EMBO/soda-nel huggingface dataset:
Myeloid leukemia with transdifferentiation plasticity developing from T-cell progenitors (2016)
Figure 5

Figure 5. Enhanced plasticity of DN2-derived myeloid leukemia depends on dynamic PU.1 expression

A. Representative FACS plots of splenocytes from diseased secondary recipients that were transplanted with 104 to 105 flow-sorted myeloid leukemia blasts (ML: CD11b+Gr1+/CD3–CD4–CD8–) from sick primary Myc/Bcl2+ LSK-, GMP- or DN2-transplanted mice. Cell percentages in indicated gates are shown. B. Table summarizing the phenotype frequencies within the spleens of secondary recipients that were retransplanted with Myc/Bcl2+ LSK-, GMP- or DN2-derived myeloid blasts as described in A. T-lymphoid leukemia: (TL: CD11b–Gr1–/CD3+CD4+CD8+), biphenotypic leukemia: BL (CD11b+Gr1+/CD3+CD4+CD8+), myeloid leukemia: ML (CD11b+Gr1+/CD3–CD4–CD8–). Numbers before the dash represent mice holding the indicated leukemia fraction; numbers after the dash indicate the total mouse count analyzed. C. Single myeloid blasts from 4 different Myc/bcl2+ DN2-derived leukemias were flow-sorted from transplanted mice and grown on irradiated OP9:OP9DL1 stroma. The clones were analyzed at two different time points. FACS plots of two representative clones, one with strong (top) and one with weak (bottom) lineage switching potential are shown. D. Table summarizing the lineage switching outcome of 20 independent clones that were grown from single DN2-derived myeloid blasts of 4 different leukemias. Percentages at the right indicate the relative amounts of cells with T-lymphoid (TL), biphenotypic (BL) or myeloid (ML) phenotype as analyzed by FACS at the end of the experiment. E. FACS plot of DN2-derived primary leukemic cells transduced with a retrovirus carrying GFP alone or GFP together with PU.1 as a bicistronic RNA. The transduced cells were grown on OP9/OP9Dll1 1:1 layer for 8-13 days and analyzed using a GFP-gate. The data are representative of 3 different experiments with similar outcome.
Myeloid leukemia with transdifferentiation plasticity developing from T-cell progenitors (2016)
Figure 6

Figure 6. A cohort of immature human AML patients resembles mouse DN2-leukemia

A. Heat map depicting all human AML samples from the Haferlach study (Haferlach et al., 2010) whose transcriptomes resembled DN2-leukemia by a Spearman's rank correlation coefficient ≥ 0.5. The top 100 up- and downregulated gene signature of Myc/Bcl2+ DN2-derived myeloid blasts was applied (see also Figure 3B). B. Hierarchical clustering of all FAB M2 AML samples from the Verhaak study (Verhaak et al., 2009) based of the Myc/Bcl2+ DN2-derived myeloid blast top 100 upregulated (UP) gene signature (see Figure 3B). C,E. GSEA confirming enrichment of the Myc/Bcl2+ DN2-derived myeloid blast top 100 UP (C) and pan-DC (Schonheit et al., 2013) (E) gene signatures in the human FAB M2 DN2-like AML samples as compared to all residual M2 AMLs from the Verhaak study (Verhaak et al., 2009). D. Relative transcript levels of the T cell receptor alpha locus (TRAC) in human DN2-like M2 AML compared to residual M2 AML patient samples from the Verhaak study (Wilcoxon rank-sum test, **=p≤0.01).
Myeloid leukemia with transdifferentiation plasticity developing from T-cell progenitors (2016)
Figure 7

Figure 7. A targeted shRNA in vivo screen reveals requirements of DN2-leukemia to Jak2-Stat3 signaling

