EMBO/soda-nel huggingface dataset:
Error-free DNA damage tolerance pathway is facilitated by the Irc5 translocase through cohesin (2018)
Figure 6

Figure 6. Cohesin loading complex allows replication completion in the presence of MMS-induced DNA damage by facilitating cohesin association with chromatin at stalled replication forks.

A, B ChIP-qPCR analysis of Scc1 association with early replication origin ARS305 or ARS607. HA-tagged Scc1 interaction with chromatin was monitored in wild type (Y39) and scc2-4 (Y4366) cells. Error bars represent mean value ± standard deviations of mean (n=3).

C, D ChIP-qPCR analysis of Pol2 association with early replication origin ARS305 or ARS607. HA-tagged Pol2 interaction with chromatin was monitored in wild type (TB067) and scc2-4 (TB068) cells. Error bars represent mean value ± standard deviations of mean (n=3).

E Cohesin loading complex is required for completion of chromosome replication after MMS treatment. Intact chromosomes were isolated from wild type (Y39) and scc2-4 (Y4366) cells cultivated at permissive temperature (25°C) and processed for PFGE. M, MMS.

F Quantification of data presented in E. Error bars represent mean value ± standard deviations of mean for the intensity values of three distinct chromosome bands representing chromosome XII or III (n=3). Values for G1-synchronized cells were set to 100.

G Sensitivity of scc2-4 to DNA damaging agents at permissive temperature (25°C) or semi-permissive temperature (30°C). Logarithmically growing cultures of wild type (Y39), and scc2-4 (Y4366) strains were 10-fold serially diluted and plated onto solid YPD containing indicated concentrations of DNA damaging agents.
Error-free DNA damage tolerance pathway is facilitated by the Irc5 translocase through cohesin (2018)
Figure 7

Figure 7. Irc5 and cohesin work in a common pathway during MMS-induced replication stress.

A Sensitivity of scc2-4 and scc2-4 irc5-Δ1 mutants to DNA damaging agents at permissive temperature 25°C. Logarithmically growing cultures of wild type (W303-1a), irc5-Δ1 (IL012), scc2-4 (TB069) and irc5-Δ1 scc2-4 (TB070) strains were 10-fold serially diluted and plated onto solid YPD containing indicated concentrations of DNA damaging agents.

B, C Levels of DNA repair foci in scc2-4 and scc2-4 irc5-Δ1 mutants. Logarithmically growing cultures of scc2-4 (TB071 or TB073) and scc2-4 irc5-Δ1 (TB072 or TB074) were treated with MMS for 1 hour at 25°C. Next, cells were washed and processed for microscopic analysis. Error bars represent mean value ± standard deviations of mean (n=3).

D Sensitivity of scc1-73 and scc1-73 irc5-Δ1 mutants to DNA damaging agents at permissive temperature 25°C or semi-permissive temperature 30°C. Logarithmically growing cultures of wild type (W303-1a), irc5-Δ1 (IL012), scc1-73 (JC1339) and irc5-Δ1 scc1-73 (TB075) strains were 10-fold serially diluted and plated onto solid YPD containing indicated concentrations of DNA damaging agents.

E, F Levels of Rfa1-YFP and Rad52-YFP foci in scc1-73 and irc5-Δ1 scc1-73 mutants. Logarithmically growing cultures of scc1-73 (TB076 or TB078) and irc5-Δ1 scc1-73 (TB077 or TB079) were treated with MMS for 1 hour at 25°C. Next, cells were washed and processed for microscopic analysis. Error bars represent mean value ± standard deviations of mean (n=3).

G Replication completion defect of scc1-73 is not exacerbated by IRC5 disruption. Intact chromosomes were isolated from scc1-73 (JC1339) and irc5-Δ1 scc1-73 cells (TB075), cultured at 25°C, and processed for PFGE. M, 0.03% MMS.

