EMBO/soda-nel huggingface dataset:
Role of Angiomotin-Like2 mono-ubiquitination on YAP inhibition (2015)
Figure 6

Figure 6. AMOTL2 is ubiquitinated at K347 and K408.

A. Flag-AMOTL2 constructs were designed to express regions spanning from the N-terminus to the coiled-coil domain (AA 1-306), the coiled-coil domain (AA 307-581), and the C-terminal portion (AA 582-781). These constructs were co-transfected into 293T cells with Myc-Ub, and an in vivo ubiquitination assay was conducted. *, non-specific band.

B. The SBP-AMOTL2 coiled-coil domain was expressed in 293T cells, subjected to tandem affinity purification, fractionated by SDS-PAGE and stained with Coomassie blue. Bands corresponding to the AMOTL2 coiled-coil domain were excised and analyzed by mass spectrometry for identification of ubiquitinated sites (K-G-G linkages). Shown are MS/MS spectra recoded using an LTQ-Orbitrap mass spectrometer for the doubly charged peptide, IEK*LESEIQR (MH+ = 1358.72, z = 2+; upper), and the triply charged peptide, LASK*TQEAQAGSQDMVAK (MH+ = 1992.96, z = 3+; lower). These peptides contain the K347 and K408 ubiquitination sites, respectively (marked by asterisks). Fragmented ions are annotated according to the nomenclature for peptide fragmentation in mass spectrometry.

C. The indicated Flag-AMOTL2 WT or mutant proteins were subjected to an in vivo ubiquitination assay in 293T cells.

D. RPE or MCF10A cells were transduced with Flag-AMOTL2 WT or Flag-AMOTL2 K347/408R, and immunoprecipitated with an anti-Flag antibody. Immunoprecipitated and WCL samples were analyzed by Western blotting for the indicated proteins.

E. RPE or MCF10A cells were transduced with Flag-AMOTL2 WT or Flag-AMOTL2 Y213A, and processed as (D).
Role of Angiomotin-Like2 mono-ubiquitination on YAP inhibition (2015)
Figure 7

Figure 7. AMOTL2 ubiquitination is required for its function.

A. MCF10A cells expressing shRNA against AMOTL2 were complemented with shRNA-resistant Flag-AMOTL2 WT or the K347/408R mutant. The indicated cells were harvested and analyzed by Western blotting for the indicated proteins.

B. mRNAs isolated from cells in (A) were analyzed by RT-qPCR against indicated EMT marker genes (n=4). Error bars indicate the S.E.M. (*p< 0.05, **p < 0.01, ***p < 0.001; paired Student’s t-test).

C. Transwell migration assay was performed using cells in (A) (n=8). We analyzed 5 random fields for each sample. Error bars indicate the S.E.M. (**p< 0.01, paired Student’s t-test).

D. Representative images of soft agar assay results using cells in (A). Scale bar, 1 mm.

E. Soft agar assay was performed using cells in (A) (n=8). Error bars indicate the S.E.M. (**p < 0.01, ***p < 0.001, paired Student’s t-test).
Role of Angiomotin-Like2 mono-ubiquitination on YAP inhibition (2015)
Figure 8

Figure 8. Ubiquitinated AMOTL2 can bind to the LATS UBA domain.

A. Schematics of human LATS1 and LATS2. PY, PY motif.

B, C. 293T cells were transfected with the indicated DNAs and immunoprecipitated with an anti-Flag antibody or pulled down with streptavidin-binding beads.

D, E. 293T cells were transfected as indicated and immunoprecipitated with an anti-HA antibody. LATS2 immune complexes were subjected to a kinase assay with His-YAP and cold ATP.

F. Lats1 -/-; Lats2 fl/fl; SV40 mouse embryonic fibroblasts complemented with vector, LATS2 WT or ΔUBA mutant were infected with empty (control) or Zeocin-Cre retroviruses and selected with 400 μg/ml of Zeocin for 4 days. Harvested cells were analyzed by Western blotting for the indicated proteins.
A small targeting domain in Ty1 integrase is sufficient to direct retrotransposon integration upstream of tRNA genes (2020)
Figure 1

Figure 1 Mutations in the IN1 bNLS linker sequence abolish the interaction with AC40.

