EMBO/SourceData huggingface dataset:
Figure 8(A) CD68 coverage in the old APPPS1 tissue (14 DIV) treated with GM-CSF and vehicle Ctr. CD68 coverage is increased upon treatment with GM-CSF. The values are normalized to CD68 coverage of the Ctr and represent mean ± SEM from 3 independent experiments, each experiment including 2 independent slice culture dishes (*P < 0.05).(B) Quantitative analysis of core-only plaques in the old APPPS1 tissue (14 DIV) treated with GM-CSF and Ctr reveals an increased number of core-only plaques upon treatment with GM-CSF. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments, each experiment including 3 independent slice culture dishes (***P < 0.001).(C) CD68 coverage in the old APPPS1 tissue (14 DIV) incubated with conditioned media collected from young WT slices (young CM) or with non-conditioned slice culture media (Ctr). CD68 coverage is increased upon incubation of the APPPS1 tissue with the young CM. The values are normalized to CD68 coverage of the Ctr and represent mean ± SEM from 3 independent experiments, each experiment including 2 independent slice culture dishes (**P < 0.01).(D) Immunofluorescence analysis of the old APPPS1 slice cultured alone (old alone) or in co-culture with young GM-CSF-/- brain slices (old co-culture) at 14 DIV and immunostained with CD68 (red) and M3.2 (green) reveals an increased number of core-only plaques upon co-culturing with young GM-CSF-/- brain slices. Scale bar: 50 µm.(E) Quantitative analysis of core-only plaques in the old APPPS1 slice cultured alone or in co-culture with young GM-CSF-/- brain slices (14 DIV) reveals an increased number of core-only plaques upon co-culturing of old APPPS1 brain slices together with young GM-CSF-/- brain slices. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques. The values represent mean ± SEM from 3 independent experiments, each experiment including 3 independent slice culture dishes (***P < 0.001).(F) CD68 coverage in the old APPPS1 tissue (14 DIV) cultured alone or in co-culture with young GM-CSF-/- brain slices. CD68 coverage is increased upon co-culturing of old APPPS1 brain slices together with young GM-CSF-/- brain slices. The values are normalized to CD68 coverage of the old slice cultured alone and represent mean ± SEM from 3 independent experiments, each experiment including 2 independent slice culture dishes (*P < 0.05).
Figure 9(A) Immunofluorescence analysis of the old APPPS1 slice (14 DIV) treated with GM-CSF and vehicle Ctr and immunostained with CD68 (red), cell proliferation marker Ki67 (green) and M3.2 (blue). Scale bar: 50 µm.(B) Quantitative analysis of Ki67 and CD68 double positive microglial cells in the old APPPS1 tissue (14 DIV) treated with GM-CSF and Ctr reveals increased microglial proliferation upon treatment with GM-CSF. The values are expressed as percentages of CD68 and Ki67 double positive microglial cells from the total number of CD68 positive cells and represent mean ± SEM from 2 independent experiments, each experiment including 4 independent slice culture dishes (***P < 0.001).(C) Immunofluorescence analysis of the old APPPS1 tissue cultured alone or in co-culture with young WT brain slices (14 DIV) and immunostained with CD68 (red), Ki67 (green) and M3.2 (blue) Scale bar: 50 µm.(D) Quantitative analysis of Ki67 and CD68 double positive microglial cells in the old APPPS1 tissue cultured alone or in co-culture with young brain slices (14 DIV) reveals increased microglial proliferation upon co-culturing of old and young brain slices. The values are expressed as percentages of CD68 and Ki67 double positive microglial cells from the total number of CD68 positive cells and represent mean ± SEM from 3 independent experiments, each experiment including 2 independent slice culture dishes (**P < 0.01).(E) Old APPPS1 tissue was incubated with conditioned media collected from young WT slices and in addition treated with proliferation inhibitor AraC (young CM + AraC) or vehicle Ctr (young CM + Ctr). Immunofluorescence analysis was performed at 14 DIV using CD68 (red) and M3.2 (green). Scale bar: 50 µm.(F) Quantitative analysis of core-only plaques in the old APPPS1 tissue (14 DIV) incubated with conditioned media from young WT slices and in addition treated with AraC or Ctr reveals a decreased number of core-only plaques upon AraC treatment. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments, each including 2 independent slice culture dishes (***P < 0.001).
