assays nebnext poly a mrna magnetic isolation module new england biolabs (New England Biolabs)

New England Biolabs assays nebnext poly a mrna magnetic isolation module new england biolabs

Figure 1. PRRC2B deﬁciency in oligodendroglia cells leads to developmental hypomyelination (A) Immunostaining of PRRC2B/PDGFRa/OLIG2 in the corpus callosum (CC) of mice with indicated genotypes. Scale bars, 25 mm. (B) Left: western blotting analysis of the protein levels of PRRC2B in the cerebral cortex from Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice at P6. Right: quantiﬁcation of PRRC2B protein levels. Values are shown as means ± SEMs, *p < 0.05, unpaired two-tailed Student’s t test (n = 3 mice). (C) Quantitative real-time PCR detected the <t>mRNA</t> levels of Prrc2b in the cerebral cortex of P6 mice. Values are means ± SEMs. ***p < 0.001, unpaired two-tailed Student’s t test (each group, n = 9 replicates from >3 mice). (D) Pictures of Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice and brains at P15. Scale bars, 5 mm (top), 2 cm (bottom).

Assays Nebnext Poly A Mrna Magnetic Isolation Module New England Biolabs, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mcxcl9 cloning plasmid (Sino Biological)

Sino Biological mcxcl9 cloning plasmid

LM2 Cells Transduced with <t>Lenti-mCXCL9</t> Have Reduced Tumorigenicity In Vivo Compared with LM2 (A) Concentration of mCXCL9 levels in the supernatants of LM2 cells transduced with Lenti-mCXCL9. ELISA data are shown at 24 h after plating in triplicate + standard deviation (****p < 0.0001). (B) Viability of LM2-Lenti-mCXCL9 compared with LM2 cells in vitro . Data are shown as mean percent cell viability in comparison with mock-infected cells at 24, 48, and 72 h postinfection + standard deviation. (C) Survival of mice bearing LM2 or Lenti-transduced LM2 tumors was evaluated using Kaplan-Meier survival curves (*p < 0.05).

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mouse cadherin 11 expression plasmide (Sino Biological)

Sino Biological mouse cadherin 11 expression plasmide

A & B. Primary human fibroblasts were pre-labeled by DiI (red) and MDA-MB-231 (A) or BT549 cells (B) were pre-labeled by DiD (blue). Cells were cocultured for overnight before fixation for IF staining. <t>Cadherin-11</t> protein was stained by the specific monoclonal primary antibody (clone 16A) followed by the secondary antibody staining (green). Purple arrows are pointing to the cadherin-11 AJs between cancer cells and fibroblasts. Confocal Z-stacks were scanned from the top of the cell to the bottom of the cell. All 2-D images shown were from 3-D Z-stacks maximum projections. C. The schematic to describe the cell invasion assay. D. Fibroblasts and MDA-MB-231 cells were pre-labeled as before. Fibroblasts alone (middle panels), MDA-MB-231 alone (right panels) or Fibroblasts and MDA-MB-231 (left panels) together in a 1:1 ratio were subjected to the cell invasion assay as described in (C). The whole cell population invasion displacements on the X-axis with a direction to the left were labeled between the yellow lines of 0 hr and 16 hr. The green fluorescence from the matrigel was omitted to clearly visualize the cells. Size bar, 100 μm. E, F & G. Cell invasion speed (Distance/time), invasion velocity (Displacement on the X-axis/time) and invasion persistence (Displacement/Distance) were quantitated. *, P < 0.05. H. Time-lapse zoom-in panels from Video 3. Green arrows are pointing at one cancer cell that was invading back-n-forth by attaching to and sliding on the cell bodies of fibroblasts. The red channel imaging offsets were elevated to visualize the long but thin invasive protrusions of fibroblasts. Size bar, 100 μm. I. Cropped and zoom-in panels from (H) to present the details of the invasive protrusions of fibroblasts (yellow arrow heads).

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mouse epha4 sirna (Sino Biological)

Sino Biological mouse epha4 sirna

( A – F ) Low-dimensional representation of single cells from mouse cerebral cortex, based on UMAP embedding of single-cell RNA-seq data [Data ref: (Di Bella et al, )] are shown. Cells are coloured based on animal age ( A ), or the expression of Phf6 ( B ), <t>EphA4</t> ( C ), EphA7 ( D ), EphB1 ( E ), or EphB2 ( F ). ( G ) Heatmap representation of the Pearson correlation coefficients between Phf6 and EphR across various cell types are shown. Correlation values were calculated using imputed gene expression profiles after applying MAGIC (Van Dijk et al, ). ( H ) UMAP embedding of cells are coloured by cell type. UMAP coordinates and cell type annotations are from [Data ref: (Di Bella et al, ) (GEO GSE153164)].

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human ace2 (Sino Biological)

Sino Biological human ace2

Immunogenicity of NVX-CoV2373 vaccine in rhesus macaques (A–C) Serum anti-spike (S) IgG titer (A), nasal wash (B), and bronchoalveolar lavage (BAL) (C) samples were collected 31/32 days after the first immunization and before challenge and analyzed for S-specific mucosal IgG (n = 4–5/group). (D) Pseudovirus-neutralizing titer (ID 50 ). (E) SARS-CoV-2 neutralizing-antibody titer (99% inhibition of cytopathic effect [99% CPE]) study day 31/32. (F) <t>hACE2</t> receptor-blocking antibody titer (study day 31/32). The geometric mean titers (GMTs) are indicated by the white bars. Hollow arrows indicate prime/boosting with NVX-CoV2373. The error bars indicate the 95% confidence interval (95% CI). Individual animal values are indicated by colored symbols. A Student’s t test (unpaired, two-tailed) was used to compare antibody levels between groups immunized with one and two doses. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001. The horizontal dashed lines indicate the LODs for each assay.

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human serpinb3 (Sino Biological)

Sino Biological human serpinb3

( A ) Normalized <t>SERPINB3</t> transcript in cervical tumor biopsies from RNAseq was distributed by reads per kilobase of transcript per million mapped reads (RPKM). ( B ) Boxplots along with individual data points show xCell immune scores in recurrent (R)/non-recurrent (NR) SERPINB3-low (B3/L) and SERPINB3-high (B3/H) tumors. * P < 0.05, one-way ANOVA test. ( C ) Heatmap of enriched immune cell subpopulation was generated through xCell immune infiltrate prediction. Color intensity is proportional to average xCell score for each population across samples. ( D-G ) Spearman’s correlation of SERPINB3 with the expression of ( D ) CXCL1, ( E ) CXCL8, ( F ) S100A8, ( G ) S100A9 was performed using RNAseq from 66 cervical tumor biopsies collected prior to (chemo)- RT. ( H ) SERPINB3 expression correlated with CXCL1, CXCL8, S100A8, S100A9 expression in multiple cancer types. Analysis was performed using TCGA PanCancer Atlas and numeric values indicate Spearman’s correlation coefficient. BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; HNSC, head and neck squamous cell carcinoma; LUSC, lung squamous cell carcinoma; PRAD, prostate adenocarcinoma; UCEC, uterine corpus endometrial carcinoma

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neurofibromin 1 nf1 (ATCC)

ATCC neurofibromin 1 nf1

Figure 1. Loss of <t>neurofibromin</t> enhances glycolysis and glucose utilization via GLUT1 in macrophages that results in elevated relative mRNA expression of inflammatory cytokines, NF- κB, and NOS2 and polarizes macrophages to mixed phenotypes (A–F) Representative measurements of extracellular acidification rate (ECAR) performed on bone marrow-derived macrophages (BMDMs) derived from long bones of Nf1f/f and Nf1ΔMϕ mice treated with 20 ng/mL LPS and IFNγ or 20 ng/mL IL-4 to polarize the macrophages to inflammatory (MLPSIFNγ) or reparative (MIL4) phenotypes for 16 h, respectively. Subsequent addition of glucose, the ATP synthase inhibitor oligomycin, and the hexokinase inhibitor 2-deoxy-glucose (2-DG) were carried out where indicated (A, C, and E); n = 4 mice/genotype; each data point represents technical replicate. Data (mean ± SEM) were normalized by total protein content at the end of experiment. p values were calculated using one-way ANOVA followed by Tukey’s multiple comparison t test (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; ns, not significant). (G–J) Quantification of percentage of glucose uptake by viable BMDMs derived from bones of Nf1f/f and Nf1ΔMϕ mice in the absence or presence of the GLUT1 inhibitor BAY876. BMDMs were labeled with 2-NBDG at a concentration of 100 μM for 30 min in the absence or presence of BAY876 (50 nM) to measure glucose

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human bt474 (ATCC)

ATCC human bt474

Identification and verification of direct target genes of ZNF408 (A) Schematic diagrams of ZNF408 targets identified by cross analysis of CUT&Tag, ATAC-seq, and RNA-seq. (B) Volcano plots showing differently expressed genes detected by RNA-seq upon knockdown of ZNF408 in MCF-7 cells. Cutoff: log 2 fold change >0.4, p < 0.05. (C) Genome tracks showing CUT&Tag results of ZNF408, SETD1A, and H3K4me3 visualized by the IGV tool at STING1 promoter. (D) MCF-7 cells were transfected with control siRNA, siZNF408, or siSETD1A. Total cellular RNA was isolated and analyzed by RT-qPCR to examine the expression of indicated genes. (E) MCF-7 cells were transfected with ZNF408, ZNF408-Δ1, ZNF408-Δ5, or ZNF408 + si SETD1A. Total RNA was isolated from cells and analyzed by RT-qPCR. (F) RT-qPCR (left) analysis and western blotting analysis (right) of human MDA-MB-231 cells, BT549 cells, or <t>BT474</t> cells with or without depletion of ZNF408. In (D–F), each bar represents the mean ± SD for triplicate experiments (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, two-tailed t test). See <xref ref-type=

Table S2 for the target genes of ZNF408-SETD1A complex. See Table S3 for primer information in the RT-qPCR assay. " width="250" height="auto" />
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vero (ATCC)

ATCC vero

Fig. 1 | DMS of C-terminal region of ZIKV E protein. a, Schematic of ZIKV E protein and workflow of DMS screening for the C-terminal 204 codons of ZIKV E protein. Amplicons generated using forward (red arrows) and reverse (blue arrows) mutagenesis primers were combined with other ZIKV cDNA fragments to generate an infectious cDNA library by CPER. The CPER cDNA library was then transfected into mammalian <t>(Vero)</t> or <t>mosquito</t> <t>(C6/36)</t> cells; recovered viruses were harvested in culture supernatants, deep sequenced and analysed to identify preferentially selected viral mutants. dpt, days post-transfection. b, Graphical representation of deep sequencing data showing codon variant heatmap. c, Mutation frequency of cDNA amplicon library illustrating the extent of mutagenesis in the E-DIII stem-anchor region. Mutation frequency is defined as the percentage at which codon mutants are detected in sequenced molecules at each site. For example, at each site, if mutations are present in 100% of sequenced molecules, the frequency is 1; if not present in any sequenced molecules, the frequency is 0. d, Mutation frequency of cDNA library, mammalian (Vero) cell-selected virus population and mosquito (C6/36) cell-selected virus population. Identities of preferentially selected amino acid mutations are indicated above each peak.

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fth1 (Cell Signaling Technology Inc)

Cell Signaling Technology Inc fth1

( A ) Increased Tfrc transcript levels in 500 nM Baf- and 500 nM Sal-treated mouse embryonic fibroblasts relative to untreated fibroblasts. Western blot showing decreased <t>FTH1</t> and FTL1 protein levels in Baf- and Sal-treated fibroblasts (n = 6). GAPDH is used as loading control. Tfrc expression is depicted as bars representing mean ± SEM, n = 6; shown as black dots. p value is determined by the Welch’s one-way ANOVA as differences between untreated group, and Baf- and Sal-treatments. ( B ) Representative images of FerroOrange staining of cytoplasmic labile iron pools in control, 500 nM Baf-treated and 500 nM Baf-treated fibroblasts with 150 µM iron citrate supplementation. Note the reduced staining in Baf-treated fibroblasts relative to the other conditions. ( C ) mRNA levels of Tfrc in control, 500 nM Baf-treated and 500 nM Baf-treated fibroblasts with 150 µM iron citrate supplementation (left) or in control, 500 nM Sal-treated and 500 nM Sal-treated fibroblasts with 150 µM iron citrate supplementation (right) for 24 hr. Bar graphs depict mean ± SEM of four independent experimental measures (shown as black dots). p values represent Welch’s one-way ANOVA with Dunnett’s correction for multiple comparisons, estimated as differences between Baf- or Sal-treated cells and other experimental groups. ( D ) Whole cell immunoblots of HIF-1α and FTH1 in fibroblasts treated with 500 nM Baf or 500 nM Baf + 150 µM iron citrate (left) or with 500 nM Sal and 500 nM Sal + 150 µM iron citrate (n = 4). GAPDH is used as loading control. ( E ) Transcript levels of HIF-1α target genes in fibroblasts treated with 500 nM Baf or with 500 nM Baf + 150 µM iron citrate. The mean ± SEM of four biological replicates (black dots) is shown. p-values are determined by Welch’s one-way ANOVA with Dunnett’s correction for multiple comparisons (all experimental groups compared to Baf-treated cells). ( F–G ) Gaa KO fibroblasts display impaired lysosomal acidification. Representative spinning-disk microscopy images for Gaa WT and Gaa KO MEFs co-stained with Dextran-Oregon Green 488 and Dextran-TMRM are shown with Baftreatment in Gaa WT used as a positive control for impaired acidification. Scale bar 2 µm. ( G ) Quantification of the intensity of Dextran-Oregon Green in Dextran-TMRM positive punta shows increased Dextran-Oregon Green intensity in Gaa KO fibroblasts and in Baf-treated Gaa WT fibroblasts. Bar graphs depict mean ± SEM, n = 3 independent experiments, with 50 cells per condition from each experiment. ***p<0.001, determined by the unpaired two-tailed t-test with Welch’s correction ( H ) Increased Tfrc expression (left) in Gaa -/- fibroblasts (n = 6, depicted as black dots). p value is determined by the unpaired two-tailed t-test with Welch’s correction. Whole cell immunoblots of FTL1 and HIF-1α (right) in Gaa -/- fibroblasts (n = 6). GAPDH is used as loading control. ( I ) Total, ferrous and ferric iron concentrations in fibroblasts prepared from Gaa -/- and their wild type littermate controls. Results are summarized as mean ± SEM of experimental measures shown as black dots. Differences between means shown as actual p values are determined by the unpaired two-tailed t-test with Welch’s correction.

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invivomab anti mouse cd8 (Bio X Cell)

Bio X Cell invivomab anti mouse cd8

( A , B ) Plasmids expressing human YAP5SA (2 mg/kg) plus RFP control (2 mg/kg) or mEPDR1 (2 mg/kg), together with plasmids expressing PB transposase (0.8 mg/kg), were delivered into mice by hydrodynamic injection ( n = 6 male mice per group). Liver tumors were analyzed 110 days after injection. Photographs show livers ( A ) and tumor numbers ( B ) were determined. Data were presented as the mean ± SEM. ( C ) Dimensionality reduction and visualization based on T-distributed stochastic neighbor embedding (t-SNE) analysis of a subset of mouse liver immunocytes from the indicated group in ( A ). ( D ) Statistical difference analysis for immune cell subsets was obtained from dimensionality reduction analysis in panel ( C ). n = 5 male mice per group and data were presented as the mean ± SD. ( E ) Flow cytometry analysis of the ratio of the immune co-suppressive molecules (PD-1, TIM-3) and immune effector molecules (IFNγ, GzmB) positive cells in liver <t>CD8</t> + T cells from the indicated group in ( A ). n = 6 male mice per group and data were presented as the mean ± SD. ( F ) Schema of coculture of human CD8 + T cells with HepG2 cells expressing Flag-EV or Flag-EPDR1. ( G ) Flow cytometry analysis of the ratio of immunosuppressive molecules (PD-1, TIM-3) and immune effector molecules (IFNγ, GzmB) positive cells in CD8 + T cells after coculture with the indicated tumor cells. n = 3 independent experiments and the data were presented as the mean ± SD. ( H – J ) Hepa 1-6 cells stably expressing Flag-EV or Flag-mEPDR1 were injected subcutaneously into C57BL/6 J mice ( n = 6 male mice per group), and α-CD8 (4 mg/kg) neutralizing antibody was injected intraperitoneally four times (twice a week starting at 10 days after inoculation) to block CD8 + T cells and IgG2b was used as control. Tumor size was measured starting at 10 days after inoculation. Photographs show xenografts ( H ), growth curves ( I ), and relative tumor burdens ( J ) determined at the end of the experiment (day 25). Data were presented as the mean ± SEM. Data information: Statistical significance was determined by two-way ANOVA ( D , I ), one-way ANOVA ( J ), and two-tailed unpaired Student’s t -test ( B , E , G ). .

Invivomab Anti Mouse Cd8, supplied by Bio X Cell, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rnase h1 cdna (Addgene inc)

Addgene inc rnase h1 cdna

Inducible <t>RNase</t> <t>H1</t> transgene expression in vitro. A) Schematic of hiPSC differentiation from fibroblasts to NPCs and neurons. Left panel: Immunofluorescent staining of the neuronal marker MAP2 (green) in hiPSC-neurons (top) and the NPC markers SOX2 (green) and Nestin (red) in hiPSC-NPCs (bottom); scale bar: 100 mm. Right panel: Immunofluorescent staining of S9.6 (red) and DAPI (blue) in each respective cell type; scale bar: 100 mm. B) Schematic of the effect of <t>RNase</t> <t>H1-mediated</t> knockdown of R-loops, or D145N mutant that is binding- competent but catalytically inactive. C) Overview of the lentiviral constructs used in this study, including the rtTA transactivator, RH1ΔMLS, RH1ΔMLS D145N catalytically-inactive mutant, and BFP-expressing lentiviral backbone control. D) (Top) Representative Western Blot of hiPSC-neurons expressing either BFP, RH1ΔMLS, or RH1ΔMLS D145N lentiviruses. Gradient triangles indicate concentration of virus. RNase H1 protein is 5.1-fold increased in RH1ΔMLS cells relative to BFP controls (normalized to β-actin loading control). (Bottom) Immunocytochemistry for S9.6 (red) and RNase H1 (green) in hiPSC-neurons expressing BFP, RH1ΔMLS D145N , or RH1ΔMLS transgenes. RNase H1 protein is robustly increased in the cell nucleus (DAPI, blue) in RH1ΔMLS and RH1ΔMLS D145N cells relative to BFP controls, indicating successful nuclear expression of the transgene-encoded protein; scale bars = 20 mm. E) Experimental timeline. NPCs are transduced with RH1ΔMLS or a control (RH1ΔMLS D145N or BFP backbone) lentivirus. After 4 days in vitro , puromycin (1:1,000) is added to the culture media to select for transgene-expressing cells. After 6 days in culture, puromycin concentration is dropped to 1:5,000. On day 0 (indicating transition from NPC to neuron differentiation), cells are replated and fed every 2 days with neuron differentiation medium containing 1:1,000 doxycycline to activate the transgene. Every week from week 2, electrophysiological recordings are captured by multielectrode array (MEA). After 6 weeks in culture, cells are harvested for DRIP-seq, bulk- and scRNA-seq. F) (top to bottom) DRIP-seq tracks for hiPSC-derived neurons transduced with BFP (blue tracks, BFP Neuron cultures 1-3) or RH1ΔMLS (bright red tracks, cultures 1-3) constructs over a well-defined, conserved R-loop hotspot region , demonstrating robustness of our DRIP-seq experimental and computational processing. Note that y-axis scaling 0-90 for BFP Neuron DRIP-seq is wider than RH1 Neuron scale, 0-10. RNA alone processed by the DRIP-seq protocol (RNA-only DRIP, magenta track) shows no discernable peaks, indicating that our S9.6 immunoprecipitation is specific for DNA/RNA hybrids and does not represent single-stranded or double-stranded RNA. Orange track: in silico HindIII/EcoRI/XbaI/ SSPI restriction enzyme digest showing expected cutting sites of the restriction enzyme cocktail used for DRIP-seq. G) Left: Volcano plot displaying the 6,380 differential promoter-bound R-loop peaks (DRIP-seq) between hiPSC-differentiated cultures expressing RH1ΔMLS (n = 3) or BFP (n = 3) (FDR < 0.05, pink dots) after 6 weeks in culture. Of these, 4,805 DRIP-seq peaks were enriched in BFP controls (log 2 fold change > 1) and 474 DRIP-seq peaks were enriched RH1ΔMLS cells (log 2 fold change < -1), indicating successful knockdown of R-loops in the latter. Right: Volcano plot displaying the 1,753 differential promoter-bound R-loop peaks between RH1ΔMLS (n = 3) and RH1ΔMLS D145N (n = 2) (FDR < 0.1, pink dots) cells after 6 weeks in culture. Of these, 1,709 DRIP-seq peaks were enriched in RH1ΔMLS D145N controls (log 2 fold change > 1) and 35 DRIP-seq peaks were enriched in RH1ΔMLS cells (log 2 fold change < -1), again indicating successful knockdown of R-loops in the latter. H) Representative DRIP-seq tracks over two neuronal genes for BFP-, RH1ΔMLS D145N -, and RH1ΔMLS- expressing hiPSC-neurons after 6 weeks in culture. Pink highlighted region and inset displays magnified promoter region.

