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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human adiponectin elisa test kit (Boster Bio)

Boster Bio human adiponectin elisa test kit

Human Adiponectin Elisa Test Kit, supplied by Boster Bio, 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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human pgrmc1 (Sino Biological)

Sino Biological human pgrmc1

Contribution of <t>PGRMC1</t> to the resistance to erlotinib in lung adenocarcinomas cancer. ( a ) Immunoblotting of PGRMC1 in PC9 and PC9/ER cells. The upper panel shows a representative blot. The lower panel shows means ± SD (n = 3); * P < 0.05 vs. PC9 (two-tailed Student t -test). ( b ) Immunoblotting analysis of the efficiency of transfection of PC9 and PC9/ER cells with PGRMC1 siRNA (siPGRMC1). NC, negative control. The results shown are representative of 2 independent experiments. ( c ) WST-8 assay of the effects of various concentrations of erlotinib on cell viability of PGRMC1 siRNA–treated PC9 and PC9/ER cells. Data are means ± SD (n = 3); ** P < 0.01, * P < 0.05 vs. PC9 (negative control); † P < 0.05, †† P < 0.01 vs. PC9/ER (negative control) (Two-Way ANOVA followed by Tukey correction). NC, negative control. ( d ) Immunoblotting analysis of the efficiency of transfection of PC9 cells with a PGRMC1-expressing plasmid. ( e ) WST-8 assay of the effects of various concentrations of erlotinib on PC9 cells transfected with empty vector or pCMV3-PGRMC1. Data are means ± SD (n = 3); ** P < 0.01 vs. PC9 with empty vector (Two-Way ANOVA followed by Bonferroni correction).

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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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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.

Human Ace2, supplied by Sino Biological, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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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human adult normal testis cdna (BioChain Institute)

BioChain Institute human adult normal testis cdna

Human Adult Normal Testis Cdna, supplied by BioChain Institute, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mx2 cdna (OriGene)

OriGene mx2 cdna

a Variants that were tested in MPRA from the Chr21q22.3 melanoma locus are shown relative to the genomic position of <t>MX2</t> . Only the 19 variants located in the first intron of MX2 coming from the primary GWAS signal are shown (the other three from a secondary signal are located upstream of the MX2 genic region). ChromHMM annotation (Primary Core Marks segmentation) of Penis Foreskin Melanocyte Primary Cells from Roadmap Epigenomics Project is shown (Red/OrangeRed: Active_TSS/Flanking_Active_TSS, Yellow/GreenYellow: Enhancers/Genic_enhancers, Green/DarkGreen: Strong_transcription/Weak_transcription). Melanoma FAIRE-seq track of 11 samples is from a study by Verfaillie and colleagues . Ensembl predicted transcripts from archive 75 are shown. b Transcriptional activity of 145 bp sequences encompassing rs398206 from MPRA are shown as normalized tag counts (log 2 (RNA TPM/DNA TPM)). Results from UACC903 melanoma cells are shown for both alleles in forward (For) and reverse (Rev) directions, where results from promoter and enhancer constructs were combined. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. Density is reflected in the width of the shape. c Individual luciferase activity assays of 145 bp sequences encompassing rs398206 is shown for UACC903. pGL4.23 construct including minimal TATA promoter was used. One representative set is shown from three biological replicates. Mean with SEM, n = 6. All constructs are significantly higher than pGL4.23 (TATA) control ( P < 0.0001, two-tailed, unpaired t-test assuming unequal variance). d eQTL plot of MX2 levels in primary melanocytes in relation to rs398206 is shown for three genotype groups. Center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. P -value and slope were derived from linear regression with no multiple-testing correction applied. Source data are provided as a Source Data file.

Mx2 Cdna, supplied by OriGene, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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chronic myeloid leukemia k562 cells (ATCC)

ATCC chronic myeloid leukemia k562 cells

a Differential abundance of isodecoders of cyto-tRNAs and mt-tRNAs of <t>K562</t> cells as identified by Induro-tRNAseq (n = 2, biological replicates) and by mim-tRNAseq ( n = 2). The Induro workflow detected 266 cyto-tRNAs and the TGIRT workflow detected 265 cyto-tRNAs, while both detected all 22 isodecoders of mt-tRNAs. Shown in each comparison are log-transformed read counts normalized by DESeq2, showing the Pearson correlation coefficient r . b Differential abundance of isodecoders of cyto-tRNAs and mt-tRNAs starting with total RNA vs. total tRNA from K562 cells by Induro-tRNAseq ( n = 2, biological replicates). The dataset consisted of cyto-tRNAs (266, 281 in two duplicates) and mt-tRNAs (22 in each sample). c Differential changes of abundance of isoacceptors of cyto-tRNAs and mt-tRNAs of HEK293T cells in sodium arsenite (SA)-induced oxidative stress as detected by the Induro workflow. The abundance of each isoacceptor in the presence of 100 µM or 300 µM of SA relative to the absence of SA was calculated ( n = 4, two biological replicates and two technical replicates). d Bar graphs represent the abundance of mt-Met(CAT) and iMet(CAT) ( n = 4) of data in ( c ). Individual data points are indicated. Error bars, mean ± 95% confidence interval. e Differential changes of tRNA charging levels of HEK293T cells in SA-induced oxidative stress as detected by the Induro workflow. The charging level of each isoacceptor was the percentage of the charged reads with 3’-CCA end relative to the total reads of the isoacceptor. X -axis and Y- axis indicate the difference in charging at 100 µM and 300 µM of SA ( n = 4, two biological replicates and two technical replicates). f Bar graphs showing the percentage of charged reads of mt-Met(CAT) and iMet(CAT) ( n = 4) of data in ( e ). Individual data points are indicated. Error bars, mean ± 95% confidence interval.

