EMBO/soda-nel huggingface dataset:
The deubiquitinase Usp27x stabilizes the BH3-only protein Bim and can enhance apoptosis (2016)
Figure 2

Figure 2 Expression of Usp27x stabilizes the expression of the phosphorylated form of BimEL and reduces Bim-ubiquitination in PMA-stimulated 293FT cells

A Usp27x inhibits the PMA-induced degradation of BimEL in 293FT cells. 293FT-TetR-3xFlagUsp27x or TetR-3xFlag-Usp27xC87A cells were treated with dox to induce 3xFlag-Usp27x or Usp27xC87A, or with PMA [16.2 nM] (to induce BimEL degradation), or with the combination of dox, PMA and QVD [10µM] (to inhibit apoptosis) as indicated for 24 h (left) or 48 h (right). Cell lysates were analysed by Western blotting. The blots are representative of n=4 similar experiments. See also Appendix Fig. S5B. * Probably modified form of wild-type 3xFlag-Usp27x (such as dimer, ubiquitinated Usp27x or a stable complex of Usp27x with another interacting partner); this form is only seen if high expression levels of catalytically active Usp27x are reached (see Appendix Fig. S5A where Usp27x was transfected into 293FT cells) or if wt Usp27x is enriched during immunoprecipitation.** Probably degradation product of 3xFlag-Usp27x. These forms of Usp27x were not seen in every experiment.

B 293FT-TetR-3xFlagUsp27x or TetR-3xFlag-Usp27xC87A cells treated as in A were analysed using conditions of higher resolution. A band very likely corresponding to phosphorylated BimEL is detectable. Similar results were obtained in n=2 separate experiments.

C Usp27x stabilizes expression of p-BimEL (Ser69) in Bim-degrading conditions without reducing β-TrCP levels

293FT-TetR-3xFlagUsp27x were treated with dox, PMA [16.2 nM], UO126 [10µM] or QVD [10µM] as indicated for 24h, and levels of phosphorylated BimEL at serine69 were analysed by Western blotting using a phospho-Bim (Ser69) specific antibody. Scans are from the same membrane and exposure times. Similar results were obtained in n=2 separate experiments.

D UO126 reduces phosphorylation-associated shift of BimEL

293FT-TetR-3xFlagUsp27x were treated for 24h with the indicated drug combinations and Bim was detected by Western blotting. UO126 was added 30 minutes prior other stimulation. Similar results were obtained in n=3 separate experiments (see Appendix Fig. S5D).

E Usp27x-expression reduces ubiquitination of Bim

293FT-TetR-3xFlagUsp27x cells were transfected with a vector coding for 6His-ubiquitin (left) or GFP (right). After 24h cells were treated with PMA and QVD, and 3xFlag-Usp27x was induced by dox at the same time. After additional 20h cells were treated for 4h with MG132 (40 µM) to block proteasomal degradation of ubiquitinated proteins. Cells were lysed under denaturing conditions and His-ubiquitin-labelled proteins or proteins from only GFP expressing cells were purified by Ni2+-NTA affinity chromatography. Ubiquitinated Bim (Bim-Ubn) was detected by Western blot using anti-Bim antibodies (left). Similar results were obtained in n=3 separate experiments (another experiment including a Coomassie stain is shown in Appendix Fig. S3E). *Indicates non-His-ubiquitinated BimEL that was bound unspecifically to the Ni2+-agarose beads.
The deubiquitinase Usp27x stabilizes the BH3-only protein Bim and can enhance apoptosis (2016)
Figure 3

Figure 3 Usp27x expression sensitizes 293FT cells to apoptosis induction by stimulation with PMA

A 293FT-TetR-3xFlag-Usp27x or 3xFlag-Usp27xC87A, a polyclonal derivative where the Bim-locus had been targeted by CRISPR/Cas9- and four single clones obtained by serial dilution from the polyclonal line were treated as indicated (PMA [16.2 nM], QVD [10 µM]). Apoptosis was measured after 24 h of treatment by staining for active caspase-3, followed by flow cytometric analysis. Data (means/SEM) are from n=14 (3xFlag-Usp27x line and 3xFlag-Usp27xBim2KO polyclonal line), n=5 (3xFlag-Usp27xC87A, and 3xFlag-Usp27xBim2KO clone #14), n=7 (3xFlag-Usp27xBim2KO clone #11) or n=4 (3xFlag-Usp27xBim2KO clone #4 and #7) separate experiments. P-values (t-test) for statistically significant differences are shown.

