tu bcx 2 k1 tumors  (Worthington Biochemical)


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  • 93
    Name:
    Deoxyribonucleic Acid E coli
    Description:
    Supplied as a dried powder purified from E coli Type B cells ATCC 11303 as described by Marmur J Mol Biol 3 208 1961
    Catalog Number:
    ls004449
    Price:
    104
    Size:
    10 mg
    Source:
    E. coli
    Cas Number:
    9007.49.2
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    Structured Review

    Worthington Biochemical tu bcx 2 k1 tumors
    Characterization of <t>TU-BCx-2</t> K1. a TU-BCx-2 K1 was derived from the biopsy specimen of a 59-year-old African-American female. This PDX tumor was categorized as a TNBC PAM50 molecular subtype and was diagnosed as an invasive ductal carcinoma. There was no evidence of lymph node nor distal metastases at the time of resection. b Representative gross images of lower and higher passage TU-BCx-2 K1 as well as the tumor thawed from cryopreservation. The tumor was homogenous and solid in cross-section. c After implantation into the mammary fat pads of SCID/Beige mice, TU-BCx-2 K1 tumor reached 1000mm 3 after approximately 40 days. Days to tumor take did not vary significantly throughout various passages. At the end of the PDX model name, ‘T’ denotes the passage of the tumor in mice; for example, ‘2K1T2’ means the tumor was passaged two times in mice before analysis. d Comparison of growth rates of tumors implanted in mice at different passages (T2-T6). e H E staining of TU-BCx-2 <t>K1</t> tumors revealed cells with aberrant mitoses surrounded by areas of fibrosis. This histologic appearance did not change dramatically between passages in mice. f Representative H E images of lungs and livers harvested from mice implanted with TU-BcX-2 K1 (passage 3 in mice). Minimal metastatic lesions found in both lungs and livers. Inserts are shown at 200X magnification
    Supplied as a dried powder purified from E coli Type B cells ATCC 11303 as described by Marmur J Mol Biol 3 208 1961
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    1) Product Images from "Drug resistance profiling of a new triple negative breast cancer patient-derived xenograft model"

    Article Title: Drug resistance profiling of a new triple negative breast cancer patient-derived xenograft model

    Journal: BMC Cancer

    doi: 10.1186/s12885-019-5401-2

    Characterization of TU-BCx-2 K1. a TU-BCx-2 K1 was derived from the biopsy specimen of a 59-year-old African-American female. This PDX tumor was categorized as a TNBC PAM50 molecular subtype and was diagnosed as an invasive ductal carcinoma. There was no evidence of lymph node nor distal metastases at the time of resection. b Representative gross images of lower and higher passage TU-BCx-2 K1 as well as the tumor thawed from cryopreservation. The tumor was homogenous and solid in cross-section. c After implantation into the mammary fat pads of SCID/Beige mice, TU-BCx-2 K1 tumor reached 1000mm 3 after approximately 40 days. Days to tumor take did not vary significantly throughout various passages. At the end of the PDX model name, ‘T’ denotes the passage of the tumor in mice; for example, ‘2K1T2’ means the tumor was passaged two times in mice before analysis. d Comparison of growth rates of tumors implanted in mice at different passages (T2-T6). e H E staining of TU-BCx-2 K1 tumors revealed cells with aberrant mitoses surrounded by areas of fibrosis. This histologic appearance did not change dramatically between passages in mice. f Representative H E images of lungs and livers harvested from mice implanted with TU-BcX-2 K1 (passage 3 in mice). Minimal metastatic lesions found in both lungs and livers. Inserts are shown at 200X magnification
    Figure Legend Snippet: Characterization of TU-BCx-2 K1. a TU-BCx-2 K1 was derived from the biopsy specimen of a 59-year-old African-American female. This PDX tumor was categorized as a TNBC PAM50 molecular subtype and was diagnosed as an invasive ductal carcinoma. There was no evidence of lymph node nor distal metastases at the time of resection. b Representative gross images of lower and higher passage TU-BCx-2 K1 as well as the tumor thawed from cryopreservation. The tumor was homogenous and solid in cross-section. c After implantation into the mammary fat pads of SCID/Beige mice, TU-BCx-2 K1 tumor reached 1000mm 3 after approximately 40 days. Days to tumor take did not vary significantly throughout various passages. At the end of the PDX model name, ‘T’ denotes the passage of the tumor in mice; for example, ‘2K1T2’ means the tumor was passaged two times in mice before analysis. d Comparison of growth rates of tumors implanted in mice at different passages (T2-T6). e H E staining of TU-BCx-2 K1 tumors revealed cells with aberrant mitoses surrounded by areas of fibrosis. This histologic appearance did not change dramatically between passages in mice. f Representative H E images of lungs and livers harvested from mice implanted with TU-BcX-2 K1 (passage 3 in mice). Minimal metastatic lesions found in both lungs and livers. Inserts are shown at 200X magnification

    Techniques Used: Derivative Assay, Mouse Assay, Staining

    Mesenchymal and cancer stem-cell like features in TU-BcX-2 K1 cells. a Panel of epithelial ( CD24, CDH1 ) and mesenchymal ( CDH2, VIM, FRA1, SNAI1, TWIST, cFOS, cMYC and SLUG ) genes analyzed by qRT-PCR in TU-BcX-2 K1 tumors passaged in mice (T2, T3, T4, T6). There were no endogenous levels of CD44 in any TU-BcX-2 K1 tumors passaged in mice and very low levels of CDH2 in T3 and T6 and CD24 in T3. Due to limited tissue availability, one sample was obtained per passage and analyzed. Data was normalized to β-actin. b Immunohistochemistry staining for CDH1 protein expression in the TU-BcX-2 K1 T3 tumor. Representative images in inserts are shown at 200X magnification. c Flow cytometry of circulating tumor cells and matched TU-BCx-2 K1 tumor explants. Mouse cells are HLA- and human cells are HLA+; N = 2
    Figure Legend Snippet: Mesenchymal and cancer stem-cell like features in TU-BcX-2 K1 cells. a Panel of epithelial ( CD24, CDH1 ) and mesenchymal ( CDH2, VIM, FRA1, SNAI1, TWIST, cFOS, cMYC and SLUG ) genes analyzed by qRT-PCR in TU-BcX-2 K1 tumors passaged in mice (T2, T3, T4, T6). There were no endogenous levels of CD44 in any TU-BcX-2 K1 tumors passaged in mice and very low levels of CDH2 in T3 and T6 and CD24 in T3. Due to limited tissue availability, one sample was obtained per passage and analyzed. Data was normalized to β-actin. b Immunohistochemistry staining for CDH1 protein expression in the TU-BcX-2 K1 T3 tumor. Representative images in inserts are shown at 200X magnification. c Flow cytometry of circulating tumor cells and matched TU-BCx-2 K1 tumor explants. Mouse cells are HLA- and human cells are HLA+; N = 2

    Techniques Used: Quantitative RT-PCR, Mouse Assay, Immunohistochemistry, Staining, Expressing, Flow Cytometry, Cytometry

    2) Product Images from "The 18 kDa Translocator Protein (Peripheral Benzodiazepine Receptor) Expression in the Bone of Normal, Osteoprotegerin or Low Calcium Diet Treated Mice"

    Article Title: The 18 kDa Translocator Protein (Peripheral Benzodiazepine Receptor) Expression in the Bone of Normal, Osteoprotegerin or Low Calcium Diet Treated Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0030623

    TSPO mRNA expression in bone. (A) Lanes 1 and 5 show 100 bp DNA ladders. TSPO mRNA is detected in the whole bone tissue of a normal mouse (lane 4). Restriction enzyme assay was used to confirm the specificity of the result. PCR products of expected band sizes are produced from the digestion by NcoI (lane 2) and PvuII (lane 3). (B) shows the expressions of TRAP, (C) the expression of ALP and (D) the expression of TSPO mRNA in primary osteoclast and osteoblast cultures and in skeletal muscle (N = 3). OB = primary osteoblast culture from calvaria; OC = primary osteoclast culture from spleen cells treated with M-CSF/RANKL. Error bar = standard deviation.
    Figure Legend Snippet: TSPO mRNA expression in bone. (A) Lanes 1 and 5 show 100 bp DNA ladders. TSPO mRNA is detected in the whole bone tissue of a normal mouse (lane 4). Restriction enzyme assay was used to confirm the specificity of the result. PCR products of expected band sizes are produced from the digestion by NcoI (lane 2) and PvuII (lane 3). (B) shows the expressions of TRAP, (C) the expression of ALP and (D) the expression of TSPO mRNA in primary osteoclast and osteoblast cultures and in skeletal muscle (N = 3). OB = primary osteoblast culture from calvaria; OC = primary osteoclast culture from spleen cells treated with M-CSF/RANKL. Error bar = standard deviation.

    Techniques Used: Expressing, Enzymatic Assay, Polymerase Chain Reaction, Produced, ALP Assay, Standard Deviation

    3) Product Images from "Sex Bias in Susceptibility to MCMV Infection: Implication of TLR9"

    Article Title: Sex Bias in Susceptibility to MCMV Infection: Implication of TLR9

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0045171

    Differences in the proportion of MZ B cells in MCMV-infected WT male and female mice. WT and TLR9 −/− male and female mice were left uninfected or infected i.p. with 1×10 5 PFU of MCMV and spleens were harvested 36 hours later. Erythrocyte-depleted splenocytes were analyzed by flow cytometry for the expression of B220, CD19, CD23, CD21, IgM and IgD. The MZ B cell population, B220 + CD19 + CD23 lo CD21 hi on (A) or IgM high IgD low CD21 high on (B), of MCMV-infected WT male and TLR9 −/− male and female mice was dramatically reduced compared to uninfected counterparts, while this reduction was minor in MCMV-infected female mice. (A) Plots show percentage of MZ B cells (CD23 lo CD21 hi ) on B220 + gated lymphocytes. (B) Histograms show expression of CD21 on IgM high IgD low B cells. (C) Graph with the numeric data of all 3 mice per group that were used for the experiments in (A) and (B). *p
    Figure Legend Snippet: Differences in the proportion of MZ B cells in MCMV-infected WT male and female mice. WT and TLR9 −/− male and female mice were left uninfected or infected i.p. with 1×10 5 PFU of MCMV and spleens were harvested 36 hours later. Erythrocyte-depleted splenocytes were analyzed by flow cytometry for the expression of B220, CD19, CD23, CD21, IgM and IgD. The MZ B cell population, B220 + CD19 + CD23 lo CD21 hi on (A) or IgM high IgD low CD21 high on (B), of MCMV-infected WT male and TLR9 −/− male and female mice was dramatically reduced compared to uninfected counterparts, while this reduction was minor in MCMV-infected female mice. (A) Plots show percentage of MZ B cells (CD23 lo CD21 hi ) on B220 + gated lymphocytes. (B) Histograms show expression of CD21 on IgM high IgD low B cells. (C) Graph with the numeric data of all 3 mice per group that were used for the experiments in (A) and (B). *p

    Techniques Used: Infection, Mouse Assay, Flow Cytometry, Cytometry, Expressing

    Increased proportion of splenic pDCs in MCMV-infected WT male mice. WT and TLR9 −/− male and female mice were left uninfected or infected i.p. with 1×10 5 PFU of MCMV and spleens were harvested 36 h later. Erythrocyte-depleted splenocytes were analyzed by flow cytometry for the expression of B220, CD11c and Ly6C. Upon MCMV-infection the percentage of pDCs (B220 + Ly6C hi CD11c lo ) is increased in all four mouse groups, while the percentage of cDCs (B220 − Ly6C − CD11c hi ) is slightly decreased. Interestingly, upon MCMV infection WT male mice showed statistically significant increased percentage of pDCs compared to WT females. Percentages on pDCs and cDCs are presented as mean ± SEM *p
    Figure Legend Snippet: Increased proportion of splenic pDCs in MCMV-infected WT male mice. WT and TLR9 −/− male and female mice were left uninfected or infected i.p. with 1×10 5 PFU of MCMV and spleens were harvested 36 h later. Erythrocyte-depleted splenocytes were analyzed by flow cytometry for the expression of B220, CD11c and Ly6C. Upon MCMV-infection the percentage of pDCs (B220 + Ly6C hi CD11c lo ) is increased in all four mouse groups, while the percentage of cDCs (B220 − Ly6C − CD11c hi ) is slightly decreased. Interestingly, upon MCMV infection WT male mice showed statistically significant increased percentage of pDCs compared to WT females. Percentages on pDCs and cDCs are presented as mean ± SEM *p

    Techniques Used: Infection, Mouse Assay, Flow Cytometry, Cytometry, Expressing

    4) Product Images from "Constitutive Gs activation using a single-construct tetracycline-inducible expression system in embryonic stem cells and mice"

    Article Title: Constitutive Gs activation using a single-construct tetracycline-inducible expression system in embryonic stem cells and mice

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/scrt52

    A single-vector tetracycline construct allows doxycycline-regulated expression . (A) Overview showing the regulator plasmid containing the EF1α-tTA cassette (pEntL1L3-EF1α-tTA, left) and the responder plasmid containing the TetO-mCh-Rs1 cassette (pEntR3L2 TetO-mCh-Rs1, right). The mCherry and Rs1 cistrons are separated by a P2A ribosomal skip sequence to allow simultaneous expression of both peptides. The entry plasmids were recombined using Gateway technology into the desired destination vector containing the AttR1 and AttR2 Gateway sites. The TetO and EF1α-tTA portions are in opposite orientation (indicated by upside-down text) to minimize steric hindrance between the two promoters, as well as potential cross-activation of the TetO by the EF1α promoter. In addition, flanking insulator sequences are included to minimize any read-through activation of the constructs by surrounding promoters (such as Rosa26) that may lead to
    Figure Legend Snippet: A single-vector tetracycline construct allows doxycycline-regulated expression . (A) Overview showing the regulator plasmid containing the EF1α-tTA cassette (pEntL1L3-EF1α-tTA, left) and the responder plasmid containing the TetO-mCh-Rs1 cassette (pEntR3L2 TetO-mCh-Rs1, right). The mCherry and Rs1 cistrons are separated by a P2A ribosomal skip sequence to allow simultaneous expression of both peptides. The entry plasmids were recombined using Gateway technology into the desired destination vector containing the AttR1 and AttR2 Gateway sites. The TetO and EF1α-tTA portions are in opposite orientation (indicated by upside-down text) to minimize steric hindrance between the two promoters, as well as potential cross-activation of the TetO by the EF1α promoter. In addition, flanking insulator sequences are included to minimize any read-through activation of the constructs by surrounding promoters (such as Rosa26) that may lead to "leakiness" or steric interference from endogenous promoter activity. (B, C) HEK-293 cells carrying the Exp-pcDNA3.2(EF1α-tTA/TetO-mCh-Rs1) expression cassette and cultured in doxycycline (suppressed expression) or in the absence of doxycycline (transgene expression allowed) demonstrate doxycycline-dependent mCherry expression. (D) Schematic of targeted Rosa26 locus and Southern screening strategy. The Rosa26 locus in E14 ES cells was targeted by homologous recombination with the Exp-R26(EF1α-tTA/TetO-mCh-Rs1) construct. Regions in hatch marks indicate the 5' and 3' homology regions of the targeting vector and the endogenous Rosa26 locus (abbreviated R26 in the figure). The location of the 5' recombination Southern probe and HindIII restriction sites are indicated. (E) Southern blots of genomic DNA digested with HindIII and probed as in (D). Heterozygous ES cells at the Rosa26 locus are indicated by the two bands.

    Techniques Used: Plasmid Preparation, Construct, Expressing, Sequencing, Activation Assay, Activity Assay, Cell Culture, Homologous Recombination

    A single copy of the EF1α-tTA regulator region weakly drives expression of a TetO transgene in mice . (A) Areal bone mineral density by DEXA of nine-week-old mice shows that the ColI(2.3)-tTA x R26(EF1α-tTA/TetO-mCh-Rs1) mice have increased bone mass. N = 9 WT, 5 R26(EF1α-tTA/TetO-mCh-Rs1), and 9 ColI(2.3)-tTA x R26(EF1α-tTA/TetO-mCh-Rs1) mice. ***, P
    Figure Legend Snippet: A single copy of the EF1α-tTA regulator region weakly drives expression of a TetO transgene in mice . (A) Areal bone mineral density by DEXA of nine-week-old mice shows that the ColI(2.3)-tTA x R26(EF1α-tTA/TetO-mCh-Rs1) mice have increased bone mass. N = 9 WT, 5 R26(EF1α-tTA/TetO-mCh-Rs1), and 9 ColI(2.3)-tTA x R26(EF1α-tTA/TetO-mCh-Rs1) mice. ***, P

    Techniques Used: Expressing, Mouse Assay

    5) Product Images from "Stop codons preceded by rare arginine codons are efficient determinants of SsrA tagging in Escherichia coli"

    Article Title: Stop codons preceded by rare arginine codons are efficient determinants of SsrA tagging in Escherichia coli

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.052707199

    Identification of SsrA(ANDH 6 D)-tagging sites in RbsK. ( A ) RbsK was expressed from the P trc promoter with (+) or without (−) IPTG induction in cells containing either wild-type SsrA or SsrA(ANDH 6 D). Cell lysates were electrophoresed on SDS-polyacrylamide gels and stained with Coomassie blue. The positions of full-length RbsK and SsrA(ANDH 6 D)-tagged RbsK are indicated. Densitometry indicated that as much as 25% of total RbsK may be tagged. ( B ) The nucleotide sequence encoding the 3′ end of wild-type rbsK and the 5′ end of rbsR with the corresponding amino acid sequence (in three letter code) is depicted. Positions of SsrA(ANDH 6 D) tagging in ribokinase are indicated by arrows. ( C ) Reverse-phase HPLC chromatogram of Ni 2+ -NTA-purified SsrA(ANDH 6 D)-RbsK fusion peptides. Electrospray mass spectrometry gave masses of 2001.87 Da for peptide 1, 2286.03 Da for peptide 2, and 2442.38 Da for peptide 3. The calculated mass for the sequences shown are 1 (2001.64 Da), 2 (2286.03 Da), and 3 (2442.12 Da). The N-terminal sequences of peptides 1 and 2 were I-D-A-F-L-D-A-A-N-D-H-H-H-H-H and I-D-A-F-L-D-R-Q-A-A-N-D-H-H-H, respectively.
    Figure Legend Snippet: Identification of SsrA(ANDH 6 D)-tagging sites in RbsK. ( A ) RbsK was expressed from the P trc promoter with (+) or without (−) IPTG induction in cells containing either wild-type SsrA or SsrA(ANDH 6 D). Cell lysates were electrophoresed on SDS-polyacrylamide gels and stained with Coomassie blue. The positions of full-length RbsK and SsrA(ANDH 6 D)-tagged RbsK are indicated. Densitometry indicated that as much as 25% of total RbsK may be tagged. ( B ) The nucleotide sequence encoding the 3′ end of wild-type rbsK and the 5′ end of rbsR with the corresponding amino acid sequence (in three letter code) is depicted. Positions of SsrA(ANDH 6 D) tagging in ribokinase are indicated by arrows. ( C ) Reverse-phase HPLC chromatogram of Ni 2+ -NTA-purified SsrA(ANDH 6 D)-RbsK fusion peptides. Electrospray mass spectrometry gave masses of 2001.87 Da for peptide 1, 2286.03 Da for peptide 2, and 2442.38 Da for peptide 3. The calculated mass for the sequences shown are 1 (2001.64 Da), 2 (2286.03 Da), and 3 (2442.12 Da). The N-terminal sequences of peptides 1 and 2 were I-D-A-F-L-D-A-A-N-D-H-H-H-H-H and I-D-A-F-L-D-R-Q-A-A-N-D-H-H-H, respectively.

    Techniques Used: Staining, Sequencing, High Performance Liquid Chromatography, Purification, Mass Spectrometry

    6) Product Images from "Deficiency in adipocyte chemokine receptor CXCR4 exacerbates obesity and compromises thermoregulatory responses of brown adipose tissue in a mouse model of diet-induced obesity"

    Article Title: Deficiency in adipocyte chemokine receptor CXCR4 exacerbates obesity and compromises thermoregulatory responses of brown adipose tissue in a mouse model of diet-induced obesity

    Journal: The FASEB Journal

    doi: 10.1096/fj.14-249797

    HFD feeding does not potentiate obesity in MyeCXCR4ko mice. WT C57BL/6 control ( n =40) and MyeCXCR4ko ( n =40) mice were fed a CD for 18 wk or an HFD for 24 wk. Animals were weighed 1×/wk and euthanized at the end of the feeding regimen. A ) Visceral
    Figure Legend Snippet: HFD feeding does not potentiate obesity in MyeCXCR4ko mice. WT C57BL/6 control ( n =40) and MyeCXCR4ko ( n =40) mice were fed a CD for 18 wk or an HFD for 24 wk. Animals were weighed 1×/wk and euthanized at the end of the feeding regimen. A ) Visceral

    Techniques Used: Mouse Assay

    Obesity in AdCXCR4ko mice is not a result of hyperphagia but positively correlates with increased adiposity, mass, and hypertrophy of BAT and WAT. WT C57BL/6 control ( n =15), AdCXCR4ko ( n =15), and MyeCXCR4ko ( n =15) mice were fed a CD or an HFD for 24 wk.
    Figure Legend Snippet: Obesity in AdCXCR4ko mice is not a result of hyperphagia but positively correlates with increased adiposity, mass, and hypertrophy of BAT and WAT. WT C57BL/6 control ( n =15), AdCXCR4ko ( n =15), and MyeCXCR4ko ( n =15) mice were fed a CD or an HFD for 24 wk.

    Techniques Used: Mouse Assay

    Adipocyte CXCR4 deficiency alters ATM and lymphocyte contents in WAT. WT C57BL/6 control ( n =15), AdCXCR4ko ( n =15), and MyeCXCR4ko ( n =15) mice were fed an HFD for 24 wk and then euthanized. Blood was collected and plasma separated. Visceral mesenteric,
    Figure Legend Snippet: Adipocyte CXCR4 deficiency alters ATM and lymphocyte contents in WAT. WT C57BL/6 control ( n =15), AdCXCR4ko ( n =15), and MyeCXCR4ko ( n =15) mice were fed an HFD for 24 wk and then euthanized. Blood was collected and plasma separated. Visceral mesenteric,

    Techniques Used: Mouse Assay

    AdCXCR4ko mice display impaired adaptive thermogenesis. WT C57BL/6 control ( n =5), AdCXCR4ko ( n =5), and MyeCXCR4ko mice were fed either a CD or an HFD for 24 wk. Body temperature was measured at 25°C or at the indicated times ( D ) at 4°C.
    Figure Legend Snippet: AdCXCR4ko mice display impaired adaptive thermogenesis. WT C57BL/6 control ( n =5), AdCXCR4ko ( n =5), and MyeCXCR4ko mice were fed either a CD or an HFD for 24 wk. Body temperature was measured at 25°C or at the indicated times ( D ) at 4°C.

    Techniques Used: Mouse Assay

    7) Product Images from "3?-Exonuclease resistance of DNA oligodeoxynucleotides containing O6-[4-oxo-4-(3-pyridyl)butyl]guanine"

    Article Title: 3?-Exonuclease resistance of DNA oligodeoxynucleotides containing O6-[4-oxo-4-(3-pyridyl)butyl]guanine

    Journal: Nucleic Acids Research

    doi:

    MALDI-TOF mass spectra of SVPDE digests of modified DNA 16mers d(AACAGCCATATGXCCC): ( A ) X = O 6 -POB-dG, time-controlled digest; ( B ) X = O 6 -POB-dG, complete digest conditions; ( C ) O 6 -Me-dG-containing oligomers, controlled digest conditions. Arrows indicate the portion of the sequence represented in the spectra, and doubly charged ions are marked with #.
    Figure Legend Snippet: MALDI-TOF mass spectra of SVPDE digests of modified DNA 16mers d(AACAGCCATATGXCCC): ( A ) X = O 6 -POB-dG, time-controlled digest; ( B ) X = O 6 -POB-dG, complete digest conditions; ( C ) O 6 -Me-dG-containing oligomers, controlled digest conditions. Arrows indicate the portion of the sequence represented in the spectra, and doubly charged ions are marked with #.

