EMBO/soda-nel huggingface dataset:
A dual role for PI4Kβ in endocytosis and phragmoplast dynamics during plant somatic cytokinesis (2018)
Figure 9

Figure 9. Synergistic genetic interaction of Arabidopsis PI4Kβ1 and/or PI4Kβ2 with MPK4 during cytokinesis.

The functional relations of PI4Kβ isoforms with MPK4 were tested by assessing phenotypes of crosses of the corresponding mutants.

(a) Crosses of pi4kβ1 pi4kβ2 and mpk4-2 mutants resulted in combined genotypes with increased growth defects, as indicated. 14-day-old seedlings are shown. Scale bars, 1 cm.

(b) Top, The severe growth defects of the combined genotypes were accompanied by increased tissue distortion, as shown by 3D projections after propidium iodide staining of root tips of 14-day-old seedlings. Scale bars, 100 µm. Bottom, Multinucleation of cells was increased in the combined genotypes, as evident from immunofluorescence of microtubules (red) counterstained with DAPI (blue). Asterisks, multiple nuclei in one cell. Dashed lines indicate the outlines of exemplary multinucleated cells. Scale bars, 10 µm.

(c) Quantification of cytokinetic defects, as shownin the bottom panels of (B). Cytokinetic defects were enhanced in pi4kβ1(-/-) pi4kβ2(+/-) mpk4-2(-/-) (for multinucleated cells, including the percentage of multinucleated cells and the average number of nuclei in multinucleated cells, both according to a Welch ANOVA with Games-Howell post-hoc test; ***, p < 0.001; **,p < 0.01 (wild type, n = 1650 cells, 13 roots; pi4kβ1 pi4kβ2, n = 1441 cells, 19 roots; mpk4-2, n = 1429 cells, 16 roots; pi4kβ1(-/-) pi4kβ2(+/-) mpk4-2(-/-), n = 574 cells, 16 roots); and the incidence of cells displaying KNOLLE fluorescence at the cell plate according to a one-way ANOVA with post-hoc Tukey HSD; p < 0.05, lowercase letters (wild type, n = 1981 cells, 13 roots; pi4kβ1 pi4kβ2, n = 1758 cells, 19 roots; mpk4-2, n = 1721 cells, 16 roots, pi4kβ1(-/-) pi4kβ2(+/-) mpk4-2(-/-), n = 660 cells, 16 roots).
A dual role for PI4Kβ in endocytosis and phragmoplast dynamics during plant somatic cytokinesis (2018)
Figure 10

Figure 10. Functional association of PI4Kβ1 and MPK4 in a protein complex at the cell plate.

Experiments were performed to assess possible physical interaction, colocalization at the cell plate, and functional interplay of PI4Kβ1 and MPK4.

(a) PI4Kβ1 interacts with MPK4 in yeast two hybrid tests. MPK4 was used as a bait, while PI4Kβ1 was divided into two fragments to be used as prey. The results are representative for three independent experiments.

(b) In vivo co-immunoprecipitation of MPK4 and PI4Kβ1 from 14-day-old plants. PI4Kβ1 was specifically co-precipitated with MPK4-myc using anti-myc antibodies. Arrowhead, Migration of PI4Kβ1. The results are representative for three independent experiments.

(c) Coordination of MPK4-YFP and mCherry-PI4Kβ1 during cytokinesis in five-day-old root meristem cells. MPK4 co-localized with PI4Kβ1 from early to late cytokinesis and gradually concentrated at the leading edges of cell plates. Images were obtained with an LSM880 in Airyscan super-resolution (SR) mode. Scale bars, 10 µm.

(d) Live cell time lapse of MPK4-YFP and mRFPFAPP1-PH during cytokinesis, obtained with an LSM880 in Airyscan resolution versus sensitivity (R-S) mode. The time series is representative for 6 cells from 5 roots. Arrowheads indicate the initiation and leading edges of the growing cell plate. Scale bar, 10 µm.