B The same cells as in A (except Usp27xC87A mutant) were treated as in A and stained for active Bax. Data (means/SEM) are from n=8 (3xFlag-Usp27x line and 3xFlag-Usp27xBim2KO polyclonal line), n=5 (3xFlag-Usp27xBim2KO clone #11 and #14) or n=4 (3xFlag-Usp27xBim2KO clone #4 and #7) separate experiments. P-values (t-test) for statistically significant differences are shown. Expression levels of Bim, β-TrCP and Flag-Usp27x of the different cell lines are shown in Appendix Fig. S5E.
The deubiquitinase Usp27x stabilizes the BH3-only protein Bim and can enhance apoptosis (2016)
Figure 4

Figure 4 Usp27x but not Usp22 can counter Bim destabilization through the Raf-ERK pathway

A Lines based on the human melanoma cell line 1205Lu (BRAF-V600E-positive) were made to carry GFP-Usp27x, GFP-Usp27xC87A or GFP-Usp22 under the control of a doxycycline (dox)-inducible promoter. GFP-Usp27x, GFP-Usp27xC87A or GFP-Usp22 were induced with dox for 24 or 48h. Levels of Bim were determined by Western blotting. The two left panels are from the same membrane and exposure times. Similar results were obtained in n=4 separate experiments (left) and n=3 experiments (right). See also Appendix Fig. S6A where 3xFlag-Usp22 is also shown to be unable to increase Bim levels.

B A cell line derived from the BRAF-V600E-positive human melanoma line WM1158 was generated to carry inducible GFP-Usp27x. GFP-Usp27x was induced, and Bim was detected as in A. Similar results were obtained in n=3 separate experiments.

C Derivatives of the NSCLC cell line HCC827 (expressing constitutively active EGFR) were generated that only express the TetR-repressor or carrying in addition either GFP-Usp27x or GFP-Usp27xC87A. Cells were treated with dox for 72h. Bim was detected by Western blotting. Similar results were obtained in n=2 separate experiments. GFP-Usp27x, GFP-Usp27xC87A, or GFP-Usp22 expression (A-C) was detected using an antibody against GFP.

D Caco2 cells carrying a dox inducible HA-BRAF-V600E [37] (left two lanes) or the same cells stably expressing 3xFlag-Usp27x were treated with dox for 48h to induce expression of HA-BRAF-V600E. Bim was detected by Western blotting. The amount of BimEL was quantified from the shown immunoblot using the expression levels of the uninduced control cells set to 100% (normalized to the tubulin signal for each condition). Similar results were obtained in n=3 separate experiments with varying induction times. See also Appendix Fig. S6D.
The deubiquitinase Usp27x stabilizes the BH3-only protein Bim and can enhance apoptosis (2016)
Figure 5

Figure 5 Endogenous Usp27x stabilizes Bim in Caco2 and 293FT cells

A Loss of endogenous Usp27x enhances destabilization of BimEL in response to BRAF-V600E

Caco2 cells carrying inducible HA-BRAF-V600E were transfected with control siRNA (siCo3) or siRNA directed against Usp27x (combination of three siRNAs targeting Usp27x mRNA) for 24h prior to BRAF-V600E expression with dox for 2h (left) or 4h (right). BimEL was detected by Western blotting. Similar results were also observed in n=2 more experiments after 3h induction (not shown, see Appendix Fig. S6E for siRNA efficacy).

