EMBO/SourceData huggingface dataset:
Figure 4Volcano plot displaying DEGs in starved cct3A(RNAi) planarians compared to controls at 72hR (q-value < 0.05). Y-axis indicates the negative log10 of the false discovery rate (FDR) (q- value). X-axis indicates the beta values (b), a biased estimator of the fold change. For better visualization, 13 dots with -log10 (q-value) > 20 are not displayed. This includes cct3A with q = 0. Notice that when down-regulating cct3A in starved planarians, xbp1, atf6, bip-1 and a repertoire of ER chaperones are down-regulated, whereas dnajb9, a repressor of the UPR, and other ccts are up-regulated.Live images show that xbp1/atf6(RNAi) planarians in starved conditions form a minimal blastema when compared to controls while at the feeding condition most of them can regenerate as controls. The remaining planarians in the starved condition are dead by the time point of regeneration shown. In the feeding condition, the remaining planarians at 8-10dR are either dead (13/145) or have a tiny blastema and died or regenerated later (40/145) while at 21-27dR are dead.Maximum projections of representative regenerating trunks after FISH for smedwi-1 in starved conditions. The dashed line delimits the body of the planarian.TUNEL maximum projections of representative blastemas from regenerating trunks in starved conditions. The dashed line delimits the body of the planarian. The graph shows the number of TUNEL+ cells per mm2 at the same time points in anterior (A) and posterior (P) blastemas. n ≥ 6 planarians per time point.Quantification of the percentage of stem cells in different mitotic phases and the percentage of defective mitotic figures in 72hR anterior blastemas of xbp1/atf6 RNAi and controls in starved conditions after double immunostaining with anti-tyrosine-tubulin and anti-H3P (****P < 0.0001, ***P < 0.001, *P < 0.05, n.s. indicates not significant using two-sided Chi-square test); n ≥ 5 planarians. Representative images are shown. Arrows indicate abnormal organization or number of spindle poles. j8 displays asymmetrical distribution of chromosome content.
Figure 6Volcano plots displaying shRNA-Cct3- and shRNA-luciferase-infected LSK cells cultured either in normal glucose (upper graph) or low glucose conditions (lower graph). Dots represent all the NanoString analyzed transcripts. Red dots are UPR and ER stress related genes. The UPR and ER stress genes significantly down-regulated only under low glucose conditions in shCct3 cells compared to controls are indicated near the lower graph. For better visualization two dots with q ≤ 0.001 have been removed from the volcano plot (see them in Dataset EV4). Y-axis indicates the false discovery rate (FDR) (q- value), and the X-axis indicates the log2 fold changes. The FDR-based method of p-value adjustment was conducted to calculate the q-values by nSolver software using Benjamini-Hochberg methods. Significance is established by q-value < 0.05 and indicated by the dotted horizontal line. n= 3 mice.
Figure 7The Kaplan-Meier curve demonstrates increased survival of starved cct3A(RNAi) animals when treated with palmitic acid/palmitoylcarnitine (PA/PC). Percentages indicate the number of survivals at the indicated time points. Three asterisks indicate P < 0.001 with log-rank (Mantel-Cox) test.
Figure 1(A) Western blot of SMC3-LAP expressing HeLa cells treated with siRNA as indicated and synchronized in G2-phase. Efficiency of RNA interference and SMC3 acetylation levels were analyzed from total extracts and after chromatin fractionation (asterisks indicate unspecific signals that were depleted from antibody dilutions used in later experiments, presumably by repeated usage of the antibody samples). Note that ESCO1 levels were increased on chromatin after depletion of ESCO2, raising the possibility that a decrease in chromatin bound ESCO2 might be compensated for by ESCO1 recruitment to chromatin.(B) Still images of an inverse fluorescence recovery after photobleaching (iFRAP) experiment. Fluorescence intensities are false-colored as indicated. Size bar, 5 μm.(C) Graph depicting the normalized intensity after photobleaching from two biological replicates to quantify chromatin dissociation kinetics. Error bars denote standard error of the mean (s.e.m.), n = 10 cells per condition.(D) Quantification of the relative abundance of SMC3-LAP in the stable chromatin binding mode.
