EMBO/soda-nel huggingface dataset:
SEUSS Integrates Transcriptional and Epigenetic Control 
of Root Stem Cell Organizer Specification (2020)
Figure 5

Figure 5. SEU cooperates with SCR and SDG4 to promote WOX5 expression and QC specification.

A-G Representative confocal images of the indicated genotypes at 5 DAG. The white dashed lines indicate the QC region. Scale bars: 20 μm.

H Domain mapping of SEU involved in interactions with SCR and SDG4. The yeast transformants were plated on SD/-2 and SD/-4 media to assess protein-protein interactions.

I SEU associates with SCR and SDG4 in N. benthamiana leaves, as shown by Co-IP assays. SDG4-myc, SCR-HA, and SEU-GFP were coinfiltrated into N. benthamiana leaves. Protein samples were immunoprecipitated with anti-GFP antibody and immunoblotted with anti-myc and anti-HA antibody. N. benthamiana leaves coinfiltrated with SDG4-myc and SCR-HA were used as a negative control.

J Co-IP assays of SCR with SEU and SDG4 in Arabidopsis. Proteins extracted from SDG4-myc and SDG4-myc SCR-GFP plants were immunoprecipitated using anti-GFP antibody, and immunoblotted using anti-myc and anti-SEU antibodies.

K Schematic representation of the role of SEU in SCR-mediated activation of WOX5 expression. In WT roots, SEU is recruited to the promoter of WOX5 through the SEU-SCR interaction. SEU interacts with SDG4, which promotes H3K4me3 deposition in the WOX5 promoter. The SCR-SEU-SDG4 ternary complex plays an essential role in activating WOX5 expression and QC specification. Mutation of SEU impairs the formation of the SCR-SEU-SDG4 complex, which leads to dramatically reduced WOX5 expression and defective QC identity.

Expanded View Figure Legends
Integrated requirement of non-specific and sequence-specific DNA binding in MYC-driven transcription (2021)
Figure 2

Figure 2. MycHEA and MycRA are defective in sustaining cell proliferation.

A Colony formation for cb9Δmyc cells infected with retroviral vectors expressing the indicated Myc proteins; all cells were expanded with doxycycline prior to the final plating step, upon which the compound was either maintained (+dox), or removed (-dox) to switch off the tet-Myc transgene. One representative experiment out of 3 is shown.

B Cumulative cell counts for cb9Δmyc cells upon serial passaging with or without dox (removed at day 2). The data are presented as mean ± SD; n = 3 (biological replicates)

C Percentage of BrdU positive cb9Δmyc cells, in the presence or absence (24h after removal) of doxycycline. Data are presented as mean ± SD; n = 3. Two-tailed Student t-test was used to compare between two groups and expressed as p-values.

D Immunoblot analysis of c-myc-/- HO15.19 rat fibroblasts infected with retroviral vectors expressing the indicated Myc proteins. Parental TGR1 cells serve as control for endogenous levels of the MycWT protein.

E Colony formation for the same cells as in (D). One representative experiment out of 3 is shown.

F Cumulative cell counts for the same cells as in (D). Data are presented as mean ± SD; n = 3 (biological replicates). Two-tailed Student t-test was used to compare between two groups and expressed as p-values.
Integrated requirement of non-specific and sequence-specific DNA binding in MYC-driven transcription (2021)
Figure 4

Figure 4. MycRA shows reduced chromatin association and increased nuclear mobility.

A 3T9 fibroblasts expressing the indicated MycER proteins and control cells (EV) were treated with OHT (4h) and subjected to biochemical fractionation in three independent experiments. The quantity of MycER protein in the different fractions was quantified by western blotting (shown in Fig. EV4A) and the relative proportion in the various fractions plotted as average and standard deviation.

B Single molecule tracking at high frame rate: the time of ttl = 10ms between two images allows to estimate the distribution of displacements, that is then fit by a three-component diffusion model to estimate the fraction of molecules immobilized on chromatin. Inset: Average bound fraction (see Fig. EV4C and Methods). Note that MycRA displays a significantly lower bound fraction than MycWT and MycHEA (ncells= 34, 30 and 35, and ndisplacements= 78550, 59783 and 83801 for MycWT, MycRA and MycHEA, respectively). Error-bar: SD. Statistical significance evaluated by permutation tests.

