EMBO/soda-nel huggingface dataset:
ARF2-PIF5 interaction controls transcriptional reprogramming in the ABS3-mediated plant senescence pathway (2022)
Figure 3

Figure 3. PIF5 interacts directly with ARF2.

A, Interaction between ARF2 and PIF5 in the yeast two-hybrid assay. Growth of yeast cells co-transformed with indicated BD and AD vectors was monitored on quadruple dropout medium (QDO, SD-Ade/-His/-Leu/-Trp) and QDO medium containing X-α-gal.

B, Interaction between ARF2 and PIF5 in protoplast BiFC assays. YN-ARF2 and PIF5-YC were coexpressed with the nuclear marker NLS-mCherry in Arabidopsis mesophyll protoplasts. Coexpression of YN-ARF2 and YC alone or YN alone with PIF5-YC served as negative controls. Bars, 10 μm.

C, Direct interaction between GST-ARF2 and MBP-PIF5-His in in vitro GST pull-down assays.

D, Co-immunoprecipitation of ARF2-HA with PIF5-GFP in vivo. ARF2-HA was coexpressed with PIF5-GFP or GFP alone in protoplasts. Proteins co-immunoprecipitated by GFP-Trap beads were immunoblotted with anti-GFP and anti-HA antibodies.
ARF2-PIF5 interaction controls transcriptional reprogramming in the ABS3-mediated plant senescence pathway (2022)
Figure 4

Figure 4. ARF2 and PIF5 regulate senescence interdependently and independently

A, Seedlings of WT, pARF2:ARF2-GFP OEs in the WT background, pif4-2 pif5-10, and pARF2:ARF2-GFP OEs in the pif4-2 pif5-10 background before and after 6 d C-deprivation.

B, RT-qPCR analysis of ARF2 expression levels in 7-d-old seedlings of the same genotypes as shown in A. Fold changes were calculated with respect to the expression level in the WT.

C, Chlorophyll and total cellular protein content reduction in genotypes shown in A after 6 d C-deprivation. Color legends in C are the same as in B.

D, Seedlings of WT, pPIF5:PIF5-GFP OEs in the WT background, arf2-20, and pPIF5:PIF5-GFP OEs in the arf2-20 background before and after 6 d C-deprivation.

E, RT-qPCR analysis of PIF5 expression levels in 7-d-old seedlings of the same genotypes as shown in D. Fold changes were calculated with respect to the expression level in the WT.

F, Chlorophyll and total cellular protein content reduction in genotypes shown in D after 6 d C-deprivation. Color legends in F are the same as in E.

G, Seedlings of WT, arf2-20, pif5-10, and arf2-20 pif5-10 before and after 12 d C-deprivation.

H, Chlorophyll and total cellular protein content reduction in genotypes shown in G after 12 d C-deprivation.

I, Seedlings of WT, abs3-1D, arf2-20 abs3-1D, pif5-10 abs3-1D, and arf2-20 pif5-10 abs3-1D before and after 8 d C-deprivation.

J, Chlorophyll and total cellular protein content reduction in genotypes shown in I after 8 d C-deprivation.

In B, C, E, F, H, and J, data are means ± s.d. of 3 biological replicates.
ARF2-PIF5 interaction controls transcriptional reprogramming in the ABS3-mediated plant senescence pathway (2022)
Figure 5

Figure 5. ORE1 and SGR1 are common target genes of ARF2 and PIF5 in the ABS3-mediated senescence pathway

A, The venn diagram of putative ARF2 target genes and PIF5/4 target genes.

B, Diagrams of the promoter regions of ORE1 and SGR1. Orange arrow heads indicate the positions of G-box and purple arrow heads indicate the positions of the core sequence (5'-TGTC-3') for ARF2 binding. Horizontal lines indicate fragments amplified in ChIP-qPCRs.

C, ChIP-qPCR analysis of ARF2-GFP binding to the promoters of ORE1 and SGR1 in the WT and pif4-2 pif5-10 backgrounds.

D, RT-qPCR analysis of ORE1 and SGR1 expression levels in seedlings of WT, pARF2:ARF2-GFP OEs in the WT background, pif4-2 pif5-10, and pARF2:ARF2-GFP OEs in the pif4-2 pif5-10 background before and after 4 d C-deprivation. Fold changes were calculated with respect to the expression level in the WT before C-deprivation.

