tu bcx 2 k1 tumors  (Worthington Biochemical)


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    Neonatal Cardiomyocyte Isolation System
    Description:
    Kit for performing five separate tissue dissociations each containing up to twelve hearts Contains single use vials of purified collagenase and trypsin CMF HBSS Leibovitz L 15 media and Falcon cell strainers along with a detailed protocol The kit is use tested by Worthington to assure performance
    Catalog Number:
    lk003300
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    256
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    1 kt
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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
    Kit for performing five separate tissue dissociations each containing up to twelve hearts Contains single use vials of purified collagenase and trypsin CMF HBSS Leibovitz L 15 media and Falcon cell strainers along with a detailed protocol The kit is use tested by Worthington to assure performance
    https://www.bioz.com/result/tu bcx 2 k1 tumors/product/Worthington Biochemical
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    Images

    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 "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

    8) 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

    9) 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

    10) Product Images from "Breast cancer endocrine therapy exhausts adipocyte progenitors promoting weight gain and glucose intolerance"

    Article Title: Breast cancer endocrine therapy exhausts adipocyte progenitors promoting weight gain and glucose intolerance

    Journal: bioRxiv

    doi: 10.1101/2020.08.21.259440

    Proposed model of endocrine therapy effects on adipose tissue. ERα signaling maintains adipocyte progenitor pools and inhibits preadipocyte expansion, adipocyte differentiation, and hypertrophy. Disruption of E 2 signaling through either tamoxifen treatment or withdrawal of E 2 depletes the adipocyte progenitor pool causing adipocyte hypertrophy consistent with a phenotype that precedes insulin resistance and the development of type 2 diabetes.
    Figure Legend Snippet: Proposed model of endocrine therapy effects on adipose tissue. ERα signaling maintains adipocyte progenitor pools and inhibits preadipocyte expansion, adipocyte differentiation, and hypertrophy. Disruption of E 2 signaling through either tamoxifen treatment or withdrawal of E 2 depletes the adipocyte progenitor pool causing adipocyte hypertrophy consistent with a phenotype that precedes insulin resistance and the development of type 2 diabetes.

    Techniques Used:

    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 "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

    13) 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

    14) 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:

    15) 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

    16) 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

    17) 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

    18) 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

    19) 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

    20) 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

    21) 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

    22) 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:

    23) 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

    24) 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

    25) 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:

    26) 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

    27) 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

    28) 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

    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

    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

    30) 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

    31) 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

    32) 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

    33) 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

    34) 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

    35) 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

    36) Product Images from "Modular output circuits of the fastigial nucleus for diverse motor and nonmotor functions of the cerebellar vermis"

    Article Title: Modular output circuits of the fastigial nucleus for diverse motor and nonmotor functions of the cerebellar vermis

