Journal: PLoS ONE

Article Title: Type VI collagen promotes lung epithelial cell spreading and wound-closure

doi: 10.1371/journal.pone.0209095

Figure Lengend Snippet: Quantification of 10hr 16HBE wound-width relative to 0hr controls on COL6, COL1, Matrigel coated wells, and uncoated tissue-culture wells after treatment with inhibitors of PI3K (LY294002, 5 μM), CDC42 (ZCL 278, 55 μM), RHOA (CCG 1423, 3 μM), FAK (PF 573228, 40 nM), RAC (EHT 1864, 600 nM), and ERK (FR 180204, 3 μM). (a) Heat map indicating the effect of inhibitor relative to control for cells on each matrix. A lighter color represents more wound closure relative to 0hr. A yellow circle represents p<0.05. Plots present wound-healing rate of cells on COL6, Matrigel, COL1, and plastic after treatment with (b) PI3K inhibitor, (c) CDC42 inhibitor, and (d) RAC1 inhibitor. ** p<0.01, *** p<0.001. n = 6.

Article Snippet: Plasmids containing GFPmPA-GFP-N1 (a gift from Michael Davidson, Addgene plasmid # 54712), and constitutively active FAK (pGFP FAK Y397F, a gift from Kenneth Yamada, Addgene plasmid # 50516), CDC42 (a gift from Joan Brugge, Addgene plasmid #14568), and RAC1 (pcDNA3-EGFP-RAC1-Q61L, a gift from Gary Bokoch, Addgene plasmid #12981) all contain GFP and were transfected individually [ , ].

Techniques: Control

Journal: PLoS ONE

Article Title: Type VI collagen promotes lung epithelial cell spreading and wound-closure

doi: 10.1371/journal.pone.0209095

Figure Lengend Snippet: (a) Representative images of GFP-only, Rac1, Cdc42 and Pi3k-overexpressing cell spreading on Matrigel. Quantification of GFP-positive 16HBE spreading 3 hours after plating on (b) Matrigel, (c) COL1, (d) uncoated tissue-culture wells, and (e) COL6 after transfection with constitutively active signaling constructs for FAK, RAC1, ERK, RHOA, CDC42, or PI3K. Black lines represent spreading of GFP-transfected cells on each matrix, respectively. Grey lines represent spreading of GFP transfected cells on COL6. N = 6. * p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: Plasmids containing GFPmPA-GFP-N1 (a gift from Michael Davidson, Addgene plasmid # 54712), and constitutively active FAK (pGFP FAK Y397F, a gift from Kenneth Yamada, Addgene plasmid # 50516), CDC42 (a gift from Joan Brugge, Addgene plasmid #14568), and RAC1 (pcDNA3-EGFP-RAC1-Q61L, a gift from Gary Bokoch, Addgene plasmid #12981) all contain GFP and were transfected individually [ , ].

Techniques: Transfection, Construct

Journal: Nature genetics

Article Title: Characterizing the molecular regulation of inhibitory immune checkpoints with multi-modal single-cell screens.

doi: 10.1038/s41588-021-00778-2

Figure Lengend Snippet: a) Single-cell mRNA expression heatmap showing 20 differentially expressed genes for each mixscape -classified perturbation. For visualization purposes we downsampled our dataset to include 30 cells from each class in the heatmap. b) Violin plots of PD-L1 protein expression for all identified regulators. BRD4 (p-value=4.37e −28 ), CUL3 (p-value=2.81e −11 ) and MYC (p-value=4.51e −7 ) are negative regulators, while the remaining are positive (p-value < 1e −6 in all cases, two-sided Wilcox Rank sum). NT, non-targeted. c) Flow cytometry measurements of PD-L1 protein expression across experimental conditions. JQ1 inhibitor treatment (24 hours, 1μM) reduces stimulation-induced PD-L1 expression. Control=20,000, stim=20,000 and JQ1+stim=20,000 cells. d) Flow cytometry measurements of PD-L1 protein expression in BRD4 gRNA expressing cells, validating our ECCITE-seq findings. BRD4g2=9,100, BRD4g5=15,000 and NT=4,800 cells. e) Violin plots showing elevated expression of PD-L1 transcript in CUL3 KO cells, in comparison to non-targeting controls. f) Barplot summarizing gene set enrichment analysis results for 300 genes upregulated in CUL3 KO cells. Analysis was performed using the Human WikiPathways database from the EnrichR package and shows strong enrichment for the NRF2 pathway.

Article Snippet: NRF2 over-expression plasmid was purchased from Addgene (#21549).

Techniques: Expressing, Flow Cytometry, Control, Comparison

Journal: Nature genetics

Article Title: Characterizing the molecular regulation of inhibitory immune checkpoints with multi-modal single-cell screens.