E, ChIP-qPCR analysis of PIF5-GFP binding to the promoters of ORE1 and SGR1 in the WT and arf2-20 backgrounds.

F, RT-qPCR analysis of ORE1 and SGR1 expression levels in seedlings of WT, pPIF5:PIF5-GFP OEs in the WT background, arf2-20, and pPIF5:PIF5-GFP OEs in the arf2-20 background before and after 4 d C-deprivation. Fold changes were calculated as in D.

G, Hypocotyl elongation in seedlings of WT, pPIF5:PIF5-GFP OEs in the WT background, arf2-20, and pPIF5:PIF5-GFP OEs in the arf2-20 background. Seedlings were grown under continuous light for one week.

H, RT-qPCR analysis of PIL1 and YUC8 expression levels in seedlings shown in G. Fold changes were calculated with respect to the expression level in the WT. Color legends in H are the same as in F.

I, Diagrams of the promoter regions of PIL1 and YUC8. Orange arrow heads indicate the positions of G-box. Horizontal lines indicate fragments amplified in ChIP-qPCR.

J, ChIP-qPCR analysis of PIF5 binding to the promoters of PIL1 and YUC8 in the WT and arf2-20 backgrounds.

In D, F, and H, data are means ± s.d. of 3 biological replicates. In C, E and J, fold enrichments were calculated with respect to the input. PP2A served as a non-binding control. Data are means ± s.d. of 3 technical replicates. two-tailed t test, n.s., not significant, ****, p<0.0001. The experiment has been independently repeated with similar results.
ARF2-PIF5 interaction controls transcriptional reprogramming in the ABS3-mediated plant senescence pathway (2022)
Figure 6

Figure 6. ABS3 subfamily MATEs are required for ARF2 and PIF5 to promote senescence.

A, RT-qPCR analysis of ARF2 expression levels in 7-d-old seedlings of WT, pARF2:ARF2-GFP OEs in the WT background, mateq, and pARF2:ARF2-GFP OEs in the mateq background. Fold changes were calculated with respect to the expression level in the WT.

B, RT-qPCR analysis of PIF5 expression levels in 7-d-old seedlings of WT, pPIF5:PIF5-GFP OEs in the WT background, mateq, and pPIF5:PIF5-GFP OEs in the mateq background. Fold changes were calculated as in A.

C, Seedlings of the same genotypes as in A before and after 6 d and 9 d C-deprivation.

D, Seedlings of the same genotypes as in B before and after 6 d and 11 d C-deprivation.

E and F, Chlorophyll and total cellular protein content reduction in genotypes shown in A (E) after 6 d and 9 d C-deprivation and in genotypes shown in B (F) after 6 d and 11 d C-deprivation.

G and H, RT-qPCR analysis of ORE1 and SGR1 expression levels in genotypes shown in A (G) and B (H) before and after 4 d C-deprivation. Fold changes were calculated with respect to the expression level in the WT before C-deprivation.

I, RT-qPCR analysis of PIL1 and YUC8 expression levels in 7-d-old seedlings of the same genotypes as shown in B. Fold changes were calculated as in A.

Color legends in E and G are the same as in A. Color legends in F, H and I are the same as in B. In A, B, E-I, data are means ± s.d. of 3 biological replicates.
ARF2-PIF5 interaction controls transcriptional reprogramming in the ABS3-mediated plant senescence pathway (2022)
Figure 7

Figure 7. Feedback transcriptional repression of ABS3 by ARF2.

A, A diagram of the promoter region of ABS3. Purple arrow heads indicate the positions of the core sequence (5'-TGTC-3') for ARF2 binding. Horizontal orange lines indicate the fragments amplified in ChIP-qPCR. Brackets indicate the deleted regions in pABS3∆R1, pABS3∆R2, pABS3∆R3, and pABS3∆R4, respectively.

B, ChIP-qPCR analysis of ARF2-GFP binding to the promoters of ABS3. Fold enrichments were calculated with respect to the input. PP2A served as a non-binding control. Data are means ± s.d. of 4 technical replicates. The experiment has been independently repeated 3 times with similar results.