    Journal: eLife

    doi: 10.7554/eLife.58613

    Analysis of retrograde tracing revealing discrete projections of F2 and F4. ( A ) Mapping of retrograde tracing results from injections to the parafascicular (PF) thalamus (n = 5 males and n = 3 females), zona incerta (ZI) (n = 3 males), superior colliculus (SC) (n = 3 males), nucleus prepositus hypoglossi (NPH) (n = 3 females), and lateral periaqueductal gray (LPAG) (n = 2 males and n = 2 females). Each dot indicates an individual labeled neuron. Labeled fastigial neurons were roughly evenly distributed at both the cFN and cDLP. ( B ) Examples of retrogradely labeled fastigial neurons that are large in size and/or either SPP1+ or NEFH-rich, which thus are identified as F2 neurons. Retrograde tracing cases from PF, ZI, SC, NPH, LPAG, and nucleus reticularis gigantocellularis (NRG) are shown. ( C ) Mapping of retrograde tracing results from injections to centrolateral (CL) thalamus (n = 2 males and n = 2 females), supramammillary region (SuM) (n = 2 males), substantia nigra pars compacta (SNc) (n = 3 males), laterodorsal tegmental nucleus (LDTg) (n = 2 males), and nucleus incertus (NI) (n = 2 females). Distribution of these labeled fastigial neurons was significantly biased to the cFN. ( D ) Examples of retrogradely labeled fastigial neurons that are small in size and either SNCA+ or NEFH-poor, which thus are identified as F4 neurons. Retrograde tracing cases from CL, SuM, SNc, and LDTg are shown. Abbreviations, CL, centrolateral thalamic nucleus; LDTg, laterodorsal tegmental nucleus; lPAG, lateral periaqueductal gray; NI, nucleus incertus; NRG, nucleus reticularis gigantocellularis; NPH, nucleus prepositus hypoglossi; PF, parafascicular thalamic nucleus; SC, superior colliculus; SNc, substantia nigra, pars compacta; SuM, supramammillary region; ZI, zona incerta.
    Figure Legend Snippet: Analysis of retrograde tracing revealing discrete projections of F2 and F4. ( A ) Mapping of retrograde tracing results from injections to the parafascicular (PF) thalamus (n = 5 males and n = 3 females), zona incerta (ZI) (n = 3 males), superior colliculus (SC) (n = 3 males), nucleus prepositus hypoglossi (NPH) (n = 3 females), and lateral periaqueductal gray (LPAG) (n = 2 males and n = 2 females). Each dot indicates an individual labeled neuron. Labeled fastigial neurons were roughly evenly distributed at both the cFN and cDLP. ( B ) Examples of retrogradely labeled fastigial neurons that are large in size and/or either SPP1+ or NEFH-rich, which thus are identified as F2 neurons. Retrograde tracing cases from PF, ZI, SC, NPH, LPAG, and nucleus reticularis gigantocellularis (NRG) are shown. ( C ) Mapping of retrograde tracing results from injections to centrolateral (CL) thalamus (n = 2 males and n = 2 females), supramammillary region (SuM) (n = 2 males), substantia nigra pars compacta (SNc) (n = 3 males), laterodorsal tegmental nucleus (LDTg) (n = 2 males), and nucleus incertus (NI) (n = 2 females). Distribution of these labeled fastigial neurons was significantly biased to the cFN. ( D ) Examples of retrogradely labeled fastigial neurons that are small in size and either SNCA+ or NEFH-poor, which thus are identified as F4 neurons. Retrograde tracing cases from CL, SuM, SNc, and LDTg are shown. Abbreviations, CL, centrolateral thalamic nucleus; LDTg, laterodorsal tegmental nucleus; lPAG, lateral periaqueductal gray; NI, nucleus incertus; NRG, nucleus reticularis gigantocellularis; NPH, nucleus prepositus hypoglossi; PF, parafascicular thalamic nucleus; SC, superior colliculus; SNc, substantia nigra, pars compacta; SuM, supramammillary region; ZI, zona incerta.

