Journal: bioRxiv

Article Title: Engineering a Scalable and Orthogonal Platform for Synthetic Communication in Mammalian Cells

doi: 10.1101/2023.01.18.524631

Figure Lengend Snippet: a Schematic representation of CC-functionalised GEMS receptor resulting in activation of target genes, upon dimerization. CC peptides are N-terminally fused, through linker α x to the extracellular and transmembrane domains of the erythropoietin receptor (EpoR) cluster that can induce transgene expression following activation of an intracellular signalling pathway. Complementary CC modules are indicated as A:A’, B:B’ and Γ:Γ’ (see Supplementary Table S1). Downstream receptor signalling can be modulated by exchanging the intracellular activation domains (indicated here as switch A, B and C) and desired output can be expressed by replacing the reporter gene to a transgene of choice. b Receptor activation can be achieved by soluble ditopic CC ligands (upper-left panel) the properties of which can be utilised to achieve Boolean logic gate operators (AND/OR – upper-right panel). Intercellular communication (bottom panel) can be achieved by engineering sender cells that express the ligand under the control of an ON/OFF switch and a receiver cell population expressing the cognate receptor and reporter genes. c Schematic overview of the reporter system to monitor receptor activation using JAK/STAT (Janus kinase/signal transducer and activator of transcription) intracellular signalling (left panel). Each receptor monomer bears a cognate CC (A and A’ with linker α 2 ) that can cause the receptor to heterodimerize. Phosphorylated STAT3 results in the production of the reporter protein: human placental secreted alkaline phosphatase (SEAP). SEAP catalyses the hydrolysis of a chromogenic substrate (p-Nitrophenylphosphate (pNPP) (see Methods and Supplementary Figure S1). Substrate conversion is only observed when the cognate receptors A:A’ are expressed on the cell surface, while non-cognate pairs (A:B) do not result in receptor activation and subsequent substrate conversion (right panel). The experiment is performed in independent triplicates; solid line indicates the mean and shaded area indicates standard deviation. d Normalised SEAP activity in HEK293T cells transiently transfected with receptor pairs with varied linker lengths α x of zero, four, eight and 27 amino acids (aa; GS or GSS repeats, Supplementary Table S2 and Supplementary Figures S2 and S3b). SEAP activity was measured 48 hours following transfection (see Methods). Only the correct combination of cognate CCs (A:A’) results in receptor activation, while non-cognate pairs (A:A, A’:A’ and A:B) result in no activation. Bars indicate mean activity; individual data points represent independent triplicates. SEAP activity is normalised with respect to the SEAP activity of the A:A’ receptor heterodimer with linker α 2 .

Article Snippet: The next day, cells were transfected with 500 ng plasmid DNA (193.8 ng per receptor dimer for a total of 387.6 ng, 96.1 ng STAT3-induced secreted alkaline phosphatase (SEAP) reporter plasmid pLS13 and 16.3 ng STAT3 expression vector pLS15; see Supplementary Table S2) for five hours, using 1.25 μl of the transfection reagent lipofectamine 2000 (Life Technologies) in a total of 200 μl Opti-MEM™ I reduced serum medium (Thermo Fisher Scientific), according to manufacturer’s instructions.

Techniques: Activation Assay, Expressing, Standard Deviation, Activity Assay, Transfection

Journal: Science signaling

Article Title: STAT3 palmitoylation initiates a positive feedback loop that promotes the malignancy of hepatocellular carcinoma cells in mice

