EMBO/SourceData huggingface dataset:
Figure 2Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB-WT-FLAG incubated with the indicated kinase inhibitors for 1 h prior to the addition of 200 μM NaAsO2 for 1 h. Quantification of immunoblot data shown in (A). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (ns) not significant, and (****)p<0.0001 from three independent experiments. Data information: n = 3 biological replicates (each dot represents a biological replicate).Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB-WT-FLAG incubated with either 200 μM NaAsO2 for 1 h, EBSS for 4 h, 100 ng/ml EGF for the indicated times or 37 μM Anisomycin for 1 h. Before the addition of EGF or Anisomycin, cells were serum starved for 8 h.Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB-WT-FLAG exposed to 30J/m2 of UV-C and allowed to recover in complete medium for the indicated times. Cells were incubated with p38 MAPK inhibitor (20 μM, SB203580) for 1 h before UV-C irradiation and allowed to recover for 30 min in the presence of the inhibitor.Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB-WT-FLAG depleted of either p38 MAPK (α+β) or JNK1 or JNK2 or JNK(1+2) and incubated with 200 μM NaAsO2 for 1 h. Immunoblots are representative of at least three independent experiments.Immunoblot analysis of in vitro p38 MAPK kinase assay using GST-TFEB-PRD as substrate in the presence or absence of either recombinant human active p38α MAPK or ATP. Quantification of immunoblot data shown in (F). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (*)p<0.05 from three independent experiments. Data information: n = 3 biological replicates (each dot represents a biological replicate).
Figure 3Immunoblot analysis of protein lysates from Raw 264.7 cells stably expressing TFEB-WT-FLAG incubated with 1μg/ml LPS for the indicated times.Immunoblot analysis of protein lysates from Raw 264.7 cells stably expressing TFEB-WT-FLAG incubated with either 20 μM SB203580 or 250 nM Torin-1 for 1 h prior to the addition of 1 μg/ml LPS for 30 min.Quantification of immunoblot data shown in (B). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (ns) not significant, (*)p<0.05 and (**)p<0.01 from three independent experiments. Data information: n = 3 biological replicates (each dot represents a biological replicate).Immunoblot analysis of protein lysates from THP1 cells incubated with 1 μg/ml LPS for the indicated times.Immunoblot analysis of protein lysates from THP1 cells incubated with either vehicle (DMSO) or 20 μM SB203580 for 1 h prior to the addition of 1μg/ml LPS for the indicated times.Immunoblot analysis of protein lysates from THP1 cells depleted of p38 MAPK (α), p38 MAPK (β) or p38 MAPK (α+β) and incubated with 1 μg/ml LPS for 1 h. Immunoblots are representative of at least three independent experiments. Quantification of immunoblot data shown in (F). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (*)p<0.05 from three independent experiments. Data information: n = 3 biological replicates (each dot represents a biological replicate).
Figure 4Immunoblot analysis of protein lysates from naïve THP1-WT or TFEB-S401A knock-in (clone M17) cells treated with 50 ng/ml PMA for the indicated times, PMA-differentiated THP1 (Rested) cells treated without or with 250 nM Torin-1 for 1 h.Immunoblot analysis of proteins from nuclear and cytosolic fractions from naïve THP1-WT or TFEB-S401A knock-ins (clones I11 and M17) cells treated with 50 ng/ml PMA for the indicated times and PMA-differentiated THP1 (Rested) cells.Immunoblot analysis of proteins from nuclear and cytosolic fractions from naïve THP1-WT or TFEB-S401A knock-in (clone I11) cells treated with 50 ng/ml PMA for the indicated times.Immunoblot analysis of proteins from nuclear fractions from naïve THP1-WT or TFEB-S401A knock-in (clone I11) cells treated with 50 ng/ml PMA or 250 nM Torin-1 for 1 h. Immunoblots are representative of at least three independent experiments.
Figure 5Immunoblot analysis of proteins from nuclear and cytosolic fractions from naïve THP1-WT or TFEB-S401A knock-in (clone I11) cells treated with 50 ng/ml PMA for 6 h.
