Journal: Nature Cell Biology

Article Title: Ca 2+ -driven PDIA6 biomolecular condensation ensures proinsulin folding

doi: 10.1038/s41556-025-01794-8

Figure Lengend Snippet: Real-time monitoring of PDIA6 (50 μM) condensate formation after the addition of 4 mM CaCl 2 . Data were obtained by 3D holographic imaging of the refractive index.

Article Snippet: Following this, the membranes were blocked with 5% skim milk or 5% BSA and incubated with anti-PDIA6 (18233-AP, Proteintech) or anti-insulin (L6B10, 8138, Cell Signaling Technology) and horseradish peroxidase-conjugated anti-rabbit IgG (711-035-152, Jackson Immuno Research).

Techniques:

Journal: Nature Cell Biology

Article Title: Ca 2+ -driven PDIA6 biomolecular condensation ensures proinsulin folding

doi: 10.1038/s41556-025-01794-8

Figure Lengend Snippet: Real-time monitoring of PDIA6 (50 μM) condensate formation after the addition of 4 mM CaCl 2 . Data were obtained by 3D holographic imaging of the refractive index.

Article Snippet: Primary antibodies to PDIA6 (1:1,000; Proteintech, 18233-1-AP), CNX (1:1,000; MBL, M178-3), mCherry (1:1,000; Proteintech, 26765-1-AP; 1:1,000; Proteintech, 68088-1-Ig), BiP (1:1,000; Abcam, ab21685) and PDIA3 (1:1,000; Proteintech, 15967-1-AP) were used.

Techniques:

Journal: Nature Cell Biology

Article Title: Ca 2+ -driven PDIA6 biomolecular condensation ensures proinsulin folding

doi: 10.1038/s41556-025-01794-8

Figure Lengend Snippet: a , Mean diameter of ensemble particles in solution ( Z -average) of PDIA1, PDIA3, PDIA4, PDIA6, PDIA10 and PDIA15 under various Ca 2+ concentrations. The values are the mean ± s.d. of three independent experiments. b , Liquid droplets observed by DIC microscopy when 50 μM PDIA6 and 4 mM CaCl 2 were mixed at pH 7.2. This experiment was replicated three times independently. c , PDIA6 phase diagram obtained by DIC microscopy when 5–100 μM PDIA6 and 0.5–10 mM CaCl 2 were mixed at pH 7.2. Dominant PDIA6 states at varying protein and Ca²⁺ concentrations are indicated by symbols: black circles, dispersed state; black triangles, condensed state. The dashed black line represents the critical droplet concentration. Three independent experiments were performed. d , Confocal fluorescence images of PDIA6 droplets before and after photobleaching. The white arrowhead indicates the laser irradiation area. Rapid recovery of mCherry–PDIA6 fluorescence after photobleaching (left). Increases in the normalized fluorescence intensity of mCherry–PDIA6 after photobleaching (five replicates; right). The fluorescence recovery t 1/2 was calculated from the normalized fluorescence intensity of the five replicates. e , Liquid droplets observed by DIC microscopy when 50 μM PDIA6 and 4 mM CaCl 2 were mixed with (right) or without (left) NaCl. This experiment was replicated three times independently. f , Liquid droplets observed by DIC microscopy when 50 μM PDIA6 and 4 mM CaCl 2 were mixed in solutions with different pH values. This experiment was replicated three times independently. g , Time course of representative two-dimensional (2D) RI distribution (top), and bright-field (middle) and fluorescence images (bottom) of FUS and PDIA6 droplets monitored by 3D holographic imaging (green, ThT fluorescence; three independent experiments). [Ca 2+ ], Ca 2+ concentration; BF, bright field; FI, fluorescence image; [PDIA6], PDIA6 concentration.

Article Snippet: Primary antibodies to PDIA6 (1:1,000; Proteintech, 18233-1-AP), CNX (1:1,000; MBL, M178-3), mCherry (1:1,000; Proteintech, 26765-1-AP; 1:1,000; Proteintech, 68088-1-Ig), BiP (1:1,000; Abcam, ab21685) and PDIA3 (1:1,000; Proteintech, 15967-1-AP) were used.