Figure 1A. Hcp release assay. HA-tagged Hcp (HcpHA) release was assessed by separating cells (C) and cell-free culture supernatant (S) fractions from 109 wild-type (WT), ΔtssM cells or ΔtssM cells carrying the AHT-inducible FLAG-tagged tssM-borne plasmid (tssM+) treated (bulgecin) or not (NT) with bulgecin A prior to tssM gene induction. Proteins were separated by 12.5%-acrylamide SDS-PAGE and the periplasmic TolB protein (control for cell lysis), HcpHA and FLTssM were immunodetected using anti-TolB (middle panel), anti-HA (lower panel) and anti-FLAG (upper panel) antibodies. Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in duplicate and a representative result is shown.B. Hcp release assay. HA-tagged Hcp (HcpHA) release was assessed by separating cells (C) and cell-free culture supernatant (S) fractions from 109 wild-type (WT) cells before washing cells (before wash) and after washing and growth (after wash) in absence (NT) or presence (bulgecin) of bulgecin A. Proteins were separated by 12.5%-acrylamide SDS-PAGE and the periplasmic TolB protein (control for cell lysis) and HcpHA were immunodetected using anti-TolB (upper panel) and anti-HA (lower panel) antibodies. Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in duplicate and a representative result is shown.
Figure 3A. Hcp release assay. FLAG-tagged Hcp (HcpFL) release was assessed by separating cells (C) and cell-free culture supernatant (S) fractions from 109 WT, ΔmltE cells or ΔmltE cells producing wild-type (mltE+) or E64Q mutant (mltEE64Q) VSV-G-tagged MltE (MltEV) from arabinose-inducible plasmids. Proteins were separated by 12.5%-acrylamide SDS-PAGE and TolB, HcpFL and MltEV were immunodetected using anti-TolB (upper panel), anti-FLAG (middle panel) and anti-VSV-G (lower panel) antibodies, respectively. Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in triplicate and a representative result is shown.B. Anti-bacterial activity. E. coli K-12 prey cells (W3110 gfp+, kanR) were mixed with the indicated attacker cells, spotted onto Sci-1 inducing medium (SIM) agar plates and incubated for 4 hours at 37°C. The image of a representative bacterial spot and the average and standard deviation (n=3) of the relative fluorescence of the bacterial mixture (in arbitrary unit, AU) are shown in the upper graph. The number of recovered E. coli prey cells (counted on selective kanamycin medium) is indicated in the lower graph (in log10 of colony-forming unit (cfu)). The black, dark grey and light grey circles indicate values from three independent assays, and the average is indicated by the bar. The experiment was performed in triplicate and a representative result is shown. Asterisks indicate significant differences compared to the wild-type attacker strain (NS, non significant; ***, p < 0.001).
Figure 4A-B. Bacterial two-hybrid assay. BTH101 reporter cells producing the indicated proteins or domains fused to the T18 or T25 domain of the Bordetella adenylate cyclase were spotted on X-Gal indicator plates. The blue color of the colony reflects the interaction between the two proteins. TolB and Pal are two proteins known to interact but unrelated to the T6SS or the MltE proteins. The experiment was performed in triplicate and a representative result is shown.C. Co-immunoprecipitation assay. The solubilized lysates from 2×1010 E. coli K-12 W3110 cells co-producing the indicated FLAG-tagged TssMP variants (exported in the periplasm) and VSV-G-tagged MltE protein (Total, T) were subjected to immune precipitation on anti-FLAG-coupled agarose beads. The immunoprecipitated material (IP) was subjected to 12.5%-acrylamide SDS-PAGE and immunodetected with anti-FLAG (upper panel, TssM domains) and anti-VSV-G (lower panel, MltE) antibodies. Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in triplicate and a representative result is shown.
