EMBO/SourceData huggingface dataset:
Figure 2(A) At 48 hours post transfection of the indicated protease plasmids in triplicate, BHK21 cells were incubated with a fluorogenic substrate and wtSPINK6 or loss-of-function mutSPINK6 or PBS for 2 hours and then applied to fluorescence assay. Data represent mean and SD of triplicated wells in a representative experiment performed 3 times. **, P<0.01. Student's t test.(B) At 48h after co-transfection of the indicated protease plasmids and H7 or H1 plasmid, BHK21 cells were lysed to examine HA cleavage with WB.(E) At 48h after triple-transfection of H1, the indicated proteases and wtSPINK6 or mutSPINK6, BHK21 cells were lysed for the detection of HA cleavage.
Figure 3(A) At 36h after transfection of the indicted protease and wtSPINK6 plasmids or blank vector in triplicate, BHK21 cells were inoculated with H7N9/ah at a MOI of 0.5. Cell-free culture media were harvested at 24 hpi for viral titration. Data represent the mean and SD of the triplicated wells in a representative experiment performed 3 times. **, P<0.01. Student's t test.(B) At 24h after transfection of HAT or KLK5 plasmid in sextuplicate, A549 cells were inoculated with H1N1/pdm at MOI of 0.25. The infected cells were incubated with recombinant wtSPINK6 or mutSPINK6 protein in triplicate for 24h. Culture media in each well were stored in two aliquots, one was applied to viral titration with conventional plaque assay (black bars), the other was pre-treated with TPCK-trypsin for 1 hour prior to plaque assay (grey bars). Data represent the mean and SD of the triplicated wells in a representative experiment performed 3 times. *, P<0.05; **, P<0.01; ***, P<0.001. Student's t test.
Figure 4(A) Confocal imaging (left) shows intrinsic SPINK6 (green) expression in the epithelial cells in 2D human airway organoids. Nuclei and actin filaments are counterstained and shown as blue and purple respectively. Scale, 10 µm. Flow cytometry analysis demonstrates HAT and SPINK6 expression in human airway organoids. The plots (middle) show the representative result of one experiment performed 3 times. The percentages of HAT and SPINK6 positive cells (right) are shown as mean and SD of 3 independent experiments. Data information: *, P<0.05; **, P<0.01; ***, P<0.001. Student's t test.(B) At 24h after inoculation of H1N1/pdm or mock inoculation in triplicate, 2D human airway organoids were harvested to detect mRNA expression levels of the indicated genes normalized with GAPDH. Data represent the mean and SD of a representative experiment performed three times. Data information: *, P<0.05; **, P<0.01; ***, P<0.001. Student's t test.(E) 2D airway organoids were applied to fluorescence assay after incubation with α-SPINK6 or isotypic IgG in triplicate for 4 hours (left). Data present mean and SD of triplicated samples (right). Data present the mean and SD of triplicated samples. (F) After inoculation of H1N1/pdm, 2D human airway organoids were incubated with an α-SPINK6 or isotype IgG in triplicate for 24 hours (left). Cell-free culture media were harvested for viral load detection (right) and examination of HA cleavage by WB after 10-fold concentration (middle). Viral load data present the mean and SD of triplicated samples. Data information: *, P<0.05; **, P<0.01; ***, P<0.001. Student's t test.
Figure 5(B) Survival rates of mice treated with wtSPINK6 and mutSPINK6 was analyzed with Mantel-Cox test.(C) Viral load and viral titer in the lung homogenates of mice treated with wtSPINK6 and mutSPINK6. Data represent mean ± SD. **, P < 0.01; ***, P< 0.001. Student's t test.(D) Mouse lung tissues are applied to immunofluorescence staining to identify the viral NP positive cells (green). Nuclei are counterstained with DAPI (blue). Representative confocal images of virus-infected cells in the indicated mice. Scale bar, 10 µm.
Figure 6(A) The association SNP rs1432689 genotypes are correlated to differential SPINK6 mRNA expression in over 1000 human lung tissues from three centers. The box denotes interquartile range; the thick line and diamond within the box are the median and mean respectively; whiskers are the minimum and maximum and open dots are outliers. Linear regression was used for eQTL data analysis. Data of three cohorts were applied to meta-analysis and resulted in the P-value of the association SNP.(B) rs1432689 genotypes are correlated to SPINK6 expression in human lung tissues and esophagus mucosa from GTEx database. Sample sizes for each genotype are shown in the x-axis. The y-axis shows the normalized expression of SPINK6 (Norm. expression) in the indicated trusses. Median and interquartile range are represented by a white line and dark box respectively. Data distribution is colored with light blue; a wider section of the violin plot indicates the data on the section have a higher frequency. Linear regression was used for data analysis.
