Journal: Frontiers in Immunology

Article Title: Foot-and-Mouth Disease Virus Counteracts on Internal Ribosome Entry Site Suppression by G3BP1 and Inhibits G3BP1-Mediated Stress Granule Assembly via Post-Translational Mechanisms

doi: 10.3389/fimmu.2018.01142

Figure Lengend Snippet: Confirmation of differentially expressed proteins and phosphoproteins by western blotting and Phos-tag western Blotting. (A) Analysis of ubiquitin conjugating enzyme E2 I, ubiquitin conjugating enzyme E2 L3, glyceraldehyde-3-phosphate dehydrogenase, β-actin expression levels in foot-and-mouth disease virus (FMDV)-infected and control cells by western blotting. SILAC-ratios and immunoblotting ratios (infection/control) were shown on the right side. (B) Analysis of the dynamic phosphorylation alterations of the three differentially phosphoproteins (ribosomal protein L15, chromosome 5 open reading frame 24, and FOS-like 2) in FMDV-infected and control cells by Phos-tag western blotting.

Article Snippet: To confirm the expression levels of GAPDH, ubiquitin conjugating enzyme E2 I (UBE2I), ubiquitin conjugating enzyme E2 L3 (UBE2L3), ribosomal protein L15 (RPL15), chromosome 5 open reading frame 24 (C5ORF24) and FOS-like 2 (FOSL2), anti-GAPDH antibody (Beyotime, China), anti-UBE2I antibody (Proteintech, China), anti-UBE2L3 antibody (Proteintech, China), anti-RPL15 antibody (Proteintech, China), anti-C5ORF24 antibody (Proteintech, China), and anti-FOSL2 antibody (Proteintech, China) were used for immunoblotting.

Techniques: Western Blot, Ubiquitin Proteomics, Expressing, Virus, Infection, Control, Multiplex sample analysis, Phospho-proteomics

Journal: Cell Reports

Article Title: SUMOylation and the HSF1-Regulated Chaperone Network Converge to Promote Proteostasis in Response to Heat Shock

doi: 10.1016/j.celrep.2018.12.027

Figure Lengend Snippet: Depletion of Proteostasis Factors by Chronic HSF1 Inhibition Delays Recovery to Normal SUMO2/3 Conjugation Levels after Heat Shock (A) Use of a dominant-negative, constitutively active HSF1 construct (dn-cHSF1), created via disruption of the regulatory domain (RD) by removing amino acids 186–202 combined with deletion of the transcription activation domain (TAD), for inhibition of endogenous HSF1. (B) Western blot analysis of HSP40 protein levels in response to different time periods of Dox-induced dn-cHSF1 induction in HEK293T-REx cells. (C) HEK293T-REx cells expressing Dox-inducible dn-cHSF1 were treated with Dox for 48 hr (chronic HSF1i) prior to heat shock (HS). Cells were exposed to a HS at 43°C for 75 min before returning to 37°C for a recovery period (rec) and lysed as indicated. Total amounts of SUMOylated proteins were analyzed by immunoblotting. HSP40 levels were used to confirm the induction of the heat shock response and functional HSF1 inhibition. (D) HEK293T-REx cells constitutively expressing DHFR.dn-cHSF1 were treated with trimethoprim (TMP) for 48 hr (chronic HSF1 inhibition) or 4 hr (acute HSF1 inhibition) prior to HS. Cells were subsequently exposed to HS and recovery as described in (A). Total amounts of SUMOylated proteins were determined by immunoblotting. (E) Quantification of total SUMO2/3 conjugation from (D) as compared to SUMOylation post-heat shock in basal proteostasis conditions. n = 3, error bars indicate SD. Significance was determined using ANOVA analysis followed by post hoc Tukey analysis, ∗ p < 0.05; ∗∗ p < 0.005, ∗∗∗∗ p < 0.0001. See also Figure S1 .

Article Snippet: His 10 -SUMO2-WT-IRES-GFP expressing cells were sorted by fluorescence-aided cell sorting (FACS) on the BD FACSAriaIII™ cell sorter for low GFP expression to limit His 10 -SUMO2 expression.

Techniques: Inhibition, Conjugation Assay, Dominant Negative Mutation, Construct, Activation Assay, Western Blot, Expressing, Functional Assay