H Quantification of data presented in G. Error bars represent mean value ± standard deviations of mean for the intensity values of two distinct chromosome bands representing chromosome XII or III (n=2). Values for G1 were set to 100.
Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4 (2007)
Figure 2

ROS formation occurs partially downstream of class III PI3K activation. (A) Left panel: HeLa cells were starved for 2 h in the presence or absence of either 100 nM wortmannin or 10 mM 3MA, after which they were treated with DCFDA and analyzed by a fluorimeter as described above. Right panel: the rate of degradation of long‐lived proteins was measured in cells incubated in either α‐MEM medium or EBSS medium in the absence or presence of 100 nM wortmannin or 10 mM 3MA. Values are represented as the means±s.d. of three separate determinations. (B) WT MEFs or Atg5 (−/−) MEFs from two separate clones were starved for 2 h before treatment with DCFDA and visualization or fluorometric analysis.
Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4 (2007)
Figure 3

ROS accumulation is essential for autophagy. (A) Upper panel: CHO cells stably transfected with GFP‐GATE‐16 were preincubated in the presence or absence of 10 mM NAC or 1000 μ/ml catalase for 10 min before starvation for 2 h in the presence or absence of these drugs, or grown in a control medium containing the drugs for 2 h. The cells were then fixed, permeabilized and stained with anti‐GFP monoclonal antibodies. Representative images are shown. Lower panel: HEK 293 cells were transfected with GFP‐GATE‐16. At 24 h post‐transfection, the cells were treated with NAC or catalase and starved as explained above, lysed in Ripa buffer and 100 μg of each lysate was separated on 10% SDS–PAGE and subsequently analyzed with anti‐GFP antibodies to detect the transfected GATE‐16 and anti‐tubulin antibodies as control. The data were quantified using NIH image program and are depicted as the percentage of lipidated protein from the total GATE‐16. (*) indicates non‐lipidated and (**) indicates lipidated GFP‐GATE‐16. (B) CHO cells treated with NAC or catalase and starved as detailed in (A) were incubated with DCFDA and visualized by a confocal microscope or analyzed by a fluorimeter as in Figure 1. (C) The rate of degradation of long‐lived proteins was measured in CHO cells incubated in either α‐MEM medium or EBSS medium, or following pretreatment with 10 mM NAC for 10 min or with 1000 μ/ml catalase overnight. A representative experiment is shown.
Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4 (2007)
Figure 4

The activity of endogenous Atg4 is inhibited under nutrient starvation. (A) CHO cells (for mammalian Atg8s) or cells of S. cerevisiae (for ScAtg8) were incubated in starvation medium (EBSS or SD‐N, respectively) for different time periods as indicated before isolation of RNA by the Tri‐reagent as explained in Materials and methods. For each gene, data obtained from non‐starved cells were set to an arbitrary value of 1, and results from the corresponding starved cells were normalized accordingly. Results represent the means±s.d. of three separate experiments. (B) HEK 293 cells were grown in control medium or starved for 2 h in EBSS in the absence or presence of 100 nM bafilomycin A1. Lysates (100 μg) obtained in Ripa buffer were run on 12% SDS–PAGE and analyzed by Western blot, using anti‐GATE‐16 or anti‐tubulin antibodies. (C) CHO cells were grown in a control medium in the presence or absence of 1 mM H2O2 for 1 h, or starved for 3 or 13 h. Lysates (10 μg) obtained in Ripa buffer were incubated with recombinant His6‐GATE‐16‐HA (0.3 μg) in 50 KT reaction buffer (25 mM Tris, pH 7.4, 50 mM KCl) at 30°C for 45 min, in the presence or absence of 1 mM DTT. The reaction was stopped by addition of sample buffer and boiling, after which the samples were resolved on 15% SDS–PAGE and subsequently analyzed by Western blot, using anti‐His monoclonal antibodies. The experiment was repeated six times; a representative blot is shown. (D) Left panel: HeLa cells transfected with Myc‐GATE‐16‐HA or with an empty vector as control were labeled with [35S]methionine for 10 min and lysed immediately or chased for 1 h before lysis in Ripa buffer. Lysates were immunoprecipitated using anti‐Myc antibodies and the immunoprecipitates were resolved on 15% SDS–PAGE. Middle panel: HeLa cells transfected with Myc‐GATE‐16‐HA were kept in control medium or starved for 30 min or 13 h in EBSS before labeling with [35S]methionine for 10 min, immediate lysis, analysis by SDS–PAGE and quantification (right panel) using NIH image program. Values are presented as the average percentage of unprimed form out of the total GATE‐16. (*) in all sections of this figure indicates non‐cleaved His6‐GATE‐16‐HA or Myc‐GATE‐16‐HA and (**) indicates cleaved His6‐GATE‐16 or Myc‐GATE‐16.
Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4 (2007)
Figure 5

