EMBO/soda-nel huggingface dataset:
The Spo13/Meikin Pathway Confines the Onset of Gamete Differentiation to Meiosis II in Yeast (2021)
Figure 9

Figure 9. Regulation of MP assembly and PSM formation at meiosis II

A. Inhibition of Cdk1 at anaphase I. CDC20-mAR and CDC20-mAR cdc28-as1 cells were released from the metaphase I-arrest with CuSO4 at 7 h in SPM (t = 0) and treated with 1NM-PP1 at t = 40 min to inhibit Cdc28-as1 (arrows). Top, time-lapse series from the imaging of Mpc70-GFP, SPBs (Cnm67-RFP), and nuclei (TetR-RFP). Bottom, quantification of meiotic events.

B. cdc20ts-mAR PDMC1-AMA1 PHSL1-CDH1 cells were released from the metaphase I-arrest with CuSO4 (t = 0) at 25°C. Subsequent incubation at 25°C allows progression through meiosis II (left), while a shift to 37°C at t = 50 min causes arrest at metaphase II (right). Mpc70-GFP and the PSM marker RFP-Spo2051-91 were imaged in cells fixed at the indicated times. RFP-Spo2051-91 is expressed from an episomal plasmid (Diamond et al., 2009). Images represent ≥ 90% of cells harboring the plasmid. Scale bar, 2 μm.

C. cdc20ts-mAR PDMC1-AMA1 PHSL1-CDH1 cells containing CDC28 or cdc28-as2 were released from metaphase I at t = 0 and shifted to 37°C at t = 50 min to cause arrest at metaphase II. 1Na-PP1 was added at t = 100 min to inhibit Cdc28-as2. Microtubules (GFP-Tub1), Mpc70-GFP, and RFP-Spo2051-91 were imaged in cells fixed at the indicated times. Images represent ≥90% of cells carrying the RFP-Spo2051-91-plasmid. Scale bar, 2 μm.

D. The regulatory network controlling MP assembly at different stages of meiosis. Green arrows: activation. Dashed, green arrows: latent activation overwhelmed by inhibition. Red, bar-headed lines: inhibition. Items in light grey are inactive or absent. Block arc: SPB outer layer. X: Cdc20-substrate that blocks PSM formation at metaphase II. See text for details.

Data information: data in (B) and (C) are representative of two independent experiments.

EXPANDED VIEW FIGURE LEGENDS
A multifaceted cellular damage repair and prevention pathway promotes high level tolerance to b-lactam antibiotics (2020)
Figure 5

Figure 5 | PenG induces hydrogen peroxide production and the Fur iron starvation response.

(A) Total intracellular hydrogen peroxide (H2O2) levels were measured using AmplexTM Red Hydrogen Peroxide/Peroxidase Assay Kit and normalized to total cellular protein level (measured by QubitTM). Data are average values from three independent experiments, error bars represent standard deviation. Cat, catalase (1μg/ml).

(B). To test PenG-mediated induction of Fur-regulated genes, S1 nuclease mapping assays were performed on the small RNA ryhB, the RyhB target sodB, hutA/B, coding for heme transport systems and vc2212, which encodes a component of a ferric citrate uptake system). Cells were grown to mid-exponential phase followed by exposure to H2O2 or PenG for the indicated duration. Numbers represent expression levels normalized to untreated control (averages from three independent replicates). White demarcation line between time points indicates that these data points were not adjacent in the original experiment but were cropped for presentation purposes. Source data is available as supplemental material.

(C) To confirm the induction of ryhB transcript by PenG treatment, the native ryhB promoter was fused with msfGFP and introduced into WT and ΔvxrA mutant cells. Each strain was cultured until mid-exponential phase, when 1mM EDTA or 100μg/ml PenG were added for the indicated duration. Quantified GFP fluorescence intensities are presented with average values from 3 independent biological replicates (cell, n>500), error bars represent standard deviation. ∗∗∗∗ P<0.0001 (paired t-test).
A multifaceted cellular damage repair and prevention pathway promotes high level tolerance to b-lactam antibiotics (2020)
Figure 8

Figure 8 | PenG exposure is associated with ROS production.

(A) RNA expression profiles of first line ROS defense systems in V. cholerae monitored by S1 nuclease mapping assay. Quantified average values (normalized to WT before PenG exposure) from three independent experiments are presented under the signals of each gene. White demarcation line between time points indicates that these data points were not adjacent in the original experiment but were cropped for presentation purposes. Source data is available as supplemental material. Note that the loading control is the same as in Fig. 2D, as these S1 assays were done on the same RNA preparations.