(e) Left panels, The distribution of mRFPFAPP1-PH was observed at the cell plate at the end of cytokinesis in root meristem cells of wild type controls (top) and mpk4-2 mutants (bottom). Intensity profiles were recorded as indicated by the dashed lines. Scale bars, 10 µm. Right top, intensity of mRFPFAPP1-PH at the cell plate normalized vs. the intensity at the apical plasma membrane (wild type, n = 43 cells, 34 roots; mpk4-2, n = 32 cells, 28 roots). Right bottom, intensity of mRFPFAPP1-PH at the TGN normalized vs. the intensity at the apical plasma membrane (wild type, n = 40 cells, 29 roots ; mpk4-2, n = 26 cells, 23 roots). *, a significant difference (p<0.05) according to a two-tailed Student's test.
Whole chromosome loss and associated breakage-fusion-bridge cycles transform mouse tetraploid cells (2017)
Figure 1

Figure 1: Generation of ICL in MEFs by Cre recombination

(A) Schematic of the inverted lox P (iLoxP) site, flanked by sortable GFP and hCD2 markers. (B) Model of reciprocal recombination yielding parental configurations 1/2' and 1'/2 and dicentric/acentric configurations as a result of inverted recombination yielding configurations 1/1' and 2/2'. (C) Representative FACS plot showing loss of hCD2 marker expression of Ch14 ICL MEFs after treatment with 4-hydroxy tamoxifen (4OHT) for 14 days to induce Cre, compared to Ethanol treated control cells (See also Appendix figure S1A). (D) Shallow whole genome sequencing (copy number profiles) of large T antigen immortalized, MEFs after exposure to Cre recombinase and sorted for control (hCD2 plus) and ICL (hCD2 Minus) cells for chromosomes 12, 14 and 9 and 10, without any in vitro culturing, post FACS sort.
Whole chromosome loss and associated breakage-fusion-bridge cycles transform mouse tetraploid cells (2017)
Figure 2

Figure 2: Validation and tumorigenic potential of early passage aneuploid cells

(A) Shallow whole genome sequencing (copy number profiles) of large T antigen immortalized, early passage MEFs after exposure to Cre recombinase and sorted for control (hCD2 plus) and ICL (hCD2 Minus) cells for chromosomes 12, 14 and 9 and 10. (B) Tumor growth curve after early passage ICL cells were injected into flanks of athymic nude mice (n = 5 per group and error bars denote SEM, p<0.05 for Ch9 and Ch14).
Whole chromosome loss and associated breakage-fusion-bridge cycles transform mouse tetraploid cells (2017)
Figure 7

Figure 7: Effects of ploidy on the tumorigenic potential of ICL cells  (A) Flow cytometry analysis of tail blood from Ch10/+ (control) at 30 days post tamoxifen administration, Ch10/+; Id1 Cre/+ at 30 days post tamoxifen administration, and Ch10/+; Id1Cre/+ at 80 days post tamoxifen administration. (B) Analysis from the Mitelman database for the fraction of human solid tumors that have lost (top) or gained (bottom) individual chromosomes, that have modal chromosome number >2N or ~2N (top, p<0.0001 for chromosome 1-22, X and p<0.05 for chromosome Y; bottom, p<0.0001 for all chromosomes). (C) Analysis from the Mitelman database for the fraction of human solid tumors that have lost or gained any chromosome, that have modal chromosome number >2N or ~2N (p<0.0001 for both modal number >2N and ~2N).
Whole chromosome loss and associated breakage-fusion-bridge cycles transform mouse tetraploid cells (2017)
Figure 8

Figure 8: Increased chromosomal instability and DNA damage in ICL in vitro lines and tumors (A) Chromosomal instability readouts, of later passage lines, assessed by average chromosomes loss per metaphase, average number of structural rearrangements per metaphase and percentage of metaphases with marker chromosomes (n=20 for each dataset and error bars denote SD). (B) Determination of number of γH2AX foci in ICL and control nuclei and quantification (scale bars denote 10μm, p<0.0001 for Ch9, Ch10 and Ch12, p<0.05 for Ch14; n=25 for each dataset and error bars denote SD). (See also Figures S5A and S5B) (C) Number of γH2AX positive cells in formalin fixed ICL and control tumor sections by immunofluorescence staining and quantification (Scale bars denote 20μm, p<0.0001 for Ch10 and p<0.05 for Ch14).
Neurotransmitter signaling regulates distinct phases of multimodal human interneuron migration (2021)
Figure 1

