EMBO/SourceData huggingface dataset:
Figure 4percentage of mAb-positive live Pb-PfCSP sporozoites (C) for the junction cross-reactive mAb 1961, C-linker-specific mAb 3764 and the control mAbs 2A10 and mGO53 at the indicated concentrations (n=3-4).Representative mean fluorescence intensity (MFI) (D) for the junction cross-reactive mAb 1961, C-linker-specific mAb 3764 and the control mAbs 2A10 and mGO53 at the indicated concentrations (n=3-4).(E) Pf sporozoite hepatocyte traversal inhibitory capacity of the indicated C-CSP reactive, C-CSP cross-reactive (1961), and control (2A10, mGO53 and anti-NANP 317 mAbs at the indicated concentrations (n=3-6).(F) Capacity of the indicated passively-transferred antibodies (100 μg) to protect mice from parasitemia after the bite of three PbPfCSP(mCherry)-infected mosquitoes (n=10 for mAb 317 , n=8 for mAb 1961, and n=9 for mAb 1710 Data show the percentage of parasite-free mice in two independent experiments (only mice with detectable serum IgG concentrations of the indicated mAbs at the time of the challenge are shown Groups marked with the same letter were not statistically significantly different
Figure 5(A) Crystal structure showing the variable regions of the C-linker specific mAb 3764 and P2, with the mAb 3764 heavy chain illustrated in green and the kappa chain in blue. HCDR1, 2 and 3 (dark, pea and light green, respectively) as well as KCDR1 and 3 (dark teal and aqua, respectively) contribute to epitope recognition, but KCDR2 (teal) does not. The central DPN core (deep blue) of the C-CSP peptide (magenta) interacts with HCDR2 and 3 similarly to other VH3-33 antibodies (inset I), forming hydrogen bonds (H-bonds, shown with black dotted lines) with HCDR2 and 3. C-CSP residues M285 (inset II), N287 (inset III) and R291 (inset IV) contribute substantial buried surface area (BSA) to the complex. (II) M285 does not contribute any H-bonds, but does contribute to BSA. N287 (III) and R291 (IV) are responsible for hydrogen bonding with HCDR1 or HCDR3, KCDR1 and KCDR3 respectively.
Figure 1FLMycER cells, primed or not with OHT (48h), were treated with 135 nm IACS-010759 (IACS) for 48 hours (B) H2O2 quantification, expressed as fold-increase of the 405/488 nm fluorescence ratio in treated vs. untreated FLMycER cells, expressing either the cytoplasmic (left) or mitochondrial (right) roGFP2-ORP1 biosensor. Data information: * p ≤ 0.05 (one-way ANOVA). Each point in the graphs in B from an independent biological replicate, each representing the average of thousands of events (single cells) in a distinct cell population, normalized to the untreated condition.FLMycER cells, primed or not with OHT (48h), were treated with 135 nm IACS-010759 (IACS) for 48 hours (C) Superoxide anion O2•- production in treated vs. untreated FLMycER cells, based on dihydroethidium staining. Data information: * p ≤ 0.05 (one-way ANOVA). Each point in the graphs in B and C is from an independent biological replicate, each representing the average of thousands of events (single cells) in a distinct cell population, normalized to the untreated condition.
Figure 2FLMycER cells were primed with100 nM OHT (48h) and/or treated with 135nM IACS-010759, as indicated. (B) glutathione quantification in FLMycER cells, measured after 40 hours of IACS-010759 treatment. Data information: n=3 biological replicates; error bars: SD. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001 (one-way ANOVA).(C) Cell viability, as determined by PI staining after 48 hours of IACS-010759 treatment, with or without addition of 10 mM N-acetyl-cysteine (NAC). (D) Same as (C), with 50 µM buthionine-sulfoximine (BSO). Data information: n=3 biological replicates; error bars: SD. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001 (one-way ANOVA).(F) Immunoblot on lysates from FLMycER cells treated as indicated, with the addition of 10 mM NAC concomitantly with IACS-010759.
