EMBO/SourceData huggingface dataset:
Figure 8C, Localization of GFP-SnRK1α1 and GFP-SnRK1α2 in root cells of 5-d-old Arabidopsis WT seedlings incubated at 39°C and imaged at the indicated time points. Scale bars = 10 μm. D, Quantification of GFP-SnRK1α1 and GFP-SnRK1α2 foci in the experiment shown in C. Data represent means ± SD of at least 16 replicate measurements. The experiment was repeated three times with similar results. ***P < 0.05 (two-tailed t-test). E, F, Immunoblot analysis with indicated antibodies of protein extracts prepared from root tips of 10-d-old Arabidopsis tsn1 tsn2 (E) or tsn1 tsn2 expressing ProTSN2:GFP-TSN2 (F) heat-stressed seedlings (39ºC) 0, 20, 40 and 60 min after the onset of HS. The charts show SnRK1 activity, expressed as the ratio of phosphorylated to total SnRK1 protein. The data represent mean ratios of integrated band intensities (for both isoforms) normalized to 0 min ± SD from at least four different experiments. P values denote statistically significant differences for comparisons to 0 min (two-tailed t-test).G, Expression levels of DIN2 and DIN6 in Arabidopsis WT, tsn1 tsn2, tsn1 tsn2;TSN2 and snrk1α1-/- snrk1α2-/+ 10-d-old heat-stressed seedlings relative to unstressed controls. For CHX treatment, the WT seedlings were pre-treated with 200 ng μL-1 CHX for 30 min before HS. Upper and lower box boundaries represent the first and third quantiles, respectively. Horizontal lines mark the median of five replicate measurements, and whiskers mark the highest and lowest values. Means with different letters are significantly different at P < 0.05 (one-way ANOVA).
Figure 1(D) HeLa cells were transfected with 10 nM control (n=84) or Nup50 siRNA (n=171), 20 nM control (n=101) or Nup50 siRNA (n=157) and 40 nM control (n=86) or Nup50 siRNA (n=204). 72 h post transfection, cells were fixed with 4% PFA and stained with antibodies against Nup50 and mAB414, chromatin was labeled with DAPI. Scale bars: 50 µm. Quantitation of the mAB414 rim intensity at three different Nup50 RNAi and control oligo concentrations. The means are indicated as diamonds, error bars show the standard deviations. P-values have been calculated from a student t-test comparing the mean between the experimental conditions.
Figure 2(A) Mouse 3T3-NIH cells were transfected with 20 nM control, Nup50A, Nup50B or a combination of Nup50A and Nup50B siRNA. After 72h cells were fixed and stained with mAB414 (green) and antibodies against mouse Nup50 (red). Chromatin is stained with DAPI (blue). The merge of the three channels is shown on the left column. Scale bar: 50 µm. An insert shows a zoom on a representative nucleus for each picture. Scale bar: 10 µm.(B) Quantitation of the mAB414 rim intensity from n=4 independent experiments done as in (A), each represented by a different color. The mean of each individual experiment and condition is represented by a diamond, the mean of all four experiments by a horizontal black line. P-values have been calculated from a paired ratio t-test comparing the mean between the experimental conditions. Error bars show the standard deviations of the means.
Figure 3(A, B) Demembranated sperm chromatin was preincubated in Xenopus egg extract, depleted for MEL28/ELYS or Nup50 (B) or control treated (Mock, A). After 10 min, membranes were added to the reaction. Reactions were stopped at the indicated time points by fixation and analyzed by confocal microscopy after immunostaining with α-MEL28/ELYS (A), α-Nup50 (B), and mAb414 (A, B). Chromatin was stained with DAPI (blue in the overlay). Scale bar: 10 μm.
Figure 43 µM recombinant EGFP, EGFP-tagged MEL28/ELYS (aa 2290-2408) or EGFP-tagged Nup50 were incubated with empty or DNA-coated magnetic beads, which were chromatinized with Xenopus egg extracts (right panel) or unchromatinized DNA-beads (left panel). After 3h the beads were re-isolated, washed, co-stained with DAPI and analyzed by confocal microscopy. Scale bar: 10 µm.
Figure 5(C) HeLa cells were transfected with EGFP-Nup50 comprising different point mutations in the minimal NPC binding region and analyzed as in (A). Scale Bar: 10 µm.(D) GST-fusion constructs of the Xenopus Nup50 minimal NPC binding fragment (aa 144-189) comprising no or single point mutations were incubated with Xenopus egg extracts. Starting material as well as bound proteins were analyzed by Western blotting with indicated antibodies. The quantitation shows the average MEL28/ELYS and Nup153 bead bound signal from three independent experiments, normalized to the signal of their respective wild-type GST fusion Data points from individual experiments are indicated.
