EMBO/soda-nel huggingface dataset:
The phyB-dependent induction of HY5 promotes iron uptake by systemically activating FER expression (2021)
Figure 7

Figure 7. Exposure to red light at night is linked to Fe accumulation in tomato fruits.

A. Accumulation of the HY5 protein in WT and phyB1B2 (phyB) plants grown under 20 μM Fe-EDTA in nutrient solution in a growth room. Rubisco and β-Actin were used as loading controls for leaves and roots in the western blot analysis, respectively. The values shown above each lane indicate the relative abundance of the HY5 protein.

B. The abundance of FER transcripts in the roots of WT and phyB plants grown under 20 μM Fe-EDTA in nutrient solution in a growth room.

C. The relative FCR activities in the roots of WT and phyB plants grown under 20 μM Fe-EDTA in nutrient solution in a growth room.

D. Fe content in the fruits of WT and phyB plants. Plants were grown in soil in a greenhouse.

Data information: Dark: 8 h white light (8 AM~4 PM); Red: 8 h white light plus supplementary red light (660 nm at 20 μmol m-2s-1) from 6 h at 4 PM~10 AM. Values are the means ± SD, n = 4 (biological replicates), P < 0.05 (Tukey's test).

Expanded View Figure Legends
The cryo-EM structure of the CENP-A nucleosome in complex with ggKNL2 (2023)
Figure 1

Figure 1. ggKNL2 recognizes the CENP-A nucleosome via the CENP-C like motif

A) Schematic diagram of the domain organization of chicken KNL2 (ggKNL2). The CENP-C like motif for CENP-A binding (aa 523-543) is colored in orange. Sequence alignment of the CENP-C motif of KNL2 or CENP-C from various species: Gg, chicken; Xl, frog; Dr, zebrafish; and Hs, human. The identical and similar residues are colored in red and orange, respectively. The KNL2 fragments, which were used for in vitro CENP-A nucleosome binding assays, are diagramed.

B) Electrophoretic mobility shift assay (EMSA) to examine binding affinities of the GST-KNL2518-560 fragment for the CENP-A or H3 nucleosome. The molar ratio of KNL2518-560 against nucleosomes are indicated.

C) Biolayer interferometry (BLI) assay for the interaction of KNL2518-560 with the CENP-A nucleosome (upper panel) or the H3 nucleosome (lower panel). The representative BLI sensorgrams for an analyte (CENP-A or H3 nucleosome) concentration range from 0 to 1 μM are plotted.

D)-F) EMSA to examine binding affinities of the GST-KNL2 fragment for the CENP-A nucleosome: D, GST-KNL2347-560; E, GST-KNL2456-560; F, GST-KNL2456-516 (CC-upstream).
The cryo-EM structure of the CENP-A nucleosome in complex with ggKNL2 (2023)
Figure 3

Figure 3. ggKNL2 recognizes the acidic patch of histone H2A/H2B

A) The magnified view of the H2A/H2B binding site of KNL2. R527 of ggKNL2 forms hydrogen bonds with the H2A acidic residues. Side chains of the key residues are indicated as a stick model. Dotted lines represent the possible hydrogen bonds (<3.0 Å).

B) EMSA to examine contribution of the ggKNL2 R527 residue to the CENP-A nucleosome binding.

C) Biolayer interferometry analyses of the CENP-A nucleosome binding of GST-KNL2518-560 WT or GST-KNL2518-560 R527A mutant. Each GST-fused KNL2 fragment was immobilized onto anti-GST prob. 750 nM of CENP-A nucleosome was used as an analyte.

D) Expression of the WT or R527A mutant GFP-KNL2 in KNL2 conditional knock out chicken DT40 cells (cKO-KNL2). Expression of full-length KNL2 WT was conditionally turned off by tetracycline (tet) addition in cKO-KNL2 cells (None). GFP-fused KNL2 WT or R527A mutant was stably expressed in the cKO-KNL2 cells. These cells were cultured in the presence or absence of tet (+tet: KNL2 OFF or −tet: KNL2 ON) for 48 h. α-Tubulin (Tub) was probed as a loading control.

