Journal: iScience

Article Title: β-adrenergic signaling modulates breast cancer cell mechanical behaviors through a RhoA-ROCK-myosin II axis

doi: 10.1016/j.isci.2025.112676

Figure Lengend Snippet: βAR activation results in increased cellular force generation (A) Schematic illustration showing the traction force microscopy (TFM) assays. (i) TFM-Pillars Assay. Gold disks (yellow) are embedded on top of polydimethylsiloxane (PDMS) pillars to facilitate imaging lateral displacements due to cellular traction forces. (ii) TFM-Beads Assay. Gold nanoparticles (yellow) are embedded in a PDMS matrix to facilitate imaging lateral displacements due to cellular traction forces. (B) Representative images of MDA-MB-231 HM cells on micropillars with superimposed vector force map. Color scale indicates the force per pillar. Scale, 4 μm. (C) Traction forces of MDA-MB-231 HM cells using TFM-Pillars assay after treatment for 24 h with: vehicle, Veh; βAR agonist isoproterenol, Iso (100 nM); βAR antagonist propranolol, Pro (10 μM); or 100 nM Iso and 10 μM Pro. Each dot represents an individual pillar from at least 16 single cells across 3 independent experiments. Bars show the median; error bars represent standard error. (D) Representative images from traction force measurements of TNBC and MCF10A cells treated with isoproterenol (Iso) for 24 h using the TFM-Beads assay. Each arrow indicates the direction of force and the color gradient corresponds to the magnitude of stress. Scale: 10 μm. (E) Quantification of traction forces. Each data point represents the average traction force per bead for an individual cell, which is averaged over multiple beads that are within the boundary of each individual cell. Data shown here represent at least 7 individual cells across 3 independent experiments. Bars show the median; error bars represent standard error. (F) Western blotting against mono-phosphorylated myosin light chain 2 (pMLC2), non-phosphorylated MLC2, and GAPDH from MDA-MB-231 HM cells treated with 100 nM of isoproterenol for up to 48 h. Quantification of band intensity for pMLC2/MLC2 was normalized to 0 h sample. (G) Western blotting against di-phosphorylated MLC2 (ppMLC2), non-phosphorylated MLC2, and GAPDH after MDA-MB-231 HM cells were treated with increasing concentrations of isoproterenol for 2 h. The ratio of ppMLC2 to MLC2 was normalized to vehicle control. (H) Expression levels of ADRB2 transcripts in TNBC, MCF10A, and MCF7 cells measured by qRT-PCR. Images in (A) are adapted from Servier Medical Art by Servier and are published a Creative Commons BY license ( https://creativecommons.org/licenses/by-nc/3.0/ ). Unless otherwise stated, all error bars represent mean ± s.e.m ( N = 3). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001 [one-way ANOVA with Tukey’s test (F–H) and statistical significance in (C and E) is determined using a permutation test to evaluate the difference in medians between control and treatment conditions. ∗ p < 0.05].

Article Snippet: Gene transcripts were examined by RT-qPCR with a CFX96 Touch Real-Time PCR Detection System (Bio-Rad), using one-step assay reagents (Qiagen Quantitect Probe RT-PCR) and TaqMan Gene Expression Assay primer-probes for human beta-2 adrenergic receptor, i.e., ADRB2 (Hs00240532_s1 from Thermo Fisher).

Techniques: Activation Assay, Microscopy, Imaging, Plasmid Preparation, Western Blot, Control, Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Implication of β2-adrenergic receptor and miR-196a correlation in neurite outgrowth of LNCaP prostate cancer cells

