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mesorhizobium loti (ATCC)

Name: mesorhizobium loti
Brand: ATCC
Rating: 85 (13 reviews)

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ATCC mesorhizobium loti
Mesorhizobium Loti, supplied by ATCC, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 85 stars, based on 1 article reviews

mesorhizobium loti - by Bioz Stars, 2025-07

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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.

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

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.

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.

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

a Top: schematic of the experimental protocol. Bottom: representative picture of a coronal brain section from an 8-week-old mouse stained with antibodies against DCX (red), showing neuroblasts in the V-SVZ. Higher magnifications of the boxed areas (dashed yellow lines) are shown on the right. a1 High-magnification image from the area indicated with the yellow dashed rectangle in a, showing DCX and BrdU immunostainings. a2 High-magnification image from the area indicated with the yellow dashed rectangle in a1 . Newborn neuroblasts (DCX+/BrdU+) in the ventral V-SVZ and NAc. a3 – a5 . Left: high-magnification maximum-projection images of newborn neuroblasts (DCX+/BrdU+) in the NAc. Right: orthogonal views. b TEM image showing a neuroblast in mitosis. b1 High-magnification image from the area indicated with the yellow dashed box in b . c Schematic of the experimental protocol. d Left: schematic illustrates the imaged brain area containing the NAc with both core and shell. Right: quantification of newborn neurons in the core and shell of the NAc in adult mice injected with BrdU as depicted in c ( n = 8 mice). d Example of newborn cells labeled by BrdU+ and the mature neuronal marker NeuN+. e Example of newborn neurons triple-labeled with BrdU, NeuN, and the MSN marker DARPP-32. f , g Confocal pictures showing BrdU labeling in D1 receptor (EGFP in f ) or D2 receptor (EGFP in g ) expressing neurons. Abbreviations: ac, anterior commissure; dSt, dorsal striatum; lv, lateral ventricle; NAc, nucleus accumbens; Sp, septum. Scale bars in µm: 1000 ( a ), 100 ( a1 ), 50 ( a2 ), 5 ( a3 – a5 , e – g ), 2 ( b ), 500 nm ( b1 ). See Supplementary Fig. .

Journal: Molecular Psychiatry

Article Title: Neurogenesis of medium spiny neurons in the nucleus accumbens continues into adulthood and is enhanced by pathological pain

doi: 10.1038/s41380-020-0823-4

Figure Lengend Snippet: a Top: schematic of the experimental protocol. Bottom: representative picture of a coronal brain section from an 8-week-old mouse stained with antibodies against DCX (red), showing neuroblasts in the V-SVZ. Higher magnifications of the boxed areas (dashed yellow lines) are shown on the right. a1 High-magnification image from the area indicated with the yellow dashed rectangle in a, showing DCX and BrdU immunostainings. a2 High-magnification image from the area indicated with the yellow dashed rectangle in a1 . Newborn neuroblasts (DCX+/BrdU+) in the ventral V-SVZ and NAc. a3 – a5 . Left: high-magnification maximum-projection images of newborn neuroblasts (DCX+/BrdU+) in the NAc. Right: orthogonal views. b TEM image showing a neuroblast in mitosis. b1 High-magnification image from the area indicated with the yellow dashed box in b . c Schematic of the experimental protocol. d Left: schematic illustrates the imaged brain area containing the NAc with both core and shell. Right: quantification of newborn neurons in the core and shell of the NAc in adult mice injected with BrdU as depicted in c ( n = 8 mice). d Example of newborn cells labeled by BrdU+ and the mature neuronal marker NeuN+. e Example of newborn neurons triple-labeled with BrdU, NeuN, and the MSN marker DARPP-32. f , g Confocal pictures showing BrdU labeling in D1 receptor (EGFP in f ) or D2 receptor (EGFP in g ) expressing neurons. Abbreviations: ac, anterior commissure; dSt, dorsal striatum; lv, lateral ventricle; NAc, nucleus accumbens; Sp, septum. Scale bars in µm: 1000 ( a ), 100 ( a1 ), 50 ( a2 ), 5 ( a3 – a5 , e – g ), 2 ( b ), 500 nm ( b1 ). See Supplementary Fig. .