Chronic Myeloid Leukemia K562 Cells, supplied by ATCC, 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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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" />
Human Bt474, supplied by ATCC, 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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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

Contribution of PGRMC1 to the resistance to erlotinib in lung adenocarcinomas cancer. ( a ) Immunoblotting of PGRMC1 in PC9 and PC9/ER cells. The upper panel shows a representative blot. The lower panel shows means ± SD (n = 3); * P < 0.05 vs. PC9 (two-tailed Student t -test). ( b ) Immunoblotting analysis of the efficiency of transfection of PC9 and PC9/ER cells with PGRMC1 siRNA (siPGRMC1). NC, negative control. The results shown are representative of 2 independent experiments. ( c ) WST-8 assay of the effects of various concentrations of erlotinib on cell viability of PGRMC1 siRNA–treated PC9 and PC9/ER cells. Data are means ± SD (n = 3); ** P < 0.01, * P < 0.05 vs. PC9 (negative control); † P < 0.05, †† P < 0.01 vs. PC9/ER (negative control) (Two-Way ANOVA followed by Tukey correction). NC, negative control. ( d ) Immunoblotting analysis of the efficiency of transfection of PC9 cells with a PGRMC1-expressing plasmid. ( e ) WST-8 assay of the effects of various concentrations of erlotinib on PC9 cells transfected with empty vector or pCMV3-PGRMC1. Data are means ± SD (n = 3); ** P < 0.01 vs. PC9 with empty vector (Two-Way ANOVA followed by Bonferroni correction).

Journal: Scientific Reports

Article Title: Progesterone receptor membrane component 1 leads to erlotinib resistance, initiating crosstalk of Wnt/β-catenin and NF-κB pathways, in lung adenocarcinoma cells

doi: 10.1038/s41598-020-61727-3

Figure Lengend Snippet: Contribution of PGRMC1 to the resistance to erlotinib in lung adenocarcinomas cancer. ( a ) Immunoblotting of PGRMC1 in PC9 and PC9/ER cells. The upper panel shows a representative blot. The lower panel shows means ± SD (n = 3); * P < 0.05 vs. PC9 (two-tailed Student t -test). ( b ) Immunoblotting analysis of the efficiency of transfection of PC9 and PC9/ER cells with PGRMC1 siRNA (siPGRMC1). NC, negative control. The results shown are representative of 2 independent experiments. ( c ) WST-8 assay of the effects of various concentrations of erlotinib on cell viability of PGRMC1 siRNA–treated PC9 and PC9/ER cells. Data are means ± SD (n = 3); ** P < 0.01, * P < 0.05 vs. PC9 (negative control); † P < 0.05, †† P < 0.01 vs. PC9/ER (negative control) (Two-Way ANOVA followed by Tukey correction). NC, negative control. ( d ) Immunoblotting analysis of the efficiency of transfection of PC9 cells with a PGRMC1-expressing plasmid. ( e ) WST-8 assay of the effects of various concentrations of erlotinib on PC9 cells transfected with empty vector or pCMV3-PGRMC1. Data are means ± SD (n = 3); ** P < 0.01 vs. PC9 with empty vector (Two-Way ANOVA followed by Bonferroni correction).

Article Snippet: pCMV3 plasmid encoding human PGRMC1 was purchased from Sino Biological Inc. (Beijing, China).

Techniques: Western Blot, Two Tailed Test, Transfection, Negative Control, Expressing, Plasmid Preparation

Effects of combination treatment of erlotinib and AG-205 on cell viability and EGFR–PGRMC1 binding in lung adenocarcinoma cells. ( a ) Effects of combination treatment of erlotinib and AG-205 on cell viability measured by WST-8 assay. Data are means ± SD (n = 3); ** P < 0.01 PC9 vs. PC9/ER, †† P < 0.01 vs . P C9/ER cells treated with erlotinib alone, ‡‡ P < 0.01 vs. PC9 cells treated with erlotinib alone (Two-Way ANOVA followed by Bonferroni correction); N.S; not significant. ( b ) Immunoprecipitation assay of binding between EGFR and PGRMC1 in PC9 and PC9/ER cells cotreated with AG-205 (1 μM) plus erlotinib (20 nM) for 72 h. Data are means ± SD (n = 3).

Journal: Scientific Reports

Article Title: Progesterone receptor membrane component 1 leads to erlotinib resistance, initiating crosstalk of Wnt/β-catenin and NF-κB pathways, in lung adenocarcinoma cells

doi: 10.1038/s41598-020-61727-3

Figure Lengend Snippet: Effects of combination treatment of erlotinib and AG-205 on cell viability and EGFR–PGRMC1 binding in lung adenocarcinoma cells. ( a ) Effects of combination treatment of erlotinib and AG-205 on cell viability measured by WST-8 assay. Data are means ± SD (n = 3); ** P < 0.01 PC9 vs. PC9/ER, †† P < 0.01 vs . P C9/ER cells treated with erlotinib alone, ‡‡ P < 0.01 vs. PC9 cells treated with erlotinib alone (Two-Way ANOVA followed by Bonferroni correction); N.S; not significant. ( b ) Immunoprecipitation assay of binding between EGFR and PGRMC1 in PC9 and PC9/ER cells cotreated with AG-205 (1 μM) plus erlotinib (20 nM) for 72 h. Data are means ± SD (n = 3).