(B and C) Quantification of the SOD and catalase activity values from the gel shown in Appendix Figure S9. Data are average values from three independent experiments normalized to untreated wild type.

(D) Detection of protein oxidation after PenG treatment. To measure total cellular protein oxidation, OxyBlot Protein Oxidation Detection Kit (Sigma-Aldrich) was used. For positive protein oxidation control, oxidized standard marker (OXM) was used. Both DNP-derivatized or nonderivatized (negative control) protein samples were separated by polyacrylamide gel electrophoresis followed by Western blotting with-DNP specific antibody.

(E) Time-dependent killing of respiratory chain mutants (∆nqrA, encoding NADH dehydrogenase and ∆ubiA, encoding an early step of ubiquinone synthesis) in the presence of PenG (100 µg/ml, 10xMIC). All strains were grown in LB 0.2 % glucose.

(F) Growth-phase dependent killing assay of ROS detoxification systems (∆fur, ∆sodB, and ∆oxyR1∆katG∆katB). At designated time-points, a 5 ml aliquot was withdrawn and exposed to 100 µg/ml PenG for 3 h. CFU/mL for each time point was assessed before (solid lines) and after addition of PenG (dotted lines).

Data information: Data are mean of three independent biological replicates and error bars represent standard deviation.
Homeostatic and pathogenic roles of GM3 ganglioside molecular species in TLR4 signaling in obesity (2020)
Figure 2

Figure 2 Alterations of relative abundance of GM3 species are involved in disease progression and chronic inflammation.

(A) Heat map analysis of serum GM3 species in various pathological phases: control (n=24), VFA (n=38), lipidemia (n=28), glycemia (n=15), and lipidemia + glycemia (n=17). Colors indicate fold change average of each species relative to control (defined as 1), as shown in key at right. Order of pathological phases corresponds to increments of HOMA-IR and serum CRP.

(B-D) Properties of various GM3 species as a function of pathological phases: control (n=24), VFA (n=38), lipidemia (n=28), glycemia (n=15), and lipidemia + glycemia (n=17). Data shown are relative abundances of total LCFA species (16:0, 18:0, 20:0) (B), total VLCFA species (22:0, 23:0, 24:0, h24:0) (C), and total unsaturated VLCFA species (22:1, 24:1, h24:1) (D) relative to total of ten major GM3 species (defined as 1) in each subject.

(E-H) Properties of various GM3 species as a function of BMI: LCFA-GM3 (E), VLCFA-GM3 (F), unsaturated VLCFA-GM3 (G), and α-hydroxyVLCFA-GM3 (h24:0) (H). Colors indicate disease severity: light blue, no abnormal scores (n=25); orange, early-phase obesity (n=74); purple, severe obesity (n=23).

(I, J) Spearman's correlations for GM3 h24:0 vs. ALT (I) and vs. HOMA-IR (J).

(K) Plots of α-hydroxylation rate (h24:0/24:0) vs. serum CRP. Colors indicate range of CRP value (mg/dL): light blue, 0.01-0.02 (n=21); orange, 0.03-0.09 (n=56); gray, 0.10-0.29 (n=29); red, 0.3-1.0 (diagnostically abnormal; n=15).

(L) Association between serum GM3 species and progression of metabolic disorders (schematic).

Data information: Data shown are individual values and mean ± SD, analyzed by two-tailed unpaired t-test with Bonferroni's correction. *p< 0.05, **p< 0.01, ***p< 0.001 for comparisons between indicated groups.
Homeostatic and pathogenic roles of GM3 ganglioside molecular species in TLR4 signaling in obesity (2020)
Figure 4

Figure 4 Positive- and negative regulation of innate immune responses by serum GM3 species in an acyl-chain dependent manner.

(A) Profiling of bioactivities of serum GM3 species in LPS-mediated monocyte activation (schematic).

(B, C) GM3-mediated enhancement and inhibition of proinflammatory cytokine production from LPS-stimulated monocytes (LPS: 0.06, 0.13, 0.25 ng/mL). TNF-α (B) and IL-6 (C) production in culture supernatant was measured by ELISA.