Rnase H1 Cdna, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results

Journal: Cell reports

Article Title: PRRC2B modulates oligodendrocyte progenitor cell development and myelination by stabilizing Sox2 mRNA.

doi: 10.1016/j.celrep.2024.113930

Figure Lengend Snippet: Figure 1. PRRC2B deﬁciency in oligodendroglia cells leads to developmental hypomyelination (A) Immunostaining of PRRC2B/PDGFRa/OLIG2 in the corpus callosum (CC) of mice with indicated genotypes. Scale bars, 25 mm. (B) Left: western blotting analysis of the protein levels of PRRC2B in the cerebral cortex from Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice at P6. Right: quantiﬁcation of PRRC2B protein levels. Values are shown as means ± SEMs, *p < 0.05, unpaired two-tailed Student’s t test (n = 3 mice). (C) Quantitative real-time PCR detected the mRNA levels of Prrc2b in the cerebral cortex of P6 mice. Values are means ± SEMs. ***p < 0.001, unpaired two-tailed Student’s t test (each group, n = 9 replicates from >3 mice). (D) Pictures of Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice and brains at P15. Scale bars, 5 mm (top), 2 cm (bottom).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 2X SanTaq PCR Master Mix Sangon Biotech Cat# B532061 Trypsin Inhibitor, Soybean Sangon Biotech Cat# A003587 Papain, Suspension Sangon Biotech Cat# A003124 Hoechst33342 Sangon Biotech Cat# E607328 2x RealStar Green Fast Mixture with ROX II Genstar Cat# A304-10 Recombinant RNasin Ribonuclease Inhibitor Promega Cat# N2518 Proteinase K, recombinant PCR Grade Roche Cat# 03115828001 HA-tag Magnetic Beads Thermo Scientific Cat# 88836 Streptavidin Beads Life Technologies Cat# 11206D L-cysteine Hydrochloride, Monohydrate Amresco Cat# 0206-500G Puromycin InvivoGen Cat# ant-pr-1 Poly-D-lysine Hydrobromide Sigma Cat# P6407 Critical commercial assays NEBNext Poly(A) mRNA Magnetic Isolation Module New England Biolabs Cat# E7490 NEBNext Magnesium RNA Fragmentation Module New England Biolabs Cat# E6150S DynabeadsTM Antibody Coupling Kit Thermo Scientific Cat# 14311D Black-Gold II Myelin Staining Kit Millipore Cat# AG105 TransScript Uni All-in-One First-Strand Cdna Synthesis SuperMix for qPCR (One-Step gDNA Removal) TransGen Biotech Cat# AU341 BCA Protein Assay Kit Beyotime Biotechnology Cat# P0009 Western LightningTM Plus Chemiluminescence Reagent PerkinElmer Cat# NEL104001EA Deposited data Raw and analyzed data (MeRIP-seq) This paper GEO: GSE243310 Raw and analyzed data (RIP-seq) This paper GEO: GSE243309 Raw and analyzed data (RNA-seq) This paper GEO: GSE222394 (NSC) and GSE222395 (OPC) Experimental models: Cell lines Oli-neu Dr. Wei Mo Gift from Dr. Wei Mo O4 Dr. Wei Mo Gift from Dr. Wei Mo GalC Dr. Wei Mo Gift from Dr. Wei Mo Experimental models: Organisms/strains Mouse: Prrc2bf/f This paper Gift from Dr. Fengchao Wang Mouse: Olig2-Cre The Jackson Laboratory Gift from Dr. Bo Xiao, Stock: 011103 Mouse: Nestin-Cre The Jackson Laboratory Stock: 003771 Mouse: Prrc2b-HA This paper Center for Excellence in Molecular Cell Science of the Chinese Academy of Sciences Mouse: Prrc2af/f This paper Gift from Dr. Fengchao Wang Mouse: C57BL/6J This paper SPF(Beijing) Biotechnology Co., Ltd. Oligonucleotides RT-qPCR Primers Sangon Biotech Table S14 Recombinant DNA pCMV-N-mCherry Beyotime Biotechnology Cat# D2711 pCMV-N-mCherry-Sox2-CDS+30-UTR-wt This paper Nanjingjinsirui Science & Technology Biology Corp (Continued on next page) Cell Reports 43, 113930, March 26, 2024 17

Techniques: Immunostaining, Western Blot, Two Tailed Test, Real-time Polymerase Chain Reaction

Figure 2. PRRC2B promotes OL progenitor cell differentiation (A) Immunoﬂuorescence of PDGFRa/OLIG2 in the CC of Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice at P15 or P40. Scale bars, 30 mm. (B) Quantiﬁcation of PDGFRa/OLIG2-positive OPCs in the CC of mice, with the indicated geno- types at P15 or P40. Images of brain slices from >3 mice were gained and analyzed. Values are shown as means ± SEMs, **p < 0.01, NS, not statistically signiﬁcant, unpaired two-tailed Student’s t test (P15: f/f, n = 19, f/f; Olig2, n = 13; P40: f/f, n = 14, f/f; Olig2, n = 6). (C) Immunostainings of CC1/OLIG2 in the CC of Prrc2bf/f, and Prrc2bf/f; Olig2Cre+/ mice at P15 or P40. Scale bars, 30 mm. (D) Quantiﬁcation of CC1/OLIG2-positive OLs in the CC of mice, with the indicated genotypes at P15 or P40. Images of brain slices from >3 mice were gained and analyzed. Values are shown as means ± SEMs, **p < 0.01, ***p < 0.001, unpaired two-tailed Student’s t test (P15: f/f, n = 18, f/f; Olig2, n = 12; P40: f/f, n = 7, f/f; Olig2, n = 6). (E) Left: western blotting analysis of OPC devel- opment-related protein in the CC of mice at P15. Right: quantiﬁcation of protein levels of OLIG2, MAG, MBP, and ALDH1L1. Values are shown as means ± SEMs, *p < 0.05, NS, not statistically signiﬁcant, unpaired two-tailed Student’s t test (n = 3 mice). (F) Quantitative real-time PCR detection of the mRNA levels of OPC-related genes in the CC of Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice at P15. Values were shown as means ± SEMs, *p < 0.05, **p < 0.01, ***p < 0.001, unpaired two-tailed Stu- dent’s t test (n R 4 independent biological repli- cates for each genotype).

Journal: Cell reports

Article Title: PRRC2B modulates oligodendrocyte progenitor cell development and myelination by stabilizing Sox2 mRNA.

doi: 10.1016/j.celrep.2024.113930

Figure Lengend Snippet: Figure 2. PRRC2B promotes OL progenitor cell differentiation (A) Immunoﬂuorescence of PDGFRa/OLIG2 in the CC of Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice at P15 or P40. Scale bars, 30 mm. (B) Quantiﬁcation of PDGFRa/OLIG2-positive OPCs in the CC of mice, with the indicated geno- types at P15 or P40. Images of brain slices from >3 mice were gained and analyzed. Values are shown as means ± SEMs, **p < 0.01, NS, not statistically signiﬁcant, unpaired two-tailed Student’s t test (P15: f/f, n = 19, f/f; Olig2, n = 13; P40: f/f, n = 14, f/f; Olig2, n = 6). (C) Immunostainings of CC1/OLIG2 in the CC of Prrc2bf/f, and Prrc2bf/f; Olig2Cre+/ mice at P15 or P40. Scale bars, 30 mm. (D) Quantiﬁcation of CC1/OLIG2-positive OLs in the CC of mice, with the indicated genotypes at P15 or P40. Images of brain slices from >3 mice were gained and analyzed. Values are shown as means ± SEMs, **p < 0.01, ***p < 0.001, unpaired two-tailed Student’s t test (P15: f/f, n = 18, f/f; Olig2, n = 12; P40: f/f, n = 7, f/f; Olig2, n = 6). (E) Left: western blotting analysis of OPC devel- opment-related protein in the CC of mice at P15. Right: quantiﬁcation of protein levels of OLIG2, MAG, MBP, and ALDH1L1. Values are shown as means ± SEMs, *p < 0.05, NS, not statistically signiﬁcant, unpaired two-tailed Student’s t test (n = 3 mice). (F) Quantitative real-time PCR detection of the mRNA levels of OPC-related genes in the CC of Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice at P15. Values were shown as means ± SEMs, *p < 0.05, **p < 0.01, ***p < 0.001, unpaired two-tailed Stu- dent’s t test (n R 4 independent biological repli- cates for each genotype).

Techniques: Cell Differentiation, Two Tailed Test, Western Blot, Real-time Polymerase Chain Reaction

Figure 3. PRRC2B deﬁciency affects OL progenitor cell differentiation (A) Immunostainings of PRRC2B/PDGFRa/OLIG2 in OPCs isolated from brain tissues of Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice at P4. Scale bars, 50 mm. (B) Western blotting analysis of the protein levels of PRRC2B in OPC-derived cells cultured in prolifera- tion medium for 4 days and quantiﬁcation of PRRC2B protein levels. Values are shown as means ± SEMs, **p < 0.01, unpaired two-tailed Student’s t test (n = 4 mice). (C) Quantitative real-time PCR analysis of the mRNA levels of Prrc2b in OPCs isolated from Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice. Values are means ± SEMs, ***p < 0.001, unpaired two-tailed Student’s t test (n = 6 independent biological replicates for each group). (D) Western blotting analysis the protein levels of OLIG2, ALDH1L1, and MBP in OPC-derived cells cultured in differentiation medium for 4 days. (E) Quantiﬁcation of OLIG2, MBP, and ALDH1L1 protein levels. Values are shown as means ± SEMs, *p < 0.05, **p < 0.01, unpaired two-tailed Student’s t test (n = 3 mice). (F) Quantitative real-time PCR detection of the mRNA levels of OL-related genes in OPCs-derived cells isolated from Prrc2bf/f and Prrc2bf/f; Olig2Cre+/

Journal: Cell reports

Article Title: PRRC2B modulates oligodendrocyte progenitor cell development and myelination by stabilizing Sox2 mRNA.

doi: 10.1016/j.celrep.2024.113930

Figure Lengend Snippet: Figure 3. PRRC2B deﬁciency affects OL progenitor cell differentiation (A) Immunostainings of PRRC2B/PDGFRa/OLIG2 in OPCs isolated from brain tissues of Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice at P4. Scale bars, 50 mm. (B) Western blotting analysis of the protein levels of PRRC2B in OPC-derived cells cultured in prolifera- tion medium for 4 days and quantiﬁcation of PRRC2B protein levels. Values are shown as means ± SEMs, **p < 0.01, unpaired two-tailed Student’s t test (n = 4 mice). (C) Quantitative real-time PCR analysis of the mRNA levels of Prrc2b in OPCs isolated from Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice. Values are means ± SEMs, ***p < 0.001, unpaired two-tailed Student’s t test (n = 6 independent biological replicates for each group). (D) Western blotting analysis the protein levels of OLIG2, ALDH1L1, and MBP in OPC-derived cells cultured in differentiation medium for 4 days. (E) Quantiﬁcation of OLIG2, MBP, and ALDH1L1 protein levels. Values are shown as means ± SEMs, *p < 0.05, **p < 0.01, unpaired two-tailed Student’s t test (n = 3 mice). (F) Quantitative real-time PCR detection of the mRNA levels of OL-related genes in OPCs-derived cells isolated from Prrc2bf/f and Prrc2bf/f; Olig2Cre+/

Techniques: Cell Differentiation, Isolation, Western Blot, Derivative Assay, Cell Culture, Two Tailed Test, Real-time Polymerase Chain Reaction

Figure 4. PRRC2B is an m6A-speciﬁc bind- ing protein in the brain (A) Schematic diagram of RNA pull-down showing that the unmethylated oligo(A) and methylated oligo-m6A RNA were used to capture potential m6A binding proteins in vitro. (B) Western blotting displaying endogenous PRRC2B and YTHDF1 pulled down in the brains of P4 mice. (C) LC-MS/MS quantiﬁcation of the m6A/A ratio in RNA isolated from OPCs of Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice. Values are means ± SEMs. Un- paired two-tailed Student’s t test (n = 4 experi- ments for each group). (D) Left: HA-tag was added to the C terminus of Prrc2b in mice. Right: western blotting of the efﬁ- ciency of HA-tag expression in whole brains of WT and Prrc2b-HA mice at P4. (E) Schematic diagram of the RIP assay for LC-MS/ MS and sequencing. (F) Quantiﬁcation of the m6A/A ratio in mRNA iso- lated from the cerebral cortex of WT and Prrc2b- HA mice at P4 by RIP. Values are means ± SEMs of 3 independent experiments, *p < 0.05, unpaired two-tailed Student’s t test (n = 3 independent biological replicates). (G) Pie chart presenting the distribution of PRRC2B-binding peaks in the 30 UTR, 50 UTR, CDS, or noncoding regions. (H) Enrichment chart depicting the proportion of PRRC2B-binding peaks in the corresponding 4 regions. (I) Binding motif identiﬁed by HOMER with PRRC2B-binding peaks (p = 1e12).

Journal: Cell reports

Article Title: PRRC2B modulates oligodendrocyte progenitor cell development and myelination by stabilizing Sox2 mRNA.

doi: 10.1016/j.celrep.2024.113930

Figure Lengend Snippet: Figure 4. PRRC2B is an m6A-speciﬁc bind- ing protein in the brain (A) Schematic diagram of RNA pull-down showing that the unmethylated oligo(A) and methylated oligo-m6A RNA were used to capture potential m6A binding proteins in vitro. (B) Western blotting displaying endogenous PRRC2B and YTHDF1 pulled down in the brains of P4 mice. (C) LC-MS/MS quantiﬁcation of the m6A/A ratio in RNA isolated from OPCs of Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice. Values are means ± SEMs. Un- paired two-tailed Student’s t test (n = 4 experi- ments for each group). (D) Left: HA-tag was added to the C terminus of Prrc2b in mice. Right: western blotting of the efﬁ- ciency of HA-tag expression in whole brains of WT and Prrc2b-HA mice at P4. (E) Schematic diagram of the RIP assay for LC-MS/ MS and sequencing. (F) Quantiﬁcation of the m6A/A ratio in mRNA iso- lated from the cerebral cortex of WT and Prrc2b- HA mice at P4 by RIP. Values are means ± SEMs of 3 independent experiments, *p < 0.05, unpaired two-tailed Student’s t test (n = 3 independent biological replicates). (G) Pie chart presenting the distribution of PRRC2B-binding peaks in the 30 UTR, 50 UTR, CDS, or noncoding regions. (H) Enrichment chart depicting the proportion of PRRC2B-binding peaks in the corresponding 4 regions. (I) Binding motif identiﬁed by HOMER with PRRC2B-binding peaks (p = 1e12).

Techniques: Methylation, Binding Assay, In Vitro, Western Blot, Liquid Chromatography with Mass Spectroscopy, Isolation, Two Tailed Test, Expressing, Sequencing

Figure 5. PRRC2B regulates OL develop- ment in an m6A modiﬁcation-dependent manner (A) Distribution of m6A peaks across the length of the mRNA (50 UTR, CDS, and 30 UTR). (B) Enriched motif identiﬁed by HOMER with m6A peaks in OPCs isolated from C57BL/6J mice at P4 (p = 1e122). (C) GO terms in the biological process category enriched in transcripts with downregulated expression levels in OPCs isolated from Prrc2bf/f

Journal: Cell reports

Article Title: PRRC2B modulates oligodendrocyte progenitor cell development and myelination by stabilizing Sox2 mRNA.

doi: 10.1016/j.celrep.2024.113930

Figure Lengend Snippet: Figure 5. PRRC2B regulates OL develop- ment in an m6A modiﬁcation-dependent manner (A) Distribution of m6A peaks across the length of the mRNA (50 UTR, CDS, and 30 UTR). (B) Enriched motif identiﬁed by HOMER with m6A peaks in OPCs isolated from C57BL/6J mice at P4 (p = 1e122). (C) GO terms in the biological process category enriched in transcripts with downregulated expression levels in OPCs isolated from Prrc2bf/f

Techniques: Isolation, Expressing

Figure 6. Sox2 is the target gene of PRRC2B during OPC development (A) Primary OPCs isolated from Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice at P4 were exposed to ActD, and RNA was extracted at the indicated time points. Quantitative real-time PCR was per- formed to assess the half-life of Sox2 mRNA. The data are shown as means ± SEMs, *p < 0.05, **p < 0.01, unpaired two-tailed Student’s t test (T1/2(f/f) = 44.99 min; T1/2(f/f; Olig2) = 20.02 min). (B) Integrative Genomics Viewer (IGV) tracks showing the read distributions of RNA-seq (top, n = 3), PRRC2B RIP-seq (center, n = 2), and MeRIP-seq (bottom, n = 3) proﬁling of Sox2 gene, with signiﬁcant peaks highlighted in yel- low. Dark green indicates normalized RNA-seq proﬁles of OPCs isolated from Prrc2b cKO mice at P4, and gray shows that of OPCs of Prrc2bf/f

Journal: Cell reports

Article Title: PRRC2B modulates oligodendrocyte progenitor cell development and myelination by stabilizing Sox2 mRNA.

doi: 10.1016/j.celrep.2024.113930

Figure Lengend Snippet: Figure 6. Sox2 is the target gene of PRRC2B during OPC development (A) Primary OPCs isolated from Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice at P4 were exposed to ActD, and RNA was extracted at the indicated time points. Quantitative real-time PCR was per- formed to assess the half-life of Sox2 mRNA. The data are shown as means ± SEMs, *p < 0.05, **p < 0.01, unpaired two-tailed Student’s t test (T1/2(f/f) = 44.99 min; T1/2(f/f; Olig2) = 20.02 min). (B) Integrative Genomics Viewer (IGV) tracks showing the read distributions of RNA-seq (top, n = 3), PRRC2B RIP-seq (center, n = 2), and MeRIP-seq (bottom, n = 3) proﬁling of Sox2 gene, with signiﬁcant peaks highlighted in yel- low. Dark green indicates normalized RNA-seq proﬁles of OPCs isolated from Prrc2b cKO mice at P4, and gray shows that of OPCs of Prrc2bf/f

Techniques: Isolation, Real-time Polymerase Chain Reaction, Two Tailed Test, RNA Sequencing

LM2 Cells Transduced with Lenti-mCXCL9 Have Reduced Tumorigenicity In Vivo Compared with LM2 (A) Concentration of mCXCL9 levels in the supernatants of LM2 cells transduced with Lenti-mCXCL9. ELISA data are shown at 24 h after plating in triplicate + standard deviation (****p < 0.0001). (B) Viability of LM2-Lenti-mCXCL9 compared with LM2 cells in vitro . Data are shown as mean percent cell viability in comparison with mock-infected cells at 24, 48, and 72 h postinfection + standard deviation. (C) Survival of mice bearing LM2 or Lenti-transduced LM2 tumors was evaluated using Kaplan-Meier survival curves (*p < 0.05).

Journal: Molecular Therapy Oncolytics

Article Title: Generation of a Tumor-Specific Chemokine Gradient Using Oncolytic Vesicular Stomatitis Virus Encoding CXCL9

doi: 10.1016/j.omto.2019.12.003

Figure Lengend Snippet: LM2 Cells Transduced with Lenti-mCXCL9 Have Reduced Tumorigenicity In Vivo Compared with LM2 (A) Concentration of mCXCL9 levels in the supernatants of LM2 cells transduced with Lenti-mCXCL9. ELISA data are shown at 24 h after plating in triplicate + standard deviation (****p < 0.0001). (B) Viability of LM2-Lenti-mCXCL9 compared with LM2 cells in vitro . Data are shown as mean percent cell viability in comparison with mock-infected cells at 24, 48, and 72 h postinfection + standard deviation. (C) Survival of mice bearing LM2 or Lenti-transduced LM2 tumors was evaluated using Kaplan-Meier survival curves (*p < 0.05).

Article Snippet: Mouse cxcl9 was PCR amplified from a mCXCL9 cloning plasmid (catalog number MG50155-M; Sino Biological, Wayne, PA, USA).

Techniques: Transduction, In Vivo, Concentration Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation, In Vitro, Infection

Murine and Human CXCL9 Transgenes Engineered in Recombinant VSVs Do Not Alter the Growth Kinetics or Viral Killing Ability In Vitro Schematic depiction of the genomes of recombinant VSVs encoding murine (A) or human (B) CXCL9, CXCLi, and GFP. (C) Replication kinetics of VSVs encoding murine or human CXCL9 were performed in a multistep viral growth curve in Vero cells. Data are shown from duplicate experiments as average titer + standard deviation. (D) Viability of VSV-mCXCL9-, VSV-mCXCLi-, and VSV-GFP-infected Vero and murine LM2 tumor cells was assessed at 24 and 48 h postinfection at an MOI of 10. (E) Viability of VSV-M51R-hCXCL9- and VSV-M51R-hCXCLi-infected Vero and human FaDu-Luc tumor cells was measured at 24 and 72 h postinfection at an MOI of 10. Data are presented as duplicate experiments as average percent viability compared with mock-infected cells + standard deviation. Significance was determined by paired t test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Journal: Molecular Therapy Oncolytics

Article Title: Generation of a Tumor-Specific Chemokine Gradient Using Oncolytic Vesicular Stomatitis Virus Encoding CXCL9

doi: 10.1016/j.omto.2019.12.003

Figure Lengend Snippet: Murine and Human CXCL9 Transgenes Engineered in Recombinant VSVs Do Not Alter the Growth Kinetics or Viral Killing Ability In Vitro Schematic depiction of the genomes of recombinant VSVs encoding murine (A) or human (B) CXCL9, CXCLi, and GFP. (C) Replication kinetics of VSVs encoding murine or human CXCL9 were performed in a multistep viral growth curve in Vero cells. Data are shown from duplicate experiments as average titer + standard deviation. (D) Viability of VSV-mCXCL9-, VSV-mCXCLi-, and VSV-GFP-infected Vero and murine LM2 tumor cells was assessed at 24 and 48 h postinfection at an MOI of 10. (E) Viability of VSV-M51R-hCXCL9- and VSV-M51R-hCXCLi-infected Vero and human FaDu-Luc tumor cells was measured at 24 and 72 h postinfection at an MOI of 10. Data are presented as duplicate experiments as average percent viability compared with mock-infected cells + standard deviation. Significance was determined by paired t test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Article Snippet: Mouse cxcl9 was PCR amplified from a mCXCL9 cloning plasmid (catalog number MG50155-M; Sino Biological, Wayne, PA, USA).

Techniques: Recombinant, In Vitro, Standard Deviation, Infection

CXCL9 Expressed from VSV-mCXCL9 Is Biologically Active (A) Murine CXCL9 secretion was evaluated in vitro in the LM2 non-small cell lung cancer cell line. Supernatants of VSV-infected LM2 cells (MOI 0.1) were collected 24 h postinfection, and chemokine concentration was determined by ELISA. Concentrations are presented as average concentration + standard deviation. (B) Chemotactic activity of virally encoded mCXCL9 was assessed in an in vitro migration assay adapted from Campanella et al. Numbers of migrated cells are presented as average percent increase in migration compared with mock treated + standard deviation. Significance was determined by paired two-tailed t test (**p < 0.01).

Journal: Molecular Therapy Oncolytics

Article Title: Generation of a Tumor-Specific Chemokine Gradient Using Oncolytic Vesicular Stomatitis Virus Encoding CXCL9

doi: 10.1016/j.omto.2019.12.003

Figure Lengend Snippet: CXCL9 Expressed from VSV-mCXCL9 Is Biologically Active (A) Murine CXCL9 secretion was evaluated in vitro in the LM2 non-small cell lung cancer cell line. Supernatants of VSV-infected LM2 cells (MOI 0.1) were collected 24 h postinfection, and chemokine concentration was determined by ELISA. Concentrations are presented as average concentration + standard deviation. (B) Chemotactic activity of virally encoded mCXCL9 was assessed in an in vitro migration assay adapted from Campanella et al. Numbers of migrated cells are presented as average percent increase in migration compared with mock treated + standard deviation. Significance was determined by paired two-tailed t test (**p < 0.01).

Article Snippet: Mouse cxcl9 was PCR amplified from a mCXCL9 cloning plasmid (catalog number MG50155-M; Sino Biological, Wayne, PA, USA).

Techniques: In Vitro, Infection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation, Activity Assay, Migration, Two Tailed Test

Oncolytic Activity of VSV-mCXCL9 Survival of mice bearing 5TGM1 or LM2 tumors was evaluated using Kaplan-Meier survival curves. (A) 5 × 10 6 5TGM1 cells were implanted subcutaneously on the right flanks of C57Bl6/KaLwRij mice; once tumors had grown to ∼6-mm diameter, mice were injected intravenously with PBS, VSV-mCXCL9, or VSV-GFP (n = 10 for PBS and VSV-mCXCL9 treatments, n = 9 for VSV-GFP treatment). (B) 1 × 10 6 LM2 cells were implanted subcutaneously on the right flanks of A/J mice, and established tumors (∼6-mm diameter) were injected intratumorally with PBS, VSV-mCXCL9, or VSV-GFP (n = 10 for all treatment groups). Survival analysis was performed using log rank statistics (***p < 0.001, ****p < 0.0001).

Journal: Molecular Therapy Oncolytics

Article Title: Generation of a Tumor-Specific Chemokine Gradient Using Oncolytic Vesicular Stomatitis Virus Encoding CXCL9

doi: 10.1016/j.omto.2019.12.003

Figure Lengend Snippet: Oncolytic Activity of VSV-mCXCL9 Survival of mice bearing 5TGM1 or LM2 tumors was evaluated using Kaplan-Meier survival curves. (A) 5 × 10 6 5TGM1 cells were implanted subcutaneously on the right flanks of C57Bl6/KaLwRij mice; once tumors had grown to ∼6-mm diameter, mice were injected intravenously with PBS, VSV-mCXCL9, or VSV-GFP (n = 10 for PBS and VSV-mCXCL9 treatments, n = 9 for VSV-GFP treatment). (B) 1 × 10 6 LM2 cells were implanted subcutaneously on the right flanks of A/J mice, and established tumors (∼6-mm diameter) were injected intratumorally with PBS, VSV-mCXCL9, or VSV-GFP (n = 10 for all treatment groups). Survival analysis was performed using log rank statistics (***p < 0.001, ****p < 0.0001).