C Single molecule tracking at lower frame rate (ttlspanning between 200 ms and 2s) allows to quantify the distribution of residence times (i. e. the duration of binding events). Inset: Average residence times (see Fig. EV4E-F and Methods). The data reveal a significantly shorter average for MycRA, relative to either MycWT or MycHEA (ncells= 35, 35 and 31, and nBound − molecules= 2452, 2084 and 2171 for MycWT, MycRA and MycHEA, respectively). Error-Bar: SD. Statistical test: Anova-Tukey.

Data information: In B and C, the statistical significances are indicated relative to the WT control (or RA vs. HEA, as indicated): * P < 10-4; # P < 10-3; + P = 3.1x10-3; ns not significant
Integrated requirement of non-specific and sequence-specific DNA binding in MYC-driven transcription (2021)
Figure 5

Figure 5. DNA sequence recognition determines transcriptional regulation. 3T9 fibroblasts expressing the indicated MycER proteins and control empty vector (EV)-transduced cells were treated with OHT (4h, 8h) and profiled by RNA-seq.

A Fold-change of each annotated mRNA (log2FC, relative to the EV control), plotted against its q-value (-log10). mRNAs showing significant up- and down-regulation (qval<0.05) are marked in red and blue, respectively. The values reported in the graphs indicate the total numbers of up- and down-regulated genes (qval<0.05) and of the subset regulated above a defined threshold of |log2FC|>0.5.

B Heatmaps representing the same log2FC values as in A (restricted to those mRNAs with qval<0.05 in at least one of the MycER samples). The two heatmaps differ in the samples driving the clustering, indicated by the asterisks at the top.

C-E Scatter plots confronting fold-change values (defined as in A) in response to MycERWT (x-axis) and MycERHEA (y-axis), showing the following groups of mRNAs: (C) all of the mRNAs called as DEGs (qval<0.05) in at least one of the samples; (D) MycERWT-regulated DEGs (whether regulated or not by MycERHEA); (E) MycERHEA-specific DEGs (excluding those regulated by MycERWT).

F Percentage of promoters with the indicated DNA motifs under the ChIP-seq peak (±100 bp from the peak summit) within each regulatory class (no DEG, UP or DOWN) for either MycERWT (left) or MycERHEA (right). p-values calculated with Fisher's exact test.

G Percentage of promoter-associated ChIP-seq peaks with the indicated motifs, as a function of peak intensity (binned in deciles: 1-10) for either MycERWT (top) or MycERHEA (bottom).

H Percentage of DEG UP genes (qval<0.05) as a function of peak intensity (binned as in G). Statistical test: Chi-squared against the "no motif" condition, performed on the entire series.

Expanded View Figure Legends
MICAL2PV suppresses the formation of tunneling nanotubes and modulates mitochondrial trafficking (2021)
Figure 1

Figure 1. TNTs in lung cancer cell lines.

A, B Representative bright field images of TNTs in H1299 (A) and A549 (B) cells (black arrow). These TNTs were actin-enriched, as shown by phalloidin staining (red) and not attached to the substrate (Y-Z axes). Scale bar: 20 μm.

C, D Two types of TNTs were detected in H1299 (C) and A549 (D) cells, Type 1, containing F-actin with little or no detectable microtubules (white box) and Type2, containing both microtubules and F-actin (yellow box). Anti-β-tubulin antibody (green) and phalloidin (red) were used for fluorescent staining. Scale bar: 20 μm.

Data information: In (A-D), nuclei were stained with Hoechst 33342 (blue). Boxed areas are enlarged and shown in panels on the right.
MICAL2PV suppresses the formation of tunneling nanotubes and modulates mitochondrial trafficking (2021)
Figure 2

Figure 2. Down-regulating MICAL2PV expression increases TNT formation in lung cancer cells.