Article Snippet: pCMV3 plasmid encoding human PGRMC1 was purchased from Sino Biological Inc. (Beijing, China).

Techniques: Binding Assay, Immunoprecipitation

Proteomics result for PGRMC1 levels in PC9 and PC9/ER cells.

Journal: Scientific Reports

Article Title: Progesterone receptor membrane component 1 leads to erlotinib resistance, initiating crosstalk of Wnt/β-catenin and NF-κB pathways, in lung adenocarcinoma cells

doi: 10.1038/s41598-020-61727-3

Figure Lengend Snippet: Proteomics result for PGRMC1 levels in PC9 and PC9/ER cells.

Article Snippet: pCMV3 plasmid encoding human PGRMC1 was purchased from Sino Biological Inc. (Beijing, China).

Techniques:

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: 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

a Variants that were tested in MPRA from the Chr21q22.3 melanoma locus are shown relative to the genomic position of MX2 . Only the 19 variants located in the first intron of MX2 coming from the primary GWAS signal are shown (the other three from a secondary signal are located upstream of the MX2 genic region). ChromHMM annotation (Primary Core Marks segmentation) of Penis Foreskin Melanocyte Primary Cells from Roadmap Epigenomics Project is shown (Red/OrangeRed: Active_TSS/Flanking_Active_TSS, Yellow/GreenYellow: Enhancers/Genic_enhancers, Green/DarkGreen: Strong_transcription/Weak_transcription). Melanoma FAIRE-seq track of 11 samples is from a study by Verfaillie and colleagues . Ensembl predicted transcripts from archive 75 are shown. b Transcriptional activity of 145 bp sequences encompassing rs398206 from MPRA are shown as normalized tag counts (log 2 (RNA TPM/DNA TPM)). Results from UACC903 melanoma cells are shown for both alleles in forward (For) and reverse (Rev) directions, where results from promoter and enhancer constructs were combined. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. Density is reflected in the width of the shape. c Individual luciferase activity assays of 145 bp sequences encompassing rs398206 is shown for UACC903. pGL4.23 construct including minimal TATA promoter was used. One representative set is shown from three biological replicates. Mean with SEM, n = 6. All constructs are significantly higher than pGL4.23 (TATA) control ( P < 0.0001, two-tailed, unpaired t-test assuming unequal variance). d eQTL plot of MX2 levels in primary melanocytes in relation to rs398206 is shown for three genotype groups. Center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. P -value and slope were derived from linear regression with no multiple-testing correction applied. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: Massively parallel reporter assays of melanoma risk variants identify MX2 as a gene promoting melanoma

doi: 10.1038/s41467-020-16590-1

Figure Lengend Snippet: a Variants that were tested in MPRA from the Chr21q22.3 melanoma locus are shown relative to the genomic position of MX2 . Only the 19 variants located in the first intron of MX2 coming from the primary GWAS signal are shown (the other three from a secondary signal are located upstream of the MX2 genic region). ChromHMM annotation (Primary Core Marks segmentation) of Penis Foreskin Melanocyte Primary Cells from Roadmap Epigenomics Project is shown (Red/OrangeRed: Active_TSS/Flanking_Active_TSS, Yellow/GreenYellow: Enhancers/Genic_enhancers, Green/DarkGreen: Strong_transcription/Weak_transcription). Melanoma FAIRE-seq track of 11 samples is from a study by Verfaillie and colleagues . Ensembl predicted transcripts from archive 75 are shown. b Transcriptional activity of 145 bp sequences encompassing rs398206 from MPRA are shown as normalized tag counts (log 2 (RNA TPM/DNA TPM)). Results from UACC903 melanoma cells are shown for both alleles in forward (For) and reverse (Rev) directions, where results from promoter and enhancer constructs were combined. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. Density is reflected in the width of the shape. c Individual luciferase activity assays of 145 bp sequences encompassing rs398206 is shown for UACC903. pGL4.23 construct including minimal TATA promoter was used. One representative set is shown from three biological replicates. Mean with SEM, n = 6. All constructs are significantly higher than pGL4.23 (TATA) control ( P < 0.0001, two-tailed, unpaired t-test assuming unequal variance). d eQTL plot of MX2 levels in primary melanocytes in relation to rs398206 is shown for three genotype groups. Center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. P -value and slope were derived from linear regression with no multiple-testing correction applied. Source data are provided as a Source Data file.

Article Snippet: The MX2 cDNA clone (RC206437) in the pCMV6-entry backbone was purchased from Origene and full-length MX2 cDNA sequence was sub-cloned to pENTR-1A vector by introducing stop codons and removing 3’ Myc-DDK tag before being transferred to pINDUCER20 vector (adapter sequence is listed in Supplementary Table ).