(D, E) Co-stimulation of monocytes by LPS plus GM3 species or complex ganglioside species (1.5, 3.0, 4.5 µM). TNF-α (D) and IL-6 (E) production were shown in heat maps.

(F) Co-stimulation of monocytes by LPS plus GM3 species or precursor GSL species. TNF-α, IL-6, and IL-12/23 production were shown in heat maps.

(G) Inhibitory effect of LCFA and unsaturated VLCFA-GM3 on LVCFA-GM3 species. IL-6 production in culture supernatant was measured by ELISA.

Data information: Data shown are mean ± SD (n=3), analyzed by Tukey's multiple comparison test. **p< 0.01 for comparisons between indicated groups.
Homeostatic and pathogenic roles of GM3 ganglioside molecular species in TLR4 signaling in obesity (2020)
Figure 5

Figure 5 VLCFA-GM3 species synergistically and selectively control human TLR4/MD-2 activation.

(A) Co-stimulation of human monocytes by GM3 species plus various TLR ligands: LPS (0.13 ng/mL), TLR4/MD-2, Pam3CSK4 (0.5 µg/mL), TLR1/2, Flagellin (50 ng/mL), TLR5, R848 (0.5 µg/mL), TLR7/8, MALP-2 (1.0 ng/mL), TLR2/6. IL-6 production in culture supernatant was quantified by ELISA (shown in a heat map).

(B) Co-stimulation of monocytes by GM3 species (16:0, 24:0, 24:1) plus synthetic TLR4 ligands LA506 (15 ng/mL), LA505 (150 ng/mL), or LA504 (150 ng/mL).

(C, D) Production of IL-6 (C) and TNF-α (D) in culture supernatant following co-stimulation of monocytes by GM3 species plus human HMGB1.

(E) Overexpression of hTLR4, hTLR4/hMD-2, hTLR4 (P714H) /MD-2 in HEK293T cells, and co-stimulation by GM3 22:0 with LPS (5 ng/mL). TLR4 activation was monitored by NF-κB luciferase reporter assay (termed "Relative Luc Activity" on Y-axis).

(F, G) Co-stimulation of hTLR4/hMD-2 by GM3 species plus LPS (5 ng/mL) (F) and further addition of soluble human CD14 (1 µg/mL) (G).

(H) Stimulation of Mal-overexpressing HEK293T cells by GM3 species.

(I) Co-stimulation of hTLR4/hMD-2 by LPS (5 ng/mL) plus various mixtures of GM3 species.

(J) Regulation of hTLR4/hMD-2 by GM3-species balance (schematic).

Data information: Data shown are mean ± SD (A-D and F-I, n=3; E, n=4) analyzed by Tukey's multiple comparison test. *p< 0.05, **p< 0.01 for comparisons between indicated groups.
Homeostatic and pathogenic roles of GM3 ganglioside molecular species in TLR4 signaling in obesity (2020)
Figure 6

Figure 6 GM3 species selectively modulate mouse TLR4/MD-2 signaling.

(A) Co-stimulation of RAW macrophages by GM3 species plus various TLR ligands: LPS (0.5, 1.0 ng/mL), bovine thymus HMGB1 (0.25 µg/mL), Pam3CSK4 (50 ng/mL), Poly I:C (10 µg/mL), R848 (4 ng/mL), and CpG type-B (20 nM). TNF-α production in culture supernatant was quantified by ELISA.

(B) Co-stimulation of RAW macrophages by low- and high-dose LPS (0, 5, 50 ng/mL) plus GM3 22:0 (5 µM). Time course of TNF-α production in culture supernatant was quantified by ELISA.

(C) Co-stimulation of BMDMs from C3H/HeN or C3H/HeJ mice by GM3 species plus LPS (0.5 ng/mL), and TNF-α production in culture supernatant.

(d) Co-stimulation of mTLR4/mMD-2-expressing HEK293T cells by GM3 species plus LPS (2.5 ng/mL), and further addition of soluble mouse CD14-Fc fusion protein (1 µg/mL).

(E, F) Co-stimulation of BMDMs from C3H/HeN mice by LPS plus GM3 species and complex ganglioside species (E; 2.5, 5.0, 10 µM), or by LPS plus GM3 species and precursor GSL species (F; 2.5, 5.0, 10 µM) (shown in heat maps).

(G) Structure-bioactivity relationships of GM3 species with human or mouse TLR4.