Figure 2(A) Quantification of iFRAP experiments using HeLa cells expressing SMC3-LAP in the wild-type form (wt) or with lysines 105-106 mutated to glutamines (QQ) or arginines (RR) and synchronized in G1-phase. Error bars denote s.e.m., n > 25 cells per condition.(B) Quantification of chromatin residence time of SMC3-LAP alleles in G1-phase.(C) Quantification of iFRAP experiments using cells synchronized in G2-phase. Error bars denote s.e.m., n > 26 cells per condition.(D) Quantification of stably bound SMC3-LAP alleles in G2-phase.(E) Western blot of mouse fibroblasts expressing SMC3-LAP in the presence or absence of endogenous Smc3 after gene deletion. Extracts were prepared after subculturing two times. Cells without SMC3-LAP do not proliferate after deletion of Smc3.(F) Western blot showing HeLa cell extracts after treatment with siRNA as indicated and synchronized in G2-phase. Note that ESCO1 immunoblot signals were also reduced in sororin depleted cells. We currently do not know if this is an effect of sororin depletion, an off-target effect or an artefact of unequal sample loading.(G) iFRAP quantification of stably chromatin bound SMC3-LAP after siRNA treatment using cells synchronized in G2-phase. Error bars denote s.e.m., n > 11 cells per condition.
Figure 3(A) FACS profile of cells transfected with nondegradable KEN-box mutant sororin-FLAG (sorKBM-FLAG) after synchronization in G1-phase and propidium iodide staining.(B) Immunofluorescence microscopy experiment to determine transfection efficiency.(C) Western blot of fractionated lysates to detect sororinKBM-FLAG.(D) Quantification of SMC3-LAP residence time on chromatin after transfecting cells with sorKBM-FLAG in G1-phase. Error bars denote s.e.m.; n > 15 cells per condition.(E-F) Analysis of cell cycle distribution and transfection efficiency using cells synchronized in G2-phase.(G) Quantification of stably chromatin-bound SMC3-LAP from cells expressing nondegradable sororin in G2-phase. Cells were treated with siRNA as indicated.(H) Western blot showing fractionated and nuclease digested chromatin from cells synchronized in G1 and G2-phase after immunoprecipitation with FLAG antibodies.
Figure 5(A) Schematic representation of wild-type (wt), targeted (flx) and deleted (Δ) Cdca5 alleles (left) and Southern blot of mouse genomic DNA (right). Red triangles, loxP sites.(B) Observed and expected genotype distribution among offspring of heterozygous Cdca5 Δ/+ mice (newborn mice, n = 136; E9.5, n = 54).(D) Western blot of cell extracts to analyze depletion of sororin protein from fibroblasts after tamoxifen-induced Cre expression. Extracts were prepared either one or two days after release from the arrest as indicated. Chr, fractionated chromatin; sol, soluble proteins.(E) Proliferation curve of cells with the indicated genotype after tamoxifen treatment.(F) Observed cell cycle distribution of fibroblasts treated with tamoxifen under starvation and subcultured in rich medium.(G) Quantification of mitotic cells with misaligned chromosomes after Cdca5 deletion and proliferation for 36 to 60 hours. Size bar, 10 µm; error bars denote s.e.m.; n > 500 cells per condition.(H) Phenotypic classification of nuclear morphology after Cdca5 deletion and proliferation for 5 to 7 days. Size bar, 10 µm; error bars denote s.e.m.; n > 500 cells per condition.(I) Western blot showing protein depletion efficiency. Cre expression was induced with tamoxifen for 24 hours in proliferating Smc3 and Cdca5 conditional knockout immortalized fibroblasts.(J) Analysis of chromosome spreads after Smc3 or Cdca5 deletion and nocodazole treatment. Representative images from every category are shown; size bar, 10 µm; error bars denote s.e.m.; n > 500 per genotype.
Figure 6(A) Schematic illustration of the LAP-AID-tag integrated into the bacterial artificial chromosome containing the mouse Cdca5 locus.(B) Immunofluorescence staining of cells expressing sor-LAP-AID and the F-box helper protein after treatment with auxin. Antibodies against GFP mark the fusion protein and Aurora B antibody stains cells in G2/M-phase. Scale bar, 10 µm.(C) Quantification of immunofluorescence after treating cells with auxin. Error bars denote s.e.m.; a.u., arbitrary units.(D) Time course quantification of GFP immunofluorescence signal decrease after auxin addition.(E) Analysis of chromosome spreads after auxin treatment.(F) Western blot showing cell extracts after treatment with auxin.