B 293FT cells deficient for Usp27x show enhanced destabilization of BimEL in response to PMA

293FT wt cells or 293FT-Usp27xKO (clone 2/10, generated using CRISPR-Cas-9) were treated with PMA [16.2 nM] for 16h or 24h to induce BimEL degradation. Western blots show protein levels of n=3 independently performed experiments (similar results were obtained in one more experiment, not shown). See Appendix Fig. S6G for verification of the Usp27xKO cell line by DNA sequence analysis.
The deubiquitinase Usp27x stabilizes the BH3-only protein Bim and can enhance apoptosis (2016)
Figure 6

Figure 6 Usp27x stabilizes Bim protein levels in the ERK-degradation pathway in 293FT cells, 1205Lu melanoma and HCC827 NSCLC cells

A 293FT-TetR-3xFlagUsp27x cells were treated with PMA [16.2 nM] plus QVD [10 µM] and 3xFlag-Usp27x was induced by dox at the same time for 24h followed by addition of cycloheximide (CHX). Cells were harvested at the indicated time points, and levels of BimEL were determined by Western blotting. Similar results were obtained in n=3 separate experiments. Cut membranes shown were from one membrane with same exposure time. For each condition BimEL-levels were quantified and normalized to the tubulin-signal. Percent BimEL gives the expression relative to the starting point.

B 1205Lu melanoma cells carrying dox-inducible GFP-Usp27x were treated with dox for 24h. Cycloheximide (CHX, 1µg/ml) was then added. Cells were harvested at the indicated time points, and levels of BimEL were determined by Western blotting. GFP-Usp27x was detected with anti-GFP-antibodies. Similar results were obtained in n=3 separate experiments. A second experiment is shown in the right panel also including 1205Lu melanoma carrying dox-inducible GFP-Usp27xC87A mutant. For each condition BimEL-levels were quantified and normalized to the tubulin-signal. Percent BimEL gives the expression relative to the starting point set to 100%. The starting point for the non-Usp27x/C87A inducing cells (-dox) is shown in lane 1 for experiment one and two (-dox, - CHX). Cut membranes shown were from one membrane with same exposure time. Same results were obtained for WM1158 melanoma cells (data not shown).

C HCC827 cells were induced to express GFP-Usp27x with dox as indicated. 24h later cycloheximide (CHX, 1µg/ml) was added for the indicated time. BimEL was detected after the indicated times of treatment by Western blotting. For each condition BimEL-levels were quantified and normalized to the tubulin-signal. Percent BimEL gives the expression relative to the starting point. Similar results were obtained in n=2 separate experiments. Cut membranes shown were from one membrane with same exposure time.
The deubiquitinase Usp27x stabilizes the BH3-only protein Bim and can enhance apoptosis (2016)
Figure 7

Figure 7 Usp27x sensitizes 1205Lu melanoma and HCC827 NSCLC cells to apoptosis induction by inhibition of the Raf-ERK-pathway

A 1205Lu melanoma cells carrying dox-inducible GFP, GFP-Usp27x or GFP-Usp27xC87A were treated with doxycycline (dox) as indicated. After 48 h UO126 (10µM) was added for another 48 h. Apoptosis was measured by staining for active caspase-3, followed by flow cytometric analysis. Data (means/SEM) are from n=3 (GFP) or from n≥6 separate experiments (GFP-Usp27x and GFP-Usp27xC87A). P-values (t-test) for statistically significant differences are shown. N.s., p>0.05. The addition of QVD inhibited cells from active Caspase-3 positive staining (not shown). A stain for active Bax is shown in Appendix Fig. S7B.

B HCC827 NSCLC cells carrying dox-inducible GFP-Usp27xC87A, GFP-Usp27x, or two separate lines established from these GFP-Usp27x-cells where the Bim-locus had been targeted for deletion using CRISPR/Cas9 (Bim-KO), were treated with combinations of dox and the EGFR-inhibitor gefitinib as indicated. Apoptosis was measured after 72h of treatment by staining for active caspase-3, followed by flow cytometric analysis. Data (means/SEM) are from n=≥15 (maternal GFP-Usp27x line) or from n≥5 (for Bim1KO) or from n≥6 (for Bim2KO), or n=4 (for GFP-Usp27xC87A) separate experiments. P-values (t-test) for statistically significant differences are shown. Again, the addition of QVD inhibited cells from active Caspase-3 positive staining (not shown). The inset shows Bim-knock-out-efficiency (n=2).
The deubiquitinase Usp27x stabilizes the BH3-only protein Bim and can enhance apoptosis (2016)
Figure 8