Article Snippet: Brain sections were stained for the following: EGFP/YFP (rabbit, Invitrogen, Cat# A-6455, 1 : 1000), BrdU (mouse, BD, Cat# 347580, 1 : 1000), NeuN (rabbit and mouse, Millipore, Cat# MAB377, 1 : 1000; chicken, Synaptic System, Cat# 266 006, 1 : 250), DARPP-32 (rabbit, Santa Cruz, Cat# ab40801, 1 : 300), Calbindin (rabbit, Swant, Cat# 300, 1 : 3000), DCX (goat, Santa Cruz, Cat# sc-8066, 1 : 500), Iba1 (rabbit, Abcam, Cat# EPR16588, 1 : 500), Olig2 (rabbit, Invitrogen, Cat# PA5–85734, 1 : 500), PSA-NCAM (mouse, Thermo Fisher Scientific, Cat# 14–9118–80, 1 : 250), Collagen IV (rabbit, Abcam, Cat# ab19808, 1 : 500), Nestin (chicken, Novus Biological, Cat# NB100–1604, 1 : 250), GFAP (rabbit, DAKO, Cat# GA52461–2, 1 : 500), Vimentin (goat, Santa Cruz, Cat# sc-7557, 1 : 500), Sox2 (Santa Cruz, Cat# sc-17320, 1 : 500), Ki67 (rabbit, Abcam, Cat# ab15580, 1 : 250).

Techniques: Staining, Injection, Labeling, Marker, Expressing

a Experimental schematic: P4-day-old pups were injected with a RFP-expressing retrovirus in the V-SVZ and analyzed 4–8 weeks later. b Coronal section of P56 wild-type mouse brain immunostained for RFP, DARPP-32, and NeuN. Inset: schematic of the anatomical region in a coronal section. The red rectangle denotes the area in the picture. c , d High-magnification images from the area indicated with the yellow dashed rectangle in b . d1 , d2 Magnifications show segments of a dendrite bearing spines. d3 High-magnification image of the retrovirus-infected soma, indicating that this cell expresses typical markers for mature MSNs in the NAc (DARPP-32 and NeuN). e Top: morphological reconstruction of a biocytin-filled virus-infected neuron in the NAc. Bottom: representative firing patterns of virus-infected neurons recorded in the NAc. f Schematic of the experimental protocol. g Overview of the NAc immunostained for DAPI, biocytin, DARPP-32, and YFP. Inset: low-magnification image of a coronal brain section of a P130 wild-type mouse immunostained for DAPI. h High-magnification image of the yellow dashed rectangle. h1 – h4 Enlargement of the areas indicated in the picture, showing dendritic spines. h5 High-magnification image of the biocytin-filled cell body, showing positive expression for YFP and DARPP-32. i Left: morphological reconstruction of biocytin-filled YFP-expressing neuron shown in h . Right: representative firing pattern of the YFP-labeled MSN recorded in the NAc. Abbreviations: ac, anterior commissure; NAc, nucleus accumbens; lv, lateral ventricle; Sp, septum. Scale bars in µm: 500 (onset in g ), 200 ( g ) 50 ( b , e , i ), 20 ( c , d , h ), 5 ( d3 , h1 – h5 ), 1 ( d1 , d2 ). See also Supplementary Fig. and Supplementary Movie .

Journal: Molecular Psychiatry

Article Title: Neurogenesis of medium spiny neurons in the nucleus accumbens continues into adulthood and is enhanced by pathological pain