    Techniques Used: Retrograde Tracing, Labeling

    Representative results of anterograde tracing from injections localized to caudal regions of the fastigial nucleus. An AAV9 injection case that hit cFN and cDLP (F2 and F4 regions) of both sides is shown (green from the left and magenta from the right). These two injections resulted in a highly reproducible pattern. At medullary and pontine levels ( A–C ), labeled axons are prominent in the dorsomedial regions of the reticular nuclei, including nucleus reticularis gigantocellularis (NRG), pontine reticular nucleus caudal part (PnC), and pontine reticular nucleus oral part (PnO). They also innervate more dorsal nuclei, including the nucleus prepositus hypoglossi (NPH), nucleus incertus (NI), laterodorsal tegmental nucleus (LDTg), and ventrolateral periaqueductal gray (vlPAG). Asterisks in ( A ) are Purkinje cell axons that were labeled from virus spread from the injection site. At midbrain levels ( D–E ), these fastigial axons strongly innervated lateral periaqueductal gray (LPAG), and perioculomotor nuclei including interstitial nucleus of Cajal (INC). Significant projections are revealed at interpeduncular nucleus lateral subnucleus (IPL), retrorubral field (RRF) and intermediate/deep layers of the superior colliculus (SC). Projection to substantia nigra pars compacta is sparse at its caudalmost level ( E ). No obvious projections are detectable in the red nucleus (RN; D,E ) and ventral tegmental area (VTA; E ). At a prethalamic level ( F ), the projections are revealed in the nuclei of mesodiencephalic junction (MDJ) and supramammillary region (SuM). Scale bar applies to all panels.
    Figure Legend Snippet: Representative results of anterograde tracing from injections localized to caudal regions of the fastigial nucleus. An AAV9 injection case that hit cFN and cDLP (F2 and F4 regions) of both sides is shown (green from the left and magenta from the right). These two injections resulted in a highly reproducible pattern. At medullary and pontine levels ( A–C ), labeled axons are prominent in the dorsomedial regions of the reticular nuclei, including nucleus reticularis gigantocellularis (NRG), pontine reticular nucleus caudal part (PnC), and pontine reticular nucleus oral part (PnO). They also innervate more dorsal nuclei, including the nucleus prepositus hypoglossi (NPH), nucleus incertus (NI), laterodorsal tegmental nucleus (LDTg), and ventrolateral periaqueductal gray (vlPAG). Asterisks in ( A ) are Purkinje cell axons that were labeled from virus spread from the injection site. At midbrain levels ( D–E ), these fastigial axons strongly innervated lateral periaqueductal gray (LPAG), and perioculomotor nuclei including interstitial nucleus of Cajal (INC). Significant projections are revealed at interpeduncular nucleus lateral subnucleus (IPL), retrorubral field (RRF) and intermediate/deep layers of the superior colliculus (SC). Projection to substantia nigra pars compacta is sparse at its caudalmost level ( E ). No obvious projections are detectable in the red nucleus (RN; D,E ) and ventral tegmental area (VTA; E ). At a prethalamic level ( F ), the projections are revealed in the nuclei of mesodiencephalic junction (MDJ) and supramammillary region (SuM). Scale bar applies to all panels.

    Techniques Used: Anterograde Tracing, Injection, Labeling

    Overview of disynaptic fastigial input to the forebrain. ( A–G ) Widespread cortical labeling obtained from AAV1-mediated anterograde transsynaptic tracing from the fastigial nucleus. These cortical labeling most likely reflect projections from thalamic neurons that are postsynaptic to the fastigial nucleus. Approximate rostrocaudal levels are indicated in ( H ). ( H ) Schematic of the dorsal view of the cortex that are color-coded for functional areas, which was redrawn from Watson et al., 2012 . ( I ) No mono- or di-synaptic fastigial input to the amygdala. Photos are taken at similar rostrocaudal levels from three representative AAV1 transsynaptic tracing cases in which injections were localized in the FN. Abbreviations, Aud, auditory cortex; BLA, basolateral amygdala; BMA, basomedial amygdala; CeA, central amygdala; Cg, cingulate cortex; FrA, frontal association cortex; IL, infralimbic cortex; LA, lateral amygdala; LO, lateral orbital cortex; M1 and M2, primary and secondary motor cortex; MO, medial orbital cortex; PrL, prelimbic cortex; Ptl, parietal association cortex; Rsp, retrosplenial cortex; S1 and S2, primary and secondary sensory cortex; S1BF, barrel field of primary sensory cortex; Tem, temporal association cortex; V1 and V2, primary and secondary visual cortex; VO, ventral orbital cortex.
    Figure Legend Snippet: Overview of disynaptic fastigial input to the forebrain. ( A–G ) Widespread cortical labeling obtained from AAV1-mediated anterograde transsynaptic tracing from the fastigial nucleus. These cortical labeling most likely reflect projections from thalamic neurons that are postsynaptic to the fastigial nucleus. Approximate rostrocaudal levels are indicated in ( H ). ( H ) Schematic of the dorsal view of the cortex that are color-coded for functional areas, which was redrawn from Watson et al., 2012 . ( I ) No mono- or di-synaptic fastigial input to the amygdala. Photos are taken at similar rostrocaudal levels from three representative AAV1 transsynaptic tracing cases in which injections were localized in the FN. Abbreviations, Aud, auditory cortex; BLA, basolateral amygdala; BMA, basomedial amygdala; CeA, central amygdala; Cg, cingulate cortex; FrA, frontal association cortex; IL, infralimbic cortex; LA, lateral amygdala; LO, lateral orbital cortex; M1 and M2, primary and secondary motor cortex; MO, medial orbital cortex; PrL, prelimbic cortex; Ptl, parietal association cortex; Rsp, retrosplenial cortex; S1 and S2, primary and secondary sensory cortex; S1BF, barrel field of primary sensory cortex; Tem, temporal association cortex; V1 and V2, primary and secondary visual cortex; VO, ventral orbital cortex.