Figure 8 Loss of Usp27x reduces gefitinib-induced apoptosis in HCC827 NSCLC

HCC827 NSCLC cells and two separate polyclonal lines established from these cells where the Usp27x-locus had been targeted for deletion using CRISPR/Cas9 with two different guide RNA against Usp27x (Usp27xKO-1 and Usp27xKO-4), were treated with gefitinib [10µM] for 48h. Apoptosis was measured by staining for active caspase-3 (A) or by staining for activated Bax (antibody: 6A7 clone) followed by flow cytometric analysis. Data shown in A, B (means/SEM) are from n=4 separate experiments. P-values (t-test) for statistically significant differences are shown.
miR-132 loss de-represses ITPKB and aggravates amyloid and TAU pathology in Alzheimer’s brain (2016)
Figure 1

Figure 1. Efficiency and specificity of in vivo down- and upregulation of miR-132.

A. Experimental scheme of antagomiR-132 and miR-132 mimic injections into the lateral ventricle of 2-month old APPPS1 mice. B. Semi-quantitative PCR of miR-132 and negative control miRNAs in the hippocampus of antagomiR-132-injected mice (ant-132) in comparison to control-injected animals (aCSF and scramble) at six months of age. Sample size, n=9 per group. C. FISH of miR-132 and negative control miR-124 in the hippocampus of ant-132- and scramble-injected mice. Scramble probes were used as FISH negative controls. Scale bar, 500μm. D. Semi-quantitative PCR of miR-132 and miR-212 in the hippocampus of 3-month old miR-132 mimic-injected mice (miR-132) compared to animals injected with a negative control oligonucleotide (Ctr). Sample size, n=6 per group. Values were normalized to scramble- (B) or control-injected group (D) and presented as mean ± SEM. In B, one-way ANOVA was used, while in D, Student’s t-test was applied.
miR-132 loss de-represses ITPKB and aggravates amyloid and TAU pathology in Alzheimer’s brain (2016)
Figure 2

Figure 2. Effect of miR-132 on Aβ accumulation.

A. Amyloid staining (6E10) combined with miR-132 FISH in ant-132- and scramble-injected mice at six months of age. Scale bar, 500 μm. B. Quantification of amyloid burden in hippocampus and cortex of ant-132-injected and control animals (see Materials and Methods). Sample size, n=4 per group. C, D. ELISA of soluble (TBS) and insoluble (Formic acid) Aβ40 and Aβ42 levels in the hippocampus of ant-132- (C) and miR-132-injected (D) animals at six and three months of age, respectively. Sample size, n=6 per group. In B, C and D, values were normalized to scramble- (B, C) or control-injected group (D) and presented as mean ± SEM. Student’s t-test was used.
miR-132 loss de-represses ITPKB and aggravates amyloid and TAU pathology in Alzheimer’s brain (2016)
Figure 3

Figure 3. Regulatory effect of miR-132 on TAU expression and phosphorylation.

Western blot analysis of total TAU and pTAU (AT8, AT270) levels upon miR-132 knockdown (A) or overexpression (B) at six and three months of age, respectively. Sample size, n=9 per group (A) and n=6 per group (B). Values were normalized to the respective control groups and presented as mean ± SEM. Student’s t-test was used.
miR-132 loss de-represses ITPKB and aggravates amyloid and TAU pathology in Alzheimer’s brain (2016)
Figure 5

Figure 5. miR-132 regulation over ITPKB.

