EMBO/SourceData huggingface dataset:
Figure 9A) DNA damage sensitivity to MMS and phleomycin of a Cdk phospho-mimic mutant of Spc110. Ten-fold serial dilutions from mid-log phase cultures of wild-type, spc110S36-91A and spc110S36-91D cells grown on solid rich media containing mock DMSO, MMS or Phleomycin at 30ºC.B) Schematic representation of the genomic background and the approximation used to measure DSB-SPB tethering. Wild-type Spc110 and spc110S36-91D were labelled with the RFP. Ddc2 was tagged with the GFP to be used as DSB reporter. D1: inter-SPB distance; D2: DSB distance to proximal SPB; D3: DSB distance to distal SPB. At least 100 cells from three independent experiments were scored using the maximum projection of nine z-planes. Graph shows the mean ± S.D. P values were calculated using a 2-tailed unpaired Student t test.C) Time-lapse experiments of living cells after induction of the HO endonuclease in wild-type and spc110S36-91D cells. Three z-planes every 10-second intervals over a period of 5 minutes were captured to measure the average SPBs track length and velocity. Spc110 and spc110S36-91D were labelled with the RFP and used to measured SPBs kinetics. At least 100 cells from three independent experiments were scored.D) Southern blot of wild-type and spc110S36-91D mutant bearing the inter-chromosomal gene conversion assay depicted. Mid-log phase cells were HO-induced at 32ºC by adding galactose to the media. Samples were collected at different time-points and the genomic DNA was extracted. Preps were digested with EcoRI and analysed by Southern blot. Blots were hybridized with MATa-only and actin probes. FACS profiles for DNA content are displayed. Graphs show quantification of gene conversion, DSB induction and crossover vs non-crossover ratio. All data were normalized using the actin signal. Graphs represent the mean ± S.D. from three independent experiments. P values were calculated using a 2-tailed unpaired Student t test. Asterisk denotes an overexposed film to visualize the formation of crossover products.D) Southern blot of wild-type and spc110S36-91D mutant bearing the inter-chromosomal gene conversion assay depicted. Mid-log phase cells were HO-induced at 32ºC by adding galactose to the media. Samples were collected at different time-points and the genomic DNA was extracted. Preps were digested with EcoRI and analysed by Southern blot. Blots were hybridized with MATa-only and actin probes. Graphs show quantification of gene conversion, DSB induction and crossover vs non-crossover ratio. All data were normalized using the actin signal. Graphs represent the mean ± S.D. from three independent experiments. P values were calculated using a 2-tailed unpaired Student t test. Asterisk denotes an overexposed film to visualize the formation of crossover products.
Figure 1(B, C) (B) Growth inhibition assay of E. coli DH10B cells harbouring pTrc200 vector or each of its derivatives expressing Tde1 variants with IPTG induction. (C) Growth inhibition assay of E. coli DH10B cells co-expressing the Tde1 variants expressed from pTrc200 plasmid and Tdi1 immunity gene expressed from pRL662 plasmid. Growth curve was determined at OD600. Graphs of panels B and C show mean ± SD of three biological replicates (n=3), each averaged with 3 technical repeats. One way ANOVA was used for the analysis of statistical significance followed by the Tukey's multiple comparison. Different letters indicate statistically different groups of strains (p value, 4.6x10-5 and 5.19x10-8 for panels B and C respectively).