Article Snippet: Mouse cxcl9 was PCR amplified from a mCXCL9 cloning plasmid (catalog number MG50155-M; Sino Biological, Wayne, PA, USA).

Techniques: Activity Assay, Injection

Generation of a Tumor to Blood CXCL9 Chemokine Gradient after Intratumoral VSV-mCXCL9 Administration Intratumoral and serum mCXCL9 protein concentrations were determined after intratumoral injection of VSV-mCXCL9 or VSV-GFP in LM2 tumor-bearing mice by ELISA (n = 3 mice/group for each time point). Values are presented as average chemokine concentration in pg/mL + standard deviation. Significance was determined by paired two-tailed t test (**p < 0.01).

Journal: Molecular Therapy Oncolytics

Article Title: Generation of a Tumor-Specific Chemokine Gradient Using Oncolytic Vesicular Stomatitis Virus Encoding CXCL9

doi: 10.1016/j.omto.2019.12.003

Figure Lengend Snippet: Generation of a Tumor to Blood CXCL9 Chemokine Gradient after Intratumoral VSV-mCXCL9 Administration Intratumoral and serum mCXCL9 protein concentrations were determined after intratumoral injection of VSV-mCXCL9 or VSV-GFP in LM2 tumor-bearing mice by ELISA (n = 3 mice/group for each time point). Values are presented as average chemokine concentration in pg/mL + standard deviation. Significance was determined by paired two-tailed t test (**p < 0.01).

Article Snippet: Mouse cxcl9 was PCR amplified from a mCXCL9 cloning plasmid (catalog number MG50155-M; Sino Biological, Wayne, PA, USA).

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Concentration Assay, Standard Deviation, Two Tailed Test

VSV-mCXCL9 Does Not Increase CD8 + or Th1 Cell Intratumoral Infiltration or CD8 + Inflammatory Cytokine Production LM2 tumors were harvested and processed 7 and 10 days after intratumoral (i.t.) injection of VSV-mCXCL9, VSV-GFP, or PBS, and flow data were gated on (A) CD45 + CD3 + CD8 + T cells or (C) CD45 + CD3 + CD4 + T-bet + (Th1) cells. T cell numbers are presented as average number of immune cells/mg tumor ± standard deviation (n = 4 mice/group/day). (B) Cytokine production was gated on TNF-α- and IFN-γ-producing cells from (A). Statistical significance was determined by paired two-tailed t test (*p < 0.05).

Journal: Molecular Therapy Oncolytics

Article Title: Generation of a Tumor-Specific Chemokine Gradient Using Oncolytic Vesicular Stomatitis Virus Encoding CXCL9

doi: 10.1016/j.omto.2019.12.003

Figure Lengend Snippet: VSV-mCXCL9 Does Not Increase CD8 + or Th1 Cell Intratumoral Infiltration or CD8 + Inflammatory Cytokine Production LM2 tumors were harvested and processed 7 and 10 days after intratumoral (i.t.) injection of VSV-mCXCL9, VSV-GFP, or PBS, and flow data were gated on (A) CD45 + CD3 + CD8 + T cells or (C) CD45 + CD3 + CD4 + T-bet + (Th1) cells. T cell numbers are presented as average number of immune cells/mg tumor ± standard deviation (n = 4 mice/group/day). (B) Cytokine production was gated on TNF-α- and IFN-γ-producing cells from (A). Statistical significance was determined by paired two-tailed t test (*p < 0.05).

Article Snippet: Mouse cxcl9 was PCR amplified from a mCXCL9 cloning plasmid (catalog number MG50155-M; Sino Biological, Wayne, PA, USA).

Techniques: Injection, Standard Deviation, Two Tailed Test

Journal: bioRxiv

Article Title: Post-EMT: Cadherin-11 mediates cancer hijacking fibroblasts

doi: 10.1101/729491

Figure Lengend Snippet: A & B. Primary human fibroblasts were pre-labeled by DiI (red) and MDA-MB-231 (A) or BT549 cells (B) were pre-labeled by DiD (blue). Cells were cocultured for overnight before fixation for IF staining. Cadherin-11 protein was stained by the specific monoclonal primary antibody (clone 16A) followed by the secondary antibody staining (green). Purple arrows are pointing to the cadherin-11 AJs between cancer cells and fibroblasts. Confocal Z-stacks were scanned from the top of the cell to the bottom of the cell. All 2-D images shown were from 3-D Z-stacks maximum projections. C. The schematic to describe the cell invasion assay. D. Fibroblasts and MDA-MB-231 cells were pre-labeled as before. Fibroblasts alone (middle panels), MDA-MB-231 alone (right panels) or Fibroblasts and MDA-MB-231 (left panels) together in a 1:1 ratio were subjected to the cell invasion assay as described in (C). The whole cell population invasion displacements on the X-axis with a direction to the left were labeled between the yellow lines of 0 hr and 16 hr. The green fluorescence from the matrigel was omitted to clearly visualize the cells. Size bar, 100 μm. E, F & G. Cell invasion speed (Distance/time), invasion velocity (Displacement on the X-axis/time) and invasion persistence (Displacement/Distance) were quantitated. *, P < 0.05. H. Time-lapse zoom-in panels from Video 3. Green arrows are pointing at one cancer cell that was invading back-n-forth by attaching to and sliding on the cell bodies of fibroblasts. The red channel imaging offsets were elevated to visualize the long but thin invasive protrusions of fibroblasts. Size bar, 100 μm. I. Cropped and zoom-in panels from (H) to present the details of the invasive protrusions of fibroblasts (yellow arrow heads).

Article Snippet: Mouse cadherin-11 expression plasmide was from Sino biological Inc. (Wayne, PA). pGL4.51[luc2/CMV/Neo] vector encoding the firefly luciferase reporter gene luc2 was from Promega (Madison, WI).

Techniques: Labeling, Staining, Invasion Assay, Fluorescence, Imaging

A. Primary human fibroblasts were pre-labeled by DiI (red) and BT549 cells were pre-labeled by DiO (green). BT549 alone spheroid with negative control siRNA or CDH11 siRNA (top panels), and fibroblasts alone spheroid with negative control siRNA or CDH11 siRNA (bottom panels) were subjected to the 3D spheroid cell invasion assay. B. Quantitation for images as in (A). Experiments were repeated 6 times (n=6). *, P < 0.05. C. BT549 and fibroblasts coculture (1:1) spheroid with negative control siRNA or CDH11 siRNA were subjected to the 3D spheroid cell invasion assay. D, E & F. Quantitation for images as in (C). Experiments were repeated 6 times (n=6). *, P < 0.05. Total cell numbers maintained the same in every spheroid. Confocal Z-stacks were scanned from the top of the spheroid to the bottom. All 2-D images shown were from 3-D Z-stacks maximum projections.

Journal: bioRxiv

Article Title: Post-EMT: Cadherin-11 mediates cancer hijacking fibroblasts

doi: 10.1101/729491

Figure Lengend Snippet: A. Primary human fibroblasts were pre-labeled by DiI (red) and BT549 cells were pre-labeled by DiO (green). BT549 alone spheroid with negative control siRNA or CDH11 siRNA (top panels), and fibroblasts alone spheroid with negative control siRNA or CDH11 siRNA (bottom panels) were subjected to the 3D spheroid cell invasion assay. B. Quantitation for images as in (A). Experiments were repeated 6 times (n=6). *, P < 0.05. C. BT549 and fibroblasts coculture (1:1) spheroid with negative control siRNA or CDH11 siRNA were subjected to the 3D spheroid cell invasion assay. D, E & F. Quantitation for images as in (C). Experiments were repeated 6 times (n=6). *, P < 0.05. Total cell numbers maintained the same in every spheroid. Confocal Z-stacks were scanned from the top of the spheroid to the bottom. All 2-D images shown were from 3-D Z-stacks maximum projections.

Techniques: Labeling, Negative Control, Invasion Assay, Quantitation Assay

A. CDH11 (Cadherin-11), ESR1(Estrogen Receptor 1), PGR (Progesterone Receptor) & ERBB2 (Receptor tyrosine-protein kinase erbB-2) expression data in breast cancer cell lines from CCLE (Cancer Cell Line Encyclopedia, The Broad Institute of MIT & Harvard) were plotted into a heat map. B. Kaplan-Meier analysis for breast cancer patients stratified by CDH11 expression for 1075 patients from The Human Protein Atlas database. C-E. Kaplan-Meier plots of overall survival (C, n=626), relapse-free survival (D, n=1764) and distant metastasis free survival (E, n=664) of breast cancer patients in relation to CDH11 expression according to The KM-plotter database. F. Kaplan-Meier plots of distant metastasis free survival (n=68) of ER negative breast cancer patients in relation to CDH11 expression according to The KM-plotter database.

Journal: bioRxiv

Article Title: Post-EMT: Cadherin-11 mediates cancer hijacking fibroblasts

doi: 10.1101/729491

Figure Lengend Snippet: A. CDH11 (Cadherin-11), ESR1(Estrogen Receptor 1), PGR (Progesterone Receptor) & ERBB2 (Receptor tyrosine-protein kinase erbB-2) expression data in breast cancer cell lines from CCLE (Cancer Cell Line Encyclopedia, The Broad Institute of MIT & Harvard) were plotted into a heat map. B. Kaplan-Meier analysis for breast cancer patients stratified by CDH11 expression for 1075 patients from The Human Protein Atlas database. C-E. Kaplan-Meier plots of overall survival (C, n=626), relapse-free survival (D, n=1764) and distant metastasis free survival (E, n=664) of breast cancer patients in relation to CDH11 expression according to The KM-plotter database. F. Kaplan-Meier plots of distant metastasis free survival (n=68) of ER negative breast cancer patients in relation to CDH11 expression according to The KM-plotter database.

Techniques: Expressing

A. Primary human fibroblasts were transduced by GIPZ lentiviral CDH11 shRNA or non-silencing shRNA with a GFP reporter. Stable transductant cells were sorted out by FACS based on the GFP signal. Silencing of CDH11 in these cells was then quantified by RT-qPCR. B. Effect of CDH11 silencing in human fibroblasts on cancer cell growth in the cancer with fibroblast co-implantation xenograft mouse model. 1 × 10 6 of MDA-MB-231-luc cells mixed with 1 × 10 6 of stable non-silencing (blue) or CHD11 silencing (red) primary human fibroblasts were co-implanted into the left fourth mammary fat pad of NOD/SCID mice. The bioluminescence of the MDA-MB-231-Luc cells were measured every 2 weeks. C. Representative in vivo bioluminescence images from (B) at 14 weeks after cancer with fibroblast co-implantation. D. Comparison of tumor volume in mice co-implanted with MDA-MB-231-luc cells and non-silencing or CDH11 silencing primary human fibroblasts. Data were presented as mean ± SD (n=8). P values were determined by two-tailed Student’s t tests (NS, not significant; *, 0.01 < p < 0.05).

Journal: bioRxiv

Article Title: Post-EMT: Cadherin-11 mediates cancer hijacking fibroblasts

doi: 10.1101/729491

Figure Lengend Snippet: A. Primary human fibroblasts were transduced by GIPZ lentiviral CDH11 shRNA or non-silencing shRNA with a GFP reporter. Stable transductant cells were sorted out by FACS based on the GFP signal. Silencing of CDH11 in these cells was then quantified by RT-qPCR. B. Effect of CDH11 silencing in human fibroblasts on cancer cell growth in the cancer with fibroblast co-implantation xenograft mouse model. 1 × 10 6 of MDA-MB-231-luc cells mixed with 1 × 10 6 of stable non-silencing (blue) or CHD11 silencing (red) primary human fibroblasts were co-implanted into the left fourth mammary fat pad of NOD/SCID mice. The bioluminescence of the MDA-MB-231-Luc cells were measured every 2 weeks. C. Representative in vivo bioluminescence images from (B) at 14 weeks after cancer with fibroblast co-implantation. D. Comparison of tumor volume in mice co-implanted with MDA-MB-231-luc cells and non-silencing or CDH11 silencing primary human fibroblasts. Data were presented as mean ± SD (n=8). P values were determined by two-tailed Student’s t tests (NS, not significant; *, 0.01 < p < 0.05).

Techniques: shRNA, Quantitative RT-PCR, In Vivo, Two Tailed Test

A. CDH11 stable overexpression in 4T1 cells (4T1-CDH11) was quantified by RT-qPCR against CDH11 expression levels in 4T1 wildtype cells (4T1-WT). B. 4T1-WT cells or 4T1-CDH11 cells were pre-labeled by DiD (red). NIH3T3 mouse fibroblasts were pre-labeled by DiO (green). 4T1 alone spheroids (top panels), or 4T1 and NIH3T3 coculture (1:1) spheroids (bottom panels) were subjected to the 3D spheroid cell invasion assay as in . Total cell numbers maintained the same in every spheroid. Confocal Z-stacks were scanned from the top of the spheroid to the bottom. All 2-D images shown were from 3-D Z-stacks maximum projections. C. Quantitation for images as in (B). Experiments were repeated 5 times (n=5). NS, not significant; *, P < 0.05. D. Comparison of tumor volume in BALB/c mice implanted with 4T1-WT cells or 4T1-CDH11 cells. 1 × 10 6 of 4T1 cells were implanted into the left fourth mammary fat pad in BALB/c mice. Data were presented as mean ± SD (n=8). E. Comparison of 4T1-WT cell and 4T1-CDH11 cell proliferation in 2D culture in vitro. Same number of cells (46,000 cells) of each group were seeded in one well of a 6-well plate. Cell number was counted at 24 hrs, 48 hrs & 72 hrs (n=3 for each cell group at each time point) after cell seeding. NS, not significant. Note all cells were still not confluent at 72 hrs in each well of a 6-well plate. F. Kaplan-Meier survival curve of BALB/c mice implanted with either 4T1-WT cells or 4T1-CDH11 cells as in (D), n=8. G. micro-MRI imaging of tumor-bearing BALB/c mice from (D) on the 21 st day after implantation of 4T1-WT cells or 4T1-CDH11 cells. Multiple distal metastatic sites in the dorsal neck region lymph nodes (denoted by small arrows) were detected in mice with 4T1-CDH11 tumors. Large arrowheads denote the original tumors at the left fourth mammary fat pad. micro-MRI image Z-stacks were scanned from the dorsal side to the ventral side of the mice. Single plane image section across the dorsal neck region lymph nodes from two representative mice from each group is shown. No distal metastasis was detected in any mice with 4T1-WT tumors in all micro-MRI image Z-stacks.

Journal: bioRxiv

Article Title: Post-EMT: Cadherin-11 mediates cancer hijacking fibroblasts

doi: 10.1101/729491

Figure Lengend Snippet: A. CDH11 stable overexpression in 4T1 cells (4T1-CDH11) was quantified by RT-qPCR against CDH11 expression levels in 4T1 wildtype cells (4T1-WT). B. 4T1-WT cells or 4T1-CDH11 cells were pre-labeled by DiD (red). NIH3T3 mouse fibroblasts were pre-labeled by DiO (green). 4T1 alone spheroids (top panels), or 4T1 and NIH3T3 coculture (1:1) spheroids (bottom panels) were subjected to the 3D spheroid cell invasion assay as in . Total cell numbers maintained the same in every spheroid. Confocal Z-stacks were scanned from the top of the spheroid to the bottom. All 2-D images shown were from 3-D Z-stacks maximum projections. C. Quantitation for images as in (B). Experiments were repeated 5 times (n=5). NS, not significant; *, P < 0.05. D. Comparison of tumor volume in BALB/c mice implanted with 4T1-WT cells or 4T1-CDH11 cells. 1 × 10 6 of 4T1 cells were implanted into the left fourth mammary fat pad in BALB/c mice. Data were presented as mean ± SD (n=8). E. Comparison of 4T1-WT cell and 4T1-CDH11 cell proliferation in 2D culture in vitro. Same number of cells (46,000 cells) of each group were seeded in one well of a 6-well plate. Cell number was counted at 24 hrs, 48 hrs & 72 hrs (n=3 for each cell group at each time point) after cell seeding. NS, not significant. Note all cells were still not confluent at 72 hrs in each well of a 6-well plate. F. Kaplan-Meier survival curve of BALB/c mice implanted with either 4T1-WT cells or 4T1-CDH11 cells as in (D), n=8. G. micro-MRI imaging of tumor-bearing BALB/c mice from (D) on the 21 st day after implantation of 4T1-WT cells or 4T1-CDH11 cells. Multiple distal metastatic sites in the dorsal neck region lymph nodes (denoted by small arrows) were detected in mice with 4T1-CDH11 tumors. Large arrowheads denote the original tumors at the left fourth mammary fat pad. micro-MRI image Z-stacks were scanned from the dorsal side to the ventral side of the mice. Single plane image section across the dorsal neck region lymph nodes from two representative mice from each group is shown. No distal metastasis was detected in any mice with 4T1-WT tumors in all micro-MRI image Z-stacks.

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Labeling, Invasion Assay, Quantitation Assay, In Vitro, Micro-MRI, Imaging

1 × 10 6 of 4T1 mouse triple negative breast cancer cells expressing firefly luciferase with or without CDH11 overexpression were implanted into the left fourth mammary fat pad of immunocompetent BALB/c mice (A-C) or NOD/SCID mice (E-G). A. Comparison of whole tumor growth volume between the 4T1-WT-luc cells implantation group and the 4T1-CDH11-luc cells implantation group in BALB/c mice. B. Comparison of cancer growth (as detected by the firefly luciferase bioluminescence) between the 4T1-WT-luc cells implantation group and the 4T1-CDH11-luc cells implantation group at 4 weeks after implantation in BALB/c mice. Data were presented as mean ± SD (n=7). C. Representative in vivo bioluminescence images from (B). D. Comparison of 4T1-WT-luc cell and 4T1-CDH11-luc cell proliferation in 2D culture in vitro. Same number of cells (46,000 cells) of each group were seeded in one well of a 6-well plate. Cell number was counted at 24 hrs, 48 hrs & 72 hrs (n=3 for each cell group at each time point) after cell seeding. NS, not significant. Note all cells were still not confluent at 72 hrs in each well of a 6-well plate. E. Comparison of whole tumor growth volume between the 4T1-WT-luc cells implantation group and the 4T1-CDH11-luc cells implantation group in NOD/SCID mice. F. Comparison of cancer growth (as detected by the firefly luciferase bioluminescence) between the 4T1-WT-luc cells implantation group and the 4T1-CDH11-luc cells implantation group at 4 weeks after implantation in NOD/SCID mice. Data were presented as mean ± SD (n=5). G. Representative in vivo bioluminescence images from (F).

Journal: bioRxiv

Article Title: Post-EMT: Cadherin-11 mediates cancer hijacking fibroblasts

doi: 10.1101/729491

Figure Lengend Snippet: 1 × 10 6 of 4T1 mouse triple negative breast cancer cells expressing firefly luciferase with or without CDH11 overexpression were implanted into the left fourth mammary fat pad of immunocompetent BALB/c mice (A-C) or NOD/SCID mice (E-G). A. Comparison of whole tumor growth volume between the 4T1-WT-luc cells implantation group and the 4T1-CDH11-luc cells implantation group in BALB/c mice. B. Comparison of cancer growth (as detected by the firefly luciferase bioluminescence) between the 4T1-WT-luc cells implantation group and the 4T1-CDH11-luc cells implantation group at 4 weeks after implantation in BALB/c mice. Data were presented as mean ± SD (n=7). C. Representative in vivo bioluminescence images from (B). D. Comparison of 4T1-WT-luc cell and 4T1-CDH11-luc cell proliferation in 2D culture in vitro. Same number of cells (46,000 cells) of each group were seeded in one well of a 6-well plate. Cell number was counted at 24 hrs, 48 hrs & 72 hrs (n=3 for each cell group at each time point) after cell seeding. NS, not significant. Note all cells were still not confluent at 72 hrs in each well of a 6-well plate. E. Comparison of whole tumor growth volume between the 4T1-WT-luc cells implantation group and the 4T1-CDH11-luc cells implantation group in NOD/SCID mice. F. Comparison of cancer growth (as detected by the firefly luciferase bioluminescence) between the 4T1-WT-luc cells implantation group and the 4T1-CDH11-luc cells implantation group at 4 weeks after implantation in NOD/SCID mice. Data were presented as mean ± SD (n=5). G. Representative in vivo bioluminescence images from (F).

Techniques: Expressing, Luciferase, Over Expression, In Vivo, In Vitro

Journal: EMBO Reports

Article Title: PHF6-mediated transcriptional control of NSC via Ephrin receptors is impaired in the intellectual disability syndrome BFLS

doi: 10.1038/s44319-024-00082-0

Figure Lengend Snippet: ( A – F ) Low-dimensional representation of single cells from mouse cerebral cortex, based on UMAP embedding of single-cell RNA-seq data [Data ref: (Di Bella et al, )] are shown. Cells are coloured based on animal age ( A ), or the expression of Phf6 ( B ), EphA4 ( C ), EphA7 ( D ), EphB1 ( E ), or EphB2 ( F ). ( G ) Heatmap representation of the Pearson correlation coefficients between Phf6 and EphR across various cell types are shown. Correlation values were calculated using imputed gene expression profiles after applying MAGIC (Van Dijk et al, ). ( H ) UMAP embedding of cells are coloured by cell type. UMAP coordinates and cell type annotations are from [Data ref: (Di Bella et al, ) (GEO GSE153164)].

Article Snippet: Transient KD of Phf6 and EphA4/A7/B1/B2 using an siRNA approach was performed with ON TARGET-plus SMART pool mouse Phf6 siRNA (Dharmacon, #L-058690-01-0005), mouse EphA4 siRNA (Sino Biological, #MG50575-M), mouse EphA7 siRNA (Sino Biological, #MG50587-M, mouse EphB1 siRNA (Sino Biological, #MG50479-M), mouse EphB2 siRNA (Santa Cruz, #sc-39950), and ON TARGET-plus non-targeting pool (Santa Cruz, #sc-36869). siRNA (100 nM) were nucleofected into eNSCs (10 6 cells) and cultured in eNSC media at 37 °C in a humidified atmosphere of 5% CO 2 .

Techniques: RNA Sequencing Assay, Expressing

( A – E ) Dot plots showing expression of Phf6 ( A ), EphA4 ( B ), EphA7 ( C ), EphB1 ( D ), and EphB2 ( E ) in the mouse cerebral cortex during development where the colour of each dot represents the mean normalized expression values per cell type for a given timepoint. The size of the circle represents the percentage of cells expressing each gene. Single cell mouse RNA-seq data was obtained from GEO GSE153164 [Data ref: (Di Bella et al, )]. ( F – J ) Analysis of PHF6 and EPHR expression in the human cortex. Average reads per kilobase million (RPKM) values over human developmental time (post-conceptual weeks; pcw) for gene analysis of PHF6 ( F ), EPHB1 ( G ), EPHA4 ( H ), EPHA7 ( I ), and EPHB2 ( J ) are shown. Gene analysis was taken from publicly available RNA-seq data taken from the human ventral frontal cortex (VFC) of the Allen Brain Atlas BrainSpan dataset [Data ref: (BrainSpan Atlas of the Developing Human Brain, )].