Journal: Cell Reports

Article Title: SUMOylation and the HSF1-Regulated Chaperone Network Converge to Promote Proteostasis in Response to Heat Shock

doi: 10.1016/j.celrep.2018.12.027

Figure Lengend Snippet: Proteomic Identification of SUMOylated Proteins Whose Recovery to Normal SUMO-Conjugation Levels Post Heat Shock Is Delayed by Chronic HSF1 Inhibition (A) Workflow for Figure 2 . HEK293T-REx cells stably co-expressing His10-SUMO2 and either Dox-inducible dn-cHSF1 or Dox-inducible GFP were treated with Dox for 48 hr (chronic HSF1i) prior to heat shock (HS). Cells were exposed to HS at 43°C for 75 min before returning to 37°C for a recovery period (rec). Red arrows indicate time points at which cells were lysed. SUMOylated proteins were purified by means of His10 pull-down, proteins were trypsinized, and peptides were analyzed by mass spectrometry. (B) Immunoblotting of cell lysates from (A) using antibodies against SUMO2/3, HSP70, and GFP. Total amounts of proteins in each lane were visualized by Ponceau S staining. (C) Volcano plots depicting statistical differences in abundance between proteins identified by mass spectrometry. Dashed lines indicate a cut-off at a p value ≤ 0.05 (–log10 p value ≤ 1.3) and a fold change of 2 (log2 = 1). The left panel shows SUMOylated proteins identified immediately post-HS in the Dox-inducible dn-cHSF1 cell line, specifically in the Dox-treated sample compared to the untreated sample. The right panel shows SUMOylated proteins identified 4-hr post-HS in the Dox-inducible dn-cHSF1 cell line compared to the untreated sample. Proteins marked in red are either HSF1 or proteins that were further validated. (D) Selection of enriched gene ontology terms of biological processes and molecular functions for selected proteins that significantly retained SUMOylation 4-hr post-HS when HSF1 activity was chronically inhibited. See also and and , , , and .

Article Snippet: His 10 -SUMO2-WT-IRES-GFP expressing cells were sorted by fluorescence-aided cell sorting (FACS) on the BD FACSAriaIII™ cell sorter for low GFP expression to limit His 10 -SUMO2 expression.

Techniques: Conjugation Assay, Inhibition, Stable Transfection, Expressing, Purification, Mass Spectrometry, Western Blot, Staining, Selection, Activity Assay

Journal: Cell Reports

Article Title: SUMOylation and the HSF1-Regulated Chaperone Network Converge to Promote Proteostasis in Response to Heat Shock

doi: 10.1016/j.celrep.2018.12.027

Figure Lengend Snippet: Chronic HSF1 Inhibition Interferes with Degradation of SUMOylated and Ubiquitinated Proteins during Heat Shock Recovery (A) HEK293T-REx cells expressing Dox-inducible dn-cHSF1 were treated with Dox for 48 hr (chronic HSF1i), with 100 nM bortezomib (proteasome inhibition) immediately prior to heat shock (HS), or with a combination of both. Cells were exposed to HS at 43°C for 75 min before returning to 37°C for a recovery period (rec) and lysed as indicated. Total amounts of SUMOylated proteins were analyzed by immunoblotting. HSP40 levels were used to confirm induction of the heat shock response and functional HSF1 inhibition. (B) HEK293T-REx cells expressing Dox-inducible dn-cHSF1 were treated as in (A). Total amounts of ubiquitinated proteins were analyzed by immunoblotting. (C) HEK293T-REx cells stably co-expressing His10-SUMO2 and Dox-inducible dn-cHSF1 were treated as in (A). SUMOylated proteins were purified by means of His10 purification. Elutions and inputs were analyzed by immunoblotting for FoxM1. Ponceau S stain was used as a loading control for inputs. (D) HEK293T-REx cells stably co-expressing His10-Ub and Dox-inducible dn-cHSF1 were treated as in (A). Ubiquitinated proteins were purified by means of His10-purification. Elutions and inputs were analyzed by immunoblotting for FoxM1. Ponceau S stain was used as a loading control for inputs. See also and .

Article Snippet: His 10 -SUMO2-WT-IRES-GFP expressing cells were sorted by fluorescence-aided cell sorting (FACS) on the BD FACSAriaIII™ cell sorter for low GFP expression to limit His 10 -SUMO2 expression.

Techniques: Inhibition, Expressing, Western Blot, Functional Assay, Stable Transfection, Purification, Staining

Journal: Cell Reports

Article Title: SUMOylation and the HSF1-Regulated Chaperone Network Converge to Promote Proteostasis in Response to Heat Shock

doi: 10.1016/j.celrep.2018.12.027

Figure Lengend Snippet: Chronic HSF1 Inhibition Selectively Impairs Degradation of SUMO2/3 and Ub Co-modified Proteins during Heat Shock Recovery (A) HEK293T-REx cells stably co-expressing His10-SUMO2 and Dox-inducible dn-cHSF1 were treated with Dox for 48 hr (chronic HSF1i) prior to heat shock (HS), with 100 nM bortezomib (proteasome inhibition) immediately prior to HS, or with a combination of both. Cells were exposed to a HS at 43°C for 75 min before returning to 37°C for a recovery period (rec) and lysed as indicated. Elutions were analyzed by immunoblotting for Ub and SUMO2/3. (B) HEK293T-REx cells stably co-expressing His10-Ub and Dox-inducible dn-cHSF1 were treated as in (A). Elutions were analyzed by immunoblotting for SUMO2/3 and Ub. (C) Quantification of (B). Graph shows fold change of the ratio of SUMOylated proteins over ubiquitinated proteins in His10-Ub purified elutions as compared to the ratio after HS under basal proteostasis conditions. n = 3, error bars represent SD. Significance was determined using ANOVA followed by post hoc Tukey’s multiple comparison test, ∗ p < 0.05; ∗∗∗ p < 0.001.

Article Snippet: His 10 -SUMO2-WT-IRES-GFP expressing cells were sorted by fluorescence-aided cell sorting (FACS) on the BD FACSAriaIII™ cell sorter for low GFP expression to limit His 10 -SUMO2 expression.

Techniques: Inhibition, Modification, Stable Transfection, Expressing, Western Blot, Purification

Journal: Cell Reports

Article Title: SUMOylation and the HSF1-Regulated Chaperone Network Converge to Promote Proteostasis in Response to Heat Shock

doi: 10.1016/j.celrep.2018.12.027

Figure Lengend Snippet: HSP90 Plays a Key Role in the Recovery of SUMO2/3 Modification following Heat Shock (A) HEK293T-REx cells expressing Dox-inducible dn-cHSF1 were either treated with Dox for 48 hr (chronic HSF1i) prior to heat shock (HS), with 500 nM STA-9090 (HSP90i) 2 hr prior to HS, or with a combination of both. Cells were exposed to a HS at 43°C for 75 min before returning to 37°C for a recovery period (rec) and lysed as indicated. Total amounts of SUMOylated proteins were analyzed by immunoblotting. HSP70 levels were used to confirm induction of the heat shock response and functional HSF1 inhibition. (B) Quantification of fold change of SUMOylation from (A) as compared to SUMOylation after HS under the basal proteostasis conditions. n = 3, error bars indicate SD. Significance was determined using ANOVA analysis followed by post hoc Tukey analysis, ∗ p < 0.05; ∗∗∗ p < 0.0005. (C) HEK293T-REx cells stably co-expressing His10-SUMO2 and Dox-inducible dn-cHSF1 were treated as in (A) with a 2-hr recovery at 37°C. SUMOylated proteins were purified by means of His10-purification. Elutions and inputs were analyzed by immunoblotting for FoxM1. Ponceau S stain was used as a loading control for inputs. (D) As for (C). Elutions were analyzed by immunoblotting for Ub and SUMO2/3. (E) HEK293T-REx cells stably co-expressing His10-Ub and Dox-inducible dn-cHSF1 were treated as in (A) with a 2-hr recovery at 37°C. Ubiquitinated proteins were purified by means of His10-purification. Elutions and inputs were analyzed by immunoblotting for FoxM1. Ponceau S stain was used as a loading control for inputs. (F) As for (E). Elutions were analyzed by immunoblotting for SUMO2/3 and Ub. See also Figure S6 .

Article Snippet: His 10 -SUMO2-WT-IRES-GFP expressing cells were sorted by fluorescence-aided cell sorting (FACS) on the BD FACSAriaIII™ cell sorter for low GFP expression to limit His 10 -SUMO2 expression.

Techniques: Modification, Expressing, Western Blot, Functional Assay, Inhibition, Stable Transfection, Purification, Staining

Journal: Cell Reports

Article Title: SUMOylation and the HSF1-Regulated Chaperone Network Converge to Promote Proteostasis in Response to Heat Shock

doi: 10.1016/j.celrep.2018.12.027

Figure Lengend Snippet: SUMO2/3 Plays a Key Role as an Integrated Component of the Proteotoxic Stress Response Upon HS, a transient increase in SUMO2/3 conjugation to target proteins is observed, which may function in part to prevent irreversible aggregation of target proteins (reduced aggregation was demonstrated in vitro ). SUMO2/3-modified proteins are frequently co-modified by Ub, and a large fraction of these co-modified proteins are subsequently degraded by the proteasome during HS recovery in a chaperone-dependent manner.

Article Snippet: His 10 -SUMO2-WT-IRES-GFP expressing cells were sorted by fluorescence-aided cell sorting (FACS) on the BD FACSAriaIII™ cell sorter for low GFP expression to limit His 10 -SUMO2 expression.

Techniques: Conjugation Assay, In Vitro, Modification

Journal: Cell Reports

Article Title: SUMOylation and the HSF1-Regulated Chaperone Network Converge to Promote Proteostasis in Response to Heat Shock

doi: 10.1016/j.celrep.2018.12.027

Figure Lengend Snippet:

Article Snippet: His 10 -SUMO2-WT-IRES-GFP expressing cells were sorted by fluorescence-aided cell sorting (FACS) on the BD FACSAriaIII™ cell sorter for low GFP expression to limit His 10 -SUMO2 expression.

Techniques: Recombinant, SYBR Green Assay, Plasmid Preparation, Software