Journal: Biology Open

Article Title: Establishing an auxin-inducible GFP nanobody-based acute protein knockdown system to mimic hypomorphic mutations during early medaka embryogenesis

doi: 10.1242/bio.062081

Figure Lengend Snippet: Endogenous GFP -tagging of pmm2 does not interfere with Pmm2 function in medaka. (A) Schematic representation of the medaka pmm2 gene locus. Endogenous GFP knock-in outline at the C-terminus of the pmm2 coding sequence via CRISPR/Cas9 (scissors) and biotinylated (red stop signs) donor template [coding sequence, red boxes; untranslated region (UTR), white boxes]. Single copy integration by homology directed repair (HDR) with the right homology flank (RH) and a non-homologous end-joining (NHEJ) event of the left homology flank (LH). Transcript analysis revealed correctly spliced pmm2-GFP mRNA. (B) Representative hatchling homozygous for pmm2-GFP shows ubiquitous GFP expression and proper development. (C) Biochemical Pmm2 enzyme activity assay highlights matching levels of wild-type (wt) and GFP-tagged Pmm2. Results from three independent experiments with lysates of pooled embryos ( n =25, 34 and 34). Mean±s.d. are shown, t -test, ns, P >0.05. (D) Western blot analysis of lysates of wild-type (wt), Pmm2-GFP heterozygous (het) and Pmm2-GFP homozygous (hom) hatchlings ( n =9 pooled hatchlings per lysate for each genotype), reveal the presence of Pmm2-GFP as a stable fusion protein using Pmm2 and GFP-specific antibodies; see absence of Pmm2 band in hom hatchlings (arrowhead), Gapdh was used as loading control. Scale bars: 500 µm. *, stop-codon.

Article Snippet: 10 μg pmm2-GFP gDNA and 200 pg of GFP control plasmid were digested with 10 U of BspHI (NEB) in combination with 20 U of HindIII (NEB) or 20 U of BsaI HF (NEB) at 37°C overnight.

Techniques: Knock-In, Sequencing, CRISPR, Non-Homologous End Joining, Expressing, Enzyme Activity Assay, Western Blot, Control

Journal: Biology Open

Article Title: Establishing an auxin-inducible GFP nanobody-based acute protein knockdown system to mimic hypomorphic mutations during early medaka embryogenesis

doi: 10.1242/bio.062081

Figure Lengend Snippet: TIR1 and mAID/GFP-nanobody degron system has strong basal activity in the absence of auxin in medaka. (A) Schematic representation of an mAID and GFP-nanobody based, auxin inducible degron system planned for acute degradation of Pmm2-GFP in medaka. Upon auxin (NAA) induction, the E3 ubiquitin ligase complex SCF (Skp1, Cul1, F-box protein TIR1) dimerizes with the mAID-GFP nanobody, resulting in ubiquitination and degradation of GFP-tagged proteins by the proteasome. (B) Validation of Pmm2-GFP degradation via western blot analysis of stage 23 pmm2-GFP embryos comparing uninjected to degron-injected and induced (50 µM NAA) at 6 h post fertilization (hpf; n =35 pooled embryos per condition). Gapdh was used as loading control. (C) Time-lapse imaging of uninjected homozygous pmm2-GFP embryos and degron-injected embryos in ERM or induced with 50 µM NAA. (D) Quantification of mean GFP fluorescence following baseline correction over 18 h. Mean±s.d. are shown, one-way ANOVA test with Tukey’s post-hoc multiple comparison was performed on endpoints (grey box), adjusted P -values shown, *** P ≤0.001, **** P ≤0.0001. Uninjected homozygous pmm2-GFP ( n =7), degron-injected in ERM ( n =8) or induced with 50 µM NAA ( n =25). Scale bar: 500 µm. hpi, hours post induction.

Article Snippet: 10 μg pmm2-GFP gDNA and 200 pg of GFP control plasmid were digested with 10 U of BspHI (NEB) in combination with 20 U of HindIII (NEB) or 20 U of BsaI HF (NEB) at 37°C overnight.

Techniques: Activity Assay, Ubiquitin Proteomics, Biomarker Discovery, Western Blot, Injection, Control, Imaging, Fluorescence, Comparison

Journal: Biology Open

Article Title: Establishing an auxin-inducible GFP nanobody-based acute protein knockdown system to mimic hypomorphic mutations during early medaka embryogenesis

doi: 10.1242/bio.062081

Figure Lengend Snippet: Combination of TIR1(F74G) with mAID/GFP nanobody as degron system enables acute and inducible knockdown of Pmm2-GFP in medaka. (A) Schematic representation of the TIR1(F74G) variant combined with the mAID/GFP-nanobody (vhhGFP4) based degron ( ; ) to induce degradation of Pmm2-GFP in an auxin-analog (5-Ph-IAA) inducible manner in medaka. (B) Time-lapse imaging of uninjected pmm2-GFP , degron-injected pmm2-GFP embryos incubated in ERM or 5 µM 5-Ph-IAA at 6 h post fertilization (hpf). (C) Quantification of mean GFP fluorescence following baseline correction over 26 hpi. Mean±s.d. of triplicates shown, uninjected pmm2-gfp control ( n total=15; green), degron/mCherry injected, non-induced embryos ( n total=61; purple), induced ( n total=65; orange). (C′) Scatterplot of raw data at 26 hpi. Mean is shown as black line, one-way ANOVA test with Tukey’s post-hoc multiple comparison was performed, adjusted P -values shown, ns, P >0.05, **** P ≤0.0001. Scale bar: 500 µm. hpi, hours post induction; 5-Ph-IAA, auxin analog.