    Techniques Used: Modification, Sequencing

    8) Product Images from "Cardiac thromboxane A2 receptor activation does not directly induce cardiomyocyte hypertrophy but does cause cell death that is prevented with gentamicin and 2-APB"

    Article Title: Cardiac thromboxane A2 receptor activation does not directly induce cardiomyocyte hypertrophy but does cause cell death that is prevented with gentamicin and 2-APB

    Journal: BMC Pharmacology & Toxicology

    doi: 10.1186/2050-6511-15-73

    U46619 does not increase gene markers associated with pathological hypertrophy. Exposing AVCMs to increasing concentrations of U46619 (0.1-10 μM) did not increase the expression of early growth response 1 (EGR1) gene or the hypertrophy-associated genes atrial natriuretic peptide (ANP) after 24 h when compared to vehicle ( p > 0.05). Forty-eight-hour treatment with U46619, did not increase the expression of other hypertrophy genes β-myosin heavy chain (β-MHC) and skeletal muscle α-actin (SkAct) when compared to vehicle (n = 4-5; p > 0.05). There was also no significant increase in these genes with U46619 treatment in HL-1 cardiomyocytes.
    Figure Legend Snippet: U46619 does not increase gene markers associated with pathological hypertrophy. Exposing AVCMs to increasing concentrations of U46619 (0.1-10 μM) did not increase the expression of early growth response 1 (EGR1) gene or the hypertrophy-associated genes atrial natriuretic peptide (ANP) after 24 h when compared to vehicle ( p > 0.05). Forty-eight-hour treatment with U46619, did not increase the expression of other hypertrophy genes β-myosin heavy chain (β-MHC) and skeletal muscle α-actin (SkAct) when compared to vehicle (n = 4-5; p > 0.05). There was also no significant increase in these genes with U46619 treatment in HL-1 cardiomyocytes.

    Techniques Used: Expressing, Aqueous Normal-phase Chromatography

    U46619 increases cell death in cardiomyocytes. A . HL-1 cardiomyocytes were incubated with increasing concentrations of U46619 (0.1-10 μM) and vehicle for 24 h. U46619 increased cell death at 5 and 10 M as measured by trypan blue staining (n = 3; p
    Figure Legend Snippet: U46619 increases cell death in cardiomyocytes. A . HL-1 cardiomyocytes were incubated with increasing concentrations of U46619 (0.1-10 μM) and vehicle for 24 h. U46619 increased cell death at 5 and 10 M as measured by trypan blue staining (n = 3; p

    Techniques Used: Incubation, Staining

    TXA2R mRNA and protein are present in AVCMs. A . 10x and 40x images of isolated AVCMs from male mice after 24 h in culture. Cells remain in high density; maintain membrane integrity and show clear striations associated with healthy cardiomyocytes following 24 h in culture. B . Real-time RT-PCR of RNA isolated from AVCMs showing the presence of TXA2R. Similar TXA2R gene expression was also observed with HL-1 cells. Inset shows TXA2R protein from isolated AVCMs detected by western blot. C . Representative data of 10 μM U46619-induced increases in intracellular Ca 2+ in cardiomyocytes as measured by Fura-2 AM. This demonstrates that the TXA2R in mouse AVCMs is functional and behaves similarly to our previous reports in the rabbit [ 16 ].
    Figure Legend Snippet: TXA2R mRNA and protein are present in AVCMs. A . 10x and 40x images of isolated AVCMs from male mice after 24 h in culture. Cells remain in high density; maintain membrane integrity and show clear striations associated with healthy cardiomyocytes following 24 h in culture. B . Real-time RT-PCR of RNA isolated from AVCMs showing the presence of TXA2R. Similar TXA2R gene expression was also observed with HL-1 cells. Inset shows TXA2R protein from isolated AVCMs detected by western blot. C . Representative data of 10 μM U46619-induced increases in intracellular Ca 2+ in cardiomyocytes as measured by Fura-2 AM. This demonstrates that the TXA2R in mouse AVCMs is functional and behaves similarly to our previous reports in the rabbit [ 16 ].

    Techniques Used: Isolation, Mouse Assay, Quantitative RT-PCR, Expressing, Western Blot, Functional Assay

    U46619-induced DNA fragmentation is receptor mediated and inhibited with gentamicin and 2-APB. A . Image of TUNEL positive AVCMs in culture. AVCMs were cultured in the absence of serum for 24 h during treatment with U46619 (0.1-10 μM) or vehicle and then stained with TUNEL (green nuclei) and DAPI (blue nuclei). Cell membranes were pseudo-colored to enhance visualization (red). B . Summary data showing increasing concentrations of U46619 (0.1-10 μM) increased the number TUNEL positive cardiomyocytes when compared to vehicle (n = 4; p
    Figure Legend Snippet: U46619-induced DNA fragmentation is receptor mediated and inhibited with gentamicin and 2-APB. A . Image of TUNEL positive AVCMs in culture. AVCMs were cultured in the absence of serum for 24 h during treatment with U46619 (0.1-10 μM) or vehicle and then stained with TUNEL (green nuclei) and DAPI (blue nuclei). Cell membranes were pseudo-colored to enhance visualization (red). B . Summary data showing increasing concentrations of U46619 (0.1-10 μM) increased the number TUNEL positive cardiomyocytes when compared to vehicle (n = 4; p

    Techniques Used: TUNEL Assay, Cell Culture, Staining

    U46619 does not induce hypertrophy or increase protein synthesis. A . Flow cytometry forward-scatter (FSC-H) data was performed on more than 10,000 live gated cells/sample (n = 3). HL-1 cardiomyocytes were treated for 48 h with vehicle, increasing concentrations of U46619 (0.1-10 μM) [T(X)
    Figure Legend Snippet: U46619 does not induce hypertrophy or increase protein synthesis. A . Flow cytometry forward-scatter (FSC-H) data was performed on more than 10,000 live gated cells/sample (n = 3). HL-1 cardiomyocytes were treated for 48 h with vehicle, increasing concentrations of U46619 (0.1-10 μM) [T(X)

    Techniques Used: Flow Cytometry, Cytometry

    9) Product Images from "A genome-wide view of the de-differentiation of central nervous system endothelial cells in culture"

    Article Title: A genome-wide view of the de-differentiation of central nervous system endothelial cells in culture

    Journal: eLife

    doi: 10.7554/eLife.51276

    In vivo analysis of transcripts from FACS-purified pituitary ECs that include or omit Ctnnb1 exon 3. Analysis of Ctnnb1 transcripts that include or omit exon 3 from FACS-purified anterior and posterior pituitary ECs from WT control mice (red; four RNA-seq data sets) or following Pdgfb-CreER mediated excision of Ctnnb1 exon 3 from the floxed allele (blue; four RNA-seq data sets). The RNA-seq data come from Wang et al. (2019) . The four WT data sets (two each from anterior and posterior pituitary ECs) showed no RNA-seq reads that join exons 2+4, whereas the four Ctnnb1 flex3/+ ;Pdgfb-CreER;Tie2-GFP data sets (two each from anterior and posterior pituitary ECs) produced a mean of ~50 RNA-seq reads that join exons 2+4 (representing exon 3 deletion by Cre-mediated recombination). The ~50 exon 2+4 reads correspond to ~25% as many reads as spanned exons 2+3; the ratios for each sample are shown in the lower left panel. One of the four Ctnnb1 flex3/+ ;Pdgfb-CreER;Tie2-GFP samples showed no exon 2+4 reads.
    Figure Legend Snippet: In vivo analysis of transcripts from FACS-purified pituitary ECs that include or omit Ctnnb1 exon 3. Analysis of Ctnnb1 transcripts that include or omit exon 3 from FACS-purified anterior and posterior pituitary ECs from WT control mice (red; four RNA-seq data sets) or following Pdgfb-CreER mediated excision of Ctnnb1 exon 3 from the floxed allele (blue; four RNA-seq data sets). The RNA-seq data come from Wang et al. (2019) . The four WT data sets (two each from anterior and posterior pituitary ECs) showed no RNA-seq reads that join exons 2+4, whereas the four Ctnnb1 flex3/+ ;Pdgfb-CreER;Tie2-GFP data sets (two each from anterior and posterior pituitary ECs) produced a mean of ~50 RNA-seq reads that join exons 2+4 (representing exon 3 deletion by Cre-mediated recombination). The ~50 exon 2+4 reads correspond to ~25% as many reads as spanned exons 2+3; the ratios for each sample are shown in the lower left panel. One of the four Ctnnb1 flex3/+ ;Pdgfb-CreER;Tie2-GFP samples showed no exon 2+4 reads.

    Techniques Used: In Vivo, FACS, Purification, Mouse Assay, RNA Sequencing Assay, Produced

    10) Product Images from "Three-Dimensional Culture Alters Primary Cardiac Cell Phenotype"

    Article Title: Three-Dimensional Culture Alters Primary Cardiac Cell Phenotype

    Journal: Tissue Engineering. Part A

    doi: 10.1089/ten.tea.2009.0458

    Assessment of homogeneity between two-dimensional (2D) and three-dimensional (3D) cultures. Samples were collected on day 1 or 6 of culture as indicated and assessed for bulk differences in culture composition, hypertrophy, and metabolic function. ( A ) Cell attachment efficiency was calculated as the number of cells attached to tissue culture polystyrene (TCPS) surfaces after 24 h divided by the total number of viable cells in the original inoculum. The proportion of attached cardiomyocytes (CMs), which are positive for myosin heavy chain (MyHC) immunostaining, per total adherent cells was also calculated. ( B ) On day 6, MyHC and filamentous actin (f-Actin) levels, which are indicative of CM hypertrophic status, were determined in fixed samples by fluorescence in situ quantitation assay and are presented as fluorescence ratios normalized to DNA. Total protein content, which is a key indicator of tissue hypertrophy, was measured in culture homogenates and normalized to DNA. Units presented are μg protein per 10 ng DNA. ( C ) Intermediary metabolic enzyme activities in day 6 cell extracts calculated as units of enzyme activity per mg of total protein per minute. No significant differences were found in ( A ), ( B ), or ( C ). Data are mean values ± standard deviation for n = 6 samples.
    Figure Legend Snippet: Assessment of homogeneity between two-dimensional (2D) and three-dimensional (3D) cultures. Samples were collected on day 1 or 6 of culture as indicated and assessed for bulk differences in culture composition, hypertrophy, and metabolic function. ( A ) Cell attachment efficiency was calculated as the number of cells attached to tissue culture polystyrene (TCPS) surfaces after 24 h divided by the total number of viable cells in the original inoculum. The proportion of attached cardiomyocytes (CMs), which are positive for myosin heavy chain (MyHC) immunostaining, per total adherent cells was also calculated. ( B ) On day 6, MyHC and filamentous actin (f-Actin) levels, which are indicative of CM hypertrophic status, were determined in fixed samples by fluorescence in situ quantitation assay and are presented as fluorescence ratios normalized to DNA. Total protein content, which is a key indicator of tissue hypertrophy, was measured in culture homogenates and normalized to DNA. Units presented are μg protein per 10 ng DNA. ( C ) Intermediary metabolic enzyme activities in day 6 cell extracts calculated as units of enzyme activity per mg of total protein per minute. No significant differences were found in ( A ), ( B ), or ( C ). Data are mean values ± standard deviation for n = 6 samples.

    Techniques Used: Cell Attachment Assay, Immunostaining, Fluorescence, In Situ, Quantitation Assay, Activity Assay, Standard Deviation

    Distribution of cells in 3D aggregates. ( A ) Phase contrast image of a standard 2D culture on TCPS culture wells. Arrows indicate patches of elongated cells (lower arrow) indicative of cardiac muscle cells interspersed with nonmuscle cells (upper arrow). ( B ) Phase contrast image of 3D aggregates initiated on TCPS support beads. Arrow indicates typical 3D cell mass. Inset is a lower magnification view of a multi-cell/multi-bead aggregate. ( C ) Two-dimensional culture stained for MyHC to indicate CMs and DNA. Similar to ( A ), patches of cardiac muscle cells are interspersed with nonmuscle cells (arrows) in a mosaic pattern. ( D ) Three-dimensional culture stained for MyHC to indicate CMs and DNA. The exterior-most cells do not contain MyHC (arrows indicate nuclei of these cells). ( E ) Three-dimensional culture stained for vimentin and DNA, verifying the presence of mesenchymal/endothelial cells (ECs) on the aggregate surface (arrows); this staining is consistent with an EC layer. ( F ) Two-dimensional culture stained for platelet/endothelial cell adhesion molecule (PECAM) (CD31) and DNA showing an island of PECAM-positive ECs amidst PECAM-negative cells (arrow). ( G ) Three-dimensional culture stained for PECAM showing that exterior-most nuclei are within cells positive for PECAM (arrow), a marker for ECs in contact with each other. ( H ) Scanning electron micrograph (SEM) of a 2D culture. Lower arrow indicates an elongated, binucleate cell, which is consistent with a CM. Upper arrow indicates a mononucleated cell with a more spread morphology. Nuceoli are readily apparent. ( I ) SEM of a 3D culture surface showing only cells with an endothelial appearance are present (arrows). ( J ) SEM of the interior aspect of a rat heart lumen showing the configuration of the ECs lining the organ lumen. Bar = 50 μm except for inset figure in ( B .
    Figure Legend Snippet: Distribution of cells in 3D aggregates. ( A ) Phase contrast image of a standard 2D culture on TCPS culture wells. Arrows indicate patches of elongated cells (lower arrow) indicative of cardiac muscle cells interspersed with nonmuscle cells (upper arrow). ( B ) Phase contrast image of 3D aggregates initiated on TCPS support beads. Arrow indicates typical 3D cell mass. Inset is a lower magnification view of a multi-cell/multi-bead aggregate. ( C ) Two-dimensional culture stained for MyHC to indicate CMs and DNA. Similar to ( A ), patches of cardiac muscle cells are interspersed with nonmuscle cells (arrows) in a mosaic pattern. ( D ) Three-dimensional culture stained for MyHC to indicate CMs and DNA. The exterior-most cells do not contain MyHC (arrows indicate nuclei of these cells). ( E ) Three-dimensional culture stained for vimentin and DNA, verifying the presence of mesenchymal/endothelial cells (ECs) on the aggregate surface (arrows); this staining is consistent with an EC layer. ( F ) Two-dimensional culture stained for platelet/endothelial cell adhesion molecule (PECAM) (CD31) and DNA showing an island of PECAM-positive ECs amidst PECAM-negative cells (arrow). ( G ) Three-dimensional culture stained for PECAM showing that exterior-most nuclei are within cells positive for PECAM (arrow), a marker for ECs in contact with each other. ( H ) Scanning electron micrograph (SEM) of a 2D culture. Lower arrow indicates an elongated, binucleate cell, which is consistent with a CM. Upper arrow indicates a mononucleated cell with a more spread morphology. Nuceoli are readily apparent. ( I ) SEM of a 3D culture surface showing only cells with an endothelial appearance are present (arrows). ( J ) SEM of the interior aspect of a rat heart lumen showing the configuration of the ECs lining the organ lumen. Bar = 50 μm except for inset figure in ( B .

    Techniques Used: Staining, Marker

    11) Product Images from "PD-L2 modulates asthma severity by directly decreasing dendritic cell IL-12 production"

    Article Title: PD-L2 modulates asthma severity by directly decreasing dendritic cell IL-12 production

    Journal: Mucosal immunology

    doi: 10.1038/mi.2012.111

    PD-L2 expression is enhanced in the airways of asthmatic individuals and allergen-exposed mice Mice were treated with PBS (PBS - intratracheally on days 0, and 14), intratracheal OVA (OVA i.t. - 100 μg on days 0, 14 and 21), intraperitoneal/intratracheal OVA (OVA i.p./i.t. - 10 μg OVA i.p. on day 0 followed by 100 μg i.t. on days 14 and 21), or intratracheal HDM (HDM - 100 μg on days 0 and 14). (A) Mice were sacrificed 72 hours after final allergen exposure to measure AHR via the airway pressure time index (APTI) method. (B) PD-L2 expression (normalized to mouse ribosomal protein s14) was measured by RT-PCR. n = 4 mice per group. 1 representative experiment of 2 shown. Mean + SEM shown. *** and ** indicate p
    Figure Legend Snippet: PD-L2 expression is enhanced in the airways of asthmatic individuals and allergen-exposed mice Mice were treated with PBS (PBS - intratracheally on days 0, and 14), intratracheal OVA (OVA i.t. - 100 μg on days 0, 14 and 21), intraperitoneal/intratracheal OVA (OVA i.p./i.t. - 10 μg OVA i.p. on day 0 followed by 100 μg i.t. on days 14 and 21), or intratracheal HDM (HDM - 100 μg on days 0 and 14). (A) Mice were sacrificed 72 hours after final allergen exposure to measure AHR via the airway pressure time index (APTI) method. (B) PD-L2 expression (normalized to mouse ribosomal protein s14) was measured by RT-PCR. n = 4 mice per group. 1 representative experiment of 2 shown. Mean + SEM shown. *** and ** indicate p

    Techniques Used: Expressing, Mouse Assay, Reverse Transcription Polymerase Chain Reaction

    12) Product Images from "Sustained Endothelial Expression of HoxA5 In Vivo Impairs Pathological Angiogenesis And Tumor Progression"

    Article Title: Sustained Endothelial Expression of HoxA5 In Vivo Impairs Pathological Angiogenesis And Tumor Progression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0121720

    De novo neoplastic progression is delayed with sustained expression of HoxA5 in EC. ( A ) Real time PCR analysis of Thrombospondin-2 (TSP-2) and VEGF-A mRNA levels in ear tissue harvested from 5 month old control tTA (-LM), K14-HPV16 (HPV16) or K14-HPV16/HoxA5-tTA (HPV16/HoxA5) mice (n = 5). All mice were given Dox (+Dox) for 3 weeks during gestation, and subsequently removed from Dox (-Dox) for the remainder of the study. ( B ) Vascular density analysis of CD31+ vessels following staining of ear tissue harvested from 5 month old control tTA (-LM), K14-HPV16 (HPV16) or K14-HPV16/HoxA5-tTA (HPV16/HoxA5) mice (p
    Figure Legend Snippet: De novo neoplastic progression is delayed with sustained expression of HoxA5 in EC. ( A ) Real time PCR analysis of Thrombospondin-2 (TSP-2) and VEGF-A mRNA levels in ear tissue harvested from 5 month old control tTA (-LM), K14-HPV16 (HPV16) or K14-HPV16/HoxA5-tTA (HPV16/HoxA5) mice (n = 5). All mice were given Dox (+Dox) for 3 weeks during gestation, and subsequently removed from Dox (-Dox) for the remainder of the study. ( B ) Vascular density analysis of CD31+ vessels following staining of ear tissue harvested from 5 month old control tTA (-LM), K14-HPV16 (HPV16) or K14-HPV16/HoxA5-tTA (HPV16/HoxA5) mice (p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Mouse Assay, Staining

    HoxA5 is expressed in EC in TRE-HoxA5/TIE2-tTA mice. ( A ) Schematic of the TRE-HoxA5-TIE2-tTA system for inducible and restricted expression of Hox A5 in ECs in FvBn mice. When the double transgenic mice are maintained on a Dox diet the activator cannot bind the TRE promoter. However in the absence of Dox, tTA binds the TRE activating transcription and HoxA5 expression. Expression of the transgene is restricted to EC by driving tTA using the TIE-2 promoter enhancer [ 12 ]. ( B ) Real Time PCR analysis of HoxA5 transgene mRNA expression in mouse liver at various times following withdrawal of Dox from the diet in control TIE-2-tTA (tTA) and TRE-HoxA5-TIE2-tTA (HoxA5-tTA) mice. Results are expressed relative to the housekeeping gene GUSB (n = 3). ( C ) HoxA5 expression levels in ECs isolated from lungs of tTA and HoxA5-tTA mice. Histogram shows HoxA5 mRNA levels measured by real time PCR in the presence of Dox (1μg/ml) or 48 hours following removal of Dox in the cell culture media. Insert shows corresponding Western blot of protein lysates extracted from lung EC isolated from tTA and HoxA5-TA mice and detected via polyclonal antibodies against HoxA5. ( D ) Real time PCR analysis of relative mRNA expression levels of thrombospndin-2 (TSP-2) and VEGF-A levels one month after removal of Dox from the diet of HoxA5-tTA mice. Results are expressed relative to mRNA levels in age-matched tTA control mice lacking the HoxA5 transgene or Dox in the diet. ( E ) Vascular permeability in tTA or HoxA5-tTA mice. Measurement of extravasated Evans Blue dye 30 minutes following topical application of mineral oil (control, left panel) or Mustard oil to induce an acute leakage (right panel; n = 4).
    Figure Legend Snippet: HoxA5 is expressed in EC in TRE-HoxA5/TIE2-tTA mice. ( A ) Schematic of the TRE-HoxA5-TIE2-tTA system for inducible and restricted expression of Hox A5 in ECs in FvBn mice. When the double transgenic mice are maintained on a Dox diet the activator cannot bind the TRE promoter. However in the absence of Dox, tTA binds the TRE activating transcription and HoxA5 expression. Expression of the transgene is restricted to EC by driving tTA using the TIE-2 promoter enhancer [ 12 ]. ( B ) Real Time PCR analysis of HoxA5 transgene mRNA expression in mouse liver at various times following withdrawal of Dox from the diet in control TIE-2-tTA (tTA) and TRE-HoxA5-TIE2-tTA (HoxA5-tTA) mice. Results are expressed relative to the housekeeping gene GUSB (n = 3). ( C ) HoxA5 expression levels in ECs isolated from lungs of tTA and HoxA5-tTA mice. Histogram shows HoxA5 mRNA levels measured by real time PCR in the presence of Dox (1μg/ml) or 48 hours following removal of Dox in the cell culture media. Insert shows corresponding Western blot of protein lysates extracted from lung EC isolated from tTA and HoxA5-TA mice and detected via polyclonal antibodies against HoxA5. ( D ) Real time PCR analysis of relative mRNA expression levels of thrombospndin-2 (TSP-2) and VEGF-A levels one month after removal of Dox from the diet of HoxA5-tTA mice. Results are expressed relative to mRNA levels in age-matched tTA control mice lacking the HoxA5 transgene or Dox in the diet. ( E ) Vascular permeability in tTA or HoxA5-tTA mice. Measurement of extravasated Evans Blue dye 30 minutes following topical application of mineral oil (control, left panel) or Mustard oil to induce an acute leakage (right panel; n = 4).

