sirna sequences targeting mouse flot2, klf4, klf6 and klf15 and control-sirna  (Ribobio co)

Journal: bioRxiv

Article Title: Flotillin-2 regulates EGFR activation, degradation, and cancer growth

doi: 10.1101/2022.03.11.483779

Figure Lengend Snippet: (A) SILAC MS scheme shows that HeLa cell proteins were labeled with stable isotopes to compare Cbl complexes without EGF (light, L) and with 30 min of 100 ng/mL EGF stimulation (heavy, H). Cbl was immunoprecipitated (IP) from differentially treated cell lysates and the IPs were mixed at 1:1 ratio. The mixed IP was either directly used to generate peptides for MS (1) or was separated by SDS-PAGE (2), divided into fractions as indicated and each fraction was analyzed separately with MS. (B) The heat maps show SILAC ratios comparing abundance of specific proteins in the complex with Cbl in EGF treated and control samples (H/L) in the descending order. The arrows indicate the candidates selected for further analysis with in-cell pEGFR ELISA. This data is from five independent experiments. (C) HeLa cells were transfected with control siRNA (siNeg) or siRNA targeting Cbl/Cbl-b (positive control), FLOT1 or FLOT2 and seeded on 96-well plate. Phospho-EGFR ELISA was used to detect the levels of EGFR phosphorylation at Y1173 upon 100 ng/mL EGF stimulation for indicated time periods. The pEGFR Y1173 signal was normalized to the Janus-green whole-cell staining signal and the average values for three experiments (± SEM) were plotted versus EGF stimulation time. (D) HeLa cells were transfected with control siRNA (siNeg) or siRNA targeting FLOT1, FLOT2 separately and together for 48 hours and then treated with 25 ng/mL EGF for the indicated time periods. The lysates were subjected to western blot analysis with the indicated antibodies. (E) EGFR phosphorylation upon EGF stimulation is shown as an average ratio of pEGFR/EGFR ± SEM calculated by densitometry analysis of western blot (as shown in D) for three independent experiments. (F) Degradation of EGFR upon EGF stimulation was calculated by densitometry analysis of western blot in D from seven independent experiments and plotted as an average ratio of EGFR/HSC70 ± SEM. EGFR levels in untreated samples (0 EGF) were set as 1. Asterisk (*) denotes p < 0.05, (**) indicates p < 0.01 as compared to the corresponding time points for siNeg using Student’s t -test. MW in kDa is shown to the left of the western blot panels.

Article Snippet: When indicated, transfected H441 cells were treated with 10 μM erlotinib for 24 hours before harvesting. siRNAs for FLOT1 (s19913, s19914, s19915), FLOT2 (s5284, s5285, s5286), Cbl (s2476), Cbl-b (s2479) and negative control (4390844) all were purchased from ThermoFisher Scientific.

Techniques: Multiplex sample analysis, Labeling, Immunoprecipitation, SDS Page, Control, Enzyme-linked Immunosorbent Assay, Transfection, Positive Control, Phospho-proteomics, Staining, Western Blot

Journal: eLife

Article Title: Syncytin-mediated open-ended membrane tubular connections facilitate the intercellular transfer of cargos including Cas9 protein

doi: 10.7554/eLife.84391

Figure Lengend Snippet: ( A–D , F ) Donor cells: HEK293T wild-type (WT) with stable overexpression of his tagged Cas9-GFP/gRNA (His-Cas9/gRNA), with stable overexpression of SBP tagged Cas9-GFP/gRNA (SBP-Cas9/gRNA) or with stable overexpression of SBP-Cas9-GFP/gRNA and Myc-streptavidin-CD63-mCherry (Str-CD63/SBP-Cas9/gRNA). The recipient cell line was MDA-MB-231 with a reporter plasmid. Nluc/Fluc assays were performed and normalized to an aliquot of co-cultured WT donor and reporter cells. ( A ) Nluc/Fluc activities were measured after donor cells and acceptor cells were co-cultured for 3 days with DMSO or different inhibitors. ( B ) The indicated genes were knocked down in recipient cells via siRNA and then co-cultured with donor cells for 3 days. siCtrl represents a negative control for the siRNA knockdown. CLTC, clathrin heavy chain; AP2B1, adaptor-related protein complex 2 subunit beta 1; CAV-1, caveolin 1; FLOT2, flotillin 2. ( C ) The donor cells and acceptor cells were co-cultured for 3 days with either DMSO, 40, 80, or 200 nM latrunculin A (LatA), or 1, 2.5, or 5 μM latrunculin B (LatB). The Nluc/Fluc signal detected after co-culture suggested that more than half of the cells remained viable during drug treatment. ( D ) The indicated genes were knocked down in recipient cells via shRNA that were then co-cultured with donor cells for 3 days. siCtrl represents negative control for the siRNA knockdown. ( E ) Donor cells: HEK293T with stable overexpression of SBP tagged Cas9-GFP/gRNA (SBP-Cas9/gRNA). The recipient cell line was MDA-MB-231 with the reporter plasmid. The indicated genes were knocked down in donor cells via shRNA that were then co-cultured with recipient cells for 3 days. ( F ) Donor cells and acceptor cells were co-cultured for 4 days with either DMSO, 10, or 25 μM formin inhibitor, SMIFH2. The Nluc/Fluc signal detected after co-culture suggested that more than 70% of the cells remained viable during drug treatment. Nluc/Fluc assays were performed and normalized to an aliquot of co-cultured WT donor and reporter cells. Data in this figure represent mean ± SEM, n ≥ 3. ****p<0.0001, one-way ANOVA.