H2O2 directly inhibits the activity of HsAtgA. (A) Cleavage activity was tested by incubation of recombinant His6‐HsAtg4A (0.1 μg) and His6‐GATE‐16‐HA (0.3 μg) in 50 KT reaction buffer at 30°C for 45 min in the presence of indicated concentrations of DTT followed by Western blot analysis, using anti‐His monoclonal antibodies. The resulting bands from three separate experiments were quantified with a densitometer using the Bio‐Rad Multi‐Analyst program and are presented as the average percentage of cleaved form out of the total GATE‐16. (*) indicates non‐cleaved His6‐GATE‐16‐HA and (**) indicates cleaved His6‐GATE‐16. (B) His6‐HsAtg4A was incubated in the presence of 200 μM DTT at 4°C for 10 min. Reduced His6‐HsAtg4A (0.1 μg) was then incubated in 50 KT (to obtain 15 μM DTT) with the indicated concentrations of H2O2 at 25°C for 5 min, after which recombinant His6‐GATE‐16‐HA (0.3 μg) was added and incubation proceeded at 30°C for 45 min. Reaction mixtures from three separate experiments were analyzed and are presented as explained in (A). (C) His6‐HsAtg4A (0.1 μg) was incubated with recombinant His6‐GATE‐16‐HA (0.3 μg) after the following procedures: no treatment (lane 1); pretreatment with 200 μM DTT at 25°C for 5 min (lane 2); treatment with 200 μM DTT followed by treatment with 1 mM H2O2 at 25°C for 5 min (lane 3); and treatment with 200 μM DTT followed by treatment with 1 mM H2O2 for 5 min and then 2 mM DTT (lane 4). Reaction mixtures were analyzed by Western blot using anti‐His monoclonal antibodies.
Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4 (2007)
Figure 7

Mutation in Cys81 reduces the redox sensitivity of HsAtg4A in vitro. (A) Recombinant His6‐HsAtg4AWT, His6‐HsAtg4AC77A or His6‐HsAtg4AC81S (0.1 μg) was incubated with His6‐GATE‐16‐HA (0.3 μg) in 50 KT reaction buffer at 30°C for 45 min in the presence or absence of 1 mM DTT. Reaction mixtures were analyzed by Western blot using anti‐His monoclonal antibodies. (*) indicates non‐cleaved His6‐GATE‐16‐HA and (**) indicates cleaved His6‐GATE‐16. (B) His6‐HsAtg4AC81S (0.1 μg) was incubated with recombinant His6‐GATE‐16‐HA (0.3 μg) following the same procedure as detailed in Figure 5C. Reaction mixtures were analyzed and are presented as explained in (A).
Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4 (2007)
Figure 8

