sirna mix  (Santa Cruz Biotechnology)

Journal: Science Advances

Article Title: Mesothelial cells promote peritoneal invasion and metastasis of ascites-derived ovarian cancer cells through spheroid formation

doi: 10.1126/sciadv.adu5944

Figure Lengend Snippet: ( A ) Representative images of collagen invasion of spheroids composed of HPMCs or TGF-β1–stimulated HPMCs. Scale bars, 200 μm. ( B ) The bar graph showing TGF-β1–stimulated HPMCs showed a higher invasion ability into the collagen layer. ( C ) Images of migration or invasion cells using the Transwell assay. Scale bar, 200 μm. ( D ) Linear plots showing that TGF-β1–stimulated HPMCs had a higher migration and invasion ability compared with the control HPMCs. ( E ) Bar graph showing the concentration of TGF-β1 in the supernatant in EOC cells when inhibiting TGF-β1 with siRNA. ( F ) Representative images of spheroid collagen invasion. Scale bars, 100 μm. ( G ) Bar graphs showing that inhibition of TGF-β1 by siRNA in OV90 cells or the TGF-β1 receptor blocker in HPMCs reduced the invasion ability of ACMSs compared with the control. ( H and I ) Differences in the spheroids in ascites when mice were injected with OV90 (green) and HPMCs (red). The number of spheroids was significantly decreased when OV90 cells were treated with siRNA for TGF-β1 than those with si-control. Scale bars, 100 μm. ( J and K ) Representative images of metastases on the omentum and bar graph showing the metastasis area on the omentum. Scale bars, 1000 μm. ( L ) Bar graph showing the TGF-β1 concentration in the culture supernatant in sh-control– or TGF-β1–transduced OV90 cells. ( M and N ) Representative images and bar graph showing the omental metastatic area ( n = 8). Scale bars, 1000 μm. ( O and P ) Representative images of confocal imaging of the omental micrometastasis area and bar graph showing the invasion depth of mesothelial cells from the metastatic border. Scale bars, 100 μm. ( Q and R ) Representative images and bar graph showing that both the number and size of spheroids in ascites were significantly decreased in mice injected with sh-TGF-β1 no. 1 or 2 O90 cells compared with sh-control mice. Scale bars, 100 μm. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: As none of these siRNAs achieved significant mRNA suppression in EOC cells, we used a commercially available siRNA mix (Santa Cruz Biotechnology, sc-270322) for subsequent experiments (no. 4).

Techniques: Migration, Transwell Assay, Control, Concentration Assay, Inhibition, Injection, Imaging

Journal: Biogerontology

Article Title: Regulation of endothelial cell senescence via the miR-217/FOXO3 axis

doi: 10.1007/s10522-025-10290-3

Figure Lengend Snippet: Overexpression of miR-217 in HUVECs downregulated FOXO3 and FOXO3 as a direct target of miR-217. RT-qPCR analysis shows ( a ) increased expression of miR-217 and ( b ) reduction of FOXO3 mRNA expression after transfection with 50 nM miR-217 mimic for 48 h. U6 snRNA and B2M were used as normalisation, respectively. ( c ) Representative immunoblots and densitometry analysis showed downregulation of FOXO3 and protein expression in miR-217 mimic-transfected HUVECs. ( d ) TargetScanHuman 7.2 predicted the consequential pairing of target region between FOXO3 3′UTR and miR-217. The solid lines represent standard base pairing. ( e ) Bar charts show the firefly luciferase activity from WT and Mut experiment groups, respectively. The Firefly luciferase signal was normalised to internal Renilla luciferase signal. Statistical significance as analysed by Student’s t -test is expressed as * P < 0.05, *** P < 0.001 versus mimic negative control. ns: not significant

Article Snippet: The 982 bp wild-type FOXO3 3′UTR and mutant FOXO3 3′UTR fragment were synthesised and amplified (Integrated DNA Technologies, US), followed by fragment ligation into pmirGLO Dual-Luciferase miRNA Target Expression vector (Promega, #PROYY-E1330). jetPRIME transfection mix (Polyplus, #101000015) was prepared according to manufacturer’s protocol, where DNA to jetPRIME ratio (w/v) was maintained at 1:2.

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Transfection, Western Blot, Luciferase, Activity Assay, Negative Control

Journal: Biogerontology

Article Title: Regulation of endothelial cell senescence via the miR-217/FOXO3 axis

doi: 10.1007/s10522-025-10290-3

Figure Lengend Snippet: miR-217 inhibitor upregulated FOXO3 and induced p16INK4a selectively in early-stage senescent (ES) HUVECs. RT-qPCR analysis shows ( a ) decreased expression of miR-217 and ( b ) increased FOXO3 mRNA expression in RS and ES HUVECs after transfection with 50 nM miR-217 inhibitor for 48 h. U6 snRNA and B2M were used for normalisation. Representative immunoblots and densitometry analysis show ( c ) upregulation of FOXO3 protein expression in miR-217 inhibitor-transfected RS HUVECs and ( d ) upregulation of FOXO3 and p16INK4a protein expression in miR-217 inhibitor-transfected ES HUVECs. Statistical significance as analysed by Student’s t -test is expressed as * P < 0.05, ** P < 0.01, *** P < 0.001 versus inhibitor negative control. ns: not significant

Article Snippet: The 982 bp wild-type FOXO3 3′UTR and mutant FOXO3 3′UTR fragment were synthesised and amplified (Integrated DNA Technologies, US), followed by fragment ligation into pmirGLO Dual-Luciferase miRNA Target Expression vector (Promega, #PROYY-E1330). jetPRIME transfection mix (Polyplus, #101000015) was prepared according to manufacturer’s protocol, where DNA to jetPRIME ratio (w/v) was maintained at 1:2.