Journal: EMBO Reports

Article Title: PHF6-mediated transcriptional control of NSC via Ephrin receptors is impaired in the intellectual disability syndrome BFLS

doi: 10.1038/s44319-024-00082-0

Figure Lengend Snippet: ( A – E ) Dot plots showing expression of Phf6 ( A ), EphA4 ( B ), EphA7 ( C ), EphB1 ( D ), and EphB2 ( E ) in the mouse cerebral cortex during development where the colour of each dot represents the mean normalized expression values per cell type for a given timepoint. The size of the circle represents the percentage of cells expressing each gene. Single cell mouse RNA-seq data was obtained from GEO GSE153164 [Data ref: (Di Bella et al, )]. ( F – J ) Analysis of PHF6 and EPHR expression in the human cortex. Average reads per kilobase million (RPKM) values over human developmental time (post-conceptual weeks; pcw) for gene analysis of PHF6 ( F ), EPHB1 ( G ), EPHA4 ( H ), EPHA7 ( I ), and EPHB2 ( J ) are shown. Gene analysis was taken from publicly available RNA-seq data taken from the human ventral frontal cortex (VFC) of the Allen Brain Atlas BrainSpan dataset [Data ref: (BrainSpan Atlas of the Developing Human Brain, )].

Techniques: Expressing, RNA Sequencing Assay

( A , B ) eNSCs were cultured from Phf6 -/Y / Nestin-CreERT2 + and control Phf6 loxP/Y / Nestin-CreERT2 - at ~E15 and mRNA and protein expression of EphR were analyzed by RT-qPCR ( A ) and immunoblotting ( B ). ( C , D ) mRNA and protein of brain tissue obtained from E14 R342X and wild-type control mice were analyzed as described in ( A , B ). ( E , F ) Cerebral cortical tissues were isolated from WT and R342X mice at E14 ( E ) or at P0 ( F ). Samples were subjected to ChIP-qPCR using a PHF6 antibody. Zfp735 loci was used as negative control for the PCR. ( G ) Dual luciferase reporter assay was performed in WT or R342X eNSC cultures 48 h following electroporation with pGL4.23- EphA4 , pGL4.23- EphA7 , pGL4.23- EphB1 or pGL4.23-basic reporter plasmids. RLU Relative luminescence units. Data information: Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. Two-tailed unpaired student t -test ( A , C , G ), one-way ANOVA ( E , F ). Representative data of n > 3 independent replicates are shown in panels ( B , D ). Data in panels ( A , C , E – G ) are plotted with n > 3 mean ± SEM. n represents an independent biological sample. .

Journal: EMBO Reports

Article Title: PHF6-mediated transcriptional control of NSC via Ephrin receptors is impaired in the intellectual disability syndrome BFLS

doi: 10.1038/s44319-024-00082-0

Figure Lengend Snippet: ( A , B ) eNSCs were cultured from Phf6 -/Y / Nestin-CreERT2 + and control Phf6 loxP/Y / Nestin-CreERT2 - at ~E15 and mRNA and protein expression of EphR were analyzed by RT-qPCR ( A ) and immunoblotting ( B ). ( C , D ) mRNA and protein of brain tissue obtained from E14 R342X and wild-type control mice were analyzed as described in ( A , B ). ( E , F ) Cerebral cortical tissues were isolated from WT and R342X mice at E14 ( E ) or at P0 ( F ). Samples were subjected to ChIP-qPCR using a PHF6 antibody. Zfp735 loci was used as negative control for the PCR. ( G ) Dual luciferase reporter assay was performed in WT or R342X eNSC cultures 48 h following electroporation with pGL4.23- EphA4 , pGL4.23- EphA7 , pGL4.23- EphB1 or pGL4.23-basic reporter plasmids. RLU Relative luminescence units. Data information: Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. Two-tailed unpaired student t -test ( A , C , G ), one-way ANOVA ( E , F ). Representative data of n > 3 independent replicates are shown in panels ( B , D ). Data in panels ( A , C , E – G ) are plotted with n > 3 mean ± SEM. n represents an independent biological sample. .

Techniques: Cell Culture, Expressing, Quantitative RT-PCR, Western Blot, Isolation, Negative Control, Luciferase, Reporter Assay, Electroporation, Two Tailed Test

( A , B ) mRNA and protein of E14 C99F-m and wild-type control mice were subjected to RT-qPCR and immunoblotting analysis ( n ≥ 3). ( C ) GFP or PHF6-GFP expressing N2A cells were subjected to ChIP using an antibody to PHF6 or IgG control followed by PCR analysis using primers to EphA4 , EphA7 and EphB1 . Zfp locus was used as control ( n = 3). ( D ) GFP or PHF6-GFP- expressing cells were electroplated with a luciferase reporter plasmid driven by a promoter containing 583 bp of the EphA4 gene (pGL4.23- EphA4 ), 550 bp of the EphA7 gene (pGL4.23- EphA7 ) or 709 bp of the EphB1 gene (pGL4.23- EphB1 ). The pGL4.23-basic reporter plasmid (pGL4.23) was used as a control. Renilla expression plasmid was used as an internal control for all samples. RLU Relative luminescence unit. Dual luciferase reporter assay was performed 48 h following electroporation ( n = 3). ( E ) N2A cells were electroporated with siRNA against Phf6 (si Phf6 ) or control siRNA (siCtl) followed by dual luciferase reporter assay at 48 h ( n = 3). ( F ) EPHA4, EPHA7 and PHF6 levels were analyzed by immunoblotting in PHF6-GFP- expressing N2A cells. TUBULIN was used as a loading control. ( G ) Densitometric quantification of PHF6, EPHA4 and EPHA7 protein level normalized to TUBULIN is shown ( n = 3). ( H ) E14-Cerebral cortical tissues from WT and C99F-m mice were subjected to ChIP-PCR analysis, as described in panel ( C ). ( I ) eNSCs cultured from Phf6 -/Y / Nestin-CreERT2 + and control Phf6 loxP/Y / Nestin-CreERT2 - ~E15 mouse brains were subjected to immunoprecipitation (IP) using PHF6 antibody or IgG as control followed by immunoblotting analysis using a PHF6 antibody. ( J ) eNSCs from Phf6 -/Y / Nestin-CreERT2 + and control Phf6 loxP/Y / Nestin-CreERT2 - mouse brains at ~E15, were subjected to ChIP-PCR using a PHF6 antibody. Zfp735 loci was used as control for the PCR ( n = 2). ( K , L ) Protein expression of EPHB1 ( K ), EPHB2 ( L ), SOX2 and NESTIN were analyzed by immunoblotting in EphB1 and EphB2 knockdown (KD) cells. Loading controls of ß-ACTIN and GAPDH were used ( n = 2). Data information: Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. [( C , H ) one-way ANOVA, ( A , D , E , G ) two-tailed unpaired student t -test]. n represents an independent biological sample.

Journal: EMBO Reports

Article Title: PHF6-mediated transcriptional control of NSC via Ephrin receptors is impaired in the intellectual disability syndrome BFLS

doi: 10.1038/s44319-024-00082-0

Figure Lengend Snippet: ( A , B ) mRNA and protein of E14 C99F-m and wild-type control mice were subjected to RT-qPCR and immunoblotting analysis ( n ≥ 3). ( C ) GFP or PHF6-GFP expressing N2A cells were subjected to ChIP using an antibody to PHF6 or IgG control followed by PCR analysis using primers to EphA4 , EphA7 and EphB1 . Zfp locus was used as control ( n = 3). ( D ) GFP or PHF6-GFP- expressing cells were electroplated with a luciferase reporter plasmid driven by a promoter containing 583 bp of the EphA4 gene (pGL4.23- EphA4 ), 550 bp of the EphA7 gene (pGL4.23- EphA7 ) or 709 bp of the EphB1 gene (pGL4.23- EphB1 ). The pGL4.23-basic reporter plasmid (pGL4.23) was used as a control. Renilla expression plasmid was used as an internal control for all samples. RLU Relative luminescence unit. Dual luciferase reporter assay was performed 48 h following electroporation ( n = 3). ( E ) N2A cells were electroporated with siRNA against Phf6 (si Phf6 ) or control siRNA (siCtl) followed by dual luciferase reporter assay at 48 h ( n = 3). ( F ) EPHA4, EPHA7 and PHF6 levels were analyzed by immunoblotting in PHF6-GFP- expressing N2A cells. TUBULIN was used as a loading control. ( G ) Densitometric quantification of PHF6, EPHA4 and EPHA7 protein level normalized to TUBULIN is shown ( n = 3). ( H ) E14-Cerebral cortical tissues from WT and C99F-m mice were subjected to ChIP-PCR analysis, as described in panel ( C ). ( I ) eNSCs cultured from Phf6 -/Y / Nestin-CreERT2 + and control Phf6 loxP/Y / Nestin-CreERT2 - ~E15 mouse brains were subjected to immunoprecipitation (IP) using PHF6 antibody or IgG as control followed by immunoblotting analysis using a PHF6 antibody. ( J ) eNSCs from Phf6 -/Y / Nestin-CreERT2 + and control Phf6 loxP/Y / Nestin-CreERT2 - mouse brains at ~E15, were subjected to ChIP-PCR using a PHF6 antibody. Zfp735 loci was used as control for the PCR ( n = 2). ( K , L ) Protein expression of EPHB1 ( K ), EPHB2 ( L ), SOX2 and NESTIN were analyzed by immunoblotting in EphB1 and EphB2 knockdown (KD) cells. Loading controls of ß-ACTIN and GAPDH were used ( n = 2). Data information: Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. [( C , H ) one-way ANOVA, ( A , D , E , G ) two-tailed unpaired student t -test]. n represents an independent biological sample.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Luciferase, Plasmid Preparation, Reporter Assay, Electroporation, Cell Culture, Immunoprecipitation, Two Tailed Test

( A – D ) WT eNSCs cultured at E14 were electroporated with siRNA targeting each of the EphR followed by self-renewal analysis. ELDA plots are presented for EphA4 (A) ( p > 0.00001), EphA7 ( B ) ( p = 0.0219), EphB1 ( C ) ( p = 0.426), and EphB2 ( D ) ( p = 0.569). ( E , F ) Protein expression of each EPHR, SOX2 and NESTIN were analyzed by immunoblotting. B-ACTIN was used as loading control. ( G – J ) E14 WT eNSCs were electroporated with pLVX.GFP and pLVX. EphA4 -GFP constructs followed by ELDA ( G ) ( p = 0.00355), and stem cell frequency analysis ( H ) ( p = 0.0527), immunoblotting using EPHA4, NESTIN, SOX2, and GFP antibodies ( I ), and sphere diameter analysis ( J ) ( p = 0.0017). ( K – P ) R342X and WT eNSCs cultured at E14 were electroporated with pLVX.GFP, pLVX. EphA4 -GFP, and pLVX. EphA7 -GFP and samples were subjected to immunoblotting analysis with EPHA4, EPHA7, and GFP antibodies ( K , L ), ELDA ( M , O ), and sphere analysis ( N , P ) following 7 days in culture [ p = 0.00264 ( M ) and p = 0.00255 ( O )]. Data information: Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. ( H , J ) two-tailed unpaired student t -test, ( N , P ) One-way ANOVA with Tukey’s multiple comparisons test. Representative data of n > 3 independent replicates are shown in panels ( A – G , I , K – M , O ). Data in panels ( H , J , N , P ) are plotted with n > 3 mean +/− SEM. n represents an independent biological sample. .

Journal: EMBO Reports

Article Title: PHF6-mediated transcriptional control of NSC via Ephrin receptors is impaired in the intellectual disability syndrome BFLS

doi: 10.1038/s44319-024-00082-0

Figure Lengend Snippet: ( A – D ) WT eNSCs cultured at E14 were electroporated with siRNA targeting each of the EphR followed by self-renewal analysis. ELDA plots are presented for EphA4 (A) ( p > 0.00001), EphA7 ( B ) ( p = 0.0219), EphB1 ( C ) ( p = 0.426), and EphB2 ( D ) ( p = 0.569). ( E , F ) Protein expression of each EPHR, SOX2 and NESTIN were analyzed by immunoblotting. B-ACTIN was used as loading control. ( G – J ) E14 WT eNSCs were electroporated with pLVX.GFP and pLVX. EphA4 -GFP constructs followed by ELDA ( G ) ( p = 0.00355), and stem cell frequency analysis ( H ) ( p = 0.0527), immunoblotting using EPHA4, NESTIN, SOX2, and GFP antibodies ( I ), and sphere diameter analysis ( J ) ( p = 0.0017). ( K – P ) R342X and WT eNSCs cultured at E14 were electroporated with pLVX.GFP, pLVX. EphA4 -GFP, and pLVX. EphA7 -GFP and samples were subjected to immunoblotting analysis with EPHA4, EPHA7, and GFP antibodies ( K , L ), ELDA ( M , O ), and sphere analysis ( N , P ) following 7 days in culture [ p = 0.00264 ( M ) and p = 0.00255 ( O )]. Data information: Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. ( H , J ) two-tailed unpaired student t -test, ( N , P ) One-way ANOVA with Tukey’s multiple comparisons test. Representative data of n > 3 independent replicates are shown in panels ( A – G , I , K – M , O ). Data in panels ( H , J , N , P ) are plotted with n > 3 mean +/− SEM. n represents an independent biological sample. .

Techniques: Cell Culture, Expressing, Western Blot, Construct, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Fab and Fc contribute to maximal protection against SARS-CoV-2 following NVX-CoV2373 subunit vaccine with Matrix-M vaccination

doi: 10.1016/j.xcrm.2021.100405

Figure Lengend Snippet: Immunogenicity of NVX-CoV2373 vaccine in rhesus macaques (A–C) Serum anti-spike (S) IgG titer (A), nasal wash (B), and bronchoalveolar lavage (BAL) (C) samples were collected 31/32 days after the first immunization and before challenge and analyzed for S-specific mucosal IgG (n = 4–5/group). (D) Pseudovirus-neutralizing titer (ID 50 ). (E) SARS-CoV-2 neutralizing-antibody titer (99% inhibition of cytopathic effect [99% CPE]) study day 31/32. (F) hACE2 receptor-blocking antibody titer (study day 31/32). The geometric mean titers (GMTs) are indicated by the white bars. Hollow arrows indicate prime/boosting with NVX-CoV2373. The error bars indicate the 95% confidence interval (95% CI). Individual animal values are indicated by colored symbols. A Student’s t test (unpaired, two-tailed) was used to compare antibody levels between groups immunized with one and two doses. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001. The horizontal dashed lines indicate the LODs for each assay.

Article Snippet: After washing, 30 ng mL -1 histidine-tagged human ACE2 (Sino Biologics, Beijing, CHN) was added to wells for 1 h at room temperature.

Techniques: Inhibition, Blocking Assay, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Fab and Fc contribute to maximal protection against SARS-CoV-2 following NVX-CoV2373 subunit vaccine with Matrix-M vaccination

doi: 10.1016/j.xcrm.2021.100405

Figure Lengend Snippet:

Article Snippet: After washing, 30 ng mL -1 histidine-tagged human ACE2 (Sino Biologics, Beijing, CHN) was added to wells for 1 h at room temperature.

Techniques: Recombinant, Binding Assay, Blocking Assay, Mutagenesis, Software

( A ) Normalized SERPINB3 transcript in cervical tumor biopsies from RNAseq was distributed by reads per kilobase of transcript per million mapped reads (RPKM). ( B ) Boxplots along with individual data points show xCell immune scores in recurrent (R)/non-recurrent (NR) SERPINB3-low (B3/L) and SERPINB3-high (B3/H) tumors. * P < 0.05, one-way ANOVA test. ( C ) Heatmap of enriched immune cell subpopulation was generated through xCell immune infiltrate prediction. Color intensity is proportional to average xCell score for each population across samples. ( D-G ) Spearman’s correlation of SERPINB3 with the expression of ( D ) CXCL1, ( E ) CXCL8, ( F ) S100A8, ( G ) S100A9 was performed using RNAseq from 66 cervical tumor biopsies collected prior to (chemo)- RT. ( H ) SERPINB3 expression correlated with CXCL1, CXCL8, S100A8, S100A9 expression in multiple cancer types. Analysis was performed using TCGA PanCancer Atlas and numeric values indicate Spearman’s correlation coefficient. BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; HNSC, head and neck squamous cell carcinoma; LUSC, lung squamous cell carcinoma; PRAD, prostate adenocarcinoma; UCEC, uterine corpus endometrial carcinoma

Journal: bioRxiv

Article Title: SCCA1/SERPINB3 promotes suppressive immune environment via STAT-dependent chemokine production, blunting the therapy-induced T cell responses

doi: 10.1101/2023.02.01.526675

Figure Lengend Snippet: ( A ) Normalized SERPINB3 transcript in cervical tumor biopsies from RNAseq was distributed by reads per kilobase of transcript per million mapped reads (RPKM). ( B ) Boxplots along with individual data points show xCell immune scores in recurrent (R)/non-recurrent (NR) SERPINB3-low (B3/L) and SERPINB3-high (B3/H) tumors. * P < 0.05, one-way ANOVA test. ( C ) Heatmap of enriched immune cell subpopulation was generated through xCell immune infiltrate prediction. Color intensity is proportional to average xCell score for each population across samples. ( D-G ) Spearman’s correlation of SERPINB3 with the expression of ( D ) CXCL1, ( E ) CXCL8, ( F ) S100A8, ( G ) S100A9 was performed using RNAseq from 66 cervical tumor biopsies collected prior to (chemo)- RT. ( H ) SERPINB3 expression correlated with CXCL1, CXCL8, S100A8, S100A9 expression in multiple cancer types. Analysis was performed using TCGA PanCancer Atlas and numeric values indicate Spearman’s correlation coefficient. BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; HNSC, head and neck squamous cell carcinoma; LUSC, lung squamous cell carcinoma; PRAD, prostate adenocarcinoma; UCEC, uterine corpus endometrial carcinoma

Article Snippet: SERPINB3 stable expression cells were generated using pULTRA lentiviral vector (Addgene #24129) containing human SERPINB3 or pLV-C-GFPSpark vector (Sino Biological LVCV-35) containing mouse Serpinb3a GFP-tagged fusion proteins.

Techniques: Generated, Expressing

( A ) Caski and SW756 cells were transduced with pUltra vector (Caski/Ctrl, SW756/Ctrl) or pUltra-SERPINB3 (Caski/B3, SW756/B3) and CXCL1/8 and S100A8/A9 mRNA expression was examined by qPCR. ( B ) Caski cells were transfected with scrambled negative control shRNA (Caski/shCtrl) or shRNAs specific SERPINB3 (Caski/shB3); SW756 cells were transduced with CRISPR control vector (SW756/CRISPR-Ctrl) or CRISPR-Cas9 for SERPINB3 knockdown (SW756/CRISPR-B3KO). The expression of CXCL1/8 and S100A8/A9 was examined by qPCR. Gene expression were normalized to GAPDH and fold changes were calculated by comparing to the expression levels in parental cells (Caski WT or SW756 WT). ( C ) Intracellular chemokine proteinexpressionin cell lysateswas measured by ELISA. The chemokine levels were normalized to total protein concentration. ( D ) Supernatant was collected from adherent cells in monolayer and chemokine secretion was measured by ELISA. Data in A-D are presentedas mean ± SEMof n = 4 independent experiments, * P < 0.05, ** P < 0.01, *** P < 0.001 using unpaired two-tailed Student’s t-test. ( E-G ) PBMC migration towards supernatant collected from cancer cells was examined by Transwell assays and the migrated PMBC populations were analyzed by flow cytometry. Fold changes were calculated as the percentage of migrated ( E ) T and myeloid cells, ( F ) T cell subsets and ( G ) myeloid cell subsets in Caski/B3 or SW756/B3 relative to Caski/Ctrl or SW756/Ctrl supernatant. Data are shown as mean ± SEM, ns, no significance; * P < 0.05, ** P < 0.01, *** P < 0.001 using two-tailed one sample T test against 1. Each dot representsthe mean of duplicate values for a single donor sample (n=7).

Journal: bioRxiv

Article Title: SCCA1/SERPINB3 promotes suppressive immune environment via STAT-dependent chemokine production, blunting the therapy-induced T cell responses

doi: 10.1101/2023.02.01.526675

Figure Lengend Snippet: ( A ) Caski and SW756 cells were transduced with pUltra vector (Caski/Ctrl, SW756/Ctrl) or pUltra-SERPINB3 (Caski/B3, SW756/B3) and CXCL1/8 and S100A8/A9 mRNA expression was examined by qPCR. ( B ) Caski cells were transfected with scrambled negative control shRNA (Caski/shCtrl) or shRNAs specific SERPINB3 (Caski/shB3); SW756 cells were transduced with CRISPR control vector (SW756/CRISPR-Ctrl) or CRISPR-Cas9 for SERPINB3 knockdown (SW756/CRISPR-B3KO). The expression of CXCL1/8 and S100A8/A9 was examined by qPCR. Gene expression were normalized to GAPDH and fold changes were calculated by comparing to the expression levels in parental cells (Caski WT or SW756 WT). ( C ) Intracellular chemokine proteinexpressionin cell lysateswas measured by ELISA. The chemokine levels were normalized to total protein concentration. ( D ) Supernatant was collected from adherent cells in monolayer and chemokine secretion was measured by ELISA. Data in A-D are presentedas mean ± SEMof n = 4 independent experiments, * P < 0.05, ** P < 0.01, *** P < 0.001 using unpaired two-tailed Student’s t-test. ( E-G ) PBMC migration towards supernatant collected from cancer cells was examined by Transwell assays and the migrated PMBC populations were analyzed by flow cytometry. Fold changes were calculated as the percentage of migrated ( E ) T and myeloid cells, ( F ) T cell subsets and ( G ) myeloid cell subsets in Caski/B3 or SW756/B3 relative to Caski/Ctrl or SW756/Ctrl supernatant. Data are shown as mean ± SEM, ns, no significance; * P < 0.05, ** P < 0.01, *** P < 0.001 using two-tailed one sample T test against 1. Each dot representsthe mean of duplicate values for a single donor sample (n=7).

Techniques: Transduction, Plasmid Preparation, Expressing, Transfection, Negative Control, shRNA, CRISPR, Enzyme-linked Immunosorbent Assay, Protein Concentration, Two Tailed Test, Migration, Flow Cytometry

( A-B ) Chemokine ( A ) CXCL1 and ( B ) S100A8/A9 levels in tumor homogenates was examined by ELISA. Data was normalized to the protein concentration for each tumor homogenate and shown as mean ± SEM; n=7 for 7-day sham-LL2/mB3a and n=6 for all other groups. ( C-H ) Cumulative data from FACSanalysis show alteration of immune cell infiltration by SERPINB3 expression and radiation in LL2 tumors. The graphs represent the frequencies of ( C ) CD11b+Ly6G-Ly6Chigh Mo-MDSCs, ( D ) CD11b+Ly6G+ PMN-MDSCs, ( E ) CD11b+Ly6G-F4/80+ TAMs, ( F ) CD11b+Ly6G-F4/80+CD163+ M2 macrophages, ( G ) CD3+CD4+ T cells, and ( H ) CD3+CD8+ T cells in total tumor infiltrating leukocytes (TILs). ( I ) The ratioof CD8/Treg represented the infiltrating percentage of CD8+ T cells relative to CD4+CD25+FoxP3+ regulatory T (Treg) cells. Data are shown as mean ± SEM and each dot represents a biologically independent animal; * indicates comparisons between LL2/Ctrl and LL2/mB3a; ✝ indicates comparisons between sham-treated and RT; * P < 0.05, ** P < 0.01, *** P < 0.001 using one-way ANOVA with Tukey’s post-test for multiple comparisons.

