Journal: Molecular Cancer

Article Title: t-DARPP regulates phosphatidylinositol-3-kinase-dependent cell growth in breast cancer

doi: 10.1186/1476-4598-9-240

Figure Lengend Snippet: t-DARPP expression directly correlates with activation of AKT pathway in breast cancer cell lines, and knockdown of endogenous t-DARPP suppresses cell growth . A , Protein extracts from a panel of 8 breast cancer cell lines were subjected to western blot analysis of t-DARPP, p-AKT ser473 , AKT, p-GSK3β ser9 , GSK3β, and β-actin. 10 μg of total protein per lane were resolved on 10% SDS-PAGE and transferred onto Hybond-P PVDF membranes for immunodetection with the corresponding specific antibodies. B&C , HCC-1569 cells were transfected with control scrambled siRNA or t-DARPP siRNA oligonucleotides and grown for 48 h. Protein extracts were subjected to western blot analysis of t-DARPP, p-AKT ser473 , AKT, p-GSK3β ser9 , and GSK3β (panel B). Knockdown of t-DARPP in HCC-1569 cells led to down-regulation of the AKT signaling pathway as indicated by a significant decrease of p-AKT ser473 and p-GSK3β ser9 protein levels. The cells were subjected to Cell-Titer-Glo Luminescent Cell Viability Assay. Overall, cell growth was significantly lower in HCC-1569 cells transfected with t-DARPP siRNA than control cells (panel C). Results are representative of three experiments and shown as the mean ± SD. Significance of difference was calculated using Student's t test analysis.

Article Snippet: The HCC-1569 cells were transfected with control scrambled siRNA (sc-37007) or t-DARPP siRNA (sc-35173) using siRNA transfection reagent (sc-29528) and transfection medium (sc-36868) following the manufacturer's instructions (Santa Cruz Biotechnology).

Techniques: Expressing, Activation Assay, Western Blot, SDS Page, Immunodetection, Transfection, Cell Viability Assay

Journal: Molecular Cancer

Article Title: t-DARPP regulates phosphatidylinositol-3-kinase-dependent cell growth in breast cancer

doi: 10.1186/1476-4598-9-240

Figure Lengend Snippet: t-DARPP-induced cell growth is dependent on PI3K/AKT signaling pathway . A , MCF-7 cells stably expressing t-DARPP or pcDNA3.1 vector were treated with either vehicle or 40 μM of LY294002 for 30 min or 2 h. The cells were washed with PBS and grown for 24 h, and then subjected to Cell-Titer-Glo Luminescent Cell Viability Assay. The results indicated a significant increase of cell growth in t-DARPP-expressing cells relative to control cells after treatment with vehicle. This growth increase was almost completely abrogated after treatment with LY294002. B , MCF-7/t-DARPP and MCF-7/pcDNA3.1 cells were treated with vehicle and 40 μM of LY294002 for 30 min or 2 h, and then grown for 24 h. Protein extracts were subjected to western blot analysis of t-DARPP, p-AKT ser473 , and AKT. The results showed that p-AKT ser473 protein level was significantly higher in t-DARPP-expressing cells than control cells, and treatment with LY294002 blocked phosphorylation of AKT ser473 in all cells. C , MCF-7/t-DARPP and MCF-7/pcDNA3.1 cells were transfected with scrambled siRNA or Akt siRNA and grown for 48 h. The cells were then subjected to Cell-Titer-Glo Luminescent Cell Viability Assay. The results indicated a significant increase of cell growth in t-DARPP-expressing cells relative to control cells after transfection with scrambled siRNA. This growth increase was partially abrogated after transfection with Akt siRNA. D , MCF-7/t-DARPP and MCF-7/pcDNA3.1 cells were transfected with scrambled siRNA or Akt siRNA and cultured for 48 h. Protein extracts were analyzed by Western blot for p-AKT ser473 , AKT, t-DARPP, and β-actin. Results are representative of four experiments and shown as the mean ± SD. Significance of difference was calculated using Student's t test analysis.

Article Snippet: The HCC-1569 cells were transfected with control scrambled siRNA (sc-37007) or t-DARPP siRNA (sc-35173) using siRNA transfection reagent (sc-29528) and transfection medium (sc-36868) following the manufacturer's instructions (Santa Cruz Biotechnology).

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Cell Viability Assay, Western Blot, Transfection, Cell Culture

Journal: Frontiers in Immunology

Article Title: HMGB1 Promotes the Release of Sonic Hedgehog From Astrocytes

doi: 10.3389/fimmu.2021.584097

Figure Lengend Snippet: The effect of HMGB1 on expression and release of shh in astrocytes. (A) The protein from astrocytes was obtained and detected by western blot (1μg/ml recombinant HMGB1 was used). (B) The level of shh in supernatant from astrocytes after recombinant HMGB1 (1μg/ml) or (C) brain homogenate of EAE onset stage (100μg/ml) with/without HMGB1 Ab/IgG (5μg/ml) stimulation for 24h were detected by ELISA. EAE homogenate (cell-free) group here indicates interstitial fluid (100μg/ml) from the onset stage of EAE mice without cultured astrocytes. All the data are shown as mean ± SD (*** P < 0.001 compared with medium; # P < 0.05 compared with each other).

