p fgfr2  (Proteintech)

Journal: Molecular Therapy. Nucleic Acids

Article Title: Targeted allele-specific FGFR2 knockdown via human recombinant ferritin nanoparticles for personalized treatment of Crouzon syndrome

doi: 10.1016/j.omtn.2024.102427

Figure Lengend Snippet: siRNA design and in vitro selection (A–C) Tables show the sequences of siRNA designed to target three different heterozygous mutations in the FGFR2 gene. siRNA fully matching the mutant FGFR2 c.983A>G mRNA of CS patient 1 are reported in (A). The mismatch C:A with wild-type FGFR2 mRNA has been introduced at positions 8–12, 14–15 from the 5′ end of siRNAs’ guide strand (A). Table (B) displays siRNA sequences designed against mutant FGFR2 transcript (c.1025G>A) of patient 2 with the mismatch located at position 10–13 and 15–16. Table (C) shows wild-type and mutant FGFR2 transcripts (c.1024T>C) and the set of designed siRNAs with the mismatch located from position 10 to 13. (D–F) Bar graphs show wild-type (in gray) and mutant (in black) FGFR2 expression analysis in CS patient-derived CMSCs treated with selected siRNAs for 48 h. Each siRNA was transfected into cells by cationic lipids and the effects was compared with cells treated with transfection reagent alone (with Lipofectamine). Data were normalized to β-actin and are expressed as fold change calculated according to 2 −ΔΔCt method. Data were analyzed using GraphPad Prism, employing Student’s t test for statistical significance evaluation. ∗ p < 0.0119 (D). ∗ p < 0.0079 (F). ns, not significant. Results are shown as mean with error bars (SD) of at least three experiments ( n = 3 in D and E; n = 4 in F).

Article Snippet: After incubation with 5% nonfat milk in TBST (10 mM Tris pH 8.0, 150 mM NaCl, 0.2% Tween 20) for 60 min, the membrane was washed three times with TBST and incubated with primary antibodies against t-FGFR2 (1:1,000; Cell Signaling Technology, Danvers, MA, USA, #23328), p -FGFR2 (1:1,000; Thermo Fisher Scientific, #PA5-64796), ERK1/2 (1:1,000; Cell Signaling Technology, #9102), p -ERK1/2 (1:1,000; Cell Signaling Technology, #9101), RUNX2 (1:1,000; Cell Signaling Technology; #12556) and GAPDH (1:1,000; Thermo Fisher Scientific) at 4°C for 12 h (O/N), followed by secondary peroxidase-conjugated antibodies (1:2,000).

Techniques: In Vitro, Selection, Mutagenesis, Expressing, Derivative Assay, Transfection

Journal: Molecular Therapy. Nucleic Acids

Article Title: Targeted allele-specific FGFR2 knockdown via human recombinant ferritin nanoparticles for personalized treatment of Crouzon syndrome

doi: 10.1016/j.omtn.2024.102427

Figure Lengend Snippet: Effects of siRNAs on FGFR2 signaling pathway Representative western blot images (A) and relative densitometric bar graphs (B) of total FGFR2 (t-FGFR2), p -FGFR2/t-FGFR2, phospho-ERK ( p -ERK)/total-ERK (t-ERK), and RUNX2 levels in patient 1-derived CMSCs treated with 1 nM of selected siRNA (si4) for 48 h, using Lipofectamine as transfection reagent. Cells grown with only Lipofectamine were used as controls (with Lipofectamine). t-FGFR2 and RUNX2 expression levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and are represented as fold changes to untreated control (with Lipofectamine). All phosphorylation levels were evaluated by the ratio of phosphoprotein to total protein. The data shown are representative of four independent experiments ( n = 4) and were analyzed by Student’s t test. ∗∗∗∗ p < 0.0001; ∗∗ p = 0.0071 ( p -FGFR2/t-FGFR2); ∗∗ p = 0.0056 (RUNX2).

Article Snippet: After incubation with 5% nonfat milk in TBST (10 mM Tris pH 8.0, 150 mM NaCl, 0.2% Tween 20) for 60 min, the membrane was washed three times with TBST and incubated with primary antibodies against t-FGFR2 (1:1,000; Cell Signaling Technology, Danvers, MA, USA, #23328), p -FGFR2 (1:1,000; Thermo Fisher Scientific, #PA5-64796), ERK1/2 (1:1,000; Cell Signaling Technology, #9102), p -ERK1/2 (1:1,000; Cell Signaling Technology, #9101), RUNX2 (1:1,000; Cell Signaling Technology; #12556) and GAPDH (1:1,000; Thermo Fisher Scientific) at 4°C for 12 h (O/N), followed by secondary peroxidase-conjugated antibodies (1:2,000).