Cys 81 is required for redox regulation of HsAtg4A in cells. (A) CHO cells stably expressing GFP‐GATE‐16 were transiently transfected with HsAtg4AWT‐Myc‐His6 or HsAtg4AC81S‐Myc‐His6. At 24 h post‐transfection, cells were starved for 2.5 h after which they were fixed, permeabilized and incubated with anti‐Myc monoclonal antibodies. Upper panel: typical images of cells transfected with the above constructs, as visualized using a confocal microscope; representative autophagosomes are indicated by arrows. Lower panel: quantification of the average number of autophagosomes per cell in the different transfectants. Images of the fixed cells were visualized using a Nikon eclipse TE300 fluorescent microscope and used for quantification of the number of autophagosomes per cell. The results presented are the means±s.d. of a total of 100 cells from three separate experiments. (*) indicates significance at P0.001. (B) Left panel: HEK 293 cells were cotransfected with GFP‐GATE‐16 and each of the Atg4A constructs mentioned in (A). At 40 h post‐transfection, the cells were starved for 2.5 h, lysed in Ripa buffer and 100 μg of each lysate was loaded to 10% SDS–PAGE and subsequently analyzed with anti‐GFP antibodies to detect the transfected GATE‐16, anti‐Myc antibodies to detect the transfected HsAtg4A and anti‐tubulin antibodies as control. (*) indicates non‐lipidated GFP‐GATE‐16 and (**) indicates lipidated GFP‐GATE‐16. Right panel: results from three separate experiments, as detailed in the left panel, were analyzed using NIH image program and quantified as follows: the amount of lipidated GFP‐GATE‐16 out of the total of GFP‐GATE‐16 was calculated for each mutant in each experiment. The value obtained for HsAtg4AWT in each experiment was set to 100% and the relative lipidation in cells transfected with the mutant was calculated accordingly.
Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4 (2007)
Figure 9

HsAtg4B is redox regulated during starvation in a similar mechanism to HsAtg4A. (A) HeLa cells were transiently transfected with GFP‐HsAtg4BWT or GFP‐HsAtg4BC78S. At 24 h post‐transfection, cells were starved for 2.5 h in the presence of 4 mM H2O2 after which they were fixed, permeabilized and incubated with anti‐LC3 polyclonal antibodies. Cells were visualized (upper panel) and quantified (lower panel) as explained in Figure 8. Representative autophagosomes are indicated in the upper panel by arrows. The results presented in the lower panel are the means±s.d. of a total of 100 cells from three separate experiments. (*) indicates significance at P0.001. (B) Left panel: HeLa cells transfected and treated as in (A) were lysed in Ripa buffer and 40 μg of each lysate was loaded to 12% SDS–PAGE and subsequently analyzed with anti‐LC3 antibodies and anti‐GFP antibodies to detect the transfected HsAtg4B and anti‐tubulin antibodies as control. (*) indicates non‐lipidated LC3 and (**) indicates lipidated LC3. Right panel: results from three separate experiments, as detailed in the left panel, were analyzed as explained in Figure 8B.
Subcellular localization of Type VI secretion system assembly in response to cell-cell contact (2022)
Figure 3

Figure 3. A periplasmic protein, TslA, is important for contact-dependent T6SS assembly in A. baylyi. (A) Main T6SS cluster of A. baylyi. Genes marked in yellow are proteins with potential regulatory functions.

(B) Time-lapse 3D-SIM imaging of sheath dynamics (TssB-sfGFP) in the absence of TslA. Larger field of view is shown as the third field of Movie EV6, left panel.

(C) Time-lapse 3D-SIM imaging of sheath dynamics (TssB-sfGFP) in ΔtslA A. baylyi strain with ectopically expressed tslA (tssB-sfGFP clpV-mCherry2 ΔtslA ampC::PtslA-tslA-KanR). Larger field of view is shown as the first field of Movie EV6, right panel. Yellow arrows indicate examples of contact-dependent sheath assembly.

(D) The frequency of contact-dependent assembly (left panel) and number of assemblies per cell per min (right panel) in cells from different strains as indicated on x-axis. Three biological replicates were performed for each strain. At least 500 cells were analyzed per replicate on 3D-SIM time-lapse imaging of sheath dynamics (TssB-sfGFP) in 3 min period. Bar graph (left panel) and scatter dot plot (right panel) show mean ± SD. Unpaired t-test was performed.