Figure 2(A) Growth inhibition assay of E. coli DH10B cells harbouring pTrc200 vector or each of its derivatives expressing Tde1 variants with IPTG inducible expression. The growth of E. coli was monitored by CFU counting every 1 hr. Data information: Graphs of panels A, show mean ± SD of three biological replicates (n=3), each averaged with 3 technical repeats. One way ANOVA was used for the analysis of statistical significance followed by the Tukey's multiple comparison.For membrane permeabilization assays, BW25113 WT alone or ∆lacY(pYTA-lacZ) cells harbouring pTrc200 vector or each of its derivatives expressing Tde1 variants were carried out for (C) propidium iodide permeability with cells treated with Propidium iodide and Hoechst for detection by fluorescence microscope (Scale bar = 5 μm). For quantification of cells with PI signals, a total of 6 randomly selected images obtained from two biological repeats were used to quantify the number of PI-stained cells / number of Hoechst-stained cells as indicated.(D) Bacteriostatic activity assay. E. coli DH10B cells harbouring pTrc200 vector or each of its derivatives expressing Tde1 variants were cultured with or without IPTG induction for 1 hr. The IPTG-induced cells were further centrifuged and resuspended in the fresh medium with or without IPTG. Cell density was measured again before continuous growth for additional 1 hr. Data information: Graphs of panels show mean ± SD of three biological replicates (n=3), each averaged with 3 technical repeats. One way ANOVA was used for the analysis of statistical significance followed by the Tukey's multiple comparison. Different letters indicate statistically different groups of strains (p value, 1×10-16 and 2×10-16 for panels D respectively).
Figure 3(A) Co-immunoprecipitation (Co-IP) in A. tumefaciens. A. tumefaciens C58 ∆tde1 harbouring pTrc200 vector or its derivatives expressing HA-tagged Tde1 variants. Anti-HA resin was used to coprecipitate the Tde1 variants and Tap1.(B) Secretion assay for HA-tagged Tde1 variants. Western blot for the cellular and extracellular fractions of A. tumefaciens C58 ∆tdei and ∆tdei∆tssK expressing the HA-tagged Tde1 variants. Hcp secretion was detected as a positive control for active T6SS secretion. Representative western blot results of three biological repeats were shown with antibody against HA, Hcp, or EF-Tu where EF-Tu serve as a loading and non-secreted protein control. Protein markers are indicated in kDa.
Figure 4(A) Fluorescence microscopy for Tde1 translocation. A. tumefaciens C58 ∆tdei expressing Tde1 variants fused with sfGFP (in green) and E. coli DH10B carrying mCherry (false coloured in blue) were co-cultured for 20 hrs. A cyan fluorescence with merged blue and green signals represented the translocation of Tde1 variants from A. tumefaciens to E. coli (Scale bar = 5 μm).(B) The number of cells with overlayed GFP and mCherry fluorescence was quantified from a total of 6 randomly selected images obtained from three biological repeats (number of cells with cyan fluorescence/ total E. coli cells counted).
Figure 5(A) Interbacterial competition of A. tumefaciens C58 ∆tdei and ∆tdei∆tssK expressing the Tde1 variants against E. coli cells was carried out on LB medium and E. coli survival rate was quantified by CFU counting. (B) Interbacterial competition between various A. tumefaciens C58 strains and A. tumefaciens 1D1609 on AK medium and the competition outcome was shown by competitive index. Data information: Data in panel A are mean ± SD of four biological repeats of two independent experiments (n=4). Panels B show mean ± SD of three biological repeats (n=3). One way ANOVA was used for the analysis of statistical significance followed by the Fishers Least Significant Difference (LSD) test for panel A and B Different letters indicate statistically different groups of strains (p value, 3.63×10-4, 2.70 × 10-3, 2.3 × 10-15 for panels A, B respectively).(C) Secretion assay for Tde1 and its variants co-expressed with its immunity protein Tdi1 in A. tumefaciens C58 ∆tdei and ∆tdei∆tssK. Data information: Western blots were detected with specific antibody against Tde1, Hcp, or EF-Tu serving as a loading and non-secreted protein control. Protein markers are indicated in kDa. Results in panel C are representative of three biological repeats.
Figure 1D. The relative abundance of valine (Val) and leucine-isoleucine (Leu-Ile). N=18; Boxplot, central band stands for median, boxes stand for 50% of the data, whiskers stand for min or max of the data E. The relative abundance of 3-hydroxybutyrate (3-HOB) and acetylcarnitine (AC). N=18; Boxplot, central band stands for median, boxes stand for 50% of the data, whiskers stand for min or max of the data Data information: Error bars stand for SEM of biological repeats. The p value was calculated by two-tailed t-test with 2-way ANOVA correction.F. The concentration of FGF21 in serum. Control, N=18; POI, N=20; Boxplot, central band stands for median, boxes stand for 50% of the data, whiskers stand for min or max of the data G. The concentration of GDF15 in serum. Control, N=14; POI, N=9; Boxplot, central band stands for median, boxes stand for 50% of the data, whiskers stand for min or max of the data. Data information: Error bars stand for SEM of biological repeats. The p value was calculated by two-tailed t-test with 2-way ANOVA correction.
Figure 2A and B. The serum concentration of FSH and the changes of follicles in mice on low BCAA diet for 1.5 months. A. Control, N=10; low BCAA, N=8; B. Control, N=4; low BCAA, N=4; Data information: S1, Primordial; S2, Primary; S3, Secondary; S4, Antral; S5, Atretic. Error bars stand for SEM of biological repeats. The p value was calculated by two-tailed t-test with 2-way ANOVA correction.C. The serum concentration of FSH in mice on a different batch of the low BCAA diet. N=5; D. The serum concentration of FSH in mice on the low BCAA diet from Research Diet. N=10; Data information: The p value was calculated by two-tailed t-test with 2-way ANOVA correction.E and F. The serum concentration of AMH and E2 in mice on the low BCAA diet for 1.5 months. Control, N=5; low BCAA, N=3 Data information: The p value was calculated by two-tailed t-test with 2-way ANOVA correction.
Figure 3A-B. Elevation of serum ceramide in POI patients from the Fudan Cohort or the Shandong Cohort. Left, the relative abundance of total ceramide; right, the relative abundance of ceramide with specific acyl chain. A. N=18; B. N=10; Truncated violin plot, central band stands for median, dotted lines stand for the upper quartile or the lower quartile of the data; Data information: The p value was calculated by two-tailed t-test with 2-way ANOVA correction.D. Elevation of ceramide in the ovaries of mice on a low BCAA diet. N=10; Truncated violin plot, central band stands for median, dotted lines stand for the upper quartile or the lower quartile of the data Data information: The p value was calculated by two-tailed t-test with 2-way ANOVA correction.F. The serum concentration of FSH in mice with ceramide treatment. N=7 Data information: The p value was calculated by two-tailed t-test with 2-way ANOVA correction.H. The changes in follicle count from mice with ceramide treatment. N=7. Data information: S1, Primordial; S2, Primary; S3, Secondary; S4, Antral; S5, Atretic. Error bars stand for SEM of biological repeats. The p value was calculated by two-tailed t-test with 2-way ANOVA correction.
Figure 4C, Relative expression of genes related to ROS in granulosa cells Data information: Error bars stand for SEM of biological repeats. The p value was calculated by two-tailed t-test with 2-way ANOVA correction.E-F. Relative abundance of GSH and GSSG. N=3; Data information: Error bars stand for SEM of biological repeats. The p value was calculated by two-tailed t-test with 2-way ANOVA correction.G. The concentration of E2 secreted by KGN cells with ceramide treatment w/o NAC. N=4; Data information: Error bars stand for SEM of biological repeats. The p value was calculated by two-tailed t-test with 2-way ANOVA correction.H- J. Serum concentration of FSH, AMH, and E2. H. Control, N=10; BCAA, N=5; TG=9; TG+BCAA=8; I. Control, N=5; BCAA, N=5; TG=6; TG+BCAA=6; J. N=5; Data information: Error bars stand for SEM of biological repeats. The p value was calculated by two-tailed t-test with 2-way ANOVA correction.K. The changes in follicle count. Control, N=5; BCAA, N=4; TG=5; TG+BCAA=5; Data information: Error bars stand for SEM of biological repeats. The p value was calculated by two-tailed t-test with 2-way ANOVA correction.
