alexafluor647 labeled anti pro insulin  (R&D Systems)

Journal: EMBO Molecular Medicine

Article Title: A new form of diabetes caused by INS mutations defined by zygosity, stem cell and population data

doi: 10.1038/s44321-025-00362-9

Figure Lengend Snippet: Unrelated control (black), heterozygous R6C (HET, yellow) and isogenic corrected (HET CORR, blue) iPSCs were differentiated into β cells. ( A ) iPSC-islet lysates were analyzed by SDS-PAGE under reducing conditions, electrotransfer to nitrocellulose, and immunoblotting with anti-human proinsulin. The blot was cropped and rearranged for clarity. The left panel is reused in Fig. as it represents a shared control. ( B ) Quantification of preproinsulin to proinsulin ratio from ( A ) (unrelated control n = 3, HET CORR n = 3, HET n = 4). ( C ) iPSC-islet lysates were resolved by nonreducing 12%-NuPAGE, and the completed gel was then treated with 100 mM DTT at 60 °C for 10 min before electrotransfer to nitrocellulose and immunoblotting with anti-proinsulin. n = 2. Medium from Min6 β cells transfected with human proinsulin was used as a positive control (INS, lane next to marker, M). The blot was cropped and rearranged for clarity. The left panel is reused in Fig. as it represents the shared marker and positive control. ( D ) Proinsulin and ( E ) insulin content (ng) normalized to total protein content (μg). ( F ) Proinsulin to insulin content ratio from ( D , E ). ( D – F ) HET CORR n = 4, HET n = 7. All panels: Unpaired t -test. In box plots, the median of independent experiments is shown by a horizontal line; 25 th and 75 th percentiles are at the bottom and top of the boxes; whiskers represent the minimum and maximum values. .

Article Snippet: Mouse anti-human proinsulin B-C junction sequence KTRREAEDLQ , Abmart , Cat# B-C junction; RRID: AB_2921300.

Techniques: Control, SDS Page, Electrotransfer, Western Blot, Transfection, Positive Control, Marker

Journal: EMBO Molecular Medicine

Article Title: A new form of diabetes caused by INS mutations defined by zygosity, stem cell and population data

doi: 10.1038/s44321-025-00362-9

Figure Lengend Snippet: Unrelated control (black), homozygous R6C (HOM, pink) and isogenic corrected (HOM CORR, blue) iPSCs were differentiated into β cells. ( A ) iPSC-islet lysates were analyzed by SDS-PAGE under reducing conditions, electrotransferred to nitrocellulose, and immunoblotted with anti-human proinsulin. The blot was cropped and rearranged for clarity. The left panel is reused as in Fig. , as it represents a shared control. ( B ) Quantification of preproinsulin to proinsulin ratio from ( A ) (unrelated control n = 3, HOM CORR n = 3, HOM n = 8) and ( C ) of homozygous R6C iPSC-β cells treated with vehicle DMSO or MG132 (10 μM) for 30 min ( n = 4). ( D ) iPSC-islet lysates were resolved by nonreducing 12%-NuPAGE and the completed gel then treated with 100 mM DTT at 60 °C for 10 min before electrotransfer to nitrocellulose and immunoblotting with anti-proinsulin. n = 2. Medium from Min6 β cells transfected with human proinsulin was used as a positive control (INS, lane next to marker, M). The blot was cropped and rearranged for clarity. The left panel is reused as in Fig. as it represents a shared marker and a positive control. ( E ) Proinsulin and ( F ) insulin content normalized to total protein content. ( G ) Proinsulin to insulin content ratio from ( E , F ). ( E – G ) HOM CORR n = 8, HOM n = 12–13. ( H ) Insulin content normalized to total protein content along stage 7 (S7, HOM CORR n = 5, HOM n = 12), 1 week (LC1W, HOM CORR n = 3, HOM n = 3), 2 weeks (LC2W, HOM CORR n = 3, HOM n = 3), 3 weeks (LC3W, HOM CORR n = 3, HOM n = 3), and 4 weeks (LC4W) of long culture (LC, HOM CORR n = 12, HOM n = 15). All panels: Unpaired t -test. In box plots, the median of independent experiments is shown by a horizontal line; 25 th and 75 th percentiles are at the bottom and top of the boxes; whiskers represent the minimum and maximum values. .

Article Snippet: Mouse anti-human proinsulin B-C junction sequence KTRREAEDLQ , Abmart , Cat# B-C junction; RRID: AB_2921300.

