injection mix  (World Precision Instruments)

Journal: bioRxiv

Article Title: An optogenetic toolkit for robust activation of FGF, BMP, & Nodal signaling in zebrafish

doi: 10.1101/2025.04.17.649426

Figure Lengend Snippet: A,B,C) Schematic of constructs used here to activate FGF (A), BMP (B), and Nodal (C) signaling. Myr = myristoylation motif, GS = glycine/serine linker, LOV = light oxygen voltage-sensing domain, HA = hemagglutinin tag, FLAG = FLAG epitope. The single-transcript bOpto-2A-Nodal construct (C) follows the same design as -FGF and -BMP, except the type I (Acvr1ba) and type II (Acvr2ba) components are connected via a 2A peptide sequence (gray). A’,B’,C’) Optogenetic strategy to activate FGF (A’), BMP (B’), and Nodal (C’) signaling. Blue light-dimerizing LOV domains are fused to myristoylated receptor kinase domains. Blue light exposure should lead to receptor kinase interactions, signaling effector phosphorylation, and activation of target genes.

Article Snippet: The mRNA amounts injected for each optogenetic tool are listed below: bOpto-FGF (Addgene #232639): 3.5 pg bOpto-BMP (combined in injection mix) bOpto-Bmpr1aa (Addgene # 207614): 23.4 pg bOpto-Acvr1l (Addgene # 207615): 23.4 pg bOpto-Bmpr2a (Addgene # 207616): 40.2 pg

Techniques: Construct, FLAG-tag, Sequencing, Phospho-proteomics, Activation Assay

Journal: bioRxiv

Article Title: An optogenetic toolkit for robust activation of FGF, BMP, & Nodal signaling in zebrafish

doi: 10.1101/2025.04.17.649426

Figure Lengend Snippet: Measured spectra for A) the 455 nm light used in uniform blue light exposure experiments (all figures except ), B) the 495+ nm used in and Supp. Figs. 2, 3, 4, & 12, and C) the 600 nm light source used to avoid inadvertent signaling activation during handling of embryos expressing bOpto constructs.

Article Snippet: The mRNA amounts injected for each optogenetic tool are listed below: bOpto-FGF (Addgene #232639): 3.5 pg bOpto-BMP (combined in injection mix) bOpto-Bmpr1aa (Addgene # 207614): 23.4 pg bOpto-Acvr1l (Addgene # 207615): 23.4 pg bOpto-Bmpr2a (Addgene # 207616): 40.2 pg

Techniques: Activation Assay, Expressing, Construct

Journal: bioRxiv

Article Title: An optogenetic toolkit for robust activation of FGF, BMP, & Nodal signaling in zebrafish

doi: 10.1101/2025.04.17.649426

Figure Lengend Snippet: A) Uninjected (-) embryos and embryos injected (+) at the one-cell stage with the indicated mRNA were exposed to dark, 495+ nm light (18.51 W/m 2 ), or 455 nm light (50 W/m 2 ) starting ∼2 hours post-fertilization (hpf). Phenotypes were scored at 1 day post-fertilization (dpf) (N = 3; ; Mean +/- SD). B,C,D) Uninjected embryos and embryos injected with the indicated bOpto + GFP mRNA were exposed to dark, 495+ nm light, or 455 nm light starting at early gastrulation (50% epiboly - shield) for 30 min. HCR-IF was used to detect phosphorylated signaling effectors (ppERK, pSmad1, and pSmad2 reflect FGF, BMP, and Nodal signaling, respectively). Scale bar is 200 µm. B’,C’,D’) Quantification of experiments shown in B,C,D. Linear-mixed model-predicted least squared means of GFP-normalized phosphorylated effector signal +/- SEM. D’ shows nuclear signal only. (N = 3, * indicates p < 0.05; & ).