Figure 6Immunoblot analysis of protein lysates and cell culture medium from PMA-differentiated (Rested) THP1-WT or TFEB-S401A knock-ins (clones I11 and M17) cells incubated with 0.1 μg/ml LPS for 4 h prior to the addition of 15 μM Nigericin for 45 min. C-F. Quantification of immunoblot data shown in (B). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (ns) not significant, (*)p<0.05, (**)p<0.01, (***)p< 0.001 and (****)p< 0.0001 from three independent experiments. Data information: n ≥ 3 biological replicates (each dot represents a biological replicate).Fluorescence confocal microscopy of PMA-differentiated (Rested) THP1-WT or TFEB-S401A knock-ins (clones I11 and M17) cells incubated with 1 μg/ml LPS for 6 h prior to the detection of dead cells with LIVE/DEAD fixable blue dead cell stain. Scale bars, 10 μm. Quantification of the percentage of dead cells shown in (G). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (ns) not significant and (****)p< 0.0001 as compared to their corresponding untreated (Control) cells, with >200 cells counted per trial from three independent experiments. Data information: n ≥ 3 biological replicates (each dot represents a biological replicate).
Figure 1A. Blood-stage growth rate of concavin(-) parasites in comparison to wild type. Data points represent parasites growing in individual mice, n indicates total number. P-value is calculated using the Mann Whitney test. Shown is the mean ± SEM.B. Gametocyte to ookinete conversion of 3 biological replicates. P-value calculated using the Mann Whitney test. Shown is the mean ± SEM. C. Average speed of moving ookinetes. Data points represent individual ookinetes, n indicates total number of cells from 3 biological replicates. P-values are calculated using the Kruskal Wallis test followed by Dunns multiple comparison. D. Ookinete images of wild type and concavin(-) parasites revealing similar cell shapes. Scale bar 5 µm.E. Oocyst development of concavin(-) parasites compared to wild type. Data points represent individual midguts observed between d12-17 post infection from 3 independent cage feeds. Shown is the mean ± SEM. P-values are calculated using the Kruskal Wallis test followed by Dunns multiple comparison.F. Mosquito infections resulted in deformed sporozoites in the salivary gland. Shown are example images of different sporozoites arranged to illustrate their rounding up over time. G. Quantification of sporozoite rounding up over time in the midgut (MG) the haemolymph (HL) and salivary gland (SG) at the indicated days post infection from three biological replicates. Sporozoites were classified as either normal, deformed or round as illustrated in F.H. Average speed of salivary gland sporozoites. Data points represent individual sporozoites, n indicates total number from 3 biological replicates. P-values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test.I. Selected trajectories of manually tracked sporozoites.
Figure 2A. Oocyst development of concavin(-) parasites complemented with either the P. berghei gene or the P. falciparum orthologue fused to GFP. Data points represent individual midguts observed between d12-17 post infection of 3 independent cage feeds. Shown is the mean ± SEM. P-values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test.B. Average speed of salivary gland sporozoites. Data points represent individual sporozoites of 3 independent biological replicates. Shown is the mean ± SEM. P-values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test.C. Sum table of mosquito infections of wild type, concavin(-) and complemented lines expressing either P. berghei (Pb) or P. falciparum (Pf) concavin-GFP. Numbers determined on d17 post mosquito infection. Infection rate contains 3 different countings of at least 20 mosquitoes from different mosquito infections. Note the difference of normally shaped and total sporozoites for concavin(-) parasites. Images show infection of salivary glands. Scale bar: 100 µmD. Pb concavin-GFP and Pf concavin-GFP localization in blood and mosquito stages. Nuclei (blue) stained with Hoechst. Scale bar: 5 µm.E. Localization of P. berghei concavin-GFP in liver stages. Nuclei (blue) stained with Hoechst. Scale bar: 5 µm.F. Immunofluorescence images of permeabilized and unpermeabilized concavin-GFP expressing salivary gland sporozoites stained with an anti-GFP antibody. Note that a GFP signal could only be detected after permeabilization, excluding concavin localization on the parasite surface as illustrated in the model. Scale bar: 5 µm.
Figure 3A, B. Time series of concavin-GFP (A) PhiL1-GFP (B) sporozoites before and after bleaching and quantification of the fluorescence signal over time of 3 technical replicates. * indicates time of bleaching. Shown is the mean ± SD.C. Super resolution (STED) imaging of PhiL1-GFP and CSP as well as concavin-GFP and CSP. Cells were stained with an anti-GFP antibody in combination with Atto-594 (green) in addition to an anti-CSP staining in combination with Atto-647. Images were deconvolved using the Richardson-Lucy algorithm. The distance between the 2 signal peaks was measured using the plot profile of the respective channels in Fiji. Data points represent distance in individual sporozoites at the center of the cell from 2 independent biological replicates. P-values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test. Scale bar: 1 µm.D. Localization of PhiL1-GFP (green) in concavin(-) parasites. Nuclei (blue) stained with Hoechst. Scale bar 5 µm. E. Localization of SiR-Tubulin (red) in concavin(-) parasites. Nuclei (blue) stained with Hoechst. Scale bar 5 µm. F. Localization of CSP (red) in concavin(-) parasites. Nuclei (blue) stained with Hoechst. Scale bar 5 µm. Data information: Asterisks in D-F indicate apical part of the sporozoite.