Figure 1A Electron microscopy analyses to see autophagic bodies in the vacuoles under both growth and nitrogen starvation conditions. Autophagic bodies typically contain cytosolic ribosomes. pep4∆prb1∆ and pep4∆prb1∆atg2∆ cells were grown in SD to mid‐log phase (OD600 = 1) and transferred to SD(‐N) for 5 h. The cells were examined by transmission electron microscopy as described in . Scale bar, 500 nm.B-D Time‐dependent changes in nucleoside contents under nitrogen starvation (B), rapamycin treatment (C), and carbon starvation (D) conditions. Wild‐type and atg2Δ cells were grown in SD and transferred to SD(‐N), SD medium with 0.2 μM rapamycin, or SD(‐C) at time 0. At the indicated time points, nucleosides were analyzed by LC/MS as described in . Results are presented as normalized intensities on the basis of peak height of each metabolite in wild‐type cells. All data are means of triplicate samples. The error bars represent the standard deviation.
Figure 2A Time‐dependent changes in nucleoside contents under nitrogen starvation. Nucleosides in wild‐type, atg2Δ, atg7Δ, atg11Δ, atg17Δ, atg19Δ, atg32Δ, nvj1Δ, and pep4prb1Δ cells under nitrogen starvation. All data are means of triplicates. The error bars represent the standard deviation.B Alkaline phosphatase (ALP) assay (Pho8∆60). Wild‐type, atg2∆, atg11Δ, atg17Δ, and atg19Δ cells expressing Pho8∆60 were grown in SD to mid‐log phase and transferred to SD(‐N) at time 0. At the indicated time points, lysates were prepared and subjected to the ALP assay. The bars represent the standard deviation of three independent experiments. *P 0.05; **P 0.005; ***P 0.0005 (paired t‐test, two‐tailed)
Figure 3A-C Wild‐type, atg2Δ, pho8Δ, and rny1Δ cells were grown in SD to mid‐log phase and transferred to SD(‐N) at time 0. At the indicated time points, nucleosides (A) were analyzed by LC/MS as described in . The results are presented as normalized intensities on the basis of the peak height of each metabolite in wild‐type cells. Note that 3′‐NMPs in pho8Δ are presented in a different scale. All data are means of triplicates. The error bars represent the standard deviation.A-C Wild‐type, atg2Δ, pho8Δ, and rny1Δ cells were grown in SD to mid‐log phase and transferred to SD(‐N) at time 0. At the indicated time points, 5′‐NMPs (B) were analyzed by LC/MS as described in . The results are presented as normalized intensities on the basis of the peak height of each metabolite in wild‐type cells. Note that 3′‐NMPs in pho8Δ are presented in a different scale. All data are means of triplicates. The error bars represent the standard deviation.A-C Wild‐type, atg2Δ, pho8Δ, and rny1Δ cells were grown in SD to mid‐log phase and transferred to SD(‐N) at time 0. At the indicated time points, 3′‐NMPs (C) were analyzed by LC/MS as described in . The results are presented as normalized intensities on the basis of the peak height of each metabolite in wild‐type cells. Note that 3′‐NMPs in pho8Δ are presented in a different scale. All data are means of triplicates. The error bars represent the standard deviation.
Figure 4A, B Expression and localization of Rny1 and Pho8. Wild‐type cells and atg2Δ cells expressing Rny1‐GFP or Pho8‐GFP were grown in SD to mid‐log phase and transferred to SD(‐N). After 2 h of starvation, GFP‐tagged proteins were observed by fluorescence microscopy (A). Scale bar, 5 μm.A, B Expression and localization of Rny1 and Pho8. Wild‐type cells and atg2∆ cells expressing Rny1‐GFP or Pho8‐GFP were grown in SD to mid‐log phase and transferred to SD(‐N). After 2 h of starvation, GFP‐tagged proteins were analyzed by Western blot with anti‐GFP antibody (B).C Detection of free RNA. Wild‐type, atg2∆, rny1∆, and rny1∆atg2∆ cells grown in SD (left) or SD(‐N) for 2 h (right) were stained with FM4‐64 and GR Green and observed by fluorescence microscopy. Scale bar, 5 μm.