    Techniques Used: Labeling, Functional Assay, Transmission Electron Microscopy

    Convergent Purkinje cell projections from multiple lobules to fastigial subregions. ( A ) Coronal section images from the same retrograde tracing case in which PCs projecting to the rFN are labeled. BDA was injected to the rFN as a retrograde tracer. Labeled PCs (red) were distributed across the anterior (top) and posterior lobes (bottom) but confined within the same cerebellar module (1-//1-/2-), which was identified by immunonegativity for ALDOC (cyan) and mediolateral level of those PCs. ( B ) Similarly, images from a retrograde tracing case with retrobead or BDA from the vlFN show that the retrogradely labeled PCs spread across the anterior and posterior lobes but were confined within the same module (2+//3+) (n = 2 males), as identified by immunonegativity for PCLb4 (cyan), which is expressed in subsets of PCs in a complemental pattern to Aldoc ( Sarna et al., 2006 ), and the mediolateral level of those PCs. A tracing case with retrobead is shown. ( C ) Schematic illustration of the distribution of PCs upstream to the rFN (orange) or vlFN (green). PCs projecting to these fastigial subregions are localized in longitudinal stripes of the cerebellar cortex, which correspond to those demarcated by Aldoc expression (gray and white) Scale bar in B applies to all panels in A and B.
    Figure Legend Snippet: Convergent Purkinje cell projections from multiple lobules to fastigial subregions. ( A ) Coronal section images from the same retrograde tracing case in which PCs projecting to the rFN are labeled. BDA was injected to the rFN as a retrograde tracer. Labeled PCs (red) were distributed across the anterior (top) and posterior lobes (bottom) but confined within the same cerebellar module (1-//1-/2-), which was identified by immunonegativity for ALDOC (cyan) and mediolateral level of those PCs. ( B ) Similarly, images from a retrograde tracing case with retrobead or BDA from the vlFN show that the retrogradely labeled PCs spread across the anterior and posterior lobes but were confined within the same module (2+//3+) (n = 2 males), as identified by immunonegativity for PCLb4 (cyan), which is expressed in subsets of PCs in a complemental pattern to Aldoc ( Sarna et al., 2006 ), and the mediolateral level of those PCs. A tracing case with retrobead is shown. ( C ) Schematic illustration of the distribution of PCs upstream to the rFN (orange) or vlFN (green). PCs projecting to these fastigial subregions are localized in longitudinal stripes of the cerebellar cortex, which correspond to those demarcated by Aldoc expression (gray and white) Scale bar in B applies to all panels in A and B.