Techniques: Microscopy, Concentration Assay, Fluorescence, Irradiation, Imaging

Journal: Nature Cell Biology

Article Title: Ca 2+ -driven PDIA6 biomolecular condensation ensures proinsulin folding

doi: 10.1038/s41556-025-01794-8

Figure Lengend Snippet: a , Purity of the recombinant PDI family proteins. Representative data from three independent biological experiments are shown. b , Z -average (the mean diameter of ensemble particles in solution) of Ca 2+ -driven PDIA6 droplets with/without EDTA. The values are the mean ± s.d. of three independent experiments. c , Liquid droplets observed by DIC microscopy when 5–100 μM PDIA6 and 0–7.5 mM CaCl 2 were mixed at pH 7.2. This experiment was performed three times independently. d , Critical concentration of PDIA6 for droplet formation. The values are the mean ± s.d. of three independent experiments. e , Critical concentration of PDIA6 for droplet formation in the presence of 10 mM NaCl. The values are the mean ± s.d. of three independent experiments. f , Z -average of PDIA6 under Ca 2+ at various pH values. The values are the mean ± s.d. of three independent experiments.

Article Snippet: Primary antibodies to PDIA6 (1:1,000; Proteintech, 18233-1-AP), CNX (1:1,000; MBL, M178-3), mCherry (1:1,000; Proteintech, 26765-1-AP; 1:1,000; Proteintech, 68088-1-Ig), BiP (1:1,000; Abcam, ab21685) and PDIA3 (1:1,000; Proteintech, 15967-1-AP) were used.

Techniques: Recombinant, Microscopy, Concentration Assay

Journal: Nature Cell Biology

Article Title: Ca 2+ -driven PDIA6 biomolecular condensation ensures proinsulin folding

doi: 10.1038/s41556-025-01794-8

Figure Lengend Snippet: a , U2OS cells were stained for endogenous PDIA6 and CNX. b , Rapid recovery of the fluorescence of mCherry–PDIA6 foci after photobleaching in U2OS cells. The graph shows the recovery in normalized mCherry–PDIA6 fluorescence for ten foci after photobleaching, obtained from three biologically independent experiments. The fluorescence recovery t 1/2 was calculated from the normalized fluorescence intensity of three replicates. c , U2OS cells stably expressing mCherry–PDIA6 were stained with Mag-Fluo4 AM and treated with thapsigargin. Three independent biological experiments were performed. d , U2OS cells were treated with thapsigargin. After thapsigargin treatment, the cells were stained for PDIA6 and CNX. Five independent biological experiments were performed. e , U2OS cells stably expressing mCherry–PDIA6 were treated with thapsigargin. After thapsigargin treatment, the cells were stained for mCherry and BiP. f , U2OS cells stably expressing mCherry–PDIA6 were treated with thapsigargin. After thapsigargin treatment, the cells were stained for mCherry and CNX. Magnified views of the dashed boxes are provided (right). a , e , f , Representative images from five independent biological experiments are shown. g , Following treatment with thapsigargin or A23187, the signal intensities of mCherry–PDIA6 and CNX were statistically analysed using line scan data ( n = 6 individual experiments). h , Average number of PDIA6 foci in a cell after thapsigargin (control, n = 27 cells, 759 total foci; 0 h, n = 31 cells, 269 total foci; 2 h, n = 24 cells, 950 total foci; 4 h, n = 29 cells, 926 total foci) and A23187 treatment (control, n = 19 cells, 509 total foci; 0 h, n = 25 cells, 212 total foci; 2 h, n = 19 cells, 559 total foci; 4 h, n = 20 cells, 545 total foci) pooled from five independent biological experiments. Box plots show the median (centre line), the 25th and 75th percentiles (bounds of box) and the minimum and maximum values (whiskers). g , h , Statistical significance was examined using a one-way analysis of variance (ANOVA) with Tukey’s honest significant difference post-hoc test; the test was two-sided. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS, not significant. i , Following thapsigargin treatment, mCherry–PDIA6 foci in U2OS cells were analysed using FRAP. Normalized fluorescence intensity curves are shown for ten (thapsigargin) and seven (dimethylsulfoxide, DMSO) foci obtained from three biologically independent experiments. The fluorescence recovery t 1/2 was calculated from the mean of these three independent biological replicates. b , g , i , Data are presented as the mean ± s.d. TG, thapsigargin.