Data information: Data shown are mean ± SD (A and B, n=3; C, E, and F, n=4; D, n=6) analyzed by Tukey's multiple comparison test (A, C, and D) or two-tailed unpaired t-test (B). *p< 0.05, **p< 0.01 for comparison with stimulation by TLR-ligand without GM3 species.
Homeostatic and pathogenic roles of GM3 ganglioside molecular species in TLR4 signaling in obesity (2020)
Figure 7

Figure 7 α-hydroxyVLCFA-GM3 species in adipose tissue showed increased abundance in obesity.

(A) GM3 molecular species of 6-wk-old control C57/BL6 mice and ob/ob mice were analyzed respectively by LC-MS/MS (n=3).

(B) GM3 molecular species of normal-diet (ND) and high-fat-diet (HFD) C57/BL6 mice were analyzed by LC-MS/MS (n=4).

(C) TLC analysis of acidic GSL fraction (equivalent to 0.1 mg protein) from epididymal fat pads of C3H/HeN (A) and C3H/HeJ mice (B) on ND or HFD for 8 wk.

(D) GM3 molecular species of C3H/HeN mice (ND, HFD) and C3H/HeJ mice (ND, HFD) were analyzed by LC-MS/MS (n=4).

(E, F) Body weight and weight gain (e), blood glucose and epididymal fat pad weight (f) of GM3S -/+ mice (ND, n=4; HFD, n=5) and GM3S -/- mice (HFD, n=4).

(G) Comparison of increased GM3 species in human serum and mouse adipose tissue.

(H) Feedback loop mediated by TLR4 and GM3 species, promoting disease progression (schematic).

Data information: Data shown are mean ± SD analyzed by two-tailed unpaired t-test (A, B) or by Tukey's multiple comparison test (D). *p< 0.05, **p< 0.01 for comparisons between indicated groups.
Homeostatic and pathogenic roles of GM3 ganglioside molecular species in TLR4 signaling in obesity (2020)
Figure 8

Figure 8 GM3 recognition by TLR4/MD-2 induces receptor oligomerization.

(A, B) Surface electrostatic potentials of reported crystal structure of human TLR4/MD-2/LPS complex (3FXI), and mapping of putative GM3-binding pocket (A). Candidate basic residues and a hydrophobic pocket recognizing sialic acid and ceramide structure of GM3 are indicated (schematic) (B).

(C, D) Alanine scanning for basic residues involved in signal transduction via VLCFA-GM3 (n=5) (C), and combinations of effective mutations (n=6) (D). Signal transduction was monitored by NF-κB reporter assay.

(E-G) Comparative inhibitory effects of GM3 16:0 on mTLR4/mMD-2 (E), mTLR4/hMD-2 (domain-swapped complex) (F), and hTLR4/hMD-2 (G) (n=3).

(H) Cross-linked SDS-PAGE analysis of recombinant mTLR4 (extracellular domain)/mMD-2 complexed with GM3 18:0, GM3 species enhancing mTLR4 activation.

Data information: Data shown are mean ± SD analyzed by Tukey's multiple comparison test. *p< 0.05, **p< 0.01 for comparisons between indicated groups.
MITOL prevents ER stress-induced apoptosis by IRE1α ubiquitylation at ER-mitochondria contact sites (2019)
Figure 1

Figure 1. Anti-apoptotic role of MITOL against ER stress

(A) MITOL-KO MEFs was vulnerable to ER stress. Control MEFs (MITOLF/F) and MITOL-KO MEFs (MITOL-/-) were treated with DMSO as control, 0.8 µM thapsigargin (Tg), 0.7 µg/mL tunicamycin (Tu) or 1.2 µg/mL brefeldin A (Br) for 24 hours. All experiments using Tu below were performed at concentration of 0.7 µg/mL. The relative cell viability shows a ratio between the survival of MEFs in the absence and presence of ER stress inducers. Viable cells were detected by cell viability assay using cell counting kit-8. Error bars represent SD (n=6). n is the number of independent experiments. ***p<0.001 (Student's t test).

(B) MITOL KO increased ER stress-induced apoptosis. MEFs were treated with indicated ER stress inducers at same concentration as Figure 1A for 18 hours and stained with Annexin V-FITC for the detection of apoptosis using flow cytometry. Error bars represent SD (n=3). *p<0.05 (Student's t test).