Figure 1 - Labelling migrating human interneurons in cerebral organoid fusions

A. Schematic overview of four different protocols for dorsal or ventral forebrain organoid generation. NI, Neural induction medium; Imp-A, Improved differentiation medium without vitamin A; MG, Matrigel; VP, Ventral patterning (2.5μM IWP2 and 100nM SAG).

B. RT-PCR analysis for forebrain (FOXG1), optic cup (VSX2), dorsal forebrain (PAX6 and EMX2) and ventral forebrain (NKX2-1) identity in organoids. All expression values (2-ΔCt) calculated relative to reference gene TBP. Analysis was performed on 5-6 organoids from multiple independent differentiations - control (7), dorsal (7), extended ventral (9) and short ventral (5). Significance values, * = <0.05, ** = <0.01, *** = <0.001. Statistical analysis was performed using one-way ANOVA and post-hoc Tukey's comparison of means. The central band depicts the median, the boxes depict values between lower and upper quartile and the whiskers display the minimum and maximum values.

C. Schematic overview of TALEN-based insertion of an interneuron-specific DLXi56-eGFP reporter into the AAVS1 locus.

D. Overview of organoid fusion co-culture method at Day 20 and 40. All images taken on a widefield cell-culture microscope. Scale bars, 500μm.

E. Whole organoid scan of a Dlxi56-eGFP ventral : dorsal organoid fusion cryosection, day 60, immunostained for GFP marking DLXi56 reporter. Scale bar, 250μm.

F. Magnified (60x) dorsal regions from organoids fusions at day 60; Co-immunostaining of GFP with markers of interneuronal (GABA), GABAergic (DLX2) and mature neuronal (NeuN) identity. Scale bars, 10μm. White arrows indicate GFP+ cells with positive labeling.

G. Quantification of co-expression of GFP with markers mentioned in 1F in dorsal and ventral regions of organoid fusions at day 60. Quantification was performed on a total of 3 organoid fusions (technical replicates) from two independent differentiations each (biological replicates), with 2 regions of interest (ROI; 10x field of view) in the dorsal and ventral regions of each fusion analyzed. Each dot represents the quantification of co-expression for both ROI analyzed per organoid fusion. Significance values, * = <0.05, ** = <0.01, *** = <0.001. Statistical analysis was performed using an unpaired two-tailed Student's t-test. The central band displays the mean and error bars depict the SD.
Neurotransmitter signaling regulates distinct phases of multimodal human interneuron migration (2021)
Figure 2

Figure 2 - scRNAseq identifies migration-dependent maturation of cortical interneurons

A. Schematic overview of experimental setup for single cell RNA-sequencing of human cortical interneurons from organoids. GFP+ cells from the dorsal and ventral regions of 70- or 90-day old organoid fusions were obtained by flow cytometry and sequenced individually.

B. Visualization of single cell RNA-sequencing data from GFP+ cells from the different groups mentioned in 2A using UMAP after removal of stressed cells (EV3A-D). Clusters are color-coded according to the cell type.

C. UMAPs depicting the expression of GABAergic markers DLX2 and DLX5, progenitor marker TOP2A, MGE marker SOX6, CGE marker NR2F2, and striatal marker ZFHX3.

D. Proportions (Y-axis) and numbers (within bars) of cells arising from the different regions of a fusion are listed for each cluster. Numbers are down-sampled to 941 cells in the smallest region group for proper representation. UMAP depicting the fusion region from which the cells were isolated.