doi: 10.1038/s41380-020-0823-4

Figure Lengend Snippet: a Experimental schematic: P4-day-old pups were injected with a RFP-expressing retrovirus in the V-SVZ and analyzed 4–8 weeks later. b Coronal section of P56 wild-type mouse brain immunostained for RFP, DARPP-32, and NeuN. Inset: schematic of the anatomical region in a coronal section. The red rectangle denotes the area in the picture. c , d High-magnification images from the area indicated with the yellow dashed rectangle in b . d1 , d2 Magnifications show segments of a dendrite bearing spines. d3 High-magnification image of the retrovirus-infected soma, indicating that this cell expresses typical markers for mature MSNs in the NAc (DARPP-32 and NeuN). e Top: morphological reconstruction of a biocytin-filled virus-infected neuron in the NAc. Bottom: representative firing patterns of virus-infected neurons recorded in the NAc. f Schematic of the experimental protocol. g Overview of the NAc immunostained for DAPI, biocytin, DARPP-32, and YFP. Inset: low-magnification image of a coronal brain section of a P130 wild-type mouse immunostained for DAPI. h High-magnification image of the yellow dashed rectangle. h1 – h4 Enlargement of the areas indicated in the picture, showing dendritic spines. h5 High-magnification image of the biocytin-filled cell body, showing positive expression for YFP and DARPP-32. i Left: morphological reconstruction of biocytin-filled YFP-expressing neuron shown in h . Right: representative firing pattern of the YFP-labeled MSN recorded in the NAc. Abbreviations: ac, anterior commissure; NAc, nucleus accumbens; lv, lateral ventricle; Sp, septum. Scale bars in µm: 500 (onset in g ), 200 ( g ) 50 ( b , e , i ), 20 ( c , d , h ), 5 ( d3 , h1 – h5 ), 1 ( d1 , d2 ). See also Supplementary Fig. and Supplementary Movie .

Techniques: Injection, Expressing, Infection, Labeling

a Schematic of the experimental protocol. b Quantification of newborn neuroblasts in the NAc 7 days after sham or SNI surgery (left) (unpaired t- test * p = 0.0026, n = 10 mice per group) or after PBS or CFA paw injection (right) (unpaired t- test * p = 0.0131, n = 7 and 8 mice per group, respectively). c , d Representative pictures of coronal brain sections from mice subjected to either sham ( c ) or SNI surgery ( d ), stained with antibodies against BrdU (white) and DCX (red). d1 , d2 High-magnification images of the dashed yellow rectangles are shown on the right. e Experimental schematic to test the effect of chronic pain in the inflammatory model (CFA/PBS injection) or in the neuropathic pain model (SNI/sham) in the NAc after 6 weeks. f Quantification of mature postnatally born neurons (NeuN+/BrdU+) in the NAc, 4 weeks after the last BrdU injection (left: unpaired t- test * p = 0.041, n = 8 mice per group for sham/SNI; right: unpaired t- test **** p < 0.0001, n = 10 mice per group for PBS/CFA injections). g Quantification of mature postnatally born neurons (NeuN+/BrdU+) in the NAc counterstained with the MSNs marker DARPP-32, 6 weeks after CFA or PBS injection (unpaired t- test *** p = 0.0003, n = 5 mice per group). h Representative picture of a coronal brain section from a CFA-treated mouse showing examples of BrdU+ (gray)/ NeuN+ (green)/ DARPP-32+ (magenta) neurons in the NAc. h1 Higher magnification of the dashed yellow rectangle for each channel is shown on the right. The inset indicates the imaged brain area (dashed yellow rectangle). i Experimental schematic to test the origin of NAc adult-born neurons after inflammatory pain. j Representative pictures of coronal sections comprising the lateral ventricles showing YFP-labeled neural stem cells in the V-SVZ of control (left) and CFA-treated mice (right). Pictures on the right are enlargements of the indicated dashed yellow rectangles showing YFP-labeled cells (green) positive for Sox2 (magenta). k Confocal picture showing newborn (BrdU in white) neuroblasts (DCX in magenta) positive for YFP (green) with migratory appearance in the NAc of a CFA-treated mouse. k1 Enlarged pictures of the boxed area in k for each channel. l Quantification of (DCX+/BrdU+) newborn neuroblasts in the NAc positive for YFP in control (PBS) or pain (CFA)-treated animals ( t -test * p = 0.035, n = 6 mice per group). m Representative image of a coronal brain section from a CFA-treated mouse stained with antibodies against YFP (green) and NeuN (magenta), showing newborn neurons in the NAc 72 days after the first tamoxifen injection. m1 Enlarged picture of the dashed yellow rectangle in m for the YFP+ cell. m2 Enlarged pictures of the dashed yellow rectangle in m for each channel, including DAPI (blue). Scale bars in µm: 100 ( c , d , m , j ), 50 ( h , k ), 25 ( d1 , d2 , m1 ), 10 ( k1 ), 5 ( m2 ). Abbreviations: ac, anterior commissure; lv, lateral ventricle; NAc, nucleus accumbens; Sp, septum; St, striatum. See also Supplementary Figs. and .