A, B MICAL2PV is down-regulated in H1299 (A) and A549 (B) cells. H1299 and A549 cells stably expressing shRNAs against MICAL2PV (shM2) or a control shRNA (shCtr) were examined by Western blotting using an anti-MICAL2PV antibody. MICAL2PV mRNA levels were detected by quantitative RT-PCR.

C, D TNT formation (marked by black triangles and white arrowheads) was imaged in control and shM2 H1299 (C) and shM2 A549 (D) cells by confocal microscopy. Scale bar: 60 μm. Boxed areas are enlarged and shown at the bottom of the corresponding images.

E, F Quantification of the percentage of cells forming TNTs in control and shM2 H1299 (E) and shM2 A549 (F) cells. 13 images per group (E) and 10 per group (F) were captured for quantification.

Three independent experiments were conducted.

G H1299 cells were transfected with a GFP-tagged MICAL2PVb vector (GFP-M2) or control vector (Ctr) followed by Western blotting with an anti-GFP antibody. The mRNA levels of MICAL2PV were measured by quantitative RT-PCR. Three independent experiments were conducted.

H,I H1299 cells stably expressing the Ctr or GFP-M2 were cultured in the presence of 20 μM 2-DG for 24 h to induce TNT formation. TNTs were imaged by confocal microscopy (A) and the percentage of cells forming TNTs was quantified (B). n=21, 20 and 19 images were captured from Ctr, GFP-M2#1 and GFP-M2#2, respectively. Three independent experiments were performed. Scale bar: 60 μm.

Data information: In (A, B, E, F, I), data were analyzed by one-way ANOVA with Bonferroni post-test and shown as mean ±SEM; *** P < 0.001. Three independent experiments were performed
MICAL2PV suppresses the formation of tunneling nanotubes and modulates mitochondrial trafficking (2021)
Figure 3

Figure 3. Down-regulation of MICAL2PV promotes TNT-mediated mitochondrial transfer.

A Time-lapse microscopic images showing that H1299 shCtr or shM2 cells stably expressing Mito-GFP were connected by TNTs containing mitochondria. Cells were seeded in collagen-coated dishes and cultured for 24 h before time-lapse life cell images were acquired by confocal microscopy (also see Movies EV1 and EV2). Fluorescent images (with bright field) at different time points show the mitochondria moving along the TNT and entering in one of the two connected cells. Scale bar: 20 μm. The moving mitochondria are marked by the white arrows. Boxed areas (shCtr in yellow dotted boxes; shM2 in red dotted boxes) are enlarged and shown in the middle panel.

B Confocal microscopic images of control and shM2 H1299 cells following immunofluorescence staining with anti-Tom20 antibody (green) and staining using phalloidin (red). The white arrows marked TNTs, whereas the white triangles marked mitochondria inside TNTs. Scale bar: 25 μm.

C Quantification of TNTs containing mitochondria in shCtr and shM2 H1299 cells. The percentage of TNTs containing mitochondria was determined. n=168, 187 and 192 for shCtr, shM2#1 and shM2#2 groups, respectively; three independent experiments.

D A diagram showing the co-culture experiments. shCtr and shM2 H1299 cells stably expressing Mito-GFP were used as donor cells to co-culture with H1299 cells expressing mCherry as recipient cells at 1 : 1 ratio either in direct contact or separated by a filter in transwells.

E Representative flow cytometry plot of shCtr and shM2 cells following co-culture for 2 days. Experiments were conducted under three conditions: co-cultured in direct contact in normal glucose medium or in galactose medium or co-cultured through a filter.

F The percentage of mCherry-expressing recipient cells containing Mito-GFP was quantified. Twenty thousand events were acquired for each condition and data were analyzed using FlowJo analysis software. When co-cultured through the filter in transwells, only 0.01% or fewer recipient cells contained Mito-GFP, and this group is not included in the graph to avoid an overcrowded figure. Three independent experiments were conducted.

