EMBO/soda-nel huggingface dataset:
A Heterotypic Assembly Mechanism Regulates CHIP E3 Ligase Activity (2022)
Figure 3

Figure 3: UFD-2 stabilizes an open/active CHN-1 conformation and HSP-1 stabilizes a closed/non-active CHN-1 conformation.

A) Auto-Ub of recombinant CHN-1 using UBE2D1 was performed alone or complexed with UFD-2 in the presence of recombinant DAF-21, DAF-21ΔEEVD, HSP-1, or HSP-1ΔEEVD. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies.

B) Top, schematics of the HSP-1 peptide sequence (cyan) aligned with the C-terminal sequence of full-length HSP-1 (amino acids 600-640) used in the ubiquitylation reaction. Auto-Ub of recombinant CHN-1 was performed as indicated HSP-1‑derived peptide. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies. Right, quantification of the CHN-1 modifications (unmodified, multi-monoubiquitylated, poly-ubiquitylated) when CHN-1 alone (black) or CHN-1+HSP-1 peptide (cyan). Plotted data are the mean of three technical replicates. Error bars represent the SEM; statistical significance was determined using a two-way ANOVA test. (****P < 0.0001)

C) Top, schematics of the UFD-2 peptide sequences aligned with the C-terminal sequence of full-length UFD-2 (UFD-2 amino acids 864-914) for PEPTIDE 1 (yellow) and PEPTIDE 2 (cyan) and (UFD-2 amino acids 648-678) for PEPTIDE 3 (magenta) used in the ubiquitylation reaction. Below, auto-Ub of recombinant CHN-1 was performed as indicated in the presence of UFD-2‑derived peptides. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies. Right, quantification of the CHN-1 modifications (unmodified, multi-monoubiquitylated, poly-ubiquitylated) when CHN-1 alone (black), CHN-1+PEPTIDE 1 (yellow), CHN-1+PEPTIDE 2 (cyan), or CHN-1+PEPTIDE 3 (magenta). Plotted data are the mean of three technical replicates. Error bars represent the SEM; statistical significance was determined using a two-way ANOVA test. (***P < 0.001)

D) Auto-Ub of recombinant CHN-1 was performed as indicated in the presence of recombinant HSP-1 or HSP-1EEYD. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies.

E) Auto-Ub of recombinant CHN-1 was performed as indicated alone or with UFD-2 or UFD-2EEVD. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies.

F) CHN-1R230A auto-Ub was performed as indicated in the presence of recombinant UFD-2 and HSP-1. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies.

G) Model of UFD-2 activation and HSP-1 inhibition of CHN-1. Dimeric CHN-1 with the TPR domain, U-box, and helix-turn-helix (HH) is indicated by magenta, gold, and cyan, respectively. UFD-2 and HSP-1 peptides are shown in yellow with the indicated amino acid positions in the full-length proteins.

Data information: Representative immunoblots for at least three independent experiments are shown in the panels.
A Heterotypic Assembly Mechanism Regulates CHIP E3 Ligase Activity (2022)
Figure 4

Figure 4: UFD-2 regulates CHN-1 turnover.

A) To detect CHN-1 and UFD-2, indicated lysates of young adult worms were subjected to immunoblotting with anti-CHN-1 and anti-UFD-2 antibodies. Tubulin served as a loading control. Right, quantification of the CHN-1 signals normalized to tubulin levels and plotted as N2 (wild-type; black), ufd-2(tm1380) (cyan), and UFD-2P951A (magenta). Plotted data are the mean of three biological replicates. Error bars represent the SEM; statistical significance was determined using an unpaired t-test. (*P < 0.05)

B) CHN-1 protein levels were determined in the indicated lysates of young adult worms treated with a proteasome inhibitor (MG-132, 10µM) and DUB inhibitor (NEM, 100mM). Tubulin served as a loading control. Right, quantification of the CHN-1 signals normalized to tubulin levels plotted as control (black), or MG-132 treated (magenta) in N2 (wild-type) and ufd-2(tm1380). Plotted data are the mean of three biological replicates.