Figure 5A Time‐dependent change in nucleoside levels after nitrogen starvation. Wild‐type, atg2∆, ubp3∆, and bre5∆ cells were grown in SD to mid‐log phase and transferred to SD(‐N) at time 0. Metabolites were analyzed by LC/MS as described in . The results are presented as normalized intensities on the basis of the peak height of each metabolite in wild‐type cells. All data are means of triplicates. The error bars represent the standard deviation.B Detection of free RNA within cells. rny1∆, rny1∆atg2∆, rny1∆ubp3∆, and rny1∆bre5∆ cells grown in SD(‐N) for 2 h were stained with FM4‐64 and GR Green and observed by fluorescence microscopy. Scale bar, 5 μm.
Figure 6B, C Nucleosides (B) and nucleobases (C) within the cells. Wild‐type and atg2∆ were grown in SD to mid‐log phase and transferred to SD(‐N) at time 0. At the indicated time points, the cells were collected; their metabolites were extracted, and the nucleosides and nucleobases were analyzed by LC/MS. The level of each nucleoside and base is presented as its absolute concentration, as described in and Supplementary Methods. All data are means of duplicates except the wild‐type strain at 1 h.D, E Nucleosides (D) and nucleobases (E) in the medium. The culture medium of the above cultures was filtered, and nucleosides and nucleobases were directly analyzed by LC/MS as described in and Supplementary Methods. All data are means of duplicates.F, G Destinations of nucleosides and bases. Based on the data from (B-E), the level of each nucleoside and base in cells (open circle) and in cells and medium (closed circle) of wild‐type strain were estimated. See Supplementary Methods in detail (flow chart of the calculation procedure).
Figure 7Wild‐type and atg1Δ of S. pombe were grown in SD to mid‐log phase and transferred to SD(‐N) at time 0. At the indicated time points, the cells were collected; the metabolites in these cells were extracted and analyzed by LC/MS as described in . The results are presented as normalized intensities on the basis of peak height of each metabolite in wild‐type cells. All data are means of triplicates. The error bars represent the standard deviation.
Figure 1B, C GOT2 can be acetylated. Flag‐tagged GOT2 was ectopically expressed in HEK293T cells treated with NAM (5 mM) and/or TSA (0.5 mM) for the indicated time period. Acetylation levels of Flag‐bead‐purified GOT2 were determined by Western blot analysis using a pan‐anti‐acetyllysine antibody (α‐Ac). IB and IP denote immunoblotting and immunoprecipitation, respectively. Relative GOT2 acetylation ratios were calculated after normalizing against Flag.D GOT2 acetylation enhances the interaction between ectopically expressed GOT2 and MDH2. Flag‐GOT2 and Myc‐MDH2 were co‐overexpressed in HEK293T cells treated without or with NAM (5 mM) for the indicated time period. The acetylation level of Flag‐bead‐purified GOT2 and the protein association between ectopic proteins of GOT2 and MDH2 were determined by Western blot analysis.E Acetylation enhances the interaction between ectopically expressed GOT2 and endogenous MDH2. Flag‐GOT2 was co‐overexpressed in HEK293T cells treated without or with NAM (5 mM) for the indicated time period. The acetylation level of Flag‐GOT2 and its association with endogenous MDH2 were determined by Western blot analysis.F Acetylation enhances the interaction between ectopically expressed MDH2 and endogenous GOT2. Flag‐MDH2 was co‐overexpressed in HEK293T cells treated without or with NAM (5 mM) for the indicated time period. The protein association between Flag‐MDH2 and endogenous GOT2 was determined by Western blot analysis.H Mapping the major lysine residue(s) of acetylation in GOT2 whose acetylation can affect protein interaction between GOT2 and MDH2. Putative acetylated residues were divided into six groups according to their position in the structure of GOT2. Each group of putative acetylated lysine (K) sites was mutated to arginine (R), and the deacetylated mimic K‐to‐R mutants were examined for their protein association with ectopically expressed Myc‐MDH2 by Western blot analysis.I GOT2 coupled with MDH2 shows a high level of K159 acetylation. Flag‐GOT2 was overexpressed in HEK293T cells without or with co‐overexpression of Myc‐MDH2. These transfected cells were treated with NAM (5 mM) for 5 h. Double immunoprecipitation was performed to obtain the Flag‐GOT2 from the Myc‐MDH2 immunoprecipitates (Ppt, supposed to be GOT2 bound with MDH2). The remaining supernatants (Sup, supposed to be GOT2 not bound with MDH2) were also harvested and precipitated by Flag antibody to test the K159 acetylation level of Flag‐GOT2 by Western blot analysis.J The 3KQ/R mutations do not change GOT2 enzyme activity. Wild‐type and 3K mutant GOT2 proteins were overexpressed and purified from E. coli, and the enzyme activity of GOT2 was determined as described in "". Shown are average values with standard deviation (SD) of triplicated experiments. n.s. = not significant for the indicated comparison.
