EMBO/soda-nel huggingface dataset:
Blocking STAT3/5 through direct or upstream kinase targeting in leukemic cutaneous T-cell lymphoma (2022)
Figure 2

Figure 2. STAT3/5 copy number gain correlates with disease clonality and results in STAT3/5 overexpression and hyperactivation

A-C Spearman correlation analysis using CNA log2 ratios and the percentage of clonal CD3+ cells detected in patients for (A) STAT3, (B) STAT5A and (C) STAT5B.

D-F RT-qPCR expression data of (C) STAT3, (D) STAT5A, and (E) STAT5B genes from whole PBMCs isolated from patient blood (n = 5) or healthy controls (n = 9). GAPDH expression was used for normalization. Statistical significance was calculated using two-tailed unpaired t-test. P-value: <0.01 (**), <0.0001 (****). One experiment was performed in technical triplicates. The bold dashed line in the middle of the violin plot denotes the median value, while the thin dotted lines denote the interquartile range.

G Immunoblotting for total STAT3/5 protein isolated from the respective whole PBMCs from patient blood (n = 3), or from healthy controls (n = 3). HSC70 was used as loading control. One experiment was performed.

H Representative image of H&E, CD3, pY-STAT5 single staining and CD3/pY-STAT5 double staining on skin biopsy tissue from P3 (n = 1), which demonstrates infiltration of CD3+ pY-STAT5+ (13.45%) cells into the dermal and subcutaneous layer of the skin. Arrows indicate pY-STAT5+ single and CD3+ pY-STAT5+ cells.

I-M Quantification of IHC staining from skin-biopsy sections of L-CTCL patients (n = 6) and healthy individuals (n = 5) depicted in Appendix Figure S3. As the number of STAT5A/B positive cells was negligible (less than 0.03/per mm2) in healthy skin biopsy sections, healthy control samples were considered negative for pY-STAT5, and levels were set to 0. Statistical significance was calculated using an unpaired Welch's t-test. P-value: <0.05 (*).The bold dashed line in the middle of the violin plot denotes the median value, while the thin dotted lines denote the interquartile range.
Blocking STAT3/5 through direct or upstream kinase targeting in leukemic cutaneous T-cell lymphoma (2022)
Figure 3

Figure 3. Established CTCL cell lines carrying STAT3/5 gains can be employed as patient-like models for in vitro testing

A-C Graphic depicts the aberration patterns on (A) chromosome 17 (STAT3/5), (B) chromosome 2 (STAT1) and (C) chromosome 16 (SOCS1) in the CTCL cell lines used in the study. Aberration patterns are represented as the log2 ratio of the fluorescence intensity of the tumor DNA vs. reference DNA and were generated by aCGH. The whole-chromosome aberration patterns of the cell lines are shown in Appendix Figure S4. Immunoblotting for total protein STAT1/3/5 and activation levels: phospo-Tyr (701)-STAT1/phospho-Tyr (705)-STAT3/phospho-Tyr (694/699)-STAT5, and SOCS1 protein levels. The cytokine-dependent cell line, SeAx, was collected 30 min after 5 ng/ml IL-2 and IL-4 cytokine addition. HSC70 was used as loading control. One representative out of three independent experiments is shown.
Blocking STAT3/5 through direct or upstream kinase targeting in leukemic cutaneous T-cell lymphoma (2022)
Figure 4

Figure 4. JPX-0750 and IQDMA are potent, apoptosis-inducing, and L-CTCL cell-specific compounds with distinctive modes of action

A Heatmap of IC50 values calculated from drug response analysis using CellTiter-Glo viability assays upon 48h drug treatment. One representative, out of three independent experiments performed in triplicates, is shown.

B, C Immunoblot showing total protein STAT3/5 and activation levels: phospho-Tyr (705)-STAT3/phospho-Tyr (694/699)-STAT5A/B after 24h treatment with (B) JPX-0750 and (C) IQDMA. The cytokine-dependent cell line, SeAx, was collected 30 min after 5 ng/ml IL-2 and IL-4 cytokine addition. HSC70 was used as loading control. The normalized levels of phospho- and total STAT3/5 quantified by densitometry, are shown below the respective blots. One representative, out of three independent experiments is shown.

