TGxKO n=9).Voluntary wheel running (VWR) activity shown hourly over 24-hrs at 15 wks of age (n=4 per genotype).Grip strength at 10 - 20wks of age (WT n=15, TG n=14, TGxKO n=11).Skeletal muscle mass relative to body lean mass of quadriceps (Quad) and gastrocnemius (Gastroc) from mice at 95wk of age (WT n=8, TG n=5, TGxKO n=5).Representative H&E histological staining of tibialis anterior (TA) muscle (G) (scale bars represent 50 µm), and cross-sectional area (CSA) of myofibers (H) at 95wks (WT n=12, TG n=8, TGxKO n=8).Representative traces of oxygen consumption rate (OCR) during substrate-uncoupler-inhibitor-titration (SUIT) protocol for mitochondrial respiratory capacity in permeabilized mouse soleus muscle fibers, PM (Pyruvate+Malate; LEAK respiration), ADP (OXPHOS capacity), Cyt c (cytochrome c, integrity of outer mt-membrane), G (glutamate;), S (succinate), U (Uncoupler, FCCP), Rot (rotenone), Ama (antimycin A; less than 2% residual oxygen consumption, ROX).Mitochondrial respiratory capacity (oxygen consumption rate, OCR) from oxidative soleus (SOL) (J) and glycolytic extensor digitorum longus (EDL) muscle fibers (K), and NetOXPHOS control ratio of 95wks old mice (L) (n=5per genotype).Quadriceps (Quad) relative mRNA expression of ISR components at 95wks of age (WT n=8, TG n=5, TGxKO n=5).Representative immunoblots of ISR component eIF2α in quadriceps (Quad) muscle of 95wks old mice.Gastrocnemius enzyme activity of NQO1 and GPX in 95wks old male mice (WT n=8, TG n=5, TGxKO n=5).
Figure 3Body mass (A), body lean mass (B) and body fat mass (C) development. Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) miceBody mass (D), body lean mass (E) and body fat mass (F) during aging at 20wks, 45wks and 95wks of age. Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) miceSubcutaneous white adipose tissue (sWAT) (G) and epididymal white adipose tissue (eWAT) (H) mass development at 10wks, 20wks, 45wks and 95wks of age. Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice at 10wks (WT n=6, TG n=7, TGxKO n=5), 20wks (WT n=10, TG n=10, TGxKO n=10), 45wks (WT n=5, TG n=6, TGxKO n=7) and 95wks (WT n=8, TG n=5, TGxKO n=5).
Figure 4Representative H&E histological staining of sWAT at 20wks of age (scale bars represent 50 μm) Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice.relative mRNA expression profile in sWAT of male mice (WT n=7, TG n=8, TGxKO n=8) (B). Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice.Representative immunoblots (C) and quantification (D) of UCP1 protein expression in sWAT of male mice at 20wks of age (WT n=4, TG n=6, TGxKO n=4). Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice.Relative mRNA expression profile in eWAT (WT n=7, TG n=8, TGxKO n=8). Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice.Plasma FGF21 levels from male mice at 20wks of age (n=10 per genotype). Plasma Leptin levels from male mice at 20wks of age (n=5 per genotype). Post-absorptive plasma insulin levels at 20wks of age (WT n=8, TG n=9, TG n=9). : Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice.Blood glucose (I) and insulin levels (J) with total area under the curve (AUC) of insulin (K) during oral glucose tolerance test (oGTT) at 17wks of age (WT n=8, TG n=11, TG n=11). Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice.
Figure 5Physical activity of male mice shown hourly over 24-hrs and as day and night time. Data shown are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice at 17-18wks of age (WT n=12, TG n=11, TG n=10) and shown as means + SEMenergy expenditure of male mice shown hourly over 24-hrs and as day and night time. Data shown are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice at 17-18wks of age (WT n=12, TG n=11, TG n=10) and shown as means + SEM, total assimilated energy of male mice shown hourly over 24-hrs and as day and night time. Table showing mean values of assimilated energy (kJ) at day/night time and per 24-hrs. Energy balance calculated as delta of assimilated energy (AE) and energy expenditure (EE) and energy balance (J) per 24-hrs. Data shown are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice at 17-18wks of age (WT n=12, TG n=11, TG n=10) and shown as means + SEMRespiratory quotient (RQ) shown hourly over 24-hrs (J), RQ amplitude (K) and metabolic flexibility via percentage relative cumulative frequency (PRCF) (L). Data shown are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice at 17-18wks of age (WT n=12, TG n=11, TG n=10) and shown as means + SEM
Figure 6Relative quadriceps (Quad) mRNA expression of the ISR components Atf4, Atf5, Atf6 and Chop (WT day n=9, WT night n=5, TG day n=20, TG night n=12). Data shown are from male wild type (WT) vs. Ucp1-TG (TG) mice sacrificed at day (10am) or night (10pm).Representative immunoblots of ISR component eIF2α (B) and quantification of phospho-eIF2α (p-eIF2aSer51) relative protein expression (C) in skeletal muscle (Gastroc, WT day n=4, WT night n=5, TG day n=9, TG night n=9). Data shown are from male wild type (WT) vs. Ucp1-TG (TG) mice sacrificed at day (10am) or night (10pm).Relative mRNA expression of Rev-erba (D) and Gdf15 (E) in quadriceps, liver and eWAT (WT day n=9, WT night n=5, TG day n=20, TG night n=12). Data shown are from male wild type (WT) vs. Ucp1-TG (TG) mice sacrificed at day (10am) or night (10pm).Skeletal muscle (Quad) GDF15 protein content normalized to total protein content (WT day n=4, WT night n=5, TG day n=14, TG night n=12) Circulating GDF15 plasma levels (WT day n=9, WT night n=5, TG day n=18, TG night n=12). Data shown are from male wild type (WT) vs. Ucp1-TG (TG) mice sacrificed at day (10am) or night (10pm).