Figure 5A. Remazol brilliant blue assay. The absorbance of supernatants from the reaction containing purified and RBB-labelled E. coli peptidoglycan and the indicated protein (50 µg) were measured at =595 nm after incubation for 0.5 or 4 hours at 37°C. The results shown are the average and standard deviation from triplicate reactions (n=3). Asterisks indicate significant differences compared to the buffer (NS, non significant; **, p < 0.01; ***, p < 0.001). The supernatant of the reaction after 4 hours of incubation is shown on bottom.B. Peptidoglycan hydrolysis. The decrease of the absorbance of the M. luteus peptidoglycan suspension in presence of the indicated protein (50 µg) was measured at =600 nm at 37°C over time. The experiment was performed in triplicate and a representative result is shown.
Figure 6A-B. Co-immunoprecipitation assay. The solubilized lysates from 2×1010 EAEC wild-type or ∆mltE cells producing FLAG-tagged TssM (FLTssM) and/or HA-tagged TssJ (TssJHA, panel A) or TssL (TssLHA, panel B) (Total, T) were subjected to immune precipitation on anti-FLAG-coupled agarose beads. The immunoprecipitated material (IP) was subjected to 12.5%-acrylamide SDS-PAGE and immunodetected with anti-FLAG (upper panel, TssM) and anti-HA (lower panel, TssJ or TssL) antibodies. Molecular weight markers (in kDa) are indicated on the left. The experiments were performed in triplicate and a representative result is shown.C. BS3 cross-linking assay. 2×109 cells of the indicated strain producing FLAG-tagged TssM (with the exception of ∆tssM cells) were treated (+) or not (-) with the BS3 cross-linker agent. After the cross-linking reaction, cells were boiled in Laemmli buffer and total proteins were subjected to 7%-acrylamide SDS-PAGE and immunodetected with anti-FLAG antibodies. The TssM protein (FLTssM) and its complexes (*, TssM-TssJ; **, TssM-TssL) are indicated on the right as well as the TssM dimer (arrow). Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in triplicate and a representative result is shown.D. Fluorescence microscopy. Recordings showing TssM localization using the chromosomally-encoded sfGFP-tssM fusion in wild-type (WT) or ΔmltE cells or ΔmltE cells producing the wild-type (mltE+) or catalytic variant (mltEE64Q) MltE protein. Scale bars are 1 μm. The experiment was performed in triplicate and a representative result is shown.
Figure 1B Ub chain interaction of the LUBEL-NZF and UBA2 domains. Immobilized GST-NZF or GST-UBA2 was incubated with Lys (K) 48-, K 63-linked, or linear di-Ub (Ub2) chains and bound Ub chains were subjected to immunoblotting using anti-Ub antibody or a linear linkage-specific Ub antibody (anti-linear Ub antibody). Loading of GST-fusion proteins was visualized by Ponceau S staining.
Figure 2A In vitro ubiquitination assays of LUBEL-RBR-C WT or catalytically dead C2704A in combination with Ube1 and two different Drosophila E2s, UbcD10 or Effete/UbcD1. Reactions were terminated at indicated times and synthesized Ub chains were detected by immunoblotting using anti-linear Ub antibody. Total protein loading was visualized by Ponceau S staining. * : nonspecific band.B In vitro deubiquitination of Ub chains synthesized by HOIP- and LUBEL-RBR-C. Ub chains produced by RBR-C (Human or Drosophila) with UbcH7, UbcD10 or Effete were incubated with a linear linkage-specific DUB, OTULIN (WT or catalytically dead C129A (C/A) mutant) or a Lys-linkage-specific DUB, vOTU. The DUB-treated samples were subjected to Coomassie staining or immunoblotting using anti-Ub antibody.C Thioester formation assay using Atto647-labelled Ub. Ubiquitination assay using LUBEL-RBR-C WT (left panel) or C2704A (right panel): lanes 1/2 Atto-Ub + E1, lanes 3/4 + ATP, lanes 5/6 + E2, lanes 7/8 + E3, and lanes 9/10 + Ub-His6. In the presence of N-terminally tagged Atto-Ub and C-terminally tagged Ub-His6, only Ub2 product can be synthesized and longer chain formation is restricted. Samples are run without or with DTT in odd or even numbered lanes, respectively. The gels monitor the Atto-labelled Ub.D The linear Ub chain formation activity of LUBEL-LDD mutants. Recombinant proteins of RBR-C WT, C2704A mutant, and LDD mutants (R2754A and D2755A) were assessed for their activity by in vitro ubiquitination assay. The samples were resolved on a gel and stained with Coomassie or immunoblotted using anti-linear Ub antibody.E Linear Ub chain formation by full-length LUBEL transient expression in insect cells. Full-length Myc-LUBEL was transiently expressed in Drosophila Schneider 2 (S2) cells, and Myc-HOIP alone or with HA-HOIL-1L in HEK293T cells. Total cell lysates (TCL) of control and transfected samples were incubated with immobilized GST-Linear-Tandem Ub binding entity (Linear-TUBE) containing three tandem repeats of ABIN-1-UBAN. Pulldown samples were blotted with anti-linear Ub antibody, while TCL were blotted with anti-Myc antibody for exogenous LUBEL and HOIP, anti-HA antibody for HOIL-1L, and anti-Tubulin antibody for loading. Input of GST proteins was analyzed by Ponceau S staining. * : nonspecific band.