D-G Real-Time apoptosis/necrosis assay in the (D, E) SeAx and (F, G) Myla cell lines treated with 2.5µM JPX-0750 and IQDMA. RLU, Apoptosis [RLU] = relative luminescence; Necrosis [RFU] = relative fluorescence units. Error bars represent mean +/-SD. One experiment in technical triplicates was performed.

H-I The effect of (H) JPX-0750 and (I) IQDMA on the viability of CTCL cells in comparison to viability of primary juvenile fibroblasts (JF) and keratinocytes (HaCat) at a low dose range (0.7-3µM). One experiment, in triplicates, was performed with JF and HaCat cells, and three experiments, in triplicates with the CTCL cells. Error bars represent mean +/-SD. Statistical significance was calculated by two-way ANOVA with Tukey's multiple comparisons test. P-value: <0.05 (*), <0.01 (**), <0.001 (***), <0.0001 (****). P-value summaries are provided in Appendix Table S1.
Blocking STAT3/5 through direct or upstream kinase targeting in leukemic cutaneous T-cell lymphoma (2022)
Figure 5

Figure 5. IQDMA is a multi-kinase inhibitor downregulating STAT5 nuclear levels

A Kinome screen with 10µM IQDMA.

B Subcellular fractions of SeAx cells treated with IQDMA for 24h and immunoblotted for pY-STAT5 and total STAT5. The cells were collected 30 min after 1 ng/ml IL-2 cytokine addition. α-Tubulin and Histone H3 were used as loading controls for cytoplasmic and nuclear fractions, respectively. The normalized levels of phospho- and total STAT5 in the nucleus and cytoplasm, quantified by densitometry, are shown below the respective blots. One representative out of two independent experiments is shown.

C Venn diagram showing the number of proteins that are significantly up/downregulated as a result of IQDMA treatments at 1, 2.5 and 10µM.

D-F Scatter plot depicting the profile of SeAx cells treated with (D) 1µM IQDMA, (E) 2.5µM IQDMA and (F) 10µM IQDMA. Protein abundances were determined using TMT-based quantification mass spectrometry (see Supplementary Materials and Methods for details). Significant changes were assessed by a modified t-test as implemented in the limma package, with the negative log10 p values on the x-axis, and log2 fold change shown on the y-axis.

G Heatmap of IC50 values calculated from drug response analysis using CellTiter-Blue or CellTiter-Glo viability assays upon 48h drug treatment. One representative out of three independent experiments performed in triplicates is shown. Grey represents the presence of STAT3/5 gain.
Blocking STAT3/5 through direct or upstream kinase targeting in leukemic cutaneous T-cell lymphoma (2022)
Figure 6

Figure 6. The PAK kinase inhibitor impacts downstream STAT3/5 signaling, can be used in drug combinations and is highly specific for L-CTCL cell carrying STAT3/5 gains

A Inhibitor treatment was carried out with increased dose escalation for 24h. SeAx cells were collected 2h after 7,5 ng/ml IL-2 and IL-4 cytokine addition. STAT3/5 target gene products MYC, PIM1, MCL-1, and AKT1/2/3, as well as proteins regulating the cell cycle, such as CYCLIN D2 and D3, and CDK6 were probed by Western blotting. HSC70 served as loading control. The normalized protein levels, quantified by densitometry, are shown below the respective blots. One representative out of three independent experiments is shown.

B-D Synergy analysis of the indicated two-drug combinations in the SeAx and Myla cells after 48 h treatment. In each graph the most synergistic area (MSA) is highlighted, which represents the most synergistic 3-by-3 dose-window with the respective MSA score. The Zero interaction potency model was applied to quantify the degree of synergy, according to which an MSA score below -10 indicates that drugs are antagonistic (green), a score between -10 and 10 indicates that 2 drugs are additive (white), while a score above 10 indicates a synergistic effect (red). (D) Heatmap showing MSA scores of the combination of FRAx597 with the listed drugs. One representative out of two independent experiments is shown. Synergy matrices for each drug combination are represented in Appendix Figure S5.

