EMBO/SourceData huggingface dataset:
Figure 4A. Alkaline phosphatase (AP)-shedding assays performed for Eiger in S2R+ cells expressing either GFP, Drosophila ADAM17, or activity dead mutants of Drosophila ADAM17, in the presence of either PMA, or PMA and TAPI-1 (DMSO is used as control; see methods); n=5. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01.B-C. Fluorescent images of retinas from 1 day old flies grown on either DMSO or TAPI-1, stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and photoreceptor membranes respectively. Scale bars: 10μm.D. Integrated total lipid counts from retinas corresponding to flies reared either on DMSO or TAPI-1 starting from larval stages. n=10 fly retinas. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01.E-H. Fluorescent images of 1week old adult fly retinas, stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and photoreceptor membranes respectively: (E) wild type; (F) eiger-/-; (G) grindelwald-/-; (H) wengen-/-. Scale bars: 10μm.I. Quantitation of the BODIPY signal shown as integrated total lipid measurements for the genotypes mentioned above. n=10 fly retinas. The box end points represent the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median, the square is the mean and the diamond an outlier. Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01.J-K. mRNA level transcripts of ACC and FASN measured from head lysates of one week old wild type, eiger-/- and grindelwald-/- . n=3 biological replicates, with 3 technical replicates for each genotype. The box end points represent the maximum and minimum values respectively, the central band is the median and the square is the mean Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01.L-O. TEM images of two week old retinas corresponding to (L) control; (M) eiger-/-; (N) grindelwald-/-; (O) wengen-/-. n=180 ommatidia from 3 different fly retinas for each. Scale bars: 10μm.P. Quantitation of the percentage of normal, abnormal and missing rhabdomeres from 2 week old retinas observed in TEM, corresponding to the genotypes mentioned above; n=180 ommatidia from 3 different fly retinas for each. Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01.
Figure 5A-D. Fluorescent images of 1 day old adult fly retinas, stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and photoreceptor membranes respectively: (A, B) wild type; (C, D) ADAM17-/- mutants, reared either on DMSO or AD4. Scale bars: 10μm.E. Integrated total lipid counts from retinas corresponding to the genotypes and treatments mentioned above; n=10 for each. The box end points represent the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were analysed using the Kruskal Wallis test followed by Dunn's test for post hoc analysis for significance due to unequal sample sizes. ***p<0.001.F-I. TEM images of 2 week aged retinas corresponding to either wildtype or ADAM17-/- mutants grown on either DMSO or AD4. Scale bars: 10μm.J. Percentage of normal, abnormal or missing rhabdomeres from retinas corresponding to the genotypes and treatments mentioned above. n=180 ommatidia from 3 different fly retinas for each.K-N. Fluorescent images of 1 day old adult fly retinas, stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and photoreceptor membranes respectively: (K) ADAM17-/- ; (L) glial specific SOD2 over-expression in an ADAM17-/- mutant; (M) glial specific SOD1 over-expression in an ADAM17-/- mutant; (N) neuronal specific SOD2 overexpression in ADAM17-/- mutant. Scale bars: 10μm.O. Quantitation of the BODIPY signal shown as Integrated total lipid for the genotypes mentioned above, n=10 for each. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median, the square is the mean, and the diamond an outlier. Data were analysed using the Kruskal Wallis test followed by Dunn's test for post hoc analysis for significance due to unequal sample sizes. ***p<0.001, **p<0.01, *p<0.05.P-Q. TEM analysis of 5 week aged retinas corresponding to either ADAM17-/- mutants or SOD2 over-expression in an ADAM17-/- mutant. Scale bars: 10μm.R. Percentage of normal, abnormal and missing rhabdomeres in ADAM17-/- mutant retinas, as compared to retinas overexpressing glial specific SOD2 in an ADAM17-/- mutant background observed with TEM; n=180 ommatidia from 3 different fly retinas for each.S. Oxyblot analysis of oxidative modification of proteins from retina lysates of wild type and ADAM17-/-. T. Analysis of Oxyblot intensity normalised to its respective actin controls for each genotype. n=3 biological replicates, with 3 technical replicates for each genotype. The box end points represent the maximum and minimum values respectively, the central band is the median and the square is the mean. Data were quantified for significance using Student's t test. **p<0.01. Scale bars: 10μm.