C, pABS3:GFP or pABS3∆S2:GFP was expressed in WT mesophyll protoplasts with or without p35S:ARF2-HA. p35S:mCherry was co-transfected and served as a transfection control. Protoplasts were examined with fluorescence microscopy. Experiments were independently repeated 3 times with similar results. Bars, 100 μm.

D, Dot plots of GFP relative fluorescence intensity in protoplasts expressing WT and mutant forms of pABS3:GFP with or without p35S:ARF2-HA. Data are median with interquartile range, n is indicated in parentheses, two-tailed Mann-Whitney test, n.s., not significant, ****, p<0.0001.

E, RT-qPCR analysis of ABS3 expression levels in 7-d-old seedlings of WT, pARF2:ARF2-GFP OEs in the WT background, pif4-2 pif5-10, and pARF2:ARF2-GFP OEs in the pif4-2 pif5-10 background. Fold changes were calculated with respect to the expression level in the WT. Data are means ± s.d. of 3 biological replicates.

F, A working model depicting the ARF2-PIF5/4 functional module in the ABS3-mediated senescence pathway.

EV Figure Legends
A catalog of numerical centrosome defects in epithelial ovarian cancers (2022)
Figure 1

Figure 1. Characterization of centrosome numbers in healthy ovarian tissues

(A) Schematic diagram of the workflow used to analyze ovarian tissue sections. (B) Left, orthogonal view of a 20μm tumor section labelled with antibodies against EpCAM (in red) and DNA in blue. Right, dot plot bar representing cell heights in cells from tumor sections, n=60 cells from 1 tumor. Mean±SD. (C) On the left, images of low magnification views of healthy tissues labelled with antibodies against pericentrin (PCNT) and CDK5RAP2, shown in red and green, respectively. DNA is in blue. The white dashed squares represent the regions shown in higher magnifications on the right. One centrosome was considered as such when PCNT and CDK5RAP2 signals co-localized (Inset 1). Lack of co-localization (Insets 2 and 3) was noticed and discarded during quantification. (D) Super-resolution microscopy of healthy tissues labelled for the centriole marker CEP135 (in green) and PCNT (in red). (E-F) Representative images of two different fields of the same tumor (E) and of a tumor section to highlight different centrososome phenotypes, labelled as described in (C). The pink dashed line highlights an area of cells without centrosomes. The white dashed squares represent the regions shown in higher magnifications in insets in (G and I) and the pink dashed linesurrounds nuclei without centrosomes. (G) Insets from (F). (H) Examples of tumor sections showing small (left) and large (right) regions with nuclei without centrosomes. (I) Insets from (F) showing different types of centrosome amplification events. (J) Super-resolution microscopy of tumor tissues labelled as in (C). (K) Maximum Z-projection of a tumor section labelled with EpCAM antibodies (Red) and DNA in blue. The dashed line shows the absence of EpCAM staining, arrows show a disorganized region.
A catalog of numerical centrosome defects in epithelial ovarian cancers (2022)
Figure 2

Figure 2. EOCs display highly heterogenous centrosome number alterations

(A) Images of EOC tumors labelled with antibodies against PCNT (red) and CDKRAP2 (green). Nuclei are shown in blue. The colored arrows reflect different centrosome number. The list of centrosome behaviors annotated it is not exhaustive to allow comprehension of the events. #TT72, shows a cyst-like arrangement of EOC nuclei where it is impossible to correspond centrosomes to individual nuclei. The white bars with arrows of different colors highlight possible interpretations of the same condition. In #TT79, regions without centrosomes can be identified. In the inset, a single centrosome localized in the lumen can be identified and it is not possible to know to which nucleus (white dashed lines) it corresponds to. #TT88 presents only very small clusters of centrosomes, few nuclei without centrosomes and many conditions with 2 centrosomes. In #TT61, regions without centrosomes can be distinguished, in addition to nuclei associated with single centrosomes. In #TT46, clusters containing large (inset 1) or small clusters (inset 3) can be identified. In cyst-like arrangements several centrosomes can be identified (inset 2), suggesting that all cells have at least one centrosome or, in contrast just one centrosome (inset 4), illustrating lack of centrosomes. (B-C) Plots showing the frequency of centrosome amplification events (B) and the frequency of nuclei without centrosomes (C) in tumor tissues. Note that in B and C, the order of the tumors is conserved between the two plots to allow for comparison. For each tumor, 10 random fields were chosen and analysed, with an average of 5248 nuclei counted per tumor. Bars represent the mean ±SD. (D) Tumor sections labelled with CEP135 (green) and PCNT (red) on the left and CEP135 (green) and CEP192 (red) on the right. The white squares represent the regions shown in higher magnifications on the right.
The Spo13/Meikin Pathway Confines the Onset of Gamete Differentiation to Meiosis II in Yeast (2021)
Figure 1

