Journal: Oncogene

Article Title: Wnt target IQGAP3 promotes Wnt signaling via disrupting Axin1-CK1α interaction

doi: 10.1038/s41388-025-03512-y

Figure Lengend Snippet: A Immunoblot of NUGC3 wild-type and IQGAP3 CRISPR knock-out clones. Samples were subjected to SDS-PAGE (7.5%) followed by immunoblotting using the indicated antibodies. The relative immunoblot bands of β-catenin and β-actin (βcat/βact) were quantified by densitometry. B NUGC3 cells were lysed, and RNA collected were converted to cDNA and subjected to RT-PCR to quantify the amount of Wnt target genes CCND1 and MYC . Representative data were collected and are expressed as the mean ± SD from three independent experiments. Student’s t test was performed, with ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, and ∗∗∗ p ≤ 0.001. C XTT Cell Proliferation Assay of NUGC3 wild-type and IQGAP3 CRISPR knock-out clones. Representative data were collected and are expressed as the mean % growth ±SD from three independent experiments. Two-way ANOVA was performed, with ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, and ∗∗∗ p ≤ 0.001. D Clonogenic Assay of NUGC3 wild-type and IQGAP3 CRISPR knock-out clones. E Colony-Forming Efficiency (%) of NUGC3 wild-type and IQGAP3 CRISPR knock-out clones. F Immunoblot of AGS wild-type and IQGAP3 CRISPR knock-out clones. Image is best representative of three independent experiments. Samples were subjected to SDS-PAGE (7.5%) followed by immunoblotting using the indicated antibodies. The relative immunoblot bands of β-catenin and α-Tubulin (βcat/αTub) were quantified by densitometry. G AGS cells were lysed, and RNA collected were converted to cDNA and subjected to RT-PCR to quantify the amount of Wnt target genes CCND1 and MYC . Representative data were collected and are expressed as the mean ± SD from three independent experiments. Student’s t test was performed, with ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, and ∗∗∗ p ≤ 0.001. H XTT Cell Proliferation Assay of AGS wild-type and IQGAP3 CRISPR knock-out clones. Representative data were collected and are expressed as the mean % growth ±SD from three independent experiments. Two-way ANOVA was performed, with ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, and ∗∗∗ p ≤ 0.001. I Clonogenic Assay of AGS wild-type and IQGAP3 CRISPR knock-out clones. J Colony-Forming Efficiency (%) of AGS wild-type and IQGAP3 CRISPR knock-out clones.

Article Snippet: Cell proliferation was determined using the TACS® XTT Cell Proliferation Assay (R&D Systems, Inc.), following the manufacturer’s instructions.

Techniques: Western Blot, CRISPR, Knock-Out, Clone Assay, SDS Page, Reverse Transcription Polymerase Chain Reaction, Proliferation Assay, Clonogenic Assay

Journal: PLOS ONE

Article Title: Mutation of SIVA , a candidate metastasis gene identified from clonally related bilateral breast cancers, promotes breast cancer cell spread in vitro and in vivo

doi: 10.1371/journal.pone.0302856

Figure Lengend Snippet: ( A ) Schematic of SIVA protein showing the position (red line in exon 4) of point mutation identified in the “breast to breast” spread in patient 8. Domains: Amphipathic helix (SAH), death domain homology region (DDHR), cysteine rich domains (Cys-rich). ( B ) Stable knockdown of SIVA protein expression by shRNA (KO) and reintroduction of SIVA-WT protein (WT*), compared to endogenous (end) SIVA expression in empty vector control cells (EV) was demonstrated by Western blot analysis. ( C) 231L cell migration (Boyden Chamber) and (D) 231L cell invasion (Matrigel matrix invasion) analyses were expressed as total cell counts in four fields of view (4 fov) normalized against EV control. Each bar represents the mean ± S.E.M (n = 3, total cells in 4 images/assay); One-way ANOVA ( C : p = 0.0035 and D : p = 0.0.0002) followed by Tukey’s post-hoc test. ( E ) Overexpression of SIVA-WT (WT) and mutated SIVA (D160N) protein, compared to endogenous SIVA levels in EV control 231L cells was demonstrated by Western blot analysis. ( F ) Cell migration and ( G ) cell invasion analyses of 231L cells overexpressing wild type SIVA (WT) and SIVA mutation ( D160N ) were expressed as in ( C , D ). One-way ANOVA ( F : p = 0.0002, G : p = 0.0005). ( H ) Proliferation XTT assays, each point represents the mean ± S.E.M (n = 2, 6 replicates/assay); simple linear regression ( p = 0.8788). ( I ) SIVA-D160N expressing 231L cells increased spread of breast cancer cells in immuno-deficient NSG mice. 25K cells were injected into the lateral tail veins of female NSG mice (n = 5), and the proliferation and spread monitored by serial in vivo imaging (IVIS). Exposure times are 60s and 3s for 7 days and 21 days respectively. Luminescence counts is indicated in colored bar on the y -axis. ( J ) Absolute luminescent intensity of photons emitted from spreading tumor cells in each animal in the IVIS images ( I ) was quantified. Each line represents the mean ± SEM (n = 5). Mixed-effects analysis ( p <0.0001) followed by Tukey’s post-hoc test. Post Test p values: * p <0.05, ** p <0.01, *** p <0.001.