Figure 6A-B. Fluorescent images of 1 day old adult fly retinas, stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and photoreceptor membranes respectively: (A) wild type; (B) ADAM17-/-, both reared in the dark. Scale bars: 10μm.C-F. TEM images of 4 week old retinas: (C, D) wildtype, or (E, F) ADAM17-/- flies reared from first larval instar either in total light (C, E) or total dark (D, F). Scale bars: 10μm.G. Quantitation of the BODIPY signal shown as integrated total lipid from 1 day old retinas corresponding to the genotypes described in A-B, all reared in the dark. n=10 for each. The box end points represent the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were quantified for significance using Student's t test. **p<0.01.H. Percentage of normal, abnormal and missing rhabdomeres in the genotypes and treatments mentioned above. n=180 ommatidia from 3 different fly retinas for each. Data were quantified for significance using Student's t test. **p<0.01.I. Measurement of MDA levels (in nmoles) from 1 day old retinas of wild type or ADAM17-/- , reared either in light or dark. Four biological replicates with 3 technical replicates for each genotype. The box end points represent the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were quantified for significance using Student's t test. **p<0.01.J. mRNA levels of the puckered transcripts from retinas of untreated or AD4 treated wild type and ADAM17-/- mutant retinas. n=3 biological replicates, with 3 technical replicates for each genotype. The box end points represent the maximum and minimum values respectively, the central band is the median and the square is the mean. Data were quantified for significance using Student's t test. **p<0.01.K-L. Fluorescent images of 1 day old adult fly retinas, stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and photoreceptor membranes respectively: (K) heterozygous mutant of bsk alone; or (L) in combination with ADAM17-/-. Scale bars: 10μm.M. Quantitation of the BODIPY signal shown as integrated total lipid for the genotypes mentioned in K-L, along with w1118 and ADAM17-/-. n=10 flies per genotype. The box end points represent the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were quantified for significance using Student's t test. **p<0.01.N-O. TEM images of 2 week old adult retinas, showing clusters of ommatidia for a heterozygous mutant of bsk alone (M) or in combination with ADAM17-/- (N); n=3 for each. Scale bars: 10μm.P. Percentage of normal, abnormal and missing rhabdomeres for the genotypes mentioned above; n=180 ommatidia from 3 different fly retinas for each. Data were quantified for significance using Student's t test. **p<0.01.
Figure 1Senescence-associated β-galactosidase (SA-β-gal) staining in A549, HAFF, HepG2 and H1299 cells after 48h transduction with either negative control lentiviruses (shCtrl) or those targeting GUARDIN (shGUARDIN). The graph shows percentage of SA-β-gal-positive cells.Senescence-associated heterochromatin foci formation (SAHF) in A549 cells with either shCtrl or shGUARDIN. Representative IF staining with H3K9me3 antibodies (green) and nuclear Hoechst counterstaining (blue).Secretion of SASP factors IL-6 and IL-8 in A549 cells with either shCtrl or shGUARDIN.Western blotting for p21, p16 and p15 in HepG2 cells after 48h transduction with shCtrl or shGUARDIN-1, -2, respectively (left). Knockdown efficiencies analyzed by qPCR (right).SA-β-gal staining in A549 cells after 24h transduction with either shCtrl or shGUARDIN in combination with p21 shRNA (shp21). The graph shows percentage of SA-β-gal-positive cells.qPCR measurement of p21 mRNA in A549, HepG2, HAFF and H1299 cells with either GUARDIN shRNA knockdown (top) or GUARDIN overexpression (bottom).Western blotting for p53 and p21 in A549, HepG2, HAFF and H1299 cells with either GUARDIN shRNA knockdown (top) or GUARDIN overexpression (bottom).qPCR assays of NOXA, TIGAR, BAX and p21 mRNA levels in A549 cells with either shCtrl or shGUARDIN.