Techniques: Control, SDS Page, Electrotransfer, Western Blot, Transfection, Positive Control, Marker

Journal: EMBO Molecular Medicine

Article Title: A new form of diabetes caused by INS mutations defined by zygosity, stem cell and population data

doi: 10.1038/s44321-025-00362-9

Figure Lengend Snippet: Heterozygous R6C (HET, yellow) and isogenic corrected (HET CORR, blue) iPSCs were differentiated into long-cultured β cells. ( A – C ) Static proinsulin and insulin secretion in response to 2.8 mM glucose (G2.8), 16.8 mM glucose (G16.8), or 16.8 mM glucose plus 10 μM forskolin (G16.8 + Fk). HET CORR n = 4, HET n = 7. ( A ) Proinsulin and ( B ) insulin secretion (ng) normalized to protein content (μg). ( C ) Proinsulin to insulin ratio from ( A , B ). ( D – H ) Dynamic insulin secretion upon perifusion with 2.8 mM glucose, 16.8 mM glucose (G16.8), G16.8 plus exendin-4 (Ex4, 50 ng/mL, 11.8 nM) or G2.8 plus KCl (30 mM). HET CORR n = 3, HET n = 4. ( D ) Insulin secretion normalized to protein content, with ( E – H ) area under the curve (AUC) per minute of secretion at G2.8, G16.8, G16.8 + Ex4, and G2.8 + KCl. All panels: Unpaired t -test. In box plots, the median of independent experiments is shown by a horizontal line; 25 th and 75 th percentiles are at the bottom and top of the boxes; whiskers represent the minimum and maximum values. In time course line plots, data are shown as mean ± s.e.m. .

Article Snippet: Mouse anti-human proinsulin B-C junction sequence KTRREAEDLQ , Abmart , Cat# B-C junction; RRID: AB_2921300.

Techniques: Cell Culture

Journal: EMBO Molecular Medicine

Article Title: A new form of diabetes caused by INS mutations defined by zygosity, stem cell and population data

doi: 10.1038/s44321-025-00362-9

Figure Lengend Snippet: Homozygous R6C (HOM, pink) and isogenic corrected (HOM CORR, blue) iPSCs were differentiated into long-cultured β cells. ( A – C ) Static proinsulin and insulin secretion in response to 2.8 mM glucose (G2.8), 16.8 mM glucose (G16.8), or 16.8 mM glucose plus 10 μM forskolin (G16.8 + Fk). HOM CORR n = 8, HOM n = 12 (for proinsulin), HOM n = 13 (for insulin). ( A ) Proinsulin and ( B ) insulin secretion normalized to protein content. ( C ) Proinsulin to insulin ratio from ( A , B ). ( D – I ) Dynamic insulin secretion upon perifusion with 2.8 mM glucose, 16.8 mM glucose (G16.8), G16.8 plus exendin-4 (Ex4, 50 ng/mL, 11.8 nM), or G2.8 plus KCl (30 mM). HOM CORR n = 12, HOM n = 14. ( D ) Insulin secretion normalized to protein content, with ( E ) zoom in on 16.8 mM glucose response. ( F – I ) Area under the curve (AUC) per minute of secretion at G2.8, G16.8, G16.8 + Ex4 and G2.8 + KCl. All panels: Unpaired t -test. In box plots, the median of independent experiments is shown by a horizontal line; 25 th and 75 th percentiles are at the bottom and top of the boxes; whiskers represent the minimum and maximum values. In time course line plots, data are shown as mean ± s.e.m. .

Article Snippet: Mouse anti-human proinsulin B-C junction sequence KTRREAEDLQ , Abmart , Cat# B-C junction; RRID: AB_2921300.

Techniques: Cell Culture

Journal: Molecular Metabolism

Article Title: Beta-cell-specific C3 deficiency exacerbates metabolic dysregulation and insulin resistance in obesity

doi: 10.1016/j.molmet.2025.102302

Figure Lengend Snippet: A) Pancreas sections from treated mice were stained for glucagon (green) and insulin (red) to visualize islets and assess their size. Blue: DAPI. Scale bars, 50 μm. B) Quantification of total islet size in sections from 22 islets from pancreas sections of 7 LFD beta-C3-KO mice, 33 islets from pancreas sections of 5 HFD beta-C3-KO mice, and 35 islets from pancreas sections of 6 HFD C3-flox mice. C) Fraction of total islet staining for insulin, from the same islets analysed in (B). D) Examples of staining of individual HFD islets for glucagon (green) and pro-insulin (red). E) Quantification of pro-insulin staining in islets from each group of mice. F) Quantification of pro-insulin in serum of mice at given time points, measured by pro-insulin specific ELISA. G) Transmission electron micrographs of pancreatic islet cells isolated from beta-C3-KO or C3-flox mice. Beta-C3-KO mouse beta-cells contain accumulated amounts of swollen ER or similar vesicular organelles (black arrows), while normal ER is apparent in C3-flox mouse islet cells (white arrows). See also . Increased amounts of autophagic material were also observed in beta-C3-KO cells (asterisk). H) In vitro insulin secretion from isolated islets from C3-flox or beta-C3-KO mice HFD-fed mice, at 15 or 60 min. I) Average insulin content values per islet from secretion experiment in panel (H) (average values shown for each of 6 mice: from 6 wells, 5 islets per well). J) Serum C3 levels in C3-flox or beta-C3-KO mice at the endpoint of HFD, as measured by ELISA. Statistics in E, one-way ANOVA, in F/H, two-way ANOVA, in I, Student’s T-test, and in B/C, by mixed model statistical analysis (see methods).