    Techniques Used: Mouse Assay, Expressing, Transgenic Assay, Real-time Polymerase Chain Reaction, Isolation, Cell Culture, Western Blot, Permeability

    Sustained HoxA5 expression inhibits wound healing. ( A ) Wound closure in tTA (diamonds) and HoxA5-tTA (circles) mice. Wounds were measured every 4 days. HoxA5-tTA mice showed a significant (P
    Figure Legend Snippet: Sustained HoxA5 expression inhibits wound healing. ( A ) Wound closure in tTA (diamonds) and HoxA5-tTA (circles) mice. Wounds were measured every 4 days. HoxA5-tTA mice showed a significant (P

    Techniques Used: Expressing, Mouse Assay

    Analysis of ear vessel density in mice. ( A ) Confocal images of FITC- L . esculentum lectin (green) in whole mounts of ears from tTA (upper) and HoxA5-tTA (lower) mice maintained without Dox (-Dox) during gestation and for an additional 8 weeks after birth. ( B ) Quantitative analysis of vessel area using Image J analysis of confocal images after binary conversion in mice maintained without Dox (- Dox) during gestation and for an additional 8 weeks after birth. (n = 5). ( C ) Qualitative analysis of vasculature described in A and B in tTA and HoxA5-tTa mice following 2-D skeletonization and analysis using Image J (n = 5). ( D ) Analysis of vascular branch length ( > 12 or
    Figure Legend Snippet: Analysis of ear vessel density in mice. ( A ) Confocal images of FITC- L . esculentum lectin (green) in whole mounts of ears from tTA (upper) and HoxA5-tTA (lower) mice maintained without Dox (-Dox) during gestation and for an additional 8 weeks after birth. ( B ) Quantitative analysis of vessel area using Image J analysis of confocal images after binary conversion in mice maintained without Dox (- Dox) during gestation and for an additional 8 weeks after birth. (n = 5). ( C ) Qualitative analysis of vasculature described in A and B in tTA and HoxA5-tTa mice following 2-D skeletonization and analysis using Image J (n = 5). ( D ) Analysis of vascular branch length ( > 12 or

    Techniques Used: Mouse Assay

    HoxA5 expression in EC inhibits angiogenesis and growth of allograph mammary tumors. ( A ) Tumor volume in tTA (square) and HoxA5-tTA (triangle) 8 week old mice (3 weeks + Dox, 5 weeks—Dox), 32 days following subcutaneous injection of MMTV-PyMT tumor cells into syngeneic female FVB/n mice. The analysis revealed a significant reduction (p
    Figure Legend Snippet: HoxA5 expression in EC inhibits angiogenesis and growth of allograph mammary tumors. ( A ) Tumor volume in tTA (square) and HoxA5-tTA (triangle) 8 week old mice (3 weeks + Dox, 5 weeks—Dox), 32 days following subcutaneous injection of MMTV-PyMT tumor cells into syngeneic female FVB/n mice. The analysis revealed a significant reduction (p

    Techniques Used: Expressing, Mouse Assay, Injection

    13) Product Images from "Protection of the heart by treatment with a divalent-copper-selective chelator reveals a novel mechanism underlying cardiomyopathy in diabetic rats"

    Article Title: Protection of the heart by treatment with a divalent-copper-selective chelator reveals a novel mechanism underlying cardiomyopathy in diabetic rats

    Journal: Cardiovascular Diabetology

    doi: 10.1186/1475-2840-12-123

    Depressed cardiac contractility in diabetic rats prevented by TETA-treatment.  Depressed cardiac contractility in diabetic rats was prevented by TETA-treatment, which by contrast did not modify diabetes-induced alterations in [Ca 2+ ] i  homeostasis.  (A)  Diabetic rats showed unchanged peak (i) and resting (ii) [Ca 2+ ] i  values but had concomitantly decreased peak stress (iii) (Diabetic: 10 ± 1 mN/mm 2 ; Control: 17 ± 2 mN/mm 2 ,  P =  0.02) and unchanged resting stress (iv). TETA-treatment preserved peak stress in diabetic rats (TETA-treated diabetic: 20 ± 4 mN/mm 2 ,  P =  0.04) but did not significantly modify peak or resting [Ca 2+ ] i .  (B)  The time course of the Ca 2+  transient was prolonged in diabetic rats: (time-to-peak [Ca 2+ ] i  (i); and time constant of decay of the Ca 2+  transient (ii)), as was the time course of isometric stress (time-to-peak stress (iii); and time-to-90% relaxation (iv)). However, TETA-treatment had no effect on the time course of either variable.  (C)  The maximum rate of rise in the Ca 2+  transient was unchanged (i) whereas the maximum rate of stress development was decreased in diabetic rats (ii), and this decrease was prevented by TETA-treatment. C: Control (Open bars,  n =  10); D: Diabetic (Solid bars,  n =  8); D + T: TETA-treated diabetic (Patterned bars,  n =  7). Data are means ± SEM, one-way ANOVA with application of the  post-hoc  Holm-Sidak test: * C vs D; ‡ C vs D + T; † D vs D + T;  P
    Figure Legend Snippet: Depressed cardiac contractility in diabetic rats prevented by TETA-treatment. Depressed cardiac contractility in diabetic rats was prevented by TETA-treatment, which by contrast did not modify diabetes-induced alterations in [Ca 2+ ] i homeostasis. (A) Diabetic rats showed unchanged peak (i) and resting (ii) [Ca 2+ ] i values but had concomitantly decreased peak stress (iii) (Diabetic: 10 ± 1 mN/mm 2 ; Control: 17 ± 2 mN/mm 2 , P =  0.02) and unchanged resting stress (iv). TETA-treatment preserved peak stress in diabetic rats (TETA-treated diabetic: 20 ± 4 mN/mm 2 , P =  0.04) but did not significantly modify peak or resting [Ca 2+ ] i . (B) The time course of the Ca 2+ transient was prolonged in diabetic rats: (time-to-peak [Ca 2+ ] i (i); and time constant of decay of the Ca 2+ transient (ii)), as was the time course of isometric stress (time-to-peak stress (iii); and time-to-90% relaxation (iv)). However, TETA-treatment had no effect on the time course of either variable. (C) The maximum rate of rise in the Ca 2+ transient was unchanged (i) whereas the maximum rate of stress development was decreased in diabetic rats (ii), and this decrease was prevented by TETA-treatment. C: Control (Open bars, n =  10); D: Diabetic (Solid bars, n =  8); D + T: TETA-treated diabetic (Patterned bars, n =  7). Data are means ± SEM, one-way ANOVA with application of the post-hoc Holm-Sidak test: * C vs D; ‡ C vs D + T; † D vs D + T; P

    Techniques Used:

    Alterations in SERCA2a and NCX in LV myocardium. (A)  Caffeine-induced Ca 2+  transients were recorded from cardiomyocytes isolated from each group of rats, which were exposed to a series of solution changes as described in methods. (i) shows examples of normalized caffeine-induced Ca 2+  transients from a single cell, with pooled data shown in (ii)  (iii). There was no significant difference in the time-constant of decay of caffeine-induced Ca 2+  transients among groups in either of the caffeine perfusion solutions, indicating no change of NCX activity among groups. These data suggest that the slower decay of the Ca 2+  transient in diabetic rats did not arise from differences in NCX function. Consistently, a western blotting study  (B)  showed no significant change in NCX levels (molecular weight 120 kDa, n = 8 in each group) among groups, but decreased expression of SERCA2a (molecular weight 110 kDa, n = 7 in each group) in diabetic rats. TETA had no effect on levels of either transporter. C: Control (Open bars); D: Diabetic (Solid bars); D + T: TETA-treated diabetic (Patterned bars). Data are means ± SEM, one-way Kruskal-Wallis ANOVA with  post-hoc  application of Dunn’s Multiple Comparisons test ( P =  0.0007): * C vs D,  P
    Figure Legend Snippet: Alterations in SERCA2a and NCX in LV myocardium. (A) Caffeine-induced Ca 2+ transients were recorded from cardiomyocytes isolated from each group of rats, which were exposed to a series of solution changes as described in methods. (i) shows examples of normalized caffeine-induced Ca 2+ transients from a single cell, with pooled data shown in (ii) (iii). There was no significant difference in the time-constant of decay of caffeine-induced Ca 2+ transients among groups in either of the caffeine perfusion solutions, indicating no change of NCX activity among groups. These data suggest that the slower decay of the Ca 2+ transient in diabetic rats did not arise from differences in NCX function. Consistently, a western blotting study (B) showed no significant change in NCX levels (molecular weight 120 kDa, n = 8 in each group) among groups, but decreased expression of SERCA2a (molecular weight 110 kDa, n = 7 in each group) in diabetic rats. TETA had no effect on levels of either transporter. C: Control (Open bars); D: Diabetic (Solid bars); D + T: TETA-treated diabetic (Patterned bars). Data are means ± SEM, one-way Kruskal-Wallis ANOVA with post-hoc application of Dunn’s Multiple Comparisons test ( P =  0.0007): * C vs D, P

    Techniques Used: Isolation, Activity Assay, Western Blot, Molecular Weight, Expressing

    The steady-state force-[Ca 2+ ] i  relationship and expression of TnT  TnI in LV myocardium. (A)  Representative traces of [Ca 2+ ] i  and stress during tetanic stimulation of a trabecula from a diabetic rat (at [Ca 2+ ] o : 0.5, 5, 15 and 30 mmol/L); the solid arrows indicate where stimulation started and ended; the dashed arrows indicate that the resting [Ca 2+ ] i  and the corresponding resting stress at 30 mmol/L [Ca 2+ ] o  were comparable to the tetanized [Ca 2+ ] i  and its corresponding stress at 5 mmol/L [Ca 2+ ] o .  (B)  The corresponding data obtained 4 s after commencing tetanic stimulation from this trabecula were fitted to the Hill equation as shown.  (C)  The rising aspects of the phase plots of [Ca 2+ ] i  and tetanus at different [Ca 2+ ] o  values from the same trabecula are shown (irregular grey lines), where the data used for fitting to the Hill equation (as in B) have been superimposed (black squares).  (D)  Averaged relaxation phase plots of [Ca 2+ ] i  and tetanus at [Ca 2+ ] o  20 mmol/L from numbers of trabeculae in each experimental groups (Control: black line,  n =  9; Diabetic: red line,  n =  7; TETA-treated diabetic: blue line,  n =  7). Diabetic rats showed a rightward shift of the relaxation phase, consistent with decreased myofibrillar Ca 2+  sensitivity whereas TETA-treatment preserved the Ca 2+  sensitivity in diabetic hearts.  (E)  Expression of TnT (upper left panel shows representative western blots of three animals from each group; box and whisker plots (median, range) below show normalized densitometry of both TnT bands (i)    ratios of the two TnT bands (ii) at molecular weights in the range of 40–42.5 kDa, n = 5 in each group); and TnI (right panel; iii, molecular weight 28 kDa, n = 6 in each group) in LV tissue from the three experimental groups; these showed no significant between-group differences. C: Control (Open bars); D: Diabetic (Solid bars); D + T: TETA-treated diabetic (Patterned bars). Significance was tested by one-way Kruskal-Wallis ANOVA.
    Figure Legend Snippet: The steady-state force-[Ca 2+ ] i relationship and expression of TnT TnI in LV myocardium. (A) Representative traces of [Ca 2+ ] i and stress during tetanic stimulation of a trabecula from a diabetic rat (at [Ca 2+ ] o : 0.5, 5, 15 and 30 mmol/L); the solid arrows indicate where stimulation started and ended; the dashed arrows indicate that the resting [Ca 2+ ] i and the corresponding resting stress at 30 mmol/L [Ca 2+ ] o were comparable to the tetanized [Ca 2+ ] i and its corresponding stress at 5 mmol/L [Ca 2+ ] o . (B) The corresponding data obtained 4 s after commencing tetanic stimulation from this trabecula were fitted to the Hill equation as shown. (C) The rising aspects of the phase plots of [Ca 2+ ] i and tetanus at different [Ca 2+ ] o values from the same trabecula are shown (irregular grey lines), where the data used for fitting to the Hill equation (as in B) have been superimposed (black squares). (D) Averaged relaxation phase plots of [Ca 2+ ] i and tetanus at [Ca 2+ ] o 20 mmol/L from numbers of trabeculae in each experimental groups (Control: black line, n =  9; Diabetic: red line, n =  7; TETA-treated diabetic: blue line, n =  7). Diabetic rats showed a rightward shift of the relaxation phase, consistent with decreased myofibrillar Ca 2+ sensitivity whereas TETA-treatment preserved the Ca 2+ sensitivity in diabetic hearts. (E) Expression of TnT (upper left panel shows representative western blots of three animals from each group; box and whisker plots (median, range) below show normalized densitometry of both TnT bands (i) ratios of the two TnT bands (ii) at molecular weights in the range of 40–42.5 kDa, n = 5 in each group); and TnI (right panel; iii, molecular weight 28 kDa, n = 6 in each group) in LV tissue from the three experimental groups; these showed no significant between-group differences. C: Control (Open bars); D: Diabetic (Solid bars); D + T: TETA-treated diabetic (Patterned bars). Significance was tested by one-way Kruskal-Wallis ANOVA.

    Techniques Used: Expressing, Western Blot, Whisker Assay, Molecular Weight

    Isometric force and [Ca 2+ ] i  measured from LV trabeculae.  Isometric force and [Ca 2+ ] i  were measured simultaneously in LV trabeculae at 37°C, 5 Hz stimulation frequency and 1.5 mmol/L [Ca 2+ ] o , conditions that are close to physiological.  (A)  Exemplary traces of Ca 2+  transients (fura-2/AM 340/380 ratio) and corresponding isometric stress (force normalized to the corresponding muscle’s cross-sectional area) averaged over a number of cardiac cycles in representative trabeculae from the 3 experimental groups, which typify those used for data analysis. Inserted figures are corresponding raw traces of Ca 2+  transients and corresponding isometric stress.  (B)  Averaged values from 7 trabeculae in each experimental group for (i) the Ca 2+  transient, (ii) isometric stress and (iii) phase plots of Ca 2+  transients and corresponding isometric stress. Diabetic rats showed decreased responsiveness to Ca 2+ , as indicated by the rightward shift of the relaxation phase (iii) and TETA-treatment prevented development of this defect.
    Figure Legend Snippet: Isometric force and [Ca 2+ ] i measured from LV trabeculae. Isometric force and [Ca 2+ ] i were measured simultaneously in LV trabeculae at 37°C, 5 Hz stimulation frequency and 1.5 mmol/L [Ca 2+ ] o , conditions that are close to physiological. (A) Exemplary traces of Ca 2+ transients (fura-2/AM 340/380 ratio) and corresponding isometric stress (force normalized to the corresponding muscle’s cross-sectional area) averaged over a number of cardiac cycles in representative trabeculae from the 3 experimental groups, which typify those used for data analysis. Inserted figures are corresponding raw traces of Ca 2+ transients and corresponding isometric stress. (B) Averaged values from 7 trabeculae in each experimental group for (i) the Ca 2+ transient, (ii) isometric stress and (iii) phase plots of Ca 2+ transients and corresponding isometric stress. Diabetic rats showed decreased responsiveness to Ca 2+ , as indicated by the rightward shift of the relaxation phase (iii) and TETA-treatment prevented development of this defect.

    Techniques Used:

    14) Product Images from "Natural killer cells limit the clearance of senescent lung adenocarcinoma cells"

    Article Title: Natural killer cells limit the clearance of senescent lung adenocarcinoma cells

    Journal: Oncogenesis

    doi: 10.1038/s41389-019-0133-3

    NK cells limit a p53-mediated immunoinflammatory reaction that is associated with lung adenocarcinoma regression. Cell suspensions from the lungs of KPr tumor-bearing Rag1 -/- mice treated with corn oil (Control), tamoxifen (Restored), and tamoxifen, and anti-NK1.1 (Restored NK Depleted) were subjected to multiparameter flow cytometry. a Cells gated to include live singlets that are CD45 pos. are plotted on CD11b X F4/80. b Alveolar macrophages (CD11b neg. ; F4/80 pos. ) are quantified from a . c Interstitial macrophages (CD11b pos. ;F4/80 pos. ) are quantified from a . d CD11b pos. cells from a are plotted on Ly6G X Ly6C. e Monocytes (CD11b pos. ; Ly6C pos. ; Ly6G neg. ) are quantified from d . f Neutrophils (CD11b pos. ; Ly6C pos. ; Ly6G pos. ) are quantified from d . Analysis of significance between control and restored treatment groups was performed by t -test
    Figure Legend Snippet: NK cells limit a p53-mediated immunoinflammatory reaction that is associated with lung adenocarcinoma regression. Cell suspensions from the lungs of KPr tumor-bearing Rag1 -/- mice treated with corn oil (Control), tamoxifen (Restored), and tamoxifen, and anti-NK1.1 (Restored NK Depleted) were subjected to multiparameter flow cytometry. a Cells gated to include live singlets that are CD45 pos. are plotted on CD11b X F4/80. b Alveolar macrophages (CD11b neg. ; F4/80 pos. ) are quantified from a . c Interstitial macrophages (CD11b pos. ;F4/80 pos. ) are quantified from a . d CD11b pos. cells from a are plotted on Ly6G X Ly6C. e Monocytes (CD11b pos. ; Ly6C pos. ; Ly6G neg. ) are quantified from d . f Neutrophils (CD11b pos. ; Ly6C pos. ; Ly6G pos. ) are quantified from d . Analysis of significance between control and restored treatment groups was performed by t -test

    Techniques Used: Mouse Assay, Flow Cytometry, Cytometry

    NK cells express B220 activation mark after p53 restoration. Cell suspensions from Rag1 -/- mouse lungs bearing orthotopically transplanted KPr lung adenocarcinomas treated with corn oil (Control) or tamoxifen (Restored) were subjected to multiparameter flow cytometry. a Cells gated to include live singlets that are CD45 pos. are plotted on NK1.1 x DX5. NK1.1 pos. ;DX5 pos. cells are gated (top) and then plotted for B220 pos. ;NKp46 pos. histogram. b Splenocytes also shown for comparison. c Analysis of significance between control and restored groups was performed by t -test
    Figure Legend Snippet: NK cells express B220 activation mark after p53 restoration. Cell suspensions from Rag1 -/- mouse lungs bearing orthotopically transplanted KPr lung adenocarcinomas treated with corn oil (Control) or tamoxifen (Restored) were subjected to multiparameter flow cytometry. a Cells gated to include live singlets that are CD45 pos. are plotted on NK1.1 x DX5. NK1.1 pos. ;DX5 pos. cells are gated (top) and then plotted for B220 pos. ;NKp46 pos. histogram. b Splenocytes also shown for comparison. c Analysis of significance between control and restored groups was performed by t -test

    Techniques Used: Activation Assay, Flow Cytometry, Cytometry

    Restoration of p53 in lung adenocarcinomas leads to potent immune inflammatory response in the lung. Cell suspensions from Rag1 -/- mouse lungs bearing orthotopically transplanted KPr adenocarcinomas were treated with corn oil (Control) or tamoxifen (Restored) and subjected to multiparameter flow cytometry. a Cells gated to include live singlets are plotted on histograms for CD45 and quantified at right. b CD45 pos. cells from a plotted on F4/80 X SSC. F4/80 pos. cells gated and quantified at right. c CD45 pos. cells from a plotted on CD11b X F4/80. CD11b neg. ;F4/80 pos. alveolar macrophages at bottom left and CD11b pos. F4/80 pos. CD11b pos. macrophages gated and quantified at bottom right. d CD11b pos. cells from a plotted on Ly6G X Ly6C. CD11b pos. Ly6G pos. neutrophils and CD11b pos. Ly6C pos. monocytes cells are gated and quantified at right. Analysis of significance between control and restored groups was performed by t -test
    Figure Legend Snippet: Restoration of p53 in lung adenocarcinomas leads to potent immune inflammatory response in the lung. Cell suspensions from Rag1 -/- mouse lungs bearing orthotopically transplanted KPr adenocarcinomas were treated with corn oil (Control) or tamoxifen (Restored) and subjected to multiparameter flow cytometry. a Cells gated to include live singlets are plotted on histograms for CD45 and quantified at right. b CD45 pos. cells from a plotted on F4/80 X SSC. F4/80 pos. cells gated and quantified at right. c CD45 pos. cells from a plotted on CD11b X F4/80. CD11b neg. ;F4/80 pos. alveolar macrophages at bottom left and CD11b pos. F4/80 pos. CD11b pos. macrophages gated and quantified at bottom right. d CD11b pos. cells from a plotted on Ly6G X Ly6C. CD11b pos. Ly6G pos. neutrophils and CD11b pos. Ly6C pos. monocytes cells are gated and quantified at right. Analysis of significance between control and restored groups was performed by t -test

    Techniques Used: Flow Cytometry, Cytometry

    15) Product Images from "Essential role of proteasomes in maintaining self-renewal in neural progenitor cells"

    Article Title: Essential role of proteasomes in maintaining self-renewal in neural progenitor cells

    Journal: Scientific Reports

    doi: 10.1038/srep19752

    Defective proliferation and neuronal differentiation capacities in NPCs derived from P90 and P540 mice. ( A ) SA-β-gal activity was measured in NPCs isolated from E14, P0, P90 and P540 mice. The senescent cells were stained into blue. ( B ) Representative phase-contrast images of neurospheres from E14, P0, P90 and P540 mice in cultures. ( C ) Proliferation of NPCs was determined by BrdU incorporation. The percentage of dividing cells was gradually reduced with age. ( D ) After a 5-day induction for neuronal differentiation, NPCs from E14, P0, P90 and P540 mice were stained with the antibody against Tuj1, an early neuronal marker (Red). The neuronal differentiation potential was compromised in NPCs from P90 or P540 mice, compared with that from E14 and P0 mice. DAPI (blue) was used to counterstain nuclei. ** p
    Figure Legend Snippet: Defective proliferation and neuronal differentiation capacities in NPCs derived from P90 and P540 mice. ( A ) SA-β-gal activity was measured in NPCs isolated from E14, P0, P90 and P540 mice. The senescent cells were stained into blue. ( B ) Representative phase-contrast images of neurospheres from E14, P0, P90 and P540 mice in cultures. ( C ) Proliferation of NPCs was determined by BrdU incorporation. The percentage of dividing cells was gradually reduced with age. ( D ) After a 5-day induction for neuronal differentiation, NPCs from E14, P0, P90 and P540 mice were stained with the antibody against Tuj1, an early neuronal marker (Red). The neuronal differentiation potential was compromised in NPCs from P90 or P540 mice, compared with that from E14 and P0 mice. DAPI (blue) was used to counterstain nuclei. ** p

    Techniques Used: Derivative Assay, Mouse Assay, Activity Assay, Isolation, Staining, BrdU Incorporation Assay, Marker

    Suppressed proliferation and neuronal differentiation of cultured NPCs following PSMB5 knockdown-triggered decrease in proteasomal activity. ( A ) NPCs isolated from E14 mice were transfected with si-PSMB5 for 72 hrs. The knockdown efficiency was measured by qPCR (a) and western blotting (b). (c) PSMB5 knockdown reduced proteasomal activity in NPCs. ( B ) PSMB5 knockdown prevented the neurosphere formation, as indicated by decreases in both the number and diameter of neurospheres. ( C ) The decline of dividing cells was found in NPCs transfected with si-PSMB5 by BrdU incorporation (a). (b) Levels of the cell cycle-related proteins Cyclin D1 and CDK4 were lowered in NPCs treated with si-PSMB5. ( D ) NPCs transfected with either scramble siRNA or si-PSMB5 were cultured in the differentiation medium for 5 days. Neuronal differentiation was evaluated by the Tuj1/DAPI percentage. * p
    Figure Legend Snippet: Suppressed proliferation and neuronal differentiation of cultured NPCs following PSMB5 knockdown-triggered decrease in proteasomal activity. ( A ) NPCs isolated from E14 mice were transfected with si-PSMB5 for 72 hrs. The knockdown efficiency was measured by qPCR (a) and western blotting (b). (c) PSMB5 knockdown reduced proteasomal activity in NPCs. ( B ) PSMB5 knockdown prevented the neurosphere formation, as indicated by decreases in both the number and diameter of neurospheres. ( C ) The decline of dividing cells was found in NPCs transfected with si-PSMB5 by BrdU incorporation (a). (b) Levels of the cell cycle-related proteins Cyclin D1 and CDK4 were lowered in NPCs treated with si-PSMB5. ( D ) NPCs transfected with either scramble siRNA or si-PSMB5 were cultured in the differentiation medium for 5 days. Neuronal differentiation was evaluated by the Tuj1/DAPI percentage. * p

    Techniques Used: Cell Culture, Activity Assay, Isolation, Mouse Assay, Transfection, Real-time Polymerase Chain Reaction, Western Blot, BrdU Incorporation Assay

    Appearance of senescence-like phenotypes in NPCs exposed to the proteasomal inhibitor MG132. ( A ) The number of proliferating cells in the SVZ was quantified by BrdU labeling 7 days after intraventricular injection with 10 μg MG132 or the vehicle DMSO. Notably, MG132 injection robustly inhibited NPCs proliferation in vivo (a), concurrent with a reduced proteasomal activity (b). ( B ) NPCs from E14 mice incubated with 0.2 μM MG132 for 24 hrs formed fewer neurospheres. ( C ) BrdU incorporation indicated a retarded proliferation of NPCs after an 8-hr treatment with MG132. The percentage of BrdU + cells was reduced in a concentration-dependent manner. ( D ) After exposing NPCs to MG132 for 5 hrs, the cell viability was decreased in a concentration-dependent manner as measured by CCK-8 assay. ( E ) Tuj1 staining showed that treatment with MG132 attenuated the neuronal differentiation of NPCs in vitro . ** p
    Figure Legend Snippet: Appearance of senescence-like phenotypes in NPCs exposed to the proteasomal inhibitor MG132. ( A ) The number of proliferating cells in the SVZ was quantified by BrdU labeling 7 days after intraventricular injection with 10 μg MG132 or the vehicle DMSO. Notably, MG132 injection robustly inhibited NPCs proliferation in vivo (a), concurrent with a reduced proteasomal activity (b). ( B ) NPCs from E14 mice incubated with 0.2 μM MG132 for 24 hrs formed fewer neurospheres. ( C ) BrdU incorporation indicated a retarded proliferation of NPCs after an 8-hr treatment with MG132. The percentage of BrdU + cells was reduced in a concentration-dependent manner. ( D ) After exposing NPCs to MG132 for 5 hrs, the cell viability was decreased in a concentration-dependent manner as measured by CCK-8 assay. ( E ) Tuj1 staining showed that treatment with MG132 attenuated the neuronal differentiation of NPCs in vitro . ** p

    Techniques Used: Labeling, Injection, In Vivo, Activity Assay, Mouse Assay, Incubation, BrdU Incorporation Assay, Concentration Assay, CCK-8 Assay, Staining, In Vitro

    Rejuvenation of adult NPCs by up-regulating proteasomal activity. ( A ) Intraventricular injection with the recombinant PSMB5-overexpressing lentiviral particles for 7 days enhanced proteasomal activity in the SVZ of P90 mice. ( B ) PSMB5 over-expression increased BrdU + cells in the SVZ (n = 6 mice). ( C ) NPCs derived from P90 mice were incubated with 18α-GA (2 μg/mL) for 10 days, followed by the proteasomal activity assay. ( D ) NPCs formed neurospheres with a larger size in the presence of 18α-GA. ( E ) After differentiation for 5 days, NPCs treated with 18α-GA generated more Tuj1 + cells than those treated with DMSO. * p
    Figure Legend Snippet: Rejuvenation of adult NPCs by up-regulating proteasomal activity. ( A ) Intraventricular injection with the recombinant PSMB5-overexpressing lentiviral particles for 7 days enhanced proteasomal activity in the SVZ of P90 mice. ( B ) PSMB5 over-expression increased BrdU + cells in the SVZ (n = 6 mice). ( C ) NPCs derived from P90 mice were incubated with 18α-GA (2 μg/mL) for 10 days, followed by the proteasomal activity assay. ( D ) NPCs formed neurospheres with a larger size in the presence of 18α-GA. ( E ) After differentiation for 5 days, NPCs treated with 18α-GA generated more Tuj1 + cells than those treated with DMSO. * p

    Techniques Used: Activity Assay, Injection, Recombinant, Mouse Assay, Over Expression, Derivative Assay, Incubation, Generated

    Expression of 20S proteasome in NPCs. ( A ) Representative images of coronal sections immunostained for 20S proteasome (Green) and nestin (Red) in the VZ/SVZ of E14 and the SVZ of P90 mice. Images with a higher magnification are presented. ( B ) Neurospheres from E14 and P90 mice were immunostained for 20S proteasome (Green) and nestin (Red), with nuclei counterstained by DAPI (Blue).
    Figure Legend Snippet: Expression of 20S proteasome in NPCs. ( A ) Representative images of coronal sections immunostained for 20S proteasome (Green) and nestin (Red) in the VZ/SVZ of E14 and the SVZ of P90 mice. Images with a higher magnification are presented. ( B ) Neurospheres from E14 and P90 mice were immunostained for 20S proteasome (Green) and nestin (Red), with nuclei counterstained by DAPI (Blue).