Figure 6(A) Confocal microscopy images of nuclei assembled for 120 min in mock depleted, Nup50 depleted (∆Nup50), and Nup50 depleted Xenopus egg extracts supplemented with recombinant wild-type Nup50 or different mutants. Nuclei were fixed in 4% PFA and 0.5 % glutaraldehyde, stained for NPCs (mAB414) and the chromatin (DAPI). Scale bar: 10 µm. (B) Average percentage of mAB414 positive nuclei for 100 randomly chosen chromatin substrates in each of at least three independent experiments shown in (A). Data points from the individual experiments are indicated. (C) Confocal microscopy images of nuclei assembled with N-terminal Nup50 truncations and an minimal N-terminal fragment. Samples were analyzed as in (A). (D) Average percentage of mAB414 positive nuclei for 100 randomly chosen chromatin substrates in each of at least three independent experiments shown in (C). Data points from the individual experiments are indicated.
Figure 7(B) Confocal microscopy images of fixed nuclei assembled for 120 min in mock depleted (mock) and Nup50 depleted (∆Nup50) Xenopus egg extracts supplemented with recombinant mouse Nup50 orthologues. Nuclei were stained for Nup50 (Nup50 antibody for Xenopus protein, His6 for mouse protein) and NPCs (mAB414, red) on the chromatin (DAPI). Scale bar: 10 µm. (C) The average percentage of mAB414 positive nuclei for 100 randomly chosen chromatin substrates in each of three independent experiments (performed as in B) is shown. Data points from individual experiments are indicated. (E) GST-fusion constructs of the Xenopus Nup53 RRM domain (aa 162-267, control) full-length Xenopus Nup50 and the two mouse Nup50 orthologues were incubated with Xenopus egg extracts. Starting material as well as bound proteins were analyzed by Western blotting with indicated antibodies The right panel shows the average MEL28/ELYS, Nup153, importin β and ran bead bound signal from two independent experiments normalized to their respective Xenopus signal. Data points from individual experiments are indicated.
Figure 8HEK293T cells were transfected with empty FLAG-tag vector as a control or FLAG-Nup50 N-terminal fragment (aa 1-120 of the human sequence). 24 h post transfection cells were lysed, FLAG-tagged proteins immuno-isolated and analyzed by mass spectrometry with the Volcano blot showing the identified interactors of Nup50 (yellow, data from three independent experiments) Data points from individual experiments are indicated. KPNA2 is the gene name of importin α.HEK293T cells were transfected with empty FLAG-tag vector as a control or FLAG-Nup50 N-terminal fragment (aa 1-120 of the human sequence). 24 h post transfection cells were lysed, FLAG-tagged proteins immuno-isolated and analyzed by western blotting (B), with 10% of the inputs loaded. The quantification of the western blot (B, lower panel) shows the mean signal intensity normalized on the input of at least two independent experiments. Data points from individual experiments are indicated.(E) 2 µM recombinant Ran, loaded with MANT-GDP was incubated with 2 mM GppNHp in buffer control, supplemented with 2 nM recombinant RCC1, 20 nM recombinant Xenopus Nup50 proteins, or RCC1 and Nup50 together. GDP to GppNHp exchanged was monitored by the decrease in MANT fluorescence of the liberated GDP-MANT. The lower panel shows the rate constant of each experimental condition with n=4 independent experiment per condition, bars represent the mean, individual data points are indicated.(G) Confocal microscopy images of nuclei assembled for 120 min in mock depleted, Nup50 depleted (∆Nup50), and Nup50 depleted Xenopus egg extracts supplemented with recombinant Xenopus wild-type Nup50 or RCC1 binding mutants. Nuclei were fixed in 4 % PFA and 0.5 % glutaraldehyde, stained for NPCs (mAB414) and the chromatin (DAPI). Scale bar: 10 µm. Quantitation shows the average percentage of mAB414 positive nuclei for 100 randomly chosen chromatin substrates in each of three independent experiments. Individual data points are indicated (H) Confocal microscopy images of nuclei assembled for 120 min in mock depleted, Nup50 depleted (∆Nup50), and Nup50 depleted Xenopus egg extracts supplemented with RCC1 excess as indicated. Nuclei were fixed in 4 % PFA and 0.5 % glutaraldehyde, stained for NPCs (mAB414) and the chromatin (DAPI). Scale bar: 10 µm. Quantitation shows the average percentage of mAB414 positive nuclei for 100 randomly chosen chromatin substrates in each of three independent experiments. Individual data points are indicated.