    Techniques Used: Retrograde Tracing, Labeling, Injection, Expressing

    Overlap of fastigial cell type markers with glutamatergic, glycinergic, and GABAergic neurons. ( A ) Fastigial marker expressions in glutamatergic neurons. Panels show high magnification images of glutamatergic neurons (green, visualized in VgluT2-Cre;Ai14 mouse) with immunostaining for SPP1 and/or SNCA. Examples show subtypes of glutamatergic neurons, which are VgluT2+/SPP1+ (left), VgluT2+/SNCA+ (center), and VgluT2+/SPP1-/SNCA- (right). Immunostaining signals are shown in magenta in the left and center panels, and double immunostaining signals in the right panel are shown in red (SPP1) and blue (SNCA). ( B ) Chart to show coexpression of glutamatergic marker and fastigial cell-type markers. The majority of VgluT2+ neurons examined (93%, 887/954, three mice) were colabeled with either SPP1 or SNCA. ( C–E ) Marker expressions in glutamatergic, glycinergic, or GABAergic fastigial neurons. Panels show immunostaining at the levels of rFN (top), vlFN (center), and cFN (bottom) in reporter mouse lines for transmitter type identification. ( C ) Shows examples for glutamatergic neurons, in which SPP1, CALB2, and SNCA are colabeled with 92% (43/47), 95% (19/20), and 90% (37/41) of fluorescently labeled neurons in VgluT2-Cre;Ai14 line, at rFN, vlFN, and cFN, respectively. ( D ) Shows examples for glycinergic neurons, in which SPP1, CALB2, and SNCA are colabeled with 100% (16/16; arrowheads), 9% (1/11; arrowhead), and 0% (0/3) of fluorescently labeled neurons in GlyT2-EGFP line, at rFN, vlFN, and cFN, respectively. ( E ) Shows examples for GABAergic neurons, in which SPP1, CALB2, and SNCA are colabeled with 0% (0/37), 11% (4/38; arrowheads), and 0% (0/12) of fluorescently labeled neurons in Gad2-nls-mCherry line, at rFN, vlFN, and cFN, respectively. Scale bar in A applies to all panels in A.
    Figure Legend Snippet: Overlap of fastigial cell type markers with glutamatergic, glycinergic, and GABAergic neurons. ( A ) Fastigial marker expressions in glutamatergic neurons. Panels show high magnification images of glutamatergic neurons (green, visualized in VgluT2-Cre;Ai14 mouse) with immunostaining for SPP1 and/or SNCA. Examples show subtypes of glutamatergic neurons, which are VgluT2+/SPP1+ (left), VgluT2+/SNCA+ (center), and VgluT2+/SPP1-/SNCA- (right). Immunostaining signals are shown in magenta in the left and center panels, and double immunostaining signals in the right panel are shown in red (SPP1) and blue (SNCA). ( B ) Chart to show coexpression of glutamatergic marker and fastigial cell-type markers. The majority of VgluT2+ neurons examined (93%, 887/954, three mice) were colabeled with either SPP1 or SNCA. ( C–E ) Marker expressions in glutamatergic, glycinergic, or GABAergic fastigial neurons. Panels show immunostaining at the levels of rFN (top), vlFN (center), and cFN (bottom) in reporter mouse lines for transmitter type identification. ( C ) Shows examples for glutamatergic neurons, in which SPP1, CALB2, and SNCA are colabeled with 92% (43/47), 95% (19/20), and 90% (37/41) of fluorescently labeled neurons in VgluT2-Cre;Ai14 line, at rFN, vlFN, and cFN, respectively. ( D ) Shows examples for glycinergic neurons, in which SPP1, CALB2, and SNCA are colabeled with 100% (16/16; arrowheads), 9% (1/11; arrowhead), and 0% (0/3) of fluorescently labeled neurons in GlyT2-EGFP line, at rFN, vlFN, and cFN, respectively. ( E ) Shows examples for GABAergic neurons, in which SPP1, CALB2, and SNCA are colabeled with 0% (0/37), 11% (4/38; arrowheads), and 0% (0/12) of fluorescently labeled neurons in Gad2-nls-mCherry line, at rFN, vlFN, and cFN, respectively. Scale bar in A applies to all panels in A.

