EMBO/soda-nel huggingface dataset:
Phosphorylation switches protein disulfide isomerase activity to maintain proteostasis and attenuate ER stress (2020)
Figure 3

Figure 3. Phosphorylation of PDI depends on the retention of Fam20C in the ER.

A Fluorescent photomicrographs of HepG2 cells co-transfected with PDI-mEmerald-KDEL (green) and Fam20C-mApple (red). The cells were treated with DMSO (Upper) or 5 μM Tg (Middle) for 30 min. After Tg treatment, the cells were washed with PBS and then returned to culture medium for 60 min (Lower). GM130 was immunostained (blue) as a cis-Golgi marker. Scale bar = 10 μm.

B Pearson correlation coefficient calculated from (A). Data were shown as mean ± SEM of N > 80 cells from four independent experiments. ***p < 0.001 (one-way ANOVA, the post hoc Tukey's HSD test).

C Detection of pS357-PDI and UPR signaling in HepG2 cells after Tg treatment and washout for indicated times.

D HepG2 cells were treated with 25 ng/μl CHX for indicated times and analyzed by immunoblotting for pS357-PDI and UPR signaling. 5 μM Tg or DMSO was added 30 min before cell harvest.

E Fluorescent photomicrographs of HepG2 cells co-transfected with PDI-mEmerald-KDEL (green) and Fam20C-mApple (red) pretreated with 25 ng/μl CHX for 7.5 h, followed by introduction of DMSO (Upper) or 5 μM Tg (Lower) for 30 min, respectively. GM130 was immunostained (blue) as a cis-Golgi marker. Scale bar = 10 μm.

F Fluorescent photomicrographs of HepG2 cells co-transfected with PDI-mEmerald-KDEL (green) and Fam20C-mApple-KDEL (red) treated with DMSO (Upper) or 5 μM Tg (Lower) for 30 min, respectively. Scale bar = 10 μm.

G HepG2 cells were transfected with empty vector (-), FLAG-tagged Fam20C or Fam20C-KDEL followed by introduction without or with 5 μM Tg for 30 min, respectively. PDI phosphorylation was detected by protein immunoblotting on both regular and phostag gels. p-PDI percentage was quantified based on band intensities on phostag gels.
Phosphorylation switches protein disulfide isomerase activity to maintain proteostasis and attenuate ER stress (2020)
Figure 4

Figure 4. Phosphorylation of PDI induces its functional switch from an oxidoreductase to a molecular chaperone.

A The last snapshot of the molecular dynamic simulation of pS357-PDI. PDI is composed of four thioredoxin-like domains a (orange), b (green), b' (blue), x-linker (red) and a' (cyan). The initial structure of PDI (PDB code 4EL1) is shown in gray. pSer357 and Ser357 are shown in stick.

B The time course of the distance between the geometrical centers of domains a and a′ and the angle among domains b, b′ and a′ from the simulation of pS357-PDI (red) and PDI (black) (Yang et al., 2014).

C (Left) ANS fluorescence spectra of PDI WT and ­­S357E. Controls with ANS alone was shown. A.U., arbitrary units. (Right) Enhancement factor were quantified.

D Limited proteolysis of PDI WT (Left) and S357E (Right) proteins by proteinase K (Pro K) for indicated times. Main digested fragments were indicated on the right margin. Band abb'x* is a digested product with cleavage after Met356 in the x-linker region (Wang et al., 2010).

E shCtrl and shFAM20C HepG2 cells were treated without or with 5 μM Tg for 30 min, and in some aliquots of cells Tg was removed for 30 min. Cell lysates were subjected to proteinase K digestion for 3 min and immunoblotting analysis.

F HepG2 cells expressing HA-tagged PDI and FLAG-tagged Fam20C with different ratios of plasmid concentrations were subjected to proteinase K digestion for 21 min and immunoblotting analysis.

G-J Chaperone activities of PDI WT and S357E (G, H) or phosphorylated PDI WT and S357A by Fam20C for indicated times (I, J). Denatured and reduced GAPDH (G, I) and rhodanese (H, J) were used as substrates, respectively. The chaperone activity of PDI WT was taken as 100%.