Figure 1Comparison of structural preservation in cryosections picked up with sucrose (a) and MU (b). In isolated hepatocytes, autophagy was induced for 30 min in suspension buffer before fixation. (a) Cryosection picked up with 2.3 M sucrose and labeled for SOD. Note the presence of large, "empty" gaps between sequestered contents of the AV and the surrounding cytoplasm. (b) A similar section as a, but picked up with an MU mixture. The AV are no longer distorted and the overall membranous structures are sharply delineated. The SOD-labeling density of AVi is as that of the cytosol, whereas AVd show much more intense labeling. (Inset) An AVi/d that is distinct from AVi by its single limiting membrane. The highly pleiomorphic AVd are also bound by a distinctive single membrane, but in contrast to AVi/d, their contents are characterized by debris of digested material. Bars, 250 nm.
Figure 2Labeling patterns of cathepsin D and CAIII in MU picked-up cryosections. (a) Cells treated as in Fig. 1 b, but the section was immunolabeled for cathepsin D, which is not detectable in the AVi, moderately present in the AVi/d, and abundant in AVd. Cells in b and c had endocytosed 5-nm gold tracer for 20 and 40 min, respectively, before fixation and CAIII labeling. (b) Complex fusion profile of a CAIII-positive AVi, an AVd which is without CAIII labeling, and a VE that contains an endocytosed gold probe (arrow). Close to the AVd is another tracer-bearing VE. (c) Fusion profile of a tracer-bearing MVE and an AVi/d that exhibits a single limiting membrane and a CAIII-labeling concentration similar to that of the cytoplasm. Bars, 200 nm.
Figure 3Appearance of tracer in AV. Cells were allowed to endocytose tracer particles continuously for the designated periods of time. At each time, random sections were searched for AV+, and AV+/500 cell profiles were plotted. The number of cell profiles examined (denominator) and the respective number of AV+ scored (numerator) are listed above the graph.
Figure 4Tracer distribution in AV+ types. Cells were allowed to endocytose tracer particles continuously for the designated periods of time. At each time, random sections were searched for AV+. The results are categorized according to AV types and plotted for the relative distribution of tracers. The total tracer counts are given on top of each bar. The number in each block represents the actual number of AV profiles, from which tracer counts are scored.
Figure 5Fusion profiles. Cells exposed to 5-nm gold tracers for 20 (a and c) and 25 min (b) before fixation. Sections were picked up by MU and air-dried (c) or underwent immunolabeling with anti-SOD antibodies (a and b). (a) Fusion between a small VE (arrow) and an AVi. A cluster of two gold particles (arrowhead) is also seen at another location. (b) Fusion between an electron lucent MVE (arrow) and an AVd. (c) Fusion between an MVE (arrow) and an AVd. Bars, 200 nm.
Figure 6Endosome types involved in FP+. The FP+ were classified according to MVE and VE involved. The actual numbers of FP+ analyzed are given above the bars.
Figure 7ASGP-R labeling in FP+ and endosomes. Cells exposed for 40 min to 5-nm gold tracer. Section immunolabeled for ASGP-R with 10-nm gold. (a) Low ASGP-R labeling in tracer-carrying endocytic vesicles (arrowheads), adjacent to (top) or fusing with (bottom) an AVi/d. (b) ASGP-R labeling at the plasma membrane and in endosomal elements near the cell surface (ex, extra cellular space). Vesicles of the MVE (asterisk) and VE (arrowhead) type, containing endocytosed gold, are present in the same area. (c) As b. A VE-like vesicle with endocytosed gold and low ASGP-R labeling (arrowhead) is shown connected to an ASGP-R rich element (arrow), possibly a recycling endosome. Bars, 200 nm.
Figure 8AV types involved in FP+. The same FP+ data as used in Fig. 6 were classified according to the AV types.