(E) Cellular localization of TslA in A. baylyi cells. Cells of the strain (tssB-sfGFP) were fixed by 2% paraformaldehyde and treated with GTE buffer, then probed with anti-TslA antibodies, followed by incubation with secondary antibodies conjugated with Alexa Fluor Plus 594. Signals were detected using a widefield microscope. Phase contrast, GFP, RFP and merged images are shown (GFP: TssB; RFP: TslA).

Data information: Scale bars and time intervals are indicated.
KUP9 maintains root meristem activity by regulating K+ and auxin homeostasis in response to low K (2020)
Figure 1

Figure 1. The primary root growth of kup9 mutants is inhibited under low-K+ conditions.

(A) Phenotypes of wild type (Col), kup9 mutants (kup9-1 and kup9-2) and the kup9-1/ProKUP9:KUP9 complementation lines (COM1 and COM2). Seeds were germinated and grown on low-K+ (LK, 50 μM) or high-K+ (HK, 5 mM) medium for 7 d. Scale bar, 1 cm.

(B) Primary root length of the plants tested in (A). Data are means ± SE (n = 25, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance, and "#" represents the control.

(C) Diagram of KUP9 gene structure. The T-DNA insertion site in the kup9-1 mutant and the mutant site in the kup9-2 mutant are indicated by the triangle and arrow, respectively. The filled boxes and lines represent exons and introns, respectively.

(D) RT-qPCR analysis of KUP9 transcript levels in various materials. Data are means ± SE (n = 3, biological replicates; each replicate contains 120 individual plants).

(E) and (F) K+ content of the various plant materials tested in (A). Data are means ± SE (n = 3, biological replicates; each replicate contains 120-150 individual plants).
KUP9 maintains root meristem activity by regulating K+ and auxin homeostasis in response to low K (2020)
Figure 2

Figure 2. Inhibition of meristem cell proliferation in the kup9 mutants leads to the short-root phenotype under low-K+ conditions.

(A) Root meristem zones of wild type (Col), kup9 mutants (kup9-1 and kup9-2) and the kup9-1/ProKUP9:KUP9 complementation lines (COM1 and COM2). Seeds were germinated and grown on LK or HK medium for 7 d. The meristem zone lengths are marked with red lines. Scale bars, 50 μm.

(B), (C) and (D) represent different zone lengths, different zone cell numbers, and cell length of the indicated seedlings germinated and grown on LK or HK medium for 7 d. MEZ, meristem zone; EZ, elongation zone; MAZ, maturation zone. Data are means ± SE (n = 10-15, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance, and "#" represents the control.
KUP9 maintains root meristem activity by regulating K+ and auxin homeostasis in response to low K (2020)
Figure 3

Figure 3. KUP9 is mainly expressed in root cells and targets to the ER.

(A) to (F) Expression of KUP9 as determined in the ProKUP9:GUS line. GUS staining of 5-day-old seedling (A), root maturation zone (B), transverse section of maturation zone (C) and (D), primary root tip (E), and root apex (F). Ep, epidermis; Co, cortex; En, endodermis; Pe, pericyle; Xy, xylem; Ph, phloem. Scale bars, 5 mm in (A), 1 mm in (B), 10 μm in (C), 5 μm in (D), 1 mm in (E), and 20 μm in (F).

(G) and (H) Expression of KUP9 protein as determined in the kup9-1/ProKUP9:KUP9-GFP transgenic plants. Green fluorescence shows the subcellular localization of KUP9-GFP. PI (propidium iodide) and ER tracker were used to indicate the positions of the plasma membrane (PM) and endoplasmic reticulum (ER) and are shown as red and cyan fluorescence, respectively. Scale bars for root tip, 10 μm in (G) and 5 μm in (H). Scale bars for EZ and MAZ, 20 μm.

(I) Co-localization analyses of KUP9-GFP with ER marker NIP1;1-mCherry in their crossing line. Scale bars, 20 μm.