E) Localization analyses of GFP-fused KNL2 WT or R527A mutant (green). CENP-T was stained as a kinetochore marker (red), and DNA was stained using DAPI (blue). Magnified views are shown in insets. Scale bar indicates 10 μm.

F) Growth of the cKO-KNL2 cells expressing GFP-fused KNL2 WT or R527A mutant. The upper panel shows examined cell numbers at the indicated time after tet addition (+tet: KNL2 OFF). The cell numbers were normalized to those at 0 h for each line and plotted as relative cell number. The lower panel shows examined untreated cells (-tet: KNL2 ON). Parental cKO-KNL2 chicken DT40 cells (None) were also examined.
The cryo-EM structure of the CENP-A nucleosome in complex with ggKNL2 (2023)
Figure 4

Figure 4. ggKNL2 recognizes the C-terminal region of CENP-A

A) Magnified view of the interface between the CENP-A C-terminal tail and the KNL2517-560 peptide. Side chains of the key residues are indicated as a stick model.

B) EMSA to examine contribution of the F535 and W536 residues in ggKNL2 to the CENP-A nucleosome binding.

C) Biolayer interferometry analyses of the CENP-A nucleosome binding of GST-KNL2518-560 WT or GST-KNL2518-560 F535/W536 mutant. Each GST-fused KNL2 fragment was immobilized onto anti-GST prob. 750 nM of CENP-A nucleosome was used as an analyte.

D) Expression of GFP-KNL2 WT or CENP-A C-terminal tail binding mutants in cKO-KNL2 cells. Expression of full-length KNL2 WT was conditionally turned off by tetracycline (tet) addition to the cKO-KNL2 cells (None). GFP-fused KNL2 WT, F535A, W536A or F535A/W536A was stably expressed in the cKO-KNL2 cells. These cells were cultured in the presence or absence of tet (+tet: KNL2 OFF or −tet: KNL2 ON) for 48 h. α-Tubulin (Tub) was probed as a loading control.

E) Localization analyses of GFP-fused KNL2 WT and F535A, W536A or F535A/W536A mutant (green). CENP-T was stained as a kinetochore marker (red), and DNA was stained using DAPI (blue). Magnified views are shown in insets. Scale bar indicates 10 μm.

F) Growth of the cKO-KNL2 cells expressing GFP-fused KNL2 WT or each CENP-A C-terminal tail binding mutant. The upper panel shows examined cell numbers at the indicated time after tet addition (+tet: KNL2 OFF). The cell numbers were normalized to those at 0 h for each line and plotted as relative cell number. The lower panel shows examined untreated cells (-tet: KNL2 ON). Parental cKO-KNL2 chicken DT40 cells (None) were also examined.
The cryo-EM structure of the CENP-A nucleosome in complex with ggKNL2 (2023)
Figure 6

Figure 6. ggKNL2 mutants for binding to the CENP-A nucleosome cause CENP-A reduction at centromeres.

A) Immunostaining with anti-Mis18α antibody in AID-based KNL2 knockout cells expressing GFP fused KNL2 wild type or each KNL2 mutant in the presence of IAA for 15 h. Clear colocalization of KNL2 signals with Mis18α was observed (Insets). DNA was stained with DAPI. Scale bar indicates 10 µm.

B) AID-based KNL2 knockout cells expressing GFP fused each KNL2 mutant and SNAP-tagged CENP-A were used. After addition of IAA, SNAP-CENP-A signals were visualized by TMR-Star staining. CENP-T was stained as a kinetochore marker. Representative images were presented. Mitotic centromere localization of each KNL2 mutant was shown as a merged image (Insets): SNAP-CENP-A (red) and each KNL2 mutant (green). DNA was stained with DAPI. Scale bar indicates 10 µm.

C) The levels of CENP-A in mitotic cells of the parental KNL2 knockout cell line (-) and KNL2 knockout cell line expressing GFP fused wild type (KNL2) or mutant KNL2 shown in (B) were evaluated. The signal intensities of SNAP-CENP-A on the mitotic centromeres (30-100 centromeres) were quantified in each of 10 mitotic cells at 0, 5, 24, or 48 h after IAA addition. Averages of signal intensities in each cell were plotted with their average and standard deviation.
The cryo-EM structure of the CENP-A nucleosome in complex with ggKNL2 (2023)
Figure 7

Figure 7. Centromere localization mode of ggKNL2 is altered during cell cycle progression

A) KNL2 (red) localization in CENP-C conditional knockout chicken DT40 cells (cKO-CENP-C) with or without CENP-C expression. DNA was stained with DAPI (blue). CENP-T was stained as a kinetochore marker (light blue). Scale bar indicates 10 μm.