Figure 3FLMycER cells were primed with 100 nM OHT and treated with 135 nM IACS-010759, as indicated. (B) NADPH/NADP redox state in FLMycER cells, assayed after 40 hours of IACS-010759 treatment. n=3 biological replicates. ** p ≤ 0.01; **** p ≤ 0.0001 (one-way ANOVA).FLMycER cells were primed with 100 nM OHT and treated with 135 nM IACS-010759, as indicated. (E-G) Cell viability after 40 hours of IACS-010759 treatment in the presence of (E) 50 µM dehydroepiandrosterone (DHEA), (F) 10 µM 6- aminonicotinamide (6AN) or (G) 1 mM 2-deoxyglucose (2DG). See Supplemental Figure3F for detailed statistical analysis. Data information: In E,G) n=6; in (F) n=9; all biological replicates; error bars: SD.
Figure 4(A) Viability of FLMycER and BaFMycER cells primed or not with 100 nM OHT and treated with 135 nM IACS-010759 for 48h and/or with ascorbate at the indicated concentration for 6h. n=3 biological replicates; error bars: SD.(C) Cell viability of FL5.12cells at the end of treatment (48h IACS-010759,6h Asc), in the presence or absence of 50 µM deferoxamine (DFX, added 1 hour before Asc). n=3 biological replicates; error bars: SD.(D) Quantification of lipid peroxides in FL5.12 cells treated with 135 nM IACS-010759 for 24h and/ or 400 µM Asc for 3 hours. ** p ≤ 0.01; * p ≤ 0.05 (one-way ANOVA).
Figure 5The DLBCL cell lines DoHH2, SU-DHL-6, Karpas 422 and HBL-1 were treated with the indicated concentrations of IACS-010759 and ascorbate, and cell viability determined after 24 hours. (A) Live cell counts. n=3 biological replicates; error bars: SD.
Figure 6The indicated lymphoma cell lines and PDX samples were xenografted in recipient animals and tumors allowed to develop to detectable sizes prior to randomization and treatment with IACS-010759 (IACS) by oral gavage and/or ascorbate (Asc) by intraperitoneal injection, as indicated (see Materials and methods). Daily doses and the number of animals per group (n) are indicated in parenthesis. (A, B) Tumor progression (mm3) in CD1 nude mice bearing subcutaneous Ramos (A) or DoHH2 tumors (B). Data information: In each panel, the plot on the right shows the data for individual groups on the indicated day. Error bars: SD; n = animals per group. Right: one-way between-group ANOVA. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.The indicated lymphoma cell lines and PDX samples were xenografted in recipient animals and tumors allowed to develop to detectable sizes prior to randomization and treatment with IACS-010759 (IACS) by oral gavage and/or ascorbate (Asc) by intraperitoneal injection, as indicated (see Materials and methods). Daily doses and the number of animals per group (n) are indicated in parenthesis. (C, D) Tumor progression in NSG mice injected with the luciferase-positive DHL-derived PDX lines DFBL-20954 (C) or DFBL-69487 (D), as determined by in vivo imaging and quantification of bilateral femur radiance efficiency. Examples from the control and double-treated groups are shown at the bottom. Data information: In each panel, the plot on the right shows the data for individual groups on the indicated day. Error bars: SD; n = animals per group. Right: one-way between-group ANOVA. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.
Figure 1  Bovine heart mitochondria were incubated at the indicated pH in absence or presence of 200 μM DPC, treated with 1% (w/v) digitonin and F-ATP synthase was immunoprecipitated with an anti-F-ATP synthase Ab followed by 15% SDS-PAGE. β subunit and CyPD content were detected by Western blotting. The ratio between CyPD and the corresponding β subunit is reported in the lower part of the panel, referring to pH 7.4 values as 100%. Data are an average ± s.e. of 3 independent experiments.  B,C EDTA-SMP were solubilized at the indicated pH values, extracted with 1% (w/v) digitonin and subjected to BN-PAGE in order to separate dimers (Vd) and monomers (Vm), which were identified by Coomassie blue (panel B) or in-gel activity staining (panel C) and analyzed by densitometry, which is reported in the bottom part of each panel. Values report the dimer/monomer ratio, where the ratio at pH 7.4 was taken as 100%. Data are an average ± s.e. of 3 independent experiments. The p-values calculated with the Student t test are shown * p≤0.05 *** p≤0.001.D Sequence of peptides 95-113 of OSCP before (left panel) and after reaction with DPC (right panel) obtained from tryptic digests of the SDS-PAGE band corresponding to the expected molecular mass of OSCP in immunoprecipitated F-ATP synthase. Fragments of the series b and y identified in the LC-MS/MS analysis are indicated on the sequence of the peptides. H112 and H112CeT are indicated in red boldface. Ions y4-y17 show a mass shift of +72 Da in the modified peptide.