Figure 3A Endogenous linear Ub chains enriched in total protein extracts (input, lane 1) from adult w1118 (w-) flies by GST-Linear-TUBE (lane 3). The enriched samples were further treated with recombinant DUBs, vOTU (lane 4) or OTULIN (lane 5). Ub chains were visualized by immunoblotting using anti-linear Ub antibody or anti-Ub antibody. Loading of GST proteins was analyzed by Ponceau S staining.B Endogenous linear Ub chains in total cell lysate (TCL) of S2 cells expressing human Flag-OTULIN WT or catalytically inactive C129A mutant. TCLs enriched with GST-Linear-TUBE were examined by immunoblotting using anti-linear Ub antibody. Expression of OTULIN was analyzed by anti-Flag antibody and loading of TCL was detected by anti-Tubulin antibody. Input of GST proteins was analyzed by Ponceau S staining. * : nonspecific band.C Mass spectrometry analysis using an identical sample as Figure 3B lane 6. MS/MS spectra of the prototypic linear Ub chain peptide (GGMQIFVK) is shown.
Figure 4C In vitro deubiquitination assay of dCYLD. dCYLD was incubated with all eight linkage types of Ub2 chains. dCYLD catalytically dead C284S mutant was incubated with K 63- or linear Ub2 chains. All the proteins in the reactions were resolved on Coomassie-stained SDS-PAGE gel. * : nonspecific band.D Stabilization of linear Ub chains by dCYLD C284S mutant transient expression in S2 cells. Myc-tagged dCYLD WT and C284S were transfected in S2 cells and linear Ub chains were enriched with GST-Linear-TUBE and immunoblotted by anti-linear Ub or anti-Ub antibodies. Input of GST proteins was visualized by Ponceau S staining. Expression of Myc-dCYLD was examined by anti-Myc antibody, and anti-Tubulin antibody was used for the loading control of TCL.E Linear Ub chains in dCYLD mutant flies. Endogenous levels of linear Ub chains in dCYLD mutant flies were compared with a control w- fly strain by performing GST-Linear-TUBE pulldown. Enriched linear Ub chains were immunoblotted with anti-linear Ub antibody. Input of GST proteins was visualized by Ponceau S staining. Tubulin was used for the loading control of the input.
Figure 5B Linear Ub chains in w- and LUBEL mutant fly lysates. w-, CC/SS and delR2 flies were analyzed for the level of endogenous linear Ub chains by enriching the linear ubiquitin chains by GST-Linear-TUBE. Pulldown samples were immunoblotted by anti-linear Ub antibody, while GST-proteins were analyzed by Ponceau S staining. Anti-Tubulin antibody was used for the loading control of the input. * : nonspecific band.C The lifespans of female w- and catalytically dead LUBEL mutant flies (two clones of CC/SS, indicated as #1 and #2, and delR2). Survival of three independent cohorts with approximately 80 flies each was monitored over time. Median survival time (days): w- = 78, CC/SS #1 = 68, CC/SS #2 = 65, and delR2 = 71. Total sample sizes are as follow; w- = 215, CC/SS #1 = 242, CC/SS #2 = 233, delR2 = 273. Log-rank (Mantel-cox) test between fly lines: w- and CC/SS #1 > 0.0001, w- and CC/SS #2 > 0.0001, w- and delR2 > 0.0001, CC/SS #1 and CC/SS #2 = 0.0007, CC/SS #1 and delR2 = not significant, CC/SS #2 and delR2 = not significant.D Histological analysis of muscle in LUBEL mutant flies. Hematoxylin and eosin (H&amp;amp;E) (top panels) and actin immunofluorescent staining (Actin-IF) (bottom panels) of thorax muscles from young (Day 3) or aged (Day 60) w- or CC/SS female flies. Scale bars: 50 μm.