Article Snippet: Primary antibodies to PDIA6 (1:1,000; Proteintech, 18233-1-AP), CNX (1:1,000; MBL, M178-3), mCherry (1:1,000; Proteintech, 26765-1-AP; 1:1,000; Proteintech, 68088-1-Ig), BiP (1:1,000; Abcam, ab21685) and PDIA3 (1:1,000; Proteintech, 15967-1-AP) were used.

Techniques: Staining, Fluorescence, Stable Transfection, Expressing, Control

Journal: Nature Cell Biology

Article Title: Ca 2+ -driven PDIA6 biomolecular condensation ensures proinsulin folding

doi: 10.1038/s41556-025-01794-8

Figure Lengend Snippet: a , U2OS cells stably expressing mCherry–PDIA6 were stained with Mag-Fluo4 AM and treated with A23187. Representative data from three independent biological experiments are shown. b , U2OS cells were treated with A23187. After A23187 treatment, the cells were stained for PDIA6 and CNX. Five independent biological experiments were performed. c , U2OS cells stably expressing mCherry–PDIA6 were treated with A23187. After TG treatment, the cells were stained for mCherry and GRP78/BiP. Representative images from five independent biological experiments are shown. d , U2OS cells stably expressing mCherry–PDIA6 were treated with A23187. After A23187 treatment, the cells were stained for mCherry and CNX. The dashed boxes on the left are shown in more detail on the right. Representative data from five independent biological experiments are shown.

Article Snippet: Primary antibodies to PDIA6 (1:1,000; Proteintech, 18233-1-AP), CNX (1:1,000; MBL, M178-3), mCherry (1:1,000; Proteintech, 26765-1-AP; 1:1,000; Proteintech, 68088-1-Ig), BiP (1:1,000; Abcam, ab21685) and PDIA3 (1:1,000; Proteintech, 15967-1-AP) were used.

Techniques: Stable Transfection, Expressing, Staining

Journal: Nature Cell Biology

Article Title: Ca 2+ -driven PDIA6 biomolecular condensation ensures proinsulin folding

doi: 10.1038/s41556-025-01794-8

Figure Lengend Snippet: a , Real-time images of PDIA6 (50 μM) condensate formation after addition of 4 mM CaCl 2 . b , Real-time imaging of fusion between the two droplets. The data were obtained by 3D holographic imaging of the refractive index (RI). These experiments were performed three times independently.

Article Snippet: Primary antibodies to PDIA6 (1:1,000; Proteintech, 18233-1-AP), CNX (1:1,000; MBL, M178-3), mCherry (1:1,000; Proteintech, 26765-1-AP; 1:1,000; Proteintech, 68088-1-Ig), BiP (1:1,000; Abcam, ab21685) and PDIA3 (1:1,000; Proteintech, 15967-1-AP) were used.

Techniques: Imaging, Refractive Index

Journal: Nature Cell Biology

Article Title: Ca 2+ -driven PDIA6 biomolecular condensation ensures proinsulin folding

doi: 10.1038/s41556-025-01794-8

Figure Lengend Snippet: a , Effects of NaCl on the interaction between Ca 2+ and PDIA6. ITC data (upper panel) and binding isotherm data (lower panel) for the titration of Ca 2+ against PDIA6 with and without 100 mM NaCl. The solid line in the binding isotherm represents the best fit curve based on a model with one set of binding sites. The experiments were independently repeated three times with reproducible results. b , Thermodynamic parameters of Ca 2+ binding to PDIA6. The error value of each parameter represents the fitting error. c , Effects of NaCl on Ca 2+ -driven droplet formation. Bright-field observation of PDIA6 droplets observed by 3D holographic imaging when 50 μM PDIA6 and 4 mM CaCl 2 were mixed at various NaCl concentrations. This experiment was performed three times independently. d , e , Structural analyses of PDIA6 by NMR (related to Fig. ). 1 H– 15 N TROSY-HSQC spectra ( d ) and 1 H– 13 C HMQC spectra ( e ) of [U- 2 H 15 N; Ala- 13 CH ; Met- 13 CH ; Ile-δ1- 13 CH ; Leu, Val- 13 CH / 13 CH ]-labelled full-length PDIA6. f , 1 H– 15 N TROSY-HSQC (left panel) and 1 H– 13 C HMQC spectra (right panel) of [U- 15 N; Ala- 13 CH ; Met- 13 CH ; Ile-δ1- 13 CH ; Leu, Val- 13 CH / 13 CH ]-labelled PDIA6 a 0 (orange), a (green), and b (grey) domains. g , Electrostatic surface of each PDIA6 domains.

Article Snippet: Primary antibodies to PDIA6 (1:1,000; Proteintech, 18233-1-AP), CNX (1:1,000; MBL, M178-3), mCherry (1:1,000; Proteintech, 26765-1-AP; 1:1,000; Proteintech, 68088-1-Ig), BiP (1:1,000; Abcam, ab21685) and PDIA3 (1:1,000; Proteintech, 15967-1-AP) were used.

Techniques: Binding Assay, Titration, Imaging

Journal: Nature Cell Biology

Article Title: Ca 2+ -driven PDIA6 biomolecular condensation ensures proinsulin folding

doi: 10.1038/s41556-025-01794-8

Figure Lengend Snippet: a , Concentration of Ca 2+ outside PDIA6 droplets. Values are the mean ± s.d. of three independent experiments. b , PDIA6 is composed of two redox-active domains (a 0 and a) at the N terminus and a redox-inactive domain b at the C terminus. PDIA6 forms a homodimer via a unique Val-Leu adhesive motif contained in a 0 . c – e , Evaluation of Ca 2+ binding sites on PDIA6 using NMR. c , 1 H– 15 N HSQC spectra of the three 15 N-labelled domains of PDIA6 (left, a 0 domain; middle, a domain; right, b domain) obtained in the absence (orange, a 0 domain; green, a domain; grey, b domain) and presence (red) of 20 mM CaCl 2 . Insets: expanded views of the regions outlined by dashed boxes in the spectra. d , Graphical representation of the CSPs between the resonances of domains a 0 (left), a (middle) and b (right). The solid and dotted lines indicate the average values and average values plus standard deviations, respectively. e , CSP mapping of PDIA6. The residues with remarkable CSPs after Ca 2+ binding are indicated as blue spheres.

Article Snippet: Primary antibodies to PDIA6 (1:1,000; Proteintech, 18233-1-AP), CNX (1:1,000; MBL, M178-3), mCherry (1:1,000; Proteintech, 26765-1-AP; 1:1,000; Proteintech, 68088-1-Ig), BiP (1:1,000; Abcam, ab21685) and PDIA3 (1:1,000; Proteintech, 15967-1-AP) were used.

Techniques: Concentration Assay, Adhesive, Binding Assay

Journal: Nature Cell Biology

Article Title: Ca 2+ -driven PDIA6 biomolecular condensation ensures proinsulin folding

doi: 10.1038/s41556-025-01794-8

Figure Lengend Snippet: a – c , 1 H– 15 N heteronuclear single quantum coherence (HSQC) spectra of the three 15 N-labelled domains of PDIA6 a 0 ( a ), a ( b ), and b ( c ) obtained in the absence (black) and presence of 10 (blue) or 20 mM (purple) CaCl 2 .

Article Snippet: Primary antibodies to PDIA6 (1:1,000; Proteintech, 18233-1-AP), CNX (1:1,000; MBL, M178-3), mCherry (1:1,000; Proteintech, 26765-1-AP; 1:1,000; Proteintech, 68088-1-Ig), BiP (1:1,000; Abcam, ab21685) and PDIA3 (1:1,000; Proteintech, 15967-1-AP) were used.

Techniques:

Journal: Nature Cell Biology

Article Title: Ca 2+ -driven PDIA6 biomolecular condensation ensures proinsulin folding