A. Survival assay of Myc/Bcl2+ GMP- and DN2-derived leukemia cells that were isolated from diseased mice and treated for 24 h with 0.15 μM daunorubicine or 1 μM doxorubicine in liquid culture. Cell death was measured by FACS of 7-AAD+ cells and was normalized to the solvent controls. The graphs show the mean+SEM of three different DN2- and five GMP-leukemias. **=p≤0.01. B. Experimental procedure of the shRNA library in vivo screen. Blasts from leukemic mice that were transplanted with Myc/Bcl2+ DN2-cells were infected with a retroviral shRNA library consisting of ~300 GFP- or YFP-tagged shRNAs targeting a total of 142 different genes. Cells were harvested 72 h post infection, the GFP+ and YFP+ fractions were flow-sorted and 6104 cells were transplanted into secondary recipients. The residual cells were expanded for 5 days on OP9:OP9Dll1 1:1 layers for amplification (T0 reference samples). Secondary recipients were sacrificed after development of leukemia, and T1 samples were generated from BMs, spleens and thymi, which confirmed BM infiltration >20%). T1 and T0 samples were deep sequenced and changes in individual shRNAs were calculated. C. Scatterplot depicting the averaged quantitative changes (log10) of each shRNA within the library in the T1 spleen and BM samples in reference to T0. The changes in both organs were concordant (R2=0.72). T1 samples from 11 mice were compared to 3 T0 reference samples. Blue dots represent shRNAs with a significant depletion by ≥10-fold. D. Top 5 genes with ≥10-fold shRNA depletion in BM, spleen and thymus in comparison to the reference. E. Percent cell growth of 5 different DN2-derived leukemic cell lines transduced with 2 different shRNA constructs targeting Jak2. The cells were sorted for the GFP or YFP reporter and normalized to the control shRNA targeting the lacZ gene (lacZ). Four of five DN2-derived leukemias show reduced growth with both shRNAs after the second round of sorting within 8 days in culture. The lacZ control was set to 100%. F. Proliferation curves of 3 Myc/Bcl2+ DN2- and 3 GMP-derived leukemia lines during treatment with 0.5 μM ruxolitinib. 1.5105 cells were seeded in culture and counted at the indicated time points. Results are shown as fold growth of the starting cell number which was set to 1. *=p≤0.05, **=p≤0.01, Student’s t-test. G. Western blot indicating active Jak/Stat signaling in DN2-derived leukemia with Stat3 as well as pStat3 protein expression, that diminishes after treatment with the Jak-inhibitor ruxolitinib. H. GSEA indicating significant enrichment of genes of the JAK/STAT signaling pathway of the KEGG pathway database in the DN2-like M2 AML samples as compared to the residual M2 AML samples using the Verhaak AML data set (Verhaak et al., 2009).
PGF2α facilitates pathological retinal angiogenesis by modulating endothelial FOS-driven ELR+ CXC chemokine expression (2022)
Figure 1

Figure 1. Activation of the PGF2α-PTGFR axis in pathological retinal angiogenesis.

A. Serum levels of 15-keto-dihydro-PGF2α in type 2 diabetic patients without diabetic retinopathy (no DR), with non-proliferative diabetic retinopathy (NPDR), and with proliferative diabetic retinopathy (PDR) (n=20-24).

B. Schematic diagram of the OIR mouse model. Mice on postnatal day 7 were placed in a 75% oxygen container for 5 days and were then transferred to a normal environment on postnatal day 12 for another 5 days to induce retinal vessel proliferation.

C. Retinal PGF2α production in OIR mice during postnatal days 12-17 (n=4).

D. mRNA expression of PGF2α synthases in the retinas of OIR mice during postnatal days 12-17 (n=4).

E. mRNA expression of Ptgfr in the retinas of OIR mice during postnatal days 12-17 (n=6-8).

F. mRNA expression of Ptgfr in each retinal layer from normoxic and OIR mice on postnatal day 16 (n=6). The left-hand image displays representative cross-sections from OIR and normoxic retinas, with isolectin B4-stained vessels in green, DAPI-stained nuclei in blue, and LCM isolation locations circled by white dashed circles. INL, inner nuclear layer; ONL, outer nuclear layer; RGC, retinal ganglion cells.

G. Ptgfr mRNA expression in retinal endothelial cells (CD31+CD45-) sorted by MACS from normoxic and OIR mice on postnatal day 16 (n=5).