G Representative images of co-cultured shCtr and shM2 H1299 cells. Cells were fixed after 2 days of co-culture, and confocal images were acquired with 100× objective. The boxed areas are enlarged to show the donor-derived mitochondria in recipient mCherry cells with the corresponding orthogonal views along the thin dotted lines. Scale bar: 20 μm.

Data information: In (B), nuclei were stained with Hoechst 33342 (blue). In (C, F), data were analyzed

using one-way ANOVA with Bonferroni post-test and shown as mean ± SEM; ** P < 0.01, *** P < 0.001.
MICAL2PV suppresses the formation of tunneling nanotubes and modulates mitochondrial trafficking (2021)
Figure 4

Figure 4. Down-regulating MICAL2PV promotes mitochondrial infiltration and mitochondrial localization of mitochondrial transport proteins.

A Down-regulating MICAL2PV promotes mitochondrial infiltration into the cortical cytoskeleton. Control and shM2 H1299 cells were subjected to immunofluorescence staining with anti-Tom20 antibody (green) and staining using phalloidin (red). Scale bar: 25 μm.

B Down-regulating MICAL2PV enhances mitochondrial infiltration. Mitochondrial infiltration index was quantified as the ratio of the radial distance from the nucleus to the furthest mitochondria and the radial distance from the nucleus to the plasma membrane. Fifty cells were quantified for each group.

C Down-regulating MICAL2PV enhances the mitochondrial localization of Miro1, Miro2 and KIF5B. Mitochondria were purified from control and shM2 H1299 cells and subjected to Western blotting. The purity of the mitochondrial fraction was confirmed by detection of mitochondrial ATP5B and the absence of the cytoplasmic β-tubulin protein.

D The band intensity of target proteins in the mitochondrial fraction was determined [gels shown in (C)] and expressed relative to the Control.

Data information: In (A), nuclei were stained with Hoechst 33342 (blue). In (B, D), data were analyzed using one-way ANOVA with Bonferroni post-test and shown as mean ± SEM; * P < 0.05, *** P < 0.001. Three independent experiments were conducted.
MICAL2PV suppresses the formation of tunneling nanotubes and modulates mitochondrial trafficking (2021)
Figure 6

Figure 6. Down-regulating Miro2 inhibits mitochondrial infiltration and transport in shM2 cells.

A Control and shM2 H1299 cells were transfected with control siRNA or siRNAs targeting Miro2. Following transfection, cells were stained with anti-Tom20 antibody (green) and phalloidin (red). Confocal microscopic images are shown. Scale bar: 20 μm.

B, C Miro2 protein levels were detected in different groups by Western botting with actin as a loading control (B). The Miro2 band intensity was determined and normalized to that in lane 1 in the shCtr group (C). Three independent experiments were conducted.

D Mitochondrial infiltration index was quantified as described for Figure 4. 50 cells were quantified for each group.

E The percentage of TNTs containing mitochondria was determined with 100 TNTs per group scored. F Down-regulating Miro2 inhibits mitochondrial transport in shM2 cells. Mito-GFP labelled control and shM2 H1299 cells were transfected with control siRNA or siRNAs targeting Miro2. These cells were then co-cultured with mCherry labelled recipient cells and analyzed by flow cytometry as described in Figure 3. The percentage of mCherry recipient cells containing Mito-GFP was quantified. Twenty thousand events were analyzed for each condition, as described in Figure 3.

Data information: In (A), nuclei were stained with Hoechst 33342 (blue). Data were analyzed using Student's t-test (panel D) or two-way ANOVA with Bonferroni post-test (panels C, E, F) and presented as mean ± SEM; * P < 0.05, *** P < 0.01, *** P < 0.001, ns, not significant. Three independent experiments were conducted.
MICAL2PV suppresses the formation of tunneling nanotubes and modulates mitochondrial trafficking (2021)
Figure 7

Figure 7. Down-regulation of MICAL2PV increases TNT formation between chemo-drug treated cells and non-treated cells and decreases drug-induced cell death.

A Stable H1299 cells (shCtr or shM2) were treated by 5-FU (50 μM, 24 h) and then cultured alone or co-cultured with non-drug treated vehicle-treated cells stably expressing mito-mCherry in the absence or presence of Cyto B (500 nM, 24 h). The percentage of dead cells in each group was quantified. Three independent experiments were conducted.

