Journal: Metallomics : integrated biometal science

Article Title: Differentiated Zn(II) binding affinities in animal, plant, and bacterial metallothioneins define their zinc buffering capacity at physiological pZn.

doi: 10.1093/mtomcs/mfad061

Figure Lengend Snippet: Fig. 8 Titration of metal-free BcrMT1B, XlMT, MacMT3, Ec-1, and TvMT with ZnSO 4 in the presence of ZnAF-2F. (A) 0.5 μM apo-MT and 2–3.5 μM ZnAF-2F (concentration varied depending on protein) in 50 mM Na + -HEPES, 100 mM NaCl, 500 μM TCEP. Orange line helps demonstrate titration end points. (B) Semilog plots of the same data are presented to visualize changes during titration. Blue lines help to determine the number of tight zinc sites. (C) Fluorescence responses were calibrated to obtain [Zn(II)] free concentrations and are shown as a pZn function of Zn(II)/apo-MT. Red lines demonstrate simulations based on calculated constants. −log K d values obtained from data fitting are presented in Table 3 (see section “Experimental procedures”). 47 Data are shown as means of n = 2 independent experiments + SD.

Article Snippet: All plasmids are deposited in Addgene under the following IDs: BcrMT1B (#200290), LIMT (#200291), SpMTA (#200292), XIMT (#200293), MacMT3 (#200294), OsMTI-1B (#200295), Ec-1 (#200296), PflQ2MT (#200297), SmtA (#200298), and TvMT (#200299).

Techniques: Titration, Concentration Assay, Fluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: δ-COP contains a helix C-terminal to its longin domain key to COPI dynamics and function

doi: 10.1073/pnas.1603544113

Figure Lengend Snippet: Yeast strains used in this study

Article Snippet: Ret2LD2α T158K ( BY4743 Spore ) MAT a his3 ∆ 1 leu2 ∆ 0 met15 ∆ 0 ura3 ∆ 0; YFR051c::kanMX4; p415 ret2LD2 α T158K This study Open in a separate window Yeast strains used in this study table ft1 table-wrap mode="anchored" t5 Table S2. caption a7 No. Plasmids Addgene ID Database ID Source 1. p415 CHI 1 73863 Z1280 This study 2. p415 CHI 2 73864 Z1281 This study 3. p415 CHI 3 73865 Z1283 This study 4. p415 CHI 4 73866 Z1284 This study 5. p415 CHI 5 73867 Z1285 This study 6. p415 CHI 6 73868 Z1286 This study 7. p415 CHI 7 73869 Z1287 This study 8. p415 CHI 8 73870 Z1288 This study 9. pRS303 Bt δCOP 73871 AE1540 This study 10. p415 Ret2 73872 AE1541 This study 11. p415 Ret2LD2α 73873 AE1518 This study 12. p415 δCLD2α 73874 AE1519 This study 13. p415 Ret2LD 73875 AE1520 This study 14. p415 δCLD 73876 AE1521 This study 15. p415 Ret2LD+δC2α 73877 AE1516 This study 16. p415 δCLD+Ret2-2α 73878 AE1523 This study 17. p415 CHI 9 73879 AF1551 This study 18. p415 CHI 10 73880 AF1553 This study 19. p415 CHI 12 73881 AF1557 This study 20. p415 CHI 13 73882 AH1658 This study 21. p415 CHI 14 73883 AH1660 This study 22. p415 CHI 15 73884 AH1662 This study 23. p415 Ret2 I145A 73885 AI1732 This study 24. p415 Ret2 I148A 73886 AI1733 This study 25. p415 Ret2 I149A 73887 AI1734 This study 26. p415 Ret2 I155A 73888 AI1735 This study 27. pGEX6P1- Mst7-Erp1CT 73920 This study 28. pGEX6P1-GCN4cc-Erp1CT 73921 This study 29. pGEX6P1- Mst7-Erp2CT 73922 This study 30. pGEX6P1-GCN4cc-Erp2CT 73923 This study 31. p415 Ret2LD2α Q146F 75259 This study 32. p415 Ret2LD2α N152T 75260 This study 33. p415 Ret2LD2α K153Q 75261 This study 34. p415 Ret2LD2α T158K 75262 This study 35. p416 PMP2-YFP-LRKRS — M650 14 36. p416 PMP2-YFP-KKXX — N654 14 37. p416 PMP2-YFP-NVRNRRK — R890 14 38. p416 PMP2-YFP-KKK — M643 14 39. p416 PMP2-YFP-AAXX — N655 14 40. p416 PMP2-YFP-Task3C44T — S901 27 41. pGEX6P1-MST27-LRKRS — F291 14 42. pGEX6P1-MST27-KKXX — O732 14 Open in a separate window Plasmids used in this study

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: δ-COP contains a helix C-terminal to its longin domain key to COPI dynamics and function

doi: 10.1073/pnas.1603544113

Figure Lengend Snippet: Plasmids used in this study

Article Snippet: Ret2LD2α T158K ( BY4743 Spore ) MAT a his3 ∆ 1 leu2 ∆ 0 met15 ∆ 0 ura3 ∆ 0; YFR051c::kanMX4; p415 ret2LD2 α T158K This study Open in a separate window Yeast strains used in this study table ft1 table-wrap mode="anchored" t5 Table S2. caption a7 No. Plasmids Addgene ID Database ID Source 1. p415 CHI 1 73863 Z1280 This study 2. p415 CHI 2 73864 Z1281 This study 3. p415 CHI 3 73865 Z1283 This study 4. p415 CHI 4 73866 Z1284 This study 5. p415 CHI 5 73867 Z1285 This study 6. p415 CHI 6 73868 Z1286 This study 7. p415 CHI 7 73869 Z1287 This study 8. p415 CHI 8 73870 Z1288 This study 9. pRS303 Bt δCOP 73871 AE1540 This study 10. p415 Ret2 73872 AE1541 This study 11. p415 Ret2LD2α 73873 AE1518 This study 12. p415 δCLD2α 73874 AE1519 This study 13. p415 Ret2LD 73875 AE1520 This study 14. p415 δCLD 73876 AE1521 This study 15. p415 Ret2LD+δC2α 73877 AE1516 This study 16. p415 δCLD+Ret2-2α 73878 AE1523 This study 17. p415 CHI 9 73879 AF1551 This study 18. p415 CHI 10 73880 AF1553 This study 19. p415 CHI 12 73881 AF1557 This study 20. p415 CHI 13 73882 AH1658 This study 21. p415 CHI 14 73883 AH1660 This study 22. p415 CHI 15 73884 AH1662 This study 23. p415 Ret2 I145A 73885 AI1732 This study 24. p415 Ret2 I148A 73886 AI1733 This study 25. p415 Ret2 I149A 73887 AI1734 This study 26. p415 Ret2 I155A 73888 AI1735 This study 27. pGEX6P1- Mst7-Erp1CT 73920 This study 28. pGEX6P1-GCN4cc-Erp1CT 73921 This study 29. pGEX6P1- Mst7-Erp2CT 73922 This study 30. pGEX6P1-GCN4cc-Erp2CT 73923 This study 31. p415 Ret2LD2α Q146F 75259 This study 32. p415 Ret2LD2α N152T 75260 This study 33. p415 Ret2LD2α K153Q 75261 This study 34. p415 Ret2LD2α T158K 75262 This study 35. p416 PMP2-YFP-LRKRS — M650 14 36. p416 PMP2-YFP-KKXX — N654 14 37. p416 PMP2-YFP-NVRNRRK — R890 14 38. p416 PMP2-YFP-KKK — M643 14 39. p416 PMP2-YFP-AAXX — N655 14 40. p416 PMP2-YFP-Task3C44T — S901 27 41. pGEX6P1-MST27-LRKRS — F291 14 42. pGEX6P1-MST27-KKXX — O732 14 Open in a separate window Plasmids used in this study

Techniques:

Journal: bioRxiv

Article Title: Single recipient cell tracking of tellurium-labeled extracellular vesicle proteomes (TeLEV) identifies EV-driven immunomodulation

doi: 10.1101/2025.11.01.685872

Figure Lengend Snippet: a, t-SNE projection of PBMC recipient cells across 16 conditions (PBS-HAT control plus 15 EV sources, in total 2,977,094 cells), colored according to cell type. Numerical annotations indicate the relative proportions of each cell lineage. EVs shown in were isolated from 25 mL of cell-conditioned medium (from 25M primary CLL cells or cell lines over 48 hours). The EV uptake conditions were normalized according to the input volume of cell-conditioned media (25 mL, 25M cells over 48 hours), and EV counts and buffer volumes are reported in Supplementary Table 2. b, t-SNE projection of PBMC recipients across 16 conditions showing TeLEV signal. c, Fold changes in cell type proportions across conditions, estimated using a Poisson generalized linear mixed model that accounts for confounding factors (Methods). Dot size represents the probability of change, measured as the local true sign rate (LTSR). d, t-SNE projection of myeloid cells across 16 conditions (276, 867 cells), colored according to subtypes. e, t-SNE plots showing expression of monocytic and DC lineage markers and interleukin receptors on myeloid cells. f, Two-dimensional density distributions of myeloid cells in t-SNE embedding space, stratified by conditions. g, A cell type–color–coded ternary plot depicting expression levels of CD25, CD123, and CD127. Circle size reflects the average TeLEV signal level within each cell type. Colors correspond to those in the legend shown in (a)

Article Snippet: Anti-CD25 antibody (Basiliximab, MedChemExpress, #HY-10885) was used at 10 μg/mL and compared to an isotype control (Invitrogen, #MA5-55090).

Techniques: Control, Isolation, Expressing

Journal: bioRxiv

Article Title: Single recipient cell tracking of tellurium-labeled extracellular vesicle proteomes (TeLEV) identifies EV-driven immunomodulation

doi: 10.1101/2025.11.01.685872

Figure Lengend Snippet: a, t-SNE projection of PBMC recipient cells across 11 time points (0-48h, in total 1,512,100 cells) of EV uptake, colored according to cell type. Numerical annotations indicate the relative proportions of each cell lineage. Primary MBC-EVs and secreted proteins shown in were isolated from 4,2 mL of cell-conditioned medium (from 4.2M primary CLL cells over 48 hours), resulting in 2.7 x 10 9 EVs used per time point condition. b, t-SNE plots showing TeLEV signal, CD25, CD127, and CD123 expression at selected time points (columns). c, Stacked bar plot depicting changes in cell type composition over 48h. Colors correspond to those in the legend shown in (a). d, Comparison of TeLEV signal distribution across major cell types and their subtypes at 16 h, with colors representing the average TeLEV signal level in each population. Bars above the plots show the relative proportions of each population. White lines denote the median, edges the IQR, and whiskers either 1.5 × IQR or minima/maxima (if no point exceeded 1.5 × IQR; minima/maxima are indicated by the violin plot range). e, Comparison of TeLEV signal, CD25, CD123, and CD127 distributions within the Monocyte population (including Non-classical Monocytes, Intermediate Monocytes, Classical Monocytes, IL2R/IL7R + Monocytes, and CD197 + IL2R/IL7R + Monocytes) over 48 h. Statistical significance across all time points for each marker was evaluated using the Kruskal-Wallis test. Pairwise comparisons between 00 h and 00 h 15 min were performed using a one-sided Mann–Whitney U test. p-values < 0.05 were considered significant and are indicated in the figures as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. f, Line plots depicting the mean expression levels of TeLEV signal, CD25, CD123, and CD127 within the Monocytes during the first 2 hours of EV uptake. Error bars represent mean ± SEM. g, The same analysis as in (e) for mDCs (left) and pDCs (right). h, Live-cell immunocytochemistry of purified monocytes exposed to increasing doses of Ramos-derived EVs. Representative frames from day 2 and day 7 are shown. CD25 (Alexa Fluor 488; green) and Cytotox NIR (blue; loss of membrane integrity) are displayed after automated single-cell segmentation (Incucyte built-in). Cells were seeded at 25,000 per well and imaged every 2 h for 7 days using an Incucyte SX5 (20× objective). Scale bar, 200 µm. EVs and secreted proteins shown in were isolated from 1.5 mL of cell-conditioned medium (from 1.5M Ramos cells over 48 hours), resulting in 9 x 10 8 EVs used per EV condition (3X condition, ). i, Single-cell quantification of CD25 (Alexa Fluor 488) fluorescence over 7 days corresponding to (h). Values are unnormalized arbitrary units; data shown as mean ± SEM; n = 4 fields of view. j, Flow cytometric contour plots of purified monocytes after 16 h of EV exposure across a dose titration (see Figure legend ). CD25 (APC), CD123 (PE), and CD127 (FITC) were measured on a Miltenyi MACSQuant X with compensation using BioLegend compensation beads. DAPI was used as a surface-flow viability dye. k, Live-cell immunocytochemistry of EV-exposed monocytes stimulated with cytokines. Representative images from day 7 are shown for IL-2 (20 ng mL −1 ), IL-3 (5 ng mL −1 ), or, IL-7 (25 ng mL −1 ), or IL-2 + IL-3, IL-2 + IL-7, IL-3 + IL-7, or IL-2 + IL-3 + IL-7 in RPMI with serum. CD25 (Alexa Fluor 488; green) and Cytotox NIR (blue) are shown after automated segmentation and background subtraction. Imaging on Incucyte SX5 (20×, 2 h intervals). Scale bar, 200 µm. l, Immunogold transmission electron microscopy of Ramos-EVs labeled for IL-3, CD123, and CD127 alongside an isotype control. Scale bar, 100 nm. m, Intracellular phospho-flow cytometry of purified monocytes incubated with EVs, showing total STAT5 (Alexa Fluor 488), total STAT3 (PE), pSTAT5 (PE-Cy7), and pSTAT3 (BV421). EV exposure was terminated by immediate formaldehyde fixation after 16 h prior to staining. Cleaved caspase was included as an apoptosis marker. Acquisition on a Miltenyi MACSQuant X with bead-based compensation of all markers. n, Live-cell immunocytochemistry of EV-exposed healthy-donor PBMCs stimulated for 2 days with IL-2 (20 ng mL −1 ), IL-3 (5 ng mL −1 ), or IL-7 (25 ng mL −1 ) in RPMI with serum. Representative images are shown (Incucyte SX5, 20×, 2 h intervals). Scale bar, 200 µm. o, Live-cell immunocytochemistry of EV-exposed healthy-donor PBMCs treated for 7 days with a STAT5 degrader (AK-2292) or anti-CD25 (Basiliximab), compared with DMSO and isotype controls. CD25 (Alexa Fluor 488; green), CD127 (PE; red), and Cytotox NIR (blue) are shown after automated segmentation and background subtraction. Cells were seeded at 50,000 per well and imaged on an Incucyte SX5 (20×, every 2 h). Scale bar, 200 µm. p, Flow-cytometric contour plots of healthy-donor PBMCs after 48 h of EV exposure across a dose titration (1× = 3×10 8 EVs per 10 5 cells), showing CD25 (APC), CD123 (PE), and CD127 (FITC). Acquisition and compensation as in (m).