K, M Reductase activities of PDI WT and S357E (K) or phosphorylated PDI WT and S357A by Fam20C for indicated times (M) measured by insulin reduction assay. The activity of PDI WT was taken as 100%.

L, N Isomerase activities of PDI WT and S357E (L) or phosphorylated PDI WT and S357A by Fam20C for indicated times (N) measured by scrambled RNase A reactivation assay. The activity of PDI WT was taken as 100%.

O Schematic diagram illustrating phosphorylation of PDI Ser357 by Fam20C induces its functional switch from a 'foldase' to a 'holdase'.

Data information: All data were shown as mean ± SEM of three independent experiments. In (C, G, H, K, L), **p < 0.01, ***p < 0.001 (two-tailed Student's t-test). In (I, J, M, N), ***p < 0.001 (two-way ANOVA, the post hoc Tukey's HSD test).
Phosphorylation switches protein disulfide isomerase activity to maintain proteostasis and attenuate ER stress (2020)
Figure 5

Figure 5. Phosphorylation of PDI Ser357 safeguards ER proteostasis.

A (Upper) Schematic representation of ER-located fluorogenic probe Halo-tag K73T/L172Q mutant (AgHaloER). SP: signal peptide. The carboxyl-terminal KDEL motif is the ER retention sequence. (Lower) Diagram of AgHaloER that detects ER proteostasis in live cells.

B Fluorescent photomicrographs of HepG2 cells expressing AgHaloER (TMR-labeled, red). Endogenous PDI was immunostained (green) as an ER marker. Scale bar = 10 μm.

C Confocal live cell imaging of HepG2 cells expressing AgHaloER labeled by P1 (green) and TMR (red), followed by introduction of DMSO or 5 μM Tg for 30 min. Scale bar = 10 μm.

D Confocal live cell imaging of WT and PDI KO HepG2 cells co-transfected with AgHaloER labeled by P1 (green) and TMR (red) and empty vector (-), PDI WT, S357E or S357A. Scale bar = 10 μm.

E Confocal live cell imaging of shCtrl and shFAM20C HepG2 cells expressing AgHaloER labeled by P1 (green) and TMR (red). Scale bar = 10 μm.

F (Upper) Cell viabilities of WT and PDI KO HepG2 cells co-transfected with AgHaloER and empty vector (-), PDI WT, S357E or S357A. Cells were treated by 5 μM Tg for indicated times and visualized by crystal violet staining. (Lower) Quantification of surviving fraction of cells. Data were shown as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 versus WT HepG2 (two-way ANOVA, the post hoc Tukey's HSD test).
Phosphorylation switches protein disulfide isomerase activity to maintain proteostasis and attenuate ER stress (2020)
Figure 6

Figure 6. Phosphorylated PDI interacts with IRE1α and attenuates IRE1α signaling during ER stress.

A (Left) Detection of XBP1 mRNA splicing in WT and PDI KO HepG2 cells treated with or without 5 μM Tg for 1 h. PDI KO was verified by protein immunoblotting. (Right) Quantification of XBP1 mRNA splicing levels. Data were shown as mean ± SEM of three independent experiments. *p < 0.05 (two-tailed Student's t-test).

B (Left) Detection of XBP1 mRNA splicing in PDI KO HepG2 cells expressing PDI WT or its mutants treated with or without 5 μM Tg for 1 h. PDI expression levels were shown by protein immunoblotting. (Right) Quantification of XBP1 mRNA splicing levels. Data were shown as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 (one-way ANOVA, the post hoc Tukey's HSD test).

C Detection of IRE1α, PERK and ATF6 activation by immunoblotting in PDI KO HepG2 cells expressing PDI S537A or S357E treated with 5 μM Tg for 30 min. Asterisk indicates unspecific background band.

D HepG2 cells were treated without or with 5 μM Tg for 1 h. Endogenous IRE1α-immunoprecipitates were analyzed by PDI and BiP immunoblotting.

E HepG2 cells expressing FLAG-tagged IRE1α were treated without or with 5 μM Tg for 1 h. An aliquot of Tg-treated cells was then washed with culture medium and chased for 4 h. FLAG-immunoprecipitates were analyzed by PDI and BiP immunoblotting.