A. Top. The Ty1 integrase (IN1) showing N-terminal and catalytic core domains (NTD and CCD) and the bipartite NLS at the C-terminus (CTD). Bottom. Alignment of amino acid sequences of Ty1, Ty2, and Ty4 integrase C-termini (IN1, IN2, and IN4, respectively). In bold: basic amino acids required for NLS function. In red: amino acids in the bNLS linker relatively conserved between the three integrases. *, identity; :, high similarity; ., low similarity; -, gap in sequence.

B. Two-hybrid interaction between GBD-AC40 and WT or mutant GAD-IN1578-635. Alanine substitutions in IN1578-635 are indicated. Cells were plated in two-fold serial dilutions on DO-Leu-Trp-His plates. No growth or protein expression defects were detected (Figs EV1A-B). +, interaction; -, no interaction.

C. Two-hybrid interaction between GBD-AC40 and different IN1 regions fused to GAD, as indicated. Cells were plated in ten-fold serial dilutions on DO-Leu-Trp-His plates. No growth or protein expression defects were detected (Figs EV1C-D). +, interaction; -, no interaction.

D. In vitro interaction between AC40 and IN1 proteins co-expressed in E. coli. Co-precipitation of WT or mutated (K617A, S621A, or L622A) IN1-C-tag using AC40-Twin-Strep-tag as bait from bacterial protein extracts co-expressing (+) or not (-) these proteins. Expected sizes are 41 kDa for AC40-Twin-Strep-tag and 100 kDa for IN1-C-tag (WT and mutants).
A small targeting domain in Ty1 integrase is sufficient to direct retrotransposon integration upstream of tRNA genes (2020)
Figure 2

Figure 2. Non-AC40 binding IN1 mutants do not affect Ty1 integration frequency.

A. Localization of GFP2-IN1 bNLS variants in yeast cells by direct fluorescence microscopy. Nup49-mCherry signal was used to visualize the location of the nuclear envelope. Corresponding DIC images are shown. Scale bar is 5 μm.

B. Retrotransposition frequency (log scale) of pGAL1-Ty1his3AI bearing the indicated substitutions of conserved residues in a spt3-101 rad52∆ strain. IN1 catalytic core domain mutant D154A is defective for integration and NLS mutant (KKR628-630GGT) for nuclear import. Values are mean±SD, n=4 experiments, each performed with four independent colonies. ***p ≤ 0.001, otherwise not significant (Student t-test).
A small targeting domain in Ty1 integrase is sufficient to direct retrotransposon integration upstream of tRNA genes (2020)
Figure 3

Figure 3. AC40 recruits IN1 at Pol III-transcribed genes.

A. Co-immunoprecipitation of ectopic IN1 using C160-HA as bait, from yeast protein extracts expressing WT or mutated IN1-Strep (K617A, S621A, or L622A) from the GAL1 promoter in the presence of galactose. Expected sizes are 160 kDa for C160-HA and 100 kDa for IN1-Strep (WT and mutants).

B. Quantitative ChIP analysis of HA-IN1 enrichment at Pol III-transcribed genes. Immunoprecipitated DNA from yeast cells producing ectopic IN1 is expressed as a value relative to that of the input. Pol III transcribed-genes: tDNA-Ile and tDNA-Leu families (16 and 22 genes, respectively) and the unique SCR1 gene. GAL1 ORF serves as a control. Data represent means ± SD (n ≥ 3).

C. Fold enrichment of WT HA-IN1 over input at all the genes where HA-IN1 has been detected by ChIP-seq analysis. Each dot represents a gene. Grey, tDNAs ; Red, other Pol III-transcribed genes ; Blue, Pol II-transcribed genes. The dashed line indicates the separation between Pol II and Pol III-transcribed genes.