E. Proportions (Y-axis) and numbers (within bars) of cells arising from the different fusion ages are listed for each cluster. Numbers are down-sampled to 1221 cells in the smallest age group for proper representation. UMAP depicting the fusion age at which the cells were isolated.
Neurotransmitter signaling regulates distinct phases of multimodal human interneuron migration (2021)
Figure 3

Figure 3 - Migrating interneurons express neurotransmitter receptors

A. UMAP depicting three pseudotime trajectories - two MGE trajectories and a CGE trajectory. Additionally, visualization of each trajectory on isolated UMAPs. After removal of the clusters relating to LGE/striatum and the TH cells, a total of 2968 cells were used for pseudotime analysis.

B. Visualization of the relative gene expression for characteristic genes along the pseudotime of the three trajectories. The genes visualized are color-coded to represent their respective pseudotemporal expression.

C. UMAPs depicting the relative expression of migration genes CXCR4 and EPHA5. Visualization of the gene expression of migration genes CXCR4 and EPHA5 along the pseudotime axis.

D. UMAPs depicting their relative expression of neurotransmitter genes GRIA4 and GRIA2 along with visualization of their gene expression along the pseudotime axis.

E. Heatmap depicting the expression of various neurotransmitter genes for all cells across the 7 clusters analyzed. Clusters are color-coded and labeled according to depiction in 2B. Clusters are labeled to reflect clusters with progenitors, migrating interneurons and mature interneurons.
Metabolic switch and epithelial-mesenchymal transition cooperate to regulate pluripotency (2020)
Figure 1

Figure 1

Facilitated OGS promotes reprogramming

(A-B) 5C or mES medium was used during reprogramming. On day 15, Oct4GFP+ colonies were counted (A). The percentages of Oct4GFP+ cells were also determined with FACS (B).

(C) The expression of several core pluripotency markers was determined with qPCR at different time points during reprogramming and normalized against those in MEFs.

(D-E) Energy metabolism was analyzed on day 6 during reprogramming with the Seahorse instrument. Increase in extracellular acidification rate (ECAR) after adding glucose was considered as glycolysis ability of the cells (D), while the decrease in oxygen consumption rate (OCR) was considered as the ATP production ability of the cells (E).

(F-G) Expression of Hif1α was modulated with overexpression or sh-RNA-mediated knockdown via a retrovirus system during reprogramming (F). The numbers of Oct4GFP+ colonies were determined on day 15 (G).

(H-I) Expression of Pdk1/2 was modulated with a retrovirus system, and oligomycin (1 µM) and 2-DG (5 mM) were used during reprogramming (H). The numbers of Oct4GFP+ colonies were determined on day 15 (I).

Data information: Experiments were independently repeated at least five times (n≥5). Error bars represent standard deviations. ***p < 0.001. Additional statistical information was listed in Dataset EV7.
Metabolic switch and epithelial-mesenchymal transition cooperate to regulate pluripotency (2020)
Figure 2

Figure 2

5C medium removes epigenetic barriers during reprogramming

(A-B) 5C and mES medium were used during reprogramming. Representative alkaline phosphatase (AP) staining on day 15 was provided in (A). AP+Oct4GFP- and AP+Oct4GFP+ colonies were counted on day 15 (B).

(C) Thirty-two epigenetic factors were selected because they had higher expression in 5C-Oct4GFP- than in mES-Oct4GFP-. Their expression in ESCs, iPSCs, and pre-iPSCs in two previous reported assay (GSE14012 and GSE10871) was also listed.

(D) The expression of Bmi1, Ctcf, Ezh2, Kdm2b, or Wdr5 was modulated with overexpression or sh-RNA-mediated knockdown via a retrovirus system during reprogramming. AP+Oct4GFP- and AP+Oct4GFP+ colonies were counted on day 15.

(E-G) During reprogramming with mES medium, Bmi1, Ctcf, Ezh2, Kdm2b, and Wdr5 were overexpressed simultaneously with the four Yamanaka factors (OKMS+5F) or Oct4 (O+5F). All factors were delivered simultaneously via a retrovirus system on day 0 and 1. Reprogramming with only Yamanaka factors (OKMS) or Oct4 (O) served as control. The expression of pluripotency markers was determined with qPCR on day 6 during reprogramming (E). The numbers of Oct4GFP+ colonies were determined on day 15 (F). The histone methylation on core pluripotency loci was determined on day 6 with ChIP-qPCR (G).