Figure 6A The level of poly-Ub chains in w- flies after heat shock. w- male flies were incubated in a 36 °C water bath for indicated times and the levels of poly-Ub chains in total protein extracts were compared by Ub using antibodies against linear, K 48-, and K 63-linked Ub chains. Ponceau S staining was used to visualize the protein loading.B Survival of catalytically dead LUBEL mutant flies upon heat shock. Young adult flies (15 males and 15 females) were incubated in a 36 °C water bath and immobilized flies were counted over the time indicated. Median survival time (hours): w- = 9.25, CC/SS #1 = 5.5, CC/SS #2 = 5, delR2 = 5.75, CC/SS #1/+ = 8.5, CC/SS #2/+ = 10, delR2/+ = 8. p-values calculated by Gehan-Breslow-Wilcoxon test between fly lines: w- and CC/SS #1, CC/SS#2, or delR2 > 0.0001, w- and CC/SS #1/+ = 0.0134, w- and CC/SS #2/+ = ns, w- and delR2/+ = 0.0002, CC/SS #1 and CC/SS #1/+ > 0.0001, CC/SS #2 and CC/SS #2/+ > 0.0001, delR2 and delR2/+ > 0.0001. Representative data are shown from six independent experiments.C Survival of whole body or muscle-specific LUBEL knockdown flies upon heat shock. shRNA-based knockdown (KD) of LUBEL were driven by Tub-Gal4 or Mef2-Gal4 flies. Control fly lines (Tub-Gal4/+ and Mef2-Gal4/+) were used to compare with the KD flies. 15 males and 15 females per each line were used in this assay. Median survival time (hours): Tub-Gal4/+ = 7, Tub-Gal4>LUBEL = 3.5, Mef2-Gal4/+ = 7.5, Mef2-Gal4>LUBEL= 7. p-values calculated by Gehan-Breslow-Wilcoxon test: Tub-Gal4 <0.0001 (****), Mef2-Gal4 = 0.0029 (**). Representative data are shown from three independent experiments.D Heat-induced mRNA expression of HSP70. w-, CC/SS #2 and delR2 flies were heat treated for 60 min and recovered for 1 hour, and mRNA HSP70 was quantified by qPCR. Rp49 was used as a reference and w- untreated sample was used as calibrator to calculate the expression ratio. Data are presented as meanSD (*<0.01, ***<0.001, ****<0.0001). Representative data are shown from three independent experiments.
figf1(b) Levels of spermidine in the heads of wild-type flies fed with food supplemented by 1 or 5 mM spermidine (Spd1mM+ or Spd5mM+, respectively) or food with no spermidine (Spd−). Data are shown normalized to spermidine levels of 1-d-old (1 d) Spd− flies (n = 4 independent experiments; F = 14.08 for Spd− flies, F = 5.30 for Spd1mM+ flies, F = 2.62 for Spd5mM+ flies; one-way-ANOVA with Bonferroni correction). 15 d, 15 d old; 30 d, 30 d old.(c) Levels of putrescine in the heads of aged Spd1mM+ or Spd5mM+ flies compared with Spd− flies. Data are shown normalized to putrescine levels of 1-d-old Spd− flies (n = 4 independent experiments; F = 24.31 for Spd- flies, F = 0.22 for Spd1mM+ flies, F = 6.67 for Spd5mM+ flies; one-way-ANOVA with Bonferroni correction). *P 0.05; **P 0.01; ***P 0.001; ns indicates not significant, P > 0.05. Data are presented as mean ± s.e.m.