Techniques: Activity Assay, Construct, Software, Luciferase, Control, Two Tailed Test, Derivative Assay

a Chromatin immunoprecipitation using anti-YY1 antibody or normal IgG followed by qPCR. On the top panel, genomic positions of the amplicons using five qPCR primer sets are shown relative to rs398206 (red dashed line) in the first intron of MX2 . qPCR results from three melanoma cell lines are shown on the bottom panel with genotype for rs398206 indicated in the X -axis label. Relative quantities are shown as fold over input DNA. Mean of PCR triplicates with SD are plotted. One representative set of three biological replicates for each cell line is shown. b Taqman genotyping of rs398206 using YY1 ChIP DNA in UACC647 melanoma cells (heterozygous for rs398206). The blue shaded areas mark HapMap CEU DNA controls showing separation of three genotype clusters. qPCR triplicates were plotted separately. One representative set from three biological replicates is shown. Normalized A and C allele intensity is shown as dRn values on x and y -axis, respectively. gDNA: genomic DNA. c Combined plot for A/C allelic ratio from three rounds of biological replicates of UACC647. Each dot represents A/C ratio calculated from 2 -(average dCt(alleleA-alleleC)) from qPCR triplicates. Input includes three 10-fold serial dilutions from each round. Mean with SEM are plotted. A two-tailed Mann–Whitney U test was used for n = 9 (Input) and 3 (YY1-bound). Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: Massively parallel reporter assays of melanoma risk variants identify MX2 as a gene promoting melanoma

doi: 10.1038/s41467-020-16590-1

Figure Lengend Snippet: a Chromatin immunoprecipitation using anti-YY1 antibody or normal IgG followed by qPCR. On the top panel, genomic positions of the amplicons using five qPCR primer sets are shown relative to rs398206 (red dashed line) in the first intron of MX2 . qPCR results from three melanoma cell lines are shown on the bottom panel with genotype for rs398206 indicated in the X -axis label. Relative quantities are shown as fold over input DNA. Mean of PCR triplicates with SD are plotted. One representative set of three biological replicates for each cell line is shown. b Taqman genotyping of rs398206 using YY1 ChIP DNA in UACC647 melanoma cells (heterozygous for rs398206). The blue shaded areas mark HapMap CEU DNA controls showing separation of three genotype clusters. qPCR triplicates were plotted separately. One representative set from three biological replicates is shown. Normalized A and C allele intensity is shown as dRn values on x and y -axis, respectively. gDNA: genomic DNA. c Combined plot for A/C allelic ratio from three rounds of biological replicates of UACC647. Each dot represents A/C ratio calculated from 2 -(average dCt(alleleA-alleleC)) from qPCR triplicates. Input includes three 10-fold serial dilutions from each round. Mean with SEM are plotted. A two-tailed Mann–Whitney U test was used for n = 9 (Input) and 3 (YY1-bound). Source data are provided as a Source Data file.

Techniques: Chromatin Immunoprecipitation, Two Tailed Test, MANN-WHITNEY

a YY1 was knocked down using four different siRNAs in UACC647 cells, and MX2 levels were measured. GAPDH -normalized MX2 mRNA levels are shown as fold change over those from non-targeting siRNA. Four biological replicates of n = 6 were combined (total n = 24). b CRISPRi using dCAS9-KRAB-MeCP2 and four different gRNAs targeting rs398206 in UACC903 cells. MX2 mRNA levels ( GAPDH -normalized) are shown as fold change over those from non-targeting gRNA. Three biological replicates of n = 6 were combined (total n = 18, except gRNA-3, n = 17). gRNA 1, 3, and 4 directly overlap rs398206. gRNA 2 targets ~25 bp upstream of rs398206. AAVS1 (gRNA targeting adeno-associated virus integration site on Chr19). Box: Median and 25th to 75th percentile. Whisker: 10th to 90th percentile. P -values are shown from one-sample Wilcoxon test (two-sided) for difference from non-targeting siRNA/gRNA. Dotted line denotes the MX2 levels in non-targeting siRNA/gRNA control. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: Massively parallel reporter assays of melanoma risk variants identify MX2 as a gene promoting melanoma

doi: 10.1038/s41467-020-16590-1

Figure Lengend Snippet: a YY1 was knocked down using four different siRNAs in UACC647 cells, and MX2 levels were measured. GAPDH -normalized MX2 mRNA levels are shown as fold change over those from non-targeting siRNA. Four biological replicates of n = 6 were combined (total n = 24). b CRISPRi using dCAS9-KRAB-MeCP2 and four different gRNAs targeting rs398206 in UACC903 cells. MX2 mRNA levels ( GAPDH -normalized) are shown as fold change over those from non-targeting gRNA. Three biological replicates of n = 6 were combined (total n = 18, except gRNA-3, n = 17). gRNA 1, 3, and 4 directly overlap rs398206. gRNA 2 targets ~25 bp upstream of rs398206. AAVS1 (gRNA targeting adeno-associated virus integration site on Chr19). Box: Median and 25th to 75th percentile. Whisker: 10th to 90th percentile. P -values are shown from one-sample Wilcoxon test (two-sided) for difference from non-targeting siRNA/gRNA. Dotted line denotes the MX2 levels in non-targeting siRNA/gRNA control. Source data are provided as a Source Data file.