Figure 5B. Expression and localization of concavinC7A-GFP in blood stage parasites. Scale bar 5 µm.C. Oocysts number in infected mosquitoes. Data points represent individual midguts observed between d12-17 post infection of 3 independent cage feeds. Shown is the mean ± SEM. P-value calculated using the Mann Whitney test.D. Quantification of concavinC7A-GFP cell shapes at the indicated days from midgut (MG) or salivary gland (SG) derived sporozoites. Numbers above bars indicate investigated sporozoites.E. Average speed of salivary gland sporozoites. Data points represent individual sporozoites from 3 independent biological replicates. Shown is the mean ± SEM. P-values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test.F. Localization of concavinC7A-GFP in normal and deformed sporozoites (arrowheads point to deformations). Scale bar: 5 µm.
Figure 6A. Growth curve of blood stage parasites in C57BL/6 mice infected by the bite of 10 mosquitoes (left) or 10.000 sporozoites intra venously (right) at two different times post mosquito infection as indicated. Shown is the mean ± SEM. Mice infected with Concavin(-) parasites by bite represent 2 independent biological replicates with 3 mice each. Wild type bite back and Concavin(-) intra venous injections represent one biological replicate using 3 or 4 mice respectively.C. Liver cell invasion assay of concavin-gfp and concavin(-) parasites. Parasites are positively stained for CSP in case they remain extracellular. Both, normal and deformed parasites were detected intracellularly. Graph shows quantification of CSP positive (red, hence extracellular) and negative (grey, hence intracellular) sporozoites. Small graph shows the percentage of deformed and normally shaped intracellular concavin(-) sporozoites. Data points represent the 4 individual biological replicates with n indicating the numbers of sporozoites observed. Shown is the mean ± SEM. P-values are calculated using the Mann Whitney test. Scale bar: 5 µm.D. Liver-stage development of concavin(-) parasites compared to wildtype, both expressing cytoplasmic GFP (white). Parasite size was measured 24h and 48h post infection. Data points represent individual parasites from 4 independent biological replicates. Shown is the mean ± SEM. P-values calculated using the Mann Whitney test. Scale bar: 5 µm.E. Sporozoite ejection of immobilized concavin(-) infected mosquitoes on glass slides and quantification of ejected sporozoites from 20 mosquitoes. * indicates individual sporozoites in the ejected saliva. Scale bar: 10 µm.F, G. Only normally shaped concavin(-) sporozoites released by salivary glands move on helical paths (arrows) through polyacrylamide gels that mimic the skin (F). Quantification of two individual experiments Scale bar 10 µm. (G): only normal shaped sporozoites were able to migrate through the gel.
Figure 7A. Percentage of mosquitoes depositing WT or concavin(-) sporozoites in the skin of a mouse during a bite (left) and sporozoites deposited during a mosquito bite (right). P-values are calculated using the Fishers exact test (left) and the Mann Whitney test (right). Bite transmission experiments were repeated over 3 independent mosquito infections for the KO and 2 independent mosquito infections for the WT. For each mosquito infection 2-3 mice were imaged, for a total of 3-5 ears.B. Maximum fluorescence intensity projections encoded by color for time from movies showing migrating sporozoites after mosquito-bite transmission. Graphs and camembert diagrams below show fraction of motile and immotile sporozoites after 10, 20 and 30 minutes of recording, numbers analysed as well as sporozoite speed. Pooled data from 4-5 WT or 5-8 concavin (-) bite sites per time point. Scale bar: 50 µm. P-values are calculated using the Mann Whitney test.C. Motile WT or concavin(-) sporozoites entering blood vessels during the first hour following micro-injection. Number above bars shows numbers of sporozoites observed. Pooled data from four WT or five concavin(-) experiments. P-values are calculated using the Fishers exact test.D, E (D) Deformation and (E) disintegration of concavin(-) sporozoites migrating in the skin. Individual images corresponding to the frames shown on the right are indicated in distinct colours in the maximum projection (left) of 13-min movies. Arrowheads indicate constrictions of the parasites. Graphs below time-lapse show that deformation and disintegration are preceded by a decrease in speed. Color of dots correspond to the time-points displayed in the time-lapse images. Scale bars: 10 µm.F. Percentage of deformation and disintegration events observed in 54 WT and 57 concavin(-) sporozoites that were tracked for at least 10 min. Pooled data from 5 WT or 10 concavin(-) bite sites.
