Journal: bioRxiv

Article Title: Coordinated translational control of multiple immune checkpoints by the integrated stress response pathway in lung cancer

doi: 10.1101/2024.10.23.619897

Figure Lengend Snippet: ( A ) Polysome profiling of H1944 Vehicle or Salubrinal-treated cells (100uM for 24 hours). ( B ) Quantitative real-time PCR analysis of PD-L1(CD274) and CD155(PVR) mRNA ( C ) in ribosomal fractions from ( A ). qRT-PCR analysis for each gene shown was performed with 1 primer set spanning an exon-exon junction (Primer Set 1). Data for Primers Set 2 is available in Supplementary Figure 2. Fractions associated with <3 ribosomes were grouped to represent poorly translated mRNAs, fractions associated with >3 ribosomes were grouped as efficiently translated mRNAs. PD-L1 and CD155 mRNA expression in each fraction was normalized to Luciferase and mRNA abundance was calculated as the % of total in all fractions. Luciferase control mRNA was added to each fraction prior to RNA extraction to control for variability. Error bars represent standard deviation from the mean from three independent fractions (<3 or >3 ribosomes). ( D ) Diagram of the wildtype human CD155 5′ UTR with 6 CTGs, and mutant constructs with CTGs mutated to CTCs, cloned upstream of a luciferase reporter. ( E ) Dual luciferase assay of MEFs transfected with indicated CD155 -5′ UTR-Firefly luciferase reporter constructs normalized to co-transfected control Renilla luciferase. Luciferase activity was monitored after 48h. Error bars represent standard deviation from the mean from n=3 biological replicates. Data from a single experiment are shown and representative of three independent experiments. ( F ) qRT-PCR analysis of mean luciferase mRNA normalized to actin in MEFs shown in ( E ). Error bars represent standard deviation from the mean from n=3 biological replicates. ( G ) Dual luciferase assay of the CD155 5′ UTR in MEFs, Vehicle or Salubrinal-treated (100uM for 24 hours). Error bars represent standard deviation from the mean from n=3 biological replicates. ( H ) qRT-PCR analysis of mean luciferase mRNA normalized to actin in MEFs shown in ( G ). n=3 biological replicates. A Student’s t-test was used to determine statistical significance (* p<0.05, ** p<0.005, *** p<0.0005).

Article Snippet: 50×10 3 MEF cells were seeded/well in 12-well plates in triplicate and transfected with a Renilla plasmid (20ng, Promega, E2241), Firefly luciferase pGL3 plasmid (Promega, E1751, RRID:Addgene_212936) expressing CD155 wildtype 5′ UTR or various mutant constructs (200ng) and a carrier pUC19 plasmid (400ng) per well using Fugene HD (Promega, E2311) at a 3:1 Fugene:DNA ratio.

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Luciferase, Control, RNA Extraction, Standard Deviation, Mutagenesis, Construct, Clone Assay, Transfection, Activity Assay

Journal: Neurochemistry international

Article Title: Reciprocal communication between astrocytes and endothelial cells is required for astrocytic glutamate transporter 1 (GLT-1) expression

doi: 10.1016/j.neuint.2020.104787

Figure Lengend Snippet: Rat cortical astrocytes were grown to 70-80% confluency and then transfected with a mixture of a Hes5 promoter firefly luciferase reporter and a thymidine kinase (Tk) promoter renilla luciferase reporter. Astrocytes transfected with a Notch intracellular domain construct (NICD) in addition to the luciferase reporters were used as a positive control. Astrocytes were maintained as monocultures or bEND.3 cells were added on top in the presence or absence of DAPT (10μM). Seven days later, cells were harvested for analysis of luciferase activities using dual-luciferase reporter assay. Data are the mean ± SEM of 6 independent experiments. *** p < 0.001, **** p < 0.0001, compared to astrocyte monocultures (control); ## p < 0.01 for indicated comparison.

Article Snippet: Two-three days after splitting, astrocytes in 12 well plates (70-80% confluent) were transfected with a mixture of Hes5 promoter firefly luciferase reporter construct (1μg, RRID:Addgene_26869) and HSV-thymidine kinase promoter renilla luciferase reporter construct (pRL-TK 500ng, Promega E2241) using lipofectamine 2000 according to manufacturer’s instructions.