Figure 1. APC/CCdc20 controls sporulation via Spo13/meikin and the cyclin Clb1

A. Quantification of spore formation in the wild-type (WT) and the indicated PHSL1-CDC20 strains at 23 h in sporulation medium (SPM). Error bars extend to data from two independent experiments (n = 200 cells per strain).

B. Cells at 23 h in SPM. Nuclei are labeled with TetR-RFP. Scale bar, 2 μm.

C. Imaging of SPBs (Cnm67-RFP) and Spc72-GFP or Mpc70-GFP in control and PHSL1-CDC20 cells. Top, time-lapse series. Bottom, meiotic events were quantified in cells synchronized in silico to SPB separation at entry into metaphase I (t = 0). Graphs show overlays of SPC72-GFP and MPC70-GFP strains.

D. Imaging of SPBs (Cnm67-RFP) and Spc72-GFP or Mpc70-GFP in PHSL1-CDC20 strains containing the indicated mutations. Meiotic events were analyzed as in (C).

Data information: data are representative of four (C) or two (D) independent experiments.
The Spo13/Meikin Pathway Confines the Onset of Gamete Differentiation to Meiosis II in Yeast (2021)
Figure 2

Figure 2. Inactivation of Spo13 or Cdk1-Clb1 causes exchange of Spc72 for Mp70 in metaphase I-arrested cells

A. Imaging of SPBs (Cnm67-RFP) and Spc72-GFP or Mpc70-GFP in PHSL1-CDC20 ama1∆ control cells and spo13 mutants. Top, time-lapse series. Bottom, meiotic events were quantified in cells synchronized in silico to SPB separation at entry into metaphase I (t = 0). Graphs show overlays of SPC72-GFP and MPC70-GFP strains.

B. TEM analysis of SPBs. Left, SPBs in SPO13 and spo13∆ cells lacking all APC/C activators. TEM samples were collected at 12 h in SPM. The spo13∆ mutation causes MP formation at metaphase I (P < 0.0001, Fisher's exact test). Right, SPBs at metaphase II. CDC20-mAR and CDC20-mAR mpc54∆ mpc70∆ cells were synchronized by arrest/release at metaphase I, and TEM samples were collected at metaphase II (80 min after release). MP formation is blocked in the absence of Mpc54 and Mpc70 (P < 0.0001, Fisher's exact test). Scale bar, 0.2 μm.

C. Imaging of SPBs (Cnm67-RFP) and Spc72-GFP or Mpc70-GFP in PHSL1-CDC20 ama1∆ control cells and cells containing clb1∆ or cdc28-as1, which have been treated with 1NM-PP1 at metaphase I (8 h in SPM). Meiotic events (bottom) were analyzed as in (A).

Data information: data in (A) and (C) are representative of two independent experiments.
The Spo13/Meikin Pathway Confines the Onset of Gamete Differentiation to Meiosis II in Yeast (2021)
Figure 3

Figure 3. Spc72 prevents premature MP assembly at metaphase I

A. Imaging of SPBs (Cnm67-RFP) and Mpc70-GFP in PHSL1-CDC20 ama1∆ cells containing SPC72, spc72-7, or spc72-7 plus hrr25-as. Cells were shifted from 24 to 36°C at 4 h in SPM and treated with 1NM-PP1 to inhibit Hrr25-as. Top, time-lapse series. Bottom, Mpc70 loading to SPBs was quantified in cells synchronized in silico to SPB separation at entry into metaphase I (t = 0).

B. TEM analysis of SPBs. PHSL1-CDC20 ama1∆ cells containing SPC72 or spc72-7 were shifted from 24 to 36°C at 4 h in SPM. TEM samples were collected at 12 h in SPM. Spc72 inactivation causes MP formation at metaphase I (P < 0.0001, Fisher's exact test). Scale bar, 0.2 μm.