doi: 10.1038/s41556-025-01794-8

Figure Lengend Snippet: a , Phase separating ability of each PDIA6 domain. Liquid droplets observed by DIC microscopy when 50 μM full-length PDIA6 or PDIA6 domains and 4 mM CaCl 2 were mixed. Three independent experiments were performed. b , Liquid droplets observed by bright-field (BF) microscopy when a 0 , a, or b domain containing Ca 2+ was mixed with b, a, or a 0 . Three independent experiments were performed. c , Liquid droplets observed by BF microscopy when a 0 or b domain containing Ca 2+ was mixed with b or a 0 in the presence of different NaCl concentrations. Three independent experiments were performed. d , Quantification of residual domains after each domain was mixed pairwise together. a 0 or b domain containing Ca 2+ was added to another domain, incubated for 30 min, and centrifuged, and the supernatant was separated by SDS‒PAGE for quantitative analysis. Three independent experiments were performed. Data are presented as mean ± s.d. e , Liquid droplets observed by BF microscopy when a 0 A5 (monomeric mutant) or b domain containing Ca 2+ was mixed with b or a 0 A5. Three independent experiments were performed.

Article Snippet: Primary antibodies to PDIA6 (1:1,000; Proteintech, 18233-1-AP), CNX (1:1,000; MBL, M178-3), mCherry (1:1,000; Proteintech, 26765-1-AP; 1:1,000; Proteintech, 68088-1-Ig), BiP (1:1,000; Abcam, ab21685) and PDIA3 (1:1,000; Proteintech, 15967-1-AP) were used.

Techniques: Microscopy, Incubation, Mutagenesis

Journal: Nature Cell Biology

Article Title: Ca 2+ -driven PDIA6 biomolecular condensation ensures proinsulin folding

doi: 10.1038/s41556-025-01794-8

Figure Lengend Snippet: a , The a 0 –b interaction is critical for PDIA6 condensation. Residual concentrations obtained when the domains were mixed pairwise together. The black and red open circles indicate the theoretical and measured values, respectively. The values are the mean ± s.d. of three independent experiments. b , Dimerization of a 0 is critical for PDIA6 condensation. Close-up view of dimeric motif within a 0 (top). Representative 2D RI distribution when the a 0 and b domains (bottom left) or monomeric mutated a 0 A5 and b domains (bottom right) are mixed, monitored by 3D holographic imaging (three independent experiments). BF, bright-field images. c , d , NMR investigation of the interaction between PDIA6 molecules during droplet formation. c , 1 H– 15 N transverse relaxation optimized spectroscopy-HSQC (left) and 1 H– 13 C heteronuclear multiple quantum coherence (HMQC; right) spectra of [U- 2 H 15 N; Ala- 13 CH 3 ; Met- 13 CH 3 ; Ile-δ1- 13 CH 3 ; Leu, Val- 13 CH 3 / 13 CH 3 ]-labelled full-length PDIA6 in the absence and presence of 4 mM CaCl 2 . d , 1 H– 15 N HSQC spectra of 15 N-labelled a 0 (top left) in the absence (orange) and presence (red) of b and 15 N-labelled b (top right) in the absence (grey) and presence (red) of a 0 . Insets: expanded views of the regions outlined by dashed boxes in the spectra. The intensity ratios between the resonances of 15 N-labelled a 0 (bottom left) and b (bottom right) in the absence and presence of the other domain are provided. The solid and dotted lines indicate the average and average values, minus the s.d., respectively. Residues with large changes in intensity are indicated as blue spheres in the structure of the a 0 (left) and b (right) domains. The error bar of intensity ratio (Δ R ) was estimated from signal-to-noise ratios using the following equation: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Delta R=R\sqrt{{\text{SNR}}_{\text{ref}}^{-2}+{\text{SNR}}_{\text{tit}}^{-2}}$$\end{document} Δ R = R SNR ref − 2 + SNR tit − 2 where R is the intensity ratio, and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\text{SNR}}_{\text{ref}}^{-2}$$\end{document} SNR ref − 2 and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\text{SNR}}_{\text{tit}}^{-2}$$\end{document} SNR tit − 2 are the signal-to-noise ratios of the peak on the reference spectrum and the titrated spectrum, respectively. e , Conformational change of PDIA6 induced by condensation. Arrival time distributions of 19+ charged ions of PDIA6 dimer before (top) and after (bottom) condensation observed in the IM–MS experiments. The arrival times of PDIA6 dimer with 0, 4 and 8 Ca 2+ ions before and after condensation are indicated in the plots.