Article Snippet: For pro-insulin staining, AlexaFluor647-labeled anti-pro-insulin (R&D Systems, #IC13361R, 1:100) was incubated overnight at 4 °C, in combination with glucagon staining, before washing and mounting as described above.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Transmission Assay, Isolation, In Vitro

Journal: Science Advances

Article Title: AI-directed gene fusing prolongs the evolutionary half-life of synthetic gene circuits

doi: 10.1126/sciadv.adx0796

Figure Lengend Snippet: ( A ) Design of the evolution experiment. The schematics of the variants in the first proinsulin evolution experiment are displayed for clarity. ( B ) Gene fusion affects expression at time zero. ELISA measurements of proinsulin expression in six configurations—unfused or fused to CAF20 or ARC15 with or without a leader sequence—at day 0. As previously shown, the leader sequence is essential for expressing unfused proinsulin. For fusion constructs, it substantially improves expression, potentially due to increased mRNA stability or improved protein localization in the cell. ( C ) Proinsulin production over time. ELISA measurements were taken every 5 days ( n = 3 per time point) for four variants: (i) baseline—Novo Nordisk’s patented sequence, integrated at the CAN1 locus; (ii) optimized—same structure with proinsulin and linker optimized by ESO ; (iii and iv) fusions—proinsulin fused to CAF20 or ARC15 . Fusion genes were synthesized using the full STABLES pipeline (EG selection, linker choice, leader inclusion, and codon optimization). Expression decay over time fits an exponential model, supported by high R 2 in the log-linear space ( P < 10 − 6 ). The variants exhibit significantly different decay rates [analysis of variance (ANOVA) F -test, P < 10 − 19 ], with the STABLES-designed constructs showing substantially improved stability. ( D ) Normalized cumulative proinsulin production. Integrating the fitted expression curves yields the cumulative expression per variant. All values were normalized by the 10-day cumulative expression of the baseline variant. STABLES-derived variants exhibited greatly increased cumulative yields across time points, illustrating improved production through rational EG fusion design.

Article Snippet: The proinsulin sequence was obtained from Novo Nordisk patented sequence for proinsulin manufacturing in yeast (patent WO2014195452A1).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Sequencing, Construct, Synthesized, Selection, Variant Assay, Derivative Assay

Journal: Science Advances

Article Title: AI-directed gene fusing prolongs the evolutionary half-life of synthetic gene circuits

doi: 10.1126/sciadv.adx0796

Figure Lengend Snippet: ( A ) Mutation accumulated in the in-lab evolution experiment. Nanopore sequencing revealed widespread deletions following in-lab evolution: (a) Unfused proinsulin lost most of its promoter and coding sequence; (b) ARC15 fusion showed large promoter deletions and smaller mutations in both the promoter and proinsulin; (c) CAF20 fusion retained most of its structure, with only minor mutations across the construct. ( B ) Western blot validation. Western blotting confirms that CAF20 and ARC15 fusions yield prolonged proinsulin expression compared to unfused constructs. Actin measurements were taken for control. ( C ) Leaky stop codon construct design. Schematic of the construct used to assess translation read-through rates of leaky stop codons using BFP and mCherry fusion. ( D ) Termination efficiency of leaky stop codons. Various constructs were tested, each linking BFP and mCherry via different leaky stop codons. The fluorescence intensity of each fluorophore (normalized to standalone expression) served as a proxy for read-through efficiency. Three top-performing designs [L1 to L3; see (E)] and three rejected variants (with too high or low read-through) are shown. The BFP signal represents GOI expression, and mCherry represents the C-terminal EG. ( E ) Proinsulin expression for different leaky stop codons. ARC15 fusions were built with three different leaky stop codons and without them as the control. ELISA measurements over 50 days demonstrated that all leaky stop codons improved protein stability (Student’s t test at t = 40, P < 10 − 5 ). Distinct expression profiles among codons (Kruskal H test, P < 0.003 ) emphasize the need for informed selection. All selected codons preserve the last amino acid of proinsulin, use the design principles outlined in , and displayed a read-through rate of 0.1 to 0.25 in the previous experiment. Sequences used (stop codon + 3 downstream nt): L1 – TAGGCG; L2 – TGAGCG; L3 – TGACAA.

Article Snippet: The proinsulin sequence was obtained from Novo Nordisk patented sequence for proinsulin manufacturing in yeast (patent WO2014195452A1).