C. Imaging of SPBs (Cnm67-RFP) and Mpc70-GFP in PHSL1-CDC20 ama1∆ spo13∆ control cells and cells expressing an additional copy of SPC72 from the PEST promoter at 4 h in SPM. Mpc70 loading (bottom) was analyzed as in (A). PEST-SPC72 expression delays the time of Mpc70 loading by 98 min (95% CI, 67-129; P < 0.0001; Welch's t-test).

Data information: data in (A) and (C) are representative of three independent experiments.
The Spo13/Meikin Pathway Confines the Onset of Gamete Differentiation to Meiosis II in Yeast (2021)
Figure 4

Figure 4. Co-expression of stabilized Spo13 and Clb1 delays Spc72 removal and Mpc70 recruitment at SPBs

A-D. Imaging of SPBs (Cnm67-RFP) and Spc72-GFP or Mpc70-GFP in cells containing different SPO13 alleles and/or PEST-clb1-mDK, which was induced with estradiol at 4 h in SPM. (A) Representative time-lapse series. (B) Analysis of cells containing wild-type Spo13, Spo13-mD, or Spo13-mD-m2. Meiotic events were quantified in cells synchronized in silico to SPB reduplication at entry into metaphase II (t = 0). Graphs show overlays of SPC72-GFP and MPC70-GFP strains. spo13-mD delays Spc72 removal and Mpc70 loading by 30 min (95% CI, 26-34; P < 0.0001) and 60 min (95% CI, 55-66; P < 0.0001; Welch's t-test), respectively. (C) Analysis of cells expressing Clb1-mDK and different versions of Spo13. Meiotic events were quantified as in (B). spo13-mD plus PEST-clb1-mDK delays Spc72 removal and Mpc70 loading by 72 min (95% CI, 63-80; P < 0.0001) and 114 min (95% CI, 99-129; P < 0.0001; Welch's t-test), respectively. (D) Time from SPB reduplication at metaphase II to Mpc70 loading plotted versus time from SPB reduplication to Spc72 removal for strains with the indicated genotypes. Diamonds represent mean values, and error bars indicate data range. Numbers of independent experiments used to calculate mean values are shown on the right.
The Spo13/Meikin Pathway Confines the Onset of Gamete Differentiation to Meiosis II in Yeast (2021)
Figure 5

Figure 5. Spc72 removal from SPBs requires the activities of Cdc5 and Ime2

A. Imaging of SPBs (Cnm67-RFP) and Spc72-GFP in PHSL1-CDC20 ama1∆ spo13∆ control cells and cells containing cdc5-as or ime2-as treated with CMK or 1Na-PP1, respectively, at metaphase I (7 h in SPM). Top, time-lapse series. Bottom, Spc72's presence at SPBs was quantified in cells synchronized in silico to SPB separation at entry into metaphase I (t = 0).

B. Whole-cell extracts from PHSL1-CDC20 ama1∆ CDC5-ha3 Ha3-SPO13 cells were subjected to α-Ha immunoblotting. Secondary antibodies were detected by near-infrared fluorescence imaging. Top, progression into metaphase I. Bottom, serial two-fold dilutions of the sample collected at t = 10 h in SPM. The Cdc5-ha3/Ha3-Spo13 ratio is ~20.

C. Cdc5 interacts with Ime2. Cdc5-ha3, Ime2, and Spo13 were detected by immunoblotting in whole-cell extracts and α-Ha immunoprecipitates from the indicated strains.

D. Cdc5's polo-box domain (PBD) binds to Ime2. Ha3-tagged versions of the wild-type PBD or the phosphopeptide-binding mutant PBD-FAA were expressed at t = 5.5 h in SPM in cells depleted of Cdc20 and Cdc5. PBD-ha3 and Ime2 were detected by immunoblotting in whole-cell extracts and α-Ha immunoprecipitates.

E. Cdc5-dependent modification of Ime2. PHSL1-CDC20 ama1∆ control cells and cells containing spo13∆ or cdc5-as were treated with CMK (to inhibit Cdc5-as) at t = 8 h in SPM (arrow heads). The panel shows immunoblot analysis of whole-cell extracts. C, sample from proliferating cells. Increased gel mobility of Ndt80 at t ≥ 10 h confirms inhibition of Cdc5-as. The arrow marks the modified form of Ime2.

