Journal: bioRxiv

Article Title: Small molecule modulators targeting the interactions of small GTPase ARF1 with C9orf72:SMCR8:WDR41 complexes implicated in ALS/FTD

doi: 10.64898/2026.01.24.701325

Figure Lengend Snippet: (A) 3D structural illustration of ARF1-CSW complex and its binding pocket (ARF1 protein is in transparent cyan, SMCR8 and C9orf72 proteins are in orange and green as well as WDR41 is shown in red color (PDB: 7MGE)). The C9orf72 and SMCR8 include both longin and DEN domains. The purple circle shows the binding pocket at the interface of ARF1-CSW complex. (B) A zoomed-in view of the protein structures highlighting the key residues involved in the binding pocket at interface of ARF1 and CSW complex. (C) STRING analysis revealed the interaction network of ARF1and proteins in CSW complex including C9orf72, SMCR8 and WRD41 proteins. Line thickness indicates the strength of data support. (D) Table summarizing the functional roles of the proteins in the network, their corresponding interaction scores with ARF1 from STRING analysis, and their involvement in neurodegenerative diseases.

Article Snippet: Recombinant human ARF1 protein (Creative Biomart Inc, Shirley, NY) in Tris solution (20 mM, pH 7.4) at a concentration of 1 mg/mL was diluted to 50 μg/mL with sodium acetate buffer (pH 4.0).

Techniques: Binding Assay, Functional Assay

Journal: bioRxiv

Article Title: Small molecule modulators targeting the interactions of small GTPase ARF1 with C9orf72:SMCR8:WDR41 complexes implicated in ALS/FTD

doi: 10.64898/2026.01.24.701325

Figure Lengend Snippet: ( A ) Superimpose of identified compounds MCULE-5095997944 , MCULE-2336465708 and MCULE-5055852153 into ARF1-CSW complex proteins (ARF1 protein is in transparent cyan, SMCR8 and C9orf72 proteins are in orange and green as well as WDR41 is shown in red color (PDB: 7MGE)). Identified compounds show different poses with a certain extent of overlap with each other. (B-D) 3D representation of identified compounds interactions with ARF1-CSW complex proteins. MCULE-5095997944 (yellow), MCULE-2336465708 (red) and MCULE-5055852153 (green) bind to interface of ARF1, C9orf72, SMCR8 via different interactions such as hydrogen bonds with Ile 49 , Gly 50 of ARF1 and Pro 107 of SMCR8, as well as π-π interaction with His 5 of C9orf72.

Article Snippet: Recombinant human ARF1 protein (Creative Biomart Inc, Shirley, NY) in Tris solution (20 mM, pH 7.4) at a concentration of 1 mg/mL was diluted to 50 μg/mL with sodium acetate buffer (pH 4.0).

Techniques:

Journal: bioRxiv

Article Title: Small molecule modulators targeting the interactions of small GTPase ARF1 with C9orf72:SMCR8:WDR41 complexes implicated in ALS/FTD

doi: 10.64898/2026.01.24.701325

Figure Lengend Snippet: Binding response of (A) MCULE-5095997944, (B) MCULE-5055852153, (C) MCULE-2336465708 and (D) ZCL278 to the purified ARF1 protein using multi-cycle kinetics model and varying concentrations of 4.88 nm to 1.25 µM. (E) Evaluation software provided the corresponding kinetics and affinity data for each compound.

Article Snippet: Recombinant human ARF1 protein (Creative Biomart Inc, Shirley, NY) in Tris solution (20 mM, pH 7.4) at a concentration of 1 mg/mL was diluted to 50 μg/mL with sodium acetate buffer (pH 4.0).

Techniques: Binding Assay, Purification, Software

Journal: bioRxiv

Article Title: Small molecule modulators targeting the interactions of small GTPase ARF1 with C9orf72:SMCR8:WDR41 complexes implicated in ALS/FTD

doi: 10.64898/2026.01.24.701325

Figure Lengend Snippet: Effects of MCULE-5095997944, MCULE-2336465708, MCULE-5055852153, GDPNP and BFA (50 µM) on the Golgi in HEK293 cells. Cells were fixed and stained with anti-GM130 antibody (Red). Cell nucleus was stained with DAPI (Blue). Immunofluorescent staining of GM130 in (A) Control HEK293 cells, (B) Cells treated with BFA, (C) 50uM of MCULE-5095997944, (D) 50 uM MCULE-5055852153, (E) 50 uM MCULE-2336465708 (F) GDPNP, (G) 50 uM GDPNP followed by BFA, (H) 50 uM GDPNP followed by MCULE-5095997944. Arrows point to perinuclear Golgi. Arrowheads point to dispersed Golgi in cytoplasm. Bar: 30 um (I) Bar graph showing percent dispersion of Golgi apparatus with and without all the treatments. The mean percent Golgi apparatus dispersion was compared. (J) G-LISA analysis of ARF1-GTP levels in HEK293 cells treated with MCULE-5095997944 (100 µM) and BFA for 15 min. All data are presented as mean ± SEM from duplicates from three independent experiments. ANOVA compared treatments to their respective control ( P -values *** p < 0.01, **** p < 0.0001 were considered significant).

Article Snippet: Recombinant human ARF1 protein (Creative Biomart Inc, Shirley, NY) in Tris solution (20 mM, pH 7.4) at a concentration of 1 mg/mL was diluted to 50 μg/mL with sodium acetate buffer (pH 4.0).

Techniques: Staining, Control, Dispersion

Journal: mBio

Article Title: Soluble MFGE8 mediates cell entry of Crimean-Congo hemorrhagic fever virus

doi: 10.1128/mbio.01617-25

Figure Lengend Snippet: CRISPR knockout screen identifies common host factors required for rVSV-CCHFV pseudovirus infection. ( A ). Bubble plot of genes significantly enriched in a genome-wide CRISPR knockout screen in wild-type A549 (A549-WT) cells challenged with rVSV-CCHFV pseudovirus. The virus-resistant A549-WT cells were collected for analysis, and genes were ranked according to the MAGeCK score. ( B ) KEGG (Kyoto Encyclopedia of Genes and Genomes) and Go (Gene Ontology) analysis of top 100 enriched genes. ( C and D ) Flow cytometry ( C ) and fluorescence imaging ( D ) analysis of A549-WT and A549-BAT (B3GAT3, AXL, and TIM-1 triple-knockout cells) infected with rVSV-CCHFV (MOI 3). The percentage of GFP-positive cells was analyzed at indicated time points using flow cytometer, and images were taken using fluorescence microscope at 24 h post-infection (hpi). Scale bar, 400 µm. Two-way ANOVA with Sidak’s multiple-comparison test. **** P < 0.0001.

Article Snippet: The human Brunello CRISPR knockout pooled library targeting 19,114 genes (Addgene #73178) or Calabrese activation pooled library targeting 18,885 genes (Addgene #92379) was a gift from David Root and John Doench ( ) and packaged in 293 FT cells after co-transfection with psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259) using FugeneHD (Promega).

Techniques: CRISPR, Knock-Out, Infection, Genome Wide, Virus, Flow Cytometry, Fluorescence, Imaging, Triple Knockout, Microscopy, Comparison

Journal: mBio

Article Title: Soluble MFGE8 mediates cell entry of Crimean-Congo hemorrhagic fever virus

doi: 10.1128/mbio.01617-25

Figure Lengend Snippet: CRISPR activation screen identifies MFGE8 as a proviral host factor for rVSV-CCHFV infection. ( A ) Identification of genes from CRISPR screen in A549-BAT cells. Cells transduced with the CRISPR activation library were infected with rVSV-CCHFV for 24 h. GFP-positive cells were sorted for sgRNA abundance analysis and ranked based on the MAGeCK score and P value. ( B and C ) Validation of MFGE8 gene. Gene expression was activated using two or representative sgRNAs in A549-BAT cells, followed by infection with rVSV-CCHFV (MOI 3, 18 h) ( B ) and rVSV (MOI 0.01, 15 h) ( C ). The percentage of GFP-positive cells were analyzed by flow cytometry. ( D ) Representative fluorescence images of A549-BAT cell infected with respective virus from ( B ) and ( C ) were taken before harvesting the cells. Scale bar, 400 µm. ( E ) Overexpression of MFGE8 enhances rVSV-CCHFV infection in A549-BAT cells. ( F ) Growth kinetics of rVSV-CCHFV in vector control and MFGE8-overexpressing cells. Cells were infected with rVSV-CCHFV at an MOI of 0.3, and viral titers in the supernatants at indicated time points were determined by plaque-forming assay. ( G–I ) Overexpression of MFGE8 enhances rVSV-CCHFV infection in A549-WT ( G ), Hela ( H ), and SW-13 ( I ) cells. The percentage of GFP-positive cells were analyzed by flow cytometry at 16 hpi. (J) Knockout of MFGE8 decreases the rVSV-CCHFV infection. A549-WT cells edited with two different nontargeting control or MFGE8 -specific sgRNAs were infected with rVSV-CCHFV, followed by flow cytometry analysis of GFP-positive cells at 16 hpi. Two-way ANOVA with Sidak’s multiple-comparison test. ns, not significant; *** P < 0.001; **** P < 0.0001.

Article Snippet: The human Brunello CRISPR knockout pooled library targeting 19,114 genes (Addgene #73178) or Calabrese activation pooled library targeting 18,885 genes (Addgene #92379) was a gift from David Root and John Doench ( ) and packaged in 293 FT cells after co-transfection with psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259) using FugeneHD (Promega).

Techniques: CRISPR, Activation Assay, Infection, Transduction, Biomarker Discovery, Gene Expression, Flow Cytometry, Fluorescence, Virus, Over Expression, Plasmid Preparation, Control, Knock-Out, Comparison