Figure 1B, Same as in A, with the exception that cells expressing flag-praja2rm or praja2 deletion mutants (praja21-530, praja21-630 and praja2Δ530-630) were included in the analysis.
Figure 2B. MST signal (normalized fluorescence) of P1 (red curve), P2 (green curve) and P3 (cyan curve) plotted against TBC1D31, at increasing concentrations of peptides. The threading modelled structure of the overlapping binding segment of praja2 (praja2550-570) is shown.F. MST signal of P1 plotted against increasing concentrations of TBC1D31 peptides: wild-type (red curve), R948A-R951A (violet curve) and R957-R959D-H960A (orange curve).
Figure 3D. Total lysates from mouse brain were immunoprecipitated with anti-TBC1D31 antibodies or with control IgG. The precipitates and an aliquot of lysates were immunoblotted with the indicated antibodies.
Figure 4E. Same as in C, with the exception that cells were co-transfected with flag-OFD1 vector and with control siRNA (siCNT) or siRNA targeting praja2. F. Quantitative analysis of the experiments shown in E. A mean value ± SD of three independent experiments is shown. Student's t test, *p<0,05. G
Figure 5C. HEK293 cells transfected with siRNAs were serum deprived, pre-treated with cycloheximide and stimulated with FSK at different time point. Lysates were immunoblotted for endogenous praja2 and OFD1. Hsp90 was used as loading control. D. Quantitative analysis of the experiments shown in C. A mean value ± SD of three independent experiments is shown. Student's t test * p<0,05; **<0,01 versus sipraja2 (+FSK) and basal values (0). E H. Immunoblot analysis of flag-OFD1 or flag-E97G mutant in HEK293 cells stimulated with FSK as in C. I. Quantitative analysis of the experiments shown in H. A mean value ± SD of three independent experiments is shown. Student's t test, *p<0,05 versus E97G mutant (+FSK) and basal values (0).
Figure 6A. siRNA transfected HEK293 cells were serum deprived and stained for acetylated-tubulin, TBC1D31 and DRAQ5. B. Quantitative analysis of the experiments shown in A. A mean value ± SD of four independent experiments is shown. Student's t test *p< 0,05. C C. HEK293 cells expressing flag-OFD1 or flag-S735A were serum deprived and subjected to staining analysis for acetylated α-tubulin, flag and DRAQ5. Images were collected by super-resolution microscopy (upper panels). Where indicated, flag-OFD1 or flag-S735A was co-transfected with cilia-APEX-GFP vector (middle panels). NIH3T3 expressing flag-OFD1 or flag-S735A were stained with anti-ARL13B and anti-flag antibodies. D, E. Statistical analysis of the experiments shown in D. A mean value ± SD of three independent experiments is shown. For each experimental group, a minimum of 40 cilia were analyzed. Student's t test p***<0,001. F F. HEK293 cells transiently transfected with flag-OFD1 or flag-S735A were serum deprived, treated with FSK (6 hours) and stained for flag, acetylated-tubulin and DRAQ5. G. Statistical analysis of the experiments shown in F. A mean value ± SD of three independent experiments is shown. Cells analysed for each experiment: 300 for NT, 80 for flag-OFD1 and 80 for flag-S735A. Student's t test, p*<0,05, p**<0,01. H
Figure 7B. Confocal images of cilia of the neural tube cells in the wild-type, Ol-TBC1D31 KD, Ol-TBC1D31 KD + hTBC1D31, wild-type hOFD1, Ol-TBC1D31 KD + wild-type hOFD1, hOFD1S735A, Ol-TBC1D31 KD + hOFD1S735A, Ol-TBC1D31 KD + hOFD1S735D and Ol-TBC1D31 KD + hOFD1S735D + hpraja2rm stained with anti-acetylated α-tubulin antibody (green) and DAPI (blue).
Figure 1A-E Echocardiographic analysis of (A) posterior wall thickness, (B) septum thickness, (C) left ventricle end diastolic diameter, (D) fractional area change and (E) ejection fraction. n= 8 per genotype.F Comparison of TAZ gene expression, normalized to S12, in heart, liver, and kidney, analyzed in triplicates by quantitative PCR. n= 5 per genotype.G TAZ gene expression in percent. Wild-type is set to 100%.H, I Lipid species profiles of cardiolipin (H) and monolysocardiolipin (I) in isolated cardiac mitochondria analyzed by mass spectrometry. n= 4 per genotype.J Comparison of ratios of total monolysocardiolipin and cardiolipin content in indicated tissues.