doi: 10.1126/scisignal.add2282

Figure Lengend Snippet: ( A and B ) Expression of ZDHHC7 and LYPLA2 genes in HCC (N = 369) samples from TCGA data were visualized using Gepia ( http://gepia.cancer-pku.cn/ ). (A) Kaplan-Meier curves of ZDHHC7 and LYPLA2 genes were performed in Gepia using HCC samples. (B) Expression of ZDHHC7 and LYPLA2 genes in different stages of HCC patients (N = 369) were performed in Gepia. ( C ) Differentially expressed ZDHHC7 and LYPLA2 genes in HCC (N = 369) and normal liver (N = 50) samples from TCGA data were visualized using Gepia ( http://gepia.cancer-pku.cn/ ). TPM, Transcripts Per Million. ( D ) Human cDNA reverse transcripts from 30 patients with HCC (Cohort 1), and ZDHHC7 were analyzed using rtPCR. N = 30 HCC patients. ( E ) Volcano plot of differentially expressed genes (DEGs) in HCC (N = 369) and normal liver (N = 50) samples from TCGA data was visualized. An over 1.41-fold increase and decrease in DEGs in HCC are shown in red and green respectively. ( F ) Venn diagram of both ZDHHC7 and LYPLA2 correlated genes was performed using HCC (N = 369) samples from TCGA data (the absolute Spearman’s correlation R value is above 0.5). ( G ) HCC cells were transfected with indicated Flag-STAT3,DHHC7 WT, or catalytic mutant counterparts. The cells were labeled with or without Alk14. STAT3 was pulled down, followed by in-gel fluorescence or western blot analyses (left) and quantified (right) as indicated. CBB was the coomassie brilliant blue staining of STAT3. N = 3 biological replicates over 3 independent experiments. ( H ) Western blots of ZDHHC7 knockdown HCC and control cells (left) and quantified (right). N = 3 biological replicates over 3 independent experiments. ( I ) Colony formation assay of ZDHHC7 knockdown HCC cells and control cells was performed and colony numbers were counted and normalized (top) and quantified (bottom) as indicated. N = 3 biological replicates over 3 independent experiments. Data represent the Mean ± SEM. ** p < 0.01, by Two-tailed unpaired student’s t-test.

Article Snippet: STAT3 expression vectors with different tags were obtained from Addgene (Watertown, MA, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Mutagenesis, Labeling, Fluorescence, Western Blot, Staining, Knockdown, Control, Colony Assay, Two Tailed Test

Journal: Science signaling

Article Title: STAT3 palmitoylation initiates a positive feedback loop that promotes the malignancy of hepatocellular carcinoma cells in mice

doi: 10.1126/scisignal.add2282

Figure Lengend Snippet: ( A ) HCC cells were transfected with control or Flag-CDK5 plasmids. Endogenous HIF1α was pulled down and western blot was performed. N = 3 biological replicates over 3 independent experiments. ( B ) HCC cells treated with CHX as indicated, with or without dinaciclib. Endogenous HIF1α was measured by western blot (left) and quantified (right). N = 3 biological replicates over 3 independent experiments. ( C ) HCC cells were treated with dinaciclib, with or without MG132. Endogenous protein was measured by western blot (top) and quantified (bottom). N = 3 biological replicates over 3 independent experiments. ( D ) WT and CDK5 knockout (KO) cells with or without CDK5 overexpression (OE) were treated with MG132. Endogenous protein targets were visualized by western blot analyses (top) and quantified (bottom) as indicated. N = 3 biological replicates over 3 independent experiments. ( E ) HCC cells were treated with indicated concentrations of dinaciclib. Endogenous protein was measured by western blot (top) and quantified (bottom). N = 3 biological replicates over 3 independent experiments. ( F and G ) WT and CDK5 KO HCC cells were transfected with Flag-STAT3 WT and labeled with Alk14. After treatment with inhibitors (2BP, dinaciclib or echinomycin), STAT3 was pulled down and palmitoylation of STAT3 was measured by in-gel fluorescence including coomassie brilliant blue (CBB) staining of STAT3 (left) and quantified (right). N = 3 biological replicates over 3 independent experiments. Data represent the Mean ± SEM. * p < 0.05, ** p < 0.01, by Two-tailed unpaired student’s t-test.

Article Snippet: STAT3 expression vectors with different tags were obtained from Addgene (Watertown, MA, USA).