Article Snippet: The mRNA amounts injected for each optogenetic tool are listed below: bOpto-FGF (Addgene #232639): 3.5 pg bOpto-BMP (combined in injection mix) bOpto-Bmpr1aa (Addgene # 207614): 23.4 pg bOpto-Acvr1l (Addgene # 207615): 23.4 pg bOpto-Bmpr2a (Addgene # 207616): 40.2 pg

Techniques: Injection

Journal: bioRxiv

Article Title: An optogenetic toolkit for robust activation of FGF, BMP, & Nodal signaling in zebrafish

doi: 10.1101/2025.04.17.649426

Figure Lengend Snippet: A-C”) Uninjected embryos (-) and embryos injected (+) at the one-cell stage with the bOp- to-FGF (A-A”), bOpto-BMP (B-B”), or bOpto-2A-Nodal (C-C”) mRNA were exposed to dark, 495+ nm light (18.51 W/m 2 ), or 455 nm light (50 W/m 2 ) starting 1.5-2 hours post-fertilization. Phenotypes were scored at 1 day post-fertilization. Individual repeats are shown. Numbers indicate the total number of embryos scored in each condition for that experiment. shows the combined data from these three repeats. Phenotype legend images shown here are the same images shown in .

Article Snippet: The mRNA amounts injected for each optogenetic tool are listed below: bOpto-FGF (Addgene #232639): 3.5 pg bOpto-BMP (combined in injection mix) bOpto-Bmpr1aa (Addgene # 207614): 23.4 pg bOpto-Acvr1l (Addgene # 207615): 23.4 pg bOpto-Bmpr2a (Addgene # 207616): 40.2 pg

Techniques: Injection

Journal: bioRxiv

Article Title: An optogenetic toolkit for robust activation of FGF, BMP, & Nodal signaling in zebrafish

doi: 10.1101/2025.04.17.649426

Figure Lengend Snippet: Uninjected embryos (-) and embryos injected (+) at the one-cell stage with mRNA encoding bOpto-FGF (A) , bOp- to-BMP (B) , or bOpto-2A-Nodal (C) were exposed to 455 nm light (50 W/m 2 , bottom rows) or dark (top rows) for 2 hours starting before gastrulation (dome - 30% epiboly). Multiplexed HCR-FISH was used to detect expression of the indicated pathway target genes. (N = 3; Scale bar is 200 µm).

Article Snippet: The mRNA amounts injected for each optogenetic tool are listed below: bOpto-FGF (Addgene #232639): 3.5 pg bOpto-BMP (combined in injection mix) bOpto-Bmpr1aa (Addgene # 207614): 23.4 pg bOpto-Acvr1l (Addgene # 207615): 23.4 pg bOpto-Bmpr2a (Addgene # 207616): 40.2 pg

Techniques: Injection, Expressing

Journal: bioRxiv

Article Title: An optogenetic toolkit for robust activation of FGF, BMP, & Nodal signaling in zebrafish

doi: 10.1101/2025.04.17.649426

Figure Lengend Snippet: Uninjected embryos (-) and embryos injected (+) at the one-cell stage with mRNA encoding bOpto-FGF (A) , bOpto-BMP (B) , or bOpto-2A-Nodal (C) were exposed to dark (left panel) or 455 nm light (50 W/m 2 , right panel) for 30 minutes starting at gastrulation (50% epiboly - shield). Triple IF was used to simultaneously detect phosphorylated signaling effectors (ppERK, pSmad1, and pSmad2 reflect FGF, BMP, and Nodal signaling, respectively). (N = 3; ; Scale bar is 200 µm).