Figure 1A. Representative H&E staining and immunohistochemical analysis of the androgen receptor (AR) in the dorsolateral prostate (DLP) of PtenL2/L2 and Pten(i)pe-/- mice, sham-operated (sham) or castrated (CTX) at 3 months after gene inactivation (AGI), and analyzed at 1 month after surgery. Scale bars: 100 µm. N=3 mice per condition.F. Violin plots depicting transcript levels of androgen-responsive genes in luminal-C cells of sham and CTX Pten(i)pe-/- mice. P-values were determined by Wilcoxon rank sum test.H. Violin plots depicting the transcript levels of inflammation-related genes in luminal-C cells of sham and CTX Pten(i)pe-/- mice. P-values were determined by Wilcoxon rank sum test.J, K. Violin plots depicting the transcript levels of hypoxia-related genes (J) and plasticity-related genes (K) in luminal-C cells of sham and CTX Pten(i)pe-/- mice. P-values were determined by Wilcoxon rank sum test.
Figure 2C. Gland areas in DLP of sham and CTX control and Pten/Hif1a(i)pe-/- mice. Five glands per mouse were quantified. N=3 mice/condition.G. Representative immunohistochemical detection of cleaved caspase 3 (CC3) in DLP of Pten(i)pe-/- and Pten/Hif1a(i)pe-/- mice, sham or CTX at 3 months AGI, and analyzed 1 or 1, 2, 3 and 30 days after surgery, respectively. N=3 mice/condition.I. Western blot analysis of CC3 in protein lysates of organoids generated from prostates of Pten(i)pe-/- and Pten/Hif1a(i)pe-/- mice at 3 months AGI, and treated with enzalutamide (Enz) for 24 hours at the indicated concentrations. β-actin was used as a loading control.
Figure 3B. Representative H&E staining of DLP of Pten/Hif1a(i)pe-/- mice sham and CTX at 3 months AGI and analyzed at 5 months after surgery. N=4-5 mice/condition. C. Gland areas in DLP of control and Pten/Hif1a(i)pe-/- mice, sham and CTX at 3 months AGI and analyzed at 5 months after surgery. N=3-4 mice/condition. Five glands per mouse were quantified. Data presented are mean, and error bars correspond to SEM.E. Representative immunohistochemical detection of AR and TROP2 in DLP of Pten/Hif1a(i)pe-/- mice, sham and CTX at 3 months AGI and analyzed at 5 months after surgery. N = 3-4 mice/condition.
Figure 4D. Representative western blot analysis of CPARP and CC3 in human C4-2B PCa cells treated for 24 h with PX-478, increasing concentrations of enzalutamide (Enz), or a combination of both. Beta-actin was used as loading control.F. Representative H&E staining of DLP of sham and CTX Pten(i)pe-/- mice, treated with vehicle or PX-478, as depicted in (E). Scale bar: 100 µm. N=3-4 mice/condition.H. Gland areas of DLP of sham and CTX Pten(i)pe-/- mice, treated with vehicle or PX-478, as depicted in (E). N=3-4 mice/condition. Data presented are mean ± SEM. Five glands per mouse were quantified,
Figure 1A-B Proteins of GS and GS2KR (A) and GDH and GDHK128R (B) were expressed from Salmonella in LB in the presence or absence of 50 mM NAM. : The acetylation levels of GS and GDH were assayed by immunoblotting. Quantitation of signals is shown. Immunoblots represent at least two independent experiments For all immunoblotting data, molecular weights in kD are indicated to the right.GS (C) were expressed from Salmonella in minimal media with 50 mM glucose and various concentrations of nitrate (left) and ammonium (right), as indicated. The acetylation levels of GS were assayed by immunoblotting. Quantitation of signals is shown. Immunoblots represent at least two independent experiments For all immunoblotting data, molecular weights in kD are indicated to the right.E-F GS (E) or GDH (F) were expressed from Salmonella under LB and minimal media with 50 mM glucose, 50 mM 2-DG, and 25 mM lactose as indicated. : The acetylation levels of GS and GDH were assayed by immunoblotting. Quantitation of signals is shown. Immunoblots represent at least two independent experiments For all immunoblotting data, molecular weights in kD are indicated to the right.
