Journal: Nature Communications

Article Title: ADAP1 promotes latent HIV-1 reactivation by selectively tuning KRAS–ERK–AP-1 T cell signaling-transcriptional axis

doi: 10.1038/s41467-022-28772-0

Figure Lengend Snippet: a Coomassie staining of recombinant proteins used in Alpha. b Alpha confirms binding of recombinant ADAP1 and KRAS. Data represents mean ± s.d. percent ( n = 3 independent experiments in triplicate). c Coomassie staining of recombinant proteins used in GTPase hydrolysis assay. d GTP-hydrolysis assay confirms that ADAP1 stimulates the rate of KRAS GTP hydrolysis ( n = 3). One-phase association fitted to data to calculate K obs hydrolysis rates. Data represents mean ± s.d. Relative RDU ( n = 3 independent experiments in triplicate). e Proximity ligation assay of unstimulated and stimulated (1 h anti-TCR/anti-CD28 beads) primary T M . Proteins were tested for proximity by incubating cells with ADAP1 and KRAS antibodies. As a negative control for non-specific probe ligation or amplification, samples incubated with only one antibody were included. Yellow arrows pointing to yellow puncta indicate proteins are in proximity (distances < 40 nm). Images are representative of two donors. Scale bar is 10 μm for multicell images and 1 μm for single cell images. f RAS GTP levels in primary ADAP1 CRISPR (red) and Ctrl CRISPR (gray) T M in the absence or presence of anti-TCR/anti-CD28 bead stimulation (10 min). Each graph is a representative donor. Data represents mean ± s.e.m. of Ras GTP /total protein (μg) ( n = 3). (two-way ANOVA followed by Sidak’s test for multiple comparison between ADAP1 CRISPR and Ctrl CRISPR groups), ns not significant. Source data are provided as a Source Data file.

Article Snippet: For the assays in Fig. , RAS GTP levels in cells were quantified using RAS GTP ELISA kit (Active Motif, 52097) per manufacturer’s protocol.

Techniques: Staining, Recombinant, Binding Assay, Hydrolysis Assay, Proximity Ligation Assay, Negative Control, Ligation, Amplification, Incubation, CRISPR, Comparison

Journal: Oncogenesis

Article Title: A porcine model of osteosarcoma

doi: 10.1038/oncsis.2016.19

Figure Lengend Snippet: Transformation of porcine MSCs. ( a ) Overview of stepwise transformation of genetically modified porcine MSCs. ( b ) Left: Cre-mediated excision of transcriptional termination cassette from the KRAS LSL-G12D allele. Cell types as shown. PCR amplification products across the site of the LSL-BS cassette in KRAS intron 1. Predicted fragment sizes: wild-type KRAS 167 bp; non-recombined KRAS LSL-G12D 1486 bp; Cre-excised KRAS L-G12D 201 bp. Right: Cre-mediated excision of transcriptional termination cassette from both TP53 LSL-R167H alleles. Cell types as shown. PCR amplification products across the site of LSL-NEO cassette in TP53 intron 1. Predicted fragment sizes: wild-type TP53 198 bp; non-recombined TP53 LSL-R167H 1929 bp; Cre-excised TP53 L-R167H 254 bp. ( c ) Ras activation assay and p53 western blotting analysis. Cre-recombined MSC-PKC and MSC-PKCM cells show increased levels of active GTP-bound Ras proteins (21 kDa) as well as abundant levels of mutant p53-R167H proteins (46 kDa). ( d ) EdU (5-ethynyl-2'-deoxyuridine) incorporation during S phase. Data are consistent with the enhanced proliferative capacity of genetically modified derivatives relative to wild-type MSCs. P -values: MSC-PK=0.0727; MSC-PKC=0.0042; MSC-PKCM=0.0019. ( e ) Upper row: Loss of contact inhibition. MSC-PKCM cells form multi-layered foci when cultured at higher densities. Scale bars indicate 400 μm. Lower row: Anchorage-independent growth in soft agar. Wild-type MSCs grow as single cells in semi-solid medium, whereas MSC-PK, MSC-PKC and MSC-PKCM cells form three-dimensional colonies. Scale bars indicate 400 μm. ( f ) Upper: H&E-stained section of paucicellular nodule formed by injected MSC-PKC cells. The nodule consists mainly of matrigel with fat islands, capillary sprouts and isolated cells with slightly irregular nuclei. Scale bar indicates 500 μm. The arrow indicates an area of higher cellularity. Lower: Mesenchymal cells with slightly irregular nuclei embedded in a myxoid stroma are evident at higher magnification. Scale bar indicates 100 μm. ( g ) H&E-stained sections of MSC-PKCM derived tumour. This tumour is mostly highly cellular, but a matrix-rich area is still present, for example, as indicated by an asterisk. In the cellular areas, large tumour cells with pleomorphic nuclei are evident. A chronic inflammatory reaction is also present. Scale bar indicates 200 μm.

Article Snippet: Ras activity was analysed using the Ras Activation Assay Kit (Merck, Millipore, Darmstadt, Germany).

Techniques: Transformation Assay, Genetically Modified, Amplification, Activation Assay, Western Blot, Mutagenesis, Inhibition, Cell Culture, Staining, Injection, Isolation, Derivative Assay