B shCtr and shM2 H1299 cells were treated by 5-FU (50 μM; 24 h). Then drugs were washed out and the cells were cultured in normal DMEM medium (Ctr) or conditioned medium from non-treated cells or co-cultured with non-treated cells in transwells (separated by the filter) for another 24 h. The cell viability of 5-FU treated cells was determined using MTT assay.

C Cyto B inhibits TNT formation in the co-culture assay. The percentage of cells forming TNTs was quantified in co-cultured cells in the absence or presence of Cyto B. Three independent experiments were conducted.

D Confocal microscopic images of TNTs in each group, white arrowheads and yellow triangle in enlarged views (zoom-in panels, zoom) marked the TNTs formed between non-drug treated (mCherry positive) and drug-treated cells. Scale bar: 25 μm.

E TNT formation was quantified between non-drug treated cells (yellow), chemo-drug treated cells (blue) or between non-drug treated and chemo-drug treated cells (red) following confocal microscopic imaging (non-drug treated cells: white circles, chemo-drug treated cells: black circles). In the co-culture assay, TNTs in shCtr, shM2#1 and shM2#2 groups (n=485, 618 and 581 respectively) were imaged. Three independent experiments were performed.

Data information: In (A, B, C, E), data were analyzed using two-way ANOVA with Bonferroni post-test and shown as mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant.
MICAL2PV suppresses the formation of tunneling nanotubes and modulates mitochondrial trafficking (2021)
Figure 8

Figure 8. The monooxygenase (MO) domain of MICAL2PV is sufficient for its activity in actin-depolymerization and suppression of TNT formation.

A A diagrammatic illustration of protein domain organization in MICAL2PV and its mutants.

B, C MICAL2PV mutants that contain the MO domain retain the activity in suppressing TNT formation. Stable shM2 H1299 cells were transfected with plasmids expressing different MICAL2PV mutants containing a hemagglutinin (HA) tag or the vector control (Ctr). The mutant proteins were detected by Western blotting using an anti-HA antibody (B). TNT formation was quantified in transfected cells following confocal microscopy. Expression of MICAL2PV mutants, #1, #2 or #4 significantly reduced TNT formation (C). Three independent experiments were conducted.

D MICAL2PV mutants containing the MO domain are active in depolymerizing F-actin. Following transfection of plasmids expressing different HA-MICAL2PV mutants (#1, #2, or #4) into shM2 cells, confocal microscopy was performed following staining with phalloidin (red) and anti-HA antibody (green). F-actin signals were reduced in cells expressing different mutants (HA-positive green cells marked by dashed lines). Scale bar: 20 μm.

E, F MICAL2PV mutants lacking the MO domain does not inhibit TNT formation. Stable shM2 H1299 (E) or A549 (F) cells were transfected with the corresponding MICAL2PV mutants lacking MO domain or control vectors (Ctr). Following transfection, Western blotting was carried out using an anti-HA antibody to detect expression of the mutant proteins, and confocal microscopy was performed to examine TNT formation. 10 images per group were captured for quantification. Three independent experiments were conducted.

G, H The MICAL2PV mutant containing an inactive MO domain does not inhibited TNT formation. Stable shM2 H1299 (G) or A549 (H) cells were transfected with plasmids expressing the MICAL2PV mutant in which the MO domain was inactivated by mutations in the FAD binding site (MOmut: amino acid residues 93-98 GxGxxG mutated to WxWxxW) or the control vector (Ctr). Following transfection, Western blotting and quantification of TNT formation were carried out as in panel E. 10 images were captured for quantification in each group. Three independent experiments were performed.

Data information: In (D), nuclei were stained with Hoechst 33342 (blue). Data were analyzed using Student's t-test (C) or a one-way ANOVA with Bonferroni post-test (E-H) and shown as mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant.