B) KNL2 intensities were evaluated in six cells before and after CENP-C knock out. KNL2 signal intensities on the mitotic centromeres (14-90 centromeres) in each cell were plotted with the average and the standard deviation. The ratio of KNL2 intensities between cells before and after CENP-C knock out was estimated. KNL2 intensities were reduced by ~55%, when CENP-C was knocked out.

C) Competitive pull-down assays for the CENP-A nucleosome binding of ggKNL2 and ggCENP-C. A complex of GST-KNL2456-560 with the CENP-A nucleosome (CA-nuc) was incubated in the absence or presence of MBP-CENP-C619-690 (right panel) or phosphorylated MBP-CENP-C619-690 (middle panel) and pulled down by GST affinity. The concentration of CENP-C619-690 was increased from 0 μM to 1 μM (lanes 3-7). The ratio of band intensities of pull-downed histones (*) to GST-KNL2456-560 in each lane (lanes 3-7) was estimated and presented in the bar graph (left panel). Data are mean with standard deviations calculated from the technical replicates, n=3.

D) A model of ggKNL2 localization dependency into centromeres in interphase and mitotic cells. ggKNL2 localizes at centromeres depending on the CENP-A nucleosome binding in interphase cells. In mitotic cells, centromere localization of ggKNL2 is mediated via CENP-C, not via CENP-A. CENP-C localizes at mitotic centromeres via the CENP-A nucleosome interaction.

Expanded View Figure legends
Steroid-dependent switch of OvoL/Shavenbaby controls self-renewal versus differentiation of intestinal stem cells (2020)
Figure 2

Figure 2 - Pri/proteasome processing of Svb is required for ISC/EB maintenance

A Schematic representation of the pri locus, with tested enhancers and location of the pri-Gal4 gene trap insertion.

B Expression of priA, priJ or priH enhancers in the posterior midgut as seen by lacZ reporters (X-Gal staining, blue), and pri-Gal4 gene trap expressing GFP (green).

C Schematic representation of Svb maturation by proteasome processing, which is triggered by EcR-mediated expression of pri.

D esgts midguts expressing GFP alone (control), or expressing pri-RNAi, and quantification of GFP-positive cells (green). The graph also shows the number of YFP-positive cells in ISCts midguts expressing YFP alone (ctrl), or pri-RNAi (see EV2A).

E ISCts midguts expressing YFP alone (control), or expressing two non-overlapping EcR-RNAi, and quantification of the number of YFP-positive cells (yellow). The graph also plots the number of GFP-positive cells in esgts midgut expressing GFP alone (ctrl), or expressing EcR-RNAi#1, EcR-RNAi#2, and mcherry-RNAi as an additional negative control (see EV2B).

F esgts midguts expressing GFP alone (control), or expressing EcR-DN, and EcR-DN + pri. Samples were stained for GFP (green). The graph shows quantification of the number of GFP-positive cells in the different genotypes.

G Posterior midguts containing control and Ubr3 null MARCM clones (GFP, green).

H esgts midguts expressing GFP alone (control), or expressing Ubr3-RNAi, and Ubr3-RNAi + OvoB. Samples were stained for GFP (green). H' quantification of GFP-positive cells from H.

Data information: Boxes extend from the 25th to 75th percentiles, whiskers from min to max, the horizontal line in each box is plotted at the median; data were collected from three independent replicates. P values from Mann-Whitney tests (D) and one-way ANOVA (E,F,H') are: ns:>0.5, * <0.05, **<0.01, ****<0.0001. Blue is DAPI, scale bars, 20µm.
Steroid-dependent switch of OvoL/Shavenbaby controls self-renewal versus differentiation of intestinal stem cells (2020)
Figure 3

Figure 3 - Svb activator induces stem cell proliferation

A esgts midguts expressing GFP alone (control), or expressing OvoB, and carrying a Dl-lacZ transgene that marks ISCs. Samples were stained for GFP (green) and ß-Gal (red); the graph shows quantification of ß-Gal-positive cells (ISCs).