Article Snippet: Primary antibodies to PDIA6 (1:1,000; Proteintech, 18233-1-AP), CNX (1:1,000; MBL, M178-3), mCherry (1:1,000; Proteintech, 26765-1-AP; 1:1,000; Proteintech, 68088-1-Ig), BiP (1:1,000; Abcam, ab21685) and PDIA3 (1:1,000; Proteintech, 15967-1-AP) were used.

Techniques: Imaging, Spectroscopy

Journal: Nature Cell Biology

Article Title: Ca 2+ -driven PDIA6 biomolecular condensation ensures proinsulin folding

doi: 10.1038/s41556-025-01794-8

Figure Lengend Snippet: a , Condensates observed by bright-field (BF) microscopy when 5 μM PDIA6 and 250 μM CaCl 2 were mixed in NH 4 OAc. Three independent experiments were performed. b , ESI mass spectra of 5 μM PDIA6 with 250 μM CaCl 2 in NH 4 OAc.

Article Snippet: Primary antibodies to PDIA6 (1:1,000; Proteintech, 18233-1-AP), CNX (1:1,000; MBL, M178-3), mCherry (1:1,000; Proteintech, 26765-1-AP; 1:1,000; Proteintech, 68088-1-Ig), BiP (1:1,000; Abcam, ab21685) and PDIA3 (1:1,000; Proteintech, 15967-1-AP) were used.

Techniques: Microscopy

Journal: Nature Cell Biology

Article Title: Ca 2+ -driven PDIA6 biomolecular condensation ensures proinsulin folding

doi: 10.1038/s41556-025-01794-8

Figure Lengend Snippet: a , Condensation of the green fluorescent protein (GFP)-tagged PDI family members in PDIA6 droplets. This experiment was performed three times independently. Statistical significance was examined using a one-way ANOVA with Tukey’s honest significant difference post-hoc test; the test was two-sided. **** P < 0.0001. b , Changes in the RI inside PDIA6 droplets with increasing concentrations of different PDI family members. Representative 2D RI distributions are indicated. Data were analysed for 198 particles for PDIA6 only, 217 particles for 1 µM PDIA1, 210 particles for 5 µM PDIA1, 212 particles for 10 µM PDIA1, 215 particles for 1 µM PDIA3, 220 particles for 5 µM PDIA3, 212 particles for 10 µM PDIA3, 215 particles for 1 µM PDIA4, 219 particles for 5 µM PDIA4, 212 particles for 10 µM PDIA4, 214 particles for 1 µM PDIA10, 212 particles for 5 µM PDIA10, 217 particles for 10 µM PDIA10, 217 particles for 1 µM PDIA15, 218 particles for 5 µM PDIA15 and 226 particles for 10 µM PDIA15 pooled from three independent replicates. a , b , Data are presented as the mean ± s.d. c , Representative images of the 2D RI distribution of PDIA6 droplets with untagged PDI family members, monitored by 3D holographic imaging from the same dataset analysed in Fig. 5b. d , Co-localization of endogenous PDIA3 and PDIA6 in U2OS cells (top). The fluorescence intensity (bottom) was analysed against the yellow line (top). The merged figure shows the fluorescence intensities of mCherry–PDIA6 and PDIA3 as a solid line, which means that their respective fluorescence intensities overlap (bottom). This experiment was performed three times independently.

Article Snippet: Primary antibodies to PDIA6 (1:1,000; Proteintech, 18233-1-AP), CNX (1:1,000; MBL, M178-3), mCherry (1:1,000; Proteintech, 26765-1-AP; 1:1,000; Proteintech, 68088-1-Ig), BiP (1:1,000; Abcam, ab21685) and PDIA3 (1:1,000; Proteintech, 15967-1-AP) were used.

Techniques: Imaging, Fluorescence

Journal: Nature Cell Biology

Article Title: Ca 2+ -driven PDIA6 biomolecular condensation ensures proinsulin folding

