pcmv6-ac-ha vectors  (OriGene)

Journal: American Journal of Human Genetics

Article Title: SMAD4 mutations causing Myhre syndrome are under positive selection in the male germline

doi: 10.1016/j.ajhg.2024.07.006

Figure Lengend Snippet: NsiI restriction enzyme digestion (RED)-PCR assay for enrichment of spontaneous MYHRS-associated SMAD4 mutations (A) SMAD4 sequence context around codon 500 (red), which encompasses an NsiI restriction site (yellow box—note the palindromic nature of the site, indicated by the dotted red line). PstI digestion releases a 1,332 bp gDNAfragment and wild-type sequences are also digested with NsiI to generate two smaller fragments (844 and 488 bp) ( Figure S1 ). All nucleotide substitutions (and corresponding amino acid changes) resistant to NsiI digestion are indicated and are color coded according to the amino acid involved. The two known MYHRS-causing mutations that can be enriched by this strategy are indicated in bold. The CRISPR-edited Integrant clone, a 17 bp (c.1478_1494del) heterozygous deletion (in purple within the dotted rectangle), is also resistant to NsiI digestion. (B) Mutation levels estimated in a titration-reconstruction assay with serial dilutions of two gDNA from MYHRS-affected individuals with the p.Ile500Thr (red) or p.Ile500Val (black) variant (as indicated on the figure) mixed with blood carrier gDNA and the Integrant DNA. Values plotted are means of two technical replicates and error bars represent 95% binomial confidence intervals (CIs) (see Table S3 ). (C) Comparison of the mutation levels (per million) in sperm samples for the equivalent (i.e., reciprocal) substitutions within the palindromic NsiI site (ATG|CAT)—note that for the top row (and in particular the top left panel), the c.1497C substitution levels within individual samples correlate significantly with the levels of their cognate c.1496G change. This pattern is best explained by the DNA substitutions occurring through a passive process, such as oxidative damage. By contrast for the other substitutions (middle and bottom rows), the relationships between equivalent substitutions are different, with mutation levels of reciprocal/cognate substitutions not correlating with one another. Note the variable range of the axes that have been adjusted to reflect the levels of the highest measurements. (D) Cumulative mutation levels for the 18 different substitutions enriched by the NsiI RED-PCR assay (see color chart) observed in sperm (top) and blood (bottom) samples. Mutations encoding the p.Ile500Val, p.Ileu500Thr, and p.Ileu500Leu changes dominate the landscape, while the c.1496C>A and its cognate c.1497G>T variant are found in similar proportions. Individual plots are presented in Figure S7 . (E–H) Mutation levels of four apparently selected variants at Ile500 (c.1498A>G [p.Ile500Val], c.1499T>C [p.Ile500Thr], and c.1498A>C and A>T which both encode p.Ile500Leu) plotted against the age of the donors on a log10 scale. The dotted line represents the 10 −6 limit of detection of the RED-PCR assay. Blood (red) and sperm (black) samples are plotted as the mean of three independent technical replicates and their 95% binomial CI.

Article Snippet: The SMAD4 fragment was cloned into the pCMV6-AC-HA mammalian expression vector (OriGene) using the Gibson method.

Techniques: Sequencing, CRISPR, Mutagenesis, Titration, Variant Assay, Comparison

Journal: American Journal of Human Genetics

Article Title: SMAD4 mutations causing Myhre syndrome are under positive selection in the male germline

doi: 10.1016/j.ajhg.2024.07.006

Figure Lengend Snippet: Overview of the epidemiological paternal age effect (PAE) for MYHRS and other known PAE mutations

Article Snippet: The SMAD4 fragment was cloned into the pCMV6-AC-HA mammalian expression vector (OriGene) using the Gibson method.

Techniques:

Journal: American Journal of Human Genetics

Article Title: SMAD4 mutations causing Myhre syndrome are under positive selection in the male germline

doi: 10.1016/j.ajhg.2024.07.006

Figure Lengend Snippet: Paternal age effect for de novo MYHRS-causing SMAD4 mutations Distribution of the observed difference between the ages of fathers ( n = 35) of MYHRS-affected individuals and year-matched USA population average (pooled in 3-year bins). The shift to the right side of the figure illustrates the paternal age excess. The specific SMAD4 mutation identified in the proband is denoted according to the color chart indicating the MYHRS variant and predicted amino acid change. The maternal age effect for these families is shown in Figure S5 A.