D. WT and mutant IN1 association with all Pol III-transcribed genes. Values obtained from ChIP seq analysis have been normalized in log2 RPKM (reads per kilobase per million mapped reads). Control is anti-HA immunoprecipitation of chromatin extracts in cells expressing IN1-Strep. Immunopurified DNA from 3-4 independent experiments was combined in one replicate. Data are presented as boxplots showing the median, the first quartile, and the third quartile. Error bars show minimum and maximum values.

Regions used for calculation are gene bodies.

E. Genome-wide occupancy profiles (top) and heatmaps (bottom) of WT and HA-IN1 mutants over a 1kb window upstream and downstream of all tDNAs. Pearson correlation to WT values corresponding to Fig EV2B are indicated.

F. WT and mutant IN1 association to all Pol-II transcribed genes respectively. Control, replicates and data presentation, as described for panel D. Regions used for calculation are gene bodies.

G. Recruitment of IN1 along the SCR1 gene locus. Top. ChIP‐qPCR analysis as described for panel Fig 3B. Data represent means ± SD (n = 3). Bottom. Schematic representation of the SCR1 locus with DNA amplicon positions. TATA, A and B boxes and terminator of transcription (T) are indicated.
A small targeting domain in Ty1 integrase is sufficient to direct retrotransposon integration upstream of tRNA genes (2020)
Figure 4

Figure 4. AC40 recruits IN1 at the RDN1 repeats.

A. Co-immunoprecipitation of ectopic IN1 using TAP-tagged-A190 as bait from yeast protein extracts expressing WT or the indicated IN1-HBH mutants from a pTet-Off promoter in the absence of doxycycline. Expected sizes are 204 kDa for A190-HA and 82 kDa for IN1-Strep (WT and mutants).

B. Genome browser visualization of HA-IN1 occupancy at the RDN1 locus. Occupancy of WT HA-IN1 and K617A, S621A and L622A HA-IN1 mutants is represented in each panel. Control, as described for panel 3D.

C. Top. IN1 is recruited at Pol I-transcribed genes. ChIP‐qPCR analysis as described for panel 3B. Data represent means ± SD (n = 3). Bottom. Schematic representation of the RDN1 locus with DNA amplicon positions.

D. Recruitment of WT and mutant HA-IN1 (K617A, S621A, or L622A) at the 18S rDNA locus transcribed by Pol I. ChIP‐qPCR analysis as described for panel 3B. Data represent means ± SD (n = 3).
A small targeting domain in Ty1 integrase is sufficient to direct retrotransposon integration upstream of tRNA genes (2020)
Figure 5

Figure 5. Non-AC40 binding Ty1 mutants have altered integration profiles.

A. Detection of de novo Ty1 insertions upstream of the SUF16 and SEO1 genes by PCR using a primer in HIS3 (red triangle) and a primer in the locus of interest (blue triangle). Ty1 retrotransposition was induced in cells transformed with plasmids expressing WT or mutant (IN1 K617A, S621A and L622A) Ty1his3AI from the GAL1 promoter. Total genomic DNA was extracted from His+ cells obtained from independent cultures.

B. Genome-Wide Ty1 insertion frequencies at each genomic feature are clustered in a heatmap. Score is computed in column Z-score. ORFs, all Pol II-transcribed genes except gene at subtelomeres; Pol I, one RDN37 copy; up 1kb Pol III, 1kb upstream of all Pol III transcribed genes; Subtelomeres, genomic coordinates corresponding to chromatin covered by Sir2 and Sir3, when they are co-overexpressed (Hocher et al, 2018); Random, 100 000 random Ty1 computed insertions in the genome.

C. Detection of de novo Ty1 insertions upstream of RDN5 and in RDN37 genes by PCR as described for panel 5A. Four total genomic DNA preparation from panel A were randomly tested by PCR.