Techniques: Western Blot, Derivative Assay, Transfection, Expressing, Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: Targeted allele-specific FGFR2 knockdown via human recombinant ferritin nanoparticles for personalized treatment of Crouzon syndrome

doi: 10.1016/j.omtn.2024.102427

Figure Lengend Snippet: Effects of siRNAs on osteogenic differentiation of CMSCs (A) The graphs show the transcript levels of t- FGFR2 and of the osteogenic markers such as RUNX2 , SP7 , ALPL , COL1A1 , COL1A2 , BGLAP , and SOST in cells treated with 1 nM of siRNA 4 (named si4) under osteogenic induction conditions for 5 days (see text for details). Cells grown with osteogenic medium supplemented with solely Lipofectamine were used as controls (w/Lipofectamine). Data were normalized to β-actin and are expressed as relative expression calculated according to 2 −ΔΔCt method. Results are displayed as mean ( n = 5 in t- FGFR2 , RUNX2 , SP7 , and ALPL; n = 4 in BGLAP ; n = 3 in COL1A1 , COL1A2 , and SOST ) with SD (error bars). Statistical significance was determined by Student’s t test; ∗∗∗∗ p ˂ 0.0001; ∗∗∗ p = 0.0005; ∗∗ p = 0.0043; ∗ p = 0.0340. (B) ARS staining (left images, 10×) and its quantification (right) performed in cells treated with 1 nM of si4 during osteogenic induction (5 days) and in control cells (with Lipofectamine). The graph shows ARS quantification in treated and untreated cells after 5 days of osteogenic differentiation. Graph bars represent the optical density mean values ( n = 3) with SD. Results were analyzed by Student’s t test; ∗∗ p = 0.0041.

Article Snippet: After incubation with 5% nonfat milk in TBST (10 mM Tris pH 8.0, 150 mM NaCl, 0.2% Tween 20) for 60 min, the membrane was washed three times with TBST and incubated with primary antibodies against t-FGFR2 (1:1,000; Cell Signaling Technology, Danvers, MA, USA, #23328), p -FGFR2 (1:1,000; Thermo Fisher Scientific, #PA5-64796), ERK1/2 (1:1,000; Cell Signaling Technology, #9102), p -ERK1/2 (1:1,000; Cell Signaling Technology, #9101), RUNX2 (1:1,000; Cell Signaling Technology; #12556) and GAPDH (1:1,000; Thermo Fisher Scientific) at 4°C for 12 h (O/N), followed by secondary peroxidase-conjugated antibodies (1:2,000).

Techniques: Expressing, Staining, Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: Targeted allele-specific FGFR2 knockdown via human recombinant ferritin nanoparticles for personalized treatment of Crouzon syndrome

doi: 10.1016/j.omtn.2024.102427

Figure Lengend Snippet: HFt-HIS-PASE/si4 investigation The graph shows mutant (c.983A>G) and wild-type FGFR2 expression levels in CMSCs derived from CS patient 1 treated with 5 nM of chemically modified siRNA 4 (NH 2 -si4) delivered by HFt-HIS-PASE nanocarrier for 48 h. Cells treated with naked HFt-HIS-PASE were used as controls. Results were normalized to β-actin and are expressed as relative expression calculated according to 2 −ΔΔCt method. Data are presented as mean ( n = 3) with SD. Data were analyzed using GraphPad Prism, employing Student’s t test for statistical analysis. ∗∗ p = 0.0096.

Article Snippet: After incubation with 5% nonfat milk in TBST (10 mM Tris pH 8.0, 150 mM NaCl, 0.2% Tween 20) for 60 min, the membrane was washed three times with TBST and incubated with primary antibodies against t-FGFR2 (1:1,000; Cell Signaling Technology, Danvers, MA, USA, #23328), p -FGFR2 (1:1,000; Thermo Fisher Scientific, #PA5-64796), ERK1/2 (1:1,000; Cell Signaling Technology, #9102), p -ERK1/2 (1:1,000; Cell Signaling Technology, #9101), RUNX2 (1:1,000; Cell Signaling Technology; #12556) and GAPDH (1:1,000; Thermo Fisher Scientific) at 4°C for 12 h (O/N), followed by secondary peroxidase-conjugated antibodies (1:2,000).

Techniques: Mutagenesis, Expressing, Derivative Assay, Modification

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Multiple receptor tyrosine kinases regulate dengue infection of hepatocytes

doi: 10.3389/fcimb.2024.1264525

Figure Lengend Snippet: Knockdown of EPHB4, ERBB2, FGFR2, and IGF1R results in decreased DENV infection. HepG2 cells were transduced with shRNA lentivirus targeting KiR-predicted RTKs then analyzed for protein reduction by western blot (A, B) . Viable cells with successful knockdown in protein expression were infected with DENV2 MON601, and the percent change of Env and NS3 double positive cells is shown (C) . Data represent three independent infections on a single transduction per clone, where green circle, blue square, and black diamond indicate each independent infection. Horizontal black bar represents the average of all replicates for each condition. Significance of differences in infection between knockdown lines and scramble were analyzed by Student’s t-test and are indicated on the graph, where ns = non-significant, * = p-value<0.05, ** = p-value<0.05, *** = p-value<0.0005, and **** = p-value<0.00005.

Article Snippet: Primary antibodies p-ErbB2 (MilliporeSigma #04-293) and p-IGF-1R (MilliporeSigma #ABE332) were used at a 1:1000 concentration, p-FGFR2 (CST #3471) and p-EPHB4 (Thermo ScientificTM #PA5-64792) were used at a 1:500 concentration.

Techniques: Knockdown, Infection, Transduction, shRNA, Western Blot, Expressing