Figure 2A Periodic measurement of the oxygen consumption rate (OCR) of mitochondria, isolated from 5 mice per genotype, before and after the administration of the indicated compounds (left). Quantification of OCR in independent experiments, n= 8 per genotype (right).B OCR rates of cardiac mitochondria after administration of pyruvate, malate and ADP, n= 7 per genotype.
Figure 3A Mitochondrial membranes, isolated from cardiac tissue, were solubilized in 1% digitonin and analyzed by BN-PAGE and Western- blotting with the indicated antibodies.B Cardiac mitochondria were prepared as in (A) and analyzed on a large pore BN-gel for better resolution of supercomplexes.C Cardiac mitochondria were solubilized in 0.4% DDM and complexes were separated by BN-PAGE and analyzed by Western- blotting with the indicated antibodies.D Mitochondria were isolated from kidney and liver and analyzed as in (A).E Quantification of ROS production over time in cardiac mitochondria using the ROS-specific fluorescence-based sensor H2DCFDA. One representative measurement is shown. Analyses were performed in triplicates and confirmed for three animals per genotype. Significance was calculated using Student's T-TEST. sh, shTAZ.
Figure 4A Enzymatic activity of ubiquinone-cytochrome c oxidase in mitochondria isolated from cardiac tissue. The ubiquinone analogue decylubiquinone was used in this assay. Data were standardized to malate-dehydrogenase activity and presented as percent of wild-type, n= 4 per genotype.B Cytochrome c reductase activity, standardized to malate-dehydrogenase activity in cardiac mitochondria, were plotted as % of wild-type, n= 3 per genotype.C Protein levels of Cox1 and Rieske compared to VDAC3 in three different dilutions of isolated mitochondria, separated on SDS gel and visualized by Western-blotting.D Respiratory chain complexes in cardiac mitochondria were separated on BN-PAGE and stained for activity of complex I, II, IV and F1FO- ATPase. sh, shTAZ; Q, ubiquinone.
Figure 5A Enzymatic activity of the succinate dehydrogenase in isolated cardiac mitochondria from 3 animals per genotype.B Mitochondrial membranes were solubilized in 1% digitonin and analyzed by BN-PAGE and Western-blotting using antibodies against SDH components SDHA and SDHB (upper panel). For a loading control, samples were separated on SDS gel and analyzed by Western-blotting (lower panel).C Steady state protein levels analyzed by SDS gel separation of indicated amounts of mitochondrial protein and subsequent Western- blotting with antibodies against SDHA and VDAC3 or visualizing of Flavin fluorescence after excitation with 488 nm. Lower panel: quantification of signal ratio SDHA/VDAC3 in shTAZ and control (set to 100%).D Analysis of gene expression in cardiac tissue analyzed in (C) by qPCR using primers against indicated mRNA. Data of one representative measurement, normalized to b-actin with WT set to 100% are shown. Identical results were obtained in further experiments with two different pairs of animals.E Upper panel: SDH complex of cardiac, liver and kidney were analyzed by BN-PAGE as in (B); lower panel: the same samples were analyzed by SDS gel and Western-blotting.F, G Analysis of enzymatic activity of the succinate dehydrogenase related to the malate dehydrogenase activity in mitochondria isolated from kidney (F) and liver (G). Values of 3 animals per genotype are plotted as percent of wild-type. sh, shTAZ.
Figure 6A Flow cytometry analysis of cardiac troponinT (cTNT) expression was performed at day 60 post cardiac differentiation. 98% of cTNT+ cardiomyocytes (CM) were obtained after metabolic selection.B RT-PCR showing the expression of indicated genes in undifferentiated iPSCs and differentiated iPSC-CMs of BTHS patient TAZ10 and a healthy control.C Immunostaining of control (left) and BTHS iPSC-CMs at day 60 post differentiation for cardiac troponin T (cTNT) and Connexin 43 (Cx43). Scale bars: 50µm.D Quantitation of sarcomere organization. Left panel: Immunostaining of cTNT, MLC2a (myosin light chain) and a-actinin at day 60 post cardiac differentiation. Right panel: The bar graphs at the right panel represent the sarcomere regularity from 3 independent experiments. Error bars represent standard deviation for the comparison between control and BTHS patient TAZ10. Significance was analyzed using Student's T-TEST **P = 0.002 and ***P = 0.0006. Scale bars: 50 µm.E Measurement of cell surface areas. Cell surface area was measured in single cardiomyocytes (n = at least 30 measurements each group from N = 3 independent experiments each group) at day 60 post cardiac differentiation after immunostaining of sarcomeric a-actinin. Scale bars: 50 µm.