    Techniques Used: Marker, Immunostaining, Double Immunostaining, Mouse Assay, Labeling

    Cre-dependent anterograde tracing of GABAergic neuronal projections from the fastigial nucleus. ( A ) Example low magnification image of Cre-dependent anterograde tracing experiments with Gad2Cre mouse. The image shows injection site and resultant axonal labeling in the brainstem. The injections site (circumscribed with a dotted white line) of Cre-dependent AAV, AAV9.CAG.FLEX.tdTomato, entirely included the FN (circumscribed with a white line). Significant labeling (black) in the injection site but outside of the FN reflects labeled Purkinje cells which also express Gad2 and thus labeled with this injection strategy. The labeled GABAergic output projections (black) in the extracerebellar regions are identified only in the inferior olive (IO) (n = 2 males). Asterisk indicates labeled axonal path. ( B ) A high magnification example of a fastigial neuron labeled by the injection. ( C ) Labeled GABAergic axonal terminals innervating the IO.
    Figure Legend Snippet: Cre-dependent anterograde tracing of GABAergic neuronal projections from the fastigial nucleus. ( A ) Example low magnification image of Cre-dependent anterograde tracing experiments with Gad2Cre mouse. The image shows injection site and resultant axonal labeling in the brainstem. The injections site (circumscribed with a dotted white line) of Cre-dependent AAV, AAV9.CAG.FLEX.tdTomato, entirely included the FN (circumscribed with a white line). Significant labeling (black) in the injection site but outside of the FN reflects labeled Purkinje cells which also express Gad2 and thus labeled with this injection strategy. The labeled GABAergic output projections (black) in the extracerebellar regions are identified only in the inferior olive (IO) (n = 2 males). Asterisk indicates labeled axonal path. ( B ) A high magnification example of a fastigial neuron labeled by the injection. ( C ) Labeled GABAergic axonal terminals innervating the IO.

