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 , 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