Article Snippet: The mRNA amounts injected for each optogenetic tool are listed below: bOpto-FGF (Addgene #232639): 3.5 pg bOpto-BMP (combined in injection mix) bOpto-Bmpr1aa (Addgene # 207614): 23.4 pg bOpto-Acvr1l (Addgene # 207615): 23.4 pg bOpto-Bmpr2a (Addgene # 207616): 40.2 pg

Techniques: Injection

Journal: bioRxiv

Article Title: An optogenetic toolkit for robust activation of FGF, BMP, & Nodal signaling in zebrafish

doi: 10.1101/2025.04.17.649426

Figure Lengend Snippet: A-E) Embryos injected at the one-cell stage with mRNA encoding bOpto-2A-Nodal exposed to dark (left panel) or 455 nm light (50 W/m 2 , right panel) starting at early gastrulation (50% epiboly - shield stage) for 30 min. Triple IF staining was used to simultaneously detect activated signaling effectors (pSmad1, ppERK, and pSmad2 reflecting BMP, FGF, and Nodal signaling, respectively). Only ppERK and pSmad2 shown here. Three bOpto-2A-Nodal-expressing embryos per condition from five experimental repeats are shown. (Scale bar is 200 µm).

Article Snippet: The mRNA amounts injected for each optogenetic tool are listed below: bOpto-FGF (Addgene #232639): 3.5 pg bOpto-BMP (combined in injection mix) bOpto-Bmpr1aa (Addgene # 207614): 23.4 pg bOpto-Acvr1l (Addgene # 207615): 23.4 pg bOpto-Bmpr2a (Addgene # 207616): 40.2 pg

Techniques: Injection, Staining, Expressing

Journal: bioRxiv

Article Title: An optogenetic toolkit for robust activation of FGF, BMP, & Nodal signaling in zebrafish

doi: 10.1101/2025.04.17.649426

Figure Lengend Snippet: Embryos were injected at the one-cell stage with mRNA encoding GFP and either bOpto-FGF , bOpto-BMP , or bOp- to-2A-Nodal . A,B,C) Starting at early gastrulation (50% epiboly - shield), embryos were either kept in the dark (top row) or exposed to 455 nm light (50 W/m 2 ) light for 30 min (bottom row) and fixed during and after exposure. HCR-IF was used to detect activated signaling effectors (ppERK, pSmad1, and pSmad2 reflect FGF, BMP, and Nodal signaling, respectively). (N = 3; Scale bar is 200 µm). A’,B’,C’) Quantification of experiments shown in A-C. Signal was GFP-normalized and subtracted against dark time-matched controls (N = 3; each N indicated by matched shape; & ; Mean +/- SD; * indicates p < 0.05 when compared to time = 0 min). A’’- C’’, A’’’-C’’’) A three-parameter logistic regression was fit to the 0-30 min data (A’’-C’’) or the >= 30 min data (A’’’-C’’’). (N = 3; Mean +/- SD; Supplementary Materials; solid line represents predicted curve fit

Article Snippet: The mRNA amounts injected for each optogenetic tool are listed below: bOpto-FGF (Addgene #232639): 3.5 pg bOpto-BMP (combined in injection mix) bOpto-Bmpr1aa (Addgene # 207614): 23.4 pg bOpto-Acvr1l (Addgene # 207615): 23.4 pg bOpto-Bmpr2a (Addgene # 207616): 40.2 pg

Techniques: Injection

Journal: bioRxiv

Article Title: An optogenetic toolkit for robust activation of FGF, BMP, & Nodal signaling in zebrafish

doi: 10.1101/2025.04.17.649426

Figure Lengend Snippet: A-C) Embryos were injected at the one-cell stage with mRNA encoding GFP and either bOp- to-FGF (A), bOpto-BMP (B), or bOpto-2A-Nodal (C). Starting at early gastrulation (50% epiboly - shield), embryos were exposed to 455 nm light (50 W/m 2 ) for 30 min and fixed at different time points during and after exposure. HCR-IF staining used to detect activated signaling effectors is shown in (ppERK, pSmad1, and pSmad2 reflect FGF, BMP, and Nodal signaling, respectively). HCR-IF signal was normalized voxel-wise based on co-injected GFP intensity shown here (see Supp. Fig. 7). GFP images correspond to representative IF images shown in . (Scale bar is 200 µm).