Figure 1C, Diarylquinoline and SQ31f inhibition of ATP hydrolysis by purified ATP synthase bearing truncated α subunits. Mean relative activity (filled symbols) ± s.d from n=3 separate assays (empty symbols) for each of four different inhibitors (triangles, circles, diamonds, and squares).D, Inhibition of ATP hydrolysis activity in M. smegmatis IMVs containing ATP synthase with truncated α subunits. Initial ATP hydrolysis activity of the ATP synthase is defined as the activity that is sensitive to 100 μM DCCD. Mean relative activity from n=3 separate assays is shown.E, Uncoupling activity of diarylquinoline analogs in M. smegmatis IMVs monitored with ACMA fluorescence. Each compound was assayed separately, and the initial fluorescence was set to 1.0. RFU: relative fluorescence units.
Figure 3A, Uncoupling activity of SQ31f in M. smegmatis IMVs monitored with acridine orange fluorescence.B, SQ31f inhibition of ATP hydrolysis by purified ATP synthase with truncated α subunits (blue curve) occurs at higher concentrations than inhibition of ATP synthesis by M. smegmatis IMVs (purple curve). Mean relative activity (filled symbols) ± s.d from n=3 separate assays (triangle and circles).
Figure 1(D) Using the dual color labeling system (esg-Gal4>UASp-FRT-H3-eGFP-FRT-H3-mCherry), old and new H3 distribution in prophase and prometaphase ISCs shows large, separable domains of H3-eGFP (old) and H3-mCherry (new). Note: all figure panels in this work are maximum intensity projection images to show signals from all Z-stacks. (E) Using the UASp-FRT-histone-eGFP-T2A-histone-mCherry-PolyA transgene that co-expresses H3-eGFP and H3-mCherry, significant overlap between the two signals were detected in ISCs during prophase and prometaphase. (F) Quantification of the colocalization between old and new H3, as well as between the co-expressed eGFP- and mCherry-tagged H3 in prophase and prometaphase ISCs. Pearson's correlation coefficients were measured, where 1 represents complete colocalization and 0 stands for no colocalization. Old and new H3 showed significantly less colocalization (Pearson's correlation coefficient for H3 = 0.44 + 0.02, n = 81 ISCs) when compared to the co-expressed eGFP- and mCherry-tagged H3 (Pearson's correlation coefficient for H3 co-expressed = 0.86 + 0.01, n = 19 ISCs). Individual data points and mean values are shown. Error bars represent SEM. **** p < 0.0001; unpaired t test to compare two individual datasets to each other. Data information: n for individual ISCs. Scale bar in (D), (E): 5 µm.
Figure 2(A) Old and new H4 distribution in prophase and prometaphase ISCs (esg-Gal4>UASp-FRT-H4-eGFP-FRT-H4-mCherry). Separable domains of H4-eGFP (old) and H4-mCherry (new) were observed (B) Old and new H2A distribution in prophase and prometaphase ISCs (esg-Gal4>UASp-FRT-H2A-eGFP-FRT-H2A-mCherry), showing largely overlapping signals between H2A-eGFP (old) and H2A-mCherry (new). Data information: n for individual ISCs. Scale bar in (A), (B) 5 µm.(D) Late-stage mitotic ISC showing asymmetric segregation of H3 between the two sets of sister chromatids at the opposite poles of the ISC. (E) Late-stage mitotic ISC showing less asymmetric segregation of H4, compared to H3, between the two sets of sister chromatids at the opposite poles of the ISC. (F) Late-stage mitotic ISC showing symmetric segregation of H2A between the two sets of sister chromatids at the opposite poles of the ISC. Data information: n for individual ISCs. Scale bar in (D), (E), and (F): 5 µm.
