Journal: STAR Protocols

Article Title: Streamlined high-throughput cloning protocol to generate arrayed mutant libraries

doi: 10.1016/j.xpro.2022.101930

Figure Lengend Snippet:

Article Snippet: Microplate Rack for Incubator Shaker , Eppendorf , Cat#TTR-221.

Techniques: Virus, Recombinant, Plasmid Preparation, Mutagenesis, Software, Sterility, Aerosol

Journal: Cell

Article Title: Cancer Burden Is Controlled by Mural Cell-β3-Integrin Regulated Crosstalk with Tumor Cells.

doi: 10.1016/j.cell.2020.02.003

Figure Lengend Snippet: Figure 6. Deletion of Pericyte-b3-Integrin Enhances Tumor Cell Growth via Pericyte-pAkt-p65-Regulated Cytokine Production (A) Western blot analysis of b3-null pericytes with CRISPR-Cas9 deletion of Akt1 (b3-null;AktKO PC) or HGFR (b3-null;HGFRKO PC) compared with b3-null;Cas9 controls. Hsc70, loading control. Blots representative of 3 separate experiments. Bar charts, average densitometric analysis relative to loading controls (n = 3 experimental repeats). (B) B16F0 cell survival after exposure to conditioned media from b3-null;AktKO, b3-null;HGFRKO, or b3-null;Cas9 pericytes (n = 16 technical replicates for 2 separate experiments). (C) Tumor growth in WT mice injected subcutaneously with either B16F0 cells plus b3-null;Cas9 pericytes or B16F0 plus b3-null;AktKO pericytes at 1:8 ratio (n = 10 per group). (D) Western blot analysis using mock- and Ik-Ba-SR-transfected WT or b3-null pericyte lysates. HSC70, loading control. (E) Protein-profiler cytokine arrays. Quantitation of fold difference in cytokine expression between mock- and Ik-Ba-SR-transfected b3-null pericytes (n = 4 dots from 2 independent experiments). (F) Conditioned media from mock- and Ik-Ba-SR-transfected b3-null pericytes were applied to B16F0, B16F10, and LLC cells and cell survival measured by MTS assay. Bar charts, means ± SEM relative to tumor cells treated with conditioned media from mock-transfected b3-null pericytes (n = 3 experimental repeats).

Article Snippet: SgRNAs were designed using the CRISPOR algorithm (http://crispor.tefor.net). sgRNA sequences targeting TIMP-1 (Gene ID: 21857), CXCL1 (Gene ID: 14825), CCL2 (Gene ID: 20296), Akt1 (Gene ID: 11651), and HGFR (Met) (Gene ID: 17295) (see Table S1) were cloned into the pLenti-CRISPR–EGFP plasmid (Addgene-#75159) using BsmBI enzyme site.

Techniques: Western Blot, CRISPR, Control, Injection, Transfection, Quantitation Assay, Expressing, MTS Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Cellular secretion and cytotoxicity of transthyretin mutant proteins underlie late-onset amyloidosis and neurodegeneration

doi: 10.1007/s00018-019-03357-1

Figure Lengend Snippet: Secretion pattern of WT and mutant TTRs in HEK293 cells (a–d) and HepG2 cells (e–h). Cultured HEK293 and HepG2 cells were transiently transfected with plasmids encoding WT- and mutant- TTRs for 24 h. Intracellular and secreted TTRs were analysed from the cell lysates and culture media, respectively, using western blot. a, b, e, f Western blot of both cell lysate and medium samples were analyzed using anti-human TTR antibody. c, d, g, h T119M-, V30M- and A97S-TTR had secretion efficiency similar to that of WT-TTR in both HEK293 and HepG2 cells while the secretion efficiency of D18G was dramatically decreased. Data are presented as mean ± standard error of mean from at least three independent experiments. Statistical analysis was performed by one-way analysis of variance, followed by Fisher’s least significant difference test. **p < 0.01, ***p < 0.001

Article Snippet: Plasmid constructs and antibodies TTR variants were generated from human TTR plasmid (Origene, RC204976, Origene Technologies) utilizing the quikChange Lightning site-directed mutagenesis kit procedure from Agilent (Santa Clara, CA, USA) using WT TTR DNA as template.

Techniques: Mutagenesis, Cell Culture, Transfection, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Cellular secretion and cytotoxicity of transthyretin mutant proteins underlie late-onset amyloidosis and neurodegeneration

doi: 10.1007/s00018-019-03357-1

Figure Lengend Snippet: Secretion of amyloidogenic late-onset monomeric TTRs (M-TTRs). a Western blot of both cell lysate and medium samples were analyzed using anti-human TTR antibody. b Secretion efficiency of M-TTRs of WT, T119M and other mutants. The highly unstable D18G and early-onset V30M mutants were not secreted while all late-onset mutants were secreted to the medium. c Secretion pattern of amyloidogenic late-onset M-TTRs in non-reduced, non-boiled SDS PAGE. Only WT-TTR was secreted as tetrameric form. The highly unstable D18G and V30M mutants showed no secretion while all late-onset M-TTRs were secreted as monomers to the medium. Western blot for cell lysates is shown in Fig. S3. d Immunoblot of WT, V30M and A97S M-TTRs in the lysate and medium of HEK293 cells pretreated with 2 µM Tg. e Quantification of relative secretion of WT M-TTR and A97S M-TTR to WT M-TTR vehicle (DMSO) in HEK293 cells pretreated with Tg shows a significant decrease in A97S M-TTR. *p < 0.05; **p < 0.01

Article Snippet: Plasmid constructs and antibodies TTR variants were generated from human TTR plasmid (Origene, RC204976, Origene Technologies) utilizing the quikChange Lightning site-directed mutagenesis kit procedure from Agilent (Santa Clara, CA, USA) using WT TTR DNA as template.

Techniques: Western Blot, SDS Page