doi: 10.1038/s41556-025-01794-8

Figure Lengend Snippet: a , Condensation of GFP-tagged PDI family members into PDIA6 droplets. Representative images of bright-field (BF) and fluorescence microscopy of PDIA6 droplets with GFP-tagged PDI family members in the uptake assay. Three independent experiments were performed. b , Signal-enhanced images of Extended Data Fig. 8a. c , Condensation of PDI family members into PDIA6 droplets. The average refractive index (RI) inside the droplet and droplet radius were calculated by enclosing the RI image in a circle. Data were analysed for 198 particles for PDIA6 only, 217 particles for 1 µM PDIA1, 210 particles for 5 µM PDIA1, 212 particles for 10 µM PDIA1, 215 particles for 1 µM PDIA3, 220 particles for 5 µM PDIA3, 212 particles for 10 µM PDIA3, 215 particles for 1 µM PDIA4, 219 particles for 5 µM PDIA4, 212 particles for 10 µM PDIA4, 214 particles for 1 µM PDIA10, 212 particles for 5 µM PDIA10, 217 particles for 10 µM PDIA10, 217 particles for 1 µM PDIA15, 218 particles for 5 µM PDIA15 and 226 particles for 10 µM PDIA15, pooled from three independent replicates.

Article Snippet: Primary antibodies to PDIA6 (1:1,000; Proteintech, 18233-1-AP), CNX (1:1,000; MBL, M178-3), mCherry (1:1,000; Proteintech, 26765-1-AP; 1:1,000; Proteintech, 68088-1-Ig), BiP (1:1,000; Abcam, ab21685) and PDIA3 (1:1,000; Proteintech, 15967-1-AP) were used.

Techniques: Fluorescence, Microscopy, Refractive Index

Journal: Nature Cell Biology

Article Title: Ca 2+ -driven PDIA6 biomolecular condensation ensures proinsulin folding

doi: 10.1038/s41556-025-01794-8

Figure Lengend Snippet: a , Time course of impaired PDIA6 droplet formation. PDIA6 A5 is a dimer-deficient mutant. This experiment was performed three times independently. b , Exogenously expressed insulin secretion was assessed in HCT116 PDIA6 -KO cells transfected with proinsulin- Gaussia luciferase (hINS-Gluc) along with either PDIA6 WT or the PDIA6 A5 mutant construct. The cells were treated with or without 0.3 µM thapsigargin for 1 h at 37 °C. Following thapsigargin treatment, the amount of insulin-Gluc secreted into the medium during the subsequent 2-h incubation period was quantified by measuring luminescence ( n = 3 biologically independent experiments). The specific Gluc signal induced by PDIA6 WT or its A5 mutant (shown in Extended Data Fig. ) was normalized to the PDIA6 WT or A5 protein levels, which were quantified by western blotting (shown in Extended Data Fig. ). Statistical significance was assessed using a two-tailed unpaired Student’s t -test ( n = 3). c , Intracellular proinsulin-Gluc levels in NP-40-soluble (left) and -insoluble (right) fractions were analysed by western blotting in three biologically independent experiments. d , The band intensities in c were quantified by densitometry. Statistical significance was assessed using one-way repeated-measures ANOVA, followed by Dunnett’s post-hoc test ( n = 3). P < 0.05 was considered statistically significant; the exact P value for this comparison was P = 0.03. Each biological replicate ( n ) is shown in a different colour. e , MIN6 cells were treated with 1 µM thapsigargin for 16 h. The amount of insulin secreted within 6 h after thapsigargin treatment was then quantified using ELISA and is expressed as a percentage of the amount secreted by the DMSO-treated control cells. Statistical significance was assessed using a two-tailed paired Student’s t -test ( n = 6 independent biological replicates). b , d , e , Data are presented as the mean ± s.d. *** P = 0.0001 and **** P < 0.0001. TG, thapsigargin.

Article Snippet: Primary antibodies to PDIA6 (1:1,000; Proteintech, 18233-1-AP), CNX (1:1,000; MBL, M178-3), mCherry (1:1,000; Proteintech, 26765-1-AP; 1:1,000; Proteintech, 68088-1-Ig), BiP (1:1,000; Abcam, ab21685) and PDIA3 (1:1,000; Proteintech, 15967-1-AP) were used.