    Techniques Used: Expressing, Mouse Assay

    16) Product Images from "Leukemia inhibitory factor regulates the timing of oligodendrocyte development and myelination in the postnatal optic nerve"

    Article Title: Leukemia inhibitory factor regulates the timing of oligodendrocyte development and myelination in the postnatal optic nerve

    Journal: Journal of neuroscience research

    doi: 10.1002/jnr.22173

    Proliferation of OPCs was increased and differentiation from the progenitor stage was inhibited in the optic nerve of LIF -/- mice. To determine the effects of endogenous LIF on OPC differentiation, OPCs isolated from optic nerve of P10 mice were grown
    Figure Legend Snippet: Proliferation of OPCs was increased and differentiation from the progenitor stage was inhibited in the optic nerve of LIF -/- mice. To determine the effects of endogenous LIF on OPC differentiation, OPCs isolated from optic nerve of P10 mice were grown

    Techniques Used: Mouse Assay, Isolation

    Gene Expression Analysis of P10 Wild-Type/LIF Knockout Optic Nerves
    Figure Legend Snippet: Gene Expression Analysis of P10 Wild-Type/LIF Knockout Optic Nerves

    Techniques Used: Expressing, Knock-Out

    Gene Expression Analysis of P10 Wild-Type/LIF Knockout Optic Nerves
    Figure Legend Snippet: Gene Expression Analysis of P10 Wild-Type/LIF Knockout Optic Nerves

    Techniques Used: Expressing, Knock-Out

    The population of cells in the oligodendrocyte lineage is reduced and displays a pronounced chiasm-to-retinal gradient in P10 optic nerve of LIF -/- mice. The optic nerves from LIF +/+ (A) and LIF -/- mice (C) at 10 days of age were stained with anti-Olig2
    Figure Legend Snippet: The population of cells in the oligodendrocyte lineage is reduced and displays a pronounced chiasm-to-retinal gradient in P10 optic nerve of LIF -/- mice. The optic nerves from LIF +/+ (A) and LIF -/- mice (C) at 10 days of age were stained with anti-Olig2

    Techniques Used: Mouse Assay, Staining

    Gene Expression Analysis of P10 Wild-Type/LIF Knockout Optic Nerves
    Figure Legend Snippet: Gene Expression Analysis of P10 Wild-Type/LIF Knockout Optic Nerves

    Techniques Used: Expressing, Knock-Out

    Gene Expression Analysis of P10 Wild-Type/LIF Knockout Optic Nerves
    Figure Legend Snippet: Gene Expression Analysis of P10 Wild-Type/LIF Knockout Optic Nerves

    Techniques Used: Expressing, Knock-Out

    MBP and PLP immunoreactivity are markedly reduced in optic nerve of P10 LIF -/- mice. The optic nerves from LIF +/+ mice (A) and LIF -/- mice (C) at 10 days of age were stained with anti-MBP antibody. MBP-positive myelin was observed throughout the entire
    Figure Legend Snippet: MBP and PLP immunoreactivity are markedly reduced in optic nerve of P10 LIF -/- mice. The optic nerves from LIF +/+ mice (A) and LIF -/- mice (C) at 10 days of age were stained with anti-MBP antibody. MBP-positive myelin was observed throughout the entire

    Techniques Used: Plasmid Purification, Mouse Assay, Staining

    Gene Expression Analysis of P10 Wild-Type/LIF Knockout Optic Nerves
    Figure Legend Snippet: Gene Expression Analysis of P10 Wild-Type/LIF Knockout Optic Nerves

    Techniques Used: Expressing, Knock-Out

    17) Product Images from "Human mesotrypsin is a unique digestive protease specialized for the degradation of trypsin inhibitors."

    Article Title: Human mesotrypsin is a unique digestive protease specialized for the degradation of trypsin inhibitors.

    Journal: The Journal of biological chemistry

    doi: 10.1074/jbc.M310301200

    Activation of pancreatic zymogens with wild-type and R198G mutant mesotrypsin (PRSS3). Human cationic trypsinogen (PRSS1-trypsinogen, 2 μM), human anionic trypsinogen (PRSS2-trypsinogen, 2 μM), bovine chymotrypsinogen A (9 μM) and human pro-elastase 2 (1 μM, final concentrations) were incubated at 37 °C, in 0.1 M Tris-HCl (pH 8.0), 1 mM CaCl 2 and 2 mg/mL bovine serum albumin. To test trypsinogen activation, incubations were performed in the absence of added trypsin (control) or in the presence of 120 nM (final concentration) cationic trypsin (PRSS1), anionic trypsin (PRSS2), wild-type mesotrypsin (PRSS3) or R198G mesotrypsin mutant. Bovine chymotrypsinogen A and human pro-elastase 2 were activated with 32 nM and 75 nM trypsins, respectively. Although not shown, in other experiments mesotrypsin at 125 nM concentration activated approximately 5 % of bovine chymotrypsinogen in 90 min, but had no measurable activating effect on human pro-elastase 2. Protease activities were determined as described in Experimental Procedures , and expressed as percent of maximal activity.
    Figure Legend Snippet: Activation of pancreatic zymogens with wild-type and R198G mutant mesotrypsin (PRSS3). Human cationic trypsinogen (PRSS1-trypsinogen, 2 μM), human anionic trypsinogen (PRSS2-trypsinogen, 2 μM), bovine chymotrypsinogen A (9 μM) and human pro-elastase 2 (1 μM, final concentrations) were incubated at 37 °C, in 0.1 M Tris-HCl (pH 8.0), 1 mM CaCl 2 and 2 mg/mL bovine serum albumin. To test trypsinogen activation, incubations were performed in the absence of added trypsin (control) or in the presence of 120 nM (final concentration) cationic trypsin (PRSS1), anionic trypsin (PRSS2), wild-type mesotrypsin (PRSS3) or R198G mesotrypsin mutant. Bovine chymotrypsinogen A and human pro-elastase 2 were activated with 32 nM and 75 nM trypsins, respectively. Although not shown, in other experiments mesotrypsin at 125 nM concentration activated approximately 5 % of bovine chymotrypsinogen in 90 min, but had no measurable activating effect on human pro-elastase 2. Protease activities were determined as described in Experimental Procedures , and expressed as percent of maximal activity.

    Techniques Used: Activation Assay, Mutagenesis, Incubation, Concentration Assay, Activity Assay

    18) Product Images from "Early Neoplastic Progression Is Complement Independent"

    Article Title: Early Neoplastic Progression Is Complement Independent

    Journal: Neoplasia (New York, N.Y.)

    doi:

    Infiltration of inflammatory cells during neoplastic progression is not reduced in the absence of C3. (A) Single-cell suspensions derived from negative littermate ear tissue (-LM) and hyperplastic (1 month of age), early dysplastic (4 months of age), and late dysplastic (6 months of age) ear tissue from HPV16 mice and HPV16/C3 -/- mice were analyzed by flow cytometry to determine the percentage of CD45 + cells as a percentage of total viable cells (n = 3). Results are shown as mean percentages ± SEM. No statistically significant differences were observed between age-matched neoplastic tissues of HPV16 and HPV16/C3 -/- mice (Mann-Whitney unpaired t test). (B) Quantitative analysis of mast cells at distinct stages of neoplastic progression in ear tissue from HPV16, HPV16/C3 -/- mice, and negative littermates (-LM). Mast cells were visualized by CAE histochemistry on 5-µm paraffin-embedded tissue sections. Values represent number of mast cells, averaged from five high-power fields per mouse and five mice per category. Error bars represent SEM. No statistically significant differences were observed between age-matched neoplastic tissues between HPV16 and HPV16/C3 -/- mice (Mann-Whitney unpaired t test). (C) Quantitative analysis of neutrophils at distinct stages of neoplastic progression in ear tissue from negative littermate (-)LM, HPV16, and HPV16/C3 -/- mice. Neutrophils were visualized by immunohistochemistry on 5-µm paraffin-embedded tissue sections. Values represent number of neutrophils, averaged from five highpower fields per mouse and five mice per category. Error bars represent SEM. No statistically significant differences were observed between agematched neoplastic tissues between HPV16 and HPV16/C3 -/- mice (Mann-Whitney unpaired t test).
    Figure Legend Snippet: Infiltration of inflammatory cells during neoplastic progression is not reduced in the absence of C3. (A) Single-cell suspensions derived from negative littermate ear tissue (-LM) and hyperplastic (1 month of age), early dysplastic (4 months of age), and late dysplastic (6 months of age) ear tissue from HPV16 mice and HPV16/C3 -/- mice were analyzed by flow cytometry to determine the percentage of CD45 + cells as a percentage of total viable cells (n = 3). Results are shown as mean percentages ± SEM. No statistically significant differences were observed between age-matched neoplastic tissues of HPV16 and HPV16/C3 -/- mice (Mann-Whitney unpaired t test). (B) Quantitative analysis of mast cells at distinct stages of neoplastic progression in ear tissue from HPV16, HPV16/C3 -/- mice, and negative littermates (-LM). Mast cells were visualized by CAE histochemistry on 5-µm paraffin-embedded tissue sections. Values represent number of mast cells, averaged from five high-power fields per mouse and five mice per category. Error bars represent SEM. No statistically significant differences were observed between age-matched neoplastic tissues between HPV16 and HPV16/C3 -/- mice (Mann-Whitney unpaired t test). (C) Quantitative analysis of neutrophils at distinct stages of neoplastic progression in ear tissue from negative littermate (-)LM, HPV16, and HPV16/C3 -/- mice. Neutrophils were visualized by immunohistochemistry on 5-µm paraffin-embedded tissue sections. Values represent number of neutrophils, averaged from five highpower fields per mouse and five mice per category. Error bars represent SEM. No statistically significant differences were observed between agematched neoplastic tissues between HPV16 and HPV16/C3 -/- mice (Mann-Whitney unpaired t test).

    Techniques Used: Derivative Assay, Mouse Assay, Flow Cytometry, Cytometry, MANN-WHITNEY, Immunohistochemistry

    Activation of keratinocyte hyperproliferation and angiogenesis during neoplastic progression is not affected in the absence of C3. (A) Quantitative analysis of the percentage of BrdU-positive keratinocytes (keratinocyte proliferative index) at distinct stages of neoplastic progression in ear tissue of HPV16 and HPV16/C3 -/- mice at 1, 4, and 6 months of age. BrdU + cells were visualized by immunohistochemical detection on 5-µm paraffin-embedded tissue sections. Values represent keratinocyte proliferative indices, averaged from five high-power fields per mouse and four to eight mice per category. Error bars represent SEM. No statistically significant differences were observed between age-matched neoplastic tissues between HPV16 and HPV16/C3 -/- mice (Mann-Whitney unpaired t test). (B) Immunolocalization of CD31/PECAM-1 (black staining) in 5-µm paraffin-embedded sections of age-matched neoplastic ear tissue of HPV16 and HPV16/C3 -/- mice and in ear tissue of negative littermate (-LM) mice reveals no difference between dilated and enlarged capillaries comparing HPV16 and HPV16/C3 -/- mice at all ages tested. Dashed line indicates epidermal-dermal interface. Arrowheads indicate representative blood vessels. The epidermal (e), dermal (d), and cartilage (c) regions of the tissues are indicated. Scale bar: 50 µm.
    Figure Legend Snippet: Activation of keratinocyte hyperproliferation and angiogenesis during neoplastic progression is not affected in the absence of C3. (A) Quantitative analysis of the percentage of BrdU-positive keratinocytes (keratinocyte proliferative index) at distinct stages of neoplastic progression in ear tissue of HPV16 and HPV16/C3 -/- mice at 1, 4, and 6 months of age. BrdU + cells were visualized by immunohistochemical detection on 5-µm paraffin-embedded tissue sections. Values represent keratinocyte proliferative indices, averaged from five high-power fields per mouse and four to eight mice per category. Error bars represent SEM. No statistically significant differences were observed between age-matched neoplastic tissues between HPV16 and HPV16/C3 -/- mice (Mann-Whitney unpaired t test). (B) Immunolocalization of CD31/PECAM-1 (black staining) in 5-µm paraffin-embedded sections of age-matched neoplastic ear tissue of HPV16 and HPV16/C3 -/- mice and in ear tissue of negative littermate (-LM) mice reveals no difference between dilated and enlarged capillaries comparing HPV16 and HPV16/C3 -/- mice at all ages tested. Dashed line indicates epidermal-dermal interface. Arrowheads indicate representative blood vessels. The epidermal (e), dermal (d), and cartilage (c) regions of the tissues are indicated. Scale bar: 50 µm.

    Techniques Used: Activation Assay, Mouse Assay, Immunohistochemistry, MANN-WHITNEY, Staining

    IgG deposition in the neoplastic microenvironment of HPV16 mice. Direct immunofluorescence on 10-µm OCT-embedded frozen tissue sections detects IgG antibodies (FITC signal) in negative littermate (-LM) ear skin, hyperplastic (1 month of age), early dysplastic (4 months of age), and late dysplastic (6 months of age) ear tissue from HPV16 and HPV16/C3 -/- mice. Dashed line indicates epidermal-dermal interface. The epidermis (e), dermis (d), and cartilage (c) are indicated. Scale bar: 50 µm.
    Figure Legend Snippet: IgG deposition in the neoplastic microenvironment of HPV16 mice. Direct immunofluorescence on 10-µm OCT-embedded frozen tissue sections detects IgG antibodies (FITC signal) in negative littermate (-LM) ear skin, hyperplastic (1 month of age), early dysplastic (4 months of age), and late dysplastic (6 months of age) ear tissue from HPV16 and HPV16/C3 -/- mice. Dashed line indicates epidermal-dermal interface. The epidermis (e), dermis (d), and cartilage (c) are indicated. Scale bar: 50 µm.

    Techniques Used: Mouse Assay, Immunofluorescence

    19) Product Images from "Cytosolic Phospholipase A2α Promotes Pulmonary Inflammation and Systemic Disease during Streptococcus pneumoniae Infection"

    Article Title: Cytosolic Phospholipase A2α Promotes Pulmonary Inflammation and Systemic Disease during Streptococcus pneumoniae Infection

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00280-17

    S. pneumoniae infection of airway epithelial cells triggers arachidonic acid (AA) release. (A) H292 cell monolayers were labeled with [ 3 H]AA as detailed in Materials and Methods and infected with the indicated S. pneumoniae ( S.p. ) strains (each of a different capsular serotype) or with B. subtilis strain 168 at a multiplicity of infection (MOI) of 10 (1 ×10 7 CFU/monolayer). Supernatants were collected at the indicated times postinfection, and radioactive counts released into the supernatants were measured by scintillation counting. Labeled H292 cell monolayers treated with PMA and HBSS+Ca/Mg were used as positive and negative controls, respectively. Shown are results from a representative of two experiments. (B) H292 cell monolayers labeled with [ 3 H]AA were treated with the indicated concentrations of pan-PLA 2 inhibitors ACA or ONO-RS-082 or the DAG lipase inhibitor RHC-80267 (labeled as RHC) prior to infection with S. pneumoniae TIGR4, as detailed in Materials and Methods. Labeled H292 cell monolayers treated with HBSS+Ca/Mg were used as negative controls. Radioactive counts released into supernatants were determined by scintillation counting. Shown are results from a representative of three experiments. *, P
    Figure Legend Snippet: S. pneumoniae infection of airway epithelial cells triggers arachidonic acid (AA) release. (A) H292 cell monolayers were labeled with [ 3 H]AA as detailed in Materials and Methods and infected with the indicated S. pneumoniae ( S.p. ) strains (each of a different capsular serotype) or with B. subtilis strain 168 at a multiplicity of infection (MOI) of 10 (1 ×10 7 CFU/monolayer). Supernatants were collected at the indicated times postinfection, and radioactive counts released into the supernatants were measured by scintillation counting. Labeled H292 cell monolayers treated with PMA and HBSS+Ca/Mg were used as positive and negative controls, respectively. Shown are results from a representative of two experiments. (B) H292 cell monolayers labeled with [ 3 H]AA were treated with the indicated concentrations of pan-PLA 2 inhibitors ACA or ONO-RS-082 or the DAG lipase inhibitor RHC-80267 (labeled as RHC) prior to infection with S. pneumoniae TIGR4, as detailed in Materials and Methods. Labeled H292 cell monolayers treated with HBSS+Ca/Mg were used as negative controls. Radioactive counts released into supernatants were determined by scintillation counting. Shown are results from a representative of three experiments. *, P

    Techniques Used: Infection, Labeling, Proximity Ligation Assay

    Ablation of cPLA 2 α leads to decreased bacteremia and increased survival in an otherwise lethal S. pneumoniae lung challenge. cPLA 2 α −/− mice or their littermate WT controls were intratracheally inoculated with TIGR4 (see Materials and Methods). A control group of WT mice received PBS. (A) Bacteremia was determined by plating tail vein blood at the specified time points. Shown are results from a combination of two experiments. Death of all infected WT mice is indicated by a dagger. (B) Survival of animals was monitored over a 7-day postinfection period. The log rank (Mantel-Cox) test was performed for survival curve analysis. The experiment was performed twice, with similar results. Shown are results from a combination of two experiments. A total of 8 mice were used per group; panels A and B represent the same cohorts of mice.
    Figure Legend Snippet: Ablation of cPLA 2 α leads to decreased bacteremia and increased survival in an otherwise lethal S. pneumoniae lung challenge. cPLA 2 α −/− mice or their littermate WT controls were intratracheally inoculated with TIGR4 (see Materials and Methods). A control group of WT mice received PBS. (A) Bacteremia was determined by plating tail vein blood at the specified time points. Shown are results from a combination of two experiments. Death of all infected WT mice is indicated by a dagger. (B) Survival of animals was monitored over a 7-day postinfection period. The log rank (Mantel-Cox) test was performed for survival curve analysis. The experiment was performed twice, with similar results. Shown are results from a combination of two experiments. A total of 8 mice were used per group; panels A and B represent the same cohorts of mice.

    Techniques Used: Mouse Assay, Infection

    20) Product Images from "Cardiac Niche Influences the Direct Reprogramming of Canine Fibroblasts into Cardiomyocyte-Like Cells"

    Article Title: Cardiac Niche Influences the Direct Reprogramming of Canine Fibroblasts into Cardiomyocyte-Like Cells

    Journal: Stem Cells International

    doi: 10.1155/2016/4969430

    Viral transduction and exogenous gene expressions. (a) Schematic representation of the lentiviral vectors carrying the human sequence of the transcription factor genes (GATA4-MEF2C-TBX5-HAND2), the microdystrophin gene, and the GFP reporter gene. (b) The GFP reporter gene under the control of the α -MyHC promoter was already expressed 48 hours after transduction. (c) Fibroblasts underwent morphology changes 7 days after transduction and become flattened and binucleated cells expressing GFP. (d) Immunofluorescence analysis on GHMT CF alone (top left panel) and in coculture with neonatal rat cardiomyocytes (bottom left panel), with antibodies against GFP (green) and myosin heavy chain (red), shows double positive cells and single positive cells for GFP (right panel). (e) Cell growth curve after transduction shows a decrease of cell proliferation ( n = 5/cell type). (f) Fold induction of microdystrophin ( μ dys) mRNA expression in transduced cells compared to the control; n = 5 ∗ p
    Figure Legend Snippet: Viral transduction and exogenous gene expressions. (a) Schematic representation of the lentiviral vectors carrying the human sequence of the transcription factor genes (GATA4-MEF2C-TBX5-HAND2), the microdystrophin gene, and the GFP reporter gene. (b) The GFP reporter gene under the control of the α -MyHC promoter was already expressed 48 hours after transduction. (c) Fibroblasts underwent morphology changes 7 days after transduction and become flattened and binucleated cells expressing GFP. (d) Immunofluorescence analysis on GHMT CF alone (top left panel) and in coculture with neonatal rat cardiomyocytes (bottom left panel), with antibodies against GFP (green) and myosin heavy chain (red), shows double positive cells and single positive cells for GFP (right panel). (e) Cell growth curve after transduction shows a decrease of cell proliferation ( n = 5/cell type). (f) Fold induction of microdystrophin ( μ dys) mRNA expression in transduced cells compared to the control; n = 5 ∗ p

    Techniques Used: Transduction, Sequencing, Expressing, Immunofluorescence

    21) Product Images from "Delivery of an engineered HGF fragment in an extracellular matrix-derived hydrogel prevents negative LV remodeling post-myocardial infarction"

    Article Title: Delivery of an engineered HGF fragment in an extracellular matrix-derived hydrogel prevents negative LV remodeling post-myocardial infarction

    Journal: Biomaterials

    doi: 10.1016/j.biomaterials.2014.12.021

    Interstitial fibrosis and cardiomyocyte cross-sectional area
    Figure Legend Snippet: Interstitial fibrosis and cardiomyocyte cross-sectional area

    Techniques Used:

    22) Product Images from "Exercise training during diabetes attenuates cardiac ryanodine receptor dysregulation"

    Article Title: Exercise training during diabetes attenuates cardiac ryanodine receptor dysregulation

    Journal: Journal of Applied Physiology

    doi: 10.1152/japplphysiol.91280.2008

    A : Ca 2+ -sensitive binding of [ 3 H]ryanodine to type 2 ryanodine receptor (RyR2) from sedentary control, sedentary STZ-diabetic, ExT control, and ExT STZ-diabetic rat hearts. Data are means ± SE from at least 5 different
    Figure Legend Snippet: A : Ca 2+ -sensitive binding of [ 3 H]ryanodine to type 2 ryanodine receptor (RyR2) from sedentary control, sedentary STZ-diabetic, ExT control, and ExT STZ-diabetic rat hearts. Data are means ± SE from at least 5 different

    Techniques Used: Binding Assay

    23) Product Images from "Protection of the heart by treatment with a divalent-copper-selective chelator reveals a novel mechanism underlying cardiomyopathy in diabetic rats"

    Article Title: Protection of the heart by treatment with a divalent-copper-selective chelator reveals a novel mechanism underlying cardiomyopathy in diabetic rats

    Journal: Cardiovascular Diabetology

    doi: 10.1186/1475-2840-12-123

    Alterations in SERCA2a and NCX in LV myocardium. (A) Caffeine-induced Ca 2+ transients were recorded from cardiomyocytes isolated from each group of rats, which were exposed to a series of solution changes as described in methods. (i) shows examples of normalized caffeine-induced Ca 2+ transients from a single cell, with pooled data shown in (ii) (iii). There was no significant difference in the time-constant of decay of caffeine-induced Ca 2+ transients among groups in either of the caffeine perfusion solutions, indicating no change of NCX activity among groups. These data suggest that the slower decay of the Ca 2+ transient in diabetic rats did not arise from differences in NCX function. Consistently, a western blotting study (B) showed no significant change in NCX levels (molecular weight 120 kDa, n = 8 in each group) among groups, but decreased expression of SERCA2a (molecular weight 110 kDa, n = 7 in each group) in diabetic rats. TETA had no effect on levels of either transporter. C: Control (Open bars); D: Diabetic (Solid bars); D + T: TETA-treated diabetic (Patterned bars). Data are means ± SEM, one-way Kruskal-Wallis ANOVA with post-hoc application of Dunn’s Multiple Comparisons test ( P = 0.0007): * C vs D, P
    Figure Legend Snippet: Alterations in SERCA2a and NCX in LV myocardium. (A) Caffeine-induced Ca 2+ transients were recorded from cardiomyocytes isolated from each group of rats, which were exposed to a series of solution changes as described in methods. (i) shows examples of normalized caffeine-induced Ca 2+ transients from a single cell, with pooled data shown in (ii) (iii). There was no significant difference in the time-constant of decay of caffeine-induced Ca 2+ transients among groups in either of the caffeine perfusion solutions, indicating no change of NCX activity among groups. These data suggest that the slower decay of the Ca 2+ transient in diabetic rats did not arise from differences in NCX function. Consistently, a western blotting study (B) showed no significant change in NCX levels (molecular weight 120 kDa, n = 8 in each group) among groups, but decreased expression of SERCA2a (molecular weight 110 kDa, n = 7 in each group) in diabetic rats. TETA had no effect on levels of either transporter. C: Control (Open bars); D: Diabetic (Solid bars); D + T: TETA-treated diabetic (Patterned bars). Data are means ± SEM, one-way Kruskal-Wallis ANOVA with post-hoc application of Dunn’s Multiple Comparisons test ( P = 0.0007): * C vs D, P

    Techniques Used: Isolation, Activity Assay, Western Blot, Molecular Weight, Expressing

    24) Product Images from "Stat and interferon genes identified by network analysis differentially regulate primitive and definitive erythropoiesis"

    Article Title: Stat and interferon genes identified by network analysis differentially regulate primitive and definitive erythropoiesis

    Journal: BMC Systems Biology

    doi: 10.1186/1752-0509-7-38

    Stat3 inhibition differentially affects primitive erythroblast maturation in vitro. Erythroid progenitor assays were performed on single-cell suspensions of individual, dissociated E8.5 embryos ( A ) and bone marrow ( B , C ) cultured in methylcellulose with appropriate media and cytokine supplementation. EryP-CFC ( A ) colonies were scored after 5 days, d3 BFU-E ( B ) were scored after 3 days and CFU-E ( C ) were scored after 2 – 3 days of culture. Primitive erythroblast maturation cultures were performed on cells pooled from dissociated E8.5 embryos ( D ) while definitive cultures were initiated with ESRE ( E ). All cultures were treated with DMSO as a vehicle control or 100 μM Stat3 inhibitor, S3I-201. Liquid cultures ( D , E ) were pretreated with DMSO or S3I-201 for 2 hours prior to EPO stimulation. Definitive erythroblasts are cultured for 2 days, versus 4 days for primitive erythroblasts, because of their more rapid maturation in vitro and in vivo. Images are representative of primitive erythroblasts at days 1 and 4 of culture ( D ) and definitive erythroblasts at days 0 and 2 of culture ( E ).
    Figure Legend Snippet: Stat3 inhibition differentially affects primitive erythroblast maturation in vitro. Erythroid progenitor assays were performed on single-cell suspensions of individual, dissociated E8.5 embryos ( A ) and bone marrow ( B , C ) cultured in methylcellulose with appropriate media and cytokine supplementation. EryP-CFC ( A ) colonies were scored after 5 days, d3 BFU-E ( B ) were scored after 3 days and CFU-E ( C ) were scored after 2 – 3 days of culture. Primitive erythroblast maturation cultures were performed on cells pooled from dissociated E8.5 embryos ( D ) while definitive cultures were initiated with ESRE ( E ). All cultures were treated with DMSO as a vehicle control or 100 μM Stat3 inhibitor, S3I-201. Liquid cultures ( D , E ) were pretreated with DMSO or S3I-201 for 2 hours prior to EPO stimulation. Definitive erythroblasts are cultured for 2 days, versus 4 days for primitive erythroblasts, because of their more rapid maturation in vitro and in vivo. Images are representative of primitive erythroblasts at days 1 and 4 of culture ( D ) and definitive erythroblasts at days 0 and 2 of culture ( E ).

    Techniques Used: Inhibition, In Vitro, Cell Culture, In Vivo

    IFNγ differentially modulates definitive erythroid colony formation. Erythroid progenitor assays were performed on single-cell suspensions of dissociated murine E8.5 embryos (EryP-CFC) and adult bone marrow (CFU-E) in the presence or absence of IFNγ (10 ng/ml). CFU-E and EryP-CFC colonies were identified by morphologic criteria and scored at 2–3 and 5 days, respectively. Values for IFN-treated cultures are presented as normalized to vehicle-treated (phosphate buffer) control cultures.
    Figure Legend Snippet: IFNγ differentially modulates definitive erythroid colony formation. Erythroid progenitor assays were performed on single-cell suspensions of dissociated murine E8.5 embryos (EryP-CFC) and adult bone marrow (CFU-E) in the presence or absence of IFNγ (10 ng/ml). CFU-E and EryP-CFC colonies were identified by morphologic criteria and scored at 2–3 and 5 days, respectively. Values for IFN-treated cultures are presented as normalized to vehicle-treated (phosphate buffer) control cultures.

    Techniques Used:

    25) Product Images from "Multipotent luminal mammary cancer stem cells model tumor heterogeneity"

    Article Title: Multipotent luminal mammary cancer stem cells model tumor heterogeneity

    Journal: Breast Cancer Research : BCR

    doi: 10.1186/s13058-015-0615-y

    MaCSCs have the capacity to differentiate into multiple cell types. ( a ) A single Py230 cell stained with myoepithelial marker keratin 14 (K14) and luminal marker keratin 8 (K8). ( b ) Clonal Py230 cells grown on a glass coverslip stained with K14 and K8. Scale bar 20 μm. ( c ) Clonogenic efficiency of C57Bl/6 MaCSC line Py230 and FVB/N MaCSC line Py9813. ( d ) Hollow mammospheres of Py230 cells in suspension culture. Scale bar 50 μm. ( e,f ) Py230 mammosphere stained with K14 and K8. Scale bar 20 μm. ( g ) Py230 mammosphere grown on collagen exhibits branching structures. Scale bar 100 μm. ( h ) Confluent Py230 cells spontaneously form domes. ( i ) Domes become enlarged upon treatment with lactogenic hormones dexamethasone and prolactin. Scale bar 50 μm. ( j ) Expression of beta-casein by MaCSC lines Py230 and Py9813 following treatment with lactogenic hormones. Data are means ± SEM of triplicate samples. ( k ) Py230 cells treated with retinoic acid and rosiglitazone express genes associated with adipocyte differentiation. Data are means ± SEM of triplicate samples. Inset: Py230 cells stained with oil red O. Scale bar 10 μm
    Figure Legend Snippet: MaCSCs have the capacity to differentiate into multiple cell types. ( a ) A single Py230 cell stained with myoepithelial marker keratin 14 (K14) and luminal marker keratin 8 (K8). ( b ) Clonal Py230 cells grown on a glass coverslip stained with K14 and K8. Scale bar 20 μm. ( c ) Clonogenic efficiency of C57Bl/6 MaCSC line Py230 and FVB/N MaCSC line Py9813. ( d ) Hollow mammospheres of Py230 cells in suspension culture. Scale bar 50 μm. ( e,f ) Py230 mammosphere stained with K14 and K8. Scale bar 20 μm. ( g ) Py230 mammosphere grown on collagen exhibits branching structures. Scale bar 100 μm. ( h ) Confluent Py230 cells spontaneously form domes. ( i ) Domes become enlarged upon treatment with lactogenic hormones dexamethasone and prolactin. Scale bar 50 μm. ( j ) Expression of beta-casein by MaCSC lines Py230 and Py9813 following treatment with lactogenic hormones. Data are means ± SEM of triplicate samples. ( k ) Py230 cells treated with retinoic acid and rosiglitazone express genes associated with adipocyte differentiation. Data are means ± SEM of triplicate samples. Inset: Py230 cells stained with oil red O. Scale bar 10 μm

    Techniques Used: Staining, Marker, Expressing

    26) Product Images from "Adipose‐Derived Mesenchymal Stem Cell Features in Patients with a History of Head and Neck Radiation"

    Article Title: Adipose‐Derived Mesenchymal Stem Cell Features in Patients with a History of Head and Neck Radiation

    Journal: Laryngoscope Investigative Otolaryngology

    doi: 10.1002/lio2.19

    Comparison of differentiation features of XRT and NRT ADSCs. (Left) Representative photographs of stained cells after induction of differentiation (20× magnification). (Right) Quantitation of eluted dyes by spectrometry and/or ImageJ analysis. CT = NRT cells. (A) Adipogenic differentiation; (B) osteogenic differentiation; (C) chondrogenic differentiation. ADSC = adipose‐derived stromal/stem cell; NRT = nonirradiated tissue; XRT = irradiated tissue.
    Figure Legend Snippet: Comparison of differentiation features of XRT and NRT ADSCs. (Left) Representative photographs of stained cells after induction of differentiation (20× magnification). (Right) Quantitation of eluted dyes by spectrometry and/or ImageJ analysis. CT = NRT cells. (A) Adipogenic differentiation; (B) osteogenic differentiation; (C) chondrogenic differentiation. ADSC = adipose‐derived stromal/stem cell; NRT = nonirradiated tissue; XRT = irradiated tissue.

    Techniques Used: Staining, Quantitation Assay, Derivative Assay, Irradiation

    Cellular kinetics of XRT and NRT cells from all donors during six successive passages. 2× 10 5 fresh frozen cells containing the ADSC fraction were plated in 10‐cm culture dishes and grown to confluence before re‐passaging. A) Cumulative population doubling; (B) percent viability; (C) generation time; (D) AUC analysis. AUC was calculated for each donor cell population belonging to the NRT and XRT groups as, described in Methods. XRT ADSCs from the head and neck area are labeled “Neck” in all cases. ADSC = adipose‐derived stromal/stem cell; AUC = area under curve analysis; CPD = cumulative population doubling; NRT = nonirradiated tissue; XRT = irradiated tissue.
    Figure Legend Snippet: Cellular kinetics of XRT and NRT cells from all donors during six successive passages. 2× 10 5 fresh frozen cells containing the ADSC fraction were plated in 10‐cm culture dishes and grown to confluence before re‐passaging. A) Cumulative population doubling; (B) percent viability; (C) generation time; (D) AUC analysis. AUC was calculated for each donor cell population belonging to the NRT and XRT groups as, described in Methods. XRT ADSCs from the head and neck area are labeled “Neck” in all cases. ADSC = adipose‐derived stromal/stem cell; AUC = area under curve analysis; CPD = cumulative population doubling; NRT = nonirradiated tissue; XRT = irradiated tissue.

    Techniques Used: Passaging, Labeling, Derivative Assay, Irradiation

    27) Product Images from "Exposure to Nanoscale Particulate Matter from Gestation to Adulthood Impairs Metabolic Homeostasis in Mice"

    Article Title: Exposure to Nanoscale Particulate Matter from Gestation to Adulthood Impairs Metabolic Homeostasis in Mice

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-37704-2

    Effect of nPM exposure on inflammatory gene expression and immune cell profiles in adipose tissue. Expression levels of various adipocytokines were not significantly different between nPM-exposed male mice compared to controls. Gene expression analysis was carried out by real-time quantitative PCR in quadruplicate with SYBR green assays. RNA levels for each sample were normalized to Ppia or Gapdh , as endogenous controls, and the replicates were averaged to determine differences between control and nPM exposure. ( A ) Representative flow cytometry plots for the gating strategy used to quantify numbers of regulatory T cells (Tregs), effector T cells (Teffs), and type 2 innate lymphoid cells (ILC2s). ( B ) The number of Tregs, Teffs, and ILC2s were not significantly different between male mice exposed to nPM compared to controls. ( C ) Data are shown as mean ± SE from 4–5 mice for gene expression analyses and from 3–4 mice for flow cytometric analyses in adipose tissue. Control and nPM groups are indicated by black and red bars, respectively. *p
    Figure Legend Snippet: Effect of nPM exposure on inflammatory gene expression and immune cell profiles in adipose tissue. Expression levels of various adipocytokines were not significantly different between nPM-exposed male mice compared to controls. Gene expression analysis was carried out by real-time quantitative PCR in quadruplicate with SYBR green assays. RNA levels for each sample were normalized to Ppia or Gapdh , as endogenous controls, and the replicates were averaged to determine differences between control and nPM exposure. ( A ) Representative flow cytometry plots for the gating strategy used to quantify numbers of regulatory T cells (Tregs), effector T cells (Teffs), and type 2 innate lymphoid cells (ILC2s). ( B ) The number of Tregs, Teffs, and ILC2s were not significantly different between male mice exposed to nPM compared to controls. ( C ) Data are shown as mean ± SE from 4–5 mice for gene expression analyses and from 3–4 mice for flow cytometric analyses in adipose tissue. Control and nPM groups are indicated by black and red bars, respectively. *p

    Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, SYBR Green Assay, Flow Cytometry, Cytometry

    28) Product Images from "Deficiency in adipocyte chemokine receptor CXCR4 exacerbates obesity and compromises thermoregulatory responses of brown adipose tissue in a mouse model of diet-induced obesity"

    Article Title: Deficiency in adipocyte chemokine receptor CXCR4 exacerbates obesity and compromises thermoregulatory responses of brown adipose tissue in a mouse model of diet-induced obesity

    Journal: The FASEB Journal

    doi: 10.1096/fj.14-249797

    Expression of mitochondrial genes involved in mitochondrial biogenesis and oxidative function is reduced in AdCXCR4ko mice. WT C57BL/6 control ( n =10) and AdCXCR4ko ( n =10) mice were fed an HFD for 24 wk. Some animals were maintained at 25°C, and
    Figure Legend Snippet: Expression of mitochondrial genes involved in mitochondrial biogenesis and oxidative function is reduced in AdCXCR4ko mice. WT C57BL/6 control ( n =10) and AdCXCR4ko ( n =10) mice were fed an HFD for 24 wk. Some animals were maintained at 25°C, and

    Techniques Used: Expressing, Mouse Assay

    Obesity in AdCXCR4ko mice is not a result of hyperphagia but positively correlates with increased adiposity, mass, and hypertrophy of BAT and WAT. WT C57BL/6 control ( n =15), AdCXCR4ko ( n =15), and MyeCXCR4ko ( n =15) mice were fed a CD or an HFD for 24 wk.
    Figure Legend Snippet: Obesity in AdCXCR4ko mice is not a result of hyperphagia but positively correlates with increased adiposity, mass, and hypertrophy of BAT and WAT. WT C57BL/6 control ( n =15), AdCXCR4ko ( n =15), and MyeCXCR4ko ( n =15) mice were fed a CD or an HFD for 24 wk.

    Techniques Used: Mouse Assay

    Deficiency in adipocyte CXCR4 results in lower metabolic rate. Rate of O 2 consumption was measured by indirect calorimetry during 12 h light ( A , C ) and 12 h dark ( B , D ) phases of the daily cycle in WT C57BL/6 control ( n =5) and AdCXCR4ko mice ( n =5) fed
    Figure Legend Snippet: Deficiency in adipocyte CXCR4 results in lower metabolic rate. Rate of O 2 consumption was measured by indirect calorimetry during 12 h light ( A , C ) and 12 h dark ( B , D ) phases of the daily cycle in WT C57BL/6 control ( n =5) and AdCXCR4ko mice ( n =5) fed

    Techniques Used: Mouse Assay

    Adipocyte CXCR4 deficiency alters ATM and lymphocyte contents in WAT. WT C57BL/6 control ( n =15), AdCXCR4ko ( n =15), and MyeCXCR4ko ( n =15) mice were fed an HFD for 24 wk and then euthanized. Blood was collected and plasma separated. Visceral mesenteric,
    Figure Legend Snippet: Adipocyte CXCR4 deficiency alters ATM and lymphocyte contents in WAT. WT C57BL/6 control ( n =15), AdCXCR4ko ( n =15), and MyeCXCR4ko ( n =15) mice were fed an HFD for 24 wk and then euthanized. Blood was collected and plasma separated. Visceral mesenteric,

    Techniques Used: Mouse Assay

    Ablation of adipocyte CXCR4 exacerbates HFD-induced obesity. WT C57BL/6 control ( n =40) and AdCXCR4ko ( n =40) mice were fed either a CD or an HFD. During this time, the animals were evaluated for BW and euthanized after 24 wk of CD or HFD feeding. A ) Detection
    Figure Legend Snippet: Ablation of adipocyte CXCR4 exacerbates HFD-induced obesity. WT C57BL/6 control ( n =40) and AdCXCR4ko ( n =40) mice were fed either a CD or an HFD. During this time, the animals were evaluated for BW and euthanized after 24 wk of CD or HFD feeding. A ) Detection

    Techniques Used: Mouse Assay

    AdCXCR4ko mice display impaired adaptive thermogenesis. WT C57BL/6 control ( n =5), AdCXCR4ko ( n =5), and MyeCXCR4ko mice were fed either a CD or an HFD for 24 wk. Body temperature was measured at 25°C or at the indicated times ( D ) at 4°C.
    Figure Legend Snippet: AdCXCR4ko mice display impaired adaptive thermogenesis. WT C57BL/6 control ( n =5), AdCXCR4ko ( n =5), and MyeCXCR4ko mice were fed either a CD or an HFD for 24 wk. Body temperature was measured at 25°C or at the indicated times ( D ) at 4°C.

    Techniques Used: Mouse Assay

    Ablation of adipocyte CXCR4 does not alter glucose tolerance or insulin sensitivity. WT C57BL/6 ( n =8) and AdCXCR4ko ( n =8) mice were fed the HFD for 24 wk and were denied access to food either overnight or for 5 h before they were injected with glucose
    Figure Legend Snippet: Ablation of adipocyte CXCR4 does not alter glucose tolerance or insulin sensitivity. WT C57BL/6 ( n =8) and AdCXCR4ko ( n =8) mice were fed the HFD for 24 wk and were denied access to food either overnight or for 5 h before they were injected with glucose

    Techniques Used: Mouse Assay, Injection

    29) Product Images from "Deficiency in adipocyte chemokine receptor CXCR4 exacerbates obesity and compromises thermoregulatory responses of brown adipose tissue in a mouse model of diet-induced obesity"

    Article Title: Deficiency in adipocyte chemokine receptor CXCR4 exacerbates obesity and compromises thermoregulatory responses of brown adipose tissue in a mouse model of diet-induced obesity

    Journal: The FASEB Journal

    doi: 10.1096/fj.14-249797

    Expression of mitochondrial genes involved in mitochondrial biogenesis and oxidative function is reduced in AdCXCR4ko mice. WT C57BL/6 control ( n =10) and AdCXCR4ko ( n =10) mice were fed an HFD for 24 wk. Some animals were maintained at 25°C, and
    Figure Legend Snippet: Expression of mitochondrial genes involved in mitochondrial biogenesis and oxidative function is reduced in AdCXCR4ko mice. WT C57BL/6 control ( n =10) and AdCXCR4ko ( n =10) mice were fed an HFD for 24 wk. Some animals were maintained at 25°C, and

    Techniques Used: Expressing, Mouse Assay

    HFD feeding does not potentiate obesity in MyeCXCR4ko mice. WT C57BL/6 control ( n =40) and MyeCXCR4ko ( n =40) mice were fed a CD for 18 wk or an HFD for 24 wk. Animals were weighed 1×/wk and euthanized at the end of the feeding regimen. A ) Visceral
    Figure Legend Snippet: HFD feeding does not potentiate obesity in MyeCXCR4ko mice. WT C57BL/6 control ( n =40) and MyeCXCR4ko ( n =40) mice were fed a CD for 18 wk or an HFD for 24 wk. Animals were weighed 1×/wk and euthanized at the end of the feeding regimen. A ) Visceral

    Techniques Used: Mouse Assay

    Obesity in AdCXCR4ko mice is not a result of hyperphagia but positively correlates with increased adiposity, mass, and hypertrophy of BAT and WAT. WT C57BL/6 control ( n =15), AdCXCR4ko ( n =15), and MyeCXCR4ko ( n =15) mice were fed a CD or an HFD for 24 wk.
    Figure Legend Snippet: Obesity in AdCXCR4ko mice is not a result of hyperphagia but positively correlates with increased adiposity, mass, and hypertrophy of BAT and WAT. WT C57BL/6 control ( n =15), AdCXCR4ko ( n =15), and MyeCXCR4ko ( n =15) mice were fed a CD or an HFD for 24 wk.

    Techniques Used: Mouse Assay

    Deficiency in adipocyte CXCR4 results in lower metabolic rate. Rate of O 2 consumption was measured by indirect calorimetry during 12 h light ( A , C ) and 12 h dark ( B , D ) phases of the daily cycle in WT C57BL/6 control ( n =5) and AdCXCR4ko mice ( n =5) fed
    Figure Legend Snippet: Deficiency in adipocyte CXCR4 results in lower metabolic rate. Rate of O 2 consumption was measured by indirect calorimetry during 12 h light ( A , C ) and 12 h dark ( B , D ) phases of the daily cycle in WT C57BL/6 control ( n =5) and AdCXCR4ko mice ( n =5) fed

    Techniques Used: Mouse Assay

    Adipocyte CXCR4 deficiency alters ATM and lymphocyte contents in WAT. WT C57BL/6 control ( n =15), AdCXCR4ko ( n =15), and MyeCXCR4ko ( n =15) mice were fed an HFD for 24 wk and then euthanized. Blood was collected and plasma separated. Visceral mesenteric,
    Figure Legend Snippet: Adipocyte CXCR4 deficiency alters ATM and lymphocyte contents in WAT. WT C57BL/6 control ( n =15), AdCXCR4ko ( n =15), and MyeCXCR4ko ( n =15) mice were fed an HFD for 24 wk and then euthanized. Blood was collected and plasma separated. Visceral mesenteric,

    Techniques Used: Mouse Assay

    Ablation of adipocyte CXCR4 exacerbates HFD-induced obesity. WT C57BL/6 control ( n =40) and AdCXCR4ko ( n =40) mice were fed either a CD or an HFD. During this time, the animals were evaluated for BW and euthanized after 24 wk of CD or HFD feeding. A ) Detection
    Figure Legend Snippet: Ablation of adipocyte CXCR4 exacerbates HFD-induced obesity. WT C57BL/6 control ( n =40) and AdCXCR4ko ( n =40) mice were fed either a CD or an HFD. During this time, the animals were evaluated for BW and euthanized after 24 wk of CD or HFD feeding. A ) Detection

    Techniques Used: Mouse Assay

    In adipose tissue, CXCR4 is expressed on adipocytes and adipose tissue macrophages. WT C57BL/6 control mice fed either a CD ( n =10) or an HFD ( n =10) for 24 wk were euthanized, and WAT, including subcutaneous and visceral fat pads (mesenteric, retroperitoneal
    Figure Legend Snippet: In adipose tissue, CXCR4 is expressed on adipocytes and adipose tissue macrophages. WT C57BL/6 control mice fed either a CD ( n =10) or an HFD ( n =10) for 24 wk were euthanized, and WAT, including subcutaneous and visceral fat pads (mesenteric, retroperitoneal

    Techniques Used: Mouse Assay

    AdCXCR4ko mice display impaired adaptive thermogenesis. WT C57BL/6 control ( n =5), AdCXCR4ko ( n =5), and MyeCXCR4ko mice were fed either a CD or an HFD for 24 wk. Body temperature was measured at 25°C or at the indicated times ( D ) at 4°C.
    Figure Legend Snippet: AdCXCR4ko mice display impaired adaptive thermogenesis. WT C57BL/6 control ( n =5), AdCXCR4ko ( n =5), and MyeCXCR4ko mice were fed either a CD or an HFD for 24 wk. Body temperature was measured at 25°C or at the indicated times ( D ) at 4°C.

    Techniques Used: Mouse Assay

    Ablation of adipocyte CXCR4 does not alter glucose tolerance or insulin sensitivity. WT C57BL/6 ( n =8) and AdCXCR4ko ( n =8) mice were fed the HFD for 24 wk and were denied access to food either overnight or for 5 h before they were injected with glucose
    Figure Legend Snippet: Ablation of adipocyte CXCR4 does not alter glucose tolerance or insulin sensitivity. WT C57BL/6 ( n =8) and AdCXCR4ko ( n =8) mice were fed the HFD for 24 wk and were denied access to food either overnight or for 5 h before they were injected with glucose

    Techniques Used: Mouse Assay, Injection

    30) Product Images from "A Mutant Tat Protein Provides Strong Protection from HIV-1 Infection in Human CD4+ T Cells"

    Article Title: A Mutant Tat Protein Provides Strong Protection from HIV-1 Infection in Human CD4+ T Cells

    Journal: Human Gene Therapy

    doi: 10.1089/hum.2012.176

    Nullbasic does not inhibit the production of lentiviral VLPs. (a) Schematic diagrams of the three VLP expression plasmids and an overview of the methods used to generate lentiviral VLPs. pCMVΔR8.91 plasmid expresses structural and regulatory proteins
    Figure Legend Snippet: Nullbasic does not inhibit the production of lentiviral VLPs. (a) Schematic diagrams of the three VLP expression plasmids and an overview of the methods used to generate lentiviral VLPs. pCMVΔR8.91 plasmid expresses structural and regulatory proteins

    Techniques Used: Expressing, Plasmid Preparation

    Nullbasic inhibits reverse transcription of lentiviral VLPs. (a) Using partially purified VLPs, ERT assays were performed with equivalent amounts of RT activity determined in a homopolymeric primer:template RT assay. Briefly, DNase I-treated VLP supernatant
    Figure Legend Snippet: Nullbasic inhibits reverse transcription of lentiviral VLPs. (a) Using partially purified VLPs, ERT assays were performed with equivalent amounts of RT activity determined in a homopolymeric primer:template RT assay. Briefly, DNase I-treated VLP supernatant

    Techniques Used: Purification, Activity Assay

    31) Product Images from "Novel engineered, membrane-localized variants of vascular endothelial growth factor (VEGF) protect retinal ganglion cells: a proof-of-concept study"

    Article Title: Novel engineered, membrane-localized variants of vascular endothelial growth factor (VEGF) protect retinal ganglion cells: a proof-of-concept study

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-1049-0

    Expressed eVEGF-38, eVEGF-53, or VEGF189 increases RGC number in two-dimensional and three-dimensional cultures. a Viability of primary mouse RGC in culture, as measured by counting beta III tubulin-positive cells (viable RGC) and Myc + beta III tubulin double-positive cells (viable, transgene-expressing RGC), 3 days after transduction with the VEGF constructs or GFP. RGC were isolated from P3 and P12 mice. The primary RGC were treated with VEGF121 protein (105 ng/ml for 24 h) for comparison. *** P
    Figure Legend Snippet: Expressed eVEGF-38, eVEGF-53, or VEGF189 increases RGC number in two-dimensional and three-dimensional cultures. a Viability of primary mouse RGC in culture, as measured by counting beta III tubulin-positive cells (viable RGC) and Myc + beta III tubulin double-positive cells (viable, transgene-expressing RGC), 3 days after transduction with the VEGF constructs or GFP. RGC were isolated from P3 and P12 mice. The primary RGC were treated with VEGF121 protein (105 ng/ml for 24 h) for comparison. *** P

    Techniques Used: Expressing, Transduction, Construct, Isolation, Mouse Assay

    32) Product Images from "The apical anion exchanger Slc26a6 promotes oxalate secretion by murine submandibular gland acinar cells"

    Article Title: The apical anion exchanger Slc26a6 promotes oxalate secretion by murine submandibular gland acinar cells

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.002378

    Slc26a6 mRNA expression in mouse salivary glands. A , Slc26a6 expression acquired by RNA-seq analysis for mouse PGs, SMGs, and SLGs are displayed as FPKM per 40 million mapped reads. Data from individual glands are displayed as circles ( n = 6 for PG, SMG, and SLG). B , Slc26a6 mRNA expression levels were confirmed by qPCR and normalized to β-actin ( Actb ) ( n = 6 for PG, SMG, and SLG). C , PCR band size and product amount for slc26a6 (149 bp) and Actb (147 bp) after PCR 27 cycles of cDNA amplification (100 ng). One-way ANOVA followed by Bonferroni's post hoc test was performed for statistical analysis; **, p
    Figure Legend Snippet: Slc26a6 mRNA expression in mouse salivary glands. A , Slc26a6 expression acquired by RNA-seq analysis for mouse PGs, SMGs, and SLGs are displayed as FPKM per 40 million mapped reads. Data from individual glands are displayed as circles ( n = 6 for PG, SMG, and SLG). B , Slc26a6 mRNA expression levels were confirmed by qPCR and normalized to β-actin ( Actb ) ( n = 6 for PG, SMG, and SLG). C , PCR band size and product amount for slc26a6 (149 bp) and Actb (147 bp) after PCR 27 cycles of cDNA amplification (100 ng). One-way ANOVA followed by Bonferroni's post hoc test was performed for statistical analysis; **, p

    Techniques Used: Expressing, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification

    Slc26a6 protein expression in mouse salivary glands. A , crude plasma membrane was isolated from PGs, SMGs, and SLGs. Each lane was loaded with 40 μg of protein and immunoblotted with mouse monoclonal antibodies ( Ab ) to Slc26a6 and β-actin. To more clearly visualize Slc26a6 protein expression in PGs, the blot was developed for 40 s ( first lane ). B , band intensities from A were quantified using ImageJ software and normalized to β-actin ( n = 4). One-way ANOVA followed by Bonferroni's post hoc test was performed for statistical analysis; *, p
    Figure Legend Snippet: Slc26a6 protein expression in mouse salivary glands. A , crude plasma membrane was isolated from PGs, SMGs, and SLGs. Each lane was loaded with 40 μg of protein and immunoblotted with mouse monoclonal antibodies ( Ab ) to Slc26a6 and β-actin. To more clearly visualize Slc26a6 protein expression in PGs, the blot was developed for 40 s ( first lane ). B , band intensities from A were quantified using ImageJ software and normalized to β-actin ( n = 4). One-way ANOVA followed by Bonferroni's post hoc test was performed for statistical analysis; *, p

    Techniques Used: Expressing, Isolation, Software

    33) Product Images from "Fidelity of plus-strand priming requires the nucleic acid chaperone activity of HIV-1 nucleocapsid protein"

    Article Title: Fidelity of plus-strand priming requires the nucleic acid chaperone activity of HIV-1 nucleocapsid protein

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn1045

    Schematic diagram of the RNA primers used in this study. The gray rectangle represents nt 8994–9138 from the 3′-end of the HIV-1 NL4-3 RNA genome (numbering according to GenBank accession number: AF324493) ( 70 ). The RNA primers (each 20 nt) are shown beneath the gray rectangle and the tick marks are placed according to the position of the first base in the primer sequence in the viral RNA genome. Note that in the case of the PPT, we used a primer containing the PPT plus the five downstream bases, so that all primers would be the same size. The five additional bases are removed by RNase H to generate the actual PPT primer ( 9 ). Symbols: 589R, stippled; 194R (PPT+5), open; 587R, solid; and 591R, hatched. The table below the diagram indicates the nt positions and the sequence (5′ to 3′ direction) of each primer.
    Figure Legend Snippet: Schematic diagram of the RNA primers used in this study. The gray rectangle represents nt 8994–9138 from the 3′-end of the HIV-1 NL4-3 RNA genome (numbering according to GenBank accession number: AF324493) ( 70 ). The RNA primers (each 20 nt) are shown beneath the gray rectangle and the tick marks are placed according to the position of the first base in the primer sequence in the viral RNA genome. Note that in the case of the PPT, we used a primer containing the PPT plus the five downstream bases, so that all primers would be the same size. The five additional bases are removed by RNase H to generate the actual PPT primer ( 9 ). Symbols: 589R, stippled; 194R (PPT+5), open; 587R, solid; and 591R, hatched. The table below the diagram indicates the nt positions and the sequence (5′ to 3′ direction) of each primer.

    Techniques Used: Sequencing

    Effect of HIV-1 NC on the kinetics of 591R primer extension catalyzed by WT RT and RNase H-minus RT. The data were plotted as % FL DNA versus Time (min). Symbols. WT RT: minus NC, squares; plus NC, triangles. RNase H-minus RT: minus NC, circles; plus NC, diamonds.
    Figure Legend Snippet: Effect of HIV-1 NC on the kinetics of 591R primer extension catalyzed by WT RT and RNase H-minus RT. The data were plotted as % FL DNA versus Time (min). Symbols. WT RT: minus NC, squares; plus NC, triangles. RNase H-minus RT: minus NC, circles; plus NC, diamonds.

    Techniques Used:

    Effect of HIV-1 NC on plus-strand initiation with four RNA primers. The 194R (PPT+5), 587R, 591R and 589R primers were extended by HIV-1 RT in the absence or presence of HIV-1 NC. ( A ) Gel analysis. FL DNA products synthesized during primer extension after incubation at 37°C for 30 min in the absence (No) (lanes 1, 7, 13, 19) or presence of increasing concentrations of HIV-1 NC as follows: 14 nt/NC (0.17 µM), lanes 2, 8, 14, 20; 7 nt/NC (0.34 µM), lanes 3, 9, 15, 21; 3.5 nt/NC (0.7 µM), lanes 4, 10, 16, 22; 1.75 nt/NC (1.4 µM), lanes 5, 11, 17, 23; 0.88 nt/NC (2.7 µM), lanes 6, 12, 18, 24. The positions of the primer (P) and the FL DNA products formed by 587R (55 nt), 591R (40 nt) and 589R (85 nt) are shown on the right and for 194R (80 nt), on the left. The bracketed bands are RNase H cleavage products. The sizes of the DNA products were verified with appropriate markers. ( B ) Bar graphs showing the percentage of total radioactivity in a given lane present as the FL 32 P-labeled DNA (% FL DNA) as a function of NC concentration. The numbers below each bar in the bar graph also correspond to the lane numbers of the gel. Note that the inset in the bar graph for 587R shows the values for % FL DNA on an expanded scale. Symbols: 194R (PPT+5), open bars; 587R, filled bars; 591R, hatched bars; and 589R, stippled bars.
    Figure Legend Snippet: Effect of HIV-1 NC on plus-strand initiation with four RNA primers. The 194R (PPT+5), 587R, 591R and 589R primers were extended by HIV-1 RT in the absence or presence of HIV-1 NC. ( A ) Gel analysis. FL DNA products synthesized during primer extension after incubation at 37°C for 30 min in the absence (No) (lanes 1, 7, 13, 19) or presence of increasing concentrations of HIV-1 NC as follows: 14 nt/NC (0.17 µM), lanes 2, 8, 14, 20; 7 nt/NC (0.34 µM), lanes 3, 9, 15, 21; 3.5 nt/NC (0.7 µM), lanes 4, 10, 16, 22; 1.75 nt/NC (1.4 µM), lanes 5, 11, 17, 23; 0.88 nt/NC (2.7 µM), lanes 6, 12, 18, 24. The positions of the primer (P) and the FL DNA products formed by 587R (55 nt), 591R (40 nt) and 589R (85 nt) are shown on the right and for 194R (80 nt), on the left. The bracketed bands are RNase H cleavage products. The sizes of the DNA products were verified with appropriate markers. ( B ) Bar graphs showing the percentage of total radioactivity in a given lane present as the FL 32 P-labeled DNA (% FL DNA) as a function of NC concentration. The numbers below each bar in the bar graph also correspond to the lane numbers of the gel. Note that the inset in the bar graph for 587R shows the values for % FL DNA on an expanded scale. Symbols: 194R (PPT+5), open bars; 587R, filled bars; 591R, hatched bars; and 589R, stippled bars.

    Techniques Used: Synthesized, Incubation, Radioactivity, Labeling, Concentration Assay

    34) Product Images from "Expression of neuroimmune semaphorins 4A and 4D and their receptors in the lung is enhanced by allergen and vascular endothelial growth factor"

    Article Title: Expression of neuroimmune semaphorins 4A and 4D and their receptors in the lung is enhanced by allergen and vascular endothelial growth factor

    Journal: BMC Immunology

    doi: 10.1186/1471-2172-12-30

    Regulation of lung CD72 expression by allergen and VEGF . Sema4D receptor CD72 expression in mouse lung tissue and cells was analyzed by immunohistochemistry and flow cytometry (A-B). (A) Formalin-fixed paraffin-embedded lung tissue sections obtained from either control or OVA-treated WT mice were deparaffinized and immunohistochemistry was performed using appropriate Abs as described in Materials and Methods. Frozen lung tissue sections were used for VEGF tg mice. CD72 is the most abundantly expressed molecule in the lungs of PBS-treated mice. Its expression is further upregulated with OVA treatment. Note that subsets of inflammatory granulocytes and lymphocytes express CD72 (red arrow). In VEGF tg lungs CD72 expression was mainly targeted to inflammatory macrophages (red arrow). Red arrowhead points to the lung epithelial cell CD72 expression. (B) CD72 is expressed on lung MHCII+ cells including DC and is upregulated by OVA. Both CD4+ and CD8+ T cells in the lung express CD72 at low level which is further upregulated by allergen on CD4+ T cells.
    Figure Legend Snippet: Regulation of lung CD72 expression by allergen and VEGF . Sema4D receptor CD72 expression in mouse lung tissue and cells was analyzed by immunohistochemistry and flow cytometry (A-B). (A) Formalin-fixed paraffin-embedded lung tissue sections obtained from either control or OVA-treated WT mice were deparaffinized and immunohistochemistry was performed using appropriate Abs as described in Materials and Methods. Frozen lung tissue sections were used for VEGF tg mice. CD72 is the most abundantly expressed molecule in the lungs of PBS-treated mice. Its expression is further upregulated with OVA treatment. Note that subsets of inflammatory granulocytes and lymphocytes express CD72 (red arrow). In VEGF tg lungs CD72 expression was mainly targeted to inflammatory macrophages (red arrow). Red arrowhead points to the lung epithelial cell CD72 expression. (B) CD72 is expressed on lung MHCII+ cells including DC and is upregulated by OVA. Both CD4+ and CD8+ T cells in the lung express CD72 at low level which is further upregulated by allergen on CD4+ T cells.

    Techniques Used: Expressing, Immunohistochemistry, Flow Cytometry, Cytometry, Formalin-fixed Paraffin-Embedded, Mouse Assay

    Regulation of lung Tim-2 expression by allergen and VEGF . Immunohistochemical (A-B) and flow cytometric (C) detection of Tim-2 expression in lung (A) and lymphoid (B) tissues, and on lung T cells (C). (A-B) Note positive Tim-2 staining on different lung cells besides lymphocytes in allergen- or VEGF-exposed mouse lungs. Red arrows point to Tim-2+ APC-like cells and granulocyte. Inserts show high magnification fields (100x) with marker-positive cells (lymphocytes). (C) Flow cytometry analysis of cells from lung enzymatic digests obtained from PBS- and allergen-treated mice showed an increase in lung Tim-2+ T cells in OVA-treated mice. The histograms show the percentage of Tim-2+CD3+ cells in the lung (clear histogram) as compared to the appropriate isotype control stained CD3+ cells (gray histogram).
    Figure Legend Snippet: Regulation of lung Tim-2 expression by allergen and VEGF . Immunohistochemical (A-B) and flow cytometric (C) detection of Tim-2 expression in lung (A) and lymphoid (B) tissues, and on lung T cells (C). (A-B) Note positive Tim-2 staining on different lung cells besides lymphocytes in allergen- or VEGF-exposed mouse lungs. Red arrows point to Tim-2+ APC-like cells and granulocyte. Inserts show high magnification fields (100x) with marker-positive cells (lymphocytes). (C) Flow cytometry analysis of cells from lung enzymatic digests obtained from PBS- and allergen-treated mice showed an increase in lung Tim-2+ T cells in OVA-treated mice. The histograms show the percentage of Tim-2+CD3+ cells in the lung (clear histogram) as compared to the appropriate isotype control stained CD3+ cells (gray histogram).

    Techniques Used: Expressing, Immunohistochemistry, Flow Cytometry, Staining, Marker, Cytometry, Mouse Assay

    35) Product Images from "The A12.2 Subunit Is an Intrinsic Destabilizer of the RNA Polymerase I Elongation Complex"

    Article Title: The A12.2 Subunit Is an Intrinsic Destabilizer of the RNA Polymerase I Elongation Complex

    Journal: Biophysical Journal

    doi: 10.1016/j.bpj.2018.04.015

    Development of an RNase-coupled EC dissociation assay. ( A  for detailed description of the assay. ( B ) Sequence of RNA and site of RNase A-catalyzed cleavage is shown. Red  C  represents cytosine monophosphate incorporated by Pol I during the labeling reaction. ( C ) Denaturing PAGE separation of WT EC dissociation time course collected at 0.29 M KCl using the salt-jump strategy (see text). ( D ) Same as in ( C ), except ECs were heated 5 min at 95°C and cooled to 25°C before the salt-jump. To see this figure in color, go online.
    Figure Legend Snippet: Development of an RNase-coupled EC dissociation assay. ( A for detailed description of the assay. ( B ) Sequence of RNA and site of RNase A-catalyzed cleavage is shown. Red C represents cytosine monophosphate incorporated by Pol I during the labeling reaction. ( C ) Denaturing PAGE separation of WT EC dissociation time course collected at 0.29 M KCl using the salt-jump strategy (see text). ( D ) Same as in ( C ), except ECs were heated 5 min at 95°C and cooled to 25°C before the salt-jump. To see this figure in color, go online.

    Techniques Used: Sequencing, Labeling, Polyacrylamide Gel Electrophoresis

    EC dissociation and RNase A-catalyzed RNA cleavage time courses collected as a function of [KCl]. ( A . ( B  ( A  ( B .
    Figure Legend Snippet: EC dissociation and RNase A-catalyzed RNA cleavage time courses collected as a function of [KCl]. ( A . ( B ( A ( B .

    Techniques Used:

    36) Product Images from "Pathophysiology of Lung Injury Induced by Common Bile Duct Ligation in Mice"

    Article Title: Pathophysiology of Lung Injury Induced by Common Bile Duct Ligation in Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0094550

    Real-time PCR analysis of representative genes identified with microarray analysis (a–g) was performed in pulmonary cells assembled by magnetic beads/CD31-antibody complexes in mice 3 weeks after CBDL (CBDL) and in sham operated controls (sham). (a) MMP9 and (b) TNF-α. (c) CCR1, (d) CXCL3, (e) CXCR2, (f) IL-1b, and (g) CCL9. (h) and (i) were performed in whole pulmonary cells of sham and 1–4 weeks after CBDL groups. (h) MMP9 and (i) TNF-α. Values are expressed as mean ± standard error (n = 3 in each group). Y-axis abbreviations: CCL9, chemokine CC motif ligand 9; MMP9, matrix metallopeptidase 9; TNF-α, tumor necrosis factor alpha; CCR1, CC chemokine type-1 receptor; CXCL3, chemokine CXC motif ligand 3; CXCR2, CXC chemokine receptor; IL-1b, interleukin-1 beta. *P
    Figure Legend Snippet: Real-time PCR analysis of representative genes identified with microarray analysis (a–g) was performed in pulmonary cells assembled by magnetic beads/CD31-antibody complexes in mice 3 weeks after CBDL (CBDL) and in sham operated controls (sham). (a) MMP9 and (b) TNF-α. (c) CCR1, (d) CXCL3, (e) CXCR2, (f) IL-1b, and (g) CCL9. (h) and (i) were performed in whole pulmonary cells of sham and 1–4 weeks after CBDL groups. (h) MMP9 and (i) TNF-α. Values are expressed as mean ± standard error (n = 3 in each group). Y-axis abbreviations: CCL9, chemokine CC motif ligand 9; MMP9, matrix metallopeptidase 9; TNF-α, tumor necrosis factor alpha; CCR1, CC chemokine type-1 receptor; CXCL3, chemokine CXC motif ligand 3; CXCR2, CXC chemokine receptor; IL-1b, interleukin-1 beta. *P

    Techniques Used: Real-time Polymerase Chain Reaction, Microarray, Magnetic Beads, Mouse Assay

    37) Product Images from "Coupling of a bifunctional peptide R13 to OTMCS-PEI copolymer as a gene vector increases transfection efficiency and tumor targeting"

    Article Title: Coupling of a bifunctional peptide R13 to OTMCS-PEI copolymer as a gene vector increases transfection efficiency and tumor targeting

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S59726

    Protection of OTMCS-PEI-R13 on plasmid DNA. ( A ) Protection of plasmid DNA from degradation by DNase I concentrations of 0, 9, 10.5, 12, 13.5, 15, 16.5, 18, 19.5, 21, and 23.5/μg DNA. ( B ) Protection of plasmid DNA from dissociation by serum at varying concentrations of 10%, 25%, and 50%. The lanes 10%, 25%, and 50% without “+” refer to the presence of only 10%, 25%, and 50% serum; the lanes 10%, 25%, and 50% with “+” refer to the presence of OTMCS-PEI-R13/DNA complex at a w/w ratio of 20 with different concentrations of serum. ( C ) Protection of plasmid DNA from dissociation by sodium heparin at varying concentrations of 0, 120, 160, 200, 240, 280, 300, 400, 500, and 600 μg/mL. Abbreviations: OTMCS, N-octyl-N-quaternary chitosan; PEI, polyethylenimine; R13, RGDC-TAT (49–57); w/w, weight/weight.
    Figure Legend Snippet: Protection of OTMCS-PEI-R13 on plasmid DNA. ( A ) Protection of plasmid DNA from degradation by DNase I concentrations of 0, 9, 10.5, 12, 13.5, 15, 16.5, 18, 19.5, 21, and 23.5/μg DNA. ( B ) Protection of plasmid DNA from dissociation by serum at varying concentrations of 10%, 25%, and 50%. The lanes 10%, 25%, and 50% without “+” refer to the presence of only 10%, 25%, and 50% serum; the lanes 10%, 25%, and 50% with “+” refer to the presence of OTMCS-PEI-R13/DNA complex at a w/w ratio of 20 with different concentrations of serum. ( C ) Protection of plasmid DNA from dissociation by sodium heparin at varying concentrations of 0, 120, 160, 200, 240, 280, 300, 400, 500, and 600 μg/mL. Abbreviations: OTMCS, N-octyl-N-quaternary chitosan; PEI, polyethylenimine; R13, RGDC-TAT (49–57); w/w, weight/weight.

    Techniques Used: Plasmid Preparation

    38) Product Images from "Genetic inactivation of mitochondria-targeted redox enzyme p66ShcA preserves neuronal viability and mitochondrial integrity in response to oxidative challenges"

    Article Title: Genetic inactivation of mitochondria-targeted redox enzyme p66ShcA preserves neuronal viability and mitochondrial integrity in response to oxidative challenges

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2012.00285

    p66-KO axonal mitochondria show greater preservation of mitochondrial length following oxidative stress. Mitochondrial morphology changes in week-old p66-KO and WT neurons were compared following treatment with either 25 μM H 2 O 2 , 500 μM DETA-NO, or control media for 1 h. Mitochondria were labeled with mito-GFP for visualization and quantification of morphology changes following treatment. (A) p66-KO and WT axonal mitochondria were similar in length following treatment with control media (WT 2.10 ± 0.11 μm, p66-KO 2.08 ± 0.12 μm). Following treatment with H 2 O 2 , axonal mitochondrial length was significantly more preserved in p66-KO neurons (1.89 ± 0.08 μm) compared to WT neurons (1.62 ± 0.03 μm). Similarly, following treatment with DETA-NO, axonal mitochondrial length was significantly more preserved in p66-KO neurons (2.02 ± 0.01 μm) compared to WT neurons (1.79 ± 0.02 μm). (B) Quantification of mitochondrial morphology changes following oxidative challenges demonstrated that p66 elimination in neurons was associated with significantly less changes in axonal mitochondrial length (H 2 O 2 = −0.19 ± 0.08 μm; DETA-NO = −0.06 ± 0.01 μm) compared to WT axonal mitochondria (H 2 O 2 = −0.48 ± 0.03 μm; DETA-NO = −0.31 ± 0.02 μm). ( * = p
    Figure Legend Snippet: p66-KO axonal mitochondria show greater preservation of mitochondrial length following oxidative stress. Mitochondrial morphology changes in week-old p66-KO and WT neurons were compared following treatment with either 25 μM H 2 O 2 , 500 μM DETA-NO, or control media for 1 h. Mitochondria were labeled with mito-GFP for visualization and quantification of morphology changes following treatment. (A) p66-KO and WT axonal mitochondria were similar in length following treatment with control media (WT 2.10 ± 0.11 μm, p66-KO 2.08 ± 0.12 μm). Following treatment with H 2 O 2 , axonal mitochondrial length was significantly more preserved in p66-KO neurons (1.89 ± 0.08 μm) compared to WT neurons (1.62 ± 0.03 μm). Similarly, following treatment with DETA-NO, axonal mitochondrial length was significantly more preserved in p66-KO neurons (2.02 ± 0.01 μm) compared to WT neurons (1.79 ± 0.02 μm). (B) Quantification of mitochondrial morphology changes following oxidative challenges demonstrated that p66 elimination in neurons was associated with significantly less changes in axonal mitochondrial length (H 2 O 2 = −0.19 ± 0.08 μm; DETA-NO = −0.06 ± 0.01 μm) compared to WT axonal mitochondria (H 2 O 2 = −0.48 ± 0.03 μm; DETA-NO = −0.31 ± 0.02 μm). ( * = p

    Techniques Used: Preserving, Labeling

    p66-KO neurons show greater preservation of neuronal structure compared to WT neurons following H 2 O 2 treatment. Representative images of week-old p66-KO and WT neurons at 24 h post-treatment with either control media (A,B) or 500 μM H 2 O 2 (C,D) ; viable cells have been fluorescently labeled with Calcein AM. Images were taken with a wide-field inverted fluorescence microscope using a 10X objective. Note the greater percentage of viable neurons in the p66-KO culture (D) compared to the WT culture (C) (Bar in A = 50 μm).
    Figure Legend Snippet: p66-KO neurons show greater preservation of neuronal structure compared to WT neurons following H 2 O 2 treatment. Representative images of week-old p66-KO and WT neurons at 24 h post-treatment with either control media (A,B) or 500 μM H 2 O 2 (C,D) ; viable cells have been fluorescently labeled with Calcein AM. Images were taken with a wide-field inverted fluorescence microscope using a 10X objective. Note the greater percentage of viable neurons in the p66-KO culture (D) compared to the WT culture (C) (Bar in A = 50 μm).

    Techniques Used: Preserving, Labeling, Fluorescence, Microscopy

    p66-KO neurons are more resistant to DETA-NO treatment compared to WT neurons. Week-old p66-KO and WT hippocampal neurons were treated with various concentrations of DETA-NO (100, 250, and 500 μM) for 3 h. Cell counts were obtained 24 h post-treatment using Calcein AM as a viability indicator. (A) Cell viability percentages were significantly greater for the p66-KO neurons compared to the WT neurons for the tested DETA-NO concentrations. ( * = p
    Figure Legend Snippet: p66-KO neurons are more resistant to DETA-NO treatment compared to WT neurons. Week-old p66-KO and WT hippocampal neurons were treated with various concentrations of DETA-NO (100, 250, and 500 μM) for 3 h. Cell counts were obtained 24 h post-treatment using Calcein AM as a viability indicator. (A) Cell viability percentages were significantly greater for the p66-KO neurons compared to the WT neurons for the tested DETA-NO concentrations. ( * = p

    Techniques Used:

    p66-KO neurons show greater preservation of neuronal structure compared to WT neurons following DETA-NO treatment. Representative images of week-old p66-KO and WT neurons at 24 h post-treatment with either control media (A,B) or 500 μM DETA-NO (C,D) ; viable cells have been fluorescently labeled with Calcein AM. Images were taken with a laser scanning confocal microscope using a 5X objective. Note the greater percentage of viable neurons in the p66-KO culture (D) compared to the WT culture (C) (Bar in A = 50 μm).
    Figure Legend Snippet: p66-KO neurons show greater preservation of neuronal structure compared to WT neurons following DETA-NO treatment. Representative images of week-old p66-KO and WT neurons at 24 h post-treatment with either control media (A,B) or 500 μM DETA-NO (C,D) ; viable cells have been fluorescently labeled with Calcein AM. Images were taken with a laser scanning confocal microscope using a 5X objective. Note the greater percentage of viable neurons in the p66-KO culture (D) compared to the WT culture (C) (Bar in A = 50 μm).

    Techniques Used: Preserving, Labeling, Microscopy

    p66-KO neurons generate less ROS following exposure to oxidative stress. Week-old p66-KO and WT neurons were treated with either 25 μM H 2 O 2 , 500 μM DETA-NO, or control media for 1 h, and assessed for changes in mitochondrial ROS levels. Mitochondria were visualized with mito-GFP labeling, and mitochondrial ROS was visualized following Mitosox (a fluorescent reporter of mitochondrial superoxide) incubation. Following H 2 O 2 treatment, p66-KO neurons showed significantly less increases in mitochondrial ROS levels compared to WT neurons (p66-KO = 1.03 ± 0.03X; WT = 1.48 ± 0.12X). Similarly, following DETA-NO treatment, p66-KO neurons showed significantly less increases in mitochondrial ROS levels compared to WT neurons (p66-KO = 0.96 ± 0.04X; WT = 1.37 ± 0.12X). ( * = p
    Figure Legend Snippet: p66-KO neurons generate less ROS following exposure to oxidative stress. Week-old p66-KO and WT neurons were treated with either 25 μM H 2 O 2 , 500 μM DETA-NO, or control media for 1 h, and assessed for changes in mitochondrial ROS levels. Mitochondria were visualized with mito-GFP labeling, and mitochondrial ROS was visualized following Mitosox (a fluorescent reporter of mitochondrial superoxide) incubation. Following H 2 O 2 treatment, p66-KO neurons showed significantly less increases in mitochondrial ROS levels compared to WT neurons (p66-KO = 1.03 ± 0.03X; WT = 1.48 ± 0.12X). Similarly, following DETA-NO treatment, p66-KO neurons showed significantly less increases in mitochondrial ROS levels compared to WT neurons (p66-KO = 0.96 ± 0.04X; WT = 1.37 ± 0.12X). ( * = p

    Techniques Used: Labeling, Incubation

    p66-KO neurons are more resistant to H 2 O 2 treatment compared to WT neurons. Week-old p66-KO and WT hippocampal neurons were treated with various concentrations of H 2 O 2 (100, 250, 500 μM, and 1 mM) for 15 min. Cell counts were obtained 24 h post-treatment using Calcein AM as a viability indicator. (A) Cell viability percentages were significantly greater for the p66-KO neurons compared to the WT neurons for the tested H 2 O 2 concentrations ( * = p
    Figure Legend Snippet: p66-KO neurons are more resistant to H 2 O 2 treatment compared to WT neurons. Week-old p66-KO and WT hippocampal neurons were treated with various concentrations of H 2 O 2 (100, 250, 500 μM, and 1 mM) for 15 min. Cell counts were obtained 24 h post-treatment using Calcein AM as a viability indicator. (A) Cell viability percentages were significantly greater for the p66-KO neurons compared to the WT neurons for the tested H 2 O 2 concentrations ( * = p

    Techniques Used:

    39) Product Images from "In vivo hepatogenic capacity and therapeutic potential of stem cells from human exfoliated deciduous teeth in liver fibrosis in mice"

    Article Title: In vivo hepatogenic capacity and therapeutic potential of stem cells from human exfoliated deciduous teeth in liver fibrosis in mice

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-015-0154-6

    SHED-derived HLA-ABC-positive cells purified from primary recipient livers of CCl 4 -treated mice express hepatocyte-specific genes without host-cell fusion . a Double-immunofluorescent staining patterns for HepPar and human albumin ( hALB ) in CCl 4 -injured liver tissues transplanted with SHED. b Flow cytometric analysis of magnetically sorted HLA-ABC-positive ( HLA + ) cells stained with PE-conjugated anti-human HLA-ABC and APC-conjugated anti-mouse H-K2 b antibodies. c Morphology of sorted HLA + cells. Toluidine blue staining. d Genomic DNA assay. e RT-PCR analysis of hALB and mouse albumin ( mALB ) mRNAs. f RT-PCR analysis of human hepatocyte-specific genes. ALB albumin, Alu human-specific Alu gene, CCl 4 carbon tetrachloride, CYP1A1 cytochrome P450 1A1, CYP3A7 cytochrome P450 3A7, DAPI 4′,6-diamidino-2-phenylindole, FAH fumarylacetoacetate hydrolase, GAPDH human glyceraldehyde 3-phosphate dehydrogenase, HepG2 human hepatoma cell line, HepPar1 human hepatocyte specific HepParaffin 1 antigen, HLA human leukocyte antigen, HLA – HLA-ABC-negative cells, mpf1 mouse-specific Pf1 gene, SHED stem cells from human exfoliated deciduous teeth, TAT tyrosine aminotransferase, TF transferrin, TTR transthyretin, UGT1A1 uridine 5′-diphospho-glucuronosyltransferase 1A1
    Figure Legend Snippet: SHED-derived HLA-ABC-positive cells purified from primary recipient livers of CCl 4 -treated mice express hepatocyte-specific genes without host-cell fusion . a Double-immunofluorescent staining patterns for HepPar and human albumin ( hALB ) in CCl 4 -injured liver tissues transplanted with SHED. b Flow cytometric analysis of magnetically sorted HLA-ABC-positive ( HLA + ) cells stained with PE-conjugated anti-human HLA-ABC and APC-conjugated anti-mouse H-K2 b antibodies. c Morphology of sorted HLA + cells. Toluidine blue staining. d Genomic DNA assay. e RT-PCR analysis of hALB and mouse albumin ( mALB ) mRNAs. f RT-PCR analysis of human hepatocyte-specific genes. ALB albumin, Alu human-specific Alu gene, CCl 4 carbon tetrachloride, CYP1A1 cytochrome P450 1A1, CYP3A7 cytochrome P450 3A7, DAPI 4′,6-diamidino-2-phenylindole, FAH fumarylacetoacetate hydrolase, GAPDH human glyceraldehyde 3-phosphate dehydrogenase, HepG2 human hepatoma cell line, HepPar1 human hepatocyte specific HepParaffin 1 antigen, HLA human leukocyte antigen, HLA – HLA-ABC-negative cells, mpf1 mouse-specific Pf1 gene, SHED stem cells from human exfoliated deciduous teeth, TAT tyrosine aminotransferase, TF transferrin, TTR transthyretin, UGT1A1 uridine 5′-diphospho-glucuronosyltransferase 1A1

    Techniques Used: Derivative Assay, Purification, Mouse Assay, Staining, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction

    SHED differentiate into human hepatocyte-like cells in recipient livers of CCl 4 -treated mice. a Schema of CCl 4 treatment and SHED transplantation in mice. C57BL/6 mice intraperitoneally received CCl 4 (0.5 ml/kg) or olive oil only twice a week ( red arrows ). Four weeks after the treatment, SHED (1 × 10 6 ) were transplanted into the CCl 4 -treated mice through the spleen. Phosphate-buffered saline ( PBS ) was infused as the control for the transplantation. b In vivo monitoring of transplanted DiR-labeled SHED in CCl 4 -treated mice 1 hour (1h) or 24 hours (24h) after the infusion. Dorsal position. c Enzyme-linked immunosorbent assay of human albumin ( hALB ) in the recipient serum. d – f Distribution of transplanted SHED in recipient livers. Immunohistochemistry with anti-human HLA-ABC, anti-hepatocyte paraffin 1 (Hep Par1), or anti-hALB antibody. Representative images. d Counterstaining with hematoxylin. The human HLA-ABC, hepatocyte paraffin 1, or hALB antibody positive area. Immunopositive area shown as the ratio to e the total area or f the fibrous area. c , e , f n = 5 for all groups. * P
    Figure Legend Snippet: SHED differentiate into human hepatocyte-like cells in recipient livers of CCl 4 -treated mice. a Schema of CCl 4 treatment and SHED transplantation in mice. C57BL/6 mice intraperitoneally received CCl 4 (0.5 ml/kg) or olive oil only twice a week ( red arrows ). Four weeks after the treatment, SHED (1 × 10 6 ) were transplanted into the CCl 4 -treated mice through the spleen. Phosphate-buffered saline ( PBS ) was infused as the control for the transplantation. b In vivo monitoring of transplanted DiR-labeled SHED in CCl 4 -treated mice 1 hour (1h) or 24 hours (24h) after the infusion. Dorsal position. c Enzyme-linked immunosorbent assay of human albumin ( hALB ) in the recipient serum. d – f Distribution of transplanted SHED in recipient livers. Immunohistochemistry with anti-human HLA-ABC, anti-hepatocyte paraffin 1 (Hep Par1), or anti-hALB antibody. Representative images. d Counterstaining with hematoxylin. The human HLA-ABC, hepatocyte paraffin 1, or hALB antibody positive area. Immunopositive area shown as the ratio to e the total area or f the fibrous area. c , e , f n = 5 for all groups. * P

    Techniques Used: Mouse Assay, Transplantation Assay, In Vivo, Labeling, Enzyme-linked Immunosorbent Assay, Immunohistochemistry

    40) Product Images from "NMNAT2:HSP90 Complex Mediates Proteostasis in Proteinopathies"

    Article Title: NMNAT2:HSP90 Complex Mediates Proteostasis in Proteinopathies

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1002472

    NMNAT2 reduces Ataxin1-82Q-GFP aggregates. ( A ) Immunostaining images show that cells expressing NMNAT2-WT, -ED, and -PM have fewer Ataxin1-82Q-GFP aggregates compared to untransfected cells or cells expressing mCherry, NMNAT2-ΔCT, or NMNAT2-ΔcATP ( n = 3 independent experiments). ( B ) Summary of the sizes of individual Ataxin1-82Q-GFP aggregates present in transfected cells ( C ) Example western blot showing that expression of HSP70 and NMNAT2-WT/ΔNT/ED/PM, but not NMNAT2-ΔCT/ΔcATP have reduced Ataxin1-82Q-GFP aggregates in both the soluble and RIPA-insoluble fractions. ( D ) Summary of normalized Ataxin1-82Q-GFP levels in both the soluble and insoluble fractions of cell lysates prepared from cells expressing the indicated cDNAs ( n = 3 independent experiments). GAPDH in total lysate was used to normalize GFP signals in the soluble and insoluble fractions. *and # indicate significant differences in soluble or insoluble Ataxin1-82Q-GFP levels between mCherry control and transfected test protein, respectively. ( E ) Immunoprecipitation identifies an interaction between NMNAT2 and HSP90, but not between NMNAT2 and HSP70 or HOP or CHIP in the Ataxin1-82Q-GFP and HA-NMNAT2 overexpressing cell line. ( F ) Western blot shows the levels of Ataxin1-82Q-GFP in both soluble and insoluble fractions upon treatment with scrambled siRNA, HSP90-siRNA (siRNA), KRI, or the combination of siRNA and KRI ( n = 3 independent experiments). (G) Inhibition of HSP90 expression, but not HSF1 activity, increases Ataxin1-82Q-GFP accumulation ( n = 3 independent experiments). Individual values for 5B, 5D, and 5G are provided in S1 Data . ns, not significant, * /# p
    Figure Legend Snippet: NMNAT2 reduces Ataxin1-82Q-GFP aggregates. ( A ) Immunostaining images show that cells expressing NMNAT2-WT, -ED, and -PM have fewer Ataxin1-82Q-GFP aggregates compared to untransfected cells or cells expressing mCherry, NMNAT2-ΔCT, or NMNAT2-ΔcATP ( n = 3 independent experiments). ( B ) Summary of the sizes of individual Ataxin1-82Q-GFP aggregates present in transfected cells ( C ) Example western blot showing that expression of HSP70 and NMNAT2-WT/ΔNT/ED/PM, but not NMNAT2-ΔCT/ΔcATP have reduced Ataxin1-82Q-GFP aggregates in both the soluble and RIPA-insoluble fractions. ( D ) Summary of normalized Ataxin1-82Q-GFP levels in both the soluble and insoluble fractions of cell lysates prepared from cells expressing the indicated cDNAs ( n = 3 independent experiments). GAPDH in total lysate was used to normalize GFP signals in the soluble and insoluble fractions. *and # indicate significant differences in soluble or insoluble Ataxin1-82Q-GFP levels between mCherry control and transfected test protein, respectively. ( E ) Immunoprecipitation identifies an interaction between NMNAT2 and HSP90, but not between NMNAT2 and HSP70 or HOP or CHIP in the Ataxin1-82Q-GFP and HA-NMNAT2 overexpressing cell line. ( F ) Western blot shows the levels of Ataxin1-82Q-GFP in both soluble and insoluble fractions upon treatment with scrambled siRNA, HSP90-siRNA (siRNA), KRI, or the combination of siRNA and KRI ( n = 3 independent experiments). (G) Inhibition of HSP90 expression, but not HSF1 activity, increases Ataxin1-82Q-GFP accumulation ( n = 3 independent experiments). Individual values for 5B, 5D, and 5G are provided in S1 Data . ns, not significant, * /# p

    Techniques Used: Immunostaining, Expressing, Transfection, Western Blot, Immunoprecipitation, Chromatin Immunoprecipitation, Inhibition, Activity Assay

    NMNAT2 is required to protect neurons against proteotoxicity and excitotoxicity. Cell viability was evaluated by the MTT reduction assay. ( A ) MTT reductions of NMNAT2 WT and KO neurons overexpressing GFP or NMNAT2-WT, -ED, or -ΔcATP after DMSO or MG132 treatment. *and # indicate significant differences in DMSO or in MG132-treated cells between GFP control and transfected test NMNAT2 construct, respectively ( n = 3 independent experiments). ( B ) MTT reduction by DIV14 NMNAT2 WT and KO neurons overexpressing GFP or NMNAT2-WT, -ED, or -ΔcATP after DMSO or KCl treatment ( n = 3 independent experiments). *and # indicate significant differences in DMSO or in KCl-treated cells between GFP control and transfected NMNAT2 construct, respectively. n = 3 with triplicates per summary. Individual values for 6A and B are provided in S1 Data . ns, not significant, * /# p
    Figure Legend Snippet: NMNAT2 is required to protect neurons against proteotoxicity and excitotoxicity. Cell viability was evaluated by the MTT reduction assay. ( A ) MTT reductions of NMNAT2 WT and KO neurons overexpressing GFP or NMNAT2-WT, -ED, or -ΔcATP after DMSO or MG132 treatment. *and # indicate significant differences in DMSO or in MG132-treated cells between GFP control and transfected test NMNAT2 construct, respectively ( n = 3 independent experiments). ( B ) MTT reduction by DIV14 NMNAT2 WT and KO neurons overexpressing GFP or NMNAT2-WT, -ED, or -ΔcATP after DMSO or KCl treatment ( n = 3 independent experiments). *and # indicate significant differences in DMSO or in KCl-treated cells between GFP control and transfected NMNAT2 construct, respectively. n = 3 with triplicates per summary. Individual values for 6A and B are provided in S1 Data . ns, not significant, * /# p

    Techniques Used: MTT Assay, Transfection, Construct

    NMNAT2 exerts chaperone activity independently from its NAD-synthase function. (A) Diagram illustrates simplified experimental procedure of cell-based luciferase denaturation and refolding assay. (B) Diagram showing human NMNAT2 and the mutants generated for this study. (C) Summary for the chaperone activity of mCherry, HSP70, and various NMNAT2 mutants. Blue bars show baseline luciferase activity. Red bars show luciferase activity immediately after heat shock, while blue bars show luciferase activity after recovery. * and # indicate significant differences from mCherry heat shock, and mCherry recovery, respectively ( n = 3 with triplicates per experiment). Individual values for 2C are provided in S1 Data . * /# ,** /## ,*** /### ,**** /#### indicate p
    Figure Legend Snippet: NMNAT2 exerts chaperone activity independently from its NAD-synthase function. (A) Diagram illustrates simplified experimental procedure of cell-based luciferase denaturation and refolding assay. (B) Diagram showing human NMNAT2 and the mutants generated for this study. (C) Summary for the chaperone activity of mCherry, HSP70, and various NMNAT2 mutants. Blue bars show baseline luciferase activity. Red bars show luciferase activity immediately after heat shock, while blue bars show luciferase activity after recovery. * and # indicate significant differences from mCherry heat shock, and mCherry recovery, respectively ( n = 3 with triplicates per experiment). Individual values for 2C are provided in S1 Data . * /# ,** /## ,*** /### ,**** /#### indicate p

    Techniques Used: Activity Assay, Luciferase, Generated

    NMNAT2 expression in human brain positively correlates with global cognition scores. ( A ) The scatter plot shows individual subject values for nmnat2 mRNA levels and global cognition scores proximate to death. The regression line shows the positive relationship between nmnat2 levels and cognitive scores. Units for both mRNA and cognitive scores are arbitrary (Materials and Methods for details). ( B ) Box plots show global cognition scores within each quartile of nmnat1/2 level. Each box is defined by the interquartile range, the line in the box is the median, and the whiskers are 1.5*interquartile range. ( C ) Bar graphs of nmnat2 and nmnat1 mRNA levels by clinical diagnosis. Abbreviations: NCI, no cognitive impairment; MCI, mild cognitive impairment; DEM, dementia. ( D ) Path analysis of hypothetical structural models linking nmnat2 levels with cognition, either indirectly via an effect on AD pathology (top) or directly (bottom). The arrows in the model represent the hypothetical causal directions of the effects being tested by the statistical modeling. Standardized path coefficient (standard error) is shown, revealing that 30% of the NMNAT2 effect on cognition is mediated by AD pathologic burden. ( E–F ) NMNAT2 protein levels were reduced in the soluble fractions of AD brains. The insoluble fraction contains aggregated proteins such as insoluble Tau (revealed by PHF-1 antibody, which detects p-S396/404 hTau). NMNAT2 and HSP90 shift solubility in AD brains, appearing in the insoluble fraction. *,*** indicate p
    Figure Legend Snippet: NMNAT2 expression in human brain positively correlates with global cognition scores. ( A ) The scatter plot shows individual subject values for nmnat2 mRNA levels and global cognition scores proximate to death. The regression line shows the positive relationship between nmnat2 levels and cognitive scores. Units for both mRNA and cognitive scores are arbitrary (Materials and Methods for details). ( B ) Box plots show global cognition scores within each quartile of nmnat1/2 level. Each box is defined by the interquartile range, the line in the box is the median, and the whiskers are 1.5*interquartile range. ( C ) Bar graphs of nmnat2 and nmnat1 mRNA levels by clinical diagnosis. Abbreviations: NCI, no cognitive impairment; MCI, mild cognitive impairment; DEM, dementia. ( D ) Path analysis of hypothetical structural models linking nmnat2 levels with cognition, either indirectly via an effect on AD pathology (top) or directly (bottom). The arrows in the model represent the hypothetical causal directions of the effects being tested by the statistical modeling. Standardized path coefficient (standard error) is shown, revealing that 30% of the NMNAT2 effect on cognition is mediated by AD pathologic burden. ( E–F ) NMNAT2 protein levels were reduced in the soluble fractions of AD brains. The insoluble fraction contains aggregated proteins such as insoluble Tau (revealed by PHF-1 antibody, which detects p-S396/404 hTau). NMNAT2 and HSP90 shift solubility in AD brains, appearing in the insoluble fraction. *,*** indicate p

    Techniques Used: Expressing, Solubility

    NMNAT2’s chaperone activity is required to reduce p-hTau levels both in vitro and in vivo. ( A–B ) Example western blot shows an increase of p-hTau in an inducible hTau40 cell line after doxycycline treatment. The increase in p-hTau was prevented by HSP70, NMNAT2-WT, -ED, or -PM but not -ΔCT or -ΔcATP ( n = 4 independent experiments). ( C ) Overexpression of NMNAT2-WT or -ED but not -ΔcATP, reduced the levels of p-hTau revealed by MC1 immunoreactivity in the CA1 region of the hippocampus (GFP, n = 8; WT, n = 6; ED, n = 5; ΔcATP, n = 6). ( D ) Summary of normalized MC1 immunoreactivity in the CA1 S.P. area. ( E ) Representative western blots show the levels of p-hTau species recognized by PHF-1 or AT8 antibodies, neurofilaments (NF-M), total hTau, and HA-tag (recognizes exogenous NMNAT2). ( F ) p-hTau levels were quantified and normalized to sample neurofilament levels. Individual values for 3B, 3D and 3E are provided in S1 Data . S.P, striatum pyramidale layer; S. R., striatum radiatum layer; ns, not significant.
    Figure Legend Snippet: NMNAT2’s chaperone activity is required to reduce p-hTau levels both in vitro and in vivo. ( A–B ) Example western blot shows an increase of p-hTau in an inducible hTau40 cell line after doxycycline treatment. The increase in p-hTau was prevented by HSP70, NMNAT2-WT, -ED, or -PM but not -ΔCT or -ΔcATP ( n = 4 independent experiments). ( C ) Overexpression of NMNAT2-WT or -ED but not -ΔcATP, reduced the levels of p-hTau revealed by MC1 immunoreactivity in the CA1 region of the hippocampus (GFP, n = 8; WT, n = 6; ED, n = 5; ΔcATP, n = 6). ( D ) Summary of normalized MC1 immunoreactivity in the CA1 S.P. area. ( E ) Representative western blots show the levels of p-hTau species recognized by PHF-1 or AT8 antibodies, neurofilaments (NF-M), total hTau, and HA-tag (recognizes exogenous NMNAT2). ( F ) p-hTau levels were quantified and normalized to sample neurofilament levels. Individual values for 3B, 3D and 3E are provided in S1 Data . S.P, striatum pyramidale layer; S. R., striatum radiatum layer; ns, not significant.

    Techniques Used: Activity Assay, In Vitro, In Vivo, Western Blot, Over Expression

    NMNAT2 abundance is positively correlated to the levels of synaptic proteins. ( A–B ) Levels of synaptic proteins analyzed by western blotting in DIV10 WT, HET and KO cortical neurons. n = 3 independent experiments for each genotypes. ( C–D ) Western analysis for the abundance of synaptic proteins in the hippocampi of 8-mo-old NMNAT2 HET and WT ( n = 6 per genotype). The regression lines show the relationships between NMNAT2 and SNAP25, SYPH, VGluT1, RIM1α, HSP90, NR1. Protein levels were normalized to GAPDH. Individual values for 7B and 7D are provided in S1 Data .
    Figure Legend Snippet: NMNAT2 abundance is positively correlated to the levels of synaptic proteins. ( A–B ) Levels of synaptic proteins analyzed by western blotting in DIV10 WT, HET and KO cortical neurons. n = 3 independent experiments for each genotypes. ( C–D ) Western analysis for the abundance of synaptic proteins in the hippocampi of 8-mo-old NMNAT2 HET and WT ( n = 6 per genotype). The regression lines show the relationships between NMNAT2 and SNAP25, SYPH, VGluT1, RIM1α, HSP90, NR1. Protein levels were normalized to GAPDH. Individual values for 7B and 7D are provided in S1 Data .

    Techniques Used: Western Blot

    NMNAT2 complexes with HSP90 to refold aggregated proteins. ( A ) Immunoprecipitation identifies an interaction between NMNAT2 and HSP90, but not between NMNAT2 and HSP70 or HOP or CHIP in the hTau40 cell line. ( B ) NMNAT2 complexes with hTau and HSP90 in insoluble fractions of 6-mo-old rTg4510 cortex. ( C ) A simple diagram illustrating how HSP90 regulates HSF1 activity. ( D ) Inhibition of HSP90 by siRNA prevents the NMNAT2-dependent clearance of p-hTau, while inhibition of HSF1 by KRI is ineffective. Bar graph shows the summary from three independent experiments. ( E ) Summary of luciferase assay in NMNAT2-expressing cells with various treatments ( n = 4 with triplicate). ( F ) Summary of ATPase activity of HSP90 and NMNAT2 (WT/ΔcATP) in the absence or presence of aggregated CS. ( G ) WT, but not NMNAT2-ΔcATP, can refold aggregated CS in the presence of HSP90 and ATP. Individual values for 4D, 4E, 4F, and 4G are provided in S1 Data . ns, not significant, * p
    Figure Legend Snippet: NMNAT2 complexes with HSP90 to refold aggregated proteins. ( A ) Immunoprecipitation identifies an interaction between NMNAT2 and HSP90, but not between NMNAT2 and HSP70 or HOP or CHIP in the hTau40 cell line. ( B ) NMNAT2 complexes with hTau and HSP90 in insoluble fractions of 6-mo-old rTg4510 cortex. ( C ) A simple diagram illustrating how HSP90 regulates HSF1 activity. ( D ) Inhibition of HSP90 by siRNA prevents the NMNAT2-dependent clearance of p-hTau, while inhibition of HSF1 by KRI is ineffective. Bar graph shows the summary from three independent experiments. ( E ) Summary of luciferase assay in NMNAT2-expressing cells with various treatments ( n = 4 with triplicate). ( F ) Summary of ATPase activity of HSP90 and NMNAT2 (WT/ΔcATP) in the absence or presence of aggregated CS. ( G ) WT, but not NMNAT2-ΔcATP, can refold aggregated CS in the presence of HSP90 and ATP. Individual values for 4D, 4E, 4F, and 4G are provided in S1 Data . ns, not significant, * p

    Techniques Used: Immunoprecipitation, Chromatin Immunoprecipitation, Activity Assay, Inhibition, Luciferase, Expressing

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    Worthington Biochemical caffeine induced ca 2
    Depressed cardiac contractility in diabetic rats prevented by TETA-treatment.  Depressed cardiac contractility in diabetic rats was prevented by TETA-treatment, which by contrast did not modify diabetes-induced alterations in [Ca 2+ ] i  homeostasis.  (A)  Diabetic rats showed unchanged peak (i) and resting (ii) [Ca 2+ ] i  values but had concomitantly decreased peak stress (iii) (Diabetic: 10 ± 1 mN/mm 2 ; Control: 17 ± 2 mN/mm 2 ,  P =  0.02) and unchanged resting stress (iv). TETA-treatment preserved peak stress in diabetic rats (TETA-treated diabetic: 20 ± 4 mN/mm 2 ,  P =  0.04) but did not significantly modify peak or resting [Ca 2+ ] i .  (B)  The time course of the Ca 2+  transient was prolonged in diabetic rats: (time-to-peak [Ca 2+ ] i  (i); and time constant of decay of the Ca 2+  transient (ii)), as was the time course of isometric stress (time-to-peak stress (iii); and time-to-90% relaxation (iv)). However, TETA-treatment had no effect on the time course of either variable.  (C)  The maximum rate of rise in the Ca 2+  transient was unchanged (i) whereas the maximum rate of stress development was decreased in diabetic rats (ii), and this decrease was prevented by TETA-treatment. C: Control (Open bars,  n =  10); D: Diabetic (Solid bars,  n =  8); D + T: TETA-treated diabetic (Patterned bars,  n =  7). Data are means ± SEM, one-way ANOVA with application of the  post-hoc  Holm-Sidak test: * C vs D; ‡ C vs D + T; † D vs D + T;  P
    Caffeine Induced Ca 2, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Depressed cardiac contractility in diabetic rats prevented by TETA-treatment.  Depressed cardiac contractility in diabetic rats was prevented by TETA-treatment, which by contrast did not modify diabetes-induced alterations in [Ca 2+ ] i  homeostasis.  (A)  Diabetic rats showed unchanged peak (i) and resting (ii) [Ca 2+ ] i  values but had concomitantly decreased peak stress (iii) (Diabetic: 10 ± 1 mN/mm 2 ; Control: 17 ± 2 mN/mm 2 ,  P =  0.02) and unchanged resting stress (iv). TETA-treatment preserved peak stress in diabetic rats (TETA-treated diabetic: 20 ± 4 mN/mm 2 ,  P =  0.04) but did not significantly modify peak or resting [Ca 2+ ] i .  (B)  The time course of the Ca 2+  transient was prolonged in diabetic rats: (time-to-peak [Ca 2+ ] i  (i); and time constant of decay of the Ca 2+  transient (ii)), as was the time course of isometric stress (time-to-peak stress (iii); and time-to-90% relaxation (iv)). However, TETA-treatment had no effect on the time course of either variable.  (C)  The maximum rate of rise in the Ca 2+  transient was unchanged (i) whereas the maximum rate of stress development was decreased in diabetic rats (ii), and this decrease was prevented by TETA-treatment. C: Control (Open bars,  n =  10); D: Diabetic (Solid bars,  n =  8); D + T: TETA-treated diabetic (Patterned bars,  n =  7). Data are means ± SEM, one-way ANOVA with application of the  post-hoc  Holm-Sidak test: * C vs D; ‡ C vs D + T; † D vs D + T;  P
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    Depressed cardiac contractility in diabetic rats prevented by TETA-treatment.  Depressed cardiac contractility in diabetic rats was prevented by TETA-treatment, which by contrast did not modify diabetes-induced alterations in [Ca 2+ ] i  homeostasis.  (A)  Diabetic rats showed unchanged peak (i) and resting (ii) [Ca 2+ ] i  values but had concomitantly decreased peak stress (iii) (Diabetic: 10 ± 1 mN/mm 2 ; Control: 17 ± 2 mN/mm 2 ,  P =  0.02) and unchanged resting stress (iv). TETA-treatment preserved peak stress in diabetic rats (TETA-treated diabetic: 20 ± 4 mN/mm 2 ,  P =  0.04) but did not significantly modify peak or resting [Ca 2+ ] i .  (B)  The time course of the Ca 2+  transient was prolonged in diabetic rats: (time-to-peak [Ca 2+ ] i  (i); and time constant of decay of the Ca 2+  transient (ii)), as was the time course of isometric stress (time-to-peak stress (iii); and time-to-90% relaxation (iv)). However, TETA-treatment had no effect on the time course of either variable.  (C)  The maximum rate of rise in the Ca 2+  transient was unchanged (i) whereas the maximum rate of stress development was decreased in diabetic rats (ii), and this decrease was prevented by TETA-treatment. C: Control (Open bars,  n =  10); D: Diabetic (Solid bars,  n =  8); D + T: TETA-treated diabetic (Patterned bars,  n =  7). Data are means ± SEM, one-way ANOVA with application of the  post-hoc  Holm-Sidak test: * C vs D; ‡ C vs D + T; † D vs D + T;  P
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    Depressed cardiac contractility in diabetic rats prevented by TETA-treatment.  Depressed cardiac contractility in diabetic rats was prevented by TETA-treatment, which by contrast did not modify diabetes-induced alterations in [Ca 2+ ] i  homeostasis.  (A)  Diabetic rats showed unchanged peak (i) and resting (ii) [Ca 2+ ] i  values but had concomitantly decreased peak stress (iii) (Diabetic: 10 ± 1 mN/mm 2 ; Control: 17 ± 2 mN/mm 2 ,  P =  0.02) and unchanged resting stress (iv). TETA-treatment preserved peak stress in diabetic rats (TETA-treated diabetic: 20 ± 4 mN/mm 2 ,  P =  0.04) but did not significantly modify peak or resting [Ca 2+ ] i .  (B)  The time course of the Ca 2+  transient was prolonged in diabetic rats: (time-to-peak [Ca 2+ ] i  (i); and time constant of decay of the Ca 2+  transient (ii)), as was the time course of isometric stress (time-to-peak stress (iii); and time-to-90% relaxation (iv)). However, TETA-treatment had no effect on the time course of either variable.  (C)  The maximum rate of rise in the Ca 2+  transient was unchanged (i) whereas the maximum rate of stress development was decreased in diabetic rats (ii), and this decrease was prevented by TETA-treatment. C: Control (Open bars,  n =  10); D: Diabetic (Solid bars,  n =  8); D + T: TETA-treated diabetic (Patterned bars,  n =  7). Data are means ± SEM, one-way ANOVA with application of the  post-hoc  Holm-Sidak test: * C vs D; ‡ C vs D + T; † D vs D + T;  P

    Journal: Cardiovascular Diabetology

    Article Title: Protection of the heart by treatment with a divalent-copper-selective chelator reveals a novel mechanism underlying cardiomyopathy in diabetic rats

    doi: 10.1186/1475-2840-12-123

    Figure Lengend Snippet: Depressed cardiac contractility in diabetic rats prevented by TETA-treatment. Depressed cardiac contractility in diabetic rats was prevented by TETA-treatment, which by contrast did not modify diabetes-induced alterations in [Ca 2+ ] i homeostasis. (A) Diabetic rats showed unchanged peak (i) and resting (ii) [Ca 2+ ] i values but had concomitantly decreased peak stress (iii) (Diabetic: 10 ± 1 mN/mm 2 ; Control: 17 ± 2 mN/mm 2 , P =  0.02) and unchanged resting stress (iv). TETA-treatment preserved peak stress in diabetic rats (TETA-treated diabetic: 20 ± 4 mN/mm 2 , P =  0.04) but did not significantly modify peak or resting [Ca 2+ ] i . (B) The time course of the Ca 2+ transient was prolonged in diabetic rats: (time-to-peak [Ca 2+ ] i (i); and time constant of decay of the Ca 2+ transient (ii)), as was the time course of isometric stress (time-to-peak stress (iii); and time-to-90% relaxation (iv)). However, TETA-treatment had no effect on the time course of either variable. (C) The maximum rate of rise in the Ca 2+ transient was unchanged (i) whereas the maximum rate of stress development was decreased in diabetic rats (ii), and this decrease was prevented by TETA-treatment. C: Control (Open bars, n =  10); D: Diabetic (Solid bars, n =  8); D + T: TETA-treated diabetic (Patterned bars, n =  7). Data are means ± SEM, one-way ANOVA with application of the post-hoc Holm-Sidak test: * C vs D; ‡ C vs D + T; † D vs D + T; P

    Article Snippet: Measurement of caffeine-induced Ca 2+ transients in isolated cardiomyocytes LV cardiomyocytes were isolated by enzymatic digestion with 1 mg/ml collagenase Type-II (Worthington, NJ, USA) and 0.1 mg/ml proteinase type-XXIV (Sigma, MO, USA) as previously described [ ].

    Techniques:

    Alterations in SERCA2a and NCX in LV myocardium. (A)  Caffeine-induced Ca 2+  transients were recorded from cardiomyocytes isolated from each group of rats, which were exposed to a series of solution changes as described in methods. (i) shows examples of normalized caffeine-induced Ca 2+  transients from a single cell, with pooled data shown in (ii)  (iii). There was no significant difference in the time-constant of decay of caffeine-induced Ca 2+  transients among groups in either of the caffeine perfusion solutions, indicating no change of NCX activity among groups. These data suggest that the slower decay of the Ca 2+  transient in diabetic rats did not arise from differences in NCX function. Consistently, a western blotting study  (B)  showed no significant change in NCX levels (molecular weight 120 kDa, n = 8 in each group) among groups, but decreased expression of SERCA2a (molecular weight 110 kDa, n = 7 in each group) in diabetic rats. TETA had no effect on levels of either transporter. C: Control (Open bars); D: Diabetic (Solid bars); D + T: TETA-treated diabetic (Patterned bars). Data are means ± SEM, one-way Kruskal-Wallis ANOVA with  post-hoc  application of Dunn’s Multiple Comparisons test ( P =  0.0007): * C vs D,  P

    Journal: Cardiovascular Diabetology

    Article Title: Protection of the heart by treatment with a divalent-copper-selective chelator reveals a novel mechanism underlying cardiomyopathy in diabetic rats

    doi: 10.1186/1475-2840-12-123

    Figure Lengend Snippet: Alterations in SERCA2a and NCX in LV myocardium. (A) Caffeine-induced Ca 2+ transients were recorded from cardiomyocytes isolated from each group of rats, which were exposed to a series of solution changes as described in methods. (i) shows examples of normalized caffeine-induced Ca 2+ transients from a single cell, with pooled data shown in (ii) (iii). There was no significant difference in the time-constant of decay of caffeine-induced Ca 2+ transients among groups in either of the caffeine perfusion solutions, indicating no change of NCX activity among groups. These data suggest that the slower decay of the Ca 2+ transient in diabetic rats did not arise from differences in NCX function. Consistently, a western blotting study (B) showed no significant change in NCX levels (molecular weight 120 kDa, n = 8 in each group) among groups, but decreased expression of SERCA2a (molecular weight 110 kDa, n = 7 in each group) in diabetic rats. TETA had no effect on levels of either transporter. C: Control (Open bars); D: Diabetic (Solid bars); D + T: TETA-treated diabetic (Patterned bars). Data are means ± SEM, one-way Kruskal-Wallis ANOVA with post-hoc application of Dunn’s Multiple Comparisons test ( P =  0.0007): * C vs D, P

    Article Snippet: Measurement of caffeine-induced Ca 2+ transients in isolated cardiomyocytes LV cardiomyocytes were isolated by enzymatic digestion with 1 mg/ml collagenase Type-II (Worthington, NJ, USA) and 0.1 mg/ml proteinase type-XXIV (Sigma, MO, USA) as previously described [ ].

    Techniques: Isolation, Activity Assay, Western Blot, Molecular Weight, Expressing

    The steady-state force-[Ca 2+ ] i  relationship and expression of TnT  TnI in LV myocardium. (A)  Representative traces of [Ca 2+ ] i  and stress during tetanic stimulation of a trabecula from a diabetic rat (at [Ca 2+ ] o : 0.5, 5, 15 and 30 mmol/L); the solid arrows indicate where stimulation started and ended; the dashed arrows indicate that the resting [Ca 2+ ] i  and the corresponding resting stress at 30 mmol/L [Ca 2+ ] o  were comparable to the tetanized [Ca 2+ ] i  and its corresponding stress at 5 mmol/L [Ca 2+ ] o .  (B)  The corresponding data obtained 4 s after commencing tetanic stimulation from this trabecula were fitted to the Hill equation as shown.  (C)  The rising aspects of the phase plots of [Ca 2+ ] i  and tetanus at different [Ca 2+ ] o  values from the same trabecula are shown (irregular grey lines), where the data used for fitting to the Hill equation (as in B) have been superimposed (black squares).  (D)  Averaged relaxation phase plots of [Ca 2+ ] i  and tetanus at [Ca 2+ ] o  20 mmol/L from numbers of trabeculae in each experimental groups (Control: black line,  n =  9; Diabetic: red line,  n =  7; TETA-treated diabetic: blue line,  n =  7). Diabetic rats showed a rightward shift of the relaxation phase, consistent with decreased myofibrillar Ca 2+  sensitivity whereas TETA-treatment preserved the Ca 2+  sensitivity in diabetic hearts.  (E)  Expression of TnT (upper left panel shows representative western blots of three animals from each group; box and whisker plots (median, range) below show normalized densitometry of both TnT bands (i)    ratios of the two TnT bands (ii) at molecular weights in the range of 40–42.5 kDa, n = 5 in each group); and TnI (right panel; iii, molecular weight 28 kDa, n = 6 in each group) in LV tissue from the three experimental groups; these showed no significant between-group differences. C: Control (Open bars); D: Diabetic (Solid bars); D + T: TETA-treated diabetic (Patterned bars). Significance was tested by one-way Kruskal-Wallis ANOVA.

    Journal: Cardiovascular Diabetology

    Article Title: Protection of the heart by treatment with a divalent-copper-selective chelator reveals a novel mechanism underlying cardiomyopathy in diabetic rats

    doi: 10.1186/1475-2840-12-123

    Figure Lengend Snippet: The steady-state force-[Ca 2+ ] i relationship and expression of TnT TnI in LV myocardium. (A) Representative traces of [Ca 2+ ] i and stress during tetanic stimulation of a trabecula from a diabetic rat (at [Ca 2+ ] o : 0.5, 5, 15 and 30 mmol/L); the solid arrows indicate where stimulation started and ended; the dashed arrows indicate that the resting [Ca 2+ ] i and the corresponding resting stress at 30 mmol/L [Ca 2+ ] o were comparable to the tetanized [Ca 2+ ] i and its corresponding stress at 5 mmol/L [Ca 2+ ] o . (B) The corresponding data obtained 4 s after commencing tetanic stimulation from this trabecula were fitted to the Hill equation as shown. (C) The rising aspects of the phase plots of [Ca 2+ ] i and tetanus at different [Ca 2+ ] o values from the same trabecula are shown (irregular grey lines), where the data used for fitting to the Hill equation (as in B) have been superimposed (black squares). (D) Averaged relaxation phase plots of [Ca 2+ ] i and tetanus at [Ca 2+ ] o 20 mmol/L from numbers of trabeculae in each experimental groups (Control: black line, n =  9; Diabetic: red line, n =  7; TETA-treated diabetic: blue line, n =  7). Diabetic rats showed a rightward shift of the relaxation phase, consistent with decreased myofibrillar Ca 2+ sensitivity whereas TETA-treatment preserved the Ca 2+ sensitivity in diabetic hearts. (E) Expression of TnT (upper left panel shows representative western blots of three animals from each group; box and whisker plots (median, range) below show normalized densitometry of both TnT bands (i) ratios of the two TnT bands (ii) at molecular weights in the range of 40–42.5 kDa, n = 5 in each group); and TnI (right panel; iii, molecular weight 28 kDa, n = 6 in each group) in LV tissue from the three experimental groups; these showed no significant between-group differences. C: Control (Open bars); D: Diabetic (Solid bars); D + T: TETA-treated diabetic (Patterned bars). Significance was tested by one-way Kruskal-Wallis ANOVA.

    Article Snippet: Measurement of caffeine-induced Ca 2+ transients in isolated cardiomyocytes LV cardiomyocytes were isolated by enzymatic digestion with 1 mg/ml collagenase Type-II (Worthington, NJ, USA) and 0.1 mg/ml proteinase type-XXIV (Sigma, MO, USA) as previously described [ ].

    Techniques: Expressing, Western Blot, Whisker Assay, Molecular Weight

    Isometric force and [Ca 2+ ] i  measured from LV trabeculae.  Isometric force and [Ca 2+ ] i  were measured simultaneously in LV trabeculae at 37°C, 5 Hz stimulation frequency and 1.5 mmol/L [Ca 2+ ] o , conditions that are close to physiological.  (A)  Exemplary traces of Ca 2+  transients (fura-2/AM 340/380 ratio) and corresponding isometric stress (force normalized to the corresponding muscle’s cross-sectional area) averaged over a number of cardiac cycles in representative trabeculae from the 3 experimental groups, which typify those used for data analysis. Inserted figures are corresponding raw traces of Ca 2+  transients and corresponding isometric stress.  (B)  Averaged values from 7 trabeculae in each experimental group for (i) the Ca 2+  transient, (ii) isometric stress and (iii) phase plots of Ca 2+  transients and corresponding isometric stress. Diabetic rats showed decreased responsiveness to Ca 2+ , as indicated by the rightward shift of the relaxation phase (iii) and TETA-treatment prevented development of this defect.

    Journal: Cardiovascular Diabetology

    Article Title: Protection of the heart by treatment with a divalent-copper-selective chelator reveals a novel mechanism underlying cardiomyopathy in diabetic rats

    doi: 10.1186/1475-2840-12-123

    Figure Lengend Snippet: Isometric force and [Ca 2+ ] i measured from LV trabeculae. Isometric force and [Ca 2+ ] i were measured simultaneously in LV trabeculae at 37°C, 5 Hz stimulation frequency and 1.5 mmol/L [Ca 2+ ] o , conditions that are close to physiological. (A) Exemplary traces of Ca 2+ transients (fura-2/AM 340/380 ratio) and corresponding isometric stress (force normalized to the corresponding muscle’s cross-sectional area) averaged over a number of cardiac cycles in representative trabeculae from the 3 experimental groups, which typify those used for data analysis. Inserted figures are corresponding raw traces of Ca 2+ transients and corresponding isometric stress. (B) Averaged values from 7 trabeculae in each experimental group for (i) the Ca 2+ transient, (ii) isometric stress and (iii) phase plots of Ca 2+ transients and corresponding isometric stress. Diabetic rats showed decreased responsiveness to Ca 2+ , as indicated by the rightward shift of the relaxation phase (iii) and TETA-treatment prevented development of this defect.

    Article Snippet: Measurement of caffeine-induced Ca 2+ transients in isolated cardiomyocytes LV cardiomyocytes were isolated by enzymatic digestion with 1 mg/ml collagenase Type-II (Worthington, NJ, USA) and 0.1 mg/ml proteinase type-XXIV (Sigma, MO, USA) as previously described [ ].

    Techniques: