EMBO/soda-nel huggingface dataset:
Synaptonuclear messenger PRR7 inhibits c-Jun ubiquitination and regulates NMDAmediated excitotoxicity (2016)
Figure 5

Figure 5. PRR7 interacts with c-Jun and the E3 ubiquitin ligase FBW7.

(A) Co-IPs of PRR7 with c-Jun from lysates of HEK293 cells overexpressing PRR7.

(B) Pulldown assays in vitro using purified proteins showed a direct interaction between PRR7 and c-Jun.

(C) Co-IPs of PRR7 with FBW7 from lysates of HEK293 cells overexpressing PRR7 and FBW7.

(D) Pulldown assays in vitro using purified PRR7 and FBW7.

(E) Co-IPs of endogenous PRR7 and FBW7 from lysates of rat brain.

(F) Co-IPs of PRR7 with c-Jun and FBW7 from lysates of HEK293 cells overexpressing PRR7 and FBW7. FBW7 expression increased the amount of c-Jun pulled down with PRR7 (n=4 gels for each panel, representative blots). Lower bar graphs- quantitation for Western blots show c-Jun/FBW7 binding is 41.4±13.9% greater in the presence of PRR7 (normalized for FBW7 expression). Similarly, c-Jun/PRR7 binding is 97.4±38.1% greater in the presence of FBW7 (normalized for PRR7 expression). Statistical significance was calculated using unpaired t-tests.
Synaptonuclear messenger PRR7 inhibits c-Jun ubiquitination and regulates NMDAmediated excitotoxicity (2016)
Figure 6

Figure 6. PRR7 regulates c-Jun transcriptional activity and mediates NMDAR-dependent excitotoxicity.

(A) Western blots of phospho-c-Jun in primary neuronal cultures. Quantification of phospho-c-Jun represents a ratio of phospho-c-Jun to total c-Jun and all ratios were normalized to NT. PRR7 knockdown (KD) decreased, while PRR7 overexpression (OE) increased phospho-c-Jun levels (black bars). Glutamate stimulation (gray bars) increased phospho-c-Jun/c-Jun ratio in NT but not in KD or OE neurons (n=7 gels for all groups). Significance was determined using two-way ANOVA with Sidak post-hoc test.

(B) AP-1 driven luciferase luminescence assays. PRR7 expression in HEK293 cells increased AP-1 activity (190.6 ± 13.9%, n=12; RLU= relative light units). Statistical significance was determined using t-tests.

(C) Ingenuity Pathway Analysis (IPA)-predicted cellular functions based on microarray analysis. Functions are sorted by p-value (Highest-top).

(D) Quantitation of cell death using 30 minutes of PI in-situ staining following 48h of WT-PRR7-GFP or mNLS-PRR7-GFP overexpression in primary hippocampal neuronal cultures (n=24 cells per group). Images represent a max projection of five z-stack sections across the cell body.

(E) NMDA treatment (25 min, 100 µM followed by 14h chase) caused 83.6±3.5% cell death in control neurons compared to 60.3±4.6% cell death in neurons with PRR7 expression knocked down. A c-Jun inhibitory peptide reduced NMDA-mediated cell death to 66.2±6.0% in control neurons and to 55.6±5.3% in neurons knocked down for PRR7, but this difference was not statistically significant (n=5 independent experiments). Two-way ANOVA was used in conjugation with Tukey post-hoc test. (Left) Representative images showing transduced neurons (green- GFP) and PI staining (red).

(F) Quantification of cell death in response to NMDA stimulation in the presence or absence of PRR7 and either WT c-Jun or a dominant negative c-Jun4A (4A). Overexpression of WT c-Jun caused extensive neuronal cell death (84.8±2.4%) that was reduced in neurons knocked down for PRR7 (43.8±9.2). Overexpressing 4A conferred neuroprotection to NMDA excitotoxicity (50.8±7.9%) but was not additive with PRR7 knockdown (42.3±8.1%) (n=3 independent experiments). Significance was determined using two-way ANOVA in conjugation with Tukey post-hoc test. Images are representative 10X widefield micrographs showing transduced neurons (green- GFP) and PI staining (red). Data is presented as mean±sem. All % values represent comparisons to control.
EI24 promotes cell adaption to ER stress by coordinating IRE1 signaling and calcium homeostasis (2021)
Figure 1

Figure 1. EI24 acts as an IRE1 inhibitor in the absence of ER stress.

(A) Schematic depicting the identification of EI24 and BiP as IRE1 interaction proteins. HeLa cells transfected with HA-IRE1 were subjected to anti-HA immunoprecipitation followed by mass spectrometry assay.

(B, C) Sequential dissociation of EI24 and BiP from IRE1 elicits a two-phase activation of IRE1 in response to ER stress. Cells were treated with 4 μM tunicamycin (TM) for the indicated times. The interactions of EI24 and BiP with IRE1 were analyzed by immunoprecipitation (B). IRE1 phosphorylation was analyzed by immunoblotting. The curve displays the coordination of IRE1 phosphorylation with the dissociation of EI24 and BiP (C).

(D) EI24 knockout promotes the activation of IRE1, but not PERK or ATF6. Control (sgGFP) and EI24 knockout (sgEI24) HeLa cells were treated with 4 μM TM for the indicated times. Protein expression was analyzed by immunoblotting.

(E, F) EI24 knockout promotes XBP1u splicing into XBP1s and XBP1s protein production (E), and ERdj4 and RAMP4 gene expressions (F). Cells were treated as in (D). RNA extracts were analyzed for XBP1u mRNA splicing by PCR, and for ERdj4 and RAMP4 mRNA expressions by Q-PCR. Protein extracts were analyzed for XBP1s protein production by immunoblotting. Data are mean ± SEM (n = 3 biologically independent replicates). *P < 0.05, **P < 0.01, ***P < 0.001; two-tailed Student's t-test.

(G) Expression of Flag-EI24 in EI24 knockout cells inhibits IRE1 activity. Flag-Metap2 served as the control. Cells transfected with the indicated cDNAs were treated with or without 4 μM TM for 2 hr. Protein expression was analyzed by immunoblotting.

Data information: Please see Appendix Figure S1 for information regarding replicates, quantification and statistical evaluations for all biochemical data in this figure.
EI24 promotes cell adaption to ER stress by coordinating IRE1 signaling and calcium homeostasis (2021)
Figure 2

Figure 2. EI24 binds to and inhibits unphosphorylated IRE1 via the C-terminal domain

(A) Schematic depicting ER localization of EI24. TM, transmembrane domain; N-term, N-terminus; C-term, C-terminus.

(B) EI24 persistently localizes on the ER regardless of the presence or absence of ER stress. HeLa cells stably expressing Flag-EI24 were treated with or without 4 μM tunicamycin (TM) for 2 hr. Colocalization of EI24 with ER was analyzed. KDEL-mCherry, ER marker. Scale bar, 10 μm.

(C) Schematic depicting EI24 truncation mutants. FL, full length; △, deletion of the indicated regions.

(D) The C-terminus of EI24 is required for IRE1 binding. HeLa cells transfected with the indicated EI24 mutants were treated as in (B), after which protein-protein interactions were analyzed by immunoprecipitation (IP). The star represents IgG light chains.

(E) The C-terminus of EI24 binds to IRE1 in a persistent fashion. HeLa cells transfected with the indicated EI24 domains were treated as in (B). Protein-protein interactions were analyzed by IP. The star represents IgG light chains.

(F) Expression of the EI24 C-terminus in EI24 knockout cells prevents TM-induced IRE1 activation. HeLa cells transfected with full-length EI24 or the C-terminus alone were treated with 4 μM TM for the indicated times. Protein expressions were analyzed by immunoblotting.

(G) EI24 binds to the kinase domain of IRE1. HeLa cells transfected with the indicated IRE1 domains were analyzed for protein-protein interactions by IP. FL, full-length.

(H) EI24 cannot bind to the phospho-mimicking (3SE) IRE1 mutant. HeLa cells transfected with wild-type HA-IRE1 or the 3SE mutant were treated as in (B). Protein-protein interactions were analyzed by IP.

(I) EI24 overexpression cannot prevent the activation of the phospho-mimicking (3SE) IRE1 mutant. Wild-type and IRE1 3SE knock-in HeLa cells were transfected with empty vector or Flag-EI24. Transfected cells were treated as in (B). Protein expressions were analyzed by immunoblotting.

(J, K) The TM34 domain of EI24 is required for ER stress sensing. HeLa cells transfected with the indicated EI24 mutants were treated with TM as indicated, after which protein-protein interactions were analyzed by IP. The star represents IgG light chains.

(L) Expression of full-length EI24 or the △TM34 or △C mutant in EI24 knockout HeLa cells for comparison of their functions. HeLa cells transfected with the indicated EI24 mutants were treated as in (B). Protein expressions were analyzed by immunoblotting.

(M) Schematic depicting ER stress sensing and IRE1 activation.

Data information: Please see Appendix Figure S2 for information regarding replicates, quantification and statistical evaluation for all biochemical data in this figure.
EI24 promotes cell adaption to ER stress by coordinating IRE1 signaling and calcium homeostasis (2021)
Figure 3

Figure 3. EI24 targets IP3R1 to prevent ER calcium depletion under conditions of ER stress.

(A) EI24 binds to IP3R1 in response to ER stress. HeLa cells stably expressing Flag-EI24 were treated with 4 μM tunicamycin (TM) for the indicated times. Protein-protein interactions were analyzed by immunoprecipitation (IP).

(B, C) EI24 knockout causes resting-state ER calcium reduction (B) and cytosolic calcium elevation (C) under ER stress. Control (sgGFP) and EI24 knockout (sgEI24) HeLa cells were first perfused in TM-free buffer, and switched to the buffer containing 4 μM TM to induce ER stress where indicated. Resting state calcium levels in the ER (B) and cytosol (C) with or without TM are displayed in columns.

(D, E) EI24 knockout accelerates calcium mobilization from the ER (D) to cytosol (E). Cells pretreated with or without 4 μM TM for 2 hr were subjected to calcium mobilization assay. Cells were first perfused in calcium-containing buffer, and switched to calcium-free buffer containing 100 μM histamine to stimulate calcium mobilization from the ER to cytosol. ER calcium depletion rates (D) and peak cytosolic calcium values (E) are displayed in columns.

(F) Validation of siRNA knockdown efficiency for IP3R1 and IP3R3. HeLa cells transfected with siRNA targeting IP3R1 or IP3R3 were analyzed for IP3R1 and IP3R3 protein expression by immunoblotting.

(G) Knockdown of IP3R1 prevents calcium mobilization from the ER to the cytosol. The measurements were similarly performed as in (E), and were from the same batch of cells.

(H) EI24 binds IP3R1 via the C-terminus. HeLa cells transfected with the indicated EI24 domains were treated with or without 4 μM TM for 2 hr. Protein-protein interactions were analyzed by IP. The star represents IgG light chains.

(I) Wild-type EI24, but not the TM34- or C-terminus-deleted mutant, preserves ER calcium stores in EI24 knockout cells. The measurements were similarly performed as in (E), and were from the same batch of cells.

Data information: (B-E, G, I) N = 20 cells for one replicate. Data are mean ± SEM (n = 3 technical replicates). ns, no significant difference; ***P < 0.001; two-tailed Student's t-test. Please see Appendix Figure S3 for information regarding replicates, quantification and statistical evaluation for all biochemical data in this figure.
EI24 promotes cell adaption to ER stress by coordinating IRE1 signaling and calcium homeostasis (2021)
Figure 4

Figure 4. EI24 protects cells against ER-stress-induced apoptosis.

(A) EI24 knockout promotes ER stress-induced apoptosis. Control (sgCtrl) and EI24 knockout (sgEI24) HeLa cells were treated with the indicated concentrations of tunicamycin (TM) for 8 hr. Percentages of early apoptotic cells were measured by flow cytometry.

(B) Knockdown of IP3R1 prevents ER stress-induced apoptosis in EI24 knockout cells. Cells were cultured with or without 4 μM TM for 8 hr. Percentages of early apoptotic cells were measured by flow cytometry.

(C) Administration of calcium chelator BAPTA prevents ER stress-induced apoptosis in EI24 knockout cells. Cells were cultured in the presence of 50 μM BAPTA for 1 hr and then treated with or without 4 μM TM for 8 hr. Percentages of early apoptotic cells were measured by flow cytometry.

(D) Knockdown of IP3R1 prevents ER stress-induced apoptosis. Cells were treated with 4 μM TM for the indicated times. Protein expressions were analyzed by immunoblotting.

(E) EI24 knockout promotes ER stress-induced, but not death ligand-induced, apoptosis. Control and EI24 knockout HeLa cells were treated with 4 μM TM or with 10 ng/mL TNFα plus 4 μg/mL cycloheximide (TNFα/CHX) for the indicated times. Protein expressions were analyzed by immunoblotting.

(F-H) Reintroduction of full-length EI24, but not the TM34- or C-terminus-deleted mutant (F, G) or the C-terminus alone (H), inhibited ER stress-induced apoptosis in EI24 knockout cells. Cells treated with or without TM were analyzed for apoptosis by flow cytometry (F, H) or by immunoblotting (G). Arrows in (H) denote decreased or increased apoptotic levels.

Data information: (A-C, F, H) N ≈ 10000 cells for one biological replicate. Data are mean ± SEM (n = 3 or 4 biologically independent replicates). ns, no significant difference; ***P < 0.001; two-tailed Student's t-test for (A-C, F), one-tailed Student's t-test for (H). Please see Appendix Figure S4 for information regarding replicates, quantification and statistical evaluation for all biochemical data in this figure.

Expanded View Figure legends
Metabolism-Induced Oxidative Stress and DNA Damage Selectively Trigger Genome Instability in Polyploid Fungal Cells (2019)
Figure 1

Figure 1. Tetraploid C. albicans cells undergo genome instability and cell death on PRE-SPO medium.

(A) Viability of diploid (SC5314) and tetraploid (RBY18) cells grown on PRE-SPO medium at 37°C (n=5, error bars represent S.E.M.).

(B) A tetraploid β-tubulin (TUB2)-GFP, histone H2B (HTB)-RFP strain was cultured on YPD or PRE-SPO medium at 30°C or 37°C, respectively, for 24 h. Cell images indicate maximum intensity Z projections for GFP and RFP channels, and merged images of the GFP/RFP and GFP/RFP/DIC channels.

(C) Assay to monitor for loss of GAL1 in tetraploid strain RBY18. Strain RBY18 is heterozygous for the GAL1 gene and only cells that have lost GAL1 function can grow on 2-DOG medium.

(D) Diploid strain RBY1 and tetraploid strain RBY18 were cultured on YPD or PRE-SPO medium at 30°C or 37°C, respectively, for 7 days and plated onto 2-DOG medium to monitor for GAL1 loss. (*) denotes a statistically significant difference between the indicated sample groups (p<0.05, student's t-test, n=3, error bars represent S.E.M.).

(E) 2-DOGR progeny arising from RBY18 on PRE-SPO medium were DNA stained and their ploidy determined via flow cytometry. Box extends from the 25th to 75th percentiles, center line represents the median value, and upper and lower whiskers represent the maximum and minimum values from the analyzed progeny (n=94).
Metabolism-Induced Oxidative Stress and DNA Damage Selectively Trigger Genome Instability in Polyploid Fungal Cells (2019)
Figure 4

Figure 4. C. albicans cells accumulate ROS and activate oxidative stress responses when cultured on PRE-SPO medium.

(A) Diploid and tetraploid cells were grown on YPD or PRE-SPO medium at 30°C or 37°C, respectively, for 24 h and stained with CellROX Green. Cell images indicate calcofluor white staining (cell wall; blue), GFP, and a merged image of GFP/DAPI/DIC channels. Scale bar = 10 μm.

(B) Diploid and tetraploid cells were grown on YPD or PRE-SPO medium at 30°C or 37°C, respectively, for 24 h, stained with CellROX Green, and signal analyzed via flow cytometry. (*) denotes a significant difference between the indicated groups (p<0.05, student's t-test, n=5, error bars represent S.E.M.).

(C) Diploid and tetraploid cells containing a fluorescently labeled version of the transcription factor Cap1 (Cap1-mNeonGreen) were grown on YPD or PRE-SPO medium at 30°C or 37°C, respectively, for 24 h. Cell images indicate calcofluor white staining (cell wall; blue), GFP, and a merged image of GFP/DAPI/DIC channels. Scale bar = 10 μm.

(D) Quantitation of the percentage of diploid and tetraploid cells with activated Cap1 on YPD or PRE-SPO medium. "n.d." indicates that no cells with activated Cap1 were detected. (*) denotes a significant difference between the indicated groups (p<0.05, student's t-test, n=5, error bars represent S.E.M.).
Metabolism-Induced Oxidative Stress and DNA Damage Selectively Trigger Genome Instability in Polyploid Fungal Cells (2019)
Figure 5

Figure 5. Antioxidants protect against cell death and genome instability in tetraploid cells grown on PRE-SPO medium.

(A) Analysis of cell viability in tetraploid cells after culture on PRE-SPO medium (37°C for 24 h) when supplemented with ascorbic acid, DTT, or glutathione at the indicated concentrations. (*) denotes a significant difference in viability between cells with and without antioxidant (p<0.05, student's t-test, n=5, error bars represent S.E.M.).

(B) Analysis of GAL1 loss (2-DOG resistant colonies) in tetraploid strain RBY18 after growth on PRE-SPO medium with or without antioxidants for 7 days at 37°C. (*) denotes a significant difference between the percentage of 2-DOG(+) colonies with and without antioxidant (p<0.05, student's t-test, n=3, error bars represent S.E.M.).

(C) Tetraploid cells expressing Cap1-mNeonGreen were grown on PRE-SPO medium supplemented with 20 mM ascorbic acid, 1 mM DTT, or 1 mM glutathione for 24 h at 37°C. Cell images were analyzed to determine the percentage of viable cells with activated Cap1. (*) denotes a significant difference in Cap1 activation between cells with and without antioxidant (p<0.05, student's t-test, n=5, error bars represent S.E.M.).

(D) Assay to monitor for loss of GAL1 from tetraploid RBY18 overexpressing the indicated genes. Cells were grown for 7 d at 37°C on PRE-SPO medium in the presence (gene OFF) or absence (gene ON) of doxycycline (DOX). (*) denotes a significant difference between + and - DOX conditions (p<0.05, student's t-test, n=3, error bars represent S.E.M.).
Metabolism-Induced Oxidative Stress and DNA Damage Selectively Trigger Genome Instability in Polyploid Fungal Cells (2019)
Figure 7

Figure 7. DNA damage induces chromosome instability in tetraploid C. albicans cells.

(A) 10-fold dilutions of diploid and tetraploid cells were spot inoculated onto SCD medium with or without the DNA-damaging agents hydroxyurea (HU, 20 mM) or methyl methanesulfonate (MMS, 0.01%). Cells were grown for 48 h at 30°C or 37°C and the plates imaged.

(B) Assay to monitor for loss of GAL1 (2-DOG resistant cells) arising from tetraploid strain RBY18 after culture at 37°C on SCD medium alone, SCD supplemented with HU (20 mM) or MMS (0.01%), or SCD medium with exposure to UV light for five seconds a day for 7 days. (*) denotes a significant difference between control SCD medium and the indicated treatment (p<0.05, student's t-test, n=3, error bars represent S.E.M.).
Metabolism-Induced Oxidative Stress and DNA Damage Selectively Trigger Genome Instability in Polyploid Fungal Cells (2019)
Figure 8

Figure 8. Tetraploid cells exposed to exogenous oxidative stress experience increased DSB formation and chromosome loss.

(A) Tetraploid cells were grown on SCD medium with or without the oxidative-stress inducing agents paraquat (PQT; 600 μg/mL), hydrogen peroxide (H2O2; 2 mM), or piperlongumine (PL; 100 μg/mL) for 24 h at 37°C and stained with CellROX Green. Cell images indicate calcofluor white staining (cell wall; blue), GFP, and a merged image of GFP/DAPI/DIC channels. Scale bar = 10 μm.

(B) Tetraploid cells expressing Cap1-mNeonGreen were grown on SCD medium with or without PQT (600 μg/mL), H2O2 (2 mM), or PL (100 μg/mL) for 24 h at 37°C. Cell images as in (A). Scale bar = 10 μm.

(C) Tetraploid cells expressing a Gam-GFP reporter were cultured on SCD medium with or without PQT (600 μg/mL), H2O2 (2 mM), or PL (100 μg/mL) for 24 h at 37°C and analyzed via fluorescence microscopy. Cell images as in (A). Scale bar = 10 μm.

(D) Flow cytometric analysis of the tetraploid Gam-GFP reporter strain grown on SCD with or without PQT (600 μg/mL), H2O2 (2 mM), or PL (100 μg/mL) for 24 h at 37°C. (*) denotes a significant difference in Gam-GFP values between control and test agent (p<0.05, student's t-test, n=3, error bars represent S.E.M.).

(E) 2-DOG assay to monitor for loss of GAL1 function from tetraploid strain RBY18 after culture on SCD medium with or without PQT (600 μg/mL), H2O2 (2 mM), or PL (100 μg/mL) at 37°C for 7 d. (*) denotes a significant difference between 2-DOG resistant colonies between control and the indicated agent (p<0.05, student's t-test, n=3, error bars represent S.E.M.).

Expanded View Figure Legends
Figure EV1. Transcriptional profiling of meiosis genes on YPD and PRE-SPO media. Heatmap of RNA-seq data for C. albicans homologs to "meiotic" genes in diploid and tetraploid cells on YPD and PRE-SPO media at 24 h, including programmed formation of DNA double-strand breaks (DSBs), recombination, coordination of the meiotic cell cycle, and transcription factors.
Neighboring mutation-mediated enhancement of dengue virus infectivity and spread (2022)
Figure 4

Figure 4. The T226K/G228E compensatory substitutions promote DENV2 infectivity by enhancing the affinity between DENV and C-type lectins (CTLs).

A The structure and interface of the DENV E and DC-SIGN CRD (PDB ID 1SL4) complex. The residues, whose distances to 226T or 228G were less than 10 Å, were selected for analysis. In the interface, the interactions of 309R/312R and 226T/228G were shown in the complex structure. The positively-charged residues in CRD were labelled by red color.

B The 293T cells were transfected with a pcDNA3.1 recombinant plasmid with a human DC-SIGN gene. The cells transfected by an empty pcDNA3.1 plasmid served as negative controls. Thirty-six hours after transfection, the cells were infected with DENV2 (0.01 MOI). The DENV2 viral loads were assessed at 48 h postinfection via qRT-PCR and were normalized against human GAPDH.

C hu-moDCs were infected with 0.01 MOI of DENV2. The infected cells were detected at 48 h postinfection by qRT-PCR.

D The binding affinity between DC-SIGN and purified DENV2 virions measured by SPR.

E The binding affinity between DC-SIGN (R309A/R312A) and purified DENV2 virions measured by SPR.

F The structure and interface of the DENV E and mosGCTL-AAEL011408 CRD complex. The residues, whose distances to 226T were less than 10 Å, were selected for analysis. In the interface, the interaction between 66E and 226T was shown in the structure. The negatively-charged residue in CRD was labelled by blue color.

G The binding affinity between mosGCTL-AAEL011408 and purified DENV2 virions measured by SPR.

Data information: In (B), n = 6 biological replicates. In (C), n = 5 biological replicates. In (B, C), the data are presented as means ± SEM and were analyzed statistically using an unpaired t-test. The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05).