Error bars represent the SEM; statistical significance was determined using an unpaired t-test. (**P < 0.01; ***P < 0.001)

C) Graph showing the comparison of protein solubility in the presence of 10% trichloroacetic acid (TCA) presented as TCA sensitive proteome in percentage (%) in N2 (wild-type; black), ufd-2(tm1380) (magenta), chn-1(by155); ufd-2(tm1380) (cyan), chn-1(by155) (yellow) and UFD-2P951A (grey) worms. Y-axis shows the percent of the entire protein sample sensitive to TCA treatment. Plotted data are the mean of three biological replicates. Error bars represent SEM; statistical significance was determined using a one-way ANOVA test. (*P < 0.05; **P < 0.01)

D) Measurement of the mobility of the indicated young adult worms exposed to 33 °C heat stress (2 h). Graph plotted as control (gray), or recovery after heat stress (black) for N2 (wild-type), ufd-2(tm1380), chn-1(by155); ufd-2(tm1380), chn-1(by155) and UFD-2P951A worms. Plotted data are the mean of three biological replicates. Error bars represent SEM; statistical significance was determined using a one-way ANOVA test. (**P < 0.01; ****P < 0.0001)
AATF/Che-1 localizes to paraspeckles and suppresses R-loops accumulation and interferon activation in Multiple Myeloma. (2022)
Figure 3

Figure 3: NEAT1 is required for recruiting Che-1 onto the DNA.

A: Quantitative RT-PCR (RT-qPCR) of Kms27 MM cells transiently transfected with two different LNA GapmeR NEAT1 or GapmeR Control oligonucleotides. Values were normalized to Actin expression. Error bars represent the standard error of three different experiments performed in duplicate (Two Tailed T test ****P<0.001).

B: WB analysis with indicated abs of total extracts from Kms27 MM cells transiently transfected as in A.

C: Representative confocal images of Kms27 MM cells transiently transfected with LNA GapmeR NEAT1A, GapmeR NEAT1B or GapmeR Control oligonucleotides and immunostained with anti-Che-1 antibody. Scale bar 10 μm.

D: Isolated cell compartments from Kms27 MM cells transfected with LNA GapmeR NEAT1A or GapmeR Control oligonucleotides, were subjected to WB with the indicated abs.

E: Che-1 ChIP-seq signal intensity of LNA GapmeR NEAT1A (2 replicates on right) and GapmeR Control (2 replicates on left) and at co-localizing Che-1/NEAT1 sites (N=14654 sites). Signal intensity from 1 (weak) to 12 (strongest).

F: FISH images of Kms27 MM cells depleted or not for Che-1 expression (siChe-1A) labeled with Hoechst (cell nuclei) and Quasar 670 (NEAT1 RNA). Scale bar 10 μm.

G: NEAT1 ChIRP-seq signal intensity of Kms27 MM cells transiently transfected with siChe-1A or siControl at co-localizing Che-1/NEAT1 sites (N=14654 sites). Signal intensity from 1 (weak) to 14 (strongest).
AATF/Che-1 localizes to paraspeckles and suppresses R-loops accumulation and interferon activation in Multiple Myeloma. (2022)
Figure 4

Figure 4: Che-1 and NEAT1 suppress RNA: DNA hybrids accumulation.

A: Venn diagrams showing the relationship between enriched protein in Che-1 and RNA:DNA hybrids interactome respectively obtained from this study and either Cristini et al.(Cristini et al., 2018) (Left) and Wu et al. (Wu et al., 2021) (Right).

B: Chromatin extracts from Kms27 and RPMI8226 MM cells treated or not with RNase H, were subjected to immunoprecipitation with S9.6 antibody and analyzed by WB with the indicated abs. Input corresponds to 5% of the total extract used for immunoprecipitation.

C: Representative confocal images showing the interaction between Che-1 and S9.6 revealed by PLA in Kms27 MM cells. Negative controls were performed by omitting Che-1 or S9.6 primary antibody. Scale bar 10 μm.