B Quantification of number of mitotic PH3-positive cells/midgut in esgts guts expressing GFP alone (control), or expressing OvoB and svb-RNAi; Y axis is drawn as log(10).

C esgts midguts expressing GFP alone (control) or expressing SvbACT. Samples were stained for GFP (green), and ß-catenin (purple), DE-Cadherin (white) or Scribble (yellow). Top and bottom pictures show separate channels for a same region.

D esgts midguts expressing GFP alone (control), or expressing miR8, OvoB, and mir8 + OvoB. Samples were stained for GFP (green) and ß-catenin (purple). The graph shows quantification of GFP-positive cells for each genotype. The Y axis is drawn using a log(10) scale.

E esgts midguts expressing GFP alone (control), or expressing Notch Intra Cellular Domain (NICD), and NICD + OvoB. In closeups (right), DAPI is shown in purple for improved contrast; the arrow highlights a cell with intermediate phenotype.

Data information: Boxes extend from the 25th to 75th percentiles, whiskers from min to max, the line in each box is plotted at the median; data were collected from three independent replicates. P values from Mann-Whitney tests (A) and one-way ANOVA (B,D) are: *<0.05, **<0.01, ***< 0.001, ****<0.0001. Blue is DAPI. Scale bars are 20µm.
Steroid-dependent switch of OvoL/Shavenbaby controls self-renewal versus differentiation of intestinal stem cells (2020)
Figure 4

Figure 4 - Svb acts downstream of Wnt and EGFR mitogenic signaling pathways in the adult midgut

A esgtsF/O midguts expressing GFP alone (control), or expressing RasV12, and RasV12 + svb-RNAi (top panels). Bottom panels show esgts midguts expressing GFP alone (control), or expressing EGFR-DN, and EGFR-DN + OvoB. Samples were stained for GFP (green). The graph shows quantification of the number of GFP-positive cells in esgts midguts expressing GFP alone (ctrl), or expressing EGFR-DN, EGFR-DN + OvoB, and EGFR-DN + svb-RNAi. The Y axis is plotted as log(10).

B esgts midguts expressing GFP alone (control), or expressing ArmS10, ArmS10 + svb-RNAi, ArmS10 + OvoB ,TCF-DN, and TCF-DN + OvoB. Samples were stained for GFP (green). The graph shows quantification of the number of GFP-positive cells in esgts midguts expressing GFP alone (ctrl), or expressing TCF-DN, TCF-DN + OvoB, and TCF-DN + svb-RNAi. The Y axis is plotted as log(10).

C The drawing at left schematizes the svb locus, with position of the E3N enhancer. Close-up shows E3N sequence that correspond to binding sites for Pnt (red) and TCF (green). Right subpanels are pictures of posterior midguts showing expression of wild-type E3N-lacZ (E3Nwt), or E3N-Pnt-mt, and E3N-TCF-mt, as seen from X-Gal staining (cyan blue).

Data information: Boxes extend from the 25th to 75th percentiles, whiskers from min to max,the line in each box is plotted at the median; data were collected from three independent replicates. P values from one-way ANOVA are: **<0.01, ****<0.0001. In all pictures of A and B panels, blue is DAPI. Scale bars are 20µm.
Steroid-dependent switch of OvoL/Shavenbaby controls self-renewal versus differentiation of intestinal stem cells (2020)
Figure 5

Figure 5: Svb repressor promotes enterocyte differentiation

A Drawing of apical-basal organization of the intestinal epithelium and expression of svb mRNA as revealed by in situ hybridization. The inlet shows an enlarged view.

B esg-Gal4 midguts expressing mCherry and a svb::GFP rescue mini-gene, consisting of svb-cDNA tagged by GFP (green) and driven by E and 7 svb enhancers (see Fig 1). Samples were stained for GFP (green), mCherry (red) and Prospero (white). B' shows closeup views.