Figure 2SDS-PAGE of RNA pulldown assays using biotin-labelled sense/antisense probes against GUARDIN from whole-cell lysates of A549 cells indicating putative GUARDIN-binding proteins (left); protein identities with high probabilities were determined by mass spectrometry (right).RNA pulldown assays interrogating putative GUARDIN-associated proteins identified in (A) from whole-cell lysates of A549 and HAFF cells. BRCA1, BARD1 served as positive controls and β-actin served as negative controls.RNA immunoprecipitation (RIP) assays against IgG/LRP130 antibodies in whole-cell lysates of A549 cells.Subcellular localization of GUARDIN and its co-localization with LRP130. RNA FISH for GUARDIN (red) and IF for LRP130 (green) in A549 cells with either shCtrl or shGUARDIN. Nucleus were counterstained with Hoechst (blue).RNA pulldown assays using biotin-labelled sense/antisense probes against GUARDIN from whole-cell lysates of A549 cells. GUARDIN levels were measured by RT-PCR and co-precipitated LRP130 and PGC1α detected by Western blotting. BRCA1 and β-actin served as positive and negative controls, respectively.RIP assay using IgG/PGC1α antibodies from whole-cell lysates of A549 cells. GUARDIN, LRP130 and PGC1α levels were measured as per (E).Two-step IP assays in whole-cell lysates of A549 cells transfected with FLAG-tagged PGC1α. First phase IPs were conducted with FLAG antibodies (left) and following elution with FLAG peptides, eluates were further subjected to second phase IPs with LRP130 antibodies (right). Samples were subjected to Western blotting and qPCR analysis for LRP130, PGC1α and GUARDIN, respectively.Co-immunoprecipitation (co-IP) between LRP130 and PGC1α in A549 cells after 48h transduction with shCtrl or shGUARDIN. LRP130 was precipitated and samples subjected to Western blotting analysis for LRP130, PGC1α and β-actin as loading control.Mammalian two-hybrid assays between pACT-LRP130 and pBIND-PGC1α in A549 cells after 48h transduction with shCtrl or shGUARDIN. Samples were subjected to the luciferase activity assays.LRP130/PGC1α and p21 protein expression was measured by Western blot in A549 and HAFF cells after 48h transduction with shCtrl or shLRP130 (top left) or shPGC1α (bottom left) as indicated. qPCR assays for p21 mRNA levels were performed in parallel (right panels).Western blotting analysis of LRP130, PGC1α and p21 protein levels in HAFF and A549 cells after 48h transduction with shCtrl or shGUARDIN.
Figure 3ChIP assays detecting binding of LRP130/PGC1α to the p21 promoter using qPCR and RT-PCR (top left and right, respectively). IgG and p53 served as a negative and positive controls, respectively. Schematic illustrations of the putative LRP130/PGC1α binding sites within DNase I hypersensitive regions of the p21 promoter (bottom).qPCR assays for GUARDIN, FOXO1, FOXO3a and FOXO4 in A549 cells after 24h transduction with shCtrl or shGUARDIN.Western blotting assays for FOXO4 and p21 protein expression in A549 cells after 48h transduction with shCtrl or shGUARDIN alone or in combination with shFOXO4. β-actin served as loading control.Half-life times of FOXO4 mRNA in A549 cells with shCtrl or shGUARDIN measured by qPCR after treating cells with 5 μg/ml of actinomycin (ActD) for the indicated times.ChIP assays detecting binding of LRP130/PGC1α to putative binding sites in the FOXO4 promoter using qPCR (top). Data were normalized to the IgG negative control. Schematic illustration of the LRP130/PGC1α binding sites within DNase I hypersensitive regions of the FOXO4 promoter (bottom).Luciferase assays conducted in A549 cells transfected with LRP130 or PGC1α using the pGL3 (negative control) or pGL3-FOXO4 promoter reporter plasmids.qPCR assays for LRP130 and FOXO4 mRNA levels (upper) and Western blotting analysis for LRP130 protein level (lower) in A549 cells after 48h transduction with LRP130 overexpression or knockdown.qPCR assays for PGC1α and FOXO4 mRNA level (upper) and Western blotting analysis for PGC1α protein level (lower) in A549 cells after 48h transduction with PGC1α overexpression or knockdown.Luciferase activity assays in A549 cells transduced with shCtrl or shGUARDIN using the pGL3 (negative control) or pGL3-FOXO4 promoter reporter plasmids.