doi: 10.1038/s41588-021-00778-2

Figure Lengend Snippet: a) Schematic representation describing two complementary modes of CUL3-mediated PD-L1 regulation. The CUL3-SPOP complex directly regulates PD-L1 protein stability through ubiquitination. The CUL3-KEAP1 complex regulates NRF2 protein stability, indirectly modulating NRF2-mediated PD-L1 transcription. b) Validation pooled CRISPR screen results (2 biological replicates) targeting KEAP1 , SPOP , CUL3 , BRD4 , IFNGR1 and NRF2 (including 4 non-targeting gRNAs). gRNAs targeting KEAP1 , SPOP , CUL3 and BRD4 (green) were enriched in cells expressing high levels of PD-L1 protein while NRF2 and IFNGR1 gRNAs were depleted (red) NT, non-targeted. c) Boxplots showing the PD-L1 protein geometric mean fluorescence intensity (gMFI) (n =2 for each boxplot), (d) PD-L1 transcript and (e) NRF2 transcript levels (log1p(TPM)) in control (n = 3) and NRF2 overexpression (OE) (n = 4) cells. f) Density plot showing the average log2 fold change of three CUL3 KO cell DE gene subsets in the NRF2 OE dataset. g) Boxplots showing the PD-L1 protein geometric mean fluorescence intensity (gMFI) (n=2 KEAP1 KO and n=3 NT), (h) PD-L1 transcript and (i) KEAP1 transcript levels (log1p(TPM)) in NT (n = 8) and KEAP1 KO (n = 8) cells. In c-e and g-i boxplots the middle line is the median, the lower and upper hinges correspond to the first and third quartiles, the upper whisker extends from the hinge to the largest value no further than 1.5 × IQR from the hinge (IQR = inter-quartile range) and the lower whisker extends from the hinge to the smallest value at most 1.5 × IQR of the hinge.

Article Snippet: NRF2 over-expression plasmid was purchased from Addgene (#21549).

Techniques: Ubiquitin Proteomics, Biomarker Discovery, CRISPR, Expressing, Fluorescence, Control, Over Expression, Whisker Assay

Journal: Cell

Article Title: Targeting Ras-, Rho-, and Rab-family GTPases via a conserved cryptic pocket

doi: 10.1016/j.cell.2024.08.017

Figure Lengend Snippet:

Article Snippet: pcDNA3.1(+) EGFP-Rac1 (G12C) , This study , Addgene #224279.

Techniques: Virus, Recombinant, Protease Inhibitor, Staining, Activation Assay, BIA-KA, Mass Spectrometry, Plasmid Preparation, Software, Control, Transfection, Western Blot

Journal: Journal of Biological Engineering

Article Title: Efficient reduction of synthetic mRNA induced immune activation by simultaneous delivery of B18R encoding mRNA

doi: 10.1186/s13036-019-0172-5

Figure Lengend Snippet: Investigation of eGFP expression after the co-transfection of fibroblasts with eGFP mRNA and different amounts of B18R mRNA using flow cytometry. 1 × 10 5 fibroblasts were transfected for two following days with 1.5 μg eGFP alone or with 0.2, 0.5, 1, or 1.5 μg B18R mRNA. Cells treated with only Opti-MEM or Opti-MEM and the transfection reagent Lipofectamine ® 2000 served as negative controls. The eGFP expression was analyzed 24 h after ( a ) the first transfection and ( b ) the second transfection by flow cytometry. Results are presented as means ± SD ( n = 3). Differences were analyzed using one-way ANOVA following Bonferroni’s multiple comparison test. (*** p < 0.001, **** p < 0.0001). ns: not significant

Article Snippet: The pcDNA 3.3 vector containing the coding sequence (CDS) of B18R (Aldevron, Fargo, North Dakota, US) or enhanced green fluorescent protein (eGFP) (Addgene, cat. no. 26822) [ ] was used as template.

Techniques: Expressing, Cotransfection, Flow Cytometry, Transfection, Comparison

Journal: Journal of Biological Engineering

Article Title: Efficient reduction of synthetic mRNA induced immune activation by simultaneous delivery of B18R encoding mRNA

doi: 10.1186/s13036-019-0172-5

Figure Lengend Snippet: Investigation of eGFP expression after the co-transfection of fibroblasts with eGFP mRNA and different amounts of B18R mRNA using fluorescence microscopy. 1 × 10 5 fibroblasts were transfected for two following days with 1.5 μg eGFP mRNA alone or with 0.2, 0.5, 1, or 1.5 μg B18R mRNA. The eGFP expression was analyzed by fluorescence microscopy 24 h after ( a ) the first and ( b ) second transfection. Scale bar corresponds to 200 μm

Article Snippet: The pcDNA 3.3 vector containing the coding sequence (CDS) of B18R (Aldevron, Fargo, North Dakota, US) or enhanced green fluorescent protein (eGFP) (Addgene, cat. no. 26822) [ ] was used as template.

Techniques: Expressing, Cotransfection, Fluorescence, Microscopy, Transfection

Journal: Journal of Biological Engineering

Article Title: Efficient reduction of synthetic mRNA induced immune activation by simultaneous delivery of B18R encoding mRNA

doi: 10.1186/s13036-019-0172-5

Figure Lengend Snippet: Impact of co-transfection with synthetic B18R mRNA on cell viability. Fibroblasts were transfected daily up to 7 days with 1.5 μg eGFP mRNA. Additionally, cells were transfected with 1.5 μg eGFP mRNA and 1.5 μg B18R mRNA or incubated with 200 ng/ml B18R protein after the transfection with 1.5 μg eGFP mRNA. The viability of cells after the Lipofectamine ® 2000 treatment was set to 100%. Results are presented as means ± SEM ( n = 3). Differences were analyzed using two-way ANOVA following Bonferroni’s multiple comparison test. (* p < 0.05, ** p < 0.01, **** p < 0.0001). ns: not significant

Article Snippet: The pcDNA 3.3 vector containing the coding sequence (CDS) of B18R (Aldevron, Fargo, North Dakota, US) or enhanced green fluorescent protein (eGFP) (Addgene, cat. no. 26822) [ ] was used as template.

Techniques: Cotransfection, Transfection, Incubation, Comparison