Journal: bioRxiv

Article Title: Macropinocytosis mediates neurotropism of Cryptococcus neoformans in a human organoid model of the blood-brain barrier

doi: 10.1101/2025.09.23.678106

Figure Lengend Snippet: a Representative fluorescent image of BBB organoids formed from EphA2 knockout (KO) human brain endothelial cells. EphA2 KO cells express RFP (magenta) against DAPI (blue) counterstaining. b Western blot analysis (1:1000 dilution of rabbit anti-Human EphA2; Cell signaling technology Inc. D4A2) (1:10,000 dilution of Goat Anti-Rabbit IgG H&L HRPab6721; Abcam Inc., Cambridge, MA, USA) confirming loss of EphA2 protein expression in EphA2 KO organoids, in contrast to controls (EphA2+ and EphA2 over-expression (o/e)). c Internalization of Cn ( Cryptococcus neoformans ) by BBB organoids is dependent on EphA2 expression. Organoids (EphA2+ or EphA2 KO brain endothelial cells) were exposed to CFSE-stained Cn for 48 h, after which organoids were sectioned and analyzed for Cn invasion (Welch two-sample t-test: t = 4.28, df = 13.94, p < 0.0008)(R v.4.4.2). d Comparison of GFAP expression between BBB organoids formed with EphA2 KO versus EphA2+ brain endothelial cells. Organoids deficient in EphA2 show significantly higher levels of GFAP expression compared to EphA2+ organoids. Quantification was performed on GFAP-antibody probed organoid sections by applying a uniform threshold to images and measuring the percent area in the relevant channel above the threshold (Welch two-sample t-test: t = 2.42, df = 10.68, p = 0.0343)(R v.4.2.2). e Organoids exposed to Cd ( Cryptococcus deuterogattii ) show increased GFAP expression in stark contrast to organoids exposed to Cn after 48 h of exposure (Welch two-sample t-test comparing Cn & Cd treatments: t = 3.84, df = 9.38, p = 0.003, comparing Cd & Control treatments: t = 4.25, df = 10.57, p = 0.0015)(R v.4.4.2). For panels c – e, each data point represents the average value for an organoid across several images.

Article Snippet: Two sets of plasmids were used in the CRISPR/Cas9 method - the EphA2 CRISPR/Cas9 KO plasmids and the EphA2 HDR plasmids, both of which were ordered from Santa Cruz Biotechnology.

Techniques: Knock-Out, Western Blot, Expressing, Over Expression, Staining, Comparison, Control

Journal: bioRxiv

Article Title: Macropinocytosis mediates neurotropism of Cryptococcus neoformans in a human organoid model of the blood-brain barrier

doi: 10.1101/2025.09.23.678106

Figure Lengend Snippet: a Cn induced stimulation of GTP-bound Cdc42 is contingent on EphA2 expression and b CD44 engagement. EphA2+ and EphA2 KO human brain endothelial cells were treated with either a Cn or b cps1Δ Cn deletion mutant at an MOI of 10 for 30 min and then lysed. Untreated and treated cell lysates were analyzed for GTP bound Cdc42 (active Cdc42) using the Cytoskeleton G-LISA assay. Data are expressed as a mean ± SD of three independent experiments. Statistical analysis was done using a two tailed unpaired t-test (n = 8, t = 0.2156, df = 14, **** p < 0.0001)(GraphPad Prism 10 software). c, d Expression of Cdc42 protein in lysates was confirmed by western blot analysis of nitrocellulose membrane probed with primary antibody to amino acid 150-182 of Cdc42 protein. (1:800 dilution of mouse anti-human Cdc42; cytoskeleton Inc. ACD04) (1:10,000 dilution of Goat anti-Mouse IgG H&L HRP ab6789; Abcam Inc., Cambridge, MA, USA). e, f Cn recruits a CD44-EphA2 protein complex in brain endothelial cells, detected by Proximity Labeling Assays (PLA - DuoLink) of EphA2 and CD44 in e mouse primary brain endothelial cells and f human brain endothelial cells (EphA2+, iBMEC). The in-situ interaction was quantified as the area in an image above a universally applied threshold. Significantly higher fluorescence was observed in Cn (+) versus Cn (-) treatments in both mouse primary cells (t = 4.40, df = 43.13, p < 0.0001) and human cells (t = 3.63, df = 131.88, p = 0.0004). In mouse primary cells, there was significantly higher signal in the Cn (+) compared to the Cd (+) treatments (t = 4.08, df = 43.63, p < 0.0002)(R v.4.4.2). Mouse primary brain endothelial cells were exposed to Cd , Cn media or a no primary antibody control. g Representative fluorescent images of PLA in human brain endothelial cells (EphA2+, iBMECs). Top row: iBMECs exposed to Cn for 90 min. Middle row: control panel iBMECs not exposed to Cn . Bottom row: control treatment in which iBMECs were exposed to Cn but omitting primary antibodies (anti-CD44 and anti-EphA2) Left column: Nuclear stain. Middle column: Fluorescent puncta (PLA-red probe) are indicative of target proteins (EphA2 and CD44) in proximity (within 40 nm) to each other. Right column: merged images.

Article Snippet: Two sets of plasmids were used in the CRISPR/Cas9 method - the EphA2 CRISPR/Cas9 KO plasmids and the EphA2 HDR plasmids, both of which were ordered from Santa Cruz Biotechnology.

Techniques: Expressing, Mutagenesis, Two Tailed Test, Software, Western Blot, Membrane, Labeling, In Situ, Fluorescence, Control, Staining

Journal: bioRxiv

Article Title: Macropinocytosis mediates neurotropism of Cryptococcus neoformans in a human organoid model of the blood-brain barrier

doi: 10.1101/2025.09.23.678106

Figure Lengend Snippet: Alpha-Fold-Multimer structure prediction indicates reliable binding between EphA2 and CD44 . a Cartoon representation of the AlphaFold3-predicted model of the EphA2 structure. extracellular region in complex with CD44, highlighting two distinct putative binding interfaces: the ligand-binding domain (LBD) (ipTM = 0.13; pTM = 0.49) and the fibronectin domains (FN1-FN2) (ipTM = 0.11; pTM = 0.49). b-c Representative binding conformations of the LBD: CD44 and FN: CD44 complexes obtained from molecular dynamics simulations. Shown here are the major conformational clusters derived from all replica trajectories. EphA2 is depicted in green and CD44 in red cartoon representations. For clarity, only the principle interfacial hydrogen bonds are displayed.

Article Snippet: Two sets of plasmids were used in the CRISPR/Cas9 method - the EphA2 CRISPR/Cas9 KO plasmids and the EphA2 HDR plasmids, both of which were ordered from Santa Cruz Biotechnology.

Techniques: Binding Assay, Ligand Binding Assay, Derivative Assay

Journal: bioRxiv

Article Title: Macropinocytosis mediates neurotropism of Cryptococcus neoformans in a human organoid model of the blood-brain barrier

doi: 10.1101/2025.09.23.678106

Figure Lengend Snippet: Upon binding to CD44, Cn promotes the association of CD44 with the ligand binding domain (LBD) and interactions involving both FN1 and FN2 domains, suggesting that CD44 can also engage EphA2 via a membrane-proximal site and/or the fibronectin 2 domain (FN2) (black arrows). The CD44-EphA2 molecular interaction leads to a PKC-dependent phosphorylation of EphA2, and the downstream activation of GTP-bound Cdc42 (green arrows). Active Cdc42 stimulates membrane ruffling by remodeling the cytoskeleton via actin polymerization. The ensuing macropinocytosis of Cn is a result of macropinosomes large enough to internalize and transport Cn across the brain endothelium. Cd does not cross the brain endothelium (crossed-out black arrow), but stimulates the expression of GFAP+ astrocytes by as yet an unknown mechanism (dashed arrow). Figure created in BioRender.com.

Article Snippet: Two sets of plasmids were used in the CRISPR/Cas9 method - the EphA2 CRISPR/Cas9 KO plasmids and the EphA2 HDR plasmids, both of which were ordered from Santa Cruz Biotechnology.

Techniques: Binding Assay, Ligand Binding Assay, Membrane, Phospho-proteomics, Activation Assay, Expressing

Journal: Nature Communications

Article Title: Mutant Huntingtin stalls ribosomes and represses protein synthesis in a cellular model of Huntington disease

doi: 10.1038/s41467-021-21637-y

Figure Lengend Snippet: A Representative polysome profiles of control and HD-homo cells after ribosome run-off assay using Harringtonine (2 µg/ml, 2 min). mRNA-Seq was performed using RNAs isolated from polysome containing fractions (arrows). B Volcano plot representing changes in gene expression levels of ribosome-bound mRNAs in HD-homo cells (vs control cells) after ribosome run-off assay from A . mRNA transcripts with absolute log2 fold change (log2FC) >0.3 and FDR-corrected P value <0.05 are shown with red (up, increased polysome binding, log2FC > 1), blue (down, decreased polysome binding, log2FC < 1), gray (unaltered, no difference in polysome binding) and green (selected targets). Statistical testing was done using DESeq2 with a two-tailed Wald test and adjusted for multiple comparisons using the procedure of Benjamini–Hochberg. C A bar plot representing log2FC in the mRNA levels of some known translation regulating genes within polysome fractions obtained from HD-homo cells (comparing to controls) after ribosome run-off assay (upper panel, Data are log2FC and the standard error of the log2FC value from n = 2 independent experiments), and qPCR of the indicated targets in the purified ribosome fractions (bottom panel, Data are mean ± SEM, n = 8 independent experiments, n.s. not significant by two-way ANOVA, Bonferroni’s multiple comparisons test). D , E Representative blots and mRNA levels and corresponding quantifications for Fmrp in mouse control and HD-homo striatal cells (Fmrp protein: n = 9 independent experiments, Fmrp mRNA: n = 3 independent experiments, D ), and unaffected and human HD patient striatum (Fmrp protein: n = 5 unaffected, n = 14 HD, FMRP mRNA: n = 3 unaffected, n = 6 HD, E ). Data are mean ± SEM, * P < 0.05, ** P < 0.01 by two-tailed Student’s t test, n.s not significant. Exact P values are reported in the Source Data file. Source data are provided as a Source Data file.

Article Snippet: GFP expressing CRISPR/Cas9 knockout plasmids for mouse Htt (sc-420825) and Fmr1 (sc-420392) or CRISPR/Cas9 control plasmid (sc-418922) were purchased from Santa Cruz Biotechnology.

Techniques: Control, Isolation, Gene Expression, Binding Assay, Two Tailed Test, Purification

Journal: Nature Communications

Article Title: Mutant Huntingtin stalls ribosomes and represses protein synthesis in a cellular model of Huntington disease

doi: 10.1038/s41467-021-21637-y

Figure Lengend Snippet: A Representative immunoblots performed on CRISPR/Cas9 control (Ctrl) and Fmr1 -depleted mouse striatal cells after puromycin metabolic labeling. B , C Quantification of blots from A showing FMRP levels ( B ), puromycin incorporation ( C ) in control (Ctrl) and Fmr1 -depleted control, and HD-homo striatal cells. Data are mean ± SEM, ( B n = 6; C n = 12 independent experiments), ** P < 0.01, **** P < 0.0001, by one-way ANOVA followed by Bonferroni’s multiple comparisons test, n.s not significant. D Representative immunoblots performed on Htt or Fmr1 depleted, or Htt/Fmr1 double-depleted HD-homo cells after puromycin metabolic labeling. E Quantification of the blots from D , showing puromycin incorporation. Data are mean ± SEM ( n = 3 independent experiments), ** P < 0.01, by one-way ANOVA followed by Bonferroni’s multiple comparisons test. P = 0.051 by two-tailed Student’s t test. F , G Representative polysome profiles ( F ) obtained from mHtt or Fmr1 -depleted mouse striatal cells before (basal) and after ribosome run-off assay using harringtonine (2 µg/ml, 3 min) and their corresponding quantification ( G ) of polysome to monosome (PS/MS) ratio (area under the curve). Data are mean ± SEM ( n = 3 independent experiments), by one-way ANOVA followed by Tukey’s multiple comparison test. n.s. not significant. Exact P values are reported in the Source Data file. Source data are provided as a Source Data file.

Article Snippet: GFP expressing CRISPR/Cas9 knockout plasmids for mouse Htt (sc-420825) and Fmr1 (sc-420392) or CRISPR/Cas9 control plasmid (sc-418922) were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, CRISPR, Control, Labeling, Two Tailed Test, Comparison

Journal: Cancer research

Article Title: Oncogenic functions of Gli in pancreatic adenocarcinoma are supported by its PRMT1-mediated methylation

doi: 10.1158/0008-5472.CAN-16-0715

Figure Lengend Snippet: (A) GLI1 mRNA levels according to TCGA data for patients with the seven deadliest cancers in the United States in 2014. The data are medians with the 5th and 95th percentiles and standard deviations (error bars).

Article Snippet: To generate Gli1 knockout cells, AsPC-1 cells were co-transfected with Gli1 CRISPR/Cas9 KO plasmid (Santa Cruz Biotechnology, sc-400266) and Gli1 HDR plasmid (Santa Cruz Biotechnology, sc-400266-HDR).

Techniques:

Journal: Cancer research

Article Title: Oncogenic functions of Gli in pancreatic adenocarcinoma are supported by its PRMT1-mediated methylation

doi: 10.1158/0008-5472.CAN-16-0715

Figure Lengend Snippet: (A) In vitro methylation assay with PRMT1 and wild-type (WT) or R597K-mutant Gli1. Left panel, Coomassie Blue staining. Right panel, fluorography.

Article Snippet: To generate Gli1 knockout cells, AsPC-1 cells were co-transfected with Gli1 CRISPR/Cas9 KO plasmid (Santa Cruz Biotechnology, sc-400266) and Gli1 HDR plasmid (Santa Cruz Biotechnology, sc-400266-HDR).

Techniques: In Vitro, Methylation, Mutagenesis, Staining

Journal: Cancer research

Article Title: Oncogenic functions of Gli in pancreatic adenocarcinoma are supported by its PRMT1-mediated methylation

doi: 10.1158/0008-5472.CAN-16-0715

Figure Lengend Snippet: (A) Left panel, Western blot analysis of meGli1 and total Gli1 in MIA PaCa-2 luciferase cells stably transfected with an empty vector (Vec), wild-type Gli1 (Gli1WT), or R597K-mutant Gli1 (Gli1RK). Right panel, mRNA expression levels, measured by quantitative real-time PCR, of Gli1 target genes in Vec-, Gli1WT (WT)-, and Gli1RK (RK)-transfected MIA PaCa-2 cells. Error bars represent SD (n = 3). *P < 0.05, **P < 0.01 (paired two-tailed Student’s t-test).

Article Snippet: To generate Gli1 knockout cells, AsPC-1 cells were co-transfected with Gli1 CRISPR/Cas9 KO plasmid (Santa Cruz Biotechnology, sc-400266) and Gli1 HDR plasmid (Santa Cruz Biotechnology, sc-400266-HDR).

Techniques: Western Blot, Luciferase, Stable Transfection, Transfection, Plasmid Preparation, Mutagenesis, Expressing, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: Cancer research

Article Title: Oncogenic functions of Gli in pancreatic adenocarcinoma are supported by its PRMT1-mediated methylation

doi: 10.1158/0008-5472.CAN-16-0715

Figure Lengend Snippet: (A) Responses of MIA PaCa-2 stable clones to gemcitabine with or without PRMT1 depletion. shCtrl: control shRNA; shPRMT1: PRMT1 shRNA; Vec: MIA PaCa-2 stable clone with empty vector; WT: stable clone with wild-type Gli1; RK: stable clone with Gli1R597 mutant. Error bars represent SD (n = 4).

Article Snippet: To generate Gli1 knockout cells, AsPC-1 cells were co-transfected with Gli1 CRISPR/Cas9 KO plasmid (Santa Cruz Biotechnology, sc-400266) and Gli1 HDR plasmid (Santa Cruz Biotechnology, sc-400266-HDR).

Techniques: Clone Assay, Control, shRNA, Stable Transfection, Plasmid Preparation, Mutagenesis