Data information: n.s. stands for "not significant." Data were analyzed by Kruskal-Wallis test with Dunn's multiple comparisons test (A), two-way ANOVA with Tukey's multiple comparisons test (C, D, E, F), or Mann-Whitney test (G). Scale bar: 20 μm. Data are represented as mean ± SEM.
PGF2α facilitates pathological retinal angiogenesis by modulating endothelial FOS-driven ELR+ CXC chemokine expression (2022)
Figure 2

Figure 2. Ptgfr knockout in vascular ECs attenuates angiogenesis in OIR mice.

A. Generation of endothelial cell (EC)-specific Ptgfr knockout mice. Ptgfr-floxed mice were crossed with Tie2-Cre mice to create EC-specific Ptgfr knockout mice (CKO-T).

B. Ptgfr mRNA levels in retinal microvessels of CKO-T and control mice (n=6). INL, inner nuclear layer; ONL, outer nuclear layer; RGC, retinal ganglion cells.

C. Representative images of OIR retinas from CKO-T and control mice on postnatal day 17. The green color shows the isolectin B4-stained vessels. The second-row panels are the enlarged images of white boxes from the first-row panels. The third-row images show neovascular tufts (NV, white) and the fourth-row images show the vaso-obliteration (VO, white) area.

D. Quantitation of oxygen-induced retinal neovascularization in CKO-T and control mice (n=12).

E. Quantitation of retinal vaso-obliteration in CKO-T and control mice (n=12).

Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test (B, D, E). Scale bars: 500 μm (unmagnified image), 150 μm (magnified image). Data are represented as mean ± SEM.
PGF2α facilitates pathological retinal angiogenesis by modulating endothelial FOS-driven ELR+ CXC chemokine expression (2022)
Figure 4

Figure 4. PGF2α promotes proliferation and tube formation of HRMECs through the upregulation of ELR+ CXC chemokines.

A. Heat map of upregulated genes in PGF2α-treated HRMECs (n=3 samples).

B. GO enrichment of the top five biological processes involving the upregulated genes in PGF2α-treated HRMECs.

C. Effect of PGF2α treatment on ELR+ CXC chemokine expression in HRMECs (n=3).

D-G. Relative expression levels of enriched ELR+ CXC chemokines in retinal microvascular ECs from patients with PDR (n=4) compared with those from normal subjects (n=7) (Gene Expression Omnibus Database dataset GSE94019). TPM stands for "Transcripts per kilobase million."

H. Effect of CXCR2 inhibitor SB265610 (1 μM) on PGF2α-stimulated HRMEC migration. The yellow solid line indicates the original position of the scratch, the dashed line indicates the position after cell migration, and the arrows indicate the migratory direction.

I. Quantitation of scratch-induced HRMEC migration in H (n=4).

J. Effect of SB265610 (1 μM) on PGF2α-stimulated HRMEC proliferation (n=4).

K. Effect of CXCR2 inhibitor SB265610 (1μM) on tube formation in PGF2α-treated HRMECs.

L. Statistical graph of tube formation experiments in K, comparing the total tubule length, number of junctions, number of meshes, and percentage of mesh area (n=4).

Data information: n.s. stands for "not significant." Data were analyzed by unpaired Student's t-test (C), Mann-Whitney test (D, E, F, G), or two-way ANOVA with Tukey's multiple comparisons test (I, J, L). Data in C. I. J. L are represented as mean ± SEM. Data in D-G are presented as the mean + SD. Scale bar: 100 μm (H) and 200 μm (K).
PGF2α facilitates pathological retinal angiogenesis by modulating endothelial FOS-driven ELR+ CXC chemokine expression (2022)
Figure 5

Figure 5. Retinal Cxcl1 overexpression abolishes decreased neovascularization in OIR retinas from CKO mice.

A. Effect of CXCR2 inhibitor SB265610 (1 μM) on PGF2α-promoted sprouting of aortic rings. Yellow lines show the sprouting area.

B. Quantitation of the sprouting areas in A (n=6).

C. mRNA expression of ELR+ CXC chemokines in retinas from OIR mice during postnatal days 12-17 (n=4-6).

D. Effect of PTGFR deficiency in ECs on ELR+ CXC chemokine expression in retinas from OIR mice on postnatal day 16 (n=6).