D. Ty1 insertion profile upstream of tDNAs. Total genomic DNA extracted in (B) was prepared for Ty1 de novo integration event sequencing. Ty1 insertions are computed in a 1kb window upstream of all the 275 nuclear tDNAs (position 0 in the graph). Each position is divided by the number of insertions at this position (weight). The Smoothing curves indicate the general trend. Nucleosome center positions for the three first nucleosomes upstream of all tDNAs are from (Brogaard et al, 2012).

E. Ty1 insertion frequencies for each left and right subtelomere of chromosomes are clustered in a heatmap. Score is computed in row Z-score. Random, as described for panel B. High level of WT Ty1-HIS3 integration in II-L is due to the presence of the tRNA gene tF(GAA)B.

F. Genome browser visualization of WT and IN1 K617A mutant Ty1-HIS3 insertions into chromosome V left and chromosome VI right subtelomeres compared to the subtelomere boundaries defined by (Hocher et al, 2018) and (Yue et al, 2017) respectively. Red stars indicate the first essential gene of each subtelomere. The CAN1 gene is indicated on chromosome V.
A small targeting domain in Ty1 integrase is sufficient to direct retrotransposon integration upstream of tRNA genes (2020)
Figure 6

Figure 6. IN1 targeting domain directs Ty5 integration at Pol III-transcribed genes.

A. Two-hybrid interaction between GBD-AC40 (left) or GBD-Sir4 (right) and different GAD-IN5 or GAD-IN1 constructions. Cells were plated in five-fold serial dilutions on DO-Leu-Trp-His plates. No growth or protein expression defects were detected (Figure EV3A-D).

B. Genome-wide occupancy profiles (top) and heatmaps (bottom) of IN5, WT, and indicated mutants, +/- 1kb upstream and downstream of all tDNAs.

C. Genome browser visualization of different HA-IN occupancy for chromosome V (chrV:431129..443275). Occupancy of the indicated integrase is represented in each panel. Control, as panel 3D. The region contains tH(GUG)E2, tK(CUU)E2, tV(ACC)E1, tI(AAU)E1, SNR52 and SCR1, all transcribed by Pol III.

D. Detection of Ty5, Ty5ΔTD5+bNLS or Ty1 de novo integration events at HMR and HML loci, SCR1 and upstream of all glycine tDNAs, by PCR using a primer in HIS3 and a primer in the locus of interest. Retrotransposition was induced in cells transformed with WT or mutated pGAL1-Ty5his3AI (Ty5 or Ty5ΔTD5+bNLS) and pGAL1-Ty1his3AI (Ty1). Total genomic DNA was extracted from His+ cells obtained from independent cultures. Nucleosome position (green circles) is indicated on the right of the panels (Brogaard et al, 2012).

EXPANDED VIEW FIGURE LEGENDS
Single-cell Analysis of ACE2 Expression in Human Kidneys and Bladders Reveals a Potential Route of 2019-nCoV Infection (2020)
Figure 1.

Single cell analysis of the public kidney data of three donors. A) UMAP plot revealing the cell type annotations of three samples. B) The cellular composition of three healthy kidney samples. C) Dotplot exhibition of canonical markers used for cell type classification. D) The gene expression patterns of ACE2 of all detected cell types in kidney.
Activation of LXRβ Inhibits Tumor Respiration and is Synthetically Lethal with Bcl-xL Inhibition (2019)
Figure 2

Figure 2. Combined treatment with LXR623 and BH3-mimetics yields enhanced induction of apoptosis.

A, U87, T98G and LN229 cells were treated with 1 µM ABT263, 20 µM LXR623 or the combination of both for 72h. Mewo melanoma and HCT116 colon carcinoma cells were treated with 1 µM ABT263, 10 µM LXR623 or the combination of both for 72h (MeWo) or for 48h (HCT116). Thereafter, cells were stained with annexin V/propidium iodide and analyzed by multi-parametric flow cytometry. Shown are means and SD (n = 3). ***p=0.0007, ****p<0.0001. Statistical significance was determined by one-way ANOVA.