Figure 3(A) Old and new H3 distribution in a post-mitotic pair of cells with asymmetric Dl-nLacZ labeling, showing that H3-eGFP (old) is asymmetrically inherited by the Delta-high cell, while H3-mCherry (new) is enriched in the Delta-low cell (esg-Gal4>UASp-FRT-H3-eGFP-FRT-H3-mCherry). (B) Old and new H3 distribution in a post-mitotic pair of cells with symmetric Dl-nLacZ labeling, showing that both H3-eGFP (old) and H3-mCherry (new) are more symmetrically distributed between the two cells. Scale bar in (A), (B), 5 µm; asterisk, ISC side.(E) Old and new H4 distribution in a post-mitotic pair of cells with asymmetric Dl-nLacZ labeling, showing that H4-eGFP (old) is asymmetrically inherited by the Delta-high cell, while H4-mCherry (new) is enriched toward the Delta-low cell (esg-Gal4>UASp-FRT-H4-eGFP-FRT-H4-mCherry). (F) Old and new H4 distribution in a post-mitotic pair with symmetric Dl-nLacZ labeling, showing that H4-eGFP (old) and H4-mCherry (new) are symmetrically distributed between the two cellss. Scale bar in (E), (F), 5 µm; asterisk, ISC side.(I) Old and new H2A distribution in a post-mitotic pair of cells with asymmetric Dl-nLacZ labeling, showing that H2A-eGFP (old) and H2A-mCherry (new) are symmetrically inherited by the two cells (esg-Gal4>UASp-FRT-H2A-eGFP-FRT-H2A-mCherry). (J) Old and new H2A distribution in a post-mitotic pair of cells with symmetric Dl-nLacZ labeling, showing that H2A-eGFP (old) and H2A-mCherry (new) are symmetrically distributed between the two cells. Scale bar in (I), (J), 5 µm; asterisk, ISC side.(M) The ISC-specific combination of drivers esg-Gal4, Su(H)-Gal80 drive H3 transgene [esg-Gal4, Su(H)-Gal80>UASp-FRT-H3-eGFP-FRT-H3-mCherry] with heat shock treatment and recovery (see Materials and Methods) in a Delta-asymmetric pair of cells. Old and new H3 distribution in a post-mitotic pair of cells with asymmetric Dl-nLacZ labeling, showing that H3-eGFP (old) is asymmetrically inherited by the Delta-high cell, while H3-mCherry (new) is enriched in the Delta-low cell. (N) ISC specific drivers express the H3 transgene in a Delta-symmetric pair of cells. Old and new H3 distribution in a post-mitotic pair of cells with symmetric Dl-nLacZ labeling, showing that both H3-eGFP (old) and H3-mCherry (new) are more symmetrically distributed between the two cells. Scale bar in (M), and (N): 5 µm; asterisk, ISC side.