Article Snippet: The SMAD4 fragment was cloned into the pCMV6-AC-HA mammalian expression vector (OriGene) using the Gibson method.

Techniques: Mutagenesis, Variant Assay

Journal: American Journal of Human Genetics

Article Title: SMAD4 mutations causing Myhre syndrome are under positive selection in the male germline

doi: 10.1016/j.ajhg.2024.07.006

Figure Lengend Snippet: Functional characterization of the sperm-enriched SMAD4 variants (A) Transcriptional activity (assessed by luciferase assay using SBE3 binding site in HEK293T cells) of wild-type (WT) and enriched SMAD4 protein mutants, as indicated on the figure. The graph bars represent mean ± SEM and values of 3 individual replicates are indicated as white circles. (B) Protein levels and stability of SMAD4 mutants. Immunoblot analysis shows WT and variant HA-tagged SMAD4 protein levels in transfected HEK293T cells, basally and after cycloheximide (CHX) treatment. GAPDH was used as loading control. Representative blots (below) and mean ± SEM densitometry values (above) of three independent experiments are shown. Asterisks indicate statistically significant differences compared to the WT SMAD4 ( ∗∗∗ p ≤ 0.001; ∗∗ p ≤ 0.05; two-way ANOVA followed by Dunnett’s multiple comparison test). (C and D) Serum-stimulated phospho-ERK (pERK) assay. Representative blots (below) and graphs reporting mean ± SEM densitometry values (above) of at least three independent experiments are shown. HEK293T cells were transiently transfected with the indicated HA-tagged SMAD4 or V5-tagged LZTR1 construct, serum starved (16 h), and stimulated with FBS (C) or TGF-β1 (D), in time-course experiments or left untreated. Equal amounts of total proteins from each cell lysate were resolved on 10% polyacrylamide gels. Asterisks indicate statistically significant differences in the phosphorylation levels compared to cells transfected with empty vector at the corresponding experimental points ( ∗∗ p < 0.01; ∗ p < 0.05; two-way ANOVA followed by Dunnett’s multiple comparison test).

Article Snippet: The SMAD4 fragment was cloned into the pCMV6-AC-HA mammalian expression vector (OriGene) using the Gibson method.

Techniques: Functional Assay, Activity Assay, Luciferase, Binding Assay, Western Blot, Variant Assay, Transfection, Control, Comparison, Construct, Phospho-proteomics, Plasmid Preparation

Journal: Autophagy

Article Title: SPHK1 (sphingosine kinase 1) induces epithelial-mesenchymal transition by promoting the autophagy-linked lysosomal degradation of CDH1/E-cadherin in hepatoma cells

doi: 10.1080/15548627.2017.1291479

Figure Lengend Snippet: SPHK1 induces the lysine 63-linked ubiquitination of BECN1 through TRAF2. (A-C) SPHK1 induced the lysine 63-linked ubiquitination of BECN1 and did not affect the lysine 48-linked ubiquitination of BECN1. HepG2 cells stably expressing vector or MYC-SPHK1 were transfected with a plasmid encoding HA-BECN1. The cell lysates were extracted and immunoprecipitated using an anti-HA antibody. The precipitates were then examined with anti-ubiquitin (A), anti-K63-specific ubiquitin (B) or anti-K48-specific ubiquitin (C). (D) TRAF2 was imperative for SPHK1 to induce the lysine 63-linked ubiquitination of BECN1. HepG2 cells were cotransfected with a plasmid encoding HA-BECN1 and control-siRNA (or si- TRAF2 ); then, the cell lysates were extracted and immunoprecipitated using an anti-HA antibody. The precipitates were then examined with anti-K63-specific ubiquitin antibody. WT, wild type; Ub, ubiquitin; Con-siRNA, control-siRNA.

Article Snippet: HA-tagged BECN1 was constructed in the pCMV6-AC-HA vector (PS100004, Origene) by standard subcloning.