B, Stem-like GBM cells, NCH644, NCH421k and NCH690 were treated with 1 µM ABT263, 20 µM LXR623 or the combination of both for 48h. Thereafter, cells were stained with annexin V/propidium iodide and analyzed by multi-parametric flow cytometry. Shown are means and SD (n = 3). ****p<0.0001. Statistical significance was determined by one-way ANOVA.

C-D, U87 (72h treatment) and HCT116 (48h treatment) were treated with 1 µM ABT263, 20 µM (U87) or 10 µM (HCT116) LXR623 or the combination of both, stained with TMRE and analyzed by flow cytometric analysis for dissipation of mitochondrial membrane potential. Shown are means and SD (n = 3). ****p<0.0001. Statistical significance was determined by one-way ANOVA.

E, HCT116 colonic carcinoma cells were treated with ABT263, LXR623 or the combination of both. Whole protein lysates were collected and subjected to capillary electrophoresis for the expression/cleavage of PARP, total caspase-9 (CP9) and Vinculin. CF: cleaved fragment, FL: full length.

F, HCT116 colonic carcinoma or NCH644 glioblastoma stem cells were treated with selective BH3-mimetics, WEHI-539 (Bcl-xL inhibitor), ABT199 (Bcl-2 inhibitor) or A1210477 (Mcl-1 inhibitor) in the presence or absence of LXR623 for 48h. Thereafter, cells were labeled with annexin V/propidium iodide and analyzed by multi-parametric flow cytometry.
Activation of LXRβ Inhibits Tumor Respiration and is Synthetically Lethal with Bcl-xL Inhibition (2019)
Figure 3

Figure 3. Treatment with LXR623 regulates the expression of pro-apoptotic Noxa and increased Noxa levels mediate enhanced sensitivity to BH3-mimetics.

A, LN229, T98G, U87, U87-EGFRvIII and HCT116 cells were treated with increasing concentrations of LXR623 for 72h. Thereafter, protein lysates were collected and subjected to standard western blot (LN229, T98G and U87) or capillary electrophoresis (U87-EGFR-vIII and HCT116) for the expression of Usp9X, Mcl-1, Bcl-2, Bcl-xL, Noxa, BIM and/or Actin and Vinculin. In U87-EGFRvIII and HCT116, Vinculin was used in lieu of Actin.

B, LN229, T98G, U87, U87-EGFRvIII and HCT116 cells were treated with LXR623 as described in A. Thereafter, protein lysates were collected and subjected to capillary electrophoresis for the expression of ABCA1 and Vinculin.

C, LN229 were transfected with siNT, siNoxa 1, siNoxa 2 or BAK-siRNA for 72h. Thereafter, cells were treated with the combination treatment of 1 µM ABT263 and 20 µM LXR623 for another 24h. After conclusion of the treatment, cells were harvested, fixed, stained with propidium iodide and analyzed by flow cytometry for DNA - fragmentation.

D, LN229 were transfected as described in B. Thereafter, whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of Noxa, BAK and Vinculin. E, LN229 GBM cells were treated with 10 µM LXR623 (LXR), 1 µM ABT263 or the combination for 48h. Thereafter, protein lysates were prepared and immunoprecipitated with an antibody against BAK. Standard western blotting was performed (immunoprecipitation and the corresponding inputs) with antibody against BAK and Mcl-1. The arrows highlight the specific protein bands, while stars indicate the immunoglobulin light-chains.
Activation of LXRβ Inhibits Tumor Respiration and is Synthetically Lethal with Bcl-xL Inhibition (2019)
Figure 4

Figure 4. Activation of liver-x-receptors cause energy deprivation and activate endoplasmic reticulum stress signaling, culminating in an in increase in Noxa protein levels.

A, U87 cells were treated with 20 µM LXR623 for 7h and 24h. Thereafter, cells were stained with annexin V/propidium iodide and analyzed by multi-parametric flow cytometry. The lower left quadrant indicates the percentage of viable cells.