Figure 1(G) Fold change in total RNA modification levels upon loss of m5C. Fold changes were calculated by dividing the peak area ratio of noNSUN samples by the one of wild type samples. n = 3 independent biological replicates. Multiple t-tests. Data information: In G), data are presented as mean ± SEM.
Figure 2(A, B) Site-specific methylation analysis by whole-transcriptome bisulfite sequencing. Scatter plots show individual cytosines and their respective non-conversion rates in rRNAs (A) and tRNAs (B) of wild type and noNSUN strains; pie chart showing most frequently methylated tRNA isoacceptors. Data information: In (A, B n = 3 independent biological replicates.
Figure 3(A) RNA bisulfite sequencing map for mitochondrial tRNA Met-CAU in wild type (top) and noNSUN (bottom) strains. Each row represents one sequence read and each column one cytosine. Data information: In (A), a representative map of the replicates is shown, n = 3 independent biological replicates. Similar effects were observed in all replicates analysed.
Figure 4D) Viable progeny counts of wild-type and noNSUN strains at 20°C and 25°C (D). Automatic counting was done using a Matlab script which processed plate images in real-time. Data information: In (D), data are presented as mean ± SEM, unpaired two-tailed Student's t-test.
Figure 5(D) Translation efficiency of UUG-enriched, leucine-enriched, proline-enriched and random genes in each sample. A gene was considered enriched in a certain codon when the proportion of this codon in the gene was at least 3-fold higher than the proportion of the same codon across the transcriptome. Data information: In n = 3 biological replicates. In (D), boxplots show the median (central band) and IQR (boxes) ± 1.5 x IQR (whiskers), Welch's t-test, p-value < 0.05.
Figure 1.(A) HeLa cells were fixed and stained for p62. The different patterns of p62 distribution were scored in >200 cells as cytoplasmic with small bodies, cytoplasmic with large and intense bodies, equal nuclear and cytoplasmic staining, and nuclear enriched. The percentage of cells in the respective groups is indicated.(B) Expression level of GFP-p62 in HeLa cells stably expressing GFP-p62 (S-GFP-p62) compared with the level of endogenous p62 in the parent HeLa cells.(C) Cells stably expressing GFP-p62 display a cytoplasmic punctuate localization of GFP-tagged p62 similar to endogenous p62. Video microscopy of live cells demonstrated that the small, faint bodies (thin arrows) displayed directed migration, whereas the majority of the larger, intense bodies (thick arrows) were nonmigratory (Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200507002/DC1). A still image from Video 1 is shown here.(D) Bodies containing either endogenous p62 or transiently or stably transfected GFP-p62 all contain polyubiquitin. HeLa cells were fixed and stained with p62 and polyubiquitin (clone FK1) mAbs directly coupled with AlexaFluor555 (red) and AlexaFluor488 (green), respectively. Cells expressing GFP-p62 were only stained for polyubiquitin (red). Bars, 20 μm.
Figure 2.(A) The indicated deletion constructs were fused COOH terminal to GFP and expressed in NIH3T3 fibroblasts. Asterisks indicate point mutations in the PB1 (D69A) and UBA (I431A) domains, respectively.(B) S-GFP-p62 cells were transiently transfected with the indicated myc-tagged p62 constructs, fixed, and stained for the myc tag. Bars, 20 μm.
Figure 3.(A) Localization of bodies containing endogenous p62 or transiently expressed GFP-p62 relative to EEA1-positive early endosomes. Endogenous p62 were stained green using p62 antibodies directly labeled with AlexaFluor488, and EEA1 was stained red with EEA1 mAbs directly labeled with AlexaFluor555. Alternatively, transiently expressed GFP-p62 was expressed in HeLa cells, and EEA1 was stained red with EEA1 mAb (bottom). The boxed area is shown to the right at a higher magnification.(B) Colocalization of GFP-p62 and CD63 (stained red using a mAb) in HeLa cells stably expressing GFP-p62.(C) Immunoelectron micrograph of S-GFP-p62 stained with a GFP pAb (10-nm gold particle, arrows) and monoclonal CD63 (15-nm gold particle, arrowheads).(D) Rapid detergent extraction of GFP-p62 from LysoTracker-positive acidic organelles. HeLa cells transiently expressing GFP-p62 were labeled with LysoTracker for 60 min. The detergent extractions were imaged in a time series with 15-s time intervals after adding 1% Triton X-100. The boxed area is shown in the bottom two images at a higher magnification before and after detergent extraction. Bars (A, B, and D), 20 μm.
Figure 4.(A) Immuno-EM of GFP-p62. S-GFP-p62 cells were labeled with rabbit anti-GFP (Abcam) followed by protein A-gold (15 nm). We observed labeling in membrane-free cytosolic structures and sequestosomes (arrows) as well as in endosomes (Endo).(B) Correlative immunofluorescence/EM of HeLa cells treated with 10 μM PSI for 5 h displaying typical sequestosomes. The insets show two magnifications of the sequestosome, which is labeled 1.(C) Representative image of a p62-containing autophagosome. HeLa cells transfected with GFP-p62 were immunogold labeled as in A. Note the cisternal-like membrane (arrowheads) surrounding the GFP-positive material. The arrow indicates a fused endosome.