Data information: Experiments were independently repeated at least five times (n≥5) except for dataset analysis. Error bars represent standard deviations. ***p < 0.001. Additional statistical information was listed in Dataset EV7.
Metabolic switch and epithelial-mesenchymal transition cooperate to regulate pluripotency (2020)
Figure 3

Figure 3

Early EMT and OGS induce the epigenetic changes

(A) The enrichment of HIF1α, SNAI2, TWIST1/2, and ZEB1 binding sites on the promoters of Bmi1, Ctcf, Ezh2, Kdm2b, and Wdr5 was determined by Pscan Zambelli et al, 2009(). The resulted Z-scores were listed. "Max. M" suggested the maximum Z-score generated with SNAI2, TWIST1/2, and ZEB1 binding sites on one indicated promoter. "Average" suggested the average Z-score of all transcriptional factors tested in the Pscan software on one indicated promoter.

(B-C) The expression of H1f1α, Snai2, Twist1/2, and Zeb1 was modulated with overexpression or sh-RNA-mediated knockdown via a retrovirus system during reprogramming with mES (B) or 5C medium (C). The expression of Bmi1, Ctcf, Ezh2, Kdm2b, Wdr5 was determined on day 6 with qPCR and normalized against those in MEFs. The comparisons were performed between all groups and corresponding Flag groups.

(D-E) pre-iPSCs were isolated during reprogramming with mES-Vc, mES or 5C medium. These pre-iPSCs were further cultured with a different medium (mES-Vc and mES medium) for seven days. Additional factors (Hif1α, Hif1α overexpression; TGFβ, TGFβ1/2/3 1 ng/ml each; 3F, Ctcf, Kdm2b, and Wdr5 overexpression) were used simultaneously with mES medium. The numbers of Oct4GFP+ colonies were determined on day 7 (E).

(F-K) Reprogramming was also performed with mES-Vc or mES medium. Additional factors (Hif1α, Hif1α overexpression; TGFβ, TGFβ1/2/3 1 ng/ml each; 3F, Ctcf, Kdm2b, and Wdr5 overexpression) were used simultaneously with mES medium. AP+Oct4GFP- and AP+Oct4GFP+ colonies were counted on day 15 (G). In addition, energy metabolism was determined on day 6 with Seahorse instrument (H-I). Cell migration was determined by measuring the migration rate with live-cell imaging on day 6 (J). The expression of Bmi1, Ctcf, Ezh2, Kdm2b, or Wdr5 was determined on day 6 with qPCR (K).

(L) Early EMT and facilitated OGS cooperate to induce pluripotency by removing the additional epigenetic barrier.

Data information: Experiments were independently repeated at least five times (n≥5) except binding sites analysis. Error bars represent standard deviations. *p < 0.05, **p < 0.01, ***p < 0.001. Additional statistical information was listed in Dataset EV7.
Metabolic switch and epithelial-mesenchymal transition cooperate to regulate pluripotency (2020)
Figure 4

Figure 4

Facilitated OGS and early EMT form a positive feedback loop

(A-B) Expression of Hif1α was modulated via a retrovirus system or small-molecule compounds, oligomycin (1 µM) and 2-DG (5 mM), during reprogramming. The expression of mesenchymal markers (A) and cell migration (B) was determined with qPCR and live-cell imaging, respectively, on day 6.

(C) HIF1α induces EMT by transcriptionally activating key mesenchymal transcriptional factors, including Snai1/2, Twist1/2, and Zeb1/2. Although to a less extent, inducing OGS directly with Pdk1/2 or small molecules also activate these factors.

(D-G) Repsox (1 μM) was used on day 2-7 to inhibit early EMT during reprogramming with 5C medium. TGFβ (TGFβ1/2/3, 1ng/ml each) on day 2-7, LSD1i (SP2509, 10 nM), and sequential introduction of Yamanaka factors (OK+M+S) were used to induce early EMT during reprogramming with mES medium (D). Except for OK+M+S group, the four Yamanaka factors were delivered simultaneously. Energy metabolism was determined on day 6 with Seahorse instrument (E-F). The expression of glycolysis markers was determined on day 6 with qPCR (G).