Figure 7(A) Cell cycle distribution of immortalized Cdca5 Δ/Δ sor-LAP-AID fibroblasts after 24-hour incubation with aphidicolin and release.(B) Western blot showing cell extracts after incubation with aphidicolin and release.(C) Still images of a fluorescence recovery after photobleaching experiment. Size bar, 5 μm. Bleach area radius, 2 µm.(D) Graph depicting the normalized intensity after photobleaching to quantify turnover of sororin (n = 15).(E) Quantification of the relative abundance of sororin-LAP-AID on chromatin. Error bars denote s.e.m.(F) Quantification of the chromatin residence time of sor-LAP-AID at different times. Error bars denote s.e.m.
Figure 8(B) Quantification of EdU-labeled prometaphase cells after treating cells with auxin and EdU at indicated time points. Error bars denote s.e.m.; n > 200 cells per condition.(C) Analysis of chromosome spreads after auxin and EdU treatment and Giemsa staining.(D) Representative picture of the most prominent phenotype class upon auxin and EdU treatment and immunofluorescence microscopy. Scale bar, 10 µm.(E) Analysis of chromosome spreads after auxin treatment and EdU and DAPI labeling. Error bars denote s.e.m.; n > 200 cells per condition.
Figure 1Phloroglucinol staining of wild-type seedlings inoculated with PTI-inducing Pst DC3000 hrcC-, virulent Pst DC3000, and avirulent Pst DC3000 (AvrRpm1) and Pst DC3000 (AvrRpt2). Data information: Twelve-day-old seedlings were flood-inoculated with P. syringae at 108 cfu/ml in (A hrcC-, Pst DC3000 hrcC-; DC3000, Pst DC3000; AvrRpm1, Pst DC3000 (AvrRpm1); AvrRpt2, Pst DC3000 (AvrRpt2); dpi, days post-inoculationB Phloroglucinol staining of wild-type adult leaves inoculated with PTI-inducing Pst DC3000 hrcC-, virulent Pst DC3000, and avirulent Pst DC3000 (AvrRpm1) and Pst DC3000 (AvrRpt2). The upper images are enlarged ones of the lower boxes at 2 dpi. Scale bars, 100 μm. Data information Six-week-old leaves were syringe-infiltrated with P. syringae at 108 cfu/ml in (B hrcC-, Pst DC3000 hrcC-; DC3000, Pst DC3000; AvrRpm1, Pst DC3000 (AvrRpm1); AvrRpt2, Pst DC3000 (AvrRpt2); dpi, days post-inoculation; IS, infected site; UIS, uninfected site.C Quantification of lignin content in pathogen-treated seedlings Data are shown as means ± SD (n = 4; 20-30 seedlings each). Data information: Twelve-day-old seedlings were flood-inoculated with P. syringae at 108 cfu/ml Significant differences are indicated by different letters (Tukey's HSD test; P < 0.05) Experiments were repeated 3 times with similar results. hrcC-, Pst DC3000 hrcC-; DC3000, Pst DC3000; AvrRpm1, Pst DC3000 (AvrRpm1); AvrRpt2, Pst DC3000 (AvrRpt2); dpi, days post-inoculationD Quantification of lignin content in pathogen-treated wild-type leaves Data are shown as means ± SD (n = 4; 3-9 leaves each). Data information: Six-week-old leaves were syringe-infiltrated with P. syringae at 108 cfu/ml Significant differences are indicated by different letters (Tukey's HSD test; P < 0.05) Experiments were repeated 3 times with similar results. hrcC-, Pst DC3000 hrcC-; DC3000, Pst DC3000; AvrRpm1, Pst DC3000 (AvrRpm1); AvrRpt2, Pst DC3000 (AvrRpt2); dpi, days post-inoculationE Quantification of lignin content in wild-type leaves treated with different titers of Pst DC3000 (AvrRpm1). Data are shown as means ± SD (n = 4; 3-9 leaves each). Six-week-old leaves were syringe-infiltrated with P. syringae at the indicated titers in (E). Significant differences are indicated by asterisks (t test; *P < 0.05; **P < 0.01; ***P < 0.001). Experiments were repeated 3 times with similar results. dpi, days post-inoculationF Quantification of lignin content in wild-type and rpm1-3 rps2-101c leaves treated with avirulent bacteria. Data are shown as means ± SD (n = 4; 3-9 leaves each). Six-week-old leaves were syringe-infiltrated with P. syringae at 108 cfu/ml Significant differences are indicated by asterisks (t test; *P < 0.05; **P < 0.01; ***P < 0.001). Experiments were repeated 3 times with similar results. AvrRpm1, Pst DC3000 (AvrRpm1); AvrRpt2, Pst DC3000 (AvrRpt2); dpi, days post-inoculation