E. Schematic diagram of Cxcl1-expressing lentivirus transfection in OIR mice. Neonatal mice were injected with lentivirus on postnatal day 5, followed by oxygen treatment on day 7.

F. Retinal CXCL1 protein-expression levels in Cxcl1-expressing lentivirus-injected CKO mice (n=3).

G. Quantitation of retinal CXCL1 protein in F (n=3).

H. Representative images of oxygen-induced retinal angiogenesis in Cxcl1-expressing lentivirus-injected CKO mice on postnatal day 17. Green represents the isolectin B4-stained vessels, the second-row panels display the enlarged images of white boxes in the first-row panels, the third-row images show neovascular tufts (NV, white), and the fourth-row images show the vaso-obliteration (VO, white) area.

I. Quantitation of oxygen-induced retinal neovascularization in H (n=8).

J. Quantitation of retinal vaso-obliteration in H (n=8).

Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test (B, C, D, G, I, J). Scale bar: 200 μm (A), 500 μm (H, unmagnified image), 150 μm (H, magnified image).Data are represented as mean ± SEM.
PGF2α facilitates pathological retinal angiogenesis by modulating endothelial FOS-driven ELR+ CXC chemokine expression (2022)
Figure 6

Figure 6. PGF2α induces expression of ELR+ CXC chemokines in HRMECs through transcription factor FOS.

A. Heat map of differentially expressed transcription factors in HRMECs in response to 500 nM PGF2α (n=3).

B. Relative expression levels of FOS in retinal microvascular ECs from patients with PDR (n=4) compared with the corresponding expression levels from normal subjects (n=7) (Gene Expression Omnibus Database dataset GSE94019). TPM stands for "Transcripts per kilobase million."

C. Effect of FOS knockdown on 500 nM PGF2α-induced CXCL8 expression in HRMECs (n=4).

D. Effect of FOS knockdown on 500 nM PGF2α-induced HRMEC CXCL8 secretion in the culture medium (n=4).

E. and F. Effect of RA (1 μM, AP-1 dimer inhibitor), SN-50 (100 μg/mL, NFκB inhibitor), and Inca-6 (2.5 μM, NFAT inhibitor) on 500 nM PGF2α-induced CXCL8 expression in HRMECs and HRMEC CXCL8 secretion in the culture medium (n=4).

G. Representative images of scratch-induced migration of 500 nM PGF2α-treated HRMECs with or without FOS knockdown. The yellow solid line indicates the original position of the scratch, the dashed line indicates the position of the cells after migration, and the arrows represent the migratory direction.

H. Quantitation of the cell-migration distance in H (n=4).

I. Effect of FOS knockdown on 500 nM PGF2α-induced HRMEC proliferation (n=4).

J. Representative images of tube formation by 500 nM PGF2α-treated HRMECs with or without FOS knockdown.

K. Quantitation of total tube length, number of junctions, number of meshes, and percentage of mesh area in J (n=4).

L. Fos mRNA levels in retinas from OIR mice during postnatal days 12-17 (n=3-4).

M. Effect of PTGFR deficiency in ECs on Fos expression in the retina from OIR mice on postnatal day 16 (n=6).

N. Effect of FOS inhibitor SR11302 (1 μM) on 500 nM PGF2α-promoted sprouting of mouse aortic rings.

O. Quantitation of sprouting areas in N (n=6).

P. Effect of FOS inhibitor SR11302 (1 μM) on 500 nM PGF2α-promoted CXCL1 secretion in mouse aortic ring culture medium (n=6).

Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test (B, C, D, E, F, M, P) or two-way ANOVA with Tukey's multiple comparisons test (H, I, K, L, O). Scale bar: 100 μm (G) and 200 μm (J, N). Data in B are expressed as the mean + SD. Data in C, D, E, F, H, I, K, L, M, O, P are represented as mean ± SEM.
PGF2α facilitates pathological retinal angiogenesis by modulating endothelial FOS-driven ELR+ CXC chemokine expression (2022)
Figure 7

Figure 7. PGF2α upregulates FOS expression in HRMECs through the Gq/CAMK2G/p38/ELK-1 signaling pathway.