Article Snippet: Anti-CD25 antibody (Basiliximab, MedChemExpress, #HY-10885) was used at 10 μg/mL and compared to an isotype control (Invitrogen, #MA5-55090).

Techniques: Isolation, Expressing, Comparison, Marker, MANN-WHITNEY, Immunocytochemistry, Purification, Derivative Assay, Membrane, Single Cell, Fluorescence, Titration, Imaging, Transmission Assay, Electron Microscopy, Labeling, Control, Flow Cytometry, Incubation, Staining

Journal: bioRxiv

Article Title: Single recipient cell tracking of tellurium-labeled extracellular vesicle proteomes (TeLEV) identifies EV-driven immunomodulation

doi: 10.1101/2025.11.01.685872

Figure Lengend Snippet: a, t-SNE projection of 16 conditions (in total 3,590,520 cells) coloured according to cell type. Numerical annotations indicate the relative proportions of each cell lineage. MBC-EVs shown in were isolated from 8 mL of cell-conditioned medium (from 8M Ramos cells over 48 hours), resulting in 5 x 10 9 EVs used per EV condition. b, Stacked bar plot depicting changes in cell type composition. Colors correspond to those in the legend shown in (a). c, Comparison of TeLEV signal distribution across major cell types and their subtypes. Shown are EVs (217,211 cells), STAT5d + EVs (226,884 cells), and STAT3d + EVs (206,018 cells) conditions, with colors representing the average TeLEV signal level in each population. Bars above the plots show the relative proportions of each population in the corresponding condition (left). White lines denote the median, edges the IQR and whiskers either 1.5 × IQR or minima/maxima (if no point exceeded 1.5 × IQR; minima/maxima are indicated by the violin plot range). t-SNE projection of cells for each condition showing TeLEV signal (middle). Two-dimensional density distributions of cells in t-SNE embedding space, stratified by conditions (right). d, Comparison of TeLEV signal distribution across three conditions shown in (c). Pairwise comparisons were performed using a two-sided Mann–Whitney U test. **** p < 0.0001. e, t-SNE projection of myeloid population of 16 conditions (865,413 cells), colored according to subtypes. f, t-SNE plots showing expression of monocytic and DC lineage markers and interleukin receptors on myeloid cells. g-k, t-SNE plots showing CD25, CD127, CD123, BCL-2, and PD-L1 expressions across the indicated conditions. l, Comparison of PD-L1 expression distribution across the indicated conditions in Monocytes, mo-derived DCs, mDCs, and pDCs. For each cell type, statistical significance across all conditions was evaluated using the Kruskal-Wallis test.**** p < 0.0001. m, Same as in (l) for BCL-2. n-p, Comparison of CD25 (n), Ki-67 (o), and CD69 (p) expression distributions across major cell types and their subtypes, with colors representing the average respective marker expression level in each population. Shown are IL-2 (238,611 cells) and IL-2 + EVs (207,300 cells) conditions.

Article Snippet: Anti-CD25 antibody (Basiliximab, MedChemExpress, #HY-10885) was used at 10 μg/mL and compared to an isotype control (Invitrogen, #MA5-55090).

Techniques: Isolation, Comparison, MANN-WHITNEY, Expressing, Derivative Assay, Marker