(C) MITOL re-expression in MITOL-KO MEFs rescued ER stress vulnerability. MEFs were transfected with MITOL-coding vector (WT), MITOL C65/68S-coding vector (CS), or empty vector (Vec) 24 hours prior to Tu treatment for 18 hours, followed by immunoblotting with indicated antibodies. Cleaved caspase-3 (cC3) and cleaved PARP (cPARP) were used as apoptosis markers. Error bars represent ± SD (n=3). **p<0.01, ***p<0.001 (Student's t test).

(D) MITOL KO enhanced ER stress-induced Bax translocation and cytochrome c release. Mitochondria or cytosol fraction was isolated from MEFs treated with Tu for 18 hours, followed by immunoblotting with indicated antibodies. Error bars represent SD (n=3). **p<0.01, ***p<0.001 (Student's t test).

(E) MITOL KO induced excessive mitochondrial depolarization by ER stress. MEFs transfected with indicated vectors were treated with Tu for 18 hours and then stained with TMRM, followed by flow cytometric analysis. Error bars represent SD (n=3). *p<0.05 (Student's t test).
MITOL prevents ER stress-induced apoptosis by IRE1α ubiquitylation at ER-mitochondria contact sites (2019)
Figure 2

Figure 2. MITOL KO enhances IRE1α-dependent apoptosis under ER stress

(A, B) IRE1α inhibition compensated MITOL-KO MEFs for vulnerability to ER stress. MEFs were transfected with scramble or siIRE1α (#1, #2). After 24 hours, these MEFs were exposed with Tu for 24 hours (A). MEFs were treated with 4µ8C (25 µM) for 2 hours prior to Tu treatment for 24 hours (B), followed by immunoblotting with indicated antibodies. Error bars represent SD (n=3). **p<0.01 (Student's t test).

(C) MITOL KO did not affect the autophosphorylation of IRE1α. MEFs were exposed with Tu for indicated periods and phosphorylated IRE1α was detected by immunoblotting after Phos-tag SDS-PAGE.

(D-G) MITOL KO enhanced RIDD and xbp1 splicing. MEFs were incubated with Tu for 4 hours. Expression levels of various UPR associated genes were determined by qRT-PCR (D, E, G). MEFs were transfected with either miR-17 or miR-34 luciferase reporter vector 24 hours prior to Tu treatment for 4 hours, followed by luciferase reporter assay (F). Error bars represent SD (n=3). *p<0.05, **p<0.01, ***p<0.001 (Student's t test).

(H) IRE1α-mediated JNK phosphorylation was prolonged by MITOL KO. MEFs were incubated with Tu for indicated periods and phosphorylated JNK was detected by immunoblotting with indicated antibodies. Error bars represent SD (n=3). **p<0.01 (Student's t test).

(I) JNK inhibition reduced ER stress-induced apoptosis of MITOL-KO MEFs. MEFs were treated with 20 µM JNK inhibitor-II (JNK inh) 2 hours prior to Tu treatment for 24 hours, followed by immunoblotting with indicated antibodies. Error bars represent SD (n=3). **p<0.01 (Student's t test).
MITOL prevents ER stress-induced apoptosis by IRE1α ubiquitylation at ER-mitochondria contact sites (2019)
Figure 3

Figure 3. MITOL KO stabilizes IRE1α oligomers

(A, B) MITOL KO facilitated IRE1α oligomerization. MEFs were transfected with IRE1α-GFP expression vector for 24 hours prior to Tu treatment for indicated periods (A). The right panels show 5-fold magnification images of the boxed regions. Percentages of cells with IRE1α-foci were calculated from 100 cells by visual inspection in each independent experiment (A). To evaluate the oligomerization level of endogenous IRE1α, cells without any transfection were solubilized and separated by sucrose density-gradient centrifugation, followed by immunoblotting with anti-IRE1α antibody (B). Scale bar represents 10 µm. Error bars represent SD (n=4). *p<0.05, **p<0.01 (Student's t test).

(C) IRE1α oligomers were stabilized by MITOL KO. MEFs transfected with IRE1α-GFP were treated with Tu. After 4 hours, these cells were washed with PBS and subsequently re-fed with fresh media for indicated periods. The right panels show 5-fold magnification images of the boxed regions. Percentages of cells with IRE1α-foci were calculated from 100 cells by visual inspection at indicated periods after wash out of Tu in each independent experiment. Scale bar represents 10 µm. Error bars represent SD (n=3). *p<0.05, **p<0.01, ***p<0.001 (Student's t test).