E Heatmap showing the IC50 values upon treatment of primary PBMCs isolated from L-CTCL patients with JPX-0750, IQDMA and FRAx597. One experiment performed in triplicates using CellTiter-Glo viability assays upon 48h drug treatment is shown. The relative STAT3/5 log2 ratio value is depicted in grey.

F-H Spearman correlation analysis using CNA log2 ratios and the IC50 values for the respective drug.
Blocking STAT3/5 through direct or upstream kinase targeting in leukemic cutaneous T-cell lymphoma (2022)
Figure 7

Figure 7. IQDMA and the PAK kinase inhibitor are highly effective in reducing tumor growth in an intradermal xenograft mouse model

A Graphical scheme depicting the experiment.

B Growth rate of Hut78, Myla and H9-derived tumors measured on the respective day post-injection. The mean of the respective tumor volumes for H9 group and for the vehicle-treated groups for Hut78 and Myla are shown. Tumor volume = (length x width2)/2. Error bars represent mean +/-SEM. n represents the total number of tumors that engrafted per group.

C-D Myla-derived tumor weight upon experiment termination, after 20 days of treatment with 5 mg/kg of (C) JPX-0750 (n = 5) and IQDMA (n = 6), or vehicle (n = 7), and (D) FRAx597 (n = 5) or vehicle (n = 4), with n representing the number of analyzed tumors per group. Statistical significance was calculated using two-tailed paired t-test. P- value: <0.05 (*). Error bars represent mean +/-SEM.

E-J H&E and IHC analysis of Myla-derived tumors treated with JPX-0750 (n = 6), IQDMA (n = 6) or vehicle (n = 6), and FRAx597 (n = 5) or vehicle (n = 4), stained with (E, F) H&E, (G, H) Ki67, as a marker for proliferation and (I, J) CD31, as a tumor vessel marker, with n representing the number of analyzed tumors per group. Pictures shown are from contiguous sections. Dotted rectangles indicate magnified areas. Scale bars, 200, 100 and 25 µm. Violin plots show the perimeter of the annotated tumor cell-infiltration/expansion region into the intradermal and subcutaneous region of the skin, as well as quantification of the percentage of Ki67+ cells in the tissue. CD31 staining was quantified as raw counts of vessels with lumen. Statistical significance was calculated using two-tailed paired t-test with Welch´s correction. P-value: <0.05 (*), <0.01 (**), <0.001 (***). The bold dashed line in the middle of the violin plot denotes the median value, while the thin dotted lines denote the interquartile range.

EXPANDED VIEW FIGURE LEGENDS
PPARα Alleviates Iron Overload-induced Ferroptosis in Mouse Livers. (2022)
Figure 2

Figure 2. PPARα Regulates Gpx4 Transcriptional Activity by Binding to An PPRE in Intron 3 of Gpx4, Induces Gpx4 Gene Expression In Vivo.

(A) Acox1 and Gpx4 mRNA was measured in liver of WT and PPARα-/- mice fed blank solvent (white bars, n = 5 mice/group) or GW7647 (black bars, n = 5 mice/group).

(B) Gpx4 protein was measured in liver of WT and PPARα-/- mice fed blank solvent or GW7647; n = 4 biological replicates.

(C) Schematic of the WT and mutant PPRE elements. The six nucleotides that were altered to form the mutant construct are underlined.

(D) A 123-base pair fragment of intron 3 of mouse Gpx4 (position from +7679 to +7803 with respect to the transcription start site) was inserted into the pGL3 promoter vector to generate the pGL3 promoter intron 3 reporter constructs. The PPRE in intron 3 was mutated to create the mutant construct (pGL3 promoter intron 3mut). These reporter constructs were transfected into Hep1-6 cells. The indicated PPARα ligands were added to cell cultures 24 hours before the reporter gene assay; n = 3 biological replicates. Data were calculated as the fold induction with respect to the empty vector (pGL3 promoter luciferase vector).

(E) Chromatin immunoprecipitation assays were performed on soluble formaldehyde-crosslinked chromatin isolated from untreated and GW7647-treated WT or PPARα-/- livers with polyclonal anti-PPARα antibodies (anti-PPARα) or control IgG. The final DNA extraction was polymerase chain reaction-amplified with a primer pair that covered the sequence in intron 3 of Gpx4.

Data information: mRNA levels were normalized to 36B4 and are expressed relative to the mean value of the WT group. Data are presented as means ± SDs. **p < 0.01, determined by ANOVA.
PPARα Alleviates Iron Overload-induced Ferroptosis in Mouse Livers. (2022)
Figure 3

Figure 3. PPARα deficiency influences iron metabolism in the liver.

(A) Fluorescent images of labile Fe2+ in hep1-6 cells. Hep1-6 cells were incubated with control, 20 μM probe1 for 30 min; 20 μM Cu2+ for 30 min then 20 μM probe1 for another 30 min; 20 μM Fe3+ for 30 min then 20 μM probe1 for another 30 min; 20 μM Fe2+ for 30 min then 20 μM probe1 for another 30 min; All scale bars are 20 μm. n = 3 biological replicates.

(B) Liver sections were obtained from the indicated mice and stained with probe1. All scale bars are 20 μm. n = 3 biological replicates.

(C) Hepatic Acox1 and TRF mRNA levels were measured in the WT and PPARα-/- mice with or without GW7647 treatment. n = 3-5 mice/group.

(D) TRF protein was measured in liver of WT and PPARα-/- mice fed blank solvent or GW7647. n = 4 biological replicates.

(E) Hepatic TRF mRNA levels were measured in the indicated mice. n = 3-5 mice/group.

(F) Hepatic and serum TRF content were measured in the indicated mice. n = 3-5 mice/group.

Data information: mRNA levels were normalized to 36B4 and are expressed relative to the mean value of the WT group. Data are presented as means ± SDs. *p < 0.05, **p < 0.01, determined by ANOVA.
UBR7 Acts as a Histone Chaperone for Post-Nucleosomal Histone H3 (2021)
Figure 1

Figure 1. UBR7 Associates with Histone H3.1

Schematic representation of BioID proximity biotinylation in HeLa-T-REx cells stably expressing H3.1-BirA*-HA. Randomly cycling cells were treated with biotin to label proteins proximal to nucleosomal (nucH3.1) or non-nucleosomal soluble (solH3.1). H3.1-BirA*-HA, and biotin-labeled proteins were purified on streptavidin-conjugated beads.

Representative images of parental cells or cells stably expressing H3.1-BirA*-HA after incubation with or without biotin as in (A). Coverslips were pre-extracted to visualize chromatin-bound proteins. DNA is visualized via DAPI staining shown in cyan, immunofluorescence for H3.1-BirA*-HA is shown in green, and biotinylated proteins are shown in red. Scale bar = 5 μM.

Representative images of HeLa cells pre-extracted as in (B). DNA is visualized as in (B). Immunofluorescence for endogenous H3 is shown in green. Scale bar = 10μM.

Immunoblot analysis of proteins biotinylated and isolated as in (A). Parental HeLa-T-REx cells or cells expressing H3.1-BirA*-HA and treated with biotin for indicated times.

Immunoblot analysis of TAP-H3.1 or -CENP-A pulldown of UBR7-LAP (schematic, upper). 293T cells were co-transfected with UBR7-LAP and TAP-tagged constructs, which were purified on IgG-conjugated sepharose beads.
UBR7 Acts as a Histone Chaperone for Post-Nucleosomal Histone H3 (2021)
Figure 2

Figure 2. UBR7 Exists in Non-Nucleosomal and Nucleosome-Bound Pools

Schematic representation of cellular fractionation to generate non-nucleosomal and nucleosomal fractions. WCE: whole cell extract, NF1: nuclear fraction 1, NF2: nuclear fraction 2.

Representative immunoblot analysis of NASP, UBR7, and ASF1 distribution in cellular fractions. Relative loading factors are marked e.g. 1x, 0.2x, etc. Numbers below blot indicate UBR7 immunoblot signal relative to 1x loading of non-nucleosomal fraction.

Enrichment of non-nucleosomal:nucleosomal UBR7 as estimated from 5x, 10x, and 20x non-nucleosomal loading conditions in Fig 2B (see Fig EV2). Error bars represent mean +/- standard deviation. A two-tailed one sample t-test was used to calculate p-value (italics) associated with mean difference from a hypothetical value of 1.0 (represented by dashed line). Experiment in (B-C) was performed twice.