A. Luciferase reporter assay of wild-type (wt) and mutant (mut) ITPKB 3’UTR in HEK293 cells co-transfected with a synthetic miR-132 (miR-132) or a negative control (Neg Ctr) oligonucleotide. B. ELISA of Aβ40 and Aβ42 levels in HEK293-APPswe cells transfected with a miR-132 antisense oligonucleotide (miR-132 inh), an siRNA against ITPKB (ITPKB siRNA) or both. The assays in A and B were performed in three independent experiments, each in triplicates. C. Western blot analysis of ITPKB knock down in APPPS1 hippocampus using an siRNA oligonucleotide against ITPKB. Sample size, n=6 per group. Values were normalized to control-injected group (Ctr) and presented as mean ± SEM. D. ELISA of insoluble (Formic acid-soluble) Aβ40 and Aβ42 levels in the hippocampus of ITPKB siRNA- and control-injected animals at three months of age. Sample size, n=6 per group. Values were normalized to control group and presented as mean ± SEM. E, F. Western blot analysis of ITPKB levels upon miR-132 down- (E) or up-regulation (F) at six and three months of age, respectively. Sample size, n=9 per group for miR-132 down regulation and n=6 per group for miR-132 overexpression. Values were normalized to control groups and presented as mean ± SEM. In A, C-F Student’s t-test was used, while in B one-way ANOVA was employed.
miR-132 loss de-represses ITPKB and aggravates amyloid and TAU pathology in Alzheimer’s brain (2016)
Figure 6

Figure 6. Effect of miR-132 regulation on BACE1 and ERK1/2 activity.

Western blot analysis of CTFβ (A), sAPPβ-swe (B) and phosphorylated ERK1/2 (pERK1/2) (C) levels upon miR-132 down- (ant-132) (left panel) or upregulation (miR-132) (right panel) in APPPS1 hippocampus at six and three months of age, respectively. Sample size, n=9 per group for miR-132 down regulation and n=6 per group for miR-132 overexpression. Values were normalized to respective control groups and presented as mean ± SEM. The Student’s t-test was used.
miR-132 loss de-represses ITPKB and aggravates amyloid and TAU pathology in Alzheimer’s brain (2016)
Figure 7

Figure 7. miR-132/ITPKB expression profile in human AD prefrontal cortex.

A. Double immunofluorescence staining of amyloid plaques (6E10) and ITPKB in AD prefrontal cortex. Scale bar, 50 μm. Arrowheads indicate ITPKB immunopositivity. B. miR-132 FISH coupled with double immunofluorescence against hyperphosphorylated TAU (AT8)-containing neurofibrillary tangles (NFTs) and ITPKB in AD prefrontal cortex. miR-124 was used as a control. Scale bar, 50 μm. C. Quantification of miR-132, ITPKB and hyperphosphorylated TAU (pTAU) signal in single neurons. Integrated intensity values of each signal per cell were normalized to the mean integrated density of each signal across all the cells analyzed (see Materials and Methods). Sample size (AD patients), n=3. Values are presented as mean ± SEM. Two-way Anova was used. Quantifications are summarized in the table provided. Arrow directions refer to the comparison of each normalized signal to the same signal in the other group (in the “low miR-132” group comparisons are made to the “high miR-132” group and vice versa).
miR-132 loss de-represses ITPKB and aggravates amyloid and TAU pathology in Alzheimer’s brain (2016)
Figure 8

Figure 8. miR-132/ITPKB pathway assessment in human AD hippocampus.

Semi-quantitative PCR of miR-132 and western blot analysis of ITPKB, phosphorylated (pERK1/2) and total ERK1/2, phosphorylated (AT8 , AT270) and total TAU, phosphorylated sphingosine kinase 1 (pSphK1), BACE1, full length APP (flAPP), APP CTFs and sAPPβ in human AD hippocampi (AD) compared to non demented control samples (ND). Sample size, n=39 for AD and n=15 for ND. Values were normalized to ND and presented as mean ± SEM. The Student’s t-test was used.
Interactomes of SARS-CoV-2 and human coronaviruses reveal host factors potentially affecting pathogenesis (2021)
Figure 2

Figure 2. Validation of selected host-virus protein-protein interactions.