Journal: Molecular Psychiatry

Article Title: Neurogenesis of medium spiny neurons in the nucleus accumbens continues into adulthood and is enhanced by pathological pain

doi: 10.1038/s41380-020-0823-4

Figure Lengend Snippet: a Schematic of the experimental protocol. b Quantification of newborn neuroblasts in the NAc 7 days after sham or SNI surgery (left) (unpaired t- test * p = 0.0026, n = 10 mice per group) or after PBS or CFA paw injection (right) (unpaired t- test * p = 0.0131, n = 7 and 8 mice per group, respectively). c , d Representative pictures of coronal brain sections from mice subjected to either sham ( c ) or SNI surgery ( d ), stained with antibodies against BrdU (white) and DCX (red). d1 , d2 High-magnification images of the dashed yellow rectangles are shown on the right. e Experimental schematic to test the effect of chronic pain in the inflammatory model (CFA/PBS injection) or in the neuropathic pain model (SNI/sham) in the NAc after 6 weeks. f Quantification of mature postnatally born neurons (NeuN+/BrdU+) in the NAc, 4 weeks after the last BrdU injection (left: unpaired t- test * p = 0.041, n = 8 mice per group for sham/SNI; right: unpaired t- test **** p < 0.0001, n = 10 mice per group for PBS/CFA injections). g Quantification of mature postnatally born neurons (NeuN+/BrdU+) in the NAc counterstained with the MSNs marker DARPP-32, 6 weeks after CFA or PBS injection (unpaired t- test *** p = 0.0003, n = 5 mice per group). h Representative picture of a coronal brain section from a CFA-treated mouse showing examples of BrdU+ (gray)/ NeuN+ (green)/ DARPP-32+ (magenta) neurons in the NAc. h1 Higher magnification of the dashed yellow rectangle for each channel is shown on the right. The inset indicates the imaged brain area (dashed yellow rectangle). i Experimental schematic to test the origin of NAc adult-born neurons after inflammatory pain. j Representative pictures of coronal sections comprising the lateral ventricles showing YFP-labeled neural stem cells in the V-SVZ of control (left) and CFA-treated mice (right). Pictures on the right are enlargements of the indicated dashed yellow rectangles showing YFP-labeled cells (green) positive for Sox2 (magenta). k Confocal picture showing newborn (BrdU in white) neuroblasts (DCX in magenta) positive for YFP (green) with migratory appearance in the NAc of a CFA-treated mouse. k1 Enlarged pictures of the boxed area in k for each channel. l Quantification of (DCX+/BrdU+) newborn neuroblasts in the NAc positive for YFP in control (PBS) or pain (CFA)-treated animals ( t -test * p = 0.035, n = 6 mice per group). m Representative image of a coronal brain section from a CFA-treated mouse stained with antibodies against YFP (green) and NeuN (magenta), showing newborn neurons in the NAc 72 days after the first tamoxifen injection. m1 Enlarged picture of the dashed yellow rectangle in m for the YFP+ cell. m2 Enlarged pictures of the dashed yellow rectangle in m for each channel, including DAPI (blue). Scale bars in µm: 100 ( c , d , m , j ), 50 ( h , k ), 25 ( d1 , d2 , m1 ), 10 ( k1 ), 5 ( m2 ). Abbreviations: ac, anterior commissure; lv, lateral ventricle; NAc, nucleus accumbens; Sp, septum; St, striatum. See also Supplementary Figs. and .

Techniques: Injection, Staining, Marker, Labeling

(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.

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

(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

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

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

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