Figure 4qPCR assays for GUARDIN expression in A549 cells treated with increasing doses rapamycin for 24hqPCR assays for GUARDIN expression in A549 cells treated with 100nM of rapamycin for the indicated times (B).Western blotting assays for p53, LRP130, FOXO4 and p21 expression in A459 and H1299 cells treated with 100nM of rapamycin or vehicle (DMSO) for 48h.A549 cells with shCtrl or shGUARDIN were treated with 100nM rapamycin or DMSO vehicle in the indicated combinations for 48h. Western blotting was used to measure p21 levels (left) while conducting SA-β-gal staining in parallel (middle) with the percentage of senescent cells calculated from the SA-β-gal staining (right).Mammalian two-hybrid assays between pACT-LRP130 and pBIND-PGC1α in A549 cells treated with DMSO or 100nM rapamycin. Samples were subjected to the luciferase activity assays.SIX4 and FOSL2 mRNA (left) and protein levels (right) measured by qPCR and Western blotting, respectively in A549 cells treated with DMSO or 100nM rapamycin. TBP served as a control.qPCR (upper) and Western blotting (lower) assays for GUARDIN, SIX4 and FOSL2 expression in A549 cells comparing shCtrl with shSIX4 (left), shFOSL2 (middle) or FOSL2 (right) after transduction for 48h.Luciferase assays in A549 cells transduced with shCtrl, shSIX4 or shFOSL2 using pGL3 (negative control) or pGL3-FOXO4 promoter reporter plasmids.ChIP assays comparing control IgG versus FOSL2 antibodies demonstrate specific recovery of the GUARDIN promoter by RT-PCR assay. UPe and UPk served as a positive and negative controls, respectively [54].
figf1(a) Confocal micrographs of HeLa cells expressing yellow fluorescent protein (YFP) fused to Nap1, Sintbad or Tank and infected with S. Typhimurium, stained for ubiquitin (Ub) 2 h after infection. At least 100 cells were analyzed. DAPI, 4,6-diamidino-2-phenylindole. Scale bar, 10 μm; arrowheads, bacteria shown in insets.(b-e) Normalized ratio between luciferase activity bound to beads and present in lysates. The indicated purified GST fusion proteins coupled to beads were incubated with lysates of 293ET cells expressing Nap1, Sintbad or Tank, each fused to luciferase (b)(c), lysates of 293ET cells expressing the indicated Nap1 mutants fused to luciferase(d), lysates of E. coli expressing Nap1(1-85) fused to luciferase(e) lysates of 293ET cells expressing Nap1, NEMO or Optineurin fused to luciferase
figf2(a) Flowthrough from nickel-agarose column, separated by SDS-PAGE and stained with Coomassie blue.(c,d) Normalized ratio between luciferase activity bound to beads and present in lysates. The indicated purified GST fusion proteins coupled to beads were incubated with bacterially expressed Nap1(1-85) fused to luciferase. Reactions were complemented with lysates from 293ET cells that had been depleted with the indicated (α-) antibodies (c, top). Immunoblot of lysates after depletion (c, bottom).(c,d) Normalized ratio between luciferase activity bound to beads and present in lysates. The indicated purified GST fusion proteins coupled to beads were incubated with bacterially expressed Nap1(1-85) fused to luciferase.Reactions were complemented with the indicated amounts of lysates from E. coli cultures expressing myelin basic protein (MBP) fused to NDP52 (d).(e) Lysates of 293ET cells were immunoprecipitated (IP) using the indicated antibodies or an isotype control. Lysates and precipitates were blotted (IB) for the presence of TBK1, IKKε and IKKβ.(f) Lysates of 293ET cells were immunoprecipitated using the indicated antibodies or an isotype control. Precipitates were blotted for the presence of TBK1 and NDP52. Fusion proteins used in Figure 2 are further characterized in Supplementary Figure 1. Data in panels c-f are representative of at least two independent experiments. Ub, ubiquitin; PCNA, proliferating cell nuclear antigen.
figf3(a) Confocal micrographs of HeLa cells 2 h after infection with S. Typhimurium, stained with antibodies to Cocoa, NDP52 and ubiquitin (Ub). (b) Colocalization, at the indicated time points after infection, of ubiquitin-positive S. Typhimurium with NDP52 or Cocoa.(c) Confocal micrographs of HeLa cells 2 h after infection with S. Typhimurium, stained with antibodies to NDP52 and LAMP1. (d) Colocalization, at the indicated time points after infection, of NDP52-positive S. Typhimurium with LAMP1. DAPI, 4,6-diamidino-2-phenylindole. All data are representative of at least two independent experiments. Error bars, s.d.; three coverslips per experiment, 50 marker-positive cells evaluated per coverslip. Scale bars, 10 μm; arrowheads, bacteria shown in insets.
figf4Percentage of NDP52-positive S. Typhimurium among ubiquitin-coated bacteria, determined by microscopy. HeLa cells transduced with the indicated NDP52 domain-deletion mutants fused to green fluorescent protein were infected with S. Typhimurium and stained for ubiquitin 2 h after infection. ZnF, zinc finger domain. *P 0.01, Student's t-test. Mean and s.d. of triplicate coverslips from two independent experiments; more than 50 ubiquitin-positive bacteria were evaluated per coverslip.