Techniques: Transfection, Luciferase, Construct, Positive Control, Reporter Assay, Control, Comparison

Journal: Neurochemistry international

Article Title: Reciprocal communication between astrocytes and endothelial cells is required for astrocytic glutamate transporter 1 (GLT-1) expression

doi: 10.1016/j.neuint.2020.104787

Figure Lengend Snippet: Tissue culture plates were coated with 5μg/mL of recombinant Notch ligands fused at the C-terminus to the Fc portion of human IgG1; the Fc portion of the same isotype was used as a control (Rc-Fc). Astrocytes were layered on top of the recombinant Notch ligands or a confluent monolayer of bEND.3 cells and maintained for 7-10 days. A) GLT-1 and β-actin were detected on the same membranes. A representative blot (all lanes from the same blot) and summary of quantification of GLT-1 normalized to β-actin are shown. Data are the mean ± SEM of 6 independent experiments. **** p < 0.0001 indicates comparison to astrocyte monocultures, ## p < 0.01 indicates comparison to control Rc-Fc. B) Astrocytes were plated by themselves or on top of the recombinant Notch ligands. Two days later astrocytes were transfected with a mixture of a Hes5 promoter firefly luciferase reporter and a thymidine kinase promoter renilla luciferase reporter. Where indicated, bEND.3 cells were plated on top of astrocytes two days post-transfection. Cells were harvested after 7 to 10 days, and luciferase activities were analyzed using dual-luciferase reporter assay. Data are the mean ± SEM of 5 independent experiments. *** p < 0.001 indicates comparison to astrocyte monocultures, ## p < 0.01 indicate comparisons to control Rc-Fc.

Article Snippet: Two-three days after splitting, astrocytes in 12 well plates (70-80% confluent) were transfected with a mixture of Hes5 promoter firefly luciferase reporter construct (1μg, RRID:Addgene_26869) and HSV-thymidine kinase promoter renilla luciferase reporter construct (pRL-TK 500ng, Promega E2241) using lipofectamine 2000 according to manufacturer’s instructions.

Techniques: Recombinant, Control, Comparison, Transfection, Luciferase, Reporter Assay

Journal: iScience

Article Title: Systematic fine-mapping and functional studies of prostate cancer risk variants

doi: 10.1016/j.isci.2023.106497

Figure Lengend Snippet: Key resources table

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-H3K27ac Abcam Cat# ab4729 RRID: AB_2118291 Chemicals, peptides, and recombinant proteins Biotin-dATP Thermo Fisher Cat# 19524016 Critical commercial assays TRIzol reagent Thermo Fisher Cat# 15596018 Streptavidin C-1 beads Life Technologies Cat# 65002 Dynabeads Protein G Invitrogen Cat#10004D Dual-Luciferase Reporter Assay System kit Promega Cat# E1910 PrimeScript RT reagent with a gDNA eraser Kit Takara Cat# RR047B Deposited data Prostate Cancer GWAS summary statistics GRASP https://grasp.nhlbi.nih.gov/Covid19GWASResults.aspx Epigenomic profiles CistromeDB http://cistrome.org/db/#/ 22Rv1 HiC This paper GSE197820 H3K27ac ChIP-seq This paper GSE197820 4C-seq This paper GSE197820 RNA-seq This paper GSE197820 PolII ChIA-PET (Ramanand et al., 2020) 36 GSE121020 Experimental models: Cell lines 22Rv1 ATCC Cat# CRL-2505 LNCaP ATCC Cat# CRL-1740 C4-2B ATCC Cat# CRL-3315 Oligonucleotides Primers are listed in Table S6 This paper N/A Recombinant DNA Plasmid: pGL3 Promoter vector Promega Cat# E1761 Plasmid: pGL3 basic vector Promega Cat# E1751 Plasmid: pRL-TK Renilla luciferase vector Promega Cat# E2241 Plasmid: pSpcas9n(sgRNAs) addgene Cat# 62987 Plasmid: pLVX-puro This paper N/A Software and algorithms GraphPad Prism9 GraphPad software https://www.graphpad.com ImageJ NIH https://imagej.nih.gov/ij Python (v3.7) The Python Language https://www.python.org GCTA (v1.94.0) (Yang et al., 2012) 53 https://yanglab.westlake.edu.cn/software/gcta/ FINEMAP (v1.4.1) (Benner et al., 2016) 54 http://www.christianbenner.com HiC-Pro (v3.0.0) (Servant et al., 2015) 55 https://github.com/nservant/HiC-Pro HiCRep (v0.2.6) (Yang et al., 2017) 56 https://github.com/dejunlin/hicrep FitHiC (v1.1.3) (Ay et al., 2014) 57 https://github.com/ay-lab/fithic FAN-C (Kruse et al., 2020) 58 https://github.com/vaquerizaslab/fanc VEP (release 96) (McLaren et al., 2016) 37 https://www.ensembl.org/info/docs/tools/vep/script/index.html BWA (v0.7.17) (Li and Durbin, 2009) 59 https://github.com/lh3/bwa Htseq (v0.11.3) (Anders et al., 2015) 60 https://github.com/simon-anders/htseq DEseq2 (v1.38.3) (Love et al., 2014) 61 https://bioconductor.org/packages/release/bioc/html/DESeq2.html DAVID (Huang da et al., 2009) 62 http://david.abcc.ncifcrf.gov/ Open in a separate window Key resources table .

Techniques: Recombinant, Reporter Assay, Plasmid Preparation, Luciferase, Software