Techniques: Virus, Whisker Assay, Control

a Ingenuity Pathway Analysis of differentially expressed genes from MX2 -high vs. MX2 -low melanocytes from 106 individuals. 252 differentially expressed genes (FDR < 1% and >2-fold change) between MX2 -high and MX2 -low melanocytes (top and bottom quantile based on MX2 levels; n = 28 each) from 106 individuals were used as input for the analysis. Enrichment P -values are based on a two-sided Fisher’s exact test with no multiple-testing correction applied. (B-C) Cell growth and movement of human primary melanocytes C23 ( b ) or melanoma cell line UACC2545 ( c ) infected with an inducible lentiviral construct of MX2 cDNA or Empty pINDUCER20 vector were measured on xCELLigence system. Cell Index values were normalized relative to those at the time of doxycycline addition (dotted vertical line: Dox). The amount of doxycycline (dox) is shown in ng/ml and color-coded. Mean Normalized Cell Index (colored dots) and SD (gray vertical lines) are plotted ( n = 3). A representative set of three biological replicates is shown. d Ingenuity Pathway Analysis of differentially expressed genes from RNA sequencing of MX2 overexpressed vs. control melanocytes from 3 individuals. 158 differentially expressed genes (FDR < 10%) between MX2 -overexpressing (100 ng/ml doxycycline) vs. control (no doxycycline) melanocytes using 3 biological replicates for 3 individuals were used as input for the analysis. Significantly enriched canonical pathways (P < 0.05 and |Z-score| >1) are color-coded for the direction of effect relative to MX2 -high melanocytes ( a ) or MX2 -overexpressing melanocytes ( d ). Enrichment P-values are based on a two-sided Fisher’s exact test with no multiple-testing correction applied. A weaker to stronger shade of each color represent the relative magnitude of Z -scores: Positive Z -score between 1 and 2.646 and negative Z -score between −1 and −3.464 ( a ) or positive between 1 and 1.134 and negative between −1.342 and −2.236 ( d ), where lightest red is closer to 1 and lightest blue is closer to −1. ( e ) Representative pictures of adult fish from GFP or MX2 group. Pictures were taken at week 10 post-injection. f Melanoma-free survival curves of a zebrafish melanoma model (Tg( mitfa:BRAF V600E ), p53 −/−, mitfa −/−). The fish were injected at the one cell stage with either miniCoopR mitfa:MX2 or miniCoopR mitfa:EGFP and monitored weekly for melanoma formation. The percentage of melanoma-free fish was combined from three independent experiments and plotted. Log-rank test was used. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: Massively parallel reporter assays of melanoma risk variants identify MX2 as a gene promoting melanoma

doi: 10.1038/s41467-020-16590-1

Figure Lengend Snippet: a Ingenuity Pathway Analysis of differentially expressed genes from MX2 -high vs. MX2 -low melanocytes from 106 individuals. 252 differentially expressed genes (FDR < 1% and >2-fold change) between MX2 -high and MX2 -low melanocytes (top and bottom quantile based on MX2 levels; n = 28 each) from 106 individuals were used as input for the analysis. Enrichment P -values are based on a two-sided Fisher’s exact test with no multiple-testing correction applied. (B-C) Cell growth and movement of human primary melanocytes C23 ( b ) or melanoma cell line UACC2545 ( c ) infected with an inducible lentiviral construct of MX2 cDNA or Empty pINDUCER20 vector were measured on xCELLigence system. Cell Index values were normalized relative to those at the time of doxycycline addition (dotted vertical line: Dox). The amount of doxycycline (dox) is shown in ng/ml and color-coded. Mean Normalized Cell Index (colored dots) and SD (gray vertical lines) are plotted ( n = 3). A representative set of three biological replicates is shown. d Ingenuity Pathway Analysis of differentially expressed genes from RNA sequencing of MX2 overexpressed vs. control melanocytes from 3 individuals. 158 differentially expressed genes (FDR < 10%) between MX2 -overexpressing (100 ng/ml doxycycline) vs. control (no doxycycline) melanocytes using 3 biological replicates for 3 individuals were used as input for the analysis. Significantly enriched canonical pathways (P < 0.05 and |Z-score| >1) are color-coded for the direction of effect relative to MX2 -high melanocytes ( a ) or MX2 -overexpressing melanocytes ( d ). Enrichment P-values are based on a two-sided Fisher’s exact test with no multiple-testing correction applied. A weaker to stronger shade of each color represent the relative magnitude of Z -scores: Positive Z -score between 1 and 2.646 and negative Z -score between −1 and −3.464 ( a ) or positive between 1 and 1.134 and negative between −1.342 and −2.236 ( d ), where lightest red is closer to 1 and lightest blue is closer to −1. ( e ) Representative pictures of adult fish from GFP or MX2 group. Pictures were taken at week 10 post-injection. f Melanoma-free survival curves of a zebrafish melanoma model (Tg( mitfa:BRAF V600E ), p53 −/−, mitfa −/−). The fish were injected at the one cell stage with either miniCoopR mitfa:MX2 or miniCoopR mitfa:EGFP and monitored weekly for melanoma formation. The percentage of melanoma-free fish was combined from three independent experiments and plotted. Log-rank test was used. Source data are provided as a Source Data file.