F shCtrl and shFAM20C HepG2 cells were treated without or with 5 μM Tg for 1 h. Endogenous IRE1α-immunoprecipitates were analyzed by PDI and BiP immunoblotting.

G Co-immunoprecipitation of FLAG-tagged IRE1α and HA-tagged PDI in HepG2 cells expressing V5-tagged Fam20C WT or DA.

H Co-immunoprecipitation of FLAG-tagged IRE1α, HA-tagged PDI WT or S357A in HepG2 cells expressing V5-tagged Fam20C.

I Co-immunoprecipitation of HA-tagged PDI and FLAG-tagged IRE1αNLD in HepG2 cells.

J Recombinant GST-PDI was phosphorylated by Fam20C protein for 2 h, and then used to pulldown His-IRE1αNLD protein, as visualized by Coomassie blue staining. Successful phosphorylation of GST-PDI was verified by pS357-PDI immunoblotting.

K Co-immunoprecipitation of FLAG-tagged IRE1αNLD WT or its mutant C109/148/332S with HA-tagged PDI (Left) or MYC-tagged P5 (Right) in HepG2 cells.

L Co-immunoprecipitation of FLAG-tagged IRE1α and HA-tagged PDI WT or its binding mutant W128I/L403W in HepG2 cells expressing V5-tagged Fam20C or not.
Phosphorylation switches protein disulfide isomerase activity to maintain proteostasis and attenuate ER stress (2020)
Figure 7

Figure 7. Phosphorylation of PDI protects against ER stress-induced liver damage in vivo.

A Schematic diagram illustrating the generation of PdiS359A/S359A knockin (KI) mice by CRISPR/Cas9 mediated genome editing.

B Sanger sequencing results of Pdi+/+ (WT) and PdiS359A/S359A (KI) mice.

C (Left) Detection of Xbp1 mRNA splicing in mice liver after injection of Tm (50 ng/g, i.p.) for indicated times. Each lane represents independent animals. (Right) Quantification of Xbp1 mRNA splicing levels at indicated times.

D, E Detection of Xbp1 mRNA splicing in primary hepatocytes isolated from WT and KI mice treated with 0.2 μM Tg for 2 h (D) or 0.5 μg/ml Tm for 2 h (E).

F-K Detection of Bip (F), Pdi (G), Erdj4 (H), Edem1 (I), Il6 (J), Chop (K) mRNA levels by real-time PCR in the liver of the same animals as in (C) after 24 h of Tm injection.

L Detection of serum alanine transaminase (ALT) levels of the same animals as in (C) after 24 h of Tm injection.

M Hematoxylin and eosin staining of liver tissue the same animals as in (C) after 24 h of Tm injection (magnification × 400). Immune cell infiltration was indicated with a black arrow. Scale bar = 50 μm. Representative images were shown from five animals per group analyzed.

N (Left) TUNEL assay of the same liver as in (M). Scale bar = 40 μm; Zoom box, scale bar = 5 μm. (Right) Quantification of apoptotic cells. 10 random fields were counted for each TUNEL-stained tissue sample.

O Proposed model for the role of p-PDI. (Left) Under basal conditions, PDI is responsible for the catalysis of oxidative protein folding, and IRE1α is maintained by BiP chaperone in a monomeric inactive state. (Middle) Upon ER stress, Fam20C kinase is retained in the ER for rapid phosphorylation of PDI. Phosphorylated Ser357 induces an open conformation of PDI and turns it from a 'foldase' to a 'holdase', which is critical for preventing protein misfolding in the ER. Meanwhile, BiP dissociates and IRE1α is activated for XBP1 splicing. p-PDI directly binds to IRE1α to control the amplitude of IRE1α activity for adaptive UPR signaling. (Right) In PDI S357A mutant, protein aggregates form under ER stress due to incapable phosphorylation of PDI, and sustained ER stress induces hyperactivation of IRE1α, which leads to terminal UPR and apoptosis.

Data information: All data were shown as mean ± SEM from five biological replicates. In (C, N), *p < 0.05, ***p < 0.001 (two-tailed Student's t-test). In (F-L), *p < 0.05, ***p < 0.001 (two-way ANOVA, the post hoc Tukey's HSD test).

Expanded View Figure Legends
Nucleolar c-Myc recruitment by a Vibrio T3SS effector promotes host cell proliferation and bacterial virulence (2020)
Figure 1

Fig. 1 VgpA is a T3SS2 effector that localizes to the nucleolus.

(A) Detection of VgpA in supernatants from indicated strains by immunoblotting (IB) with anti-VgpA antisera.

(B) T3SS2 translocation of VgpA into Caco-2 cells using CyaA-based assay; VopV is a known T3SS2 effector. Error bars represent mean ± standard deviation (n=3 biologically independent experiments).

(C) Localization of translocated VgpA (upper panel) and VgpAL10A (lower panel) during infection.
Nucleolar c-Myc recruitment by a Vibrio T3SS effector promotes host cell proliferation and bacterial virulence (2020)
Figure 2

Fig. 2 Identification of EBP2 as a binding partner of VgpA.

(A) Silver-stained PAGE gel of proteins from a nuclear extract that interact with either GST-VgpA or GST glutathione agarose. The protein bound by GST-VgpA (red arrow), but not GST, was identified as EBP2 by mass spectrometry; GST-VgpA is labeled with a blue arrow.

(B) Western blot using anti-EBP2 as antibody confirmed that EBP2 binds VgpA.

(C) Interaction of VgpA or VgpA mutants (VgpAΔ10 and VgpAL10A) with EBP2.

(D) Localization of VgpA (upper panel) or in VgpAL10A (lower panel) in HeLa cells after transfection. EBP2 was detected with anti-EBP2 antibody (red), VgpA and VgpAL10A were detected with anti-VgpA sera (green) and nuclei were stained with DAPI (blue). White arrows indicate co-localization of EBP2 and VgpA (upper panel). VgpAL10A localizes primarily to the nucleoplasm (white arrow, lower panel).
Nucleolar c-Myc recruitment by a Vibrio T3SS effector promotes host cell proliferation and bacterial virulence (2020)
Figure 3

Fig. 3 The VgpA-EBP2 interaction promotes Caco-2 cell proliferation.

(A) Fluorescence micrographs of uninfected Caco-2 cells or Caco-2 cells infected with the indicated V. parahaemolyticus strains. Cells were stained with DAPI (blue) and anti-BrdU antibody (green).

(B) Fluorescence micrographs of Caco-2 cells transfected with siRNA targeting EBP2 (siEBP2) or control siRNA (siNC) and subsequently infected with vopZ'ΔvscN1. Cells were stained with anti-BrdU antibody (green) and DAPI (blue).

(C) The percentage of BrdU-positive cells (green) among the total cells (green + blue) was analyzed for 100 cells in three experiments. Error bars represent mean ± standard deviation (n=3 biologically independent experiments). One-way analysis of variance (ANOVA) was used for statistical analysis. *P<0.05 (when compared to the uninfected Caco-2 cells).

(D) The percentage of BrdU+ cells was analyzed as above for cells treated with siNC or siEBP2. Error bars represent mean ± standard deviation (n=3 biologically independent experiments). Two-way ANOVA was used for statistical analysis. *P<0.05 (when compared to siNC-transfected cells that are infected with vopZ'ΔvscN1).

(E) Immunoblot of EBP2 and Actin in Caco-2 cells transfected with siEBP2 or siNC.
Nucleolar c-Myc recruitment by a Vibrio T3SS effector promotes host cell proliferation and bacterial virulence (2020)
Figure 4

Fig. 4 The VgpA-EBP2 interaction promotes EBP2-c-Myc interaction and c-Myc nucleolar localization.

(A) Localization of c-Myc in uninfected Caco-2 cells (first row), Caco-2 cells infected with V. parahaemolyticus vopZ'ΔvscN1 (wild type VgpA, second row), Caco-2 cells infected with vopZ'ΔvscN1vgpA' (third row), Caco-2 cells infected with vopZ'ΔvscN1vgpA':pvgpA (fourth row) and Caco-2 cells infected with vopZ'ΔvscN1vgpA':pvgpAL10A (fifth row). Fixed cells were stained with DAPI (blue), and anti-EBP2 (red), and anti c-Myc (green) antibodies. White arrows are examples indicating the co-localization of EBP2 and c-Myc.

(B, C) Detection of c-Myc or EBP2 in nuclear extracts from uninfected Caco-2 cells or after infection with indicated strains. Following infection, nuclear extracts were obtained and loaded on protein A/G beads cross-linked with anti-EBP2 (B, IP: anti-EBP2), or anti-C-Myc (C, IP: anti-c-Myc) antibodies, respectively. Input and eluates were blotted with anti-EBP2, anti-c-Myc or anti-VgpA antibodies, respectively. Quantified value (mean ± standard deviation) as measured by band intensity (normalized to uninfected) from three independent experiments are presented under the signals of the blot of IP samples. n.d., not determined.

(D) Purified proteins EBP2, c-Myc and GST-VgpA or GST-VgpAL10A were mixed and then pulled down with protein A/G beads cross-linked with anti-EBP2. Input and eluates were blotted with anti-EBP2, anti-c-Myc or anti-VgpA antibodies, respectively. Quantified value (mean ± standard deviation) as measured by band intensity (normalized to EBP2+c-Myc sample without GST-VgpA and GST-VgpAL10A) from three independent experiments are presented under the signals of the blot of IP samples. n.d., not determined.

(E) Caco-2 cells were infected with the indicated strains and treated with cycloheximide (CHX) for the indicated time. Whole cells lysates were analyzed by western blot using anti-c-Myc and anti-Actin antibody.
Nucleolar c-Myc recruitment by a Vibrio T3SS effector promotes host cell proliferation and bacterial virulence (2020)
Figure 5

Fig. 5 VgpA promotes rRNA transcription in vitro and in vivo.

(A) Amounts of rRNA were from uninfected Caco-2 cells or cells infected with indicated strains. *P<0.05 (when compared to the uninfected Caco-2 cells).

(B) Amounts of rRNA in uninfected or infected Caco-2 cells transfected with control (siNC) or EBP2-targeting (siEBP2) siRNAs. *P<0.05 (when compared to the siNC-transfected cells that are infected with vopZ'ΔvscN1).

(C) Amounts of RNA polymerase I (POLR1A) were from uninfected Caco-2 cells or cells infected with indicated strains. *P<0.05 (when compared to the uninfected Caco-2 cells).

(D). Amounts of rRNA in intestinal homogenates from infant rabbits infected with indicated strains 18 and 38 h after infection. *P<0.05 (when compared to the uninfected rabbits at 18 hours). ΔP<0.05 (when compared to the uninfected rabbits at 38 hours post infection).

All error bars represent mean ± standard deviation (n=3 biologically independent experiments). One-way ANOVA was used for statistical analysis for (A) and (C). Two-way ANOVA was used for statistical analysis for (B) and (D).
Nucleolar c-Myc recruitment by a Vibrio T3SS effector promotes host cell proliferation and bacterial virulence (2020)
Figure 6

Fig. 6 VgpA promotes cell proliferation within the intestine of infected rabbits.

(A) VgpA (upper panel: animals infected with WT) and VgpAL10A (lower panel: animals infected with vgpAL10A) localization in intestinal epithelial cells of infected animals. Arrows in upper panel indicate VgpA localized as punctae in the nucleus; the arrow in the lower panel indicates VgpAL10A localized diffusively in the nucleoplasm.

(B) VgpA and VgpAL10A interactions with EBP2 in intestinal epithelial cells isolated from infant rabbits infected with WT or vgpAL10A V. parahaemolyticus. Inputs represent proteins detected in intestinal epithelial cells of uninfected rabbits or rabbits infected with the indicated strains. Outputs represent eluates from protein A/G beads cross-linked with anti-EBP2 (IP: anti-EBP2) or anti-VgpA (IP: anti-VgpA) antibodies and analyzed with anti-VgpA and anti-EBP2 antibodies.

(C) Frozen sections from uninfected rabbits or rabbits infected with the indicated strains for 18 hours were stained with anti-Ki67 antibodies to detect actively dividing cells (green), phalloidin to visualize F-actin (red) and DAPI to detect nuclei (blue).

(D) Eighteen hours after infant rabbits were infected with WT (middle panel) or vgpAL10A (lower panel) V. parahaemolyticus, fixed sections from the small intestine were stained with antibodies to c-Myc (green), EBP2 (red), and DAPI (blue). Uninfected rabbits were processed similarly and were included as a control (upper panel). White arrows indicate co-localization of c-Myc and EBP2 (middle panel).
NeutrobodyPlex - monitoring SARS-CoV-2 neutralizing immune responses using nanobodies (2021)
Figure 4

Figure 4 - Biparatopic NM1267 competes with ACE2 and neutralizes SARS-CoV-2 infection.

A Coomassie staining of 1 µg of purified biparatopic Nb NM1267.

B Table summarizing the affinity (KD) determined by BLI and IC50 values of the multiplex ACE2 competition assay and virus neutralization assay obtained for NM1267.

C Sensogram of affinity measurements via BLI using four concentrations (6.25 - 50 nM) of NM1267.

D Results from multiplex ACE2 competition assay are shown for the three spike-derived antigens: RBD, S1-domain (S1) and homotrimeric spike (Spike). Color-coded beads coated with the respective antigens were co-incubated with biotinylated ACE2 and dilution series of NM1267 (8 pM to 126 nM) followed by measuring residual binding of ACE2. MFI signals were normalized to the maximum detectable signal per antigen given by the ACE2-only control. IC50 values were calculated from a four-parametric sigmoidal model. Data are presented as mean ± s.d. of three technical replicates.

E Neutralization potency of NM1267 was analyzed in Caco‑2 cells using the SARS-CoV-2-mNG infectious clones. Infection rate normalized to virus-only infection control is illustrated as percent of infection (% Infection). IC50 value was calculated from a four-parametric sigmoidal model and data are presented as mean ± s.e.m. of three biological replicates (n = 3).
NeutrobodyPlex - monitoring SARS-CoV-2 neutralizing immune responses using nanobodies (2021)
Figure 5

Figure 5 - NeutrobodyPlex: multiplex competitive binding assay to monitor a neutralizing immune response in patients.

A Schematic illustration of the NeutrobodyPlex. The displacement of serum-derived neutralizing IgGs binding to SARS-CoV-2 antigens upon addition of bipNb is measured. In presence of neutralizing IgGs, the fluorescent signal from anti-human-IgG-PE is inversely proportional to the applied bipNb concentration.

B For the NeutrobodyPlex, antigen-coated beads comprising RBD, S1 or spike were co-incubated with serum samples from 5 patients and a dilution series of NM1267 (1 µM to 6 pM) (n = 1). Mean fluorescent intensities (MFI) derived from antigen-bound IgGs in the presence of bipNb normalized to the MFI values of IgGs in the serum-only samples, illustrated as MFI (% control), are shown.

C For two patient derived serum samples (#289, #265), differences in the NM1267-mediated displacement of IgGs binding the three spike-derived antigens (RBD, S1, spike) are shown.
NeutrobodyPlex - monitoring SARS-CoV-2 neutralizing immune responses using nanobodies (2021)
Figure 7

Figure 7 - NeutrobodyPlex enables a differentiated analysis on neutralizing IgGs compared to total SARS-CoV-2 binding IgGs in individuals.

To investigate neutralizing capacities in relation to total levels of SARS-CoV-2 specific IgGs in individuals, serum samples of 112 convalescent SARS-CoV-2-infected individuals were analyzed using the NeutrobodyPlex (n = 1). Individuals displaying strong neutralizing IgGs but showed an overall low level of SARS-CoV-2 specific antibodies are highlighted with light-blue background.

A ,B Total IgGs derived from spike-binding IgGs were plotted against normalized MFI values from IgGs binding to RBD (MFI RBD (%control)) in the presence of both concentrations of NM1267 (1 µM; 1 nM,).

C, D Total IgGs derived from N-binding IgGs were plotted against normalized MFI values from IgGs binding to RBD (MFI RBD (%control)) in the presence of both concentrations of NM1267 (1 µM; 1 nM,).

Figures legends Expanded View