C The drawing at left schematizes the svb locus, with position of E3N and 9CJ2 enhancers. Close-up shows 9CJ2 sequence with binding sites for Pdm1 (orange). The right subpanels are posterior midguts showing expression of wild type 9CJ2 svb enhancer, and expression of 9CJ2-Pdm-mt in which Pdm1 binding sites have been mutated.

D esgts midguts expressing GFP alone (control), or expressing SvbREP. Samples were stained for GFP (green) and Prospero (red). Closeups correspond to boxed regions, with DAPI shown in purple and GFP-positive cells outlined in yellow.

E esgts-F/O midguts expressing GFP alone (control), or expressing SvbREP. Samples were stained for GFP (green) and cleaved DCP1 (red). E' shows quantification of the number of GFP-positive cells in esgts guts expressing GFP alone (ctrl), or DIAP, SvbREP, and SvbREP + DIAP.

F ReDDM lineage tracing in control midguts, or in midguts expressing SvbREP, and quantification of the percentage of progenitors (GFP-positive, RFP-positive) versus differentiated cells (GFP-negative, RFP-positive).

G esgts midguts expressing GFP alone (control), or expressing SvbREP. Samples were stained for GFP (green) and Pdm1 (red); arrows show enlarged GFP-positive cells which are also positive for Pdm1.

Data information: Boxes extend from the 25th to 75th percentiles, whiskers from min to max, the line in each box is plotted at the median; data were collected from three independent replicates. P values from one-way ANOVA (E') and Mann-Whitney tests (F) are: ns >0.5, ****<0.0001. Blue is DAPI, scale bars, 20µm.
Steroid-dependent switch of OvoL/Shavenbaby controls self-renewal versus differentiation of intestinal stem cells (2020)
Figure 6

Figure 6: SvbREP suppresses stem cell tumors in the gut epithelium

A esgts midguts expressing GFP alone (control), or expressing Notch-RNAi, Notch-RNAi + svb-RNAi, and Notch-RNAi + SvbREP. Samples were stained for GFP (green) and Prospero (red). The graph shows quantification of the number of GFP-positive cells.

B esgts midguts expressing GFP alone (control), or expressing STAT-RNAi, STAT-RNAi + svb-RNAi and STAT-RNAi + SvbREP. Samples were stained for GFP (green) and Prospero (red). The graph shows quantification of the number of GFP-positive cells.

C esgts midguts expressing GFP alone (control), or expressing Wg, Wg + OvoB, Wg + SvbREP, and Wg + svb-RNAi. In closeup views, nuclei are in purple.

Data information: Boxes extend from the 25th to 75th percentiles, whiskers from min to max, the line in each box is plotted at the median, data were collected from three independent replicates. P values from one-way ANOVA are: ns p>0.5, ****<0.0001. Graphs are drawn using a log(10) Y axis scale. DAPI is blue; scale bars, 20µm.
Steroid-dependent switch of OvoL/Shavenbaby controls self-renewal versus differentiation of intestinal stem cells (2020)
Figure 7

Figure 7: Svb repressor is required to maintain enterocyte differentiation

A Control MyoIAts midguts, and MyoIAts>svb-RNAi or MyoIAts>OvoB midguts. Cyan dye stains the lumen.

B MyoIAts midguts expressing GFP alone (control), or expressing svb-RNAi, and SvbACT. Samples were stained for GFP (green).

C Electron micrographs of MyoIAts control midguts, or expressing svb-RNAi and SvbACT. Brush border microvilli are pseudo-colored in green, and high magnification views are shown in inlets. Nuclei are pseudo-colored in purple, and visceral muscles located above the basement membrane are in pink. Arrowheads point to impaired cell contacts, the arrow points to a pyknotic nucleus.

D Cross sections of control MyoIAts midguts (expressing GFP and mCherry-RNAi), or expressing svb-RNAi, SvbACT, and Pri. Samples were stained for F-actin (white), GFP (green) and Tsp2a (yellow).