figf2(a) Aversive associative memory performance at 3 min after training (STM) of 3-d-old (3 d) Spd1mM+ and Spd5mM+ flies in comparison to Spd− flies (n = 8-9 independent experiments, F = 2.28, one-way-ANOVA with Bonferroni correction).(b) Aversive associative memory performance at 3 h after training (ITM), ARM and ASM of 3-d-old Spd1mM+ and Spd5mM+ compared with Spd− flies (n = 8-9 independent experiments; F = 2.05 for ITM, F = 0.02 for ARM, F = 1.74 for ASM; one-way ANOVA with Bonferroni correction).(c) STM of 30-d-old (30 d) Spd1mM+ and Spd5mM+ and Spd− flies (n = 8-9 independent experiments, F = 14.48, one-way ANOVA with Bonferroni correction).(d) ITM, ARM and ASM of 30-d-old Spd1mM+ and Spd5mM+ compared with Spd− flies (n = 7-9 independent experiments; F = 43.26 for ITM, F = 1.74 for ARM, F = 26.12 for ASM; one-way ANOVA with Bonferroni correction).(e) Spermidine feeding for 10 d before measuring memory was sufficient to suppress AMI in aged (30 d old) flies (n = 9-11 independent experiments, F = 12.32, one-way ANOVA with Bonferroni correction).(f,g) Spermidine feeding on days 21-30 resulted in a significant increase in the levels of spermidine and putrescine in 30-d-old flies (n = 3 independent experiments, F = 32.96 (f) and 64.77 (g), one-way ANOVA with Bonferroni correction). The feeding regime of the flies is presented beneath the graphs, with + and − indicating food with or without spermidine. *P 0.05, **P 0.01, ***P 0.001; ns indicates not significant, P > 0.05. Data are presented as mean ± s.e.m.
figf3(a) Expression of GCaMP3.0 in the mushroom body of an individual fly, focused on the horizontal lobes. The red line indicates a region of interest used to determine changes in fluorescence emission. Scale bar represents 50 μm. (b) False color-coded image of Ca2+ activity in the horizontal mushroom body lobes shown in a. Warm colors indicate high levels, cold colors indicate low levels or no Ca2+ activity. The numbers indicate changes in fluorescence ΔF (%).(c,d) Time course of Ca2+ increase in horizontal mushroom body lobes of 3-d-old and 30-d-old Spd− flies evoked by the odors 4-methyl-cyclohexanol (MCH) or 3-octanol (OCT) in comparison with the diluent, mineral oil (n = 5 flies, non-parametric Mann-Whitney U test found no substantial difference between the Ca2+ increase of 3-d-old and 30-d-old Spd− flies evoked by the odors). The gray bars indicate the duration of the odor stimuli. Data are presented as mean ± s.e.m.
figf4(a) Quantification of Atg8a proteins levels from head extracts normalized to α-tubulin (n = 17 or 8-9 technical replicates from two independent biological aging replicates for all data, F = 7.52, one-way-ANOVA with Tukey post-test). The full-length blot is shown in Supplementary Figure 9a.(b) Western blot analysis of poly-ubiquitinated proteins (SDS-soluble protein fraction) from head extracts from Spd− (3 and 30 d old) flies compared with Spd5mM+ (3 and 30 d old) flies. Right, quantification of poly-ubiquitinated proteins levels normalized to α-tubulin (n = 10 or 5 technical replicates from two independent biological aging replicates for all data, F = 6.32, one-way ANOVA with Tukey post-test). The full-length blot is shown in Supplementary Figure 9b.(c-f) Adult brains of 3-d-old and 30-d-oldSpd− flies, as well as 3-d-old and 30-d-oldSpd5mM+ fliesimmunostained for ref(2)P. Scale bar represents 25 μm. (g) Quantification of ref(2)P intensity in the central brain region normalized to 3-d-oldSpd− (n = 12-13 independent brains for all data, F = 26.58, one-way ANOVA with Bonferroni correction). *P 0.05, **P 0.01, ***P 0.001; P > 0.05. Data are presented as mean ± s.e.m.