figf5(a,b) Confocal micrographs of HeLa cells expressing YFP-Nap1, YFP-Sintbad or YFP-Tank (a) or YFP-TBK1(K38M), a catalytically inactive mutant (b), stained with NDP52 antiserum 2 h after infection with S. Typhimurium. All data represent at least two independent experiments. DAPI, 4,6-diamidino-2-phenylindole. Scale bars, 10 μm; arrowheads, bacteria shown in insets.
figf6(a) Lysates of cells transfected with the indicated siRNAs blotted for NDP52, TBK1, IKKε and proliferating cell nuclear antigen (PCNA). Control, a nonspecific band detected by the IKKε antibody.(b,c) Numbers of bacteria recovered from HeLa cells transfected with the indicated siRNAs and infected with S. Typhimurium. At the indicated time points after infection, cells were lysed and bacteria counted on the basis of their ability to form colonies on agar plates. Mean and s.d. of triplicate HeLa cultures and duplicate colony counts.(d) Numbers of bacteria recovered from HCT116 cells expressing the indicated shRNAs and infected with S. Typhimurium. At the indicated time points cells were lysed and bacteria counted by flow cytometry. Mean and s.d. of triplicate cultures.(e) HeLa cells transfected with the indicated siRNAs were infected with S. Typhimurium expressing green fluorescent protein (GFP). At the indicated time points after infection, cells were scored according to the number of bacteria they contained.(f) HeLa cells transfected with the indicated siRNAs were infected with S. Typhimurium expressing GFP. At the indicated time points after infection, cells were stained for ubiquitin and infected cells were scored if they contained ubiquitylated bacteria. All data represent at least two independent experiments. *P 0.05, Student's t-test.
figf7(a-d) Analysis of HeLa cells stably expressing green fluorescent protein-LC3, infected with S. Typhimurium. (a) Confocal micrographs of cells stained 1 h after infection with NDP52 antiserum. Scale bar, 10 μm; arrowheads, bacteria shown in insets. (b) Colocalization of NDP52 with LC3-positive bacteria. (c,d) Counts of LC3-positive S. Typhimurium 1 h after infection in cells transfected with the indicated siRNAs. In c, cells were stained for ubiquitin (Ub); in d, red fluorescence protein (RFP)-expressing S. Typhimurium were used. Means of duplicate counts from four independent experiments; *P 0.05, Student's t-test.(e) Binding of NDP52 to GST-LC3 and GST-ubiquitin. Beads carrying the indicated purified GST fusion proteins were incubated with lysates of E. coli expressing NDP52. Bound proteins were eluted with glutathione and stained with Coomassie blue after SDS-PAGE.(f) Normalized ratio of luciferase-LC3 binding to beads. The indicated purified GST proteins coupled to beads were incubated with bacterially expressed LC3 fused to luciferase and the indicated volumes of lysed E. coli expressing NDP52. Data in a,b,e and f are representative of at least two independent experiments.
Figure 1Phenotypes of wild type and nine microtubule-associated gene mutants. Comparison of the filament and stigma phenotypes of wild type and nine microtubule-associated gene mutants.
Figure 2Microtubule organization in wild type stamen filament cells. (D-F) Microtubule organization in the stamen filament cells of lue1 and ktn80.1234. Arrows indicate microtubule crossovers. Dotted lines indicate abnormal microtubules.
Figure 3Time-lapse images showing that KTN1 is associated with microtubule severing at crossover sites in wild type filament cells. GFP-labeled KTN1 fluorescent particles are pseudo-colored in red, and microtubules (labeled with mCherry-TUB6) are shown in green. Yellow arrows indicate KTN1 at microtubule crossover sites. Asterisks indicate the location at which a microtubule has been severed at a crossover site. Time-lapse images showing that KTN1 cannot be recruited to crossover sites and no severing was observed in ktn80.1234 filament cells. Yellow open arrows indicate microtubule crossovers, which remain stable for a long time.
Figure 4Comparison of the morphologies of the stamen filament cells in wild type and qui-2.
Figure 5EMSA showing that BZR1 binds to the E-box of the KTN80.4 promoter regions in vitro. Nonlabelled (free) native probe in 10- to 100-fold molar excess relative to the biotin-labeled native probe was used as a cold competitor. The mutant probe was used as a negative control.