Article Snippet: Cancer cell proliferation in vitro was determined by the Trevigen TACS®XTT Proliferation Assay (R&D Systems, Cat# 4891-025-K) according to manufacturer’s instructions.

Techniques: Mutagenesis, Expressing, shRNA, Plasmid Preparation, Western Blot, Migration, Over Expression, Injection, In Vivo Imaging

Journal: International Journal of Molecular Sciences

Article Title: A Fast and Reliable Process to Fabricate Regenerated Silk Fibroin Solution from Degummed Silk in 4 Hours

doi: 10.3390/ijms221910565

Figure Lengend Snippet: Cytocompatibility of silk fibroin films: Left : XTT assay for cell viability and proliferation of L929 cells cultivated on films from silk fibroin for 24 h, 48 h, and 72 h, respectively. The mean cell metabolic activity and standard error of the mean are reported for three independent silk fibroin samples dissolved with ZnCl 2 in triplicates. Right : Representative fluorescence microscopy image of live-dead staining of L929 cells after 72 h cultivated on films from silk fibroin dissolved in ZnCl 2 .

Article Snippet: The XTT Cell Proliferation Assay Kit (Serva, Heidelberg, Germany) was used to quantitatively evaluate cell metabolic activity according to the manufacturer’s protocol after 24, 48, and 72 h, respectively.

Techniques: XTT Assay, Activity Assay, Fluorescence, Microscopy, Staining

Journal: bioRxiv

Article Title: Therapeutic reversal of prenatal pontine ID1 signaling in DIPG

doi: 10.1101/2021.05.10.443452

Figure Lengend Snippet: (A) Western blot (WB) confirming ID1 knockdown in DIPG007 cells. (B) WB depicting reduction in SPARCL1 expression along with decreased ID1 expression in ID1 -knockdown DIPG007 cells. (C) Effect of ID1 knockdown on invasion as measured by Matrigel-coated Boyden chamber assay. Images show invading cells stained with crystal violet. Each data point represents an individual image; **P < 0.01, unpaired parametric t-test. (D-E) Effect of ID1 knockdown on DIPG007 migration as measured by scratch assay, quantified as percent wound closure. Images show representative scratch at 0 and 24 hours outlined in dotted red line. Experiment was completed in triplicate and data points represent mean+/-SEM, **P < 0.01; images taken with Incucyte; area measured by ImageJ. (F) WB for ID1 and ACTB expression in DIPG007 and PPK cells treated with increasing concentrations of CBD or DMSO control. (G) Viability of DIPG007 and PPK cells treated with increasing concentrations of CBD (0.5-20µM) relative to DMSO-treated control. Experiment was completed in triplicate and data points represent mean+/-SEM. (H) DIPG007 cells were treated for 2 days with DMSO (control), 2.5µM or 5µM CBD and invasion was measured by Matrigel-coated Boyden chamber. Each data point represents an individual image, mean+/-SEM; ****P < 0.0001, unpaired parametric t-test. (I) Effect of CBD treatment (5-10µM) on DIPG007 migration as measured by scratch assay, quantified as percent wound closure. Experiment was completed in triplicate and data points represent mean+/-SEM, **P < 0.005, two-way ANOVA t-test. (J) Histogram showing increase in DCF (ROS) with increasing doses of CBD. (K) Production of ROS mediates the inhibitory activity of CBD through ID1. DIPG007 cells were treated for 72 hours with vehicle (DMSO) or different concentrations of CBD (10, 8, 6, 4, 2, 1 µM) in the presence and absence of 50 µM TOC. IC 50 was 2.3µM for CBD treatment alone and 10.34µM for CBD + TOC; ****P < 0.0001, two-way ANOVA t-test. Cell proliferation was measured using XTT assay.

Article Snippet: After 72 hours, in vitro cell viability was monitored by XTT Cell Proliferation Assay kit (Cayman Chemical).

Techniques: Western Blot, Expressing, Boyden Chamber Assay, Staining, Migration, Wound Healing Assay, Activity Assay, XTT Assay