Journal: bioRxiv

Article Title: Integrative Chemical Genetics Platform Identifies Condensate Modulators Linked to Neurological Disorders

doi: 10.1101/2025.06.07.658469

Figure Lengend Snippet: (A) Drug treatment decreases nuclear MLF2-GFP foci. Torsin-deficient cells expressing MLF2-GFP (8 h) were treated at four different concentrations (2x below IC 50 , at IC 50 , 2x above IC 50 , and 8x above IC 50 ) for 3 h with either PZ, celastrol, plumbagin, or disulfiram. Nuclear MLF2-GFP foci were compared between treated and DMSO (orange boxplot) conditions. Each dot represents the number of MLF2-GFP foci for a single cell, while the horizontal lines indicate the median foci/condition. (B) Drugs modulate K48-Ub orthogonal NE condensate biomarker. Dots depict the number of MLF2-GFP foci (left, 6h Dox) and endogenous α-K48-ubiquitin (right) foci/cell after treatment with drug or DMSO. Asterisks indicate Bonferroni-corrected p-values determined by Mann-Whitney U testing (***: p <= 0.001 ****: p <= 0.0001). (C) Representative immunofluorescence confocal images of MLF2-GFP (green) and α-K48-ubiquitin (red) in torsin-deficient cells treated with DMSO compared to either PZ (3 µM) or celastrol (6 µM). (D) Schematic of inducible MLF2-GFP-P2A construct without (top) or with (bottom) Dox. (E) Confocal images of torsin-deficient cells expressing MLF2-GFP-P2A-tdTomato comparing DMSO or 1.5 µM PZ treatments (6h). Scale bars, 5 µm.

Article Snippet: We created a custom analysis pipeline to segment condensate foci into single cells using the open-source software CellProfiler 4.2.6. , and downstream statistical analysis using R Studio (Version 2023.12.1+402).

Techniques: Expressing, Biomarker Discovery, Ubiquitin Proteomics, MANN-WHITNEY, Immunofluorescence, Construct

Journal: bioRxiv

Article Title: Integrative Chemical Genetics Platform Identifies Condensate Modulators Linked to Neurological Disorders

doi: 10.1101/2025.06.07.658469

Figure Lengend Snippet: (A) Structural representation of ZNF335 with two microcephaly patient disease alleles. The p.Cys467Arg mutation (left image, purple residue) disrupts the second conserved C2H2 zinc finger motif of ZNF335. The p.Arg1111His mutation (right image, red residue) results in splicing defects. (B) Only WT ZNF335 rescues the condensate formation of ZNF335 KO cells. Hela cells with a ZNF335 KO were transfected with either WT ZNF335-HA, C467R ZNF335-HA, or R1111H ZNF335-HA. Cells were immunostained against HA (red), and MLF2-GFP was used as a condensate marker. (C) Quantification comparing rescue of WT vs hypomorphic alleles for ZNF335 KO and RNF26 KO cells. HeLa cells were transfected with either non-targeting (NT), RNF26, or ZNF335 sgRNAs before attempted rescue with empty vector control (EV), WT, or associated loss-of-function alleles. See (B) and Figure S3B for representative ZNF335 KO and RNF26 KO immunofluorescence images, respectively. (D) TPEN negates the effect of PZ treatment. Confocal images of 4TorKO cells expressing MLF2-GFP (green) and stained with Hoechst (nucleus, blue). Cells were treated with either DMSO, 6 µM TPEN, 2 µM PZ, or 6 µM TPEN + 2 µM PZ (3 h). Scale bar, 5 µm. (E) Zinc sulfate is sufficient for condensate modulation. Quantification indicates no statistically significant difference between the CNS-value of 4TorKO treated with either 2 µM PZ, 500 µM ZnSO4, 2 mM ZnSO4, or 10 mM ZnSO4. Corresponding confocal images in Figure S4A. (F) PZ modulates condensates of RNF26 KO and ZNF335 KO cells. HeLa expressing MLF2-GFP (green) were treated with NT, Tor1A/B, RNF26, or ZNF335 sgRNAs and 2 µM PZ or DMSO for 3 h. Scale bar, 5 µM. (G) Quantification of MLF2-GFP foci in CRISPR KO cells after PZ treatment. Boxplots outlined in black or blue were treated with DMSO or 2 µM PZ, respectively. Asterisks indicate Bonferroni-corrected p-values by Mann-Whitney U testing (****: p <= 0.0001). Scale bars at 10 µm (6B) and 5 µm (6D,6F).

Article Snippet: We created a custom analysis pipeline to segment condensate foci into single cells using the open-source software CellProfiler 4.2.6. , and downstream statistical analysis using R Studio (Version 2023.12.1+402).

Techniques: Mutagenesis, Residue, Transfection, Marker, Plasmid Preparation, Control, Immunofluorescence, Expressing, Staining, CRISPR, MANN-WHITNEY

Journal: bioRxiv

Article Title: Integrative Chemical Genetics Platform Identifies Condensate Modulators Linked to Neurological Disorders

doi: 10.1101/2025.06.07.658469

Figure Lengend Snippet: ( top ) Candidate genes from CRISPR KO screen are implicated in neurodevelopmental disorders. Top 50 candidate genes (left columns), their significance score (Z-Score, middle columns), and associated diseases (right columns) are color-coded based on disorders. Rows labeled yellow indicate established link to microcephaly/congenital brain malformations, orange indicates other neurodevelopmental disorders, purple indicates ophthalmological disorders, and red indicates cardiovascular, lymphatic, or other disorders. Genes denoted with an asterisk indicate evidence from genome-wide association studies. Gene relevance to disease was determined based on allelic variants obtained from OMIM and GTR. ( bottom ) Schematic representation depicting the relationship between aberrant condensates and neuro-developmental disorders. Genes involved in the etiology of primary microcephaly and encephalopathies were highly enriched, potentially indicating a role of condensate formation as a driver for these disorders. These condensates can be modulated using small molecules (such as pyrithione zinc and celastrol) or genetic perturbations. Targeting condensates could potentially lead to preserving neuronal homeostasis. Arrows indicate possible intervention points and potential outcomes.

Article Snippet: We created a custom analysis pipeline to segment condensate foci into single cells using the open-source software CellProfiler 4.2.6. , and downstream statistical analysis using R Studio (Version 2023.12.1+402).

Techniques: CRISPR, Labeling, GWAS, Preserving

Journal: STAR Protocols

Article Title: A quantitative medium-throughput assay to measure Caenorhabditis elegans development and reproduction

doi: 10.1016/j.xpro.2020.100224

Figure Lengend Snippet: Description of CellProfiler pipeline steps a In contrast to “Worms_4d” and “Worms_4d_small”, offspring objects are not expanded prior to object measurements to improve the separation of objects. b Chemical treatment may massively affect nematode growth up to a point, at which it is not possible to discriminate small 4-day-old worms (Worms_4d_small) and offspring of unaffected worms (Offspring) by size. Hence, “Worms_4d_small” need to be identified separately. Images of “Worms_4d_small” tend to have more background signal due to an increased amount of stained food bacteria remaining in the wells. In order to avoid false “Offspring” identification, “Worms_4d_small” are defined to have no offspring during data evaluation. See CellProfiler pipeline “C.elegans_assay.cpproj” (Mendeley Data, folder: CellProfiler_pipeline).

Article Snippet: Figure 7 Adaption of open-source routines: removal of the automatic grouping function (A) CellProfiler pipeline (B and C) KNIME workflow (red circle: “Treatment 1” metanode).

Techniques: Staining, Bacteria

Journal: STAR Protocols

Article Title: A quantitative medium-throughput assay to measure Caenorhabditis elegans development and reproduction

doi: 10.1016/j.xpro.2020.100224

Figure Lengend Snippet: Manual scoring of “Worms_4d” (A) Example images: 4-day-old worms with minor offspring appendage (Scoring = 0). (B) Example images: 4-day-old worms with major appendage (Scoring = 1). (C) Example images: Cut or fragmented 4-day-old worms (Scoring = 1). (D) Example images: No actual 4-day-old worms (Scoring = 1). (E) Test data: identification of "Worms_4d_2" (image numbers 17, 19, 29 f.l.t.r.). All test data output images and data CSV files can be downloaded from Mendeley Data (folder: Test_data_CellProfiler_Output).

Article Snippet: Figure 7 Adaption of open-source routines: removal of the automatic grouping function (A) CellProfiler pipeline (B and C) KNIME workflow (red circle: “Treatment 1” metanode).

Techniques:

Journal: STAR Protocols

Article Title: A quantitative medium-throughput assay to measure Caenorhabditis elegans development and reproduction

doi: 10.1016/j.xpro.2020.100224

Figure Lengend Snippet: Expected outcome of CellProfiler image analysis (A) Raw gray-scale image of a control well, recorded with the Opera Phenix high content screening system and merged using CombineTIFF software. Images are analyzed by open-source software CellProfiler. (B) Identification of the “Worm_4d” in a solvent control well (0.2% DMSO). Red number indicates worm area in pixel. (C) Identification of offspring (eggs + larvae) in a solvent control well (0.2% DMSO). Red numbers indicate the offspring count. (D) Example of a 4 μg/mL prochloraz treatment well containing a “Worm_4d_small”. Red number indicates worm area in pixel.

Article Snippet: Figure 7 Adaption of open-source routines: removal of the automatic grouping function (A) CellProfiler pipeline (B and C) KNIME workflow (red circle: “Treatment 1” metanode).

Techniques: Control, High Content Screening, Software, Solvent

Journal: STAR Protocols

Article Title: A quantitative medium-throughput assay to measure Caenorhabditis elegans development and reproduction

doi: 10.1016/j.xpro.2020.100224

Figure Lengend Snippet: Example of CellProfiler image analyses using manually recorded fluorescence images Images of whole wells were recorded with a LSM 880 microscope equipped with a motorized stage (10× objective, 570–630 nm filter). (A) Identification of the “Worm_4d” in a solvent control well (0.2% DMSO). Red number indicates worm area in pixel. (B) Identification of “Offspring” (eggs + larvae) in a solvent control well (0.2% DMSO). Red numbers indicate the offspring count. (C) Identification of the “Worm_4d” in a 4 μg/mL prochloraz treatment well. Red number indicates worm area in pixel. (D) Identification of “Offspring” (eggs + larvae) in a 4 μg/mL prochloraz treatment well. Red numbers indicate the offspring count.

Article Snippet: Figure 7 Adaption of open-source routines: removal of the automatic grouping function (A) CellProfiler pipeline (B and C) KNIME workflow (red circle: “Treatment 1” metanode).

Techniques: Fluorescence, Microscopy, Solvent, Control

Journal: STAR Protocols

Article Title: A quantitative medium-throughput assay to measure Caenorhabditis elegans development and reproduction

doi: 10.1016/j.xpro.2020.100224

Figure Lengend Snippet: Adaption of open-source routines: removal of the automatic grouping function (A) CellProfiler pipeline (B and C) KNIME workflow (red circle: “Treatment 1” metanode). Find open-source routines “C.elegans_assay.cpproj” and “C.elegans_assay.knwf” in Mendeley Data (folders: “CellProfiler_pipeline” and “KNIME_workflow”).

Article Snippet: Figure 7 Adaption of open-source routines: removal of the automatic grouping function (A) CellProfiler pipeline (B and C) KNIME workflow (red circle: “Treatment 1” metanode).

Techniques:

Journal: STAR Protocols

Article Title: A quantitative medium-throughput assay to measure Caenorhabditis elegans development and reproduction

doi: 10.1016/j.xpro.2020.100224

Figure Lengend Snippet:

Article Snippet: Figure 7 Adaption of open-source routines: removal of the automatic grouping function (A) CellProfiler pipeline (B and C) KNIME workflow (red circle: “Treatment 1” metanode).

Techniques: Virus, Recombinant, Software, Fluorescence, Microscopy, High Content Screening