cells rh30 atcc crl 2061  (ATCC)

Journal: bioRxiv

Article Title: Rhabdomyosarcoma fusion oncoprotein initially pioneers a neural signature in vivo

doi: 10.1101/2024.07.12.603270

Figure Lengend Snippet: (a) Wildtype zebrafish embryos were injected at the one-cell stage with 100 ng/μL of PAX3::FOXO1 (P3F) or equal molarity of control (CNTL) mRNA. (b) Schematic of embryonic zebrafish development with positioning of primary germ layers at key time points used in experiments. (c) Representative merged, phase contrast, and GFP images of zebrafish embryos at 3, 6, and 9 developmental hours-post-fertilization (hpf). Scale bar, 1 mm. (d) Representative western blot with protein lysate from 12 embryos per lane for each condition and 8 μg of protein lysate from RH30 cells, a PAX3::FOXO1-positive patient-derived cell line. The PAX3::FOXO1 time-course was repeated for a total of four replicates. Normalized quantification for the representative western blot of PAX3::FOXO1/αTUBULIN signal compared to RH30 cells is on the right, n=1.

Article Snippet: For spike-in normalization, PAX3::FOXO1-positive RH30 patient-derived cells RH30 (ATCC, CRL-2061) were fixed and stored like the zebrafish samples following their expansion and lifting with TrypLE Express (ThermoFisher Scientific, 12604013).

Techniques: Injection, Control, Western Blot, Derivative Assay

Journal: bioRxiv

Article Title: Rhabdomyosarcoma fusion oncoprotein initially pioneers a neural signature in vivo

doi: 10.1101/2024.07.12.603270

Figure Lengend Snippet: (a) Quantification of raw integrated density of GFP fluorescence. Each point represents an individual embryo, which were collected across three different injection days. Control-injected (CNTL) embryos: n=29 per time point, PAX3::FOXO1-injected (P3F) embryos: n=30 at 3 and 9 developmental hours post-fertilization (hpf), n=29 at 6 hpf. A Brown-Forythe and Welch ANOVA test with a Benjamini, Krieger, and Yekutieli correction for multiple comparisons was used to determine statistical significance. Asterisks represents statistics between P3F embryos. CNTL embryos across time-points had p≤0.005. Error bars indicate standard deviation. (b) Flow cytometry for GFP of 24 hpf wildtype (WT) uninjected zebrafish embryo as negative control. (c, d) Flow cytometry for GFP of zebrafish embryos injected with 50 ng/μL of PAX3::FOXO1 and 50 ng/uL pCS2-tagRFPT.zf1 at 6 and 12 hpf, respectively. (e) Protein sequence BLAST alignment between human FOXO1 and zebrafish orthologs Foxo1a and Foxo1b. (f) Western blot with protein lysate from 15 embryos per lane for each condition and 10 μg of protein lysate from RH30 cells, a PAX3::FOXO1-positive patient-derived cell line. Endogenous FOXO1 is detected in the patient-derived sample but not in zebrafish embryos. WT – Wildtype. *** denotes p<0.001 and ** denotes 0.001

Article Snippet: For spike-in normalization, PAX3::FOXO1-positive RH30 patient-derived cells RH30 (ATCC, CRL-2061) were fixed and stored like the zebrafish samples following their expansion and lifting with TrypLE Express (ThermoFisher Scientific, 12604013).

Techniques: Fluorescence, Injection, Control, Standard Deviation, Flow Cytometry, Negative Control, Sequencing, Western Blot, Derivative Assay

Journal: NAR Genomics and Bioinformatics

Article Title: mRNAid, an open-source platform for therapeutic mRNA design and optimization strategies

doi: 10.1093/nargab/lqae028

Figure Lengend Snippet: Impact of mRNAid sequence optimization on spike protein expression. Luminescence signals (normalized to a HiBiT control protein) of HiBiT-tagged spike CDS optimized by mRNaid (teal) or from the Moderna (black) and Pfizer (pink) vaccines are compared with the native spike CDS (blue) at ( A ) 24 h and ( B ) 48 h in SJCRH30 and MIA PaCa-2 cell lines at the 12.5 ng dose of the respective mRNA. Scatter plots, with bars representing the mean from two independent biological replicate experiments. ( C ) Western blot analysis of the spike sequence variants 24 or 48 h post-transfection in MIA PaCa-2 and SJCRH30 cells. HSP90 was used as a loading control. A representative blot is shown from two independent biological replicates. ( D ) Band intensities from two biological replicate experiments for HiBiT–spike (full-length, FL; and cleaved S2, S2) were normalized to HSP90 and represented as fold change over the native control. The presence of the expected product and the addition of a poly(A) tail for all mRNA constructs synthesized in this study were confirmed by gel electrophoresis ( ; ).

Article Snippet: SJCRH30 (CRL-2061) cells were cultured in RPMI with GlutaMAX™ supplement (Gibco) and 10% FBS (HyClone).

Techniques: Sequencing, Expressing, Vaccines, Western Blot, Transfection, Construct, Synthesized, Nucleic Acid Electrophoresis