Article Snippet: 10 μg pmm2-GFP gDNA and 200 pg of GFP control plasmid were digested with 10 U of BspHI (NEB) in combination with 20 U of HindIII (NEB) or 20 U of BsaI HF (NEB) at 37°C overnight.

Techniques: Knockdown, Variant Assay, Imaging, Injection, Incubation, Fluorescence, Control, Comparison

Journal: Biology Open

Article Title: Establishing an auxin-inducible GFP nanobody-based acute protein knockdown system to mimic hypomorphic mutations during early medaka embryogenesis

doi: 10.1242/bio.062081

Figure Lengend Snippet: Recovery of Pmm2-GFP expression and enzyme activity. (A) Time-lapse imaging of uninjected pmm2-GFP and degron-injected pmm2-GFP embryos incubated in 5 µM 5-Ph-IAA (lower row) from 6 h post fertilization on. (B) Quantification of mean GFP fluorescence over 143 hpi. Mean±s.d. shown, uninjected pmm2-gfp control ( n =7), degron/mCherry injected, induced ( n =18). (B′) Scatterplot of individual mean GFP levels at 31 hpi and 69 hpi. Mean is shown as black line, t -test, ns, P >0.05, * P ≤0.05. (C) Pmm2 enzyme activity assay comparing control (injected, non-induced; grey) and degron-injected and induced (5 µM 5-Ph-AA) embryos (orange). Three independent experiments with lysates of n =25, 26 and 34 pooled embryos for 31 hpi. Two independent experiments with lysates of pooled embryos in ERM ( n =19 and 23) and in 5-Ph-IAA ( n =12 and 32) for 69 hpi. Mean±s.d. are shown, t -test, ** P ≤0.01. (D) Differential GFP levels of uninjected and injected and depletion induced specimens from B plotted and correlated with measured Pmm2-GFP enzyme activity at 31 and 69 hpi from C. Estimated window of Pmm2-GFP activity below 50% indicated (dashed grey line). Scale bars: 500 µm. hpi, hours post induction; 5-Ph-IAA, auxin analog.

Article Snippet: 10 μg pmm2-GFP gDNA and 200 pg of GFP control plasmid were digested with 10 U of BspHI (NEB) in combination with 20 U of HindIII (NEB) or 20 U of BsaI HF (NEB) at 37°C overnight.

Techniques: Expressing, Activity Assay, Imaging, Injection, Incubation, Fluorescence, Control, Enzyme Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Clostridioides difficile TcdB induces expression of its receptor (CSPG4) through a noncanonical Hippo signaling mechanism

doi: 10.1016/j.jbc.2026.111137

Figure Lengend Snippet: CSPG4 is upregulated by TcdB independent of YAP . A , immunoblots from protein lysates acquired from pericytes exposed for 24 h to 1 ng/ml of TcdB and/or 10 μM XMU-MP-1. B , heatmaps depicting Z-score values for YAP regulated genes generated from RNA-seq analysis of pericytes exposed for 24 h to 1 ng/ml of TcdB and/or 10 μM XMU-MP-1. C , heatmaps showing Z-score values for MTF1 regulated genes generated from RNA-seq analysis of pericytes exposed for 24 h to 1 ng/ml of TcdB and/or 10 μM XMU-MP-1. D , immunoblots from protein lysates taken from HeLa cells transfected for 48 h with control plasmid (pCMV-GFP) or a plasmid for expressing YAP S5A (pCMV-flag YAP2 5SA). For the final 24 h, the transfected cells were exposed to 1 ng/ml of TcdB.

Article Snippet: HeLa cells were also transfected with the control plasmid pCMV-GFP, which was a gift from Connie Cepko (Addgene plasmid # 11153; http://n2t.net/addgene:11153 ) ( ).

Techniques: Western Blot, Generated, RNA Sequencing, Transfection, Control, Plasmid Preparation, Expressing

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: High-throughput optimization of antibody production in CHO cells by tuning heavy- and light-chain promoter strength

doi: 10.3389/fbioe.2025.1747473

Figure Lengend Snippet: mAb B: effects of LC/HC promoter strength on titer, cell density, and viability post-transfection (72 hpt), and model profiling. (A) Viability (%), (B) viable cell density (VCD; cells/mL), and (C) titer (mg/L) for all LC/HC combinations. Promoter levels: 5, 40, and 100 RPU for both LC and HC. Bars show mean ± SD (n = 2). Statistical analysis was based on one-way ANOVA with Tukey's HSD; different letters indicate p < 0.05. (D) Transfection efficiency (% GFP+) of a control plasmid (pMAX-GFP) quantified by flow cytometry. (E) JMP profiler for least-squares models of titer, VCD, and viability versus LC/HC promoter strength. Left: predicted means with 95% Cls. Right: composite desirability (0-1) balancing high titer with acceptable VCD and viability. Red dashed lines mark targets/constraints.

Article Snippet: CHO-S cells (R80007, Life Technologies, Waltham, MA, United States) CD CHO medium (10743029, Gibco, Thermo Fisher Scientific, Waltham, MA, United States) L-Glutamine (25030081, Thermo Fisher Scientific, Waltham, MA, United States) Freestyle MAX Transfection Reagent (16447100, Thermo Fisher Scientific, Waltham, MA, United States) OptiPRO serum free medium (12309019, Thermo Fisher Scientific, Waltham, MA, United States) Transfection positive control expressing GFP (pMax-E2F1, 16007, Addgene, Watertown, MA, United States) Solution 18 (910-3018, ChemoMetec A/S, Allerød, Denmark) NC-Slide A8 (941-0002, ChemoMetec A/S, Allerød, Denmark)

Techniques: Transfection, Control, Plasmid Preparation, Flow Cytometry

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: High-throughput optimization of antibody production in CHO cells by tuning heavy- and light-chain promoter strength

doi: 10.3389/fbioe.2025.1747473

Figure Lengend Snippet: mAb C: effects of LC/HC promoter strength on titer, cell density, and viability post-transfection (72 hpt), and model profiling. (A) Viability (6), (B) viable cell density (VCD; cells/mL), and (C) titer (mg/L) for all LC/HC combinations. Promoter levels: 5, 40, and 100 RPU for both LC and HC. Bars show mean = SD (n = 2). Statistical analysis was based on one-way ANOVA with Tukey’s HSD; different letters indicate p < 0.05. (D) JMP profiler for least-squares models of titer, VCD, and viability versus LC/HC promoter strength. Left: predicted means with 95% Cls. Right: composite desirability (0-1) balancing high titer with acceptable VCD and viability. Red dashed lines mark targets/constraints.

Article Snippet: CHO-S cells (R80007, Life Technologies, Waltham, MA, United States) CD CHO medium (10743029, Gibco, Thermo Fisher Scientific, Waltham, MA, United States) L-Glutamine (25030081, Thermo Fisher Scientific, Waltham, MA, United States) Freestyle MAX Transfection Reagent (16447100, Thermo Fisher Scientific, Waltham, MA, United States) OptiPRO serum free medium (12309019, Thermo Fisher Scientific, Waltham, MA, United States) Transfection positive control expressing GFP (pMax-E2F1, 16007, Addgene, Watertown, MA, United States) Solution 18 (910-3018, ChemoMetec A/S, Allerød, Denmark) NC-Slide A8 (941-0002, ChemoMetec A/S, Allerød, Denmark)

Techniques: Transfection

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: High-throughput optimization of antibody production in CHO cells by tuning heavy- and light-chain promoter strength

doi: 10.3389/fbioe.2025.1747473

Figure Lengend Snippet: mAb E: effects of LC/HC promoter strength on titer, cell density, and viability post-transfection (72 hpt), and model profiling. (A) Viability (%), (B) viable cell density (VCD; cells/mL), and (C) titer (mg/L) for all LC/HC combinations. Promoter levels: 5, 40, and 100 RPU for both LC and HC. Bars show mean ± SD (n = 2). Statistical analysis was based on one-way ANOVA with Tukey’s HSD; different letters indicate p < 0.05. (D) JMP profiler for least- squares models of titer, VCD, and viability versus LC/HC promoter strength. Left: predicted means with 95% CIs. Right: composite desirability (0-1) balancing high titer with acceptable VCD and viability. Red dashed lines mark targets/constraints.

Article Snippet: CHO-S cells (R80007, Life Technologies, Waltham, MA, United States) CD CHO medium (10743029, Gibco, Thermo Fisher Scientific, Waltham, MA, United States) L-Glutamine (25030081, Thermo Fisher Scientific, Waltham, MA, United States) Freestyle MAX Transfection Reagent (16447100, Thermo Fisher Scientific, Waltham, MA, United States) OptiPRO serum free medium (12309019, Thermo Fisher Scientific, Waltham, MA, United States) Transfection positive control expressing GFP (pMax-E2F1, 16007, Addgene, Watertown, MA, United States) Solution 18 (910-3018, ChemoMetec A/S, Allerød, Denmark) NC-Slide A8 (941-0002, ChemoMetec A/S, Allerød, Denmark)

Techniques: Transfection