Techniques: Quantitative RT-PCR, Expressing, Transfection, Western Blot, Negative Control

Journal: iScience

Article Title: Evolutionary conserved regulation of TFEB stability by the E3 ubiquitin ligase WWP2 modulates response to stress in vivo

doi: 10.1016/j.isci.2025.111838

Figure Lengend Snippet: Conserved interaction of HLH-30 with WWP-1 involves human ortholog WWP2 which also regulates TFEB stability (A, B, D, and E) Protein lysates from HEK293T cells transfected with the indicated plasmids were subjected to Flag immunoprecipitation (IP: Flag) prior to immunoblot (IB) analysis. Whole cell lysate (WCL) IB analysis shows total protein expression. One representative experiment of at least two biological replicates. (C) Schematic representation of TFEB protein structure showing the glutamine-rich domain (Gln rich), the activation domain (AD), the basic-helix-loop-helix domain (bHLH), the leucine zipper domain (LZ) and the proline rich domain (Pro rich), as well as the localization of the PPxY (PY) motif. (F and G) Immunoblot (IB) analysis of endogenous TFEB and WWP2 from HeLa transfected with control (siCtrl) and Wwp2 ( siWwp2 ) siRNA for 48 h, and starved for 30 min (F) or treated with cycloheximide (CHX) at 20 ng/mL for 3 or 6 h (G). IB: actin serve as loading control. (H) Representative graph of TFEB/actin levels over time after CHX treatment as shown in G. See also Figure S6 .

Article Snippet: Wwp2 siRNA mix , Santa cruz , Cat# sc-40362.

Techniques: Transfection, Immunoprecipitation, Western Blot, Expressing, Activation Assay, Control

Journal: iScience

Article Title: Evolutionary conserved regulation of TFEB stability by the E3 ubiquitin ligase WWP2 modulates response to stress in vivo

doi: 10.1016/j.isci.2025.111838

Figure Lengend Snippet: Direct ubiquitination and stabilization of TFEB by WWP2 (A and D–F) Protein lysates from HEK293T cells transfected with the indicated plasmids were subjected to histidine pull-down (His-PD) assay using TALON cobalt beads prior to immunoblot (IB) analysis. IB analysis shows the total protein expression in whole cell lysate. IB: c-myc is used as a positive control of MG132 treatment. 6His-Ub K48 and 6His-Ub K63 encode 6His-tagged form of Ubiquitin where all lysine residues, except K48 or K63, have been mutated to arginine. One representative experiment of at least two biological replicates. (B) Immunoblot analysis of endogenous TFEB (IB: TFEB) and Flag-WWP2 (IB: Flag) from HeLa transfected with Flag-WWP2 WT or the catalytic inactive mutant Flag-WWP2 C838A. IB: actin serve as loading control. (C) qPCR analysis of Tfeb mRNA level in HeLa transfected with Flag-WWP2 WT or C838A. Scatterplot with bars representing mean ± sem. ns: non-significant (One-way ANOVA with Šidák’s multiple comparisons test on ΔCt values). (G) Immunoblot (IB) analysis of in vitro ubiquitination assay of recombinant 6His-TFEB in presence of recombinant 6His-WWP2 WT or phospho-mimetic mutant Y369E. 6His-TFEB and/or 6His-WWP2 WT and Y369E were incubated for 2 h at 30°C prior to analysis. Input corresponds to the samples prior to incubation at 30°C. One representative experiment of two biological replicates. See also Figures S7 and .

Article Snippet: Wwp2 siRNA mix , Santa cruz , Cat# sc-40362.

Techniques: Ubiquitin Proteomics, Transfection, Western Blot, Expressing, Positive Control, Mutagenesis, Control, In Vitro, Recombinant, Incubation

Journal: iScience

Article Title: Evolutionary conserved regulation of TFEB stability by the E3 ubiquitin ligase WWP2 modulates response to stress in vivo

doi: 10.1016/j.isci.2025.111838

Figure Lengend Snippet: WWP2 regulates TFEB stability and TFEB-dependent host response to S. aureus in primary hMDMs (A) Immunoblot analysis of endogenous TFEB (IB: TFEB) and WWP2 (IB: WWP2) from hMDMs transfected with control or Wwp2 siRNA, incubated 72 h prior to S. aureus infection (6 h). IB: actin serves as loading control. (B–D) Graphs represent relative expression of Wwp2 (B), Tfeb (B) and TFEB target genes (C and D) measured by RT-qPCR in hMDMs transfected with control or Wwp2 siRNA prior to S. aureus infection (6 h). Scatterplot with matched values. ∗∗: p < 0.01, ∗∗∗: p < 0.001 (two-way ANOVA with uncorrected Fisher’s LSD multiple comparisons test on ΔCt values). Experiment was performed in 3 independent biological replicates ( N = 3). (E) Graph represents the fold change expression of TFEB target genes in si Wwp2 condition relative to siCtrl, in uninfected (blue) or S. aureus infected (red) conditions. See also Figure S9 .

Article Snippet: Wwp2 siRNA mix , Santa cruz , Cat# sc-40362.

Techniques: Western Blot, Transfection, Control, Incubation, Infection, Expressing, Quantitative RT-PCR

Journal: iScience

Article Title: Evolutionary conserved regulation of TFEB stability by the E3 ubiquitin ligase WWP2 modulates response to stress in vivo

doi: 10.1016/j.isci.2025.111838

Figure Lengend Snippet:

Article Snippet: Wwp2 siRNA mix , Santa cruz , Cat# sc-40362.

Techniques: Virus, Recombinant, Ubiquitin Proteomics, Luciferase, Mutagenesis, Reverse Transcription, SYBR Green Assay, Plasmid Preparation, Software, Lysis