B, U87 cells were treated with LXR623 as indicated for 7h and analyzed for total ATP levels by a luminescence assay. To account for dead cells, assays were normalized to DNA-content (cell number). Shown are means and SD (n = 3). ***p=0.0003, ****p<0.0001. Statistical significance was determined by two-sided student's t.test.

C, U87 cells were treated with LXR623 as indicated for 24h. Cell lysates were prepared and subjected to LC/MS for the determination of ATP levels. Shown are means and SD (n = 3). *p=0.0363. Statistical significance was determined by two-sided student's t.test.

D, U87 cells were treated with increasing concentrations of LXR623 for 7h and 24h. Thereafter, whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of phosphorylated AMPK (threonine 172) and total AMPK.

E, U87 cells were treated with increasing concentrations of LXR623 in the presence or absence of exogenous ATP for 7h. Thereafter, whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of ATF4.

F, U87 cells were treated with increasing concentrations of LXR623 for 24h in the presence or absence of cholesterol. Thereafter, whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of phosphorylated AMPK (threonine 172) and total AMPK.

G-H, Cells were treated as in F and whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of ATF4 and Vinculin (G) and by the standard western blot for the expression of Noxa and Actin.

I, U87 cells were treated with increasing concentrations of LXR623. Thereafter, whole cell protein lysates were prepared and analyzed for classical ER-stress markers, GRP78, ATF4 and Noxa by capillary electrophoresis.

J, U87 cells were transfected with non-targeting siRNA (n.t.-siRNA), ATF3-siRNA, ATF4 siRNA or the combination of both ATF4 and ATF3 siRNAs. After 48h, cells were treated with LXR623, whole cell protein lysates were harvested and analyzed by capillary electrophoresis for the expression of ATF3, ATF4, Noxa and Vinculin.
Activation of LXRβ Inhibits Tumor Respiration and is Synthetically Lethal with Bcl-xL Inhibition (2019)
Figure 6

Figure 6. Activation of liver-x-receptor mediates a state of energy deprivation through inhibition of oxidative phosphorylation (OXPHOS).

A-F, U87 cells were treated with LXR623 for 1h, 3h or 5h and subjected to extracellular flux analysis on the Seahorse XFp instrument in the context of a mitochondrial stress assay (A-C). From this assay, coupled respiration was calculated (D-F). Shown are means and SD (n = 3). **p=0.006. Statistical significance was determined by two-sided student's t.test.

G-I, U87 cells were treated with LXR623, cholesterol or the combination of both for 24h and subjected to extracellular flux analysis on the Seahorse XFp instrument in the context of a mitochondrial stress assay (G). From this assay, mitochondrial OCR and coupled respiration was calculated (H-I). Shown are means and SD (n = 5). ***p=0.0001, ****p<0.0001. Statistical significance was determined by one-way ANOVA.

J, U87 cells were treated with DMSO or LXR623 20 µM with the indicated time point. Thereafter, whole cell protein lysates were collected and subjected to standard western blot for the expression of the respiratory complexes (OXPHOS), Noxa, and ATF4. A star indicates an unspecific band.

K, U87 cells were treated with DMSO or LXR623 20 µM in the presence or absence of cycloheximide for 7h. Thereafter, whole cell protein lysates were collected and the protein levels of the components of complex V were analyzed by standard western blot.

L, U87 cells were treated with increasing concentration of LXR623 in the presence or absence of cholesterol. Thereafter, whole cell protein lysates were collected and analyzed the protein levels of the respiratory complexes (OXPHOS) by standard western blot.

M-P, U87 cells transduced with non-targeting or LXRβ shRNA were treated with vehicle or 20 µM LXR623 for 24h. Thereafter, extracellular flux analysis was performed in the context of a mitochondrial stress assay. From this assay, basal respiration and coupled respiration was calculated (M-N). mRNA and protein were collected and analyzed for the expression of LXRβ mRNA (O) or LXRβ protein by capillary electrophoresis (P). Shown are means and SD (n = 5). Statistical significance was determined by two-sided student's t.test.