Data information: data in (A-E) are representative of two independent experiments.
The Spo13/Meikin Pathway Confines the Onset of Gamete Differentiation to Meiosis II in Yeast (2021)
Figure 7

Figure 7. Reconstitution of Spc72 removal and MP assembly in mitotic cells

A, B. Mitotic cdc20-3 cells were shifted to 36°C for 80 min. At t = 0, cells were treated with estradiol to induce expression from the PEST promoter. Top, time-lapse series from the imaging of SPBs (Cnm67-RFP) and Spc72-GFP. Frame width, 19 μm. Bottom, quantification of cells with Spc72-GFP at SPBs. (A) Expression of Ime2-∆C induces removal of Spc72 from SPBs, which is inhibited by co-expression of Spo13. (B) Expression of Cdc5 plus Ime2-∆C causes rapid removal of Spc72, which is inhibited by co-expression of Spo13.

C. Mitotic cdc20-3 cells containing PEST-CDC5 plus PEST-IME2-∆C and/or PEST-NDT80 were shifted to 36°C for 30 min and subsequently treated with estradiol (t = 0). Top, time-lapse series from the imaging of SPBs (Cnm67-RFP) and Mpc70-GFP. Frame width, 19 μm. Bottom, quantification of cells with Mpc70-GFP at SPBs.

Data information: data are representative of three (A, B) or two (C) independent experiments.
The Spo13/Meikin Pathway Confines the Onset of Gamete Differentiation to Meiosis II in Yeast (2021)
Figure 8

Figure 8. MP assembly is promoted by Cdk1-Clb and inhibited by Cdc5-Spo13

A. PHSL1-CDC20 ama1∆ cdc28-as2 cells were treated with 1Na-PP1 at t = 3 h in SPM to inhibit Cdc28-as2. At t = 5 h (left) or t = 9 h (right), Cdc28-as2 was activated by washing cells with conditioned SPM (cSPM) lacking 1Na-PP1 (washout). Top, time-lapse series from the imaging of Mpc70-GFP and RFP-tubulin. The weak, nuclear signal from Ndt80-GFP serves to confirm entry into meiosis. Middle, quantification of spindle formation and Mpc70 loading to SPBs. Bottom, quantification of control experiments. Cells were washed with cSPM plus 1Na-PP1 at t = 5 h (left) or t = 9 h (right) in SPM.

B. TEM analysis of SPBs. PHSL1-CDC20 ama1∆ cdc28-as2 cells were treated with 1Na-PP1 at 3 h in SPM. At 9 h in SPM, cells were washed with cSPM containing (top) or lacking 1Na-PP1 (washout, bottom). TEM samples were collected at 13 h in SPM. Washout of 1Na-PP1 causes MP formation (P < 0.0001, Fisher's exact test). Scale bar, 0.2 μm.

C. PHSL1-CDC20 ama1∆ cdc28-as2 control cells and cells containing spo13∆ or cdc5-as were treated with 1Na-PP1 at t = 3 h in SPM to inhibit Cdc28-as2. At t = 9 h in SPM, Cdc28-as2 was activated by washing cells with cSPM lacking 1Na-PP1 (washout). cdc5-as cells were washed with cSPM plus CMK to activate Cdc28-as2 and inhibit Cdc5-as. Top, time-lapse series from the imaging of Mpc70-GFP and RFP-tubulin. The weak, nuclear signal originates from Ndt80-GFP. Bottom, quantification of spindle formation and Mpc70 loading to SPBs.

D. PHSL1-CDC20 ama1∆ cells containing SPC72, spc72-7, or spc72-7 plus cdc5-as were shifted from 24 to 36°C at t = 4.2 h in SPM (to inactivate Spc72-7, arrow heads) and treated with CMK at t = 6 h in SPM (to inhibit Cdc5-as). Top, time-lapse series from the imaging of SPBs (Cnm67-RFP) and Mpc70-GFP. Bottom, quantification of SPB separation and Mpc70 loading to SPBs. Inhibition of Cdc5-as in spc72-7 cells advances the time of Mpc70 loading by 119 min (95% CI, 76-162; P < 0.0001; Welch's t-test). Data are representative of two independent experiments.