Journal: bioRxiv

Article Title: SCCA1/SERPINB3 promotes suppressive immune environment via STAT-dependent chemokine production, blunting the therapy-induced T cell responses

doi: 10.1101/2023.02.01.526675

Figure Lengend Snippet: ( A-B ) Chemokine ( A ) CXCL1 and ( B ) S100A8/A9 levels in tumor homogenates was examined by ELISA. Data was normalized to the protein concentration for each tumor homogenate and shown as mean ± SEM; n=7 for 7-day sham-LL2/mB3a and n=6 for all other groups. ( C-H ) Cumulative data from FACSanalysis show alteration of immune cell infiltration by SERPINB3 expression and radiation in LL2 tumors. The graphs represent the frequencies of ( C ) CD11b+Ly6G-Ly6Chigh Mo-MDSCs, ( D ) CD11b+Ly6G+ PMN-MDSCs, ( E ) CD11b+Ly6G-F4/80+ TAMs, ( F ) CD11b+Ly6G-F4/80+CD163+ M2 macrophages, ( G ) CD3+CD4+ T cells, and ( H ) CD3+CD8+ T cells in total tumor infiltrating leukocytes (TILs). ( I ) The ratioof CD8/Treg represented the infiltrating percentage of CD8+ T cells relative to CD4+CD25+FoxP3+ regulatory T (Treg) cells. Data are shown as mean ± SEM and each dot represents a biologically independent animal; * indicates comparisons between LL2/Ctrl and LL2/mB3a; ✝ indicates comparisons between sham-treated and RT; * P < 0.05, ** P < 0.01, *** P < 0.001 using one-way ANOVA with Tukey’s post-test for multiple comparisons.

Techniques: Enzyme-linked Immunosorbent Assay, Protein Concentration, Expressing

Figure 1. Loss of neurofibromin enhances glycolysis and glucose utilization via GLUT1 in macrophages that results in elevated relative mRNA expression of inflammatory cytokines, NF- κB, and NOS2 and polarizes macrophages to mixed phenotypes (A–F) Representative measurements of extracellular acidification rate (ECAR) performed on bone marrow-derived macrophages (BMDMs) derived from long bones of Nf1f/f and Nf1ΔMϕ mice treated with 20 ng/mL LPS and IFNγ or 20 ng/mL IL-4 to polarize the macrophages to inflammatory (MLPSIFNγ) or reparative (MIL4) phenotypes for 16 h, respectively. Subsequent addition of glucose, the ATP synthase inhibitor oligomycin, and the hexokinase inhibitor 2-deoxy-glucose (2-DG) were carried out where indicated (A, C, and E); n = 4 mice/genotype; each data point represents technical replicate. Data (mean ± SEM) were normalized by total protein content at the end of experiment. p values were calculated using one-way ANOVA followed by Tukey’s multiple comparison t test (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; ns, not significant). (G–J) Quantification of percentage of glucose uptake by viable BMDMs derived from bones of Nf1f/f and Nf1ΔMϕ mice in the absence or presence of the GLUT1 inhibitor BAY876. BMDMs were labeled with 2-NBDG at a concentration of 100 μM for 30 min in the absence or presence of BAY876 (50 nM) to measure glucose

Journal: Cell reports

Article Title: Loss of neurofibromin induces inflammatory macrophage phenotypic switch and retinal neovascularization via GLUT1 activation.

doi: 10.1016/j.celrep.2025.115625

Figure Lengend Snippet: Figure 1. Loss of neurofibromin enhances glycolysis and glucose utilization via GLUT1 in macrophages that results in elevated relative mRNA expression of inflammatory cytokines, NF- κB, and NOS2 and polarizes macrophages to mixed phenotypes (A–F) Representative measurements of extracellular acidification rate (ECAR) performed on bone marrow-derived macrophages (BMDMs) derived from long bones of Nf1f/f and Nf1ΔMϕ mice treated with 20 ng/mL LPS and IFNγ or 20 ng/mL IL-4 to polarize the macrophages to inflammatory (MLPSIFNγ) or reparative (MIL4) phenotypes for 16 h, respectively. Subsequent addition of glucose, the ATP synthase inhibitor oligomycin, and the hexokinase inhibitor 2-deoxy-glucose (2-DG) were carried out where indicated (A, C, and E); n = 4 mice/genotype; each data point represents technical replicate. Data (mean ± SEM) were normalized by total protein content at the end of experiment. p values were calculated using one-way ANOVA followed by Tukey’s multiple comparison t test (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; ns, not significant). (G–J) Quantification of percentage of glucose uptake by viable BMDMs derived from bones of Nf1f/f and Nf1ΔMϕ mice in the absence or presence of the GLUT1 inhibitor BAY876. BMDMs were labeled with 2-NBDG at a concentration of 100 μM for 30 min in the absence or presence of BAY876 (50 nM) to measure glucose

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 1X Pierce RIPA buffer ThermoFisher Scientific Cat# 89900 7.5% TGX Stain-free FastCast SDS-PAGE gel kit Bio Rad Cat# 1610181 10% TGX Stain-free FastCast SDS-PAGE gel kit Bio Rad Cat# 1610183 12% TGX Stain-free FastCast SDS-PAGE gel kit Bio Rad Cat# 1610185 SuperSignal West Femto Maximum Sensitivity Substrate ThermoFisher Scientific Cat# 34095 TidyBlot HRP-conjugated Bio-Rad Cat# STAR209PA Alexa Fluor 594-conjugated Isolectin GS-IB4 Invitrogen Cat# I21413 Mounting media without DAPI Vector Laboratories Cat# H-1000 Mounting medium with DAPI Vector Laboratories Cat# H-1200 Critical commercial assays Seahorse XF glycolysis stress test kit Agilent Technologies Cat# 103020-100 Fluorescent 2-NBDG Glucose Uptake Assay Kit BioVision Cat# K682-50 Viability using Zombie NIR fixable viability kit BioLegend Cat# 423105 Pierce bicinchoninic acid (BCA) protein assay ThermoFisher Scientific Cat# 23227 RNeasy Mini kit Qiagen Cat# 74104 iScriptTM cDNA synthesis kit Bio-Rad Cat# 1708891 High-capacity M-MLV reverse transcriptase Invitrogen Cat# 28025013 iTaq Universal SYBR Green Supermix Bio-Rad Cat# 1725121 Subcellular protein fractionation kit ThermoFisher Scientific Cat# 78840 Immunoprecipitation kit ThermoFisher Scientific Cat# 10007D Deposited data X-ray crystal structures of glucose transporter 1 (GLUT1) This paper PDB: 4PYP X-ray crystal structures of Akt2 This paper PDB: 8Q61 X-ray crystal structures of neurofibromin 1 (NF1) This paper PDB: 8K4L Experimental models: Cell lines Mouse: L929 Fibroblast cell (NCTC clone 929) ATCC Cat# CCL-1; RRID: CVCL_0462 Mouse: Bone marrow-derived macrophages This paper N/A Human: THP-1 cells Gabor Csanyi’s Laboratory N/A Human: Lenti-XTM 293T packaging cells David Fulton’s Laboratory N/A Experimental models: Organisms/strains Mouse: Nf1-heterozygous (Nf1+/− ) Dr. Tyler Jacks Massachusetts Institute of Technology, Cambridge, MA Mouse: Nf1flox/flox; LysMCre− (Nf1fl/fl) Dr. Luis Parada University of Texas Southwestern Medical Center, Dallas, TX, USA Mouse: Nf1flox/flox;LysMCre+ (Nf1ΔMΦ) Dr. Tyler Jacks Massachusetts Institute of Technology, Cambridge, MA Mouse: C57BL/6J The Jackson Laboratory 000664, Jackson Laboratories, Bar Harbor, ME Mouse: LysMCre+ The Jackson Laboratory 00472, Jackson Laboratories, Bar Harbor, ME (Continued on next page) 16 Cell Reports 44, 115625, May 27, 2025

Techniques: Expressing, Derivative Assay, Comparison, Labeling, Concentration Assay

$Figure 2. Loss of neurofibromin increases facilitative GLUT1 expression in all macrophages (A–C and E–G) Representative immunoblots and quantification of GLUT1 expression (A–C) in total BMDM lysates and (E–G) in membrane and cytoplasm fractions of BMDMs derived from bones of Nf1f/f and Nf1ΔMϕ mice. BMDMs were differentiated using macrophage colony-stimulating factor (M-CSF) and polarized to MLPSIFNγ and MIL4 macrophages. GLUT1 expression was measured by immunoblotting in all subpopulations of macrophages. n = 3 mice per genotype. (D) Quantitative RT-PCR analysis of GLUT1 mRNA relative expression in all subpopulations of macrophages (MCSF, MLPSIFNγ, and MIL4) from bones of Nf1f/f and Nf1ΔMϕ mice. n = 3 mice per genotype; each data point represent the mean of two technical replicates of each mouse. (H) Representatives immunoblot of neurofibromin expression in membrane and cytoplasm fractions of all macrophage subpopulations of Nf1f/f mice. (I) Polarized BMDM lysates from Nf1f/f mice were prepared and GLUT1 was immunoprecipitated with anti-GLUT1 antibody. The immune complex was then immunoblotted with anti-NF1 antibody to identify the GLUT1:NF1 complex. Data are expressed as mean ± SD. p values were calculated using one-way ANOVA followed by Tukey’s multiple comparison test for (B)–(D) and two-tailed Student’s t test for (F) and (G) (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; ns, not significant).$

Journal: Cell reports

Article Title: Loss of neurofibromin induces inflammatory macrophage phenotypic switch and retinal neovascularization via GLUT1 activation.

doi: 10.1016/j.celrep.2025.115625

Figure Lengend Snippet: Figure 2. Loss of neurofibromin increases facilitative GLUT1 expression in all macrophages (A–C and E–G) Representative immunoblots and quantification of GLUT1 expression (A–C) in total BMDM lysates and (E–G) in membrane and cytoplasm fractions of BMDMs derived from bones of Nf1f/f and Nf1ΔMϕ mice. BMDMs were differentiated using macrophage colony-stimulating factor (M-CSF) and polarized to MLPSIFNγ and MIL4 macrophages. GLUT1 expression was measured by immunoblotting in all subpopulations of macrophages. n = 3 mice per genotype. (D) Quantitative RT-PCR analysis of GLUT1 mRNA relative expression in all subpopulations of macrophages (MCSF, MLPSIFNγ, and MIL4) from bones of Nf1f/f and Nf1ΔMϕ mice. n = 3 mice per genotype; each data point represent the mean of two technical replicates of each mouse. (H) Representatives immunoblot of neurofibromin expression in membrane and cytoplasm fractions of all macrophage subpopulations of Nf1f/f mice. (I) Polarized BMDM lysates from Nf1f/f mice were prepared and GLUT1 was immunoprecipitated with anti-GLUT1 antibody. The immune complex was then immunoblotted with anti-NF1 antibody to identify the GLUT1:NF1 complex. Data are expressed as mean ± SD. p values were calculated using one-way ANOVA followed by Tukey’s multiple comparison test for (B)–(D) and two-tailed Student’s t test for (F) and (G) (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; ns, not significant).

Techniques: Expressing, Western Blot, Membrane, Derivative Assay, Quantitative RT-PCR, Immunoprecipitation, Comparison, Two Tailed Test

$Figure 3. Loss of neurofibromin preferentially increases Akt-2 phosphorylation, but not Akt1 phosphorylation, in total and in membrane fractions of all macrophage subpopulations and promotes GLUT1 translocation to the cell membrane via Akt2 (A–H) Representative immunoblots and densitometry graphs showing the ratios of (A and B) P-PI3K/PI3K, (C and D) P-Akt-1/Akt-1, (E and F) P-Akt-2/Akt-2 expression in total BMDM lysates, and (G and H) P-Akt-2/Akt-2 expression in membrane fractions of BMDMs from long bones of Nf1f/f and Nf1ΔMϕ mice. n = 3 mice per genotype. Data are expressed as mean ± SD. p values were calculated using one-way ANOVA followed by Sidak’s multiple comparison test for (B), (D), (F), and (H) (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; ns, not significant). (I–M) Representative immunoblots and densitometry graphs showing (I–K) GLUT1 expression in membrane fractions of both inflammatory (MLPSIFNγ) and reparative macrophages (MIL4) and (L and M) P-Akt-1 and total Akt-1 expression in membrane fractions of only MIL4 macrophages from long bones of Nf1f/f and Nf1ΔMϕ mice after Akt2 inhibitor (Akt2i; CCT128930) treatment at a dose of 10 μM for 6 h (n = 3 per genotype). Data are expressed as mean ± SD. p values were calculated using one-way ANOVA followed by Sidak’s multiple comparison test (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; ns, not significant).$

Journal: Cell reports

Article Title: Loss of neurofibromin induces inflammatory macrophage phenotypic switch and retinal neovascularization via GLUT1 activation.

doi: 10.1016/j.celrep.2025.115625

Figure Lengend Snippet: Figure 3. Loss of neurofibromin preferentially increases Akt-2 phosphorylation, but not Akt1 phosphorylation, in total and in membrane fractions of all macrophage subpopulations and promotes GLUT1 translocation to the cell membrane via Akt2 (A–H) Representative immunoblots and densitometry graphs showing the ratios of (A and B) P-PI3K/PI3K, (C and D) P-Akt-1/Akt-1, (E and F) P-Akt-2/Akt-2 expression in total BMDM lysates, and (G and H) P-Akt-2/Akt-2 expression in membrane fractions of BMDMs from long bones of Nf1f/f and Nf1ΔMϕ mice. n = 3 mice per genotype. Data are expressed as mean ± SD. p values were calculated using one-way ANOVA followed by Sidak’s multiple comparison test for (B), (D), (F), and (H) (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; ns, not significant). (I–M) Representative immunoblots and densitometry graphs showing (I–K) GLUT1 expression in membrane fractions of both inflammatory (MLPSIFNγ) and reparative macrophages (MIL4) and (L and M) P-Akt-1 and total Akt-1 expression in membrane fractions of only MIL4 macrophages from long bones of Nf1f/f and Nf1ΔMϕ mice after Akt2 inhibitor (Akt2i; CCT128930) treatment at a dose of 10 μM for 6 h (n = 3 per genotype). Data are expressed as mean ± SD. p values were calculated using one-way ANOVA followed by Sidak’s multiple comparison test (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; ns, not significant).

Techniques: Phospho-proteomics, Membrane, Translocation Assay, Western Blot, Expressing, Comparison

Figure 4. Neurofibromin complexes with GLUT1 and conserved regions of Akt2 Molecular docking and atomistic simulations implicate the complex formation of NF1-GLUT1 and GLUTI-Akt2. (A–D) Surface representations of (A) NF1-GLUT1 complex having docking score of −429.60 obtained by H-dock, (B) GLUT1-Akt2 at 0 ns with docking score of −301.96, (C) GLUT1-Akt2 at 125 ns, and (D) GLUT1-Akt2 at 250 ns with binding energy of −97.5 kcal/mol obtained by simulations. (E–G) Plots showing (E) number of H-bonds present in GLUT1-Akt2 complex over a period of 250 ns, (F) radius of gyration (RG) of GLUT1-Akt2 complex over a period of 250 ns, and (G) root-mean-square deviation (RMSD) of GLUT1, Akt2, and GLUT1-Akt2 complex (also see Table S1).

Journal: Cell reports

Article Title: Loss of neurofibromin induces inflammatory macrophage phenotypic switch and retinal neovascularization via GLUT1 activation.

doi: 10.1016/j.celrep.2025.115625

Figure Lengend Snippet: Figure 4. Neurofibromin complexes with GLUT1 and conserved regions of Akt2 Molecular docking and atomistic simulations implicate the complex formation of NF1-GLUT1 and GLUTI-Akt2. (A–D) Surface representations of (A) NF1-GLUT1 complex having docking score of −429.60 obtained by H-dock, (B) GLUT1-Akt2 at 0 ns with docking score of −301.96, (C) GLUT1-Akt2 at 125 ns, and (D) GLUT1-Akt2 at 250 ns with binding energy of −97.5 kcal/mol obtained by simulations. (E–G) Plots showing (E) number of H-bonds present in GLUT1-Akt2 complex over a period of 250 ns, (F) radius of gyration (RG) of GLUT1-Akt2 complex over a period of 250 ns, and (G) root-mean-square deviation (RMSD) of GLUT1, Akt2, and GLUT1-Akt2 complex (also see Table S1).

Techniques: Binding Assay

Figure 5. Neurofibromin is sensitive to hypoxia, and loss of neurofibromin in macrophages/microglia is sufficient to induce pathological retinal neovascularization (A) Schematic of the mouse oxygen-induced retinopathy (OIR) model. (B–G) Representative immunoblots and quantifications showing (B and C) neurofibromin expression in P17 retinas from WT mice exposed to room air (−) or OIR (+) (n = 3 mice per group), (D and E) F4/80 expression in P17 retinas from WT and Nf1+/−mice exposed to OIR (n = 3 mice per genotype), and (F and G) upregulation of GLUT1 expression in P17 retinas from WT and Nf1+/−mice exposed to OIR (n = 3 mice per genotype). (H) Representative retinal whole mounts from P17 OIR retinas of Nf1f/f and Nf1ΔMϕ mice stained with isolectin-B4 (red) with highlighted neovascular areas and avascular areas (white). (I and J) Quantification of neovascular areas and avascular areas in OIR retinas were expressed as percentage of total retinal areas (n = 10 retinas). Data are expressed as mean ± SD. p values were calculated using two-tailed Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; ns, not significant).

Journal: Cell reports

Article Title: Loss of neurofibromin induces inflammatory macrophage phenotypic switch and retinal neovascularization via GLUT1 activation.

doi: 10.1016/j.celrep.2025.115625

Figure Lengend Snippet: Figure 5. Neurofibromin is sensitive to hypoxia, and loss of neurofibromin in macrophages/microglia is sufficient to induce pathological retinal neovascularization (A) Schematic of the mouse oxygen-induced retinopathy (OIR) model. (B–G) Representative immunoblots and quantifications showing (B and C) neurofibromin expression in P17 retinas from WT mice exposed to room air (−) or OIR (+) (n = 3 mice per group), (D and E) F4/80 expression in P17 retinas from WT and Nf1+/−mice exposed to OIR (n = 3 mice per genotype), and (F and G) upregulation of GLUT1 expression in P17 retinas from WT and Nf1+/−mice exposed to OIR (n = 3 mice per genotype). (H) Representative retinal whole mounts from P17 OIR retinas of Nf1f/f and Nf1ΔMϕ mice stained with isolectin-B4 (red) with highlighted neovascular areas and avascular areas (white). (I and J) Quantification of neovascular areas and avascular areas in OIR retinas were expressed as percentage of total retinal areas (n = 10 retinas). Data are expressed as mean ± SD. p values were calculated using two-tailed Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; ns, not significant).

Techniques: Western Blot, Expressing, Staining, Two Tailed Test

Figure 6. Loss of neurofibromin promotes inflammatory macrophage/microglia infiltration and localization to sites of neovascular tufts in the retina (A–C) Flow cytometric analysis of whole P17 retinas from Nf1f/f and Nf1ΔMϕ mice exposed to OIR-expressing macrophage or microglial markers (also see Figure S4). Plots represent the percentages of viable (A) inflammatory macrophage/microglia (F4/80+/CD11b+/NOS2+), (B) reparative macrophage/microglia (F4/80+/CD11b+/Arg1+), and (C) macrophage/microglia with mixed phenotype (F4/80+/CD11b+/NOS2+/Arg1+) in P17 OIR retinas (n = 5–6 mice per genotype; total of 10–12 retinas in each genotype). (D and F) Confocal images of retinal flat mounts at P17 OIR from Nf1f/f and Nf1ΔMϕ mice, stained with isolectin-B4 (vessel stain), F4/80, and either (D) CD86 (inflammatory macrophage/microglia) or (F) CD206 (reparative macrophage/microglia) to show the localization of macrophage/microglia near neovascular tufts. (E and G) Pearson’s correlation coefficient for F4/80 with either (E) CD86 or (G) CD206 to depict co-localization (n = 6 retinas in each group). Scale bars: 50 μm (D and F) and 10 μm (inset). Data are expressed as mean ± SD. p values were calculated using two-tailed Student’s t test (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; ns, not significant).

Journal: Cell reports

Article Title: Loss of neurofibromin induces inflammatory macrophage phenotypic switch and retinal neovascularization via GLUT1 activation.

doi: 10.1016/j.celrep.2025.115625

Figure Lengend Snippet: Figure 6. Loss of neurofibromin promotes inflammatory macrophage/microglia infiltration and localization to sites of neovascular tufts in the retina (A–C) Flow cytometric analysis of whole P17 retinas from Nf1f/f and Nf1ΔMϕ mice exposed to OIR-expressing macrophage or microglial markers (also see Figure S4). Plots represent the percentages of viable (A) inflammatory macrophage/microglia (F4/80+/CD11b+/NOS2+), (B) reparative macrophage/microglia (F4/80+/CD11b+/Arg1+), and (C) macrophage/microglia with mixed phenotype (F4/80+/CD11b+/NOS2+/Arg1+) in P17 OIR retinas (n = 5–6 mice per genotype; total of 10–12 retinas in each genotype). (D and F) Confocal images of retinal flat mounts at P17 OIR from Nf1f/f and Nf1ΔMϕ mice, stained with isolectin-B4 (vessel stain), F4/80, and either (D) CD86 (inflammatory macrophage/microglia) or (F) CD206 (reparative macrophage/microglia) to show the localization of macrophage/microglia near neovascular tufts. (E and G) Pearson’s correlation coefficient for F4/80 with either (E) CD86 or (G) CD206 to depict co-localization (n = 6 retinas in each group). Scale bars: 50 μm (D and F) and 10 μm (inset). Data are expressed as mean ± SD. p values were calculated using two-tailed Student’s t test (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; ns, not significant).

Techniques: Expressing, Staining, Two Tailed Test

Figure 7. Loss of neurofibromin results in GLUT1 and Akt2 co-localization within CD68+ macrophages from NF1-associated tumors (A) Representative high-magnification confocal images of NF1-associated tumor cross-section from human patients showing co-localization of GLUT1 and P-Akt2 with CD68+ macrophages, Scale bars: 10 μm. (B) Percentage of Pearson’s correlation coefficient for CD68 with either GLUT1 or P-Akt2 to depict co-localization (n = 4 patients with NF1). Each data point reflects the values to each patient (also see Table S2).

Journal: Cell reports

Article Title: Loss of neurofibromin induces inflammatory macrophage phenotypic switch and retinal neovascularization via GLUT1 activation.

doi: 10.1016/j.celrep.2025.115625

Figure Lengend Snippet: Figure 7. Loss of neurofibromin results in GLUT1 and Akt2 co-localization within CD68+ macrophages from NF1-associated tumors (A) Representative high-magnification confocal images of NF1-associated tumor cross-section from human patients showing co-localization of GLUT1 and P-Akt2 with CD68+ macrophages, Scale bars: 10 μm. (B) Percentage of Pearson’s correlation coefficient for CD68 with either GLUT1 or P-Akt2 to depict co-localization (n = 4 patients with NF1). Each data point reflects the values to each patient (also see Table S2).