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Transfection, Immunoprecipitation

Journal: Autophagy

Article Title: SPHK1 (sphingosine kinase 1) induces epithelial-mesenchymal transition by promoting the autophagy-linked lysosomal degradation of CDH1/E-cadherin in hepatoma cells

doi: 10.1080/15548627.2017.1291479

Figure Lengend Snippet: SPHK1 stimulates the lysine 63-linked ubiquitination of BECN1 to trigger autophagy by binding to TRAF2. (A) BECN1 interacted with TRAF2. Whole cell lysates were extracted from HepG2 cells and then immunoprecipitated with anti-BECN1 antibody or an equal amount of mouse IgG and blotted with anti-TRAF2 antibody. (B) SPHK1 bound to TRAF2 and BECN1. Whole cell lysates were extracted from HepG2 cells stably expressing MYC-SPHK1 and then immunoprecipitated with anti-MYC (SPHK1) antibody or an equal amount of mouse IgG and blotted with anti-BECN1 or anti-TRAF2 antibody. (C) Silencing TRAF2 inhibited the binding of SPHK1 and BECN1. HepG2 cells stably expressing MYC-SPHK1 were cotransfected with a plasmid encoding HA-BECN1 and control-siRNA (or si- TRAF2 ). Whole cell extracts were immunoprecipitated with anti-MYC (SPHK1) antibody and blotted with an anti-HA (BECN1) antibody. (D) Deletion of the RING domain of TRAF2 reduced the lysine 63-linked ubiquitination of BECN1 and blocked the interaction of BECN1 with TRAF2 in SPHK1-overexpressing cells. HepG2 cells were cotransfected with a plasmid encoding HA-BECN1 and the indicated constructs of Flag-TRAF2. The cell lysates were extracted and immunoprecipitated using an anti-HA antibody. The precipitates were then analyzed with anti-K63-specific ubiquitin or anti-Flag antibody. (E) The deletion of the RING domain of TRAF2 did not induce autophagy. HepG2 cells were transfected with the indicated constructs of Flag-TRAF2 and the cell lysates were extracted. Autophagy-related proteins were detected by western blot analysis. Data are presented as the mean ± SE of 4 independent assays. Con-siRNA, control-siRNA; FL, full-length.

Article Snippet: HA-tagged BECN1 was constructed in the pCMV6-AC-HA vector (PS100004, Origene) by standard subcloning.

Techniques: Binding Assay, Immunoprecipitation, Stable Transfection, Expressing, Plasmid Preparation, Construct, Transfection, Western Blot

Journal: Autophagy

Article Title: SPHK1 (sphingosine kinase 1) induces epithelial-mesenchymal transition by promoting the autophagy-linked lysosomal degradation of CDH1/E-cadherin in hepatoma cells

doi: 10.1080/15548627.2017.1291479

Figure Lengend Snippet: SPHK1 induces the EMT by stimulating BECN1 in HepG2 cells. (A, B) Silencing BECN1 blocked cell migration and invasion in SPHK1-overexpressing cells. HepG2 cells stably expressing vector or MYC-SPHK1 were transfected with control-siRNA or si- BECN1 and plated in the upper chamber of the filters for 24 h. Then cells migrating to the underside of the transwell insert were counted. (C) SPHK1 overexpression regulated the expression of EMT-related markers through stimulating BECN1. HepG2 cells stably expressing vector or MYC-SPHK1 were transfected with control-siRNA or si- BECN1 for 24 h and then harvested for cell lysate extraction. The level of epithelial or mesenchymal markers was detected by western blot analysis. (D) Silencing BECN1 blocked the degradation of CDH1 in SPHK1-overexpressing cells. HepG2 cells stably expressing MYC-SPHK1 were cotransfected with pCMV6-CDH1 and si- BECN1 (or control-siRNA) for 24 h and then treated with CHX (20 μmol/L) for the indicated times. The cell lysates were analyzed by western blotting using an anti-CDH1 antibody. (E) Schematic diagram of the mechanism of SPHK1-mediated autophagy and lysosomal CDH1 degradation that stimulates the EMT in HepG2 cells. Con-siRNA, control-siRNA.

Article Snippet: HA-tagged BECN1 was constructed in the pCMV6-AC-HA vector (PS100004, Origene) by standard subcloning.

Techniques: Migration, Stable Transfection, Expressing, Plasmid Preparation, Transfection, Over Expression, Western Blot