(H) Key mesenchymal transcriptional factors, including Snai1/2, Twist1/2, and Zeb1/2 induce OGS by transcriptionally activating Hif1α and glycolysis genes.

(I) Early EMT and facilitated OGS form a positive feedback loop to induce pluripotency.

Data information: Experiments were independently repeated at least five times (n≥5). Error bars represent standard deviations. ***p < 0.001. Additional statistical information was listed in Dataset EV7.
Metabolic switch and epithelial-mesenchymal transition cooperate to regulate pluripotency (2020)
Figure 5

Figure 5

5C state is a distinct pluripotency state

(A-E) On day 15 during reprogramming, the expression of E-cadherin (E-CAD) and N-cadherin (N-CAD), was determined with immunofluorescence together with the morphology in Oct4GFP+ colonies (A, Scale bar, 25 μM). After sorting Oct4GFP+ cells out (5C-Oct4GFP+ and mES-Oct4GFP+ cells), the expression of markers of different pluripotent states (B) and Xist (C), and their abilities to form chimeras (D) were determined. The characteristics of 5C-Oct4GFP+ cells were summarized and compared to naïve and primed PSCs in (E).

(F) The isolated 5C-Oct4GFP+ cells were cultured in naïve or primed medium for three days, and the morphology changed towards R1 ESCs (naïve PSCs) and R1 ESCs-primed (primed PSCs), respectively. The obtained cells were maintained for additional 20 passages. The 5C-Oct4GFP+ cells cultured in primed medium lost GFP fluorescence. R1 ESCs-primed were primed PSCs generated from R1 ESCs. Scale bar, 100 μM.

(G) PSCs mentioned in (F) were used for RNA-Seq analysis. Similarity among gene expression profiles was determined.

(H-J) The isolated 5C-Oct4GFP+ cells were cultured in naïve or primed medium for three days to generate 5C-Oct4GFP+ (naïve) and 5C-Oct4GFP+ (primed) cells. The expression of Xist (H) and the abilities to form chimeras (I-J) were determined.

Data information: Experiments were independently repeated at least five times (n≥5) except for sequencing and animal studies. Error bars represent standard deviations. *p < 0.05, **p < 0.01, ***p < 0.001. Additional statistical information was listed in Dataset EV7.
Metabolic switch and epithelial-mesenchymal transition cooperate to regulate pluripotency (2020)
Figure 6

Figure 6

EMT and OGS cooperate to inhibit reprogramming at the late stage

(A-E) The expression Oct4GFP and E-cadherin was determined with FACS on day 15 during reprogramming (A). Based on the FACS results, the expression of Oct4GFP and E-cadherin was summarized in Oct4GFP+E-cadherin+ cells (B) and in Oct4GFP-E-cadherin- cells (C). The percentage of E-cadherin+ cells in Oct4GFP+ cells was listed in (D), while the percentage of Oct4GFP+ cells in E-cadherin+ cells was listed in (E).

(F-G) Energy metabolism was analyzed in 5C-Oct4GFP+ and mES-Oct4GFP+ cells with Seahorse instrument.

(H) 5C-Oct4GFP- cells had higher expression of glycolysis and mesenchymal markers, but lower expression of epithelial markers than mES-Oct4GFP- cells. 5C-Oct4GFP+ cells had higher expression of all three groups of markers than mES-Oct4GFP+ cells.

(I-K) EMT and OGS were modulated with TGFβ (TGFβ1/2/3, 1ng/ml each), Repsox (1 μM), oligomycin (1 µM), and 2-DG (5 mM) at the late stage during reprogramming with 5C medium (I). The numbers of three types of Oct4GFP+ colonies (J) and the expression of four epithelial markers in Oct4GFP+ cells (K) were determined on day 15. "Average" in (K) suggested the average expression of four epithelia markers.

Data information: Experiments were independently repeated at least five times (n≥5). Error bars represent standard deviations. ***p < 0.001. Additional statistical information was listed in Dataset EV7.