Figure 1(A) HeLa cells were exposed to 5 Gy ionizing radiation (γIR), collected at the indicated time points and stained for RASSF1A and γH2AX. Images for representative intra-nuclear RASSF1A foci distribution are shown. Nucleolar boundaries are marked with dashed lines.(B) Quantification of the number of cells with RASSF1A foci at the time points presented in (A). Error bars represent standard deviation and derive from 3 independent experiments.(C) HeLa cells were treated with 5 Gy γIR and 2 h post treatment fixed and stained with the indicated antibodies. (D) HeLa cells were irradiated with 5 Gy and 2 h post treatment stained for RASSF1A (R1A) and NUCLEOLIN (NCL). (E) HeLa cells were treated with 50 ng/ml NCS for 30 min, washed and 2 h later fixed and stained with the indicated antibodies. The distance of RASSF1A+/γH2AX+ foci or RASSF1A-/γH2AX+ foci to the closest nucleolus (based on Fibrillarin (Fb) staining) was measured. Middle line represents the median and the boxes 25th and 75th percentiles. The whiskers mark the smallest and largest values.(F) AsiSI expression in U2OS cells was induced by OHT and cells were stained for RASSF1A and γH2AX. Boxed areas are shown in higher magnification. Nucleolar boundaries are marked with dashed lines. (G) Quantification of the number of cells with RASSF1A foci in (F). Error bars represent standard deviation and derive from 3 independent experiments.(H) U2OS cells with stable integration of a Lac operator (LacO) sequence adjacent to an I-SceI endonuclease site were transfected with Lac Repressor (LacR)-mCherry and GFP or GFP-NLS-HA-I-SceI. 24 h post transfection cells were fixed and stained for 53BP1 or RASSF1A. (I) Quantification of the LacO arrays enriched for 53BP1 or RASSF1A in (H). Error bars represent standard deviation and derive from 3 independent experiments.(J) HeLa cells were transfected with I-PpoI endonuclease and stained for RASSF1A and γH2AX. Fluorescence intensity profiles of RASSF1A (green) and γH2AX (red) signals across the HeLa nuclei are shown. Position of line scan indicated by the yellow line.
Figure 2(A) HeLa cells were transfected with I-PpoI mRNA and collected at the indicated time points. Cells were co-stained for RASSF1A (R1A) and NBS1. Boxed areas are shown in higher magnification. Nucleolar boundaries are marked with dashed lines. (B) Quantification of (A). Error bars represent standard deviation and derive from 3 independent experiments.(C) Assessment of RASSF1A localisation after induction of rDNA DSBs using the I-PpoI endonuclease in methanol fixed HeLa cells. Fluorescence intensity profile of RASSF1A (green) and LAMIN A/C (red) signals across the HeLa nuclei in the presence and absence of rDNA DSBs in control and I-PpoI treated cells. Position of line scan indicated by the yellow line.(D) HeLa cells were transfected with the I-PpoI mRNA and 6 h later stained for RASSF1A-pS131 (R1A-pS131) and γH2AX. Fluorescence intensity profile of RASSF1A-pS131 (green) and γH2AX (red) signals across the HeLa nuclei are shown. Position of line scan indicated by the yellow line.(E) Quantification of cells with RASSF1A-pS131 positive nucleolar caps 6 h post I-PpoI transfection. Error bars represent standard deviation and derive from 3 independent experiments. Representative images are shown in Fig EV3A.(F) HeLa cells were treated with the ATMi or ATRi prior to transfection with I-PpoI mRNA. 6 h post mRNA transfection cells were stained for RASSF1A-pS131 (R1A-pS131). (G) Quantification of (F). Error bars represent standard deviation and derive from 3 independent experiments.(H) HeLa cells were treated or not with ATM inhibitor (ATMi) followed by I-PpoI mRNA transfection and 6 h later cell lysates were analyzed by western blot for the indicated antibodies.(I) HeLa cells were treated or not with ATMi followed by I-PpoI mRNA transfection and 6 h later cells were fixed and RASSF1A recruitment at rDNA DSBs was assessed. (J) Quantification of (I). Error bars represent standard deviation and derive from 3 independent experiments.
Figure 3(A) HeLa cells with I-PpoI induced rDNA breaks were stained with the indicated antibodies. Intra-nucleolar cap localization for RASSF1A and 53BP1 in correlation with other proteins was accessed. Boxed areas are shown in higher magnification.(B) HeLa cells were transfected with I-PpoI mRNA and stained for RASSF1A and 53BP1. Fluorescence intensity profiles of RASSF1A (green) and 53BP1 (red) signals across the HeLa nuclei are shown. Position of line scan indicated by the yellow line.(C) HeLa cells were treated with siRNAs against LUCIFERASE (siLUC) or 53BP1 (si53BP1), 48 h later transfected with I-PpoI mRNA, fixed and stained for RASSF1A (R1A) and γH2AX. (D) Quantification of (C) and western blot analysis from siLUCIFERASE (siLUC) or si53BP1 treated cells for the indicated antibodies. Error bars represent standard deviation and derive from 3 independent experiments.(E) HeLa cells were treated with siRNAs against LUSIFERASE (siLUC) or RNF8 (siRNF8), 48 h later transfected for I-PpoI mRNA, fixed and stained for RASSF1A and γH2AX. (F) Quantification of (E) and western blot analysis from siLUCIFERASE (siLUC) or siRNF8 treated cells for the indicated antibodies. Error bars represent standard deviation and derive from 3 independent experiments.(G) HeLa cells were treated with siRNA against 53BP1, or an ATM inhibitor (ATMi) followed by 5 Gy of ionizing radiation (γIR). Cells were fixed 2 h post treatment and stained for RASSF1A. Nucleolar boundaries are marked with dashed lines. (H) Quantification of cells with RASSF1A foci in (G). Error bars represent standard deviation and derive from 3 independent experiments.(I) Western blot analysis from total cell extracts with the indicated treatments and RASSF1A immuneprecipitates (IPs) with the indicated antibodies. IgG immunoprecipitation served as a control.(J) Western blot analysis from total cell extracts and RASSF1A immunoprecipitates with the indicated treatments with the indicated antibodies. IgG immunoprecipitation served as a control.(K) U2OS cells were transfected with FLAG-RASSF1A (FLAG-R1A), FLAG-RASSF1A131A (FLAG-R1A131A) or pcDNA3 and 24 h later transfected with the I-PpoI mRNA. Cell lysates were subjected to immunoprecipitation against FLAG. Western blot analysis of total cell extracts and the IPs is shown.