Figure 2A-B GS (A) or GDH (B) were expressed from Salmonella in minimal media with 100 mM ammonium and various glucose concentrations as indicated. The specific activity of GS and GDH from 2 mM glucose was set as a control for statistical analysis. : Acetylation levels were determined by immunoblotting. The specific activities of GS and GDH were measured spectrophotometrically and compared. The states of adenylylation GS were determined by spectral analysis of total glutamyltransferase activity. Immunoblots represent at least two independent experimentsC In vivo GS-specific activity of Salmonella cultured in LB with or without NAM. The specific activity of GS from cells untreated by NAM was set as the statistical control. : Acetylation levels were determined by immunoblotting. The specific activities of GS were measured spectrophotometrically and compared. The states of adenylylation GS were determined by spectral analysis of total glutamyltransferase activity. Immunoblots represent at least two independent experimentsGSs (F) were expressed and purified from Salmonella cultured in minimal media with high (100 mM) or low (2 mM) ammonium supplemented with high (50 mM) or low (2 mM) glucose. The specific activity of GS from 2 mM glucose was set as a control for statistical analysis. Acetylation levels were determined by immunoblotting. The specific activities of GS were measured spectrophotometrically and compared. The states of adenylylation GS were determined by spectral analysis of total glutamyltransferase activity. Immunoblots represent at least two independent experiments Each symbol represents an independent biological replicate Data are from three independent biological replicates and expressed as the mean ± SDH GDHWT, GDHK128Q and GDHK128R were expressed and purified from Salmonella in LB. The specific activity of GDHWT was set as a statistical reference.K GDH-His was inserted into the chromosome of Salmonella to replace GDH-coding gene gdhA named as gdhA::gdhAHis and cultured in minimal media with glucose levels as indicated. The specific activity of GDH from 5 mM glucose was set as a statistical control. Acetylation levels were determined by immunoblotting. The specific activities GDH were measured spectrophotometrically and compared. Immunoblots represent at least two independent experiments K). Each symbol represents an independent biological replicate Data are from three independent biological replicates and expressed as the mean ± SD
Figure 3A-B GS and GDH, purified from Salmonella cultured in LB media, were treated with Pat/Ac-CoA in vitro. Specific activities and acetylation levels of GS (A) and GDH (B) were measured. Specific activities of GS and GDH without any treatment were set as controls. : Immunoblots represent at least two independent experiments. The degrees of adenylylation GS were determined by spectral analysis of total glutamyltransferase activity. Each symbol represents an independent biological replicate and data are from three independent biological replicates and expressed as the mean ± SDGS (C) proteins were expressed from Salmonella wild-type, Δpat and Δpta mutant strains cultured in LB with or without 50 mM glucose.F GSWT, GS2KQ and GS2KR were expressed from wild-type, Δpat and ΔcobB Salmonella strains in LB, respectively. The acetylation levels and specific activities of each enzyme were determined. Specific activities of GSWT, GS2KQ and GS2KR expressed from the wild-type strain were set as controls. : Immunoblots represent at least two independent experiments. The degrees of adenylylation GS were determined by spectral analysis of total glutamyltransferase activity. Each symbol represents an independent biological replicate and data are from three independent biological replicates and expressed as the mean ± SDG-H GS and GDH were expressed from the Salmonella strain cultured in LB media supplemented with 100 mM ammonium. GS (G) or GDH (H) were treated with CobB, PatWT and its catalytically inactive mutants PatA810V and PatD592N in vitro. Specific activities of GS and GDH without any treatment were set as controls. Immunoblots represent at least two independent experiments. The degrees of adenylylation GS were determined by spectral analysis of total glutamyltransferase activity. Each symbol represents an independent biological replicate and data are from three independent biological replicates and expressed as the mean ± SD