Techniques:

Table S2 for the target genes of ZNF408-SETD1A complex. See Table S3 for primer information in the RT-qPCR assay. " width="100%" height="100%">

Journal: iScience

Article Title: Loss of ZNF408 attenuates STING-mediated immune surveillance in breast carcinogenesis

doi: 10.1016/j.isci.2024.110276

Figure Lengend Snippet: Identification and verification of direct target genes of ZNF408 (A) Schematic diagrams of ZNF408 targets identified by cross analysis of CUT&Tag, ATAC-seq, and RNA-seq. (B) Volcano plots showing differently expressed genes detected by RNA-seq upon knockdown of ZNF408 in MCF-7 cells. Cutoff: log 2 fold change >0.4, p < 0.05. (C) Genome tracks showing CUT&Tag results of ZNF408, SETD1A, and H3K4me3 visualized by the IGV tool at STING1 promoter. (D) MCF-7 cells were transfected with control siRNA, siZNF408, or siSETD1A. Total cellular RNA was isolated and analyzed by RT-qPCR to examine the expression of indicated genes. (E) MCF-7 cells were transfected with ZNF408, ZNF408-Δ1, ZNF408-Δ5, or ZNF408 + si SETD1A. Total RNA was isolated from cells and analyzed by RT-qPCR. (F) RT-qPCR (left) analysis and western blotting analysis (right) of human MDA-MB-231 cells, BT549 cells, or BT474 cells with or without depletion of ZNF408. In (D–F), each bar represents the mean ± SD for triplicate experiments (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, two-tailed t test). See Table S2 for the target genes of ZNF408-SETD1A complex. See Table S3 for primer information in the RT-qPCR assay.

Article Snippet: Human: BT474 , ATCC , ATCC HTB-20; RRID: CVCL_0179.

Techniques: RNA Sequencing, Knockdown, Transfection, Control, Isolation, Quantitative RT-PCR, Expressing, Western Blot, Two Tailed Test

Journal: iScience

Article Title: Loss of ZNF408 attenuates STING-mediated immune surveillance in breast carcinogenesis

doi: 10.1016/j.isci.2024.110276

Figure Lengend Snippet:

Article Snippet: Human: BT474 , ATCC , ATCC HTB-20; RRID: CVCL_0179.

Techniques: Purification, Virus, Recombinant, Protease Inhibitor, Silver Staining, Bicinchoninic Acid Protein Assay, Transfection, Plasmid Preparation, cDNA Synthesis, Enzyme-linked Immunosorbent Assay, shRNA, Software

Journal: Nature microbiology

Article Title: Determinants of Zika virus host tropism uncovered by deep mutational scanning.

doi: 10.1038/s41564-019-0399-4

Figure Lengend Snippet: Fig. 1 | DMS of C-terminal region of ZIKV E protein. a, Schematic of ZIKV E protein and workflow of DMS screening for the C-terminal 204 codons of ZIKV E protein. Amplicons generated using forward (red arrows) and reverse (blue arrows) mutagenesis primers were combined with other ZIKV cDNA fragments to generate an infectious cDNA library by CPER. The CPER cDNA library was then transfected into mammalian (Vero) or mosquito (C6/36) cells; recovered viruses were harvested in culture supernatants, deep sequenced and analysed to identify preferentially selected viral mutants. dpt, days post-transfection. b, Graphical representation of deep sequencing data showing codon variant heatmap. c, Mutation frequency of cDNA amplicon library illustrating the extent of mutagenesis in the E-DIII stem-anchor region. Mutation frequency is defined as the percentage at which codon mutants are detected in sequenced molecules at each site. For example, at each site, if mutations are present in 100% of sequenced molecules, the frequency is 1; if not present in any sequenced molecules, the frequency is 0. d, Mutation frequency of cDNA library, mammalian (Vero) cell-selected virus population and mosquito (C6/36) cell-selected virus population. Identities of preferentially selected amino acid mutations are indicated above each peak.

Article Snippet: Secondary antibodies are validated and documented by LI-COR Biotechnology (https://www.licor.com/bio/) Eukaryotic cell lines Policy information about cell lines Cell line source(s) K-562 (ATCC CCL-243) HTR-8/SVneo (ATCC CRL-3271) C6/36 (ATCC CRL-1660) A549 (ATCC CRM-CCL-185) Vero (ATCC CCL-81) IFNAR-/- Mouse Embryonic Fibroblast (generated in the lab of Shizuo Akira by Alexander A Khromykh, and first described in Melian et al. J Gen Virol.

Techniques: Generated, Mutagenesis, cDNA Library Assay, Transfection, Sequencing, Variant Assay, Amplification, Virus

Fig. 2 | In vitro characterization of ZIKV mutants. a, Immuno-plaque assay (iPA) of WT and mutant viruses in Vero cells. b–e, Growth kinetics of WT and mutant viruses infected at a MOI of 0.1 in Vero (b), A549 (c), HTR-8 (d) and C6/36 (e) cells. f, Growth kinetics of Vero cells infected with WT or 316Q/461G mutant at a MOI of 0.1 incubated at 28 or 37 °C. g, WT and mutant viruses, as well as control dengue serotype 2 (strain TSV-01) virus, were subjected to heat treatment at the indicated temperatures for 5 h and then titred. All culture supernatants above were harvested at their indicated timepoints after infection, and virus titres determined by iPA in Vero cells; n = 3 independent experiments for all assays, and statistical analysis was performed by two-way analysis of variance with Tukey’s multiple comparisons test against WT virus. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Mean ± s.e.m. Limit of detection for iPA is 1.6 log10FFU ml–1. h, Immunoprecipitation with anti-E antibody (6B6C-1) of 35S-labelled cell lysates (C) or supernatants (S) from Vero or C6/36 cells infected with WT or 316Q/461 G mutant viruses. E protein indicated by the arrowhead. i,j, Antigen-capture enzyme-linked immunosorbent assay (ELISA) detection of extracellular (i) and intracellular (j) E protein. Dotted lines show the limit of detection, n = 3 independent experiments. k,l, Detection of extracellular (k) and intracellular (l) viral RNA by RT–qPCR. ND, not detected. Mean ± s.e.m., n = 3 independent experiments. Statistical analysis for RT–qPCR data was performed using unpaired t-test, two-tailed. ***P = 0.0005.

Journal: Nature microbiology

Article Title: Determinants of Zika virus host tropism uncovered by deep mutational scanning.

doi: 10.1038/s41564-019-0399-4

Figure Lengend Snippet: Fig. 2 | In vitro characterization of ZIKV mutants. a, Immuno-plaque assay (iPA) of WT and mutant viruses in Vero cells. b–e, Growth kinetics of WT and mutant viruses infected at a MOI of 0.1 in Vero (b), A549 (c), HTR-8 (d) and C6/36 (e) cells. f, Growth kinetics of Vero cells infected with WT or 316Q/461G mutant at a MOI of 0.1 incubated at 28 or 37 °C. g, WT and mutant viruses, as well as control dengue serotype 2 (strain TSV-01) virus, were subjected to heat treatment at the indicated temperatures for 5 h and then titred. All culture supernatants above were harvested at their indicated timepoints after infection, and virus titres determined by iPA in Vero cells; n = 3 independent experiments for all assays, and statistical analysis was performed by two-way analysis of variance with Tukey’s multiple comparisons test against WT virus. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Mean ± s.e.m. Limit of detection for iPA is 1.6 log10FFU ml–1. h, Immunoprecipitation with anti-E antibody (6B6C-1) of 35S-labelled cell lysates (C) or supernatants (S) from Vero or C6/36 cells infected with WT or 316Q/461 G mutant viruses. E protein indicated by the arrowhead. i,j, Antigen-capture enzyme-linked immunosorbent assay (ELISA) detection of extracellular (i) and intracellular (j) E protein. Dotted lines show the limit of detection, n = 3 independent experiments. k,l, Detection of extracellular (k) and intracellular (l) viral RNA by RT–qPCR. ND, not detected. Mean ± s.e.m., n = 3 independent experiments. Statistical analysis for RT–qPCR data was performed using unpaired t-test, two-tailed. ***P = 0.0005.

Techniques: In Vitro, Plaque Assay, Mutagenesis, Infection, Incubation, Control, Virus, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Two Tailed Test

Fig. 3 | Immunofluorescence and TEM of infected C6/36 or Vero cells. a, Immunofluorescence microscopy of Vero or C6/36 cells infected with WT or 316Q/461G mutant, and immunostained with 4G2 anti-E antibody (green channel) and DAPI (blue channel). White arrows indicate perinuclear localization of E protein. Scale bars, 5 µm. Representative images of n = 3 biologically independent samples. b, TEM of C6/36 or Vero cells infected with WT, 316Q/461G mutant or uninfected (mock). Virus particles are indicated by arrowheads. CM, convoluted membranes; VP, vesicle packets; V, vesicles; N, nucleus; mt, mitochondria; ER, endoplasmic reticulum. Scale bars in C6/36 images are 200 nm, and in Vero images are 500 nm; n = 3 biologically independent samples.

Journal: Nature microbiology

Article Title: Determinants of Zika virus host tropism uncovered by deep mutational scanning.

doi: 10.1038/s41564-019-0399-4

Figure Lengend Snippet: Fig. 3 | Immunofluorescence and TEM of infected C6/36 or Vero cells. a, Immunofluorescence microscopy of Vero or C6/36 cells infected with WT or 316Q/461G mutant, and immunostained with 4G2 anti-E antibody (green channel) and DAPI (blue channel). White arrows indicate perinuclear localization of E protein. Scale bars, 5 µm. Representative images of n = 3 biologically independent samples. b, TEM of C6/36 or Vero cells infected with WT, 316Q/461G mutant or uninfected (mock). Virus particles are indicated by arrowheads. CM, convoluted membranes; VP, vesicle packets; V, vesicles; N, nucleus; mt, mitochondria; ER, endoplasmic reticulum. Scale bars in C6/36 images are 200 nm, and in Vero images are 500 nm; n = 3 biologically independent samples.

Techniques: Immunofluorescence, Infection, Microscopy, Mutagenesis, Virus

Journal: eLife

Article Title: Impaired lysosomal acidification triggers iron deficiency and inflammation in vivo

doi: 10.7554/eLife.51031

Figure Lengend Snippet: ( A ) Increased Tfrc transcript levels in 500 nM Baf- and 500 nM Sal-treated mouse embryonic fibroblasts relative to untreated fibroblasts. Western blot showing decreased FTH1 and FTL1 protein levels in Baf- and Sal-treated fibroblasts (n = 6). GAPDH is used as loading control. Tfrc expression is depicted as bars representing mean ± SEM, n = 6; shown as black dots. p value is determined by the Welch’s one-way ANOVA as differences between untreated group, and Baf- and Sal-treatments. ( B ) Representative images of FerroOrange staining of cytoplasmic labile iron pools in control, 500 nM Baf-treated and 500 nM Baf-treated fibroblasts with 150 µM iron citrate supplementation. Note the reduced staining in Baf-treated fibroblasts relative to the other conditions. ( C ) mRNA levels of Tfrc in control, 500 nM Baf-treated and 500 nM Baf-treated fibroblasts with 150 µM iron citrate supplementation (left) or in control, 500 nM Sal-treated and 500 nM Sal-treated fibroblasts with 150 µM iron citrate supplementation (right) for 24 hr. Bar graphs depict mean ± SEM of four independent experimental measures (shown as black dots). p values represent Welch’s one-way ANOVA with Dunnett’s correction for multiple comparisons, estimated as differences between Baf- or Sal-treated cells and other experimental groups. ( D ) Whole cell immunoblots of HIF-1α and FTH1 in fibroblasts treated with 500 nM Baf or 500 nM Baf + 150 µM iron citrate (left) or with 500 nM Sal and 500 nM Sal + 150 µM iron citrate (n = 4). GAPDH is used as loading control. ( E ) Transcript levels of HIF-1α target genes in fibroblasts treated with 500 nM Baf or with 500 nM Baf + 150 µM iron citrate. The mean ± SEM of four biological replicates (black dots) is shown. p-values are determined by Welch’s one-way ANOVA with Dunnett’s correction for multiple comparisons (all experimental groups compared to Baf-treated cells). ( F–G ) Gaa KO fibroblasts display impaired lysosomal acidification. Representative spinning-disk microscopy images for Gaa WT and Gaa KO MEFs co-stained with Dextran-Oregon Green 488 and Dextran-TMRM are shown with Baftreatment in Gaa WT used as a positive control for impaired acidification. Scale bar 2 µm. ( G ) Quantification of the intensity of Dextran-Oregon Green in Dextran-TMRM positive punta shows increased Dextran-Oregon Green intensity in Gaa KO fibroblasts and in Baf-treated Gaa WT fibroblasts. Bar graphs depict mean ± SEM, n = 3 independent experiments, with 50 cells per condition from each experiment. ***p<0.001, determined by the unpaired two-tailed t-test with Welch’s correction ( H ) Increased Tfrc expression (left) in Gaa -/- fibroblasts (n = 6, depicted as black dots). p value is determined by the unpaired two-tailed t-test with Welch’s correction. Whole cell immunoblots of FTL1 and HIF-1α (right) in Gaa -/- fibroblasts (n = 6). GAPDH is used as loading control. ( I ) Total, ferrous and ferric iron concentrations in fibroblasts prepared from Gaa -/- and their wild type littermate controls. Results are summarized as mean ± SEM of experimental measures shown as black dots. Differences between means shown as actual p values are determined by the unpaired two-tailed t-test with Welch’s correction.

Article Snippet: Membranes were blocked in 5% Milk in TBS tween and probed with the following primary antibodies: HIF-1 alpha (Novus), GAPDH (Sigma-Aldrich),SQSTM1 (Abcam), ATP6V1H (Abcam), VAPB (Bethyl), PEX5 (Sigma), anti-HA (Abcam), LAMP1 (Sigma Aldrich), (LC3B (Cell signaling), mtTFA (Abcam), Total rodent OXPHOS cocktail (Abcam), TNFα (Abcam), cleaved caspase 3 (Cell signaling), cleaved PARP (Cell signaling), GFAP (Abcam), PLP and MBP (Kind gift of K-A Nave, MPI-EM), FTH1 (Cell signaling), Ferritin light chain (Abcam), TOM20 (Proteintech), VDAC1 (Abcam), VHL (Santa Cruz) and self-made rabbit polyclonal antibodies to COX1 and ATP5B.

Techniques: Western Blot, Control, Expressing, Staining, Microscopy, Positive Control, Two Tailed Test

( A ) Increased Tfrc mRNA levels in fibroblasts treated with lysosomal iron chelator, Dfo (300 µM) for 24 hr. Bar graphs represent mean ± SEM for six independent experimental measures (shown as black dots). p-value is unpaired two-tailed t-test with Welch’s correction. ( B ) Immunoblots of SQSTM1 and LC3B in 500 nM Baf- and 500 nM Sal-treated fibroblasts (n = 6). 300 µM Dfo is used as positive control for the independence of impaired lysosomal iron efflux on defective autophagy. GAPDH is used as loading control. ( C ) Increased Tfrc mRNA levels in fibroblasts treated with either vehicle control, 50 µM Chloroquine (CQ) or 50 nM Baf for 24 hr (left). Bar graphs represent mean ± SEM for four independent experimental measures (shown as black dots). p value is Welch’s one-way ANOVA with all experimental groups compared to vehicle control treated cells. Multiple test corrections were by the Dunnett’s method. Immunoblots of HIF-1α, FTH1 and FTL1 with GAPDH as loading control in whole cell lysates prepared from fibroblasts treated for 24 hr with either vehicle control, 50 µM Chloroquine (CQ) or 50 nM Baf (right). Blots are representative of n = 4 biological replicates. ( D ) Expression level of HIF-1a target genes in 500 nM Sal- and 500 nM Sal-treated fibroblasts with 150 µM iron citrate supplementation. p values are estimated as Welch’s one-way ANOVA with all experimental groups compared to Sal-treated cells. Multiple test corrections were by the Dunnett’s method. ( E ) Tfrc mRNA levels in fibroblasts with siRNA mediated silencing of Atp6v1h subunit of the vATPase (left). Relative Tfrc expression is shown as bars representing mean ± SEM, n = 6 biological replicates (black dots). p value is determined by the Welch’s one-way ANOVA followed by Dunnett’s correction for multiple comparisons of all groups to the siCtrl group. Efficient knockdown is shown as immunoblot of ATP6V1H (right). Immunoblots of HIF-1α, FTH1, FTL1 with GAPDH as loading control are also shown for n = 3 biological samples in fibroblasts with Atp6v1h knockdown. ( F–G ) Silencing of Atp6v1h in fibroblasts results in impaired lysosomal acidification. Representative spinning-disk microscopy images for siCtrl and si Atp6v1h fibroblasts were co-stained with Dextran-Oregon Green 488 and Dextran-TMRM with Baf treatment in siCtrl cells used as a positive control for impaired acidification. Scale bar 2 µm. ( G ) Quantification of the intensity of Dextran-Oregon Green in Dextran-TMRM positive punta shows increased Dextran-Oregon Green intensity in Atp6v1h knockdown fibroblasts and in Baf-treated siCtrl fibroblasts. Bar graphs depict mean ± SEM, n = 3 independent experiments, with 20 cells per condition from each experiment. ***p<0.001, determined by the unpaired two-tailed t-test with Welch’s correction ( H ) Expression level of HIF-1a target genes following Atp6v1h knockdown in fibroblasts. p values are estimated as Welch’s one-way ANOVA with all experimental groups compared to siCtrl cells. Multiple test corrections were by the Dunnett’s method.

Journal: eLife

Article Title: Impaired lysosomal acidification triggers iron deficiency and inflammation in vivo

doi: 10.7554/eLife.51031

Figure Lengend Snippet: ( A ) Increased Tfrc mRNA levels in fibroblasts treated with lysosomal iron chelator, Dfo (300 µM) for 24 hr. Bar graphs represent mean ± SEM for six independent experimental measures (shown as black dots). p-value is unpaired two-tailed t-test with Welch’s correction. ( B ) Immunoblots of SQSTM1 and LC3B in 500 nM Baf- and 500 nM Sal-treated fibroblasts (n = 6). 300 µM Dfo is used as positive control for the independence of impaired lysosomal iron efflux on defective autophagy. GAPDH is used as loading control. ( C ) Increased Tfrc mRNA levels in fibroblasts treated with either vehicle control, 50 µM Chloroquine (CQ) or 50 nM Baf for 24 hr (left). Bar graphs represent mean ± SEM for four independent experimental measures (shown as black dots). p value is Welch’s one-way ANOVA with all experimental groups compared to vehicle control treated cells. Multiple test corrections were by the Dunnett’s method. Immunoblots of HIF-1α, FTH1 and FTL1 with GAPDH as loading control in whole cell lysates prepared from fibroblasts treated for 24 hr with either vehicle control, 50 µM Chloroquine (CQ) or 50 nM Baf (right). Blots are representative of n = 4 biological replicates. ( D ) Expression level of HIF-1a target genes in 500 nM Sal- and 500 nM Sal-treated fibroblasts with 150 µM iron citrate supplementation. p values are estimated as Welch’s one-way ANOVA with all experimental groups compared to Sal-treated cells. Multiple test corrections were by the Dunnett’s method. ( E ) Tfrc mRNA levels in fibroblasts with siRNA mediated silencing of Atp6v1h subunit of the vATPase (left). Relative Tfrc expression is shown as bars representing mean ± SEM, n = 6 biological replicates (black dots). p value is determined by the Welch’s one-way ANOVA followed by Dunnett’s correction for multiple comparisons of all groups to the siCtrl group. Efficient knockdown is shown as immunoblot of ATP6V1H (right). Immunoblots of HIF-1α, FTH1, FTL1 with GAPDH as loading control are also shown for n = 3 biological samples in fibroblasts with Atp6v1h knockdown. ( F–G ) Silencing of Atp6v1h in fibroblasts results in impaired lysosomal acidification. Representative spinning-disk microscopy images for siCtrl and si Atp6v1h fibroblasts were co-stained with Dextran-Oregon Green 488 and Dextran-TMRM with Baf treatment in siCtrl cells used as a positive control for impaired acidification. Scale bar 2 µm. ( G ) Quantification of the intensity of Dextran-Oregon Green in Dextran-TMRM positive punta shows increased Dextran-Oregon Green intensity in Atp6v1h knockdown fibroblasts and in Baf-treated siCtrl fibroblasts. Bar graphs depict mean ± SEM, n = 3 independent experiments, with 20 cells per condition from each experiment. ***p<0.001, determined by the unpaired two-tailed t-test with Welch’s correction ( H ) Expression level of HIF-1a target genes following Atp6v1h knockdown in fibroblasts. p values are estimated as Welch’s one-way ANOVA with all experimental groups compared to siCtrl cells. Multiple test corrections were by the Dunnett’s method.

Techniques: Two Tailed Test, Western Blot, Positive Control, Control, Expressing, Knockdown, Microscopy, Staining

( A ) Mitochondrial OCR in fibroblasts with siRNA mediated silencing of Atp6v1h . Results represent mean ± SEM, n = 3. Each experimental replicate is calculated from the average of 16 technical replicates. ***p<0.001, Welch’s one-way ANOVA with Dunnett’s correction for multiple comparisons. ( B ) Immunoblots of whole cell extracts showing ATP6V1H, ATP5A, UQCRC2, mtCO1, SDHB, and NDUFB8, Atp6v1h knockdown fibroblasts. GAPDH is used as loading control, n = 3 independent experiments. ( C ) Mitochondrial biogenesis is repressed in fibroblasts with Atp6v1h silencing as assessed by the expression of nuclear-encoded mitochondrial genes. Results are shown as bar graphs representing mean ± SEM of six independent experimental replicates. **p<0.01; ***p<0.001, Welch’s one-way ANOVA with Dunnett’s correction for multiple comparisons. Mean differences compared between siCtrl and the other experimental groups. ( D ) Expression levels of Mcoln1 (left) and Slc11a2 (right) in siRNA-mediated knockdown of Slc11a2 in Mcoln1 -/- mouse embryonic fibroblast and their wild type control fibroblasts. Bars represent mean ± SEM, n = 6 biological replicates. ***p<0.0001, unpaired two-tailed t-test with Welch’s correction. ( E ) Tfrc mRNA levels in Mcoln1 WT and KO fibroblasts with siRNA-mediated silencing of Slc11a2 (left). Relative Tfrc expression is shown as bars representing mean ± SEM, n = 6 biological replicates. ***p<0.001, determined by the Welch’s one-way ANOVA followed by Dunnett’s correction for multiple comparisons of all groups to the Mcoln1 WT siCtrl group. Immunoblots of HIF-1α, FTH1, FTL1 with GAPDH as loading control in Mcoln1 WT and KO fibroblasts with siRNA-mediated silencing of Slc11a2 are also shown for n = 3 biological samples (right). ( F ) Mitochondrial OCR in Mcoln1 WT and KO fibroblasts with Slc11a2 knockdown. Results represent mean ± SEM, n = 3. Each experimental replicate is calculated from the average of 9 technical replicates. ***p<0.001, Welch’s one-way ANOVA with Dunnett’s correction for multiple comparisons ( G ) Immunoblots of ATP5A, UQCRC2, mtCO1 AND SDHB in lysates prepared from Mcoln1 WT and KO fibroblasts with siRNA-mediated knockdown of Slc11a2 . GAPDH is used as loading, n = 3 independent experiments. ( H ) Reduced levels of nuclear-encoded mitochondrial transcripts in either Mcoln1 KO fibroblasts, Slc11a2 knockdown fibroblasts or both. Results are shown as bar graphs representing mean ± SEM of six independent experimental replicates. *p<0.05; **p<0.01; ***p<0.001, determined by the Welch’s one-way ANOVA followed by Dunnett’s correction for multiple comparisons of all groups to the Mcoln1 WT siCtrl group.