Data information: Blue is DAPI. Scale bars are 20µm (A, B, D) and 5µm in C.
Steroid-dependent switch of OvoL/Shavenbaby controls self-renewal versus differentiation of intestinal stem cells (2020)
Figure 8

Figure 8: Ectopic Svb processing disrupts enterocyte differentiation

A MyoIAts midguts expressing GFP alone (control), or expressing SvbACT. Samples were stained for GFP (green), Scribble (yellow), ß-catenin (purple) and DAPI (Blue). A' pictures display cross sections of the regions shown in A.

B MyoIAts midguts expressing GFP alone (control), or expressing SvbACT. Samples were stained for GFP (green) and PH3 (red). The graph plots number of mitotic PH3-positive cells per midgut of MyoIAts guts expressing GFP alone (ctrl), or expressing SvbACT , and SvbREP + pri.

C MyoIAts midguts expressing GFP and SvbACT (green), and stained for GFP (green) and PH3 (red). The picture is a single focal plane; the arrow shows a large GFP-positive cell, which is also positive for mitotic PH3.

D Summary of the role of SvbACT and SvbREP in the control of intestinal stem cell maintenance, proliferation and differentiation.

Data information: Boxes extend from the 25th to 75th percentiles, whiskers from min to max, the horizontal line in each box is plotted at the median; data were collected from three independent replicates. P values from one-way ANOVA are : **<0.01, ****<0.0001. Scale bars are 20µm.

Expanded View Figure legends
Maternal secretin ameliorates obesity by promoting white adipose tissue browning in offspring (2022)
Figure 1

Figure 1. SCT level is regulated by maternal nutritional status during pregnancy.

A qPCR analysis of relative SCTR levels in different tissues [heart (He), intestine (Int), inguinal WAT (Ing), epididymal WAT (Ep), brown fat adipose (BAT), liver (Liv), hypothalamus (Hypo), placenta (Pla), skeletal muscle (Mus), kidney (Kid)] from C57BL/6J mice as indicated (n = 5 per group).

B Correlation of serum SCT levels and serum triglycerides (TG), total cholesterol (TC) and nonesterified fatty acids (NEFA) levels in pregnant women in the middle trimester to the early third trimester (n = 15).

C, D Plasma SCT levels of dams (C) at gestational day 18.5 (GD18.5) and embryo (D) at E18.5 (n = 8 per group).

E Representative images of SCT immunohistochemical staining of duodenum from dams on standard (CON), whole grain (WG) and high fat (HFD) diet at GD18.5. SCT expression of WG is highest, but lower in HFD compared with CON. Scale bar, 50 μm (n = 10 per group). Right part shows quantification of SCT positive cells in duodenum from dams of three groups.

Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. **p < 0.01; ***p < 0.001.
Maternal secretin ameliorates obesity by promoting white adipose tissue browning in offspring (2022)
Figure 2

Figure 2. Maternal SCT deficiency promotes HFD-induced obesity in offspring.

A Breeding scheme. M-SCT KO offspring were derived by breeding a SCT+/+, Cre-/- male and SCTf/f, Cre+/- female; M-SCT floxed offspring were derived by breeding a SCT+/+, Cre-/- male and SCTf/f, Cre-/- female.

B Change of body weight of M-SCT floxed and M-SCT KO mice upon HFD feeding (n = 8 per group). Right part shows representative photograph of M-SCT floxed and M-SCT KO mice with HFD.

C The weight of different tissues from M-SCT floxed and M-SCT KO mice with HFD (n = 8 per group).

D Random blood glucose levels in M-SCT floxed and M-SCT KO mice with HFD (n = 8 per group).

E Insulin levels in M-SCT floxed and M-SCT KO mice with HFD (n = 8 per group).

F Intraperitoneal glucose tolerance test (GTT) (1.0 g glucose/ kg body weight) in M-SCT floxed and M-SCT KO mice with HFD (n = 5 per group) and AUCs were calculated. Mice were fasted for 18h before assay.

G Intraperitoneal insulin tolerance test (ITT) (0.75 U insulin/kg body weight) in M-SCT floxed and M-SCT KO mice with HFD (n = 5 per group) and AUCs were calculated. mice were fasted for 6h before assays.

H Serum TG, TC and NEFA levels in M-SCT floxed and M-SCT KO mice with HFD (n = 8 per group).

Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. **p < 0.01; ***p < 0.001; ns, not statistically significant.