Expanded View Figure Legends
A simple magnetic nanoparticles-based viral RNA extraction method for efficient detection of SARS-CoV-2 (2020)
Figure3

RT-PCR assays towards viral RNA extracted by different pcMNPs-based methods.RT-PCR assays amplifying (A) the ORF1ab region and (B) the N gene in pseudoviral RNA extracted by a manual protocol.
A simple magnetic nanoparticles-based viral RNA extraction method for efficient detection of SARS-CoV-2 (2020)
Figure4

Automated viral RNA extraction based on pcMNPs.(A) A schematic diagram of automated extraction protocol. (B) RT-PCR assays amplifying the ORF1ab region in pseudoviral RNA extracted by automated protocol, and (B) RT-PCR assays amplifying the N gene in pseudoviral RNA extracted by an automated protocol.
A simple magnetic nanoparticles-based viral RNA extraction method for efficient detection of SARS-CoV-2 (2020)
Figure5

Sensitivity and dynamics range of pcMNPs-based viral RNA detection.(A) RT-PCR assays amplifying the N gene in pseudoviral RNA following the Conventional RT-PCR protocol, and (B) corresponding calibration curve. The red line is the linear regression fit (R2 = 0.999). (C) RT-PCR assays amplifying the N gene in pseudoviral RNA following the Direct RT-PCR protocol, and (D) corresponding calibration curve. The red line is the linear regression fit (R2 = 0.998).
EZH2 cooperates with gain-of-function p53 mutants to promote cancer growth and metastasis (2019)
Figure 1

Fig 1. EZH2 binds to 5'UTR of p53 mRNA

(A) Heat map showing a subset of mRNAs of genes immunoprecipitated by anti-EZH2 antibody in LNCaP and C4-2 prostate cancer cell lines, which was generated based on the distinct RIP-seq reads on specific gene exons. The units of the heatmap values were reads per kilobase million (RPKM).

(B) Screen shots from the UCSC genome browser showing signal profiles of p53 mRNA immunoprecipitated by anti-EZH2 antibody in LNCaP and C4-2 cells.

(C) C4-2 cells were subjected to UV-RIP assay. RT-qPCR measurement of p53 mRNA immunoprecipitated by IgG or anti-EZH2 antibody. Data shown as means ± SD (n=3). Statistical significance was determined by two-tailed Student's t-test. ** P<0.01.

(D) Schematic diagram of four GST-EZH2 recombinant proteins (EZ1 to EZ4). EBD, EED binding domain; SANT1(/2)L, "Swi3, Ada2, N-Cor, TFIIIB"1(/2) like; MCSS, motif connecting SANT1L and SANT2L; CXC, cysteine-rich domain; SET, catalytic domain.

(E) Top, RT-qPCR analysis of p53 mRNA in C4-2 cell lysate pulled down by GST or GST-EZH2 recombinant proteins EZ1-EZ4. Bottom, western blotting analysis of GST or GST-EZH2 proteins used for GST pull-down assay. Asterisks indicate the protein bands at expected molecular weight. Data shown as means ± SD (n=3). Statistical significance was determined by two-tailed Student's t-test.** P<0.01.

(F) C4-2 cells were transfected with Myc-tagged EZH2-WT or EZH2-∆336-554 for 24 h and cells were harvested for RIP with IgG or anti-Myc tag antibody. Transfected proteins and pull-down p53 mRNAs were analyzed by western blot and RT-qPCR, respectively. Data shown are means ± SD (n=3). * P<0.01. ERK2, a loading control.

(G) GST pull-down assay using in vitro transcribed different fragments of p53 mRNA and indicated GST proteins followed by RT-qPCR analysis of pull-down p53 mRNA. FL, full length; ORF, open reading frame; UTR, untranslated region.

(H, I) RNA EMSA evaluation of EZh2 binding of p53 mRNA. GST-EZH2 recombinant proteins (EZ1 to EZ4) were incubated with biotin-labeled in vitro transcribed p53 5'UTR (biotin-labeled probe) in the presence or absence of 100-fold unlabeled p53 5'UTR (unlabeled probe), followed by PAGE and immune blotting with HPR-conjugated streptavidin.