Figure 2B Quantification of lignin content in palQ leaves after the indicated treatments and pathogen infection. Data are shown as means ± SD (n = 4; 3-9 leaves each). Data information: Six-week-old leaves were inoculated with Pst DC3000 (AvrRpm1) at 108 cfu/ml for experiments. Different letters indicate significant differences (Tukey's HSD test; P < 0.05). Experiments were repeated 3 times with similar results. M, mock; PA, piperonylic acid; CA, coniferyl alcohol; dpi, days post-inoculation.C Measurements of Pst DC3000 (AvrRpm1) growth. Data are shown as means ± SD (n = 3). Data information: Six-week-old leaves were inoculated with Pst DC3000 (AvrRpm1) at 105 cfu/ml for growth assays Different letters indicate significant differences (Tukey's HSD test; P < 0.05). Experiments were repeated 3 times with similar results. M, mock; PA, piperonylic acid; CA, coniferyl alcohol; dpi, days post-inoculation.D Cell death phenotypes of leaves inoculated with Pst DC3000 (AvrRpm1). E Quantification of leaves (n ≥ 30) with spreading cell death as in (D). Data information: Six-week-old leaves were inoculated with Pst DC3000 (AvrRpm1) at 105 cfu/ml for growth assays M, mock; PA, piperonylic acid; CA, coniferyl alcohol; dpi, days post-inoculation.F Quantification of total and free SA in Col-0 and palQ leaves after the indicated treatments and pathogen infection. Data are shown as means ± SD (n = 5; 6 leaves each). Data information: Six-week-old leaves were inoculated with Pst DC3000 (AvrRpm1) at 108 cfu/ml for experiments. Different letters indicate significant differences (Tukey's HSD test; P < 0.05). Experiments were repeated 3 times with similar results. M, mock; PA, piperonylic acid dpi, days post-inoculation.
Figure 3A Colonization patterns of GFP-Pst DC3000 and Pst DC3000 (AvrRpt2) in wild-type plants. Data information: Leaves were inoculated with GFP-labeled bacteria at 108 cfu/ml for 2 days. IS, infected site; UIS, uninfected site. White dash lines indicate the boundary between IS and UIS. Scale bars, 100 μmB Time-lapse images of GFP-Pst DC3000 and Pst DC3000 (AvrRpt2) captured at 0 (magenta), 15, (yellow), and 30 (green) sec Time in seconds in the movies is shown at the top of images. Blue color was used for chlorophyll autofluorescence. Data information: Leaves were inoculated with GFP-labeled bacteria at 108 cfu/ml for 2 days. Scale bars, 10 μmC Colonization patterns of GFP-Pst DC3000 (AvrRpt2) in wild-type and palQ plants after PA and CA pretreatments. Data information: Leaves were inoculated with GFP-labeled bacteria at 108 cfu/ml for 2 days. M, mock; PA, piperonylic acid; CA, coniferyl alcohol; IS, infected site; UIS, uninfected site. White dash lines indicate the boundary between IS and UIS. Scale bars, 100 μm