Article Snippet: The mRNA amounts injected for each optogenetic tool are listed below: bOpto-FGF (Addgene #232639): 3.5 pg bOpto-BMP (combined in injection mix) bOpto-Bmpr1aa (Addgene # 207614): 23.4 pg bOpto-Acvr1l (Addgene # 207615): 23.4 pg bOpto-Bmpr2a (Addgene # 207616): 40.2 pg

Techniques: Injection, Staining

Journal: bioRxiv

Article Title: An optogenetic toolkit for robust activation of FGF, BMP, & Nodal signaling in zebrafish

doi: 10.1101/2025.04.17.649426

Figure Lengend Snippet: Embryos were injected at the one-cell stage with mRNA encoding GFP and either bOpto-FGF (A,A’), bOpto-BMP (B,B’), or bOpto-2A-Nodal (C,C’). Starting at early gastrulation (50% epiboly - shield), embryos were exposed to 455 nm light (50 W/m 2 ) for 30 min and fixed during and after exposure. Three full repeats were performed across 3-5 trials. HCR-IF staining used to detect phosphorylated signaling effectors is shown in (ppERK, pSmad1, and pSmad2 reflect FGF, BMP, and Nodal signaling, respectively). A,B,C) Raw phosphorylated signaling effector intensity was measured in each DAPI-positive nuclear pixel. Each dot represents the median nuclear pixel intensity in one embryo. Black lines represent the mean nuclear intensity of all embryos in the indicated condition. A’,B’) Phosphorylated siganling effector intensity in each GFP-positive pixel was divided by the corresponding GFP intensity. Each dot represents the median GFP-normalized pixel signal in one embryo. Black lines represent the mean GFP-normalized signal of all embryos in the indicated condition. C’) pSmad2 intensity in each DAPI + GFP-positive pixel was divided by the corresponding GFP intensity. Each dot represents the median GFP-normalized nuclear pixel signal in one embryo. Black lines represent the mean GFP-normalized nuclear signal of all embryos in the indicated condition.

Article Snippet: The mRNA amounts injected for each optogenetic tool are listed below: bOpto-FGF (Addgene #232639): 3.5 pg bOpto-BMP (combined in injection mix) bOpto-Bmpr1aa (Addgene # 207614): 23.4 pg bOpto-Acvr1l (Addgene # 207615): 23.4 pg bOpto-Bmpr2a (Addgene # 207616): 40.2 pg

Techniques: Injection, Staining

Journal: bioRxiv

Article Title: An optogenetic toolkit for robust activation of FGF, BMP, & Nodal signaling in zebrafish

doi: 10.1101/2025.04.17.649426

Figure Lengend Snippet: A) Table of irradiance and corresponding light dosage values used in B-D’. B,C,D) Embryos were injected at the one-cell stage with mRNA encoding GFP and either bOpto-FGF (B), bOpto-BMP (C), or bOpto-2A-Nodal (D). Starting at early gastrulation (50% epiboly - shield), embryos were exposed to 455 nm light at the indicated irradiance for 5 min (bOp- to-FGF, B) or 25 min (bOpto-BMP and -2A-Nodal, C and D). HCR-IF was used to detect phosphorylated signaling effectors (ppERK, pSmad1, and pSmad2 reflect FGF, BMP, and Nodal signaling, respectively; Scale bar is 200 µm). B’,C’,D’) Quantification of experiments in B-D. Signal was GFP-normalized and subtracted against dark controls. A three-parameter logistic regression was fit to the data. (N = 2-3; each N indicated by matched shape; Mean +/- SD; & ; solid line represents predicted curve fit +/- 95% CI; D’ shows nuclear signal only). Tables indicate goodness of fit (R 2 ), the predicted irradiance (with 95% CI) at which 20 (I 20 ), 50 (I 50 ), and 90% (I 90 ) of the curve’s upper asymptote is reached (Supplementary Materials).