Figure 5(A) A view of the midgut of an UAS-H3-mCherry transgene driven by esg-Gal4, Su(H)-Gal80, tub-Gal80ts at the Gal80ts permissive temperature (18°C), showing no expression of the histone transgene or the UAS-eYFP reporter that marks ISCs. (B) A view of the midgut of an UAS-H3-mCherry transgene driven by esg-Gal4, SuH-Gal80, tub-Gal80ts at the Gal80ts restrictive temperature (29°C), showing expression of the H3-mCherry transgene (red) and the UAS-eYFP reporter (green). (C) A view of the midgut of an UAS-H3T3A-mCherry transgene driven by esg-Gal4, SuH-Gal80, tub-Gal80ts at the Gal80ts permissive temperature (18°C), showing no expression of the histone transgene or the UAS-eYFP reporter. (D) A view of the midgut of an UAS-H3T3A-mCherry transgene driven by esg-Gal4, SuH-Gal80, tub-Gal80ts at the Gal80ts restrictive temperature (29°C), showing expression of the H3T3A-mCherry transgene (red) and the UAS-eYFP reporter (green). Data information: Scale bars in (A), (B), (C), and (D): 50 µm(I) Comparison of the mitotic index in H3-expressing (N = 11 midguts) and H3T3A-expressing (N = 11 midguts) midguts. Total number of mitotic ISCs were quantified per midgut by the presence of H3S10 phosphorylation mark. Bars represent mean number of mitotic cells per midgut. Error bars represent SEM. H3T3A-expressing midguts had a significantly higher mitotic index when compared to H3-expressing midguts (two sample t test, * p < 0.05).(J) Qualitative analysis of midgut phenotypes. No phenotype is called when the ISCs appear normally distributed based on eYFP reporter signal. A mild phenotype is called when there is a small region of eYFP-positive cells clustered within a region, showing mild disorganization. A strong phenotype is called when there is a large region of eYFP-positive cells clustered, showing significant disorganization. (K) Percentage of H3- expressing midguts (N = 11 midguts) and H3T3A- expressing midguts (N = 11 midguts) with no phenotype, a mild phenotype, or a strong phenotype. Data information: Scale bars in (J): 500 µm.
Figure 6(A) A view of the midgut of an UAS-H3-mCherry transgene driven by esg-Gal4, SuH-Gal80, tub-Gal80ts at the Gal80ts restrictive temperature (29°C), showing expression of the H3-mCherry transgene (grey) and the UAS-eYFP reporter (green) from the driver as well as expression of the Su(H)-LacZ reporter line (magenta). eYFP and Su(H)-LacZ signals are organized and separable. (B) A view of the midgut of an UAS-H3T3A-mCherry transgene driven by esg-Gal4, SuH-Gal80, tub-Gal80ts at the Gal80ts restrictive temperature (29°C), showing expression of the H3T3A-mCherry transgene (grey) and the UAS-eYFP reporter (green) from the driver as well as expression of the Su(H)-LacZ reporter line (magenta). eYFP and Su(H)-LacZ signals are disorganized and overlapping. Data information: Scale bars in (A) and (B): 50 µm.
Figure 1(B) Immunogold electron micrograph of GFP or ORF6-GFP expressed HeLa cells. Blue arrows mark ORF6-GFP dots are enriched on the surface of LDs. Green arrows mark GFP dots are distributed in the cytoplasm. Scale bar represents 500 nm.(C, D and E) HeLa cells stable expressing ORF6-Flag were treated with or without 200 μM OA for 12 h, then fixed and stained with anti-Flag (red). Initial LDs were labeled with GFP-GPAT4152-208 (green), mature LDs were labeled with BODIPY-493/503 (green) or GFP-Plin2 (green). Cells were imaged by confocal microscopy. Scale bar represents 10 μm.
Figure 2(B) Analysis of the homodimerization of ORF6 by cross-linking with DSS. HEK293T cells expressing ORF6-Flag were treated with 0, 0.1, 0.2 and 0.3 mM DSS for 30 min. Cell lysates were analyzed via WB.(D) The indicated Flag tagged ORF6 or its mutants were co-expressed in HEK293T cells with HA tagged ORF6. Protein interactions were analyzed by immunoprecipitation with anti-Flag beads and immunoblotting analysis.(E) Purified GST-ORF6 or indicated mutants were incubated with purified His tagged ORF6, and analysis of the self-interaction by GST pull-down.(F) HEK293T cells were transfected with ORF6-Flag or ΔNTD-Flag or ΔAH1-Flag or ΔAH2-Flag and were treated with 0.1 mM DSS for 30 min. Cell lysates were analyzed via WB.(G) HeLa cells were transfected with ORF6-Flag or indicated mutants and were treated with 200 μM OA for 12 h, and then fixed and stained with anti-Flag (red). LDs were labeled with BODIPY-493/503 (green). Cells were imaged by confocal microscopy. Scale bar represents 10 μm.