Figure 4A CANBD- and CADMAC-incorporated lignin polymerization. CANBD (green) and CADMAC (blue) fluorescence was observed in the lignified infection site at 2 dpi, as revealed by phloroglucinol staining. Data information: Bacterial inoculum was at 108 cfu/ml. IS, infected site; UIS, uninfected site White dash lines indicate the boundary between IS and UIS. Scale bars, 500 μmB Cross-sections of CANBD- and Pst DC3000 (AvrRpm1)-treated leaves. Data information: Bacterial inoculum was at 108 cfu/ml. hpi, hours post-inoculation Scale bars, 40 μmC Cross-section of the entire leaf with one half leaf infected. Data information: Bacterial inoculum was at 108 cfu/ml. IS, infected site; UIS, uninfected site; hpi, hours post-inoculation Scale bars, 120 μmD CADMAC-incorporated lignin deposition in the GFP-Pst DC3000 (AvrRpt2)-infected site at 2 dpi. Data information: Bacterial inoculum was at 108 cfu/ml. IS, infected site; UIS, uninfected site DC3000, Pst DC3000; AvrRpt2, Pst DC3000 (AvrRpt2); Chl, chlorophyll. White dash lines indicate the boundary between IS and UIS. Scale bars, 100 μmE Cellular observation of CADMAC-incorporated lignin formation against infecting GFP-Pst DC3000 (AvrRpt2). Data information: Bacterial inoculum was at 108 cfu/ml. hpi, hours post-inoculation Scale bars, 20 μmF 3D image of CANBD-polymerized structure. Images were taken at 24 hpi Data information: Bacterial inoculum was at 108 cfu/ml. Scale bars, 20 μm
Figure 5A Time course expression profiles of CASPLs in response to Pst DC3000 (AvrRpm1) treatment. Analysis was based on the Genevestigator database (http://www.genevestigator.ethz.ch/). The darker color corresponds to stronger expression. ND, not determined.Quantification of lignin content in wild-type and caspl mutant leaves after Pst DC3000 (AvrRpm1) inoculation. ata are shown as means ± SD (n = 4; 3-9 leaves each). ata information: Different letters indicate significant differences (Tukey's HSD test; P < 0.05). Experiments were repeated 3 times with similar results. AvrRpm1, Pst DC3000 (AvrRpm1); dpi, days post-inoculation.C Cell death phenotypes of leaves inoculated with Pst DC3000 (AvrRpm1). Data information: AvrRpm1, Pst DC3000 (AvrRpm1) dpi, days post-inoculation.D Quantification of leaves (n ≥ 30) with spreading cell death after Pst DC3000 (AvrRpm1) inoculation. dpi, days post-inoculation.E Measurements of Pst DC3000 (AvrRpm1) and Pst DC3000 (AvrRpt2) growth. Data are shown as means ± SD (n = 3). Data information: Different letters indicate significant differences (Tukey's HSD test; P < 0.05). Experiments were repeated 3 times with similar results. AvrRpm1, Pst DC3000 (AvrRpm1); AvrRpt2, Pst DC3000 (AvrRpt2); dpi, days post-inoculation.F Colonization patterns of GFP-Pst DC3000 (AvrRpt2) in caspl mutant plants. IS, infected site; UIS, uninfected site. White dash lines indicate the boundary between IS and UIS. Scale bars, 100 μm.
Figure 6A Visualization of CASPL4D1 proteins and lignin by mCherry and CADMAC fluorescence. Data information: pCASPL4D1::CASPL4D1-mCherry plants were pretreated with CADMAC and then inoculated with Pst DC3000 (AvrRpm1). mCh, mCherry; Chl, chlorophyll; hpi, hours post-inoculation. Scale bars, 20 μm (A)B Magnified views of CASPL4D1 and lignin localization. Images in boxes were enlarged in the 2nd to 4th columns. Arrows indicate lignin deposition between cells. Data information: pCASPL4D1::CASPL4D1-mCherry plants were pretreated with CADMAC and then inoculated with Pst DC3000 (AvrRpm1). mCh, mCherry; Chl, chlorophyll; hpi, hours post-inoculation. Scale bars 10 μm (B).C Relative fluorescence intensity profiles of CASPL1D1 (mCherry) and lignin (CADMAC) across transects indicated by dotted lines Data information: pCASPL4D1::CASPL4D1-mCherry plants were pretreated with CADMAC and then inoculated with Pst DC3000 (AvrRpm1).D A model showing morphological changes of CASPL- and lignin-deposited cells during the HR response.
Figure 2C,D,E Examples of embryos expressing the tFT-Bcd reporter with C: mCherry-sfGFP-Bcd, D: fmCherry-sfGFP-Bcd, and E: mCherry-sfGFP. For all, (i) Images of "shells" of embryos in early n.c. 14. 3D images of the embryos were generated, and then the interior of the embryo was erased, leaving only the embryo cortex to improve clarity (ii) Mean AP intensity profile of each color for the embryo in i. Shade region represents ±1 s.d. Inset shows same profiles after multiplication of red intensity by constant factor. Data binned into 10 µm bins (n=4 embryos). (iii) Ratio of green over red signal, reflecting protein age. The thin lines represent individual embryos, while the thick solid line is the mean. The solid dashed line depicts the mean green/red ratio for a line with the tFT-Bcd reporter but lacking endogenous Bcd (BcdE1 mutant, n=4).
Figure 4,B,C Injection of the proteasome inhibitor MG132 in embryos increases both the amount and the average age of Bcd (gray, open squares, N=10). Control embryos were injected with DMSO (black, filled circles, N=9). All embryos are y/w and were injected while in cycle 14 and imaged 60 min later.D The age of Bcd and its gradient are unaltered in fsd mutant. Comparison of a representative y/w embryo (left) and loss-of-function mutation fsdKG02393 (right) are shown. Both embryos have the tFT-Bcd construct in the same locus, are in early n.c. 14, were imaged side-by-side under identical conditions, and are displayed with the same settings. Embryo "shells" are displayed.E Quantification of embryos Fluorescence intensity from sfGFP (green) and mCherry (magenta) for wt (N=8, filled circles) and fsd (N=6, open squares) at early cycle 14. Inset: tFT-Bcd ratio for wt (black) and FSD (gray) embryos.
Figure 1 Immunofluorescence microscopy of HEK293T exogenously expressing ATF6α-V5 tagged. Cells were fixed and stained with anti-calnexin (green) or anti-V5 (red). An overlay of the cells is presented in the third panel. Bar = 10 µm.  Whole cell lysates from HEK293T cells, with or without stable expression of ATF6α (as indicated) were immunoisolated with mouse anti-V5. RCN1 was transiently transfected into both cell lines in the experiment depicted in panel 3. Immunoisolated material was separated by SDS-PAGE under reducing conditions followed by western blotting to determine the co-isolation of ER proteins with ATF6α. Ig-HC (anti-BiP blot) indicates immunoglobulin heavy chains. Whole cell lysates from HEK293T cells stably expressing ATF6α were either untreated (-) or treated (+) with 5 µM thapsigargin (TG) for 1 h. THBS4 was transiently transfected into cells in the experiment depicted in panel 2. ATF6α was either immunoisolated with anti-V5 (panel 1 and 3) or anti-HA (panel 2). For the experiment in panel 3 the cells were cross-linked prior to immunoisolation. Samples were separated by SDS-PAGE under reducing conditions followed by western blots to determine co-isolation of endogenous PDIR, ERp18 or exogenously expressed THBS4 with ATF6α. HC indicates immunoglobulin heavy chain.
Figure 2HEK293T cells stably expressing ATF6α were either left untransfected (UT) or transfected with substrate-trapping mutants of PDI oxidoreductases. ATF6α was immunoisolated from cell lysates with mouse anti-ATF6α, separated by SDS-PAGE under non-reducing conditions and ATF6α detected by western blotting using rabbit anti-HA. Blots confirm mixed-disulfide complexes between ATF6α and ERp18, ERp57, and PDIR indicated with arrows (lanes 2, 4, 5). M, D and O refer to ATF6α monomer, dimer and oligomer respectively. Whole cell lysates of HEK293T cells expressing an ATF6α mutant containing cysteine to alanine mutations in the lumenal domain (ATF6 DM) were transfected with PDI substrate-trapping mutants. ATF6α was immunoisolated with mouse anti-ATF6α, separated by SDS-PAGE under non-reducing conditions, and ATF6α detected with rabbit anti-HA.