Journal: eLife

Article Title: Impaired lysosomal acidification triggers iron deficiency and inflammation in vivo

doi: 10.7554/eLife.51031

Figure Lengend Snippet: ( A ) Mitochondrial OCR in fibroblasts with siRNA mediated silencing of Atp6v1h . Results represent mean ± SEM, n = 3. Each experimental replicate is calculated from the average of 16 technical replicates. ***p<0.001, Welch’s one-way ANOVA with Dunnett’s correction for multiple comparisons. ( B ) Immunoblots of whole cell extracts showing ATP6V1H, ATP5A, UQCRC2, mtCO1, SDHB, and NDUFB8, Atp6v1h knockdown fibroblasts. GAPDH is used as loading control, n = 3 independent experiments. ( C ) Mitochondrial biogenesis is repressed in fibroblasts with Atp6v1h silencing as assessed by the expression of nuclear-encoded mitochondrial genes. Results are shown as bar graphs representing mean ± SEM of six independent experimental replicates. **p<0.01; ***p<0.001, Welch’s one-way ANOVA with Dunnett’s correction for multiple comparisons. Mean differences compared between siCtrl and the other experimental groups. ( D ) Expression levels of Mcoln1 (left) and Slc11a2 (right) in siRNA-mediated knockdown of Slc11a2 in Mcoln1 -/- mouse embryonic fibroblast and their wild type control fibroblasts. Bars represent mean ± SEM, n = 6 biological replicates. ***p<0.0001, unpaired two-tailed t-test with Welch’s correction. ( E ) Tfrc mRNA levels in Mcoln1 WT and KO fibroblasts with siRNA-mediated silencing of Slc11a2 (left). Relative Tfrc expression is shown as bars representing mean ± SEM, n = 6 biological replicates. ***p<0.001, determined by the Welch’s one-way ANOVA followed by Dunnett’s correction for multiple comparisons of all groups to the Mcoln1 WT siCtrl group. Immunoblots of HIF-1α, FTH1, FTL1 with GAPDH as loading control in Mcoln1 WT and KO fibroblasts with siRNA-mediated silencing of Slc11a2 are also shown for n = 3 biological samples (right). ( F ) Mitochondrial OCR in Mcoln1 WT and KO fibroblasts with Slc11a2 knockdown. Results represent mean ± SEM, n = 3. Each experimental replicate is calculated from the average of 9 technical replicates. ***p<0.001, Welch’s one-way ANOVA with Dunnett’s correction for multiple comparisons ( G ) Immunoblots of ATP5A, UQCRC2, mtCO1 AND SDHB in lysates prepared from Mcoln1 WT and KO fibroblasts with siRNA-mediated knockdown of Slc11a2 . GAPDH is used as loading, n = 3 independent experiments. ( H ) Reduced levels of nuclear-encoded mitochondrial transcripts in either Mcoln1 KO fibroblasts, Slc11a2 knockdown fibroblasts or both. Results are shown as bar graphs representing mean ± SEM of six independent experimental replicates. *p<0.05; **p<0.01; ***p<0.001, determined by the Welch’s one-way ANOVA followed by Dunnett’s correction for multiple comparisons of all groups to the Mcoln1 WT siCtrl group.

Techniques: Western Blot, Knockdown, Control, Expressing, Two Tailed Test

Journal: eLife

Article Title: Impaired lysosomal acidification triggers iron deficiency and inflammation in vivo

doi: 10.7554/eLife.51031

Figure Lengend Snippet:

Techniques: Magnetic Beads, Iron Assay, cDNA Synthesis, Viability Assay, Sequencing, Recombinant, Plasmid Preparation, Transfection, Software, Imaging

Journal: The EMBO Journal

Article Title: EPDR1 promotes PD-L1 expression and tumor immune evasion by inhibiting TRIM21-dependent ubiquitylation of IkappaB kinase-β

doi: 10.1038/s44318-024-00201-6

Figure Lengend Snippet: ( A , B ) Plasmids expressing human YAP5SA (2 mg/kg) plus RFP control (2 mg/kg) or mEPDR1 (2 mg/kg), together with plasmids expressing PB transposase (0.8 mg/kg), were delivered into mice by hydrodynamic injection ( n = 6 male mice per group). Liver tumors were analyzed 110 days after injection. Photographs show livers ( A ) and tumor numbers ( B ) were determined. Data were presented as the mean ± SEM. ( C ) Dimensionality reduction and visualization based on T-distributed stochastic neighbor embedding (t-SNE) analysis of a subset of mouse liver immunocytes from the indicated group in ( A ). ( D ) Statistical difference analysis for immune cell subsets was obtained from dimensionality reduction analysis in panel ( C ). n = 5 male mice per group and data were presented as the mean ± SD. ( E ) Flow cytometry analysis of the ratio of the immune co-suppressive molecules (PD-1, TIM-3) and immune effector molecules (IFNγ, GzmB) positive cells in liver CD8 + T cells from the indicated group in ( A ). n = 6 male mice per group and data were presented as the mean ± SD. ( F ) Schema of coculture of human CD8 + T cells with HepG2 cells expressing Flag-EV or Flag-EPDR1. ( G ) Flow cytometry analysis of the ratio of immunosuppressive molecules (PD-1, TIM-3) and immune effector molecules (IFNγ, GzmB) positive cells in CD8 + T cells after coculture with the indicated tumor cells. n = 3 independent experiments and the data were presented as the mean ± SD. ( H – J ) Hepa 1-6 cells stably expressing Flag-EV or Flag-mEPDR1 were injected subcutaneously into C57BL/6 J mice ( n = 6 male mice per group), and α-CD8 (4 mg/kg) neutralizing antibody was injected intraperitoneally four times (twice a week starting at 10 days after inoculation) to block CD8 + T cells and IgG2b was used as control. Tumor size was measured starting at 10 days after inoculation. Photographs show xenografts ( H ), growth curves ( I ), and relative tumor burdens ( J ) determined at the end of the experiment (day 25). Data were presented as the mean ± SEM. Data information: Statistical significance was determined by two-way ANOVA ( D , I ), one-way ANOVA ( J ), and two-tailed unpaired Student’s t -test ( B , E , G ). .

Article Snippet: InVivoMAb anti-mouse CD8 , BioXcell , BE0061.

Techniques: Expressing, Control, Injection, Flow Cytometry, Stable Transfection, Blocking Assay, Two Tailed Test

( A ) Western blotting analysis of the protein level of Myc-tag mEPDR1 in liver samples from the YAP5SA-induced mouse model. Calnexin served as a loading control. ( B ) t-SNE plot showing the expression level marker genes for distinct subpopulations of lymphocytes. ( C ) Flow cytometric analysis of the ratio of immunosuppressive molecules (PD-1 + TIM-3 + ) cells in tumor CD8 + T cells from the indicated group in ( A ). Data were presented as the mean ± SD. ( D ) Schema of coculture of mouse CD8 + T cells with Hepa 1-6 cells expressing Flag-EV or Flag-mEPDR1. ( E ) Flow cytometry analysis of the ratio of immunosuppressive molecules (PD-1, TIM-3) and immune effector molecules (IFNγ, GzmB) positive cells in CD8 + T cells after coculture with the indicated tumor cells. Data were presented as the mean ± SD of three independent experiments ( n = 3). ( F ) Flowrate analysis of the amount of CD8 + T cells in blood from the mice with the indicated manipulation. ( G – I ) Hepa 1–6 cells stably expressing NTC or shEPDR1 were injected subcutaneously into C57BL/6J mice ( n = 6 male mice per group). Tumor size was measured starting at 10 days after inoculation. The figure depicts xenografts ( A ), growth curves ( B ), and tumor weights ( C ) determined at the end of the experiment (day 25). Data were presented as the mean ± SEM ( H ), and mean ± SD ( I ), respectively. ( J ) Flow cytometric analysis of the ratio of immunosuppressive molecules (PD-1, TIM-3) positive cells in tumor CD8 + T cells from the indicated group in ( G ). Data were presented as the mean ± SD ( n = 6). ( K ) Flow cytometric analysis of the ratio of immune effector molecules (IFNγ, Granzyme B) positive cells in tumor CD8 + T cells from the indicated groups in ( G ). Data were presented as the mean ± SD ( n = 6). Data information: Statistical significance was determined by two-tailed unpaired Student’s t -test ( C , E ), two-way ANOVA ( H ), and one-way ANOVA ( I – K ). .

Journal: The EMBO Journal

Article Title: EPDR1 promotes PD-L1 expression and tumor immune evasion by inhibiting TRIM21-dependent ubiquitylation of IkappaB kinase-β

doi: 10.1038/s44318-024-00201-6

Figure Lengend Snippet: ( A ) Western blotting analysis of the protein level of Myc-tag mEPDR1 in liver samples from the YAP5SA-induced mouse model. Calnexin served as a loading control. ( B ) t-SNE plot showing the expression level marker genes for distinct subpopulations of lymphocytes. ( C ) Flow cytometric analysis of the ratio of immunosuppressive molecules (PD-1 + TIM-3 + ) cells in tumor CD8 + T cells from the indicated group in ( A ). Data were presented as the mean ± SD. ( D ) Schema of coculture of mouse CD8 + T cells with Hepa 1-6 cells expressing Flag-EV or Flag-mEPDR1. ( E ) Flow cytometry analysis of the ratio of immunosuppressive molecules (PD-1, TIM-3) and immune effector molecules (IFNγ, GzmB) positive cells in CD8 + T cells after coculture with the indicated tumor cells. Data were presented as the mean ± SD of three independent experiments ( n = 3). ( F ) Flowrate analysis of the amount of CD8 + T cells in blood from the mice with the indicated manipulation. ( G – I ) Hepa 1–6 cells stably expressing NTC or shEPDR1 were injected subcutaneously into C57BL/6J mice ( n = 6 male mice per group). Tumor size was measured starting at 10 days after inoculation. The figure depicts xenografts ( A ), growth curves ( B ), and tumor weights ( C ) determined at the end of the experiment (day 25). Data were presented as the mean ± SEM ( H ), and mean ± SD ( I ), respectively. ( J ) Flow cytometric analysis of the ratio of immunosuppressive molecules (PD-1, TIM-3) positive cells in tumor CD8 + T cells from the indicated group in ( G ). Data were presented as the mean ± SD ( n = 6). ( K ) Flow cytometric analysis of the ratio of immune effector molecules (IFNγ, Granzyme B) positive cells in tumor CD8 + T cells from the indicated groups in ( G ). Data were presented as the mean ± SD ( n = 6). Data information: Statistical significance was determined by two-tailed unpaired Student’s t -test ( C , E ), two-way ANOVA ( H ), and one-way ANOVA ( I – K ). .

Article Snippet: InVivoMAb anti-mouse CD8 , BioXcell , BE0061.

Techniques: Western Blot, Control, Expressing, Marker, Flow Cytometry, Stable Transfection, Injection, Two Tailed Test

( A – C ) Hepa 1-6 cells stably expressing Flag-EV and Flag-mEPDR1 were infected with shNTC or shPD-L1, and subsequently subcutaneously injected into C57BL/6J mice ( n = 6 male mice per group). Tumor size was measured starting at 10 days after inoculation. Photographs show xenografts ( A ), growth curves ( B ), and tumor weight ( C ) determined at the end of the experiment (day 25). Data were presented as the mean ± SEM ( B ) and mean ± SD ( C ), respectively. ( D ) Flow cytometry analysis of the ratio of immunosuppressive molecules (PD-1, TIM-3) positive cells in tumor CD8 + T cells from the indicated group in ( A ). Data were presented as the mean ± SD. ( E ) Flow cytometry analysis of the ratio of immune effector molecules (IFNγ, GzmB) positive cells in tumor CD8 + T cells from the indicated groups in ( A ). Data were presented as the mean ± SD. ( F – H ) Hepa 1-6 cells stably expressing Flag-EV, Flag-mTRIM21, Flag-mEPDR1, or Flag-mTRIM21 plus Flag-mEPDR1 both were injected subcutaneously into C57BL/6 J mice ( n = 6 male mice per group). Tumor size was measured starting at 10 days after inoculation. Photographs show xenografts ( A ), growth curves ( B ), and tumor weight ( C ) determined at the end of the experiment (day 25). Data were presented as the mean ± SEM ( G ), and mean ± SD ( H ), respectively. ( I ) Flow cytometry analysis of membrane-bound PD-L1 in the indicated group in ( A ). Data were presented as the mean ± SD. ( J ) Flow cytometry analysis of the ratio of immunosuppressive molecules (PD-1, TIM-3) positive cells in tumor CD8 + T cells from the indicated group in ( A ). Data were presented as the mean ± SD ( n = 6). ( K ) Flow cytometry analysis of the ratio of immune effector molecules (IFNγ, GzmB) positive cells in tumor CD8 + T cells from the indicated group in ( A ). Data were presented as the mean ± SD ( n = 6). Data information: Statistical significance was determined by two-way ANOVA ( B , G ) and one-way ANOVA ( C – E , H – K ). .

Journal: The EMBO Journal

Article Title: EPDR1 promotes PD-L1 expression and tumor immune evasion by inhibiting TRIM21-dependent ubiquitylation of IkappaB kinase-β

doi: 10.1038/s44318-024-00201-6

Figure Lengend Snippet: ( A – C ) Hepa 1-6 cells stably expressing Flag-EV and Flag-mEPDR1 were infected with shNTC or shPD-L1, and subsequently subcutaneously injected into C57BL/6J mice ( n = 6 male mice per group). Tumor size was measured starting at 10 days after inoculation. Photographs show xenografts ( A ), growth curves ( B ), and tumor weight ( C ) determined at the end of the experiment (day 25). Data were presented as the mean ± SEM ( B ) and mean ± SD ( C ), respectively. ( D ) Flow cytometry analysis of the ratio of immunosuppressive molecules (PD-1, TIM-3) positive cells in tumor CD8 + T cells from the indicated group in ( A ). Data were presented as the mean ± SD. ( E ) Flow cytometry analysis of the ratio of immune effector molecules (IFNγ, GzmB) positive cells in tumor CD8 + T cells from the indicated groups in ( A ). Data were presented as the mean ± SD. ( F – H ) Hepa 1-6 cells stably expressing Flag-EV, Flag-mTRIM21, Flag-mEPDR1, or Flag-mTRIM21 plus Flag-mEPDR1 both were injected subcutaneously into C57BL/6 J mice ( n = 6 male mice per group). Tumor size was measured starting at 10 days after inoculation. Photographs show xenografts ( A ), growth curves ( B ), and tumor weight ( C ) determined at the end of the experiment (day 25). Data were presented as the mean ± SEM ( G ), and mean ± SD ( H ), respectively. ( I ) Flow cytometry analysis of membrane-bound PD-L1 in the indicated group in ( A ). Data were presented as the mean ± SD. ( J ) Flow cytometry analysis of the ratio of immunosuppressive molecules (PD-1, TIM-3) positive cells in tumor CD8 + T cells from the indicated group in ( A ). Data were presented as the mean ± SD ( n = 6). ( K ) Flow cytometry analysis of the ratio of immune effector molecules (IFNγ, GzmB) positive cells in tumor CD8 + T cells from the indicated group in ( A ). Data were presented as the mean ± SD ( n = 6). Data information: Statistical significance was determined by two-way ANOVA ( B , G ) and one-way ANOVA ( C – E , H – K ). .

Article Snippet: InVivoMAb anti-mouse CD8 , BioXcell , BE0061.

Techniques: Stable Transfection, Expressing, Infection, Injection, Flow Cytometry, Membrane

( A – C ) Hepa 1-6 cells stably expressing Flag-EV and Flag-mEPDR1 were injected subcutaneously into C57BL/6J mice ( n = 6 male mice per group), and α-PD-L1 (6 mg/kg) neutralizing antibody was injected intraperitoneally four times (twice a week starting at 10 days after inoculation) to block PD-L1 and IgG2b was used as control. Tumor size was measured starting at 10 days after inoculation. Photographs show xenografts ( A ), growth curves ( B ), and tumor weight ( C ) determined at the end of the experiment (day 25). Data were presented as the mean ± SEM ( B ) and mean ± SD ( C ), respectively. ( D ) Flow cytometry analysis of ratio of immunosuppressive molecules (PD-1, TIM-3) positive cells in tumor CD8 + T cells from the indicated group in ( A ). Data were presented as the mean ± SD. ( E ) Flow cytometry analysis of the ratio of immune effector molecules (IFNγ, Granzyme B) positive cells in tumor CD8 + T cells from the indicated groups in ( A ). Data were presented as the mean ± SD. ( F – H ) Hepa 1–6 cells stably expressing Flag-EV and Flag-mEPDR1 were injected subcutaneously into C57BL/6J mice ( n = 6 male mice per group). BAY11-7082 was used to inhibit the NF-Κb pathway, and the vehicle was used as a control. Tumor size was measured starting at 10 days after inoculation. Photographs show xenografts ( F ), growth curves ( G ), and final tumor weight ( H ) determined at the end of the experiment (day 25). Data were presented as the mean ± SEM ( G ), and mean ± SD ( H ), respectively. ( I ) Flow cytometry analysis of the ratio of immunosuppressive molecules (PD-1, TIM-3) positive cells in tumor CD8 + T cells from the indicated group in ( F ). Data were presented as the mean ± SD. ( J ) Flow cytometry analysis of the ratio of immune effector molecules (IFNγ, GzmB) positive cells in tumor CD8 + T cells from the indicated group in ( F ). Data were presented as the mean ± SD. Data information: Statistical significance was determined by two-way ANOVA ( B , G ) and one-way ANOVA ( C – E , H – J ). .

Journal: The EMBO Journal

Article Title: EPDR1 promotes PD-L1 expression and tumor immune evasion by inhibiting TRIM21-dependent ubiquitylation of IkappaB kinase-β

doi: 10.1038/s44318-024-00201-6

Figure Lengend Snippet: ( A – C ) Hepa 1-6 cells stably expressing Flag-EV and Flag-mEPDR1 were injected subcutaneously into C57BL/6J mice ( n = 6 male mice per group), and α-PD-L1 (6 mg/kg) neutralizing antibody was injected intraperitoneally four times (twice a week starting at 10 days after inoculation) to block PD-L1 and IgG2b was used as control. Tumor size was measured starting at 10 days after inoculation. Photographs show xenografts ( A ), growth curves ( B ), and tumor weight ( C ) determined at the end of the experiment (day 25). Data were presented as the mean ± SEM ( B ) and mean ± SD ( C ), respectively. ( D ) Flow cytometry analysis of ratio of immunosuppressive molecules (PD-1, TIM-3) positive cells in tumor CD8 + T cells from the indicated group in ( A ). Data were presented as the mean ± SD. ( E ) Flow cytometry analysis of the ratio of immune effector molecules (IFNγ, Granzyme B) positive cells in tumor CD8 + T cells from the indicated groups in ( A ). Data were presented as the mean ± SD. ( F – H ) Hepa 1–6 cells stably expressing Flag-EV and Flag-mEPDR1 were injected subcutaneously into C57BL/6J mice ( n = 6 male mice per group). BAY11-7082 was used to inhibit the NF-Κb pathway, and the vehicle was used as a control. Tumor size was measured starting at 10 days after inoculation. Photographs show xenografts ( F ), growth curves ( G ), and final tumor weight ( H ) determined at the end of the experiment (day 25). Data were presented as the mean ± SEM ( G ), and mean ± SD ( H ), respectively. ( I ) Flow cytometry analysis of the ratio of immunosuppressive molecules (PD-1, TIM-3) positive cells in tumor CD8 + T cells from the indicated group in ( F ). Data were presented as the mean ± SD. ( J ) Flow cytometry analysis of the ratio of immune effector molecules (IFNγ, GzmB) positive cells in tumor CD8 + T cells from the indicated group in ( F ). Data were presented as the mean ± SD. Data information: Statistical significance was determined by two-way ANOVA ( B , G ) and one-way ANOVA ( C – E , H – J ). .

Article Snippet: InVivoMAb anti-mouse CD8 , BioXcell , BE0061.

Techniques: Stable Transfection, Expressing, Injection, Blocking Assay, Control, Flow Cytometry

( A ) Representative immunohistochemistry images of EPDR1, PD-L1, p65, and CD8 staining in HCC specimens; scale bars, 50 μm. ( B ) Correlation analysis of EPDR1 and PD-L1 positive signal in HCC specimens, P values and R were calculated by two-tailed Person’s correlation analysis. ( C ) Correlation analysis of EPDR1 and nucleus located p65 positive signal in HCC specimens, P values and R were calculated by two-tailed Person’s correlation analysis. ( D ) Summary: In the presence of EPDR1, intracellular EPDR1 directly binds to TRIM21 in competition with IKBKB, thereby disrupting the ubiquitylation and degradation of IKBKB, leading to enhanced NF-κB signaling and downstream PD-L1 expression, which exacerbates the exhaustion of CD8 + T cells and contributes to tumor progression. .

Journal: The EMBO Journal

Article Title: EPDR1 promotes PD-L1 expression and tumor immune evasion by inhibiting TRIM21-dependent ubiquitylation of IkappaB kinase-β

doi: 10.1038/s44318-024-00201-6

Figure Lengend Snippet: ( A ) Representative immunohistochemistry images of EPDR1, PD-L1, p65, and CD8 staining in HCC specimens; scale bars, 50 μm. ( B ) Correlation analysis of EPDR1 and PD-L1 positive signal in HCC specimens, P values and R were calculated by two-tailed Person’s correlation analysis. ( C ) Correlation analysis of EPDR1 and nucleus located p65 positive signal in HCC specimens, P values and R were calculated by two-tailed Person’s correlation analysis. ( D ) Summary: In the presence of EPDR1, intracellular EPDR1 directly binds to TRIM21 in competition with IKBKB, thereby disrupting the ubiquitylation and degradation of IKBKB, leading to enhanced NF-κB signaling and downstream PD-L1 expression, which exacerbates the exhaustion of CD8 + T cells and contributes to tumor progression. .

Article Snippet: InVivoMAb anti-mouse CD8 , BioXcell , BE0061.

Techniques: Immunohistochemistry, Staining, Two Tailed Test, Expressing

Journal: The EMBO Journal

Article Title: EPDR1 promotes PD-L1 expression and tumor immune evasion by inhibiting TRIM21-dependent ubiquitylation of IkappaB kinase-β

doi: 10.1038/s44318-024-00201-6

Figure Lengend Snippet: Reagents and tools table

Article Snippet: InVivoMAb anti-mouse CD8 , BioXcell , BE0061.