Techniques: Mutagenesis, Transfection, Luciferase, Construct, Incubation, Western Blot, Two Tailed Test, Comparison, Enzyme-linked Immunosorbent Assay, Control

Journal: Nature Cell Biology

Article Title: Ca 2+ -driven PDIA6 biomolecular condensation ensures proinsulin folding

doi: 10.1038/s41556-025-01794-8

Figure Lengend Snippet: a , The specific Gluc signal induced by PDIA6 WT or PDIA6 A5 mutant was normalized to the PDIA6 WT or A5 protein levels, which were quantified by western blotting (shown in Extended Data Fig. 9b). Statistical significance was assessed using one-way ANOVA, followed by Dunnett’s post-hoc test (indicated in red) or a two-tailed unpaired t -test (indicated in black). Data are presented as mean ± s.d. ( n = 3 independent replicates). ** P < 0.01, *** P < 0.001, **** P < 0.0001. b , After collecting the medium for the Gluc assay, cells were lysed for western blotting to verify the expression of exogenous PDIA6 constructs using an antibody to PDIA6. Ponceau S staining was performed as a loading control.

Article Snippet: Primary antibodies to PDIA6 (1:1,000; Proteintech, 18233-1-AP), CNX (1:1,000; MBL, M178-3), mCherry (1:1,000; Proteintech, 26765-1-AP; 1:1,000; Proteintech, 68088-1-Ig), BiP (1:1,000; Abcam, ab21685) and PDIA3 (1:1,000; Proteintech, 15967-1-AP) were used.

Techniques: Mutagenesis, Western Blot, Two Tailed Test, Expressing, Construct, Staining, Control

Journal: Nature Cell Biology

Article Title: Ca 2+ -driven PDIA6 biomolecular condensation ensures proinsulin folding

doi: 10.1038/s41556-025-01794-8

Figure Lengend Snippet: a , Condensation of fluorescently labelled proinsulin into PDIA6 droplets. In labelled proinsulin, five Cys residues (but not Cys8) were replaced with Ser and Cys8 was selectively fluorescently labelled. This experiment was performed three times independently. BF, bright field. b , High-performance liquid chromatography profiles of oxidative proinsulin folding using 20 μM PDIA6 and 20 μM proinsulin with (right) or without 3 mM Ca 2+ (left). The oxidative folding reaction of proinsulin was quenched with 7.0 mg ml −1 2-aminoethyl methanethiosulfonate, a selective thiol functional group modification reagent, at the selected time points. Three independent experiments were performed. c , Quantification of native proinsulin, determined from three biologically independent experiments. d , The folding kinetics were determined from the formation rate of native proinsulin. The rate constants were determined from three independent experiments. e , Amyloid fibril formation of all-Cys/Ser proinsulin with or without PDIA6 droplets detected by ThT (three independent experiments); a.u., arbitrary units. d , e , Data are presented as the mean ± s.d. f , g , AFM ( f ) and TEM ( g ) images obtained with or without PDIA6 droplet. These experiments were replicated three times independently. h , Proposed model of PDIA6 condensation as a protein quality control mechanism.

Article Snippet: Primary antibodies to PDIA6 (1:1,000; Proteintech, 18233-1-AP), CNX (1:1,000; MBL, M178-3), mCherry (1:1,000; Proteintech, 26765-1-AP; 1:1,000; Proteintech, 68088-1-Ig), BiP (1:1,000; Abcam, ab21685) and PDIA3 (1:1,000; Proteintech, 15967-1-AP) were used.

Techniques: High Performance Liquid Chromatography, Functional Assay, Modification, Control

Journal: Nature Cell Biology

Article Title: Ca 2+ -driven PDIA6 biomolecular condensation ensures proinsulin folding

doi: 10.1038/s41556-025-01794-8

Figure Lengend Snippet: HPLC profiles of oxidative proinsulin folding using 20 μM PDIA6 and 20 μΜ proinsulin with (red) or without 3 mM Ca 2+ (black). The oxidative folding reaction of proinsulin was quenched with 2-aminoethyl methanethiosulfonate (7.0 mg ml −1 ), a selective thiol functional group modification reagent, at the selected time points. Three independent experiments were performed. R and N represent reduced/denatured and natively folded proinsulin respectively.

Article Snippet: Primary antibodies to PDIA6 (1:1,000; Proteintech, 18233-1-AP), CNX (1:1,000; MBL, M178-3), mCherry (1:1,000; Proteintech, 26765-1-AP; 1:1,000; Proteintech, 68088-1-Ig), BiP (1:1,000; Abcam, ab21685) and PDIA3 (1:1,000; Proteintech, 15967-1-AP) were used.

Techniques: Functional Assay, Modification