Journal: bioRxiv

Article Title: A multivalent peptide-polymer conjugate material mimics STING to therapeutically activate innate immune signaling

doi: 10.64898/2026.03.24.712780

Figure Lengend Snippet: ( A ) Sequence of the murine STING C-terminal tail peptide mSTING(340-378), highlighting motifs known to interact with TBK1 and IRF3. ( B ) Peptide-polyanion conjugate was synthesized through copper-catalyzed azide-alkyne cycloaddition of an azidolysine-mSTING(340-378) peptide and the polyanion poly(L-glutamate)-graft-alkyne. ( C ) HPLC chromatogram using size exclusion column of azidolysine-mSTING(340-378), poly(L-glutamate)-graft-alkyne, and poly(L-glutamate)-graft-mSTING(340-378). ( D ) Circular dichroism spectrum of azidolysine-mSTING(340-378), poly(L-glutamate)-graft-alkyne, and poly(L-glutamate)-graft-mSTING(340-378) in 1× PBS. ( E ) Activity of the peptide-polyanion conjugate was examined using a HEK293T-derived reporter with luciferase expression under the control of target transcription factor IRF3. IRF3 reporter signal relative to buffer treatment for HEK293T reporter cells treated with 2 μM STING or Scr peptide alone, conjugated to only C-terminus of size-matched poly(L-glutamate) to generate a monovalent peptide-polyanion conjugate control, or conjugated to side chains as specified in ( B ) to generate a multivalent peptide-polyanion conjugate. All treatments were transfected using TransIT-X2, activity was measured 24 h post treatment (N = 3 biological replicates). Data represented as geometric mean ± SD.

Article Snippet: Assays with HEK293T pGL4.45 reporter cells were performed as described previously., Briefly, 3×10 5 cells/mL were added to a 96-well plate in 100 μL of DMEM with 10% FBS and P/S, then allowed to incubate for 24 h. To each well, 20 μL of treatment specified in each figure caption was added, then allowed to incubate for 24 h. Luciferase was quantified using the Firefly Luciferase Assay Kit (Biotium).

Techniques: Sequencing, Synthesized, Circular Dichroism, Activity Assay, Derivative Assay, Luciferase, Expressing, Control, Transfection

Journal: Journal of Gastrointestinal Oncology

Article Title: Nestin overexpression reduces the sensitivity of gastric cancer cells to trastuzumab

doi: 10.21037/jgo-22-1048

Figure Lengend Snippet: Influence of Nestin on the activation of Nrf2/HO-1 and HER2/AKT/ERK1/2 of HER2-positive GC cells. The two cell lines received transfection by lentiviral-Nestin or lentiviral-empty, and then added to 10 μg/mL for 48 h. (A,B) Nestin mRNA level in MKN45 and NCI-N87 cells was detected by qRT-PCR. (C,D) WB was adopted to determine the protein level of Nestin, Nrf2, HO-1, NQO1, GST, AKT, ERK1/2, and HER2, in the MKN45 and NCI-N87 cells. (E,F) The cells were exposed to ARE luciferase reporter. 36 h post transfection, the luciferase activity was determined. Data are presented as the means ± SD of three independent experiments. *, P<0.05; **, P<0.01; ***, P<0.001. NTC, non-treated control; TRA, trastuzumab; TRA + NC, mice inoculated with cell infected with lentiviral-negative control were treated with TRA; TRA + Nestin, mice inoculated with cell infected with lentiviral-Nestin were treated with TRA; HO-1, hemeoxygenase-1; NQO1, NAD(P)H: quinone oxidoreductase-1; GST, glutathione S-transferase; AKT, protein kinase B; ERK1/2, extracellular regulated kinase 1/2; HER2, human epidermal growth factor receptor 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ARE, antioxidant response element; GC, gastric cancer; qRT-PCR, quantitative real-time polymerase chain reaction; WB, Western blotting; SD, standard deviation.

Article Snippet: After a day of culture, the cells were treated with 200 ng of ARE luciferase reporter plasmids using OriGene transfection reagent (Company, City, Country).