Figure 4(A) U2OS cells were transfected with either HA-53BP1WT (aa1-1972), HA-53BP1ΔBRCT (aa 1-1709), HA-53BP1ΔΝterminus (aa 921-1972) or empty pcDNA3 vector and 24 h later transfected with I-PpoI mRNA. Cell lysates were subjected to immunoprecipitation with an HA tag antibody. Western blot analysis of total cell extracts and the IPs is shown.(B) U2OS cells were transfected with either full length MYC-RASSF1A or with the indicated RASSF1A deletion mutants and 24 h later transfected with I-PpoI mRNA. Cell lysates were subjected to immunoprecipitation with a MYC-tag antibody. Western blot analysis of the total cell extracts and the IPs is shown.(C) HeLa cells were treated with siLUCIFERASE (siLUC) or si53BP1 and 48 h later transfected with I-PpoI mRNA and stained for ATM-pS1981. (D) Quantification of (C). Error bars represent standard deviation and derive from 3 independent experiments.(E-H) HeLa cells were treated with siLUCIFERASE (siLUC) or two different siRNAs against RASSF1A (siR1A_1 and siR1A_2) and 48 h later cells were transfected with I-PpoI mRNA to induce rDNA DSBs. 6 h post mRNA transfection, cells were stained for V5 to identify I-PpoI transfected cells and the other indicated antibodies. Representative images (E, F) and quantification of staining (G, H) for the indicated antibodies are shown. Error bars represent standard deviation and derive from 3 independent experiments.(I) HeLa cells were treated with siLUCIFERASE (siLUC), siRASSF1A or si53BP1 and 48 h later transfected with the I-PpoI mRNA. Cells were fixed and underwent ImmunoFISH against UBF and an rDNA probe. Boxed areas are shown in higher magnification. Nucleolar boundaries are marked with dashed lines. (J) HeLa cells were treated with siLUCIFERASE (siLUC), siRASSF1A or si53BP1 and 48 h later transfected with the I-PpoI mRNA. Cells were fixed and underwent ImmunoFISH against ATM-pS1981 and an rDNA probe. Boxed areas are shown in higher magnification. Nucleolar boundaries are marked with dashed lines.(K) HeLa cells were treated with siLUCIFERASE (siLUC) or siRASSF1A followed by I-PpoI mRNA transfection. 6 h later cells were stained for 53BP1. (L) Quantification of (K). Error bars represent standard deviation and derive from 3 independent experiments.(M) U2OS cells were transfected with MYC-RASSF1A (1-340) or a RASSF1A mutant that lacks the SARAH domain, MYC-RASSF1A (1-288). Cells were transfected with I-PpoI mRNA and stained with the indicated antibodies.(N) U2OS cells were transfected with MYC-RASSF1A (1-340) or MYC-RASSF1A (1-288). 24 h later cells were transfected with I-PpoI mRNA and harvested after 6 h. Cell lysates underwent cell fractionation and the chromatin bound fraction was analyzed by western blot.(O) U2OS cells were treated with siRNA against RASSF1A followed by transfection with MYC-tag, MYC-RASSF1A (1-340) or MYC-RASSF1A (1-288). 24 h later cells were transfected with I-PpoI mRNA and stained for MYC expression and ATM-pS1981.(P) Quantification of cells with ATM-pS1981 positive nucleolar caps in (O). Error bars represent standard deviation and derive from 3 independent experiments.