Techniques: Recombinant, Purification, Sequencing, Control, Modification, Cell Stimulation, Ligation, Cloning, cDNA Synthesis, SYBR Green Assay, Software

Inducible RNase H1 transgene expression in vitro. A) Schematic of hiPSC differentiation from fibroblasts to NPCs and neurons. Left panel: Immunofluorescent staining of the neuronal marker MAP2 (green) in hiPSC-neurons (top) and the NPC markers SOX2 (green) and Nestin (red) in hiPSC-NPCs (bottom); scale bar: 100 mm. Right panel: Immunofluorescent staining of S9.6 (red) and DAPI (blue) in each respective cell type; scale bar: 100 mm. B) Schematic of the effect of RNase H1-mediated knockdown of R-loops, or D145N mutant that is binding- competent but catalytically inactive. C) Overview of the lentiviral constructs used in this study, including the rtTA transactivator, RH1ΔMLS, RH1ΔMLS D145N catalytically-inactive mutant, and BFP-expressing lentiviral backbone control. D) (Top) Representative Western Blot of hiPSC-neurons expressing either BFP, RH1ΔMLS, or RH1ΔMLS D145N lentiviruses. Gradient triangles indicate concentration of virus. RNase H1 protein is 5.1-fold increased in RH1ΔMLS cells relative to BFP controls (normalized to β-actin loading control). (Bottom) Immunocytochemistry for S9.6 (red) and RNase H1 (green) in hiPSC-neurons expressing BFP, RH1ΔMLS D145N , or RH1ΔMLS transgenes. RNase H1 protein is robustly increased in the cell nucleus (DAPI, blue) in RH1ΔMLS and RH1ΔMLS D145N cells relative to BFP controls, indicating successful nuclear expression of the transgene-encoded protein; scale bars = 20 mm. E) Experimental timeline. NPCs are transduced with RH1ΔMLS or a control (RH1ΔMLS D145N or BFP backbone) lentivirus. After 4 days in vitro , puromycin (1:1,000) is added to the culture media to select for transgene-expressing cells. After 6 days in culture, puromycin concentration is dropped to 1:5,000. On day 0 (indicating transition from NPC to neuron differentiation), cells are replated and fed every 2 days with neuron differentiation medium containing 1:1,000 doxycycline to activate the transgene. Every week from week 2, electrophysiological recordings are captured by multielectrode array (MEA). After 6 weeks in culture, cells are harvested for DRIP-seq, bulk- and scRNA-seq. F) (top to bottom) DRIP-seq tracks for hiPSC-derived neurons transduced with BFP (blue tracks, BFP Neuron cultures 1-3) or RH1ΔMLS (bright red tracks, cultures 1-3) constructs over a well-defined, conserved R-loop hotspot region , demonstrating robustness of our DRIP-seq experimental and computational processing. Note that y-axis scaling 0-90 for BFP Neuron DRIP-seq is wider than RH1 Neuron scale, 0-10. RNA alone processed by the DRIP-seq protocol (RNA-only DRIP, magenta track) shows no discernable peaks, indicating that our S9.6 immunoprecipitation is specific for DNA/RNA hybrids and does not represent single-stranded or double-stranded RNA. Orange track: in silico HindIII/EcoRI/XbaI/ SSPI restriction enzyme digest showing expected cutting sites of the restriction enzyme cocktail used for DRIP-seq. G) Left: Volcano plot displaying the 6,380 differential promoter-bound R-loop peaks (DRIP-seq) between hiPSC-differentiated cultures expressing RH1ΔMLS (n = 3) or BFP (n = 3) (FDR < 0.05, pink dots) after 6 weeks in culture. Of these, 4,805 DRIP-seq peaks were enriched in BFP controls (log 2 fold change > 1) and 474 DRIP-seq peaks were enriched RH1ΔMLS cells (log 2 fold change < -1), indicating successful knockdown of R-loops in the latter. Right: Volcano plot displaying the 1,753 differential promoter-bound R-loop peaks between RH1ΔMLS (n = 3) and RH1ΔMLS D145N (n = 2) (FDR < 0.1, pink dots) cells after 6 weeks in culture. Of these, 1,709 DRIP-seq peaks were enriched in RH1ΔMLS D145N controls (log 2 fold change > 1) and 35 DRIP-seq peaks were enriched in RH1ΔMLS cells (log 2 fold change < -1), again indicating successful knockdown of R-loops in the latter. H) Representative DRIP-seq tracks over two neuronal genes for BFP-, RH1ΔMLS D145N -, and RH1ΔMLS- expressing hiPSC-neurons after 6 weeks in culture. Pink highlighted region and inset displays magnified promoter region.

Journal: bioRxiv

Article Title: R-loop landscapes in the developing human brain are linked to neural differentiation and cell-type specific transcription

doi: 10.1101/2023.07.18.549494

Figure Lengend Snippet: Inducible RNase H1 transgene expression in vitro. A) Schematic of hiPSC differentiation from fibroblasts to NPCs and neurons. Left panel: Immunofluorescent staining of the neuronal marker MAP2 (green) in hiPSC-neurons (top) and the NPC markers SOX2 (green) and Nestin (red) in hiPSC-NPCs (bottom); scale bar: 100 mm. Right panel: Immunofluorescent staining of S9.6 (red) and DAPI (blue) in each respective cell type; scale bar: 100 mm. B) Schematic of the effect of RNase H1-mediated knockdown of R-loops, or D145N mutant that is binding- competent but catalytically inactive. C) Overview of the lentiviral constructs used in this study, including the rtTA transactivator, RH1ΔMLS, RH1ΔMLS D145N catalytically-inactive mutant, and BFP-expressing lentiviral backbone control. D) (Top) Representative Western Blot of hiPSC-neurons expressing either BFP, RH1ΔMLS, or RH1ΔMLS D145N lentiviruses. Gradient triangles indicate concentration of virus. RNase H1 protein is 5.1-fold increased in RH1ΔMLS cells relative to BFP controls (normalized to β-actin loading control). (Bottom) Immunocytochemistry for S9.6 (red) and RNase H1 (green) in hiPSC-neurons expressing BFP, RH1ΔMLS D145N , or RH1ΔMLS transgenes. RNase H1 protein is robustly increased in the cell nucleus (DAPI, blue) in RH1ΔMLS and RH1ΔMLS D145N cells relative to BFP controls, indicating successful nuclear expression of the transgene-encoded protein; scale bars = 20 mm. E) Experimental timeline. NPCs are transduced with RH1ΔMLS or a control (RH1ΔMLS D145N or BFP backbone) lentivirus. After 4 days in vitro , puromycin (1:1,000) is added to the culture media to select for transgene-expressing cells. After 6 days in culture, puromycin concentration is dropped to 1:5,000. On day 0 (indicating transition from NPC to neuron differentiation), cells are replated and fed every 2 days with neuron differentiation medium containing 1:1,000 doxycycline to activate the transgene. Every week from week 2, electrophysiological recordings are captured by multielectrode array (MEA). After 6 weeks in culture, cells are harvested for DRIP-seq, bulk- and scRNA-seq. F) (top to bottom) DRIP-seq tracks for hiPSC-derived neurons transduced with BFP (blue tracks, BFP Neuron cultures 1-3) or RH1ΔMLS (bright red tracks, cultures 1-3) constructs over a well-defined, conserved R-loop hotspot region , demonstrating robustness of our DRIP-seq experimental and computational processing. Note that y-axis scaling 0-90 for BFP Neuron DRIP-seq is wider than RH1 Neuron scale, 0-10. RNA alone processed by the DRIP-seq protocol (RNA-only DRIP, magenta track) shows no discernable peaks, indicating that our S9.6 immunoprecipitation is specific for DNA/RNA hybrids and does not represent single-stranded or double-stranded RNA. Orange track: in silico HindIII/EcoRI/XbaI/ SSPI restriction enzyme digest showing expected cutting sites of the restriction enzyme cocktail used for DRIP-seq. G) Left: Volcano plot displaying the 6,380 differential promoter-bound R-loop peaks (DRIP-seq) between hiPSC-differentiated cultures expressing RH1ΔMLS (n = 3) or BFP (n = 3) (FDR < 0.05, pink dots) after 6 weeks in culture. Of these, 4,805 DRIP-seq peaks were enriched in BFP controls (log 2 fold change > 1) and 474 DRIP-seq peaks were enriched RH1ΔMLS cells (log 2 fold change < -1), indicating successful knockdown of R-loops in the latter. Right: Volcano plot displaying the 1,753 differential promoter-bound R-loop peaks between RH1ΔMLS (n = 3) and RH1ΔMLS D145N (n = 2) (FDR < 0.1, pink dots) cells after 6 weeks in culture. Of these, 1,709 DRIP-seq peaks were enriched in RH1ΔMLS D145N controls (log 2 fold change > 1) and 35 DRIP-seq peaks were enriched in RH1ΔMLS cells (log 2 fold change < -1), again indicating successful knockdown of R-loops in the latter. H) Representative DRIP-seq tracks over two neuronal genes for BFP-, RH1ΔMLS D145N -, and RH1ΔMLS- expressing hiPSC-neurons after 6 weeks in culture. Pink highlighted region and inset displays magnified promoter region.

Article Snippet: As mitochondrial R-loops are essential for cell survival, we first used the QuikChange II Site-directed mutagenesis kit (Agilent, cat. no. #200523) according to the manufacturer’s instructions to remove the 78 bp mitochondrial localization signal (MLS) from RNase H1 cDNA (AddGene; pFRT-TODestGFP_RNAseH1; Plasmid #65784), using the primer sequences in Supplementary Table 24, RH1ΔMLS.

Techniques: Expressing, In Vitro, Staining, Marker, Knockdown, Mutagenesis, Binding Assay, Construct, Control, Western Blot, Concentration Assay, Virus, Immunocytochemistry, Transduction, Derivative Assay, Immunoprecipitation, In Silico

Cell specifications and transcriptomic alterations in RH1 transgenic cultures. A, B) Gene set enrichment analysis of scRNA-seq data from RH1ΔMLS and BFP control hiPSC-differentiated cultures. (A) Cnet plot and (B) dot plot showing significant gene ontologies up- (left) and down-regulated (right) following RH1ΔMLS transgene induction. Dot color indicates fold change (cnet plot) or adjusted p- value (dot plot), dot size indicates number of genes in category. n = 2 replicates from 2 cell lines/group. C) Combination of clusters into broad cell type classes, including glia (green), neurons (purple), and NPCs (blue). Gray clusters did not show canonical markers of these major neural cell types. D) RH1ΔMLS cells have significantly (p < 0.0001, Wilcoxin Rank Sum test) higher RNASEH1 expression than BFP controls (mean normalized expression: RH1ΔMLS = 2.45, BFP = 0.94). Cells from each condition were split into two groups according to RNASEH1 normalized expression value (> 1.25 = hi; ≤ 1.25 = lo) for downstream analyses. E) Bar charts showing difference in cell proportions across BFP or RH1ΔMLS transduced cells, stratified by RNASEH1 expression level. There is a significant reduction in the proportion of glia (p = 0.015) and an increased proportion of neurons (p = 0.036) in RH1ΔMLS- RNASEH1 high relative to BFP- RNASEH1 low groups (Two-way ANOVA with Dunnett’s Multiple Comparison test; n = 2 replicates from 2 cell donors per group; error bars denote S.E.M.; n.s. = not significant.) F, G) Volcano plots showing differentially expressed genes in scRNA-seq ( F ) and bulk RNA-seq ( G ) from RH1ΔMLS hiPSC-differentiated cultures and BFP controls. Green dots indicate genes more highly expressed in control cells (FDR < 0.05, log 2 fold change < 0) and purple/magenta dots indicate genes more highly expressed in RH1ΔMLS cells (FDR < 0.05, log 2 fold change > 0). H) Venn diagram showing overlap of upregulated genes in RH1ΔMLS hiPSC-differentiated cultures from scRNA and bulk RNA-seq, and promoter-bound R-loops that are knocked down following RNase H1-transgene expression. RF = Representation factor, which signifies more overlap of the two gene sets than expected based on a background list of genes if > 1. P-values calculated via hypergeometric test. I) Gene ontology of genes with upregulated expression following promoter-bound R-loop loss in RH1ΔMLS hiPSC-differentiated cultures. Numbers within gene ontology images indicate number of genes in the enriched set that occur within the ontology class, legend denotes -log 10 enrichment FDR. J) Venn diagram showing overlap of genes that have upregulated gene expression following promoter-bound R-loop loss in RH1ΔMLS hiPSC-differentiated cultures and R-loop “primed” genes in germinal matrix . RF = Representation factor, p-values calculated via hypergeometric test. K) Heatmap indicating overlap of genes that have upregulated gene expression following promoter-bound R- loop loss in RH1ΔMLS hiPSC-differentiated cultures and disease risk genes. p-values calculated via hypergeometric test. L) Optical intensity from immunofluorescence images of the R-loop-specific antibody S9.6, and the synaptic proteins PDS-95 and synapsin, in RH1ΔMLS and RH1ΔMLS D145N hiPSC-differentiated cultures. P-values calculated via two-tailed student’s t test; Error bars denote S.E.M.; n = 25 images per cell line / group. M) Representative immunofluorescence images of the synaptic protein synapsin (green) in RH1ΔMLS hiPSC- differentiated cultures and RH1ΔMLS D145N controls, along with the nuclear marker DAPI (blue) and the neuronal marker MAP2 (magenta). Scale bar: 20 mm.

Journal: bioRxiv

Article Title: R-loop landscapes in the developing human brain are linked to neural differentiation and cell-type specific transcription

doi: 10.1101/2023.07.18.549494

Figure Lengend Snippet: Cell specifications and transcriptomic alterations in RH1 transgenic cultures. A, B) Gene set enrichment analysis of scRNA-seq data from RH1ΔMLS and BFP control hiPSC-differentiated cultures. (A) Cnet plot and (B) dot plot showing significant gene ontologies up- (left) and down-regulated (right) following RH1ΔMLS transgene induction. Dot color indicates fold change (cnet plot) or adjusted p- value (dot plot), dot size indicates number of genes in category. n = 2 replicates from 2 cell lines/group. C) Combination of clusters into broad cell type classes, including glia (green), neurons (purple), and NPCs (blue). Gray clusters did not show canonical markers of these major neural cell types. D) RH1ΔMLS cells have significantly (p < 0.0001, Wilcoxin Rank Sum test) higher RNASEH1 expression than BFP controls (mean normalized expression: RH1ΔMLS = 2.45, BFP = 0.94). Cells from each condition were split into two groups according to RNASEH1 normalized expression value (> 1.25 = hi; ≤ 1.25 = lo) for downstream analyses. E) Bar charts showing difference in cell proportions across BFP or RH1ΔMLS transduced cells, stratified by RNASEH1 expression level. There is a significant reduction in the proportion of glia (p = 0.015) and an increased proportion of neurons (p = 0.036) in RH1ΔMLS- RNASEH1 high relative to BFP- RNASEH1 low groups (Two-way ANOVA with Dunnett’s Multiple Comparison test; n = 2 replicates from 2 cell donors per group; error bars denote S.E.M.; n.s. = not significant.) F, G) Volcano plots showing differentially expressed genes in scRNA-seq ( F ) and bulk RNA-seq ( G ) from RH1ΔMLS hiPSC-differentiated cultures and BFP controls. Green dots indicate genes more highly expressed in control cells (FDR < 0.05, log 2 fold change < 0) and purple/magenta dots indicate genes more highly expressed in RH1ΔMLS cells (FDR < 0.05, log 2 fold change > 0). H) Venn diagram showing overlap of upregulated genes in RH1ΔMLS hiPSC-differentiated cultures from scRNA and bulk RNA-seq, and promoter-bound R-loops that are knocked down following RNase H1-transgene expression. RF = Representation factor, which signifies more overlap of the two gene sets than expected based on a background list of genes if > 1. P-values calculated via hypergeometric test. I) Gene ontology of genes with upregulated expression following promoter-bound R-loop loss in RH1ΔMLS hiPSC-differentiated cultures. Numbers within gene ontology images indicate number of genes in the enriched set that occur within the ontology class, legend denotes -log 10 enrichment FDR. J) Venn diagram showing overlap of genes that have upregulated gene expression following promoter-bound R-loop loss in RH1ΔMLS hiPSC-differentiated cultures and R-loop “primed” genes in germinal matrix . RF = Representation factor, p-values calculated via hypergeometric test. K) Heatmap indicating overlap of genes that have upregulated gene expression following promoter-bound R- loop loss in RH1ΔMLS hiPSC-differentiated cultures and disease risk genes. p-values calculated via hypergeometric test. L) Optical intensity from immunofluorescence images of the R-loop-specific antibody S9.6, and the synaptic proteins PDS-95 and synapsin, in RH1ΔMLS and RH1ΔMLS D145N hiPSC-differentiated cultures. P-values calculated via two-tailed student’s t test; Error bars denote S.E.M.; n = 25 images per cell line / group. M) Representative immunofluorescence images of the synaptic protein synapsin (green) in RH1ΔMLS hiPSC- differentiated cultures and RH1ΔMLS D145N controls, along with the nuclear marker DAPI (blue) and the neuronal marker MAP2 (magenta). Scale bar: 20 mm.

Techniques: Transgenic Assay, Control, Expressing, Comparison, RNA Sequencing, Gene Expression, Immunofluorescence, Two Tailed Test, Marker

RNase H1-mediated R-loop loss during neuronal differentiation in vitro results in electrophysiological deficits. A) Top left: Electrophysiological recordings were taken with an Axion multielectrode array (MEA) system to assess population-wide neuronal activity. Schematic of the 48-well MEA plate used for electrophysiological recordings. Bottom Left: Schematic of an individual well within the MEA plate, containing 16 recording electrodes. Right: Screen shot of the MEA recording on week 8, with BFP-expressing hiPSC-neurons plated on the left half of the plate and RNase H1-overexpressing neurons on the right. B) Representative spike raster plots over the 10-minute recording at week 8 for BFP-expressing (left) and RNase H1-overexpressing (right) hiPSC-neurons. Each row denotes a single recording electrode within a single well of the 48-well MEA plate. C-E) The number of spontaneous spikes ( C ), weighted mean firing rate (wMFR) in Hertz ( D ), and number of bursts ( E ) were recorded weekly from BFP-controls and RNase H1 transgene expressing hiPSC-neurons from two donor lines. Data from the two donor lines were combined and a two-way ANOVA was performed with Sidak’s multiple comparisons test to test significant differences in each metric between weeks 2-8. Each condition contained at least 14-20 viable well replicates after removing wells that had less than 10 active recording electrodes. F) Statistics were additionally calculated for each electrophysiological metric at week 8 (unpaired two-tailed student’s t test) between RNase H1-transgene hiPSC-neurons and BFP controls. Individual donors represented with either filled in or hollow shapes.

Journal: bioRxiv

Article Title: R-loop landscapes in the developing human brain are linked to neural differentiation and cell-type specific transcription

doi: 10.1101/2023.07.18.549494

Figure Lengend Snippet: RNase H1-mediated R-loop loss during neuronal differentiation in vitro results in electrophysiological deficits. A) Top left: Electrophysiological recordings were taken with an Axion multielectrode array (MEA) system to assess population-wide neuronal activity. Schematic of the 48-well MEA plate used for electrophysiological recordings. Bottom Left: Schematic of an individual well within the MEA plate, containing 16 recording electrodes. Right: Screen shot of the MEA recording on week 8, with BFP-expressing hiPSC-neurons plated on the left half of the plate and RNase H1-overexpressing neurons on the right. B) Representative spike raster plots over the 10-minute recording at week 8 for BFP-expressing (left) and RNase H1-overexpressing (right) hiPSC-neurons. Each row denotes a single recording electrode within a single well of the 48-well MEA plate. C-E) The number of spontaneous spikes ( C ), weighted mean firing rate (wMFR) in Hertz ( D ), and number of bursts ( E ) were recorded weekly from BFP-controls and RNase H1 transgene expressing hiPSC-neurons from two donor lines. Data from the two donor lines were combined and a two-way ANOVA was performed with Sidak’s multiple comparisons test to test significant differences in each metric between weeks 2-8. Each condition contained at least 14-20 viable well replicates after removing wells that had less than 10 active recording electrodes. F) Statistics were additionally calculated for each electrophysiological metric at week 8 (unpaired two-tailed student’s t test) between RNase H1-transgene hiPSC-neurons and BFP controls. Individual donors represented with either filled in or hollow shapes.

Techniques: In Vitro, Activity Assay, Expressing, Two Tailed Test

RNase H1 overexpression leads to reduced dendritic complexity and spine density. Expression of the RNASEH1 ΔMLS transgene, delivered via in utero electroporation into neural precursor cells of the ventricular zone in E15 mouse embryos, affects dendrite and spine morphology of cortical projection neurons. A) Expression plasmids of GFP combined with tdTomato are used as controls, while GFP combined with RNASEH1 ΔMLS are used to destroy neural R-loops in mice during prenatal development. B) Overview of the in utero electroporation technique. TdTomato or RH1ΔMLS along with GFP reporters are transfected and electroporated unilaterally in E15 mouse ventricular zone. In adult mouse, P28, strong unilateral GFP expression is observed. Scale bar = 1 mm. C) In utero electroporation experimental timeline. D) Expression of exogenous (human) RNASEH1 in RH1ΔMLS mice was confirmed in FAC-sorted GFP+ neurons, whereas no difference was observed for endogenous expression of (mouse) Rnaseh1 between groups via qPCR (Normalized to Gapdh expression). Student’s two-tailed t test; n.s. = not significant. E) Expression of human RH1ΔMLS reduces S9.6 immunoreactivity in the nucleus of mouse neural cells at P0. F) FlSH images showing RNA expression of RNASEH1 (red) in the P28 cortex of tdTomato controls and RH1ΔMLS mice, and colocalization of GFP (green) and RNASEH1 in RH1ΔMLS cells. Scale bar = 100 mm. G) Representative images of (left) control and (right) RH1ΔMLS-expressing frontal pyramidal neuron dendritic trees at P28. H) Sholl analysis, showing reduced dendritic complexity in (left) control and (right) RH1ΔMLS-expressing pyramidal neurons at P28 (tdTomato control n = 3, RH1ΔMLS n = 4). I) Representative images of (left) control and (right) RH1ΔMLS frontal pyramidal neuron dendritic spines at P28. J) Apical and basal spine densities, assessed by Imaris tracing + FOCM (see text), are significantly decreased in RH1ΔMLS-expressing prefrontal pyramidal neurons. N = 3/group. *P < 0.05, **P < 0.01 determined by Two-Tailed Student’s t-test.

Journal: bioRxiv

Article Title: R-loop landscapes in the developing human brain are linked to neural differentiation and cell-type specific transcription

doi: 10.1101/2023.07.18.549494

Figure Lengend Snippet: RNase H1 overexpression leads to reduced dendritic complexity and spine density. Expression of the RNASEH1 ΔMLS transgene, delivered via in utero electroporation into neural precursor cells of the ventricular zone in E15 mouse embryos, affects dendrite and spine morphology of cortical projection neurons. A) Expression plasmids of GFP combined with tdTomato are used as controls, while GFP combined with RNASEH1 ΔMLS are used to destroy neural R-loops in mice during prenatal development. B) Overview of the in utero electroporation technique. TdTomato or RH1ΔMLS along with GFP reporters are transfected and electroporated unilaterally in E15 mouse ventricular zone. In adult mouse, P28, strong unilateral GFP expression is observed. Scale bar = 1 mm. C) In utero electroporation experimental timeline. D) Expression of exogenous (human) RNASEH1 in RH1ΔMLS mice was confirmed in FAC-sorted GFP+ neurons, whereas no difference was observed for endogenous expression of (mouse) Rnaseh1 between groups via qPCR (Normalized to Gapdh expression). Student’s two-tailed t test; n.s. = not significant. E) Expression of human RH1ΔMLS reduces S9.6 immunoreactivity in the nucleus of mouse neural cells at P0. F) FlSH images showing RNA expression of RNASEH1 (red) in the P28 cortex of tdTomato controls and RH1ΔMLS mice, and colocalization of GFP (green) and RNASEH1 in RH1ΔMLS cells. Scale bar = 100 mm. G) Representative images of (left) control and (right) RH1ΔMLS-expressing frontal pyramidal neuron dendritic trees at P28. H) Sholl analysis, showing reduced dendritic complexity in (left) control and (right) RH1ΔMLS-expressing pyramidal neurons at P28 (tdTomato control n = 3, RH1ΔMLS n = 4). I) Representative images of (left) control and (right) RH1ΔMLS frontal pyramidal neuron dendritic spines at P28. J) Apical and basal spine densities, assessed by Imaris tracing + FOCM (see text), are significantly decreased in RH1ΔMLS-expressing prefrontal pyramidal neurons. N = 3/group. *P < 0.05, **P < 0.01 determined by Two-Tailed Student’s t-test.

Techniques: Over Expression, Expressing, In Utero, Electroporation, Transfection, Two Tailed Test, RNA Expression, Control

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