D: Chromatin extracts from Kms27 MM cells transfected with LNA GapmeR NEAT1A or GapmeR Control oligonucleotides were immunoprecipitated with S9.6 antibody and analyzed by WB with the indicated antibodies. Input corresponds to 5% of the total extract used for immunoprecipitation.

E: (Left) Representative confocal images of Kms27 MM cells treated or not with RNase H, transiently transfected with siChe-1A or siControl and immunostained with S9.6 antibody and. Scale bar 10 μm. (Right) Box plot showing S9.6 signal nuclear intensity per cell (Arbitrary Unit-AU). siChe-1A cells are described in green color, while siControl in grey color. 100 cells were counted in each replicate. Each dot represents a cell. Median scores per sample are written at the top of the median red line. P value: <1.02-18. ANOVA test was performed to evaluate statistical significance. The red center line denotes the median value (50th percentile) while the black box contains the 25th to 75th percentiles of data. The whiskers mark the 5th and 95th percentiles.

F: (Top) S9.6 antibody was used for dot blot analysis to evaluate RNA:DNA hybrids formation in Kms27 MM cells by serial dilutions of genomic DNA starting at 1.5 micrograms. (Bottom) Quantification of S9.6 levels by densitometric analysis. Values were normalized to a Methylene blue loading control. Error bars represent the standard error of three different biological experiments (Two Tailed T test ***P<0.005, ****P<0.001).

G: Enrichment density of DRIP-seq signals at NEAT1/Che1 co-localizing sites in Kms27 MM cells identified in siRNA control (siControl) (grey) and siRNA Che-1 (siChe-1A) (dark green) samples. Enrichment density was calculated on the relative size of each peak (start to end of each fragment) extended 2000 bp upstream and downstream. Right frame represents the signal obtained from 14654 randomly selected sites from the same experiments.
AATF/Che-1 localizes to paraspeckles and suppresses R-loops accumulation and interferon activation in Multiple Myeloma. (2022)
Figure 5

Figure 5: Che-1 and NEAT1 prevent RNA:DNA hybrids formation during transcription.

A: Signal intensity plot (Top) of DRIP-seq enrichment in control KMS27 cell (left) and upon the siChe-1A interference (right) at ribosomal gene loci. Each line represents a single genomic locus. The genomic window was extended 1.5kb upstream and downstream of each ribosomal gene locus. Normalized DRIP-seq signals at all the selected ribosomal genes were then plotted in a heatmap (Bottom) using the following color scheme: Red: high enrichment, Blue: lower enrichment.

B: Screenshots of DRIP-seq (light and dark green color) and ChIP-seq (red color) enrichment at selected ribosomal genes loci. The coordinates of each genomic window are depicted above each frame (HG38).

C: DRIP-qPCR enrichment of the indicated genes in Kms27 cells transiently transfected with siChe-1A or siControl and treated or not with CX5461. Data are expressed as percent of Input. Error bars represent the standard error of three different biological experiments performed in technical duplicate (Two Tailed T test ****P<0.001, n.s. = not significant).

D: (Top) Representative confocal images of Kms27 MM cells transfected and treated as in C and immunostained with S9.6 antibody. Scale bar 10 μm. (Bottom) Box plot showing S9.6 signal nuclear intensity per cell (Arbitrary Unit-AU). siChe-1A cells are described in dark grey, siChe1A plus CX5461 in green color, while siControl in grey color. Median scores per sample are written at the top of the median red line. P value: <1.30-54. ANOVA test was performed to evaluate statistical significance. The red center line denotes the median value (50th percentile) while the black box contains the 25th to 75th percentiles of data. The whiskers mark the 5th and 95th percentiles.

E: (Top) Dot blot analysis of decreasing amounts of genomic DNA extracts from Kms27 MM cells depleted or not for Che-1 expression (siChe-1A) and treated with CX5461, were probed using abs directed against RNA:DNA hybrids. (Bottom) Quantification of S9.6 levels by densitometric analysis. Values were normalized to a Methylene blue loading control. Error bars represent the standard error of three different biological experiments (Two Tailed T test ****P<0.001).

F: RT-qPCR analysis of 47S pre-rRNA levels of Kms27 MM cells transiently transfected with siChe-1A or siControl and treated or not with CX5461. Values were normalized to Actin expression. Error bars represent the standard error of three different biological experiments performed in technical duplicate (Two Tailed T test **P<0.01, ****P<0.001).
AATF/Che-1 localizes to paraspeckles and suppresses R-loops accumulation and interferon activation in Multiple Myeloma. (2022)
Figure 6

Figure 6: Che-1 controls Interferon activation in MM cells.

A: Differential analysis of siControl vs. siChe1A transcriptome in Kms27 MM cells. Volcano plot shows 135 significantly upregulated (dark green) and 392 downregulated genes (dark grey). x-axis reports the base-2 logarithm of fold change which is approximated to the B value coming from the statistical Wald test. y-axis reports the base-10 logarithm of Q value of significant genes.

B: Heatmap depicting pathway activity scores for each transcriptome sample according to PROGENy. The activity score is shown as shade ranging from blue to red which indicates the level of pathway activation. Blu= non-active; Red=active.

C: Gene Set Enrichment Analysis (GSEA) of upregulated significant differential genes with their relative base-10 logarithm of Q value. The analysis was based on hallmark gene sets collection.

D: WB analysis of total extracts of Kms27 MM cells depleted or not for Che-1 (siChe-1A) and probed for the indicated abs.

E: (Left) Luciferase activity of human NF-kB promoter was measured in Kms27 MM cells transiently transfected with siChe-1A or siControl oligonucleotides or with empty vector or p65/RelA protein expressing vector. Error bars represent the standard error of three different biological experiments performed in duplicate (Two Tailed T test ***P<0.005, ****P<0.001). (Right) WB analysis of total extracts of Kms27 MM cells transiently transfected as described in E and incubated with the indicated antibodies.

F and G: RT-qPCR analysis of the levels of the indicated genes in Kms27 MM cells transiently transfected with siChe-1A or siControl. Values were normalized to Actin expression. Error bars represent the standard error of three different experiments in duplicate (Two Tailed T test ***P<0.001, ****P<0.001).

H: WB analysis for the indicated antibodies of total extracts from Kms27 MM cells with siChe-1A or siControl and where indicated with GFP-tagged RNaseH expression vectors. I: RT-qPCR analysis of IFNB and IFNG levels in Kms27 MM cells transiently transfected as in H. Values were normalized to Actin expression. Error bars represent the standard error of three different biological experiments performed in duplicate (Two Tailed T test **P<0.01, ***P<0.005.)
AATF/Che-1 localizes to paraspeckles and suppresses R-loops accumulation and interferon activation in Multiple Myeloma. (2022)
Figure 8

Figure 8: UPR induced R-loops in MM cells.

A: WB analysis of total extracts from Kms27 and U266 human MM cells with the indicated abs.

B: (Left) Kms27 and U266 MM cells were subjected to immunostaining with S9.6 antibody Scale bar 10 μm. (Right) Dot plot showing relative S9.6 nuclear mean intensity cell in the nucleus. 100 cells were counted in each replicate. Median scores per sample are written at the top of the median red line. P value: 3.3-13. T-test was performed to evaluate statistical significance.

C: Different expression of IFNG and IFNB in Kms27 and U266 MM cells was assessed by RT-qPCR. Values were normalized to Actin expression. Error bars represent the standard error of three different biological experiments performed in duplicate (Two Tailed T test ***P<0.005).

D: WB analysis with the indicated abs of total extracts from U266 MM cells treated or not with Thapsigargin (THAP) 0.5 μg/ml for 16 hours.

E: (Left) U266 MM cells treated or not with THAP as in D and immunostained with S9.6 antibody Scale bar 10 μm. (Right) Dot plot showing relative S9.6 nuclear mean intensity per cell. 100 cells were counted in each replicate. Median scores per sample are written at the top of the median red line. P value: 1.1-20. T-test was performed to evaluate statistical significance.

F: RT-qPCR analysis of IFNG and IFNB in U266 MM cells treated or not with THAP. Values were normalized to Actin expression. Error bars represent the standard error of three different biological experiments performed in duplicate (Two Tailed T test ***P<0.005, ****P<0.001)

G: WB with indicated abs of total extracts from Kms27 MM cells treated or not with ISRIB 1μM for 6 hours.

H: (Left) Immunostaining with S9.6 antibody of Kms27 MM cells treated or not with ISRIB as in G Scale bar 10 μm. (Right) Dot plot showing relative S9.6 nuclear mean intensity per cell. 100 cells were counted in each replicate Median scores per sample are written at the top of the median red line. P value: 4.7-23. T-test was performed to evaluate statistical significance.

I: RT-qPCR analysis of IFNG and IFNB levels in Kms27 MM cells treated as in G. Values were normalized to Actin expression. Error bars represent the standard error of three different biological experiments performed in duplicate (Two Tailed T test ****P<0.001).

Expanded View Figure Legends
Human primordial germ cell commitment in vitro associates with a unique PRDM14 expression profile (2015)
Figure 1

Induction of OCT4+/BLIMP1+ PGC precursors from human iPSCs

Effects of ActA and BMP4 on the expression of selected pluripotency, PGC, and mesodermal genes during PGC‐precursor induction. DM: dorsomorphin, SB: SB431542. Samples were calibrated with iPSC values, and iPSC values depict 1. Y‐axes are in log2 scale.
Gene expression dynamics of selected pluripotency, PGC, and mesodermal genes during prolonged PGC‐precursor induction of up to day 7. Y‐axes are in log2 scale.
Immunofluorescence analysis for OCT4 (red), BLIMP1 (green), and T (white) on days 0, 1, and 2. Nuclei were stained with Hoechst (blue). The culture contained BMP4 (5 ng/ml), Activin A (50 ng/ml), and bFGF (20 ng/ml). Scale bar: 100 μm.
Quantification of respective cell populations shown in (C).

Data information: Data are presented as means ± SD (n = 3).
Human primordial germ cell commitment in vitro associates with a unique PRDM14 expression profile (2015)
Figure 2

Induction of PGCLCs from human iPSCs

Schematic presentation of PGC‐precursor and PGCLC induction.
FACS analysis of the concentration‐dependent effect of BMP4 on TRA‐1‐81+/cKIT+ PGCLCs on day 4. B25: 25 ng/ml BMP4; B50: 50 ng/ml BMP4; B100: 100 ng/ml BMP4.
FACS analysis of TRA‐1‐81 and cKIT during PGC induction of up to day 8.
Gene expression analysis of selected PGC markers in TRA‐1‐81+/cKIT+ and SSEA1+/cKIT+ FACS fractions of d4 PGCLCs. Samples were calibrated with iPSC values, and iPSC values depict 1.

Data information: Data are presented as means ± SD (n = 3).
Human primordial germ cell commitment in vitro associates with a unique PRDM14 expression profile (2015)
Figure 3

Characterization of PGCLCs

Gene expression dynamics of selected pluripotency and PGC genes in FACS‐sorted, specified cells during PGC induction. The value for iPSCs is set as 1, and values are on log2 scale.
Immunofluorescence analysis for OCT4 (green), BLIMP1 (red), and STELLA (yellow) in TRA‐1‐81+/cKIT+ PGCLCs on day 6. Nuclei were stained with Hoechst (blue). Scale bar: 15 μm.
Bisulfite sequence analysis of DMRs of the imprinted genes (PEG1, KvDMR1, PEG10, and NESP55) in iPSCs (top) and d6 TRA‐1‐81+/cKIT+ PGCLCs (bottom). White and black circles represent unmethylated and methylated CpG sequences, respectively.

Immunofluorescence analysis for 5mC in d6 PGCLCs.

Nuclei were stained with Hoechst (blue). Scale bar: 50 μm.

Transplantation of human iPSCs and PGCLCs into ovaries of recipient mice. (i) left ovary: control ovary without transplantation; right ovary: teratoma from iPSC‐reconstituted ovary; (ii) PGCLC‐reconstituted ovaries. Note that no teratomas had formed. Bottom panels: Immunohistochemical analysis for NUMA in sections of iPSC‐ and PGCLC‐reconstituted ovaries. (iii) iPSC‐induced teratoma shown in (i); (iv) ovary containing NUMA+ cells shown in (ii). Insert: higher magnification of selected area. Scale bars: (i) and (ii), 0.5 cm; (iii) and (iv), 50 μm.
Human primordial germ cell commitment in vitro associates with a unique PRDM14 expression profile (2015)
Figure 4

Effects of KSR and WNT3A on PGC‐precursor and PGCLC induction

Effects of KSR and WNT3A during PGC‐precursor induction on the expression of selected pluripotency, PGC, and a mesodermal gene of d2 cultures. The value for iPSCs is set as 1, and values are on log2 scale. Data are presented as means ± SD (n = 3).
Effects of KSR and WNT3A on the induction of d6 PGCLCs as analyzed by FACS gated for TRA‐1‐81 and cKIT.
Gene expression analysis of selected PGC and hematopoietic markers in d6 PGCLCs that were cultured in 0% (control) or 20% KSR (KSR) condition during PGC‐precursor induction. Samples were calibrated with iPSC values, and iPSC values depict 1. Data are presented as means ± SD (n = 3).
Immunofluorescence analysis for OCT4 (red), BLIMP1 (green), and T (yellow) in d2 precursors cultured in increasing KSR concentrations. Nuclei were stained with Hoechst (blue). Scale bar: 100 μm.
Human primordial germ cell commitment in vitro associates with a unique PRDM14 expression profile (2015)
Figure 5

Transcriptomics profiles during PGC‐precursor and PGCLC induction

Unsupervised hierarchical clustering of HuES6 ESCs, 383.2 iPSCs, d2 PGC‐precursor cultures, and FACS‐sorted PGCLCs.
Principal component analysis of HuES6 ESCs, 393.2 iPSCs, d2 PGC‐precursor cultures, and FACS‐sorted PGCLCs.
Scatter plots of global gene expression microarrays comparing d2 PGC‐precursor cultures, or d4 or d6 FACS‐sorted PGCLCs with iPSCs.
Venn diagrams of the transcriptomics meta‐analysis with the intersections of the DEGs of mouse in vitro PGCLCs, human in vivo PGCs, and human in vitro PGC‐like cells in relation to their respective pluripotent counterparts in their respective platforms.
Heatmap of pluripotent, germ cell, mesodermal, and chromatin‐related markers in mouse (left) and human (right) transcriptomics samples (Tfap2c is not targeted in the Illumina MouseRef‐8 v2). The color bars codify the gene expression in log2 scale. Red corresponds to high gene expression. Blimp1+/Stella+ PGCLCs are denoted “B+S+”, and Blimp1+/Stella− PGCLCs are denoted “B+S−”.
Effect of knockdown of BLIMP1 and PRDM14 on PGCLC induction. Knockdown of BLIMP1 (F) and PRDM14 (G) was done using the lentiviral system with two individual shRNA vectors for each gene. The knockdown efficiencies were assessed by qPCR (left panel, each). The induction of d4 PGCLCs analyzed by FACS, gated for TRA‐1‐81 and cKIT (right panel, each). Data are presented as means ± SD (n = 3).
Intersectin-s interaction with DENND2B facilitates  recycling of epidermal growth factor receptor (2017)
Figure 1

Figure 1. DENND2B binds ITSN

Purified GST-SH3-containing proteins were incubated with lysates of HEK-293T cells expressing Flag-DENND2B. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.

Purified GST-SH3 domains of ITSN were incubated with lysates of HEK-293T cells expressing Flag-DENND2B. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.

Lysates from HEK-293T cells expressing Flag-ITSN-s and GFP or GFP-DENND2B were incubated with protein A beads or protein A beads coupled to anti-GFP. Bound proteins were detected by Western blot.

D,E HeLa cells expressing the indicated constructs. Flag-ITSN-s was visualized with anti-Flag and AlexaFluor561 or AlexaFluor488 in D and E respectively. Scale bar: 5 µm. Boxed regions are magnified on the bottom. White arrows and red arrows point to colocalizing and non-colocalizing structures, respectively.