Figure 4B-C GSK164K353 and GDHK128 were purified from LB, LB+20 mM glucose, LB+20 mM citrate, and LB+50 mM NAM for 4 hr. The acetylation levels of GSK164K353 (B) and GDHK128 (C) were detected by western blotting by normalization to their fully acetylated endogenous proteins GSK164AcK353Ac and GDHK128Ac. The specific activities of GS and GDH were determined. The correlation between the individual acetylation ratio of GSK164K353 (B) or GDHK128 (C) and their specific activity was plotted. The acetylation levels of GSK164K353 or GDHK128 were set as the X axis, and the specific activities of GS or GDH were set as the Y axis. Immunoblots represent at least two independent experiments Specific activities of GS and GDH without any treatment were set as control. Data are from one experimentD-E GS and GS2KQ (D) or GDH and GDHK128Q (E) were expressed from Salmonella in LB media. Gradient proportions of GS2KQ and GDHK128Q were added with GS and GDH as indicated, respectively, and then incubated at 4 °C for 24 hours for the activity assay. Specific activities of GS and GDH without any treatment were set as controls. Immunoblots represent at least two independent experiments are from three independent biological replicates and data are expressed as the mean ± SD
Figure 5A GSWT and its mutants were purified from Salmonella cultured in LB. Specific activities of enzymes were compared. Specific activities of GSY398S and GSY398F were set as controls. Each symbol represents an independent biological replicate and data are from three independent biological replicates and expressed as the mean ± SDB GSs were expressed and purified from Salmonella wild-type and ΔglnE knockout mutants in minimal media with or without 50 mM NAM supplemented with 2 mM glucose and 2 mM ammonium. The specific activity of GS from ΔglnE with NAM treatment was set as the control. Immunoblots represent at least two independent experiments (B). The states of adenylylation GS were determined by spectral analysis of total glutamyltransferase activity (B).F The specific activities of GSWT, GS2KR and GS2KQ were compared. Each enzyme was expressed in Salmonella wild-type, Δpat and ΔcobB strains cultured in minimal media with 20 mM glucose and either 2 mM or 100 mM ammonium. Specific activities of GS from high ammonium or low ammonium were set as controls. Each symbol represents an independent biological replicate
Figure 7A-B Specific activities of endogenous GS in Salmonella wild-type, Δpat, ΔcobB strains (A) and ΔglnA::glnAC, ΔglnA::glnACΔpat, ΔglnA::glnACΔcobB strains (B) were compared before and after glucose shock. : Immunoblots represent at least two independent experiments (A and B). The specific activities of GS before shock were set as controls. Each symbol represents an independent biological replicates and data are from three independent biological replicates and expressed as the mean ± SDD GSWT, GSY398S and GSY398F were expressed from the Salmonella wild-type, Δpat and ΔcobB strains, respectively. The specific activities of GSWT, GSY398S and GSY398F were compared before and after glucose shock. The specific activities of GS before shock were set as controls. Each symbol represents an independent biological replicates and data are from three independent biological replicates and expressed as the mean ± SDE The cellular activities of GDH were determined in Salmonella ΔgltB, ΔgltBΔpat and ΔgltBΔcobB strains, as well as in the ΔgltBΔpat strain transformed by a pat-containing plasmid and the ΔgltBΔcobB strain transformed by a cobB-containing plasmid before and after glucose shock. The specific activities of GDH before shock were set as controls. Each symbol represents an independent biological replicates and data are from three independent biological replicates and expressed as the mean ± SD