Immunoblot of anti-FLAG IP of 3x-FLAG tagged GFP, or FLAG-HA tagged H3.1 or H2B, from the non-nucleosomal fraction of 293T cells.

Representative images of HeLa cells transfected with UBR7-FL. DNA is visualized via DAPI staining shown in magenta and immunofluorescence for 3xFLAG is shown in green. The area within yellow dotted box in (E) is magnified 4x in (E'). Scale bar = 5 μm.

Quantification of the nuclear anti-FLAG immunofluorescence intensity in (E). Error bars represent mean +/- standard deviation. One way ANOVA was used to calculate p-values (italics) associated with differences between treatments. Experiment in (E-F) was performed twice.
UBR7 Acts as a Histone Chaperone for Post-Nucleosomal Histone H3 (2021)
Figure 3

Figure 3. The UBR7 UBR Box and PHD Mediate Binding to the Histone H3.1 N-terminus

Immunoblot analysis of anti-H3.1-HA pulldown of UBR7-LAP after deletion of H3.1 N-terminal tail (schematic, upper). 293T cells were co-transfected with UBR7-LAP and HA-tagged constructs. Numbers below blot indicate HA-normalized anti-GFP immunoblot signal relative to full-length H3.1-HA.

Representative images of HeLa cells transfected with indicated constructs. DNA is visualized via DAPI staining shown in cyan and immunofluorescence for endogenous H3 is shown in green. Scale bar = 10 μm. Experiment was performed three times.

Immunoblot analysis of anti-FLAG pulldown of full length UBR7-FLAG (residues 1-425) mutant UBR7 bearing deletion of UBR7 C-terminus (∆C-term, residues 1-215), UBR box (∆UBR, residues 117-425), UBR box and PHD (∆UBR∆PHD, residues 216-425), or PHD and C-terminus (∆PHD∆C-term, residues 1-116). 293T cells were co-transfected with H3.1-HA and UBR7-FLAG constructs (schematic, upper). Asterisk represents non-specific band. Numbers below blot indicate FLAG-normalized anti-HA immunoblot signal relative to full length UBR7-FLAG.

Immunoblot analysis of anti-H3.1-HA pulldown of full-length UBR7-LAP and UBR7PHD fragment (residues 117-215) (schematic, upper). 293T cells were transfected as in (A). Values below pulldown represent H3.1-HA-normalized GFP-UBR7 immunoblot signal relative to UBR7-FL. Data were acquired from a continuous membrane, and the image is cropped to omit irrelevant samples. Numbers below blot indicate HA-normalized anti-GFP immunoblot signal relative to full length UBR7-LAP.
UBR7 Acts as a Histone Chaperone for Post-Nucleosomal Histone H3 (2021)
Figure 4

Figure 4. The UBR7 PHD and UBR Mediate Binding to Post-Nucleosomal Histone H3.1

Histone peptide array identified modified histone H3 N-terminal peptides bound by purified His-MBP-UBR7. Boxplots represent Z-scores of the local background corrected peptide intensity values on the array. Box limits represent 25th and 75th percentiles. Whiskers extend to 1.5x the interquartile range. Experiment was performed twice. Left: Peptides (un)modified at H3K4. Right: Peptides (un)modified at H3K9 and H3S10.

Anti-GFP immunoblot analysis of streptavidin pulldown of biotin-conjugated H3 and H4 peptides following incubation with 293T-GFP-UBR7 cell lysate. HP1α serves as a positive control for binding to H3K9me3.

Predicted structure of UBR7 PHD (a.a.133-190) using Phyre2 (Kelley et al, 2015) overlayed on the BPTF PHD2 structure (RSCB PDB: 2F6N) as reference. UBR7 PHD aromatic cage residues mutated in (D) and corresponding residues in BPTF PHD2 are labeled.

Anti-GFP immunoblot analysis of streptavidin pulldown of the indicated biotin-conjugated H3 peptides following incubation with 293T-GFP-UBR7 cell lysate after mutation of residues highlighted in (C).

Immunoblot analysis for total and modified histones in anti-FLAG pulldown of constructs in (Fig EV4A) from soluble cellular fractions demonstrates that UBR7 UBR box and PHD are necessary for binding to post-nucleosomal H3/H4.