Techniques: Activation Assay, Transfection, Quantitative RT-PCR, Luciferase, Activity Assay, Control, Infection, Negative Control, Real-time Polymerase Chain Reaction, Western Blot, Standard Deviation

Journal: bioRxiv

Article Title: Novel uAUG creating variants in the 5’UTR of ENG causing Hereditary Hemorrhagic Telangiectasia

doi: 10.1101/2022.12.18.520932

Figure Lengend Snippet: (A) cDNA of the long (NM_001114753.3) isoform of ENG transcripts. Positions of the identified 5’UTR uAUG-creating variants from this project and published studies – as well as position of the associated uStop codon at position c.125, and of main start (c.1) and stop (c.1975) codons are indicated. 5’UTR, 5’ Untranslated Region; CDS, CoDing Sequence; WT, wild-type; uoORF, overlapping upstream Open Reading Frame; eCDS, elongated CDS. (B) Schematic presentation of the pcDNA3.1-L-ENG constructs prepared and used for the evaluation of ENG steady-state levels in HeLa cells. Arrows indicate specific primers targeting the cDNA of ENG with extra sequences containing restriction sites to allow specific cloning in the expression vector. Position of the Myc-His tag is represented by the black square on the plasmid. CMV, cytomegalovirus promoter; WT, wild-type; Var, Variant; 5’UTR, 5’ UnTranslated Region; L, Long. (C and D) Western blot results on total proteins extracted from transfected HeLa cells with 1 µg of pcDNA3.1-L-ENG constructs or from transduced HUVEC cells with 20 MOI of lentiviruses containing ENG, respectively. Two bands of different molecular weights are observed for endoglin likely corresponding to more glycosylated (upper band) and less/non glycosylated (lower band) ENG monomers . Anti-Myc and anti-ENG correspond to the used antibodies for the target protein from HeLa and HUVECs, respectively, and anti-βactin corresponds to the antibody used against the reference protein. kDa, kilodalton; M, protein ladder; WT, wild-type, C-, negative control corresponding to pcDNA3.1-empty vector. Shown results are representative of 5 independent experiments. (E) Decrease of luciferase activity observed with uAUG-creating variants in ENG . Schematic presentation of the pGL3b-(ENG)-luciferase prepared and used in this assay is shown in the upper panel. Arrows indicate specific primers targeting the promoter of ENG with extra sequences containing restriction sites to allow specific cloning in the expression vector. Luc, luciferase. In the lower panel, shown results correspond to Firefly/Renilla ratios normalized to the wild-type (WT) in 5 independent experiments. ***, p-value < 10 −3 (two-factor ANOVA followed by Tukey’s multiple comparison test of variants versus WT).

Article Snippet: Four hours after transfection, cells were stimulated overnight with 5 pg/ml of BMP9 in serum-free medium (R&D Systems) and luciferase activity was measured with the twinlite Firefly and Renilla Luciferase Reporter Gene Assay System (PerkinElmer).

Techniques: Sequencing, Construct, Cloning, Expressing, Plasmid Preparation, Variant Assay, Western Blot, Transfection, Negative Control, Luciferase, Activity Assay, Comparison

Journal: bioRxiv

Article Title: Novel uAUG creating variants in the 5’UTR of ENG causing Hereditary Hemorrhagic Telangiectasia

doi: 10.1101/2022.12.18.520932

Figure Lengend Snippet: (A) Schematic presentation of the co-transfected constructs in this assay. BRE, BMP-response element; CMV, cytomegalovirus promoter; WT, wild-type; Var, variant; 5’UTR, 5’ UnTranslated Region; L, Long. (B) Decrease of BRE activity observed with all the analyzed variants in co-transfected NIH3T3 cells stimulated with BMP9 (5 pg/ml). Shown results correspond to the quantification of Firefly/Renilla and normalized to the wild-type (WT) (n = 4). ***, p-value < 10 −3 ; *, p-value < 5.10 −2 , ns, non-significant (two-factor ANOVA followed by Tukey’s multiple comparison test of variants versus WT).

Article Snippet: Four hours after transfection, cells were stimulated overnight with 5 pg/ml of BMP9 in serum-free medium (R&D Systems) and luciferase activity was measured with the twinlite Firefly and Renilla Luciferase Reporter Gene Assay System (PerkinElmer).

Techniques: Transfection, Construct, Variant Assay, Activity Assay, Comparison