doi: 10.1371/journal.pone.0253828

Figure Lengend Snippet: (A) qRT-PCR analysis of ADRB2 in stably transfected LNCaP cells, shADRB2-1, shADRB2-2 and shADRB2-3, and negative control cells, shCtrl. A representative experiment with triplicates is shown. (B) qRT-PCR analysis of miR-196a in stably transfected LNCaP cells, shADRB2-1, shADRB2-2 and shADRB2-3, and negative control cells, shCtrl. (C) qRT-PCR analysis of ADRB2 in shADRB2-2 cells transfected with a plasmid overexpressing ADRB2 (shADRB2-2 p-ADRB2) and negative control cells (shADRB2-2 p-Empty). (D) qRT-PCR analysis of miR-196a in shADRB2-2 cells transfected with a plasmid overexpressing ADRB2 (shADRB2-2 p-ADRB2) and negative control cells (shADRB2-2 p-Empty). (E) qRT-PCR analysis of miR-196a in LNCaP Ctrl cells treated with different β-blockers or vehicle for 48 hours. The experiments were performed three times and a representative experiment showing the mean ± standard deviation of three replicates is presented in the figure. miR-196a expression was normalized to the reference gene miR-16-5p. The ADRB2 mRNA levels were normalized to the reference gene ALAS1.

Article Snippet: Briefly, LNCaP shADRB2-2 cells were transfected for 48h using XtremeGene HP DNA transfection reagent (cat. no. 06366236001; Sigma, St. Louis, MO) under androgen-proficient conditions with a pcDNA3 plasmid containing the full length ADRB2 gene (p-ADRB2) (cat. no. 14697; Addgene, Watertown, MA).

Techniques: Quantitative RT-PCR, Stable Transfection, Transfection, Negative Control, Plasmid Preparation, Standard Deviation, Expressing

Journal: bioRxiv

Article Title: Discovery and Validation of Context-Dependent Synthetic Mammalian Promoters

doi: 10.1101/2023.05.11.539703

Figure Lengend Snippet: A) Volcano plots of promoter responses following receptor agonism in HEK293 cells co-transfected with the TRE-MPRA library and GPCR expression plasmids. Dashed lines indicate an FDR threshold of 5%. Negative control promoters arc indicated by dark gray data points. B) Hcatmap of promoter fold change responses to aminergic GPCR agonism in HEK293 cells. Displayed are the fold change values for all promoters that were significantly altered by agonist treatment in one comparison. C) Separated biplots displaying the treatment (left) and promoter (right) projections across two dimensions. GPCR-specific responding promoters arc highlighted. Orange and gray projection lines indicate the positive and negative directions of the treatment projection, respectively. D) Volcano plots comparing promoter responses of HEK.293 cells treated with 1 uM epinephrine and untreated cells (left) and HEK293 cells transfected with GFP or ADRB2 expression plasmids (right).Dashed lines indicate an FDR threshold of 5%. Negative control promoters arc indicated by dark gray data points. E) Transcription rate estimations (alphas) for Mafb and CREB3 units in HEK293 cells following 1 uM epinephrine treatment or ADRB2 overexpression, or both. Each connected set of data points represents a single promoter. In the min CMV CREB3comparison, the filled triangular data points represent units with spacer set 2, filled circular points arc units containing spacer set 1.

Article Snippet: Plasmids used in this study are listed in Table S5. pcDNA3.1_eGFP was generated by PCR amplifying the eGFP CDS from Arch(D95H)-eGFP (Addgene #51081) and inserting into pcDNA3.1(-)/ myc -His A using the EcoRI and NotI restriction sites. pcDNA3.1_Signal-Flag-ADRB2 was generated by PCR amplifying the ADRB2 CDS from ADRB2-Tango (Addgene #66220) (introducing a stop codon) and inserting into pcDNA3.1(-)/ myc -His A using Gibson assembly. pcDNA3.1_Signal-Flag-DRD2 was generated by PCR amplifying the DRD2 CDS from DRD2-Tango (Addgene #66269) (introducing a stop codon) and inserting into pcDNA3.1(+) using the NotI and XhoI restriction sites. pcDNA3.1_Signal-Flag-HTR2A was generated by PCR amplifying the HTR2A CDS from HTR2A-Tango (Addgene #66409) (introducing a stop codon) and inserting into pcDNA3.1(+) using the NotI and XhoI restriction sites. pcDNA3.1-GPR91 was generated by PCR amplifying the GPR91 CDS from SUCNR1-Tango (Addgene #66507) (introducing a stop codon) and inserting into pcDNA3.1(-)/ myc -His A using Gibson assembly. pTwist-PAR1 was purchased from Twist Bioscience. pcDNA3.1_Signal-Flag-MRGPRX2 was previously described .

Techniques: Transfection, Expressing, Negative Control, Comparison, Over Expression