Article Snippet: The mRNA amounts injected for each optogenetic tool are listed below: bOpto-FGF (Addgene #232639): 3.5 pg bOpto-BMP (combined in injection mix) bOpto-Bmpr1aa (Addgene # 207614): 23.4 pg bOpto-Acvr1l (Addgene # 207615): 23.4 pg bOpto-Bmpr2a (Addgene # 207616): 40.2 pg

Techniques: Injection

Journal: bioRxiv

Article Title: An optogenetic toolkit for robust activation of FGF, BMP, & Nodal signaling in zebrafish

doi: 10.1101/2025.04.17.649426

Figure Lengend Snippet: A-C) Embryos were injected at the one-cell stage with mRNA encoding GFP and either bOpto-FGF (A), bOpto-BMP (B), or bOp- to-2A-Nodal (C). Starting at early gastrulation (50% epiboly - shield), embryos were exposed to 455 nm light (50 W/m 2 ) at the indicated irradiances for 5 min (bOp- to-FGF) or 25 min (bOpto-BMP and -2A-Nodal). HCR-IF staining used to detect phosphorylated signaling effectors is shown in (ppERK, pSmad1, and pSmad2 to reflect FGF, BMP, and Nodal signaling, respectively). HCR-IF signal was normalized based on co-injected GFP intensity shown here; GFP images correspond to representative images shown in . (Scale bar is 200 µm).

Article Snippet: The mRNA amounts injected for each optogenetic tool are listed below: bOpto-FGF (Addgene #232639): 3.5 pg bOpto-BMP (combined in injection mix) bOpto-Bmpr1aa (Addgene # 207614): 23.4 pg bOpto-Acvr1l (Addgene # 207615): 23.4 pg bOpto-Bmpr2a (Addgene # 207616): 40.2 pg

Techniques: Injection, Staining

Journal: bioRxiv

Article Title: An optogenetic toolkit for robust activation of FGF, BMP, & Nodal signaling in zebrafish

doi: 10.1101/2025.04.17.649426

Figure Lengend Snippet: Embryos were injected at the one-cell stage with mRNA encoding GFP and either bOpto-FGF (A,A’), bOpto-BMP (B,B’), or bOpto-2A-Nodal (C,C’). At early gastrulation (50% epiboly - shield), embryos were exposed to 455 nm light (50 W/m 2 ) at the indicated irradiance for 5 min (bOpto-FGF) or 25 min (bOpto-BMP and -2A-Nodal). HCR-IF was used to detect phosphorylated signaling effectors (ppERK, pSmad1, and pSmad2 reflect FGF, BMP, and Nodal signaling, respectively). A,B,C) Raw phosphorylated signaling effector intensity was measured in each DAPI-positive nuclear pixel. Each dot represents the median nuclear pixel intensity in one embryo. Black lines represent the mean nuclear intensity of all embryos in the indicated condition. A’,B’) Phosphorylated siganling effector intensity in each GFP-positive pixel was divided by the corresponding GFP intensity. Each dot represents the median GFP-normalized pixel signal in one embryo. Black lines represent the mean GFP-normalized signal of all embryos in the indicated condition. C’) pSmad2 intensity in each DAPI + GFP-positive pixel was divided by the corresponding GFP intensity. Each dot represents the median GFP-normalized nuclear pixel signal in one embryo. Black lines represent the mean GFP-normalized nuclear signal of all embryos in the indicated condition.

Article Snippet: The mRNA amounts injected for each optogenetic tool are listed below: bOpto-FGF (Addgene #232639): 3.5 pg bOpto-BMP (combined in injection mix) bOpto-Bmpr1aa (Addgene # 207614): 23.4 pg bOpto-Acvr1l (Addgene # 207615): 23.4 pg bOpto-Bmpr2a (Addgene # 207616): 40.2 pg

Techniques: Injection

Journal: bioRxiv

Article Title: An optogenetic toolkit for robust activation of FGF, BMP, & Nodal signaling in zebrafish

doi: 10.1101/2025.04.17.649426

Figure Lengend Snippet: Embryos were injected at the one-cell stage with mRNA encoding the green-to-red photoconvertible fluorescent protein nls-Kaede and bOpto-FGF . At early gastrulation (shield) embryos were either kept in the dark (A) or illuminated locally with 405 and 445 nm confocal lasers (B,B’) . HCR-IF was used to detect ppERK. Dotted white line in B’ outlines photoconverted Kaede in embryo shown in B. ( ; Scale bar is 200 µm).