A. Schematic diagram of the potential PTGFR-mediated signaling pathways. All inhibitors of targeted molecules are shown in red.

B. Effect of U73122 (10 μM), BAPTA (50 μM), Y27632 (5 μM), and Ly294002 (15 μM) treatment on 500 nM PGF2α-induced FOS gene expression in HRMECs (n=4).

C. Effect of KN93 (10 μM) and RO-318220 (250 nM) treatment on 500 nM PGF2α-induced FOS expression in HRMECs (n=4).

D. Effect of SB203580 (2 μM), PD98059 (20 μM), and SP600125 (60 nM) treatment on 500 nM PGF2α-induced FOS gene expression in HRMECs (n=4).

E. Effect of U73122 (10 μM), BAPTA (50 μM), and KN93 (10 μM) treatment on 500 nM PGF2α-induced p38 phosphorylation in HRMECs.

F. Quantitation of the ratio of p38 phosphorylation to total p38 protein in E (n=3).

G. Effect of U73122 (10 μM), BAPTA (50 μM), and SB203580 (2 μM) on the phosphorylation of CAMK2 isoforms in 500 nM PGF2α-treated HRMECs.

H. Quantitation of the ratio of CAMK2G phosphorylation to total CAMK2G in G (n=3).

I. Quantitation of the ratio of phosphorylated CAMK2D to total CAMK2D in G (n=3).

J. Effect of CAMK2G or CAMK2D knockdown on 500 nM PGF2α-triggered p38 phosphorylation in HRMECs.

K. Quantitation of the ratio of p38 phosphorylation to total p38 protein in J (n=3).

L. Effect of CAMK2G or CAMK2D knockdown on 500 nM PGF2α-induced FOS expression in HRMECs (n=4).

M. Effect of U73122 (10 μM), BAPTA (50 μM), KN93 (10 μM), and SB203580 (2 μM) on ELK-1 phosphorylation in 500 nM PGF2α-treated HRMECs.

N. Quantitation of the ratio of phosphorylated ELK-1 to total ELK-1 in M (n=3).

O. Schematic diagram of the PGF2α/PTGFR axis-mediated angiogenic signaling in retinal ECs.

Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test (B, C, D and L) or unpaired student's t-test (F, H, I, K and N). Data are represented as mean ± SEM.
PGF2α facilitates pathological retinal angiogenesis by modulating endothelial FOS-driven ELR+ CXC chemokine expression (2022)
Figure 8

Figure 8. Administration of PTGFR inhibitor AL8810 attenuates retinal angiogenesis in OIR mice.

A. Schematic diagram of AL8810 administration in OIR mice. i.p. stands for "intraperitoneally injected".

B. Representative images of OIR retinas in WT and Cxcr2-/- mice with or without AL8810 treatment. The green color shows the isolectin B4-stained vessels, the second-row panels display the enlarged images of white boxes in the first-row panels, the third-row images show neovascular tufts (NV, white), and the fourth-row images show the vaso-obliteration (VO, white) area.

C. Quantitation of oxygen-induced retinal neovascularization in WT and Cxcr2-/- mice with or without AL8810 treatment (n=12).

D. Quantitation of retinal vaso-obliteration in WT and Cxcr2-/- mice with or without AL8810 treatment (n=12).

E. Effect of AL8810 on retinal Fos expression in WT and Cxcr2-/- OIR mice on postnatal day 16 (n=6).

F. Effect of AL8810 on retinal Cxcl1 expression in WT and Cxcr2-/- OIR mice on postnatal day 16 (n=6).

Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test (C, D) or Mann-Whitney test (E, F). Scale bar: 500 μm (unmagnified image), 150 μm (magnified image). Data are represented as mean ± SEM.
ZBTB2 links p53-deficiency to HIF-1-mediated hypoxia signaling to promote cancer aggressiveness (2022)
Figure 1

Figure 1. ZBTB2 links p53-deficiency to HIF-1 activation.