Article Snippet: Transfected construct (human) , siRNA to FLOT2 , QIAGEN , Hs_FLOT2_5 FlexiTube siRNA, SI02781422 , Transfected construct (human).

Techniques: Over Expression, Plasmid Preparation, Cell Culture, Negative Control, Knockdown, Co-Culture Assay, shRNA

Journal: eLife

Article Title: Syncytin-mediated open-ended membrane tubular connections facilitate the intercellular transfer of cargos including Cas9 protein

doi: 10.7554/eLife.84391

Figure Lengend Snippet:

Article Snippet: Transfected construct (human) , siRNA to FLOT2 , QIAGEN , Hs_FLOT2_5 FlexiTube siRNA, SI02781422 , Transfected construct (human).

Techniques: Cell Culture, Transfection, Construct, Transduction, Recombinant, Plasmid Preparation, shRNA, Isolation, Luciferase, Reporter Assay, SYBR Green Assay, Confocal Microscopy, Software

Journal: Diagnostic Pathology

Article Title: miR-449a targets Flot2 and inhibits gastric cancer invasion by inhibiting TGF-β-mediated EMT

doi: 10.1186/s13000-015-0435-5

Figure Lengend Snippet: miR-449a directly targeted Flot2. a Representative diagram of the predicted wild-type (WT) or mutant (Mut) binding site of miR-449a in the 3'- untranslated region (UTR) of Flot2 mRNA. The luciferase reporter plasmid containing the WT or Mut Flot2 3′-UTR was cotransfected into HEK293T cells with miR-449a mimics. Luciferase activity of the cells was assayed at 48 h after transfection, and the values were normalized to the normal control values. * P <0.01 (compared with the control). b qRT-PCR analysis revealed the effects of Flot2 siRNA and miR-449a mimics on the expression level of miR-449a. c Western blot analysis revealed the effects of Flot2 siRNA-810 and miR-449a mimics on the expression level of Flot2. Error bars represent ± S.E. and *, p < 0.01 versus control and NC

Article Snippet: The cells were incubated in an atmosphere of 5 % CO 2 at 37 °C. miR-449a mimics, negative controls (NC), and siRNA targeting human Flot2 mRNA were designed and synthesized by Shanghai GenePharma Company (Shanghai, China).

Techniques: Mutagenesis, Binding Assay, Luciferase, Plasmid Preparation, Activity Assay, Transfection, Control, Quantitative RT-PCR, Expressing, Western Blot

Journal: Diagnostic Pathology

Article Title: miR-449a targets Flot2 and inhibits gastric cancer invasion by inhibiting TGF-β-mediated EMT

doi: 10.1186/s13000-015-0435-5

Figure Lengend Snippet: MiR-449a repressed Flot2 regulates TGF-β-mediated EMT in GC. a MiR-449a induces EMT of GC cells by suppressing Flot2 expression. Western bolt analysis revealed the effects of miR-449a and Flot2 on EMT-relative protein expression. b Upregulation of Flot2 is necessary for TGF-β-induced EMT in GC. Western bolt analysis revealed the effects of TGF-β and Flot2 siRNA on EMT-relative protein expression. Error bars represent ± S.E. and *, p < 0.01 versus control. &, p < 0.01 versus TGF-β group

Article Snippet: The cells were incubated in an atmosphere of 5 % CO 2 at 37 °C. miR-449a mimics, negative controls (NC), and siRNA targeting human Flot2 mRNA were designed and synthesized by Shanghai GenePharma Company (Shanghai, China).

Techniques: Expressing, Western Blot, Control