Figure 2A, B Glucose and glutamine increase GOT2 K159 acetylation and GOT2-MDH2 association in HEK293T cells. Flag‐GOT2 and Myc‐MDH2 were overexpressed in cells treated with increased concentrations of glucose (A) or glutamine (B) for 6 h. GOT2 proteins were purified by Flag beads, and the K159 acetylation level of GOT2 and its protein association with Myc‐MDH2 were determined by Western blot analysis.C, D Glucose and glutamine increase GOT2 K159 acetylation in Panc‐1 cells. Flag‐GOT2 was overexpressed in cells treated with different concentrations of glucose (C) and glutamine (D) for 4 h. GOT2proteins were purified by Flag beads, and the K159 acetylation level of GOT2 was determined by Western blot analysis. Relative GOT2 K159 acetylation levels were normalized against Flagprotein levels.E, F Quantification of the percentage of K159‐acetylated endogenous GOT2 in Panc‐1 cells. Recombinant fully K159‐acetylated GOT2 was loaded onto the same gel, together with endogenous GOT2 from Panc‐1 cells treated without or with glucose (12 mM) (E) or glutamine (2 mM) (F) for 4 h. GOT2 protein and K159 acetylation were detected by Western blot. The percentages of K159 acetylation in GOT2 were calculated after normalizing against GOT2 protein levels.G, H 3K mutant GOT2 displays negligible response in changing protein association with MDH2 after glucose or glutamine treatment. Panc‐1 cells with GOT2 knockdown and re‐expression of wild‐type or 3K mutant GOT2 were treated with glucose (G) or glutamine (H) at the indicated concentrations for 4 h. The protein association between Flag‐tagged wild‐type or 3K mutant GOT2 and endogenous MDH2 was determined by Western blot analysis.
Figure 3A GOT2 interacts with SIRT3, but not SIRT4 and SIRT5. Flag‐tagged GOT2 was ectopically expressed in HEK293T cells together with the individual HA‐tagged SIRT as indicated. Proteins were purified by IP with Flag beads, following Western blot to detect SIRTs with an HA antibody.B Endogenous proteins of GOT2 and SIRT3 interact with each other. SIRT3 protein in HEK293T cells was purified by IP with an anti‐SIRT3 antibody, following Western blot to detect GOT2 with an anti‐GOT2 antibody.C GOT2 deacetylation is dependent on SIRT3 catalytic activity. Flag‐tagged GOT2 was ectopically expressed in HEK293T cells together with HA‐tagged wild‐type SIRT3 and a catalytically inactive mutant, SIRT3 H248Y. GOT2 proteins were purified by Flag beads, following Western blot to detect GOT2 K159 acetylation.D SIRT3 impairs the protein interaction between GOT2 and MDH2. HEK293T cells were transfected with the plasmids as indicated. GOT2 proteins were purified by Flag beads, and their protein interaction with Myc‐MDH2 was determined by Western blot.E Knockdown of SIRT3 increases GOT2 K159 acetylation and enhances GOT2-MDH2 association. HEK293T cells with or without transient SIRT3 knockdown were transfected with the plasmids as indicated. GOT2 proteins were purified by Flag beads, and the K159 acetylation level of GOT2 and its protein association with endogenous MDH2 were determined by Western blot analysis.F, G Knockdown of SIRT3 diminishes the effect of glucose or glutamine on changing GOT2 K159 acetylation. Flag‐tagged GOT2 was ectopically expressed in HEK293T cells without or with transient SIRT3 knockdown. These cells were treated with different concentrations of glucose (F) and glutamine (G) as indicated. GOT2 proteins were purified by Flag beads, and the K159 acetylation level of GOT2 was determined by Western blot analysis.H, I Up‐regulation of Sirt3 leads to decreased Got2 K159 acetylation in mouse tissues during fasting. C57BL/6 mice (male, 20 weeks; n = 2 per group) were fed a normal chow diet or fasted overnight. Upon sacrifice, mouse liver and white adipose tissues were harvested, and the levels of the indicated proteins were determined by Western blot analysis.