A, The indicated cells co-transfected with p5HRE-Luc and either pEF6/ZBTB2 (ZBTB2) or pEF6/myc-His B (empty vector: EV) were cultured under the indicated oxygen conditions for 24 hours for the luciferase assay.

B, U2OS cells co-transfected with p5HRE-Luc and the indicated siRNA were cultured under the indicated oxygen conditions for 24 hours for the luciferase assay.

C, HCT116 p53-/- cells co-transfected with p5HRE-Luc, either pEF6/ZBTB2 (ZBTB2) or pEF6/myc-His B (EV), and either pcDNA3/p53 (at two concentrations: + and ++) or pcDNA3 (EV) were cultured under the indicated oxygen conditions for 24 hours for the luciferase assay and Western blotting using the indicated antibodies.

D, HCT116 p53+/+ cells co-transfected with p5HRE-Luc, either pEF6/ZBTB2 (ZBTB2) or pEF6/myc-His B (EV), and either pcDNA3/p53 R175H, R248W, R273H, or pcDNA3 (EV) were cultured under the indicated oxygen conditions for 24 hours for the luciferase assay.

Data information: Mean ± s.d. The number of technical replicates in all of the experimental groups was 3, and reproducibility of the results was confirmed at least 3 times by biologically independent experiments (A-D). N.S.: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Student's t-test (A-D).
ZBTB2 links p53-deficiency to HIF-1-mediated hypoxia signaling to promote cancer aggressiveness (2022)
Figure 2

Figure 2. ZBTB2 promotes invasion, distant metastases, and growth of tumors when functional p53 is deficient in vivo.

A-F, The indicated cells transiently transfected with either pEF6/ZBTB2 (ZBTB2) or pEF6/myc-His B (EV) (A,C,D,F), either pcDNA3/p53 (p53) or pcDNA3 (EV) (C), or with the indicated siRNA (B,E,F) were cultured under < 0.1% O2 conditions for 24 hours and subjected to qRT-PCR for the indicated mRNA (A-C) or to the transwell invasion assay (D-F).

G, The number of metastatic colonies in lungs in the pulmonary metastasis model using the indicated cells stably transfected with ZBTB2 expression vector (ZBTB2) or its empty vector (EV) (left) and the representative images of lungs (right).

H,I, The indicated cells stably transfected with the ZBTB2 expression vector (ZBTB2) or its empty vector (EV) (H) or transiently transfected with siScr or siZBTB2 (I) were cultured under < 0.1% O2 (H) or the indicated oxygen conditions (I) for the indicated periods and subjected to the in vitro cell proliferation assay.

J,K, Growth of the indicated tumor xenografts with or without ZBTB2 overexpression was analyzed.

Data information: Mean ± s.d. The number of technical replicates was 3 (A-F,I), at least 8 (G), 5 and 4 for EV and ZBTB2, respectively (H), and 7 (J,K), and reproducibility of the results was confirmed at least 3 times by biologically independent experiments (A-K). N.S.: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Student's t-test (A-K).
ZBTB2 links p53-deficiency to HIF-1-mediated hypoxia signaling to promote cancer aggressiveness (2022)
Figure 3

Figure 3. ZBTB2 expression is associated with poor prognoses of patients with p53-deficient cancers.

A, Tissue microarrays of human lung adenocarcinoma (243 samples) stained with anti-ZBTB2 antibodies. High magnification images are shown in the left lower side of each image. Scale bar, 500 and 100 μm in the low and high magnification images, respectively.

B, Fisher's exact test for percentage of CA9-positive tumors in the four indicated groups. Percentages and absolute numbers of each group are indicated in the graph.

C, Kaplan-Meier analysis of the disease-free survival of the lung cancer patients stratified by the expression levels of ZBTB2 and p53 status.

D-F, TCGA-based Kaplan-Meier analysis of overall survival of lung adenocarcinoma patients stratified by the expression levels of ZBTB2 and those of BAX (D), ZMAT3 (E), and CEACAM1 (F).

Data information: N.S.: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, Fisher's exact test (B), log-rank tests (C-F).