Quantification of the immunoblot data in (E). Immunoblot signals were normalized to anti-FLAG signal. Error bars represent mean +/- standard deviation. A two-tailed one sample t-test was used to calculate p-values (italics) associated with group means difference from a hypothetical value of 1.0 (represented by dashed line). Experiment in (E-F) was performed twice.

Schematic representation of proximity biotinylation in stable inducible H3.1-APEX2-3xFLAG cells. To perform proximity biotinylation with newly synthesized H3.1-APEX2-3xFLAG, after doxycycline induction followed by washout, cells were immediately subjected to labeling reaction. To perform proximity biotinylation with pre-existing H3.1-APEX2-3xFLAG, following washout cells were grown for 48 h before labeling reaction. Biotin-labeled proteins were purified on streptavidin-conjugated beads.

Immunoblot analysis of streptavidin pulldown from proximity biotinylation described in (G). 4.5 h represent newly synthesized histones and 48 h represent pre-existing histones. Wedge represents loading titration: 1x > 0.1x > 0.01x > 0.001x. All other lanes are loaded at 1x concentration. Immunoblot signals were normalized to anti-FLAG signal.

Quantification of anti-FLAG (H3.1-APEX2-3xFLAG) and anti-UBR7 immunoblot represented in (G-H). Lanes 8 and 11 were quantified. Error bar represents mean +/- standard deviation. A two-tailed one-sample t-test was used to calculate p-value (italics) associated with group mean difference from a hypothetical value of 1.0 (represented by dashed line). Experiment in (G-I) was performed twice.
UBR7 Acts as a Histone Chaperone for Post-Nucleosomal Histone H3 (2021)
Figure 5

Figure 5. UBR7 Bound to Post-Nucleosomal Histone H3/H4 Interacts With NASP

Immunoblot analysis of parental HeLa cells or cells expressing UBR7-BirA*-HA confirms specific expression of UBR7-BirA*-HA.

Representative images of parental cells or cells stably expressing UBR7 fused to BirA* ligase and HA tag (UBR7-BirA*-HA) after incubation with or without biotin. DNA is visualized via DAPI staining shown in cyan, immunofluorescence for UBR7-BirA*-HA is shown in green, and biotinylated proteins are shown in red. Scale bar = 5 μm.

Quantification of log2 fold change and -log10 p-value of MS spectral counts from UBR7-BirA*-HA (heavy) compared to parental (light) cells. Positive log2 fold changes are presented. Full dataset displayed as inset bordered by dotted line. The two proteins most abundantly labeled by UBR7-BirA*-HA are UBR7 and NASP. Experiment was performed twice.

Immunoblot analysis of anti-FLAG IP. Cells were fractionated 72 h post-transfection, and 3xFLAG constructs were immunoprecipitated from non-nucleosomal fraction and eluted with FLAG peptide.

Immunoblot analysis of anti-FLAG IP. Immunoblot signals were normalized to anti-FLAG signal. Experiment was performed as in (D). Asterisk indicates non-specific bands in input. Quantification appears on the right. A two-tailed one sample t-test was used to calculate p-value (italics) associated with group mean difference from a hypothetical value of 1.0 (represented by dashed line). Experiment was performed twice.

Immunoblot analysis for total and modified H3 and H4 in anti-FLAG IP. Experiment was performed as in (D). Dot plot error bars represent mean +/- standard deviation. Immunoblot signals were normalized to anti-H4 signal. A two-tailed one sample t-test was used to calculate p-value associated with group mean difference from a hypothetical value of 1.0. Experiment was performed twice.

Representative images of HeLa cells transfected with sNASP-FL. DNA is visualized via DAPI staining shown in magenta and immunofluorescence for 3xFLAG is shown in green. Scale bar = 5 μm.

Quantification of the nuclear anti-FLAG immunofluorescence intensity in (G) demonstrating that sNASP-FL is highly susceptible to pre-extraction with Triton X-100. One way ANOVA was used to determine p-values (italics) associated with differences between treatments. Error bars represent mean +/- standard deviation. Experiment in (G-H) was performed twice.