Techniques: Infection, Construct, Plasmid Preparation, RNA Sequencing, Control, Injection

a Differential abundance of isodecoders of cyto-tRNAs and mt-tRNAs of K562 cells as identified by Induro-tRNAseq (n = 2, biological replicates) and by mim-tRNAseq ( n = 2). The Induro workflow detected 266 cyto-tRNAs and the TGIRT workflow detected 265 cyto-tRNAs, while both detected all 22 isodecoders of mt-tRNAs. Shown in each comparison are log-transformed read counts normalized by DESeq2, showing the Pearson correlation coefficient r . b Differential abundance of isodecoders of cyto-tRNAs and mt-tRNAs starting with total RNA vs. total tRNA from K562 cells by Induro-tRNAseq ( n = 2, biological replicates). The dataset consisted of cyto-tRNAs (266, 281 in two duplicates) and mt-tRNAs (22 in each sample). c Differential changes of abundance of isoacceptors of cyto-tRNAs and mt-tRNAs of HEK293T cells in sodium arsenite (SA)-induced oxidative stress as detected by the Induro workflow. The abundance of each isoacceptor in the presence of 100 µM or 300 µM of SA relative to the absence of SA was calculated ( n = 4, two biological replicates and two technical replicates). d Bar graphs represent the abundance of mt-Met(CAT) and iMet(CAT) ( n = 4) of data in ( c ). Individual data points are indicated. Error bars, mean ± 95% confidence interval. e Differential changes of tRNA charging levels of HEK293T cells in SA-induced oxidative stress as detected by the Induro workflow. The charging level of each isoacceptor was the percentage of the charged reads with 3’-CCA end relative to the total reads of the isoacceptor. X -axis and Y- axis indicate the difference in charging at 100 µM and 300 µM of SA ( n = 4, two biological replicates and two technical replicates). f Bar graphs showing the percentage of charged reads of mt-Met(CAT) and iMet(CAT) ( n = 4) of data in ( e ). Individual data points are indicated. Error bars, mean ± 95% confidence interval.

Journal: Nature Communications

Article Title: Genome-wide profiling of tRNA modifications by Induro-tRNAseq reveals coordinated changes

doi: 10.1038/s41467-025-56348-1

Figure Lengend Snippet: a Differential abundance of isodecoders of cyto-tRNAs and mt-tRNAs of K562 cells as identified by Induro-tRNAseq (n = 2, biological replicates) and by mim-tRNAseq ( n = 2). The Induro workflow detected 266 cyto-tRNAs and the TGIRT workflow detected 265 cyto-tRNAs, while both detected all 22 isodecoders of mt-tRNAs. Shown in each comparison are log-transformed read counts normalized by DESeq2, showing the Pearson correlation coefficient r . b Differential abundance of isodecoders of cyto-tRNAs and mt-tRNAs starting with total RNA vs. total tRNA from K562 cells by Induro-tRNAseq ( n = 2, biological replicates). The dataset consisted of cyto-tRNAs (266, 281 in two duplicates) and mt-tRNAs (22 in each sample). c Differential changes of abundance of isoacceptors of cyto-tRNAs and mt-tRNAs of HEK293T cells in sodium arsenite (SA)-induced oxidative stress as detected by the Induro workflow. The abundance of each isoacceptor in the presence of 100 µM or 300 µM of SA relative to the absence of SA was calculated ( n = 4, two biological replicates and two technical replicates). d Bar graphs represent the abundance of mt-Met(CAT) and iMet(CAT) ( n = 4) of data in ( c ). Individual data points are indicated. Error bars, mean ± 95% confidence interval. e Differential changes of tRNA charging levels of HEK293T cells in SA-induced oxidative stress as detected by the Induro workflow. The charging level of each isoacceptor was the percentage of the charged reads with 3’-CCA end relative to the total reads of the isoacceptor. X -axis and Y- axis indicate the difference in charging at 100 µM and 300 µM of SA ( n = 4, two biological replicates and two technical replicates). f Bar graphs showing the percentage of charged reads of mt-Met(CAT) and iMet(CAT) ( n = 4) of data in ( e ). Individual data points are indicated. Error bars, mean ± 95% confidence interval.

Article Snippet: Human embryonic kidney HEK293T cells (ATCC, #CRL-3216), chronic myeloid leukemia K562 cells (ATCC, #CL-243), cervical cancer HeLa cells (ATCC, #CCL-2), neuroblastoma SH-SY5Y (ATCC, #CRL-2266), and near-haploid HAP1 cells (Horizon, #C631) were cultured at 37 °C in Dulbecco’s Modified Eagle’s Medium (DMEM) (Corning, #10-013-CV) with 5% CO 2 and 10% fetal bovine serum (Corning, #35-010-CV).

Techniques: Comparison, Transformation Assay

a The L-shaped tRNA structure showing RT-readable modifications. b Yield of readthrough (%) in end-to-end cDNA synthesis at 25, 37, 42, and 55 °C after the Induro RT reaction for 1, 2, or 16 h. Data were collected from total RNA of K562 cells as the input ( n = 2; technical replicates). Center line: median; box limits: upper and lower quartiles; whiskers: 1.5X interquartile range; points: outliers. N.D.; Not detectable. ** p < 0.01; two-tailed unpaired t-test. c Heatmaps of the frequency (%) of the RT misincorporations and RT stops at the annotated modifications. d Left: A scatter plot showing the frequency (%) of the RT misincorporation as a function of the G9:m 1 G9 ratio of the transcript of human mt-Leu(TAA) ( n = 2; technical replicates). Right: The frequency of misincorporation of the native mt-Leu(TAA) in sequencing analysis of a total RNA sample. Data were collected from K562 cells with total RNA as the input under the RT reaction at 42 °C for 16 h ( n = 2; technical replicates). Source data containing p values and n values are provided in Source Data 2.

Journal: Nature Communications

Article Title: Genome-wide profiling of tRNA modifications by Induro-tRNAseq reveals coordinated changes

doi: 10.1038/s41467-025-56348-1

Figure Lengend Snippet: a The L-shaped tRNA structure showing RT-readable modifications. b Yield of readthrough (%) in end-to-end cDNA synthesis at 25, 37, 42, and 55 °C after the Induro RT reaction for 1, 2, or 16 h. Data were collected from total RNA of K562 cells as the input ( n = 2; technical replicates). Center line: median; box limits: upper and lower quartiles; whiskers: 1.5X interquartile range; points: outliers. N.D.; Not detectable. ** p < 0.01; two-tailed unpaired t-test. c Heatmaps of the frequency (%) of the RT misincorporations and RT stops at the annotated modifications. d Left: A scatter plot showing the frequency (%) of the RT misincorporation as a function of the G9:m 1 G9 ratio of the transcript of human mt-Leu(TAA) ( n = 2; technical replicates). Right: The frequency of misincorporation of the native mt-Leu(TAA) in sequencing analysis of a total RNA sample. Data were collected from K562 cells with total RNA as the input under the RT reaction at 42 °C for 16 h ( n = 2; technical replicates). Source data containing p values and n values are provided in Source Data 2.

Techniques: cDNA Synthesis, Two Tailed Test, Sequencing

a The frequency (%) of readthrough at m 1 A9 and m 1 A58. Data were collected from total RNA of K562 cells ( n = 2; technical replicates for individual RT conditions). b The frequency (%) of readthrough at m 1 G9 and m 1 G37. Data were collected from total RNA of K562 cells ( n = 2; technical replicates for individual RT conditions). c Top chart: the frequency (%) of RT misincorporation. Middle chart: the frequence (%) of RT stop in the presence of Mg 2+ or Mn 2+ as the only divalent metal ion. Data were collected from total RNA of K562 cells ( n = 2; technical replicates for individual RT conditions). Unmodified natural nucleotides at tRNA positions 70, 74, and 75 were used as the control. A modification is identified if misincorporation is detected over 10%, which is marked by the red lines. Bottom chart: the number of detectable modifications in the presence of Mg 2+ or Mn 2+ . a – c Center line: median; box limits: upper and lower quartiles; whiskers: 1.5X interquartile range; points: outliers. * p < 0.05, ** p < 0.01, *** p < 0.001; two-tailed unpaired t-test. All p values and n values used for statistical analysis are available in Source Data 4–6. d , e The frequency (%) of RT misincorporation in the presence of Mg 2+ vs. Mn 2+ at m 7 G ( d ) and at Ψ ( e ), showing the Pearson correlation coefficient r .

Journal: Nature Communications

Article Title: Genome-wide profiling of tRNA modifications by Induro-tRNAseq reveals coordinated changes

doi: 10.1038/s41467-025-56348-1

Figure Lengend Snippet: a The frequency (%) of readthrough at m 1 A9 and m 1 A58. Data were collected from total RNA of K562 cells ( n = 2; technical replicates for individual RT conditions). b The frequency (%) of readthrough at m 1 G9 and m 1 G37. Data were collected from total RNA of K562 cells ( n = 2; technical replicates for individual RT conditions). c Top chart: the frequency (%) of RT misincorporation. Middle chart: the frequence (%) of RT stop in the presence of Mg 2+ or Mn 2+ as the only divalent metal ion. Data were collected from total RNA of K562 cells ( n = 2; technical replicates for individual RT conditions). Unmodified natural nucleotides at tRNA positions 70, 74, and 75 were used as the control. A modification is identified if misincorporation is detected over 10%, which is marked by the red lines. Bottom chart: the number of detectable modifications in the presence of Mg 2+ or Mn 2+ . a – c Center line: median; box limits: upper and lower quartiles; whiskers: 1.5X interquartile range; points: outliers. * p < 0.05, ** p < 0.01, *** p < 0.001; two-tailed unpaired t-test. All p values and n values used for statistical analysis are available in Source Data 4–6. d , e The frequency (%) of RT misincorporation in the presence of Mg 2+ vs. Mn 2+ at m 7 G ( d ) and at Ψ ( e ), showing the Pearson correlation coefficient r .

Techniques: Control, Modification, Two Tailed Test

a Cumulative bar graphs show the frequency (%) of RT stops (pink) and RT misincorporations (blue) detected by Induro and by TGIRT at each readable modification as averaged from all tRNA isodecoders of K562 cells, in relation to the frequency (%) of correct reads (white) and of lost coverage (gray). The % of RT stops at a given position was the number of stops relative to the number of total reads. b The frequency (%) of RT stops at each readable and annotated modification detected by Induro and by TGIRT. c The number of reads at each annotated modification site among all isodecoders detected by Induro and by TGIRT. d The distribution of read identity (in %) of each readable modification as detected from total tRNA of K562 cells by Induro and by TGIRT in response to the upstream nucleotide. e , f The read identity (%) of m 1 A9 vs. m 1 A58 ( e ), and of m 1 G9 vs. m 1 G37 ( f ) generated by Induro and by TGIRT. All data were collected from K562 cells. For all of the plots, the analysis of Induro was based on this work ( n = 4, biological replicates), while that of TGIRT was extracted from the published dataset . Center line: median; box limits: upper and lower quartiles; whiskers: 1.5X interquartile range; points: outliers. Student’s t -test was performed by a two-sided analysis (* p < 0.05, ** p < 0.01). Source data are provided in Source Data 8-10, where p values and n values used for statistical analysis are available.

Journal: Nature Communications

Article Title: Genome-wide profiling of tRNA modifications by Induro-tRNAseq reveals coordinated changes

doi: 10.1038/s41467-025-56348-1

Figure Lengend Snippet: a Cumulative bar graphs show the frequency (%) of RT stops (pink) and RT misincorporations (blue) detected by Induro and by TGIRT at each readable modification as averaged from all tRNA isodecoders of K562 cells, in relation to the frequency (%) of correct reads (white) and of lost coverage (gray). The % of RT stops at a given position was the number of stops relative to the number of total reads. b The frequency (%) of RT stops at each readable and annotated modification detected by Induro and by TGIRT. c The number of reads at each annotated modification site among all isodecoders detected by Induro and by TGIRT. d The distribution of read identity (in %) of each readable modification as detected from total tRNA of K562 cells by Induro and by TGIRT in response to the upstream nucleotide. e , f The read identity (%) of m 1 A9 vs. m 1 A58 ( e ), and of m 1 G9 vs. m 1 G37 ( f ) generated by Induro and by TGIRT. All data were collected from K562 cells. For all of the plots, the analysis of Induro was based on this work ( n = 4, biological replicates), while that of TGIRT was extracted from the published dataset . Center line: median; box limits: upper and lower quartiles; whiskers: 1.5X interquartile range; points: outliers. Student’s t -test was performed by a two-sided analysis (* p < 0.05, ** p < 0.01). Source data are provided in Source Data 8-10, where p values and n values used for statistical analysis are available.

Techniques: Modification, Generated

$a Violin plots showing differences of the weighted RT misincorporation frequencies (%) at the most variable site of ASL or non-ASL modifications among isodecoders. SH-SY5Y & HeLa & K562, 2 biological replicates, each 2 technical replicates; HEK293T & HAP1, 2 biological replicates; mouse tissues, each 3 biological replicates. b Dataset of a in bar graphs. c Dataset of b in bar graphs at the major sites of ASL and non-ASL modifications in mouse tissues. d Datasets of c in bar graphs at position 32 (left) and 20 (right). e Dataset of ( b ) in bar graphs at the major sites of ASL and non-ASL modifications in human cells. f Datasets of e in bar graphs at position 32 (left) and 20 (right). Error bars, mean ± 95% standard error, by a two-sided Wilcoxon test (* p < 0.05, ** p < 0.01, *** p < 0.001). g Violin plots showing the fraction of modifications (%) in the consistency scale across human cell lines and mouse tissues. A modification of consistency of 100% means that it is present in all 5 human cell lines, while a consistency of 80% means that it is present in 4 of the 5 cell lines. Student’s t -test by a two-sided analysis. h A landscape of coordinated changes of tRNA modifications across tissues and cell types. The charged aa-tRNA is shown with the amino acid as a star; the ribosome is drawn as a cartoon with the large and small subunits, while the mRNA is drawn with abundant codons (triplets without a margin) and rare codons (triplets with a margin), where the distance between each marked codon indicates the presence of unmarked codons. a – f Individual data points, p values and n values for statistical analysis are available in Source Data 12−19.$

Journal: Nature Communications

Article Title: Genome-wide profiling of tRNA modifications by Induro-tRNAseq reveals coordinated changes

doi: 10.1038/s41467-025-56348-1

Figure Lengend Snippet: a Violin plots showing differences of the weighted RT misincorporation frequencies (%) at the most variable site of ASL or non-ASL modifications among isodecoders. SH-SY5Y & HeLa & K562, 2 biological replicates, each 2 technical replicates; HEK293T & HAP1, 2 biological replicates; mouse tissues, each 3 biological replicates. b Dataset of a in bar graphs. c Dataset of b in bar graphs at the major sites of ASL and non-ASL modifications in mouse tissues. d Datasets of c in bar graphs at position 32 (left) and 20 (right). e Dataset of ( b ) in bar graphs at the major sites of ASL and non-ASL modifications in human cells. f Datasets of e in bar graphs at position 32 (left) and 20 (right). Error bars, mean ± 95% standard error, by a two-sided Wilcoxon test (* p < 0.05, ** p < 0.01, *** p < 0.001). g Violin plots showing the fraction of modifications (%) in the consistency scale across human cell lines and mouse tissues. A modification of consistency of 100% means that it is present in all 5 human cell lines, while a consistency of 80% means that it is present in 4 of the 5 cell lines. Student’s t -test by a two-sided analysis. h A landscape of coordinated changes of tRNA modifications across tissues and cell types. The charged aa-tRNA is shown with the amino acid as a star; the ribosome is drawn as a cartoon with the large and small subunits, while the mRNA is drawn with abundant codons (triplets without a margin) and rare codons (triplets with a margin), where the distance between each marked codon indicates the presence of unmarked codons. a – f Individual data points, p values and n values for statistical analysis are available in Source Data 12−19.

Techniques: Modification

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

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