Figure 3(A) HeLa cells were transfected with vector or ORF6-Flag for 24 h, and then treated with 200 μM OA for indicated times. Cells were fixed and stained with anti-Flag (green). LDs were labeled with LipidTox Deep Red (red). The nuclei were stained with DAPI. Cells were imaged by confocal microscopy. Scale bar represents 10 μm. White ROIs indicate cells expressing ORF6 and yellow ROIs indicate the cells without ORF6 expression.(D) HeLa cells stable expressing vector or ORF6-Flag were treated or untreated with 50 μM ATGL inhibitor (ATGLi) for 24 h, and then were fixed and stained with anti-Flag (red). LDs were labeled with BODIPY-493/503 (green). Cells were imaged by confocal microscopy. Scale bar represents 10 μm. (E) Mean number of LDs in each cell in (D) was counted from 25 cells of three independent experiments. Two-tailed Unpaired Student's t-test, ***p < 0.001, ****p < 0.0001. Error bars represent the mean ± SD.(H) HeLa cells stable expressing vector or ORF6-Flag were treated or untreated with 50 μM ATGL inhibitor, along with DMSO or 1 μM DGAT1 inhibitor (DGAT1i) or/and 2 μM DGAT2 inhibitor (DGAT2i) for 24 h. LDs were labeled with BODIPY-493/503 (green). Cells were imaged by confocal microscopy. Scale bar represents 10 μm. (I) Mean number of LDs in each cell in (H) was counted from 35 cells of three independent experiments. Two-tailed Unpaired Student's t-test, ***p < 0.001, ****p < 0.0001, ns means no significance. Error bars represent the mean ± SD.(J) SARS-CoV-2-infected Vero-E6 cells were treated with 1 μM DGAT1 inhibitor or/and 2 μM DGAT2 inhibitor for 24 h. Cell viability was analyzed. Cell lysates were analyzed via WB. Supernatants were determined by plaque assays. Virus titer values represent mean ± SD for three independent experiments. Two-tailed Unpaired Student's t-test, ***p < 0.001, ns means no significance. Error bars represent the mean ± SD.
Figure 4(A) Subcellular fractions were isolated from GFP or ORF6-GFP over-expressed cells. Calnexin represent the ER, Plin2 represent LDs, Rab11 represent endosomes, VDAC represent mitochondria, and Tubulin represents cytoplasm, respectively.(C) Representative transmission electron micrograph of non-infected or SARS-CoV-2 infected Vero-E6 cells. Red arrows mark the ER. LD, lipid droplets. Scale bar represents 1 μm. Quantification of number of ER engaged in ER-LD contact per LD. 20 LDs of two independent experiments were counted. Two-tailed Unpaired Student's t-test, ****p < 0.0001. Error bars represent as the mean ± SD.(E) ORF6-Flag or the mutants were co-expressed with mCheey-RAMP4. Cells were treated with 200 μM OA for 12 h, and then fixed and stained with anti-Flag (red). The ER was visualized with mCheey-RAMP4 (red). LDs were labeled with LipidTOX Deep Red (white). Cells were imaged by confocal microscopy. Scale bar represents 10 μm.(J) HeLa cells stable expressing ORF6-Flag were transfected with si-BAP31 and/or si-USE1 for 24 h, and were further treated with 50 μM ATGL inhibitor for 24h. Cells were fixed and stained with anti-Flag (red). LDs were labeled with BODIPY-493/503 (green). The nuclei were stained with DAPI. Cells were imaged by confocal microscopy. Scale bar represents 10 μm. (K) Mean number of LDs in each cell in (J) was counted from 25 cells of three independent experiments. Two-tailed Unpaired Student's t-test, *p < 0.05, **p < 0.01. Error bars represent the mean ± SD.(L) SARS-CoV-2-infected Vero-E6 cells were transfected with control, or si-BAP31 and/or si-USE1 for 48 h. Cell lysates were analyzed via WB. Supernatants were determined by plaque assays. Virus titer values represent mean ± SD for three independent replicates. Two-tailed Unpaired Student's t-test, ****p < 0.0001, ns means no significance. Error bars represent the mean ± SD.