Figure 4A The malate-aspartate shuttle, such as LDH, also plays a key role in regulating cytosolic NADH levels in Panc‐1 cells. Cells were treated with AOA (1.6 mM, 20 min), oxamate (5 mM, 20 min), and EGCG (0.5 mM, 20 min). The cytosolic NADH level was measured as described in .B, C GOT2 3K acetylation promotes the net transfer of cytosolic NADH into mitochondria. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, the NADH level in the cytosol (B) and mitochondrion (C) was determined in cell extracts as described in .D GOT2 3K acetylation regulates mitochondrial NADH/NAD+ redox in the cell. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, the ratio of NADH/NAD+ in the mitochondria of cells was measured as described in .E GOT2 3K acetylation promotes ATP production. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, ATP production was determined in cell extracts as described in .F GOT2 3K acetylation promotes NADPH production. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, the ratio of NADPH/NADP+ was determined in cell extracts as described in .G GOT2 3K acetylation promotes GSH production. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, the GSH/GSSG ratio was determined in cell extracts as described in .H GOT2 3K acetylation suppresses cellular ROS levels. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, ROS was determined as described in in cells under non‐stressed condition or exposed to menadione (50 μM for 30 min).I GOT2 3K acetylation protects cells from oxidative damage. Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins were treated with increasing concentrations of menadione for 3 h as indicated, and cell viability was determined by counting the remaining adherent cells.J, K Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins were seeded in a 6‐well plate, and the cells were maintained under the condition of low glucose (0.5 mM, J) or high glucose (12 mM, K). Culture medium was refreshed every day, and cell numbers were counted every 1-2 days over a period of 7 days.L, M Panc‐1 stable cells in (J, K) were grown under low glucose (0.5 mM) or high glucose (12 mM) for 7 days. The level of histone H3 phosphorylation at Ser10 was determined by Western blot analysis (L). The percentages of living cells and apoptotic cells were determined by flow cytometry as described in (M).
Figure 5A-C GOT2 3K acetylation promotes xenograft tumorgrowth. Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins (2 × 106cells) were injected subcutaneously into the flanks of nude mice. The tumor volume was carefully monitored over the indicated time period (A). At 8 weeks after injection, tumors from six mice were extracted, photographed, and weighted (B, C).D GOT2 3K acetylation promotes tumor cellproliferation. Tumor sections from xenografts were prepared for IHC staining, and the PCNA‐stained tumor sections were analyzed by quantifying the PCNA‐positive area.E Clinical cases with increased GOT2 K159 acetylation in humanpancreatictumor tissues. 10 pairs of humanpancreatictumor tissue (shown as T) and adjacent normal tissue (shown as N) were lysed. The acetylation level of GOT2 at K159 was compared against GOT2protein by Western blot.F Clinical cases with decreased SIRT3protein expression in humanpancreatictumor tissues. 10 pairs of humanpancreatictumor tissue (shown as T) and adjacent normal tissue (shown as N) were lysed. The SIRT3protein levels were compared against tubulin by Western blot.G, H Quantification of GOT2 K159 acetylation and SIRT3protein expression in clinical cases as described in (E) and (F).
figf1(a) Subcellular compartments from U937 cell extracts were separated by sedimentation velocity on discontinuous sucrose gradient and probed for the indicated proteins by immunoblotting. Numbers represent fractions from the gradient (1, top; 5, bottom).(b) Immunoblot analysis of cellular compartments after purification of mitochondria by Qproteome Mitochondria Isolation Kit.(c) Immunoblot analysis of KDEL and IRGM distribution in different cellular fractions after purification of mitochondria as in b.d) Intracellular localization of IRGM analysed by confocal microscopy. Cells were stained with antibodies against the indicated proteins or vitally with MitoTracker Red (MTR) where indicated. Images on the right are merged from images on the left. A-L; endogenous IRGM localization in HeLa cells relative to calnexin, MTR, cytochrome c and COX IV. M-O; IRGM localization in primary human macrophages relative to mitochondria visualized with cytochrome c antibody. Scale bars, 5 μm.(e) Analysis of IRGM (green) and COX IV (red) intensity as a function of distance along the line shown in the inset. Image in inset corresponds to panels J-L in d. Uncropped images of blots are shown in Supplementary Information, Fig. S8.
figf2(a) Purified mitochondria were left untreated, or were treated with Proteinase K (Pr.K) following osmotic shock (OS), or solubilization of the total protein using Triton X-100 (TX-100), as indicated. Samples were analysed by immunoblotting with antibodies against the indicated proteins.(b) Mitochondrial preparations were subjected to membrane disruption by freeze-thaw cycles as indicated and accessibility of the indicated proteins to Proteinase K was examined by immunoblotting.(c) Immunoblot analysis of membranous organelles from U937 cells separated by isopycnic sucrose density-gradient centrifugation. Numbers represent fractions from the gradient (1, top; 12, bottom). Uncropped images of blots are shown in Supplementary Information, Fig. S8.