Techniques: Mutagenesis, Nanopore Sequencing, Sequencing, Construct, Western Blot, Biomarker Discovery, Expressing, Control, Fluorescence, Enzyme-linked Immunosorbent Assay, Selection

Journal: Science Advances

Article Title: AI-directed gene fusing prolongs the evolutionary half-life of synthetic gene circuits

doi: 10.1126/sciadv.adx0796

Figure Lengend Snippet: ( A ) Design of the evolution experiment. The schematics of the variants in the first proinsulin evolution experiment are displayed for clarity. ( B ) Gene fusion affects expression at time zero. ELISA measurements of proinsulin expression in six configurations—unfused or fused to CAF20 or ARC15 with or without a leader sequence—at day 0. As previously shown, the leader sequence is essential for expressing unfused proinsulin. For fusion constructs, it substantially improves expression, potentially due to increased mRNA stability or improved protein localization in the cell. ( C ) Proinsulin production over time. ELISA measurements were taken every 5 days ( n = 3 per time point) for four variants: (i) baseline—Novo Nordisk’s patented sequence, integrated at the CAN1 locus; (ii) optimized—same structure with proinsulin and linker optimized by ESO ; (iii and iv) fusions—proinsulin fused to CAF20 or ARC15 . Fusion genes were synthesized using the full STABLES pipeline (EG selection, linker choice, leader inclusion, and codon optimization). Expression decay over time fits an exponential model, supported by high R 2 in the log-linear space ( P < 10 − 6 ). The variants exhibit significantly different decay rates [analysis of variance (ANOVA) F -test, P < 10 − 19 ], with the STABLES-designed constructs showing substantially improved stability. ( D ) Normalized cumulative proinsulin production. Integrating the fitted expression curves yields the cumulative expression per variant. All values were normalized by the 10-day cumulative expression of the baseline variant. STABLES-derived variants exhibited greatly increased cumulative yields across time points, illustrating improved production through rational EG fusion design.

Article Snippet: The strains tested were the original proinsulin (as patented by Novo Nordisk), the proinsulin as optimized by the Evolutionary Stability Optimizer (ESO) , and the optimized sequence fused to CAF20 and ARC15 as fusion genes ( ).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Sequencing, Construct, Synthesized, Selection, Variant Assay, Derivative Assay

Journal: Science Advances

Article Title: AI-directed gene fusing prolongs the evolutionary half-life of synthetic gene circuits

doi: 10.1126/sciadv.adx0796

Figure Lengend Snippet: ( A ) Mutation accumulated in the in-lab evolution experiment. Nanopore sequencing revealed widespread deletions following in-lab evolution: (a) Unfused proinsulin lost most of its promoter and coding sequence; (b) ARC15 fusion showed large promoter deletions and smaller mutations in both the promoter and proinsulin; (c) CAF20 fusion retained most of its structure, with only minor mutations across the construct. ( B ) Western blot validation. Western blotting confirms that CAF20 and ARC15 fusions yield prolonged proinsulin expression compared to unfused constructs. Actin measurements were taken for control. ( C ) Leaky stop codon construct design. Schematic of the construct used to assess translation read-through rates of leaky stop codons using BFP and mCherry fusion. ( D ) Termination efficiency of leaky stop codons. Various constructs were tested, each linking BFP and mCherry via different leaky stop codons. The fluorescence intensity of each fluorophore (normalized to standalone expression) served as a proxy for read-through efficiency. Three top-performing designs [L1 to L3; see (E)] and three rejected variants (with too high or low read-through) are shown. The BFP signal represents GOI expression, and mCherry represents the C-terminal EG. ( E ) Proinsulin expression for different leaky stop codons. ARC15 fusions were built with three different leaky stop codons and without them as the control. ELISA measurements over 50 days demonstrated that all leaky stop codons improved protein stability (Student’s t test at t = 40, P < 10 − 5 ). Distinct expression profiles among codons (Kruskal H test, P < 0.003 ) emphasize the need for informed selection. All selected codons preserve the last amino acid of proinsulin, use the design principles outlined in , and displayed a read-through rate of 0.1 to 0.25 in the previous experiment. Sequences used (stop codon + 3 downstream nt): L1 – TAGGCG; L2 – TGAGCG; L3 – TGACAA.

Article Snippet: The strains tested were the original proinsulin (as patented by Novo Nordisk), the proinsulin as optimized by the Evolutionary Stability Optimizer (ESO) , and the optimized sequence fused to CAF20 and ARC15 as fusion genes ( ).

Techniques: Mutagenesis, Nanopore Sequencing, Sequencing, Construct, Western Blot, Biomarker Discovery, Expressing, Control, Fluorescence, Enzyme-linked Immunosorbent Assay, Selection