Article Snippet: The mRNA amounts injected for each optogenetic tool are listed below: bOpto-FGF (Addgene #232639): 3.5 pg bOpto-BMP (combined in injection mix) bOpto-Bmpr1aa (Addgene # 207614): 23.4 pg bOpto-Acvr1l (Addgene # 207615): 23.4 pg bOpto-Bmpr2a (Addgene # 207616): 40.2 pg

Techniques: Injection

Journal: bioRxiv

Article Title: An optogenetic toolkit for robust activation of FGF, BMP, & Nodal signaling in zebrafish

doi: 10.1101/2025.04.17.649426

Figure Lengend Snippet: Embryos were injected at the one-cell stage with mRNA encoding the green-to-red photoconvertible fluorescent proteins nls-Kaede and bOpto-FGF . At early gastrulation embryos were either kept in the dark ( A-C, J-L, S-U ) or illuminated locally with 405 and 445 nm confocal lasers. HCR-IF was used to detect ppERK. Two representative exposed embryos from three repeats shown ( D-I’, M-R’, V-AA’ , respectively). Dotted white lines outline photoconverted Kaede from images to the left. A-F’ are shown in . (Scale bar is 200 µm).

Article Snippet: The mRNA amounts injected for each optogenetic tool are listed below: bOpto-FGF (Addgene #232639): 3.5 pg bOpto-BMP (combined in injection mix) bOpto-Bmpr1aa (Addgene # 207614): 23.4 pg bOpto-Acvr1l (Addgene # 207615): 23.4 pg bOpto-Bmpr2a (Addgene # 207616): 40.2 pg

Techniques: Injection

Journal: Frontiers in Pharmacology

Article Title: Pharmacokinetics and pharmacodynamics of insulin lispro 25 versus the original preparation (Humalog ® 25) in Chinese healthy male volunteers

doi: 10.3389/fphar.2025.1533548

Figure Lengend Snippet: Mean (±SD) plasma concentration–time curves (profile of changes in the blood concentration over time) (A) and semi-logarithmic curves (B) of the test and reference products of insulin lispro 25.

Article Snippet: The test (T) formulation was a mixed protamine zinc recombinant human insulin lispro injection (3 mL: 300U), provided by Tonghua Dongbao Pharmaceutical Co., Ltd. (lot number: 2L12020100052).

Techniques: Clinical Proteomics, Concentration Assay

Journal: Frontiers in Pharmacology

Article Title: Pharmacokinetics and pharmacodynamics of insulin lispro 25 versus the original preparation (Humalog ® 25) in Chinese healthy male volunteers

doi: 10.3389/fphar.2025.1533548

Figure Lengend Snippet: Mean (±SD) glucose infusion rate versus time of insulin lispro 25 (both test and reference products).

Article Snippet: The test (T) formulation was a mixed protamine zinc recombinant human insulin lispro injection (3 mL: 300U), provided by Tonghua Dongbao Pharmaceutical Co., Ltd. (lot number: 2L12020100052).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Pharmacokinetics and pharmacodynamics of insulin lispro 25 versus the original preparation (Humalog ® 25) in Chinese healthy male volunteers

doi: 10.3389/fphar.2025.1533548

Figure Lengend Snippet: Bio-equivalence between the insulin lispro test and reference products.

Article Snippet: The test (T) formulation was a mixed protamine zinc recombinant human insulin lispro injection (3 mL: 300U), provided by Tonghua Dongbao Pharmaceutical Co., Ltd. (lot number: 2L12020100052).

Techniques: