Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay

Journal: Alzheimer's research & therapy

Article Title: Structural and functional alterations of neurons derived from sporadic Alzheimer's disease hiPSCs are associated with downregulation of the LIMK1-cofilin axis.

doi: 10.1186/s13195-024-01632-3

Figure Lengend Snippet: Fig. 2 Differentiation of hiPSCs into neurons and analysis of neuronal markers in HS and sAD neurons. A Schematic representation of the Ngn2-mediated differentiation process used to convert hiPSCs from HS and sAD donors into neurons. Neurons were induced using doxycycline (Doxy) and selected with puromycin (Puro). B Diagram of the lentiviral constructs used in the differentiation process, including Ngn2, EGFP, and Puromycin resistance genes. C Timeline of neuronal differentiation showing key steps from hiPSC plating (DIV0), doxycycline induction (DIV1), puromycin selection (DIV3), and final analysis at DIV30. D Immunofluorescence staining of neuronal markers in HS and sAD-derived neurons at DIV30. DAPI (blue) stains nuclei, EGFP (green) marks neurons, and NEUN (red) marks mature neurons. Merged images confirm co-localization of neuronal markers. Scale bars: 50 µm. E Immunofluorescence staining of the neuronal marker MAP2 (red) in HS and sAD-derived neurons, with DAPI and EGFP labeling. F Quantification of MAP2 and NEUN median fluorescence intensity (in arbitrary units, a.u.) in HS and sAD neurons, showing no significant difference (n.s.) between groups (n = 5/group). G Representative Western blot images of MAP2 and NEUN in HS and sAD neurons, with GAPDH as the loading control. H Densitometric analysis of MAP2 and NEUN expression in HS and sAD neurons showing no significant differences in fold change(n = 5/group). Each dot represents the average of three independent experiments. Data are presented as mean ± SEM. n.s. p > 0.05, assessed by Mann Whitney Rank Sum test

Article Snippet: The lentiviral constructs used for neuronal induction of hIPSCs included: pLV-TetO-hNGN2-eGFP-Puro (a gift from Kristen Brennand; Addgene plasmid #79823), FUW-M2rtTA (a gift from Rudolf Jaenisch; Addgene plasmid #20342), pRSV-Rev, pMD2.G, pMDLg/pRRE (gifts from Didier Trono; Addgene plasmid #12253, Addgene plasmid #12259, Addgene plasmid #12251, respectively).

Techniques: Construct, Selection, Immunofluorescence, Staining, Derivative Assay, Marker, Labeling, Fluorescence, Western Blot, Control, Expressing, MANN-WHITNEY

Journal: Alzheimer's research & therapy

Article Title: Structural and functional alterations of neurons derived from sporadic Alzheimer's disease hiPSCs are associated with downregulation of the LIMK1-cofilin axis.

doi: 10.1186/s13195-024-01632-3

Figure Lengend Snippet: Fig. 3 Neurons obtained from sAD-hiPSCs recapitulate the typical hallmarks of AD. A Immunofluorescence staining for Aβ using 6E10 antibody (red), DAPI (nuclei, blue), and EGFP (neurons, green) in HS and sAD hiPSC-derived neurons. sAD neurons show strong 6E10 staining, indicating the presence of Aβ pathology. Scale bars: 50 µm. B Quantification of 6E10 median fluorescence intensity (in arbitrary units, a.u.) showing a significant increase in Aβ accumulation in sAD-hNs neurons compared to HS-hNs; (n = 5/group). C Representative dot blot images of Aβ levels in DIV30 HS- and AD-hNs lysates; (D) Bar graphs showing the band intensity analysis of Aβ in HS- and sAD-hNs. Red Ponceau, RP, was used as loading index and used to normalize sample; synthetic oligomeric Aβ42 (200 nM) was used as positive control; (E) Representative WB images of p-TAU (Thr181, Ser199, Thr205, Thr217 and Thr231) levels in DIV30 HS- and sAD-hNs lysates; (F) Densitometric analysis of p-TAU (Thr181, Ser199, Thr205, Thr217 and Thr231), normalized to total human TAU (Ht7) protein levels; n = 5/group. GAPDH was used as a loading control;; norm. normalized protein levels. Each dot represents the average of three independent experiments conducted on n = 5/group. Data are presented as mean ± SEM, n.s. p > 0.05, ** p < 0.01 vs HS-hNs, assessed by Mann Whitney Rank Sum test

Article Snippet: The lentiviral constructs used for neuronal induction of hIPSCs included: pLV-TetO-hNGN2-eGFP-Puro (a gift from Kristen Brennand; Addgene plasmid #79823), FUW-M2rtTA (a gift from Rudolf Jaenisch; Addgene plasmid #20342), pRSV-Rev, pMD2.G, pMDLg/pRRE (gifts from Didier Trono; Addgene plasmid #12253, Addgene plasmid #12259, Addgene plasmid #12251, respectively).

Techniques: Immunofluorescence, Staining, Derivative Assay, Fluorescence, Dot Blot, Positive Control, Control, MANN-WHITNEY

Journal: Alzheimer's research & therapy

Article Title: Structural and functional alterations of neurons derived from sporadic Alzheimer's disease hiPSCs are associated with downregulation of the LIMK1-cofilin axis.

doi: 10.1186/s13195-024-01632-3

Figure Lengend Snippet: Fig. 6 Neurons obtained from sAD-hiPSCs show impairments in synaptic protein expression. A Representative WB images of pre- and postsynaptic proteins in DIV30 HS- and sAD-hNs lysates; Densitometric analyses of presynaptic proteins: SYN-1, SYP and postsynaptic proteins: PSD95,GluA1; GAPDH was used as loading control; n = 5/group. C Immunofluorescence staining of synaptic markers SYN-1, SYP, PSD95, and GluA1 in HS and sAD neurons. Co-localization with EGFP (neuronal marker) is shown. sAD neurons display decreased synaptic marker expression. Scale bars: 50 µm. D Quantification of median fluorescence intensity (MFI) in HS and sAD neurons shows a significant reduction in SYN-1, SYP, PSD95, and GluA1 in sAD neurons compared to HS neurons; n = 5/group. Each dot represents the average of three independent experiments conducted on n = 5/group. Data are presented as mean ± SEM, * p < 0.05, ** p < 0.01 vs HS-hNs, assessed by Mann Whitney Rank Sum test

Article Snippet: The lentiviral constructs used for neuronal induction of hIPSCs included: pLV-TetO-hNGN2-eGFP-Puro (a gift from Kristen Brennand; Addgene plasmid #79823), FUW-M2rtTA (a gift from Rudolf Jaenisch; Addgene plasmid #20342), pRSV-Rev, pMD2.G, pMDLg/pRRE (gifts from Didier Trono; Addgene plasmid #12253, Addgene plasmid #12259, Addgene plasmid #12251, respectively).

Techniques: Expressing, Control, Immunofluorescence, Staining, Marker, Fluorescence, MANN-WHITNEY

Journal: bioRxiv

Article Title: Estradiol modulates neuronal network hyperexcitability in select NDD risk genes

doi: 10.64898/2026.02.18.706588

Figure Lengend Snippet: The effect of estradiol treatment and loss-of-function mutations in neurodevelopmental disorder (NDD) risk genes is measured across three modalities: (1) single-cell RNAseq in hiPSC-derived NGN2 -induced glutamatergic neurons (iGLUTs), (2) neuronal calcium imaging in iGLUTs, and (3) sensorimotor and arousal behavior in larval zebrafish. Modalities include overlapping but independent gene sets. ASD/NDD target genes encompass myriad functions, including gene expression regulation (pink), neuronal function and communication (green), cellular signaling and protein modification (gray), and cytoskeleton (blue).

Article Snippet: H1-iCas9 hESCs were transduced with a lentivirus expressing doxycycline-inducible NGN2 (pLVX-UbC-rtTA-Ngn2:2A, Addgene #127288) modified to express blasticidin resistance in place of EGFP.

Techniques: Single Cell, RNA sequencing, Derivative Assay, Imaging, Gene Expression, Modification

Journal: bioRxiv

Article Title: Estradiol modulates neuronal network hyperexcitability in select NDD risk genes

doi: 10.64898/2026.02.18.706588

Figure Lengend Snippet: A. Schematic of experimental design and different effect comparisons. Thirty-six NDD risk genes are targeted using pooled single-cell CRISPR-knockout screening in hiPSC-derived NGN2-glutamatergic neurons (iGLUTs) with and without 24hr exposure to 100nM estradiol. Each gene was targeted with 3-4 gRNAs (i). Various effect comparisons were performed among the treated and untreated groups (ii). Main KO effect measures KO+DMSO vs Non-targeting (NT)+DMSO. Main E2 effect measures NT+E2 vs NT+DMSO. KO effect within E2 measures KO+E2 vs NT+E2. E2 effect within KO measures KO+E2 vs KO+DMSO. E2 and KO joint effect measures KO+E2 vs NT+DMSO. B. Extent of perturbation per target NDD gene (left, dot plot). Circle size indicates negative log 10 (p-value). Stem indicates the log fold change of the knockout +DMSO vs non-targeting control + DMSO. KOs were called based on enrichment of gRNA for target NDD genes and downregulation of target KO gene expression. Number of differentially expressed genes in the main KO effect are shown in the bar plot (FDR <0.05). C . Odds of convergent genes being rescued by E2 compared to nonconvergent genes, tested across NDD KO. Fisher’s exact test, ***p<0.001, **p<0.01, *p<0.05. D. Effect of estradiol reversal on biological pathways across 12 target gene knockouts in iGLUTs. Orange color indicates that the pathway was originally downregulated in main KO effect, and estradiol upregulated the pathway in reversal effect. Blue color indicates that the pathway was originally upregulated in main KO effect, and estradiol downregulated the pathway in reversal effect. Dot size indicates the –log10 of the gene set enrichment significance of E2 effect within KO. E . Effect of estradiol rescue on DEGs across NDD risk genes in iGLUTs. Impact of estradiol on number of main KO DEGs is classified into categories of exacerbated, unchanged, partial rescue, rescue, and over-corrected. For detailed description of thresholds, see Methods. Target genes are ranked from highest to lowest percentage of DEGs that are classified as rescued (nominal p-value <0.05). F-G. Select Gene Ontology terms indicating estradiol reversal among ASH1L ( F ) and SCN2A ( G ) KO in iGLUTs. Dot size indicates the –log10 of the gene set enrichment significance of E2 effect within KO (E2) or main KO effect (KO). Asterisks for Benjamini Hochberg p-adjusted: * <0.05; **<0.01, ***<0.001.

Article Snippet: H1-iCas9 hESCs were transduced with a lentivirus expressing doxycycline-inducible NGN2 (pLVX-UbC-rtTA-Ngn2:2A, Addgene #127288) modified to express blasticidin resistance in place of EGFP.

Techniques: Single Cell, CRISPR, Knock-Out, Derivative Assay, Control, Gene Expression

Journal: bioRxiv

Article Title: Estradiol modulates neuronal network hyperexcitability in select NDD risk genes

doi: 10.64898/2026.02.18.706588

Figure Lengend Snippet: A. Schematic of experimental workflow to assess impact of estradiol on baseline gene expression in scn1lab -/- larvae. B-C . Comparison of number of Gene Ontology (GO) pathways that are originally upregulated in HOM+DMSO and downregulated by estradiol ( B ) or originally downregulated in HOM+DMSO and upregulated by estradiol ( C ). Selected reversed pathways are highlighted in the bar graphs, with bars indicating -log10(FDR). Asterisk for the p-adjusted values: * <0.05,**<0.01, ***<0.001. D. Schematic of experimental workflow to assess impact of estradiol on baseline brain activity in HOM larvae. Dissected larval brains are stained with phosphorylated ERK (pERK) as a marker of active neurons and total ERK (tERK) as a pan-neuronal marker. Images undergo voxel-by-voxel division of pERK by tERK to obtain an activity ratio. Activity is mapped onto the standard zebrafish brain atlas and quantified based on a 4-region or 149-region atlas. E. Voxel-wise Z score normalized pERK/tERK values representing brain activity differences in scn1lab Δ44/Δ44 + DMSO or estradiol compared to DMSO-treated scn1lab +/+ larvae and the difference (subtraction) between the two conditions. Rescued regions from the 149-region atlas are shown. For specific rescued regions, refer to SI Fig. 7. Images are a representative slice of the axial (top) and sagittal (bottom) view. Scale bar represents Z score (red/yellow, increased in mutant; cyan/purple, decreased in mutant). F. Bar graphs showing regional differences in pERK/tERK brain activity in the rhombencephalon, diencephalon, mesencephalon, and telencephalon. G. Left: Schematic of experimental workflow to assess impact of estradiol on induced seizure activity in scn1lab -/- larvae. Right: Pentylenetetrazole (PTZ)-induced seizure activity, with and without 24 h exposure to 20 𝜇M estradiol, is measured in scn1lab -/- larval zebrafish mutants using video-tracking. Representative activity tracings of control and scn1lab -/- larvae at 5dpf before and after exposure to DMSO vehicle or 20uM estradiol and 10mM PTZ or water. Ribbon shows +/- SEM. H. Average activity 30 min before and after the addition of 10mM PTZ or water to control and scn1lab -/- larvae before and after exposure to DMSO vehicle or 20uM estradiol. scn1lab -/- + E2 + PTZ larvae show significantly decreased activity compared to scn1lab -/- + vehicle + PTZ larvae (Unpaired t-test with Welch’s correction on post values, ****p<0.0001; n=24/group). I. Interaction models assessing genotype-x-estradiol-x-PTZ effect, comparing post- vs pre-PTZ measurements. Dissection of the mutant effect (HOM vs control), estradiol effect (Estradiol vs DMSO), and PTZ effect (PTZ vs water) (i). Three-way interaction model to estimate the PTZ effect among genotype-x-estradiol groups of larvae (ii). J. Left: Schematic of experimental workflow to assess impact of estradiol on neuronal busting activity in CRISPR-engineered SCN2A +/- iGLUTs . Right: Cellular and network bursting activity, with and without exposure to acute 100nM estradiol, is measured in SCN2A +/- NGN2-iGLUTs using multielectrode array. Bar graphs show mean number of cellular bursts and network bursts in controls and SCN2A +/- iGLUTs. Unpaired t-test, cellular bursts *p=0.0415, network bursts SCN2A +/- spontaneous vs SCN2A +/- E2 *p=0.0127, SCN2A +/- spontaneous vs WT spontaneous *p=0.0450.

Article Snippet: H1-iCas9 hESCs were transduced with a lentivirus expressing doxycycline-inducible NGN2 (pLVX-UbC-rtTA-Ngn2:2A, Addgene #127288) modified to express blasticidin resistance in place of EGFP.

Techniques: Gene Expression, Comparison, Activity Assay, Staining, Marker, Mutagenesis, Control, Dissection, CRISPR

Journal: Journal of the American Chemical Society

Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors

doi: 10.1021/jacs.8b07601

Figure Lengend Snippet: Figure 4. (A) Top: Composite brightfield and fluorescence microscopy images of spheroids formed from transfected 4T1 cells grown either with (right) or without (left) 15 μg/mL cumate to induce EGFR and GFP expression. Bottom: Fluorescence images of cryotome-sectioned similarly prepared spheroids showing the distribution of fluorescence throughout the interior. All images were captured and viewed under identical settings for comparison. (B) Western blot for EGFR expression of the combined lysates of five spheroids grown either with (+) or without (−) 15 μg/mL cumate. Image shows composite overlay of both bright field imaging for display of prestained protein ladders (L) and chemiluminescence imaging for the anti-EGFR antibody. The molecular weight of each ladder band is indicated to the right.

Article Snippet: For this, we started with 4T1 mouse mammary carcinoma cells, which have been shown to readily form tumor spheroids in polyhydroxyethyl methacrylate (polyHEMA) coated round-bottom wells.29 However, these 4T1 cells did not produce detectable amounts of EGFR as determined by Western blot with an anti-EGFR antibody, so to induce EGFR expression, these 4T1 cells were next transfected with an inducible human EGFR expression cassette (PBQM812A-1, System Biosciences) in which EGFR is expressed as a fusion to green fluorescent protein (GFP) through a self-cleaving linkage.

Techniques: Microscopy, Transfection, Expressing, Fluorescence, Comparison, Western Blot, Imaging, Molecular Weight

Journal: Journal of the American Chemical Society

Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors

doi: 10.1021/jacs.8b07601

Figure Lengend Snippet: Figure 8. (A) Denaturing polyacrylamide gel electrophoresis (SDS- PAGE) showing photo-cross-linking products of either N23BP or WT to EGFR, with or without irradiation at 980 nm. Note that only 980 nm irradiation of N23BP produced a photoproduct significantly different than free EGFR (right lane). Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) Retention of fluorescently labeled affibodies (left, N23BP; right, WT) in 3D tumor spheroids grown from transfected 4T1 cells either induced with 15 μg/mL cumate. Concentrations of retained affibodies were calculated by comparing spheroid lysate fluorescence to standard curves prepared for each labeled affibody. “20 IR” designates that this sample was irradiated at 980 nm after 3 h incubation, then left to grow to a total of 20 h. Lanes without an IR designation were not irradiated.

Article Snippet: For this, we started with 4T1 mouse mammary carcinoma cells, which have been shown to readily form tumor spheroids in polyhydroxyethyl methacrylate (polyHEMA) coated round-bottom wells.29 However, these 4T1 cells did not produce detectable amounts of EGFR as determined by Western blot with an anti-EGFR antibody, so to induce EGFR expression, these 4T1 cells were next transfected with an inducible human EGFR expression cassette (PBQM812A-1, System Biosciences) in which EGFR is expressed as a fusion to green fluorescent protein (GFP) through a self-cleaving linkage.

Techniques: Polyacrylamide Gel Electrophoresis, SDS Page, Irradiation, Produced, Labeling, Transfection, Incubation