(D) Sustained IRE1α autophosphorylation after wash out of Tu in MITOL-KO MEFs. MEFs were treated with Tu for 4 hours and washed with PBS and re-fed with fresh media for indicated periods, following by immunoblotting anti-IRE1α antibody after Phos-tag SDS-PAGE. Error bars represent SD (n=3). *p<0.05 (Student's t test).

(E) MITOL deletion promoted the interaction between IRE1α and BIM under ER stress. MITOL-depleted HEK293 cells (KO) were generated using CRISPR-Cas9 system. Cells were co-transfected with indicated vectors 24 hours prior to Tu treatment for 12 hours. Cells were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with indicated antibodies. Error bars represent SD (n=3). ***p<0.001 (Student's t test).
MITOL prevents ER stress-induced apoptosis by IRE1α ubiquitylation at ER-mitochondria contact sites (2019)
Figure 4

Figure 4. MITOL interacts with and ubiquitylates IRE1α

(A-D) Interaction between MITOL and IRE1α. Lysates of HEK293 cells were subjected to immunoprecipitation with anti-IRE1α antibody, followed by immunoblotting with indicated antibodies (A). Lysates of HEK293 cells co-transfected with indicated vectors were subjected to immunoprecipitation, followed by immunoblotting with indicated antibodies (B). GST-pull-down assay of IRE1α or MITOL was performed on lysates of MEFs, followed by immunoblotting with indicated antibodies (C, D). N-ter: 1-90 aa of MITOL, Loop: 158-210 aa of MITOL, C-ter: 241-278 aa of MITOL, 465-977 aa of IRE1α.

(E-G) MITOL ubiquitylated IRE1α. Lysates of HEK293 cells (E, F) or MEFs (G) transfected with indicated vectors, were immunoprecipitated with indicated antibody, followed by immunoblotting with indicated antibodies.

(H-J) MITOL adds lysine 63-linked polyubiquitin chains to IRE1α. Lysates of HEK293 cells transfected with indicated vectors were immunoprecipitated with indicated antibody. IRE1α ubiquitylation was detected as similar to (E). WT: wild-type ubiquitin. K48R, K63R: ubiquitin mutants containing lysine (K) to arginine (R) substitution at position 48 or 63, respectively. K all R: ubiquitin mutant containing all lysine to arginine substitutions. R63K: ubiquitin mutants containing only one lysine intact at position 63.
MITOL prevents ER stress-induced apoptosis by IRE1α ubiquitylation at ER-mitochondria contact sites (2019)
Figure 5

Figure 5. IRE1α K481R induces apoptosis via abnormal clustering of IRE1α and RIDD

(A) MITOL added polyubiquitin chains to K481 of IRE1α. Lysates of HEK293 cells transfected with each lysine mutant of IRE1α and indicated vectors were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with indicated antibodies. 481: K481R, 545: K545R, 568: K568R.

(B) IRE1α K481R interacted with MITOL. Lysates of HEK293 cells transfected with indicated vectors were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with indicated antibodies.

(C, D) Overexpression of IRE1α K481R induced apoptosis. MEFs were transfected with indicated vectors. After 24 hours, these cells were stained with Annexin V-FITC (C) or subjected by immunoblotting with indicated antibodies (D). Error bars represent SD (n=3). *p<0.05 (Student's t test).

(E, F) Enhanced RIDD activity in IRE1α K481R. MEFs were transfected with indicated vectors 24 hours prior to analysis (E, F). The RIDD activity was measured by qRT-PCR. Error bars represent SD (n=3). *p<0.05, **p<0.01 (Student's t test).

(G) Hyper-oligomerization of IRE1α K481R. MEFs were transfected with IRE1α-GFP or IRE1α K481R-GFP. After 24 hours, these cells were exposed with Tu for indicated periods and GFP signals were observed. The right panels show 5.5-fold magnification images of the boxed regions. Percentages of cells with IRE1α-foci were calculated from 100 cells by visual inspection in each independent experiment. Scale bar represents 10 µm. Error bars represent SD (n=3). *p<0.05, **p<0.01 (Student's t test).

(H) Increased interaction of IRE1α K481R with BIM. Cells transfected with indicated vectors 24 hours prior to immunoprecipitation, followed by immunoblotting with indicated antibodies.