(J) Co-localization analyses of KUP9-GFP with the ER marker BIP2 using immunological staining. Scale bars, 5 μm.
KUP9 maintains root meristem activity by regulating K+ and auxin homeostasis in response to low K (2020)
Figure 4

Figure 4. The maintenance of QC activity is defective in the kup9 mutants under LK conditions.

(A) QC cell observation in wild type and the kup9 mutants using the QC marker lines ProWOX5:GFP and ProQC25:GUS. Seeds were germinated on HK medium for 4 d, then the seedlings were transferred to LK or HK medium for the indicated times. Scale bars, 10 μm.

(B) QC division rates of wild type and the kup9 mutants after LK treatment for 96 h. The statistical method is described in Materials and Methods. Data are means ± SE (n = 150, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance, and "#" represents control.
KUP9 maintains root meristem activity by regulating K+ and auxin homeostasis in response to low K (2020)
Figure 7

Figure 7. Increase of auxin level in QC cells can rescue the short-root phenotype of kup9 mutants.

(A) and (C) Phenotypes of the kup9-1/ProWOX5:AMI1 (A) and kup9-1/ProKUP9:AMI1 (C) transgenic lines. Seeds were germinated and grown on LK or HK medium with or without IAM for 7 d. Scale bars, 1 cm.

(B) and (D) Primary root length of the plants tested in (A) and (C), respectively. Data are means ± SE (n = 25, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance, and "#" represents control.
KUP9 maintains root meristem activity by regulating K+ and auxin homeostasis in response to low K (2020)
Figure 8

Figure 8. KUP9 mediates K+ and IAA efflux in BY-2 tobacco cells and Xenopus oocytes.

(A) Expression of KUP9-GFP in BY-2 tobacco cells. HDEL-mCherry was used as an ER marker. Scale bar, 10 μm.

(B) 3H-IAA accumulation in the BY-2 cells expressing the indicated proteins. PIN4-GFP and KUP10-GFP were used as positive control and negative control, respectively. Data are means ± SE (n = 3, biological replicates; each replicate contains 3-4 mL suspension cells).

(C) Expression of KUP9-GFP and OST4-GFP-KUP9 in Xenopus oocytes. Scale bar, 100 μm.

(D) 3H-IAA accumulation in the oocytes expressing KUP9-GFP and PIN4 at the indicated times. PIN4 was used as a positive control. The oocytes were incubated in the bath solution containing 3H-IAA for the indicated times. Data are means ± SE (n = 6, biological replicates; each replicate contains 6 oocytes).

(E) IAA content in the oocytes expressing the indicated proteins. The oocytes were injected with the same amount of IAA and incubated in IAA-free bath solution for 6 h. IAA content was measured using UPLC-MS. Data are means ± SE (n = 3, biological replicates; each replicate contains 6 oocytes).

(F) K+ efflux activity of the oocytes expressing KUP9 and NRT1.5. The oocytes were not injected with IAA. The oocytes were incubated in K+-free bath solution for 6 h. Data are means ± SE (n = 3, biological replicates; each replicate contains 6 oocytes).

(G) K+ efflux activity of the oocytes expressing the indicated proteins. The oocytes were injected with H2O, IAA, or BA. Data are means ± SE (n = 3, biological replicates; each replicate contains 6 oocytes).

(H) K+ efflux activity of the oocytes expressing the indicated proteins. IAA was injected into the oocytes. Then the oocytes were incubated in K+-free bath solutions at different pH for 6 h. Data are means ± SE (n = 3-6, biological replicates; each replicate contains 6 oocytes).

(I) IAA efflux activity of oocytes expressing the indicated proteins under different external K+ conditions (-K, 0 mM; +K, 50 mM). The oocytes were injected with the same amount of IAA, and then incubated in IAA-free bath solution for 6 h. IAA content was measured using UPLC-MS. Data are means ± SE (n = 3, biological replicates; each replicate contains 6 oocytes).

(B) to (I) Student's t-test (*P < 0.05, **P < 0.01) was used to analyze statistical significance, and "#" represent control.