A Pulldown and Western blot analysis validated the interaction between the viral protein ORF3a and its interactors, VPS11 and CLCC1, and the binding of S protein to SPCS2. HEK293T cells with SFB-tagged bait expression were collected and lysed. Cell lysates were subjected to pulldown assay using S-protein beads. Western blot analysis was conducted with the indicated antibodies. Cells transfected with vector or construct encoding control genes were included as controls in these experiments.

B Immunostaining analysis of protein localization. U2OS cells were transfected with construct encoding ORF3a. Cells were fixed and stained with the indicated antibodies. The green signal is CLCC1 or lysosome marker LAMP1, the red signal is flag (for SFB-ORF3a), and the blue signal indicates DAPI/nuclei. Scale bar: 10 µm.

C Overlap of HCIPs identified from SFB-tandem affinity purification and second-generation biotin ligase (BioID2) labeling experiments.

D Overlap of HCIPs reported previously and those identified in the current study.
Interactomes of SARS-CoV-2 and human coronaviruses reveal host factors potentially affecting pathogenesis (2021)
Figure 5

Figure 5. Comparison of interactomes of seven NSP1 proteins from seven human coronaviruses.

A Comparisons of NSP1 proteins from seven human coronaviruses.

B Principal Component Analysis of the seven HCIP lists with bait NSP1 from different human coronaviruses.

C Heat map of the NSP1 HCIPs obtained using SFB- TAP. NSP1 HCIPs were compared among seven human coronaviruses using the preys' spectral counts. Six areas of the heat map are manually selected, enlarged and labeled as (1), (2), (3), (4), (5), and (6). These groups of proteins are enriched by NSP1 protein specific from one or limited human coronaviruses. Functional characterization of each protein is shown with the red circles below the enlarged images.

D Pulldown and Western blot validation of the interaction between human coronavirus NSP1 proteins and the human proteins PYCR1/PYCR2. Cells transfected with vector or construct encoding GFP were included as controls in these experiments.
Interactomes of SARS-CoV-2 and human coronaviruses reveal host factors potentially affecting pathogenesis (2021)
Figure 6

Figure 6. Comparison of interactomes of N proteins from different human coronaviruses.

A Comparison of N proteins from seven human coronaviruses.

B Principal Component analysis of the seven high-confidence interacting protein (HCIP) lists with bait N protein from different human coronaviruses.

C Heat map of the N protein HCIPs identified by SFB-tandem affinity purification. N protein HCIPs were compared among seven human coronaviruses using the preys' spectral counts. Three areas of the heat map are manually selected, enlarged and labeled as (1), (2), and (3), which are enriched by all N proteins of the seven human coronaviruses. Functional characterization for each protein is shown with the red circles below the enlarged images.

D The human proteins which binding to N proteins of human coronaviruses were integrated and analyzed by STRING to illustrate the protein-protein interaction among the HCIPs. The minimum required interaction score setting in STRING is 0.9. Different function group or complex were colored differently. The filled circles are proteins, and the lines are the interactions.
Interactomes of SARS-CoV-2 and human coronaviruses reveal host factors potentially affecting pathogenesis (2021)
Figure 7

Figure 7. Analysis of HCIPs showing specific interaction with one or more N proteins of human coronaviruses.

A Preys identified showing significant enrichment by N proteins specifically from one or more human coronaviruses. PSM, peptide-spectrum match.

B Pulldown and Western blot validation of the interaction between N protein and G3BP1/G3BP2. Cells transfected with vector or construct encoding GFP or N protein from different human coronaviruses were compared in these experiments.

C,D Effect of SARS-CoV-2 N protein on stress granule formation. A549 (C) and MCF10A (D) cells were transfected with pinducer20-N protein of SARS-CoV-2. All the cells are treated with sodium arsenite but with or without Dox to induce N protein expression. Cells were fixed and stained with the indicated antibodies. The green signal is G3BP1, the red signal is HA (for N protein), and the blue signal indicates DAPI/nuclei. Scale bar: 10 µm.

E Quantification of the stress granule formation in A549 and MCF10A cells. Total of 30 cells were counted and a student's t test was used for the statistical analysis (***, p<0.001). Box limits represent 25th percentile and 75th percentile; horizontal line represents median. Whiskers display min. to max. values.

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