Article Snippet: Recombinant mouse sonic hedgehog (Shh) protein (R&D system, Minneapolis, MN, USA) in 10 μL saline or 10 μL saline was delivered over a 2-min period every day from day 11 to day 19 post immunization.

Techniques: Expressing, Western Blot, Recombinant, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Frontiers in Immunology

Article Title: HMGB1 Promotes the Release of Sonic Hedgehog From Astrocytes

doi: 10.3389/fimmu.2021.584097

Figure Lengend Snippet: The effect of HMGB1 receptors on the release of shh in astrocytes. (A) Three surface receptors for HMGB1 were analyzed by flow cytometry after HMGB1 stimulation (2μg/ml) in astrocytes. (B) The release of shh from astrocytes was detected after TLR2, TLR4 and RAGE were knocked out (2μg/ml and 1μg/ml recombinant HMGB1 was used in TLR2, TLR4 and RAGE knockout astrocytes respectively). (C) The effect of TLR4 blocker (TAK-242: 100nM) and RAGE blocker (FPS-ZM1: 148nM) on the release of shh in astrocytes (1μg/ml recombinant HMGB1 was used). Data are shown as mean ± SD (* P < 0.05, ** P < 0.01, *** P < 0.001). ns, no significance.

Article Snippet: Recombinant mouse sonic hedgehog (Shh) protein (R&D system, Minneapolis, MN, USA) in 10 μL saline or 10 μL saline was delivered over a 2-min period every day from day 11 to day 19 post immunization.

Techniques: Flow Cytometry, Recombinant, Knock-Out

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: t-Darpp activates IGF-1R signaling to regulate glucose metabolism in trastuzumab-resistant breast cancer cells

doi: 10.1158/1078-0432.CCR-17-0824

Figure Lengend Snippet: (A) Forest plots of the hazard ratios for 20 year OS, associated with expression of IGF-1R (top) and PPP1R1B (bottom). (B) Kaplan-Meier survival curves for patients with HER2+ (top) and HER2− (bottom) tumors, comparing tumors with PPP1R1B over-expression (PPP1R1Bhigh) to tumors with normal PPP1R1B (PPP1R1Blow) levels. (C) Kaplan-Meier survival curves for the effect of PPP1R1B (left) and IGF-1R (right) on 5-year OS in breast cancer patients. (D) Kaplan-Meier survival curve using an index value comprised of the mean expression of PPP1R1B and the inverse expression of IGF-1R (PPP1R1B expression + (−1) * IGF-1R expression). HR=hazard ratio; all p-values displayed are log rank values, *p<0.05, **p<0.01, ****p<0.0001.

Article Snippet: Expression knockdown was performed using validated siRNA: IGF-1R (Ambion IGF1R Silencer validated AM51331, ID:74), INSR (AM51331, ID:29) and PPP1R1B (Santa Cruz Biotechnology, sc-35173) and transfected using RNAiMAX (Invitrogen, Lipofectamine RNAiMAX, 13778–075), according to the manufacturer’s protocol.

Techniques: Expressing, Over Expression

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: t-Darpp activates IGF-1R signaling to regulate glucose metabolism in trastuzumab-resistant breast cancer cells

doi: 10.1158/1078-0432.CCR-17-0824

Figure Lengend Snippet: (A) Seahorse flux analysis comparing glycolysis and glycolytic capacity between cells transfected with t-Darpp siRNA (si-tDrp) and control siRNA (si-Scrambled). (B) ATP levels in untreated (UT) cells and in cells following 30 min exposure to2.5µM trastuzumab, 50mM 2-DG, or both, in cells transfected with t-Darpp/PPP1R1B (si-tDrp) siRNA or control siRNA (si-Scrambled). (C) Fold change in glycolysis in SK.HerR cells following siRNA knock-down of IGF-1R or IR compared to a scrambled siRNA control, with corresponding Western blots of the receptors. (D) Growth inhibition of SK-BR-3 derived cell lines by trastuzumab, NVP-AEW or combined treatment. SK-BR-3, SK.tDrp and SK.HerR were incubated for 3 days in SFM only (untreated, UT) or in the presence of either trastuzumab, NVP-AEW or both. Cell number was measured by WST-1 and normalized to untreated wells. (E) Cellular ATP levels were measured in untreated (UT) cells and in cells following 30 min exposure to 2.5µM trastuzumab, 2µM NVP-AEW, or the combination. Mean values (±S.D.) normalized to UT (=1.0) are reported for SK-BR-3 versus SK.HerR cells (left) and SK.empty versus SK.tDrp cells (right). ***p<0.001 ****p<0.0001

Article Snippet: Expression knockdown was performed using validated siRNA: IGF-1R (Ambion IGF1R Silencer validated AM51331, ID:74), INSR (AM51331, ID:29) and PPP1R1B (Santa Cruz Biotechnology, sc-35173) and transfected using RNAiMAX (Invitrogen, Lipofectamine RNAiMAX, 13778–075), according to the manufacturer’s protocol.

Techniques: Transfection, Western Blot, Inhibition, Derivative Assay, Incubation