Techniques: Transfection, Control, Western Blot, Knock-Out, Over Expression, Labeling, Fluorescence, Staining, Two Tailed Test

Journal: Science signaling

Article Title: STAT3 palmitoylation initiates a positive feedback loop that promotes the malignancy of hepatocellular carcinoma cells in mice

doi: 10.1126/scisignal.add2282

Figure Lengend Snippet: ( A ) The core hypoxia-response element (HRE) recognized by HIF1 and the sequence of the predicted HIF1 binding site in the promoter region of ZDHHC7 . ( B ) Relative luciferase activity was analyzed after ZDHHC7 reporter plasmids were cotransfected with HIF1α or echinomycin treatment as indicated in 293T cells. N = 3 biological replicates over 3 independent experiments. ( C ) ZDHHC7 reporter plasmids were mutated as indicated (Δ1 and Δ2) and the relative luciferase activity was analyzed after plasmids were transfected with or without HIF1α as indicated in 293T cells. N = 6/3/3/3 biological replicates over 3 independent experiments. ( D ) HepG2 cells were transfected with HIF1α plasmid or treated with echinomycin . ZDHHC7 mRNA was analyzed by rtPCR. N = 3 biological replicates over 3 independent experiments. ( E ) Control and HIF1α knockdown HCC cells were harvested and endogenous protein was measured by western blot (left) and quantified (right). N = 3 biological replicates over 3 independent experiments. ( F ) HepG2 cells were treated with echinomycin as indicated. Cells were harvested, and ZDHHC7 mRNA was analyzed by rtPCR. N = 3 biological replicates over 3 independent experiments. ( G ) 293T cells were transfected with Flag-STAT3 WT and labeled with Alk14. After treating with the indicated inhibitor, STAT3 was pulled down and the palmitoylation of STAT3 was visualized by in-gel fluorescence (left) and quantified (right). CBB was the coomassie brilliant blue staining of STAT3. N = 3 biological replicates over 3 independent experiments. Data represent the Mean ± SEM. * p < 0.05, ** p < 0.01, NS not significant, by Two-tailed unpaired student’s t-test.

Article Snippet: STAT3 expression vectors with different tags were obtained from Addgene (Watertown, MA, USA).

Techniques: Sequencing, Binding Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Control, Knockdown, Western Blot, Labeling, Fluorescence, Staining, Two Tailed Test

Journal: Science signaling

Article Title: STAT3 palmitoylation initiates a positive feedback loop that promotes the malignancy of hepatocellular carcinoma cells in mice

doi: 10.1126/scisignal.add2282

Figure Lengend Snippet: ( A ) Human HCC tissues from 85 patients (Cohort 2) were analyzed using IHC. The correlation between DHHC7, HIF1α and p-STAT3 abundance were analyzed. N = 85 HCC patients. Indicated P value was calculated by Pearson correlation analysis. ( B ) Kaplan-Meier curves of DHHC7, HIF1α, p-STAT3 and CDK5 for HCC patients’ overall survival were performed, and the P value of each Kaplan-Meier curves were visualized. N = 85 HCC patients. ( C ) Working model of the DHHC7-STAT3-HIF1α positive feedback loop. In HCC cells, the palmitoyltransferase DHHC7 increased the transcriptional activity of STAT3 and the transcription of its target gene HIF1A by palmitoylating STAT3 on Cys108. Conversely, as a transcription factor, HIF1α directly promotes ZDHHC7 gene expression. This creates a cycle between DHHC7 and HIF1α which is induced by STAT3 palmitoylation (left). The inhibition of DHHC7-HIF1α cycle blocks the malignancy of hepatic carcinoma cells (right).

Article Snippet: STAT3 expression vectors with different tags were obtained from Addgene (Watertown, MA, USA).

Techniques: Activity Assay, Gene Expression, Inhibition

Journal: Scientific Reports

Article Title: EGF-mediated reduced miR-92a-1-5p controls HTR-8/SVneo cell invasion through activation of MAPK8 and FAS which in turn increase MMP-2/-9 expression

doi: 10.1038/s41598-020-68966-4

Figure Lengend Snippet: Effect of STAT1 and STAT3 silencing and inhibition by Fludarabine and Stattic in HTR-8/SVneo cells on the expression of miR-92a-1-5p in presence or absence of EGF. ( a ) Expression of miR-92a-1-5p by qRT-PCR in HTR-8/SVneo cells transfected with STAT1 siRNA treated with or without EGF for 24 h as compared to untreated scrambled control siRNA transfected cells. ( b ) Expression of miR-92a-1-5p by qRT-PCR in HTR-8/SVneo cells transfected with STAT3 siRNA treated with or without EGF for 24 h as compared to untreated scrambled control siRNA transfected cells. ( c ) Expression of miR-92a-1-5p by qRT-PCR in HTR-8/SVneo cells pre-treated with or without Fludarabine (4 h) and subsequently treated with or without EGF for 24 h. ( d ) Expression of miR-92a-1-5p by qRT-PCR in HTR-8/SVneo cells pre-treated with or without Stattic (1 h) and subsequently treated with or without EGF for 24 h. Each bar represents relative expression after normalization with the miR-191 used as an internal miRNA normalizer. Data in ( a ), ( b ), ( c ) and ( d ) is mean ± SEM of three independent experiments performed in triplicates. Synthetically synthesized STAT3 binding sites present in the promoter region of miR-92a-1-5p (BS1 corresponding to 492–510 nt and BS2 corresponding to 777–795 nt position from miRNA precursor encoding region, table and ) were cloned upstream of the PKG promoter of the firefly luciferase gene in pmirGLO Dual-Luciferase vector. ( e ) shows the relative luciferase activity determined from HEK-293T cells co-transfected with clones possessing the synthetically synthesized STAT3 binding sites in pmirGLO Dual-Luciferase vector with STAT3 mammalian expression vector, as indicated. The data are represented as the mean of three experiments ± S.E.M. performed in duplicates.

Article Snippet: Alternately, HEK-293T cells were co-transfected with 250 ng of luciferase reporter plasmid harboring the STAT3 binding site along with 310 ng of STAT3 mammalian expression vector (OriGene Technologies, MD, USA) using Lipofectamine RNAiMAX reagent in OptiMEM.

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Transfection, Synthesized, Binding Assay, Clone Assay, Luciferase, Plasmid Preparation, Activity Assay

Journal: Scientific Reports

Article Title: EGF-mediated reduced miR-92a-1-5p controls HTR-8/SVneo cell invasion through activation of MAPK8 and FAS which in turn increase MMP-2/-9 expression

doi: 10.1038/s41598-020-68966-4

Figure Lengend Snippet: Graphical representation of the effect of reduced miR-92a-1-5p expression during EGF-mediated increased HTR-8/SVneo cell invasion. EGF leads to reduced expression of miR-92a-1-5p in HTR-8/SVneo trophoblastic cells; reduced miR-92a-1-5p leads to increased invasion of HTR-8/SVneo cells by targeting MAPK8 and FAS, which increases expression of MMP-2 & MMP-9, and decreases expression of TIMP1. EGF activated STAT1 and STAT3 may lead to reduced expression of miR-92a-1-5p by binding to the miRNA precursor encoding region as inhibition of STAT1 and STAT3 abrogates reduction of miR-92a-1-5p expression in EGF treated HTR-8/SVneo cells.

Article Snippet: Alternately, HEK-293T cells were co-transfected with 250 ng of luciferase reporter plasmid harboring the STAT3 binding site along with 310 ng of STAT3 mammalian expression vector (OriGene Technologies, MD, USA) using Lipofectamine RNAiMAX reagent in OptiMEM.

Techniques: Expressing, Binding Assay, Inhibition