    Techniques Used: Anterograde Tracing, Injection, Labeling

    Distinct input and output projections of the caudal medial accessory olive subnuclei ‘d’ and c. ( A–D ) Distinct input from the superior colliculus (SC) to subnuclei d and c of medial accessory olive are shown with anterograde tracing experiments from the Allen Connectivity Atlas. Labeled axons from the medial SC ( A ) were found at the most medial part of the caudal medial accessory olive (cMAO), which we designated as cMAO-d ( B ). Data were obtained from Experiment #128001349, section 49 for injection site at the SC ( A ) and section 15 for the labeling in the IO ( B ). Labeled axons from the lateral SC ( C ) are found at the cMAO-c ( D ). Data were obtained from Experiment #146078721, section 54 for injection site at the SC and section 11 for the labeling in the IO. ( E ) Schematic illustration of experiments to identify projection targets of the subnuclei d and c of the cMAO. Retrograde tracers were injected to various loci in the cerebellar cortex (n = 14 injections to n = 2 males and n = 4 females). Each circle indicates one tracer injection case. Retrobead, BDA, and AAV1.Cre, which retrogradely infects IO neurons, were used as retrograde tracer. Injection cases are color coded on the basis of downstream fastigial areas revealed in Figure 7 , in which the lobules VIc/VII and medial Crus I (blue) were upstream of the cDLP (F2 region) and the medial Simplex lobule (Sim)/Crus II (orange) were upstream of the rDLP (F1 rDLP region). ( F ) Mapping of retrogradely labeled IO neurons from the injection sites shown in ( E ). Individual labeled IO neurons are mapped onto serial coronal drawings of the inferior olive, in which 1 and 6 correspond to the most caudal and rostral levels used in this analysis, respectively. Circumscribed are two clusters of IO neurons; blue dots are those labeled by injections to the lobules VIc/VII and medial Crus I, while orange dots are those labeled by injections to the medial Sim/Crus II. The two clusters were mostly not overlapped. Projection to the medial Sim/Crus II is hallmark of the cMAO-c subnucleus (orange cluster). We termed the other IO neuronal cluster (blue cluster) as
    Figure Legend Snippet: Distinct input and output projections of the caudal medial accessory olive subnuclei ‘d’ and c. ( A–D ) Distinct input from the superior colliculus (SC) to subnuclei d and c of medial accessory olive are shown with anterograde tracing experiments from the Allen Connectivity Atlas. Labeled axons from the medial SC ( A ) were found at the most medial part of the caudal medial accessory olive (cMAO), which we designated as cMAO-d ( B ). Data were obtained from Experiment #128001349, section 49 for injection site at the SC ( A ) and section 15 for the labeling in the IO ( B ). Labeled axons from the lateral SC ( C ) are found at the cMAO-c ( D ). Data were obtained from Experiment #146078721, section 54 for injection site at the SC and section 11 for the labeling in the IO. ( E ) Schematic illustration of experiments to identify projection targets of the subnuclei d and c of the cMAO. Retrograde tracers were injected to various loci in the cerebellar cortex (n = 14 injections to n = 2 males and n = 4 females). Each circle indicates one tracer injection case. Retrobead, BDA, and AAV1.Cre, which retrogradely infects IO neurons, were used as retrograde tracer. Injection cases are color coded on the basis of downstream fastigial areas revealed in Figure 7 , in which the lobules VIc/VII and medial Crus I (blue) were upstream of the cDLP (F2 region) and the medial Simplex lobule (Sim)/Crus II (orange) were upstream of the rDLP (F1 rDLP region). ( F ) Mapping of retrogradely labeled IO neurons from the injection sites shown in ( E ). Individual labeled IO neurons are mapped onto serial coronal drawings of the inferior olive, in which 1 and 6 correspond to the most caudal and rostral levels used in this analysis, respectively. Circumscribed are two clusters of IO neurons; blue dots are those labeled by injections to the lobules VIc/VII and medial Crus I, while orange dots are those labeled by injections to the medial Sim/Crus II. The two clusters were mostly not overlapped. Projection to the medial Sim/Crus II is hallmark of the cMAO-c subnucleus (orange cluster). We termed the other IO neuronal cluster (blue cluster) as "cMAO-d " subnucleus that are medial to the cMAO-c and project to the lobules VIc/VII and medial Crus I.

    Techniques Used: Anterograde Tracing, Labeling, Injection

    Correlation of neurofilament gene expression with axonal caliber and the expression of ion channels and synaptic molecules. ( A–C ) High magnification analysis of fastigial axons that originate from rostral or caudal parts of the FN. As illustrated in ( A ), small, localized injections of AAV9.RFP to rostral and caudal parts of the FN anterogradely labeled subsets of fastigial axons, which were then imaged at the level of the superior cerebellar peduncle (scp). ( B ) Shows a case of rostral FN injection (n = 2 males), where majority of labeled axons had large axonal calibers. In contrast, ( C ) shows a case of caudal FN injection (n = 2 males), where the calibers of the labeled axons were significantly smaller than those in ( B ). Because the rostral and caudal parts of the FN are dominated by NEFH-rich and -poor neurons, respectively, these results are in accordance with a positive correlation between neurofilament expression level and axonal diameter previously reported ( Hoffman et al., 1987 ). ( D and E ) Expression levels (in qPCR Ct) of Nefm ( D ) or Nefh ( E ) in the molecularly defined cell types are plotted against cell body areas of corresponding cell types measured from Nissl images in combination with marker immunostaining (n = 210). Plots are color-coded for the cell-type as indicated in inset (F1, orange; F2, blue; F3, green; F4, purple; this color-code also applies to E and G-I of this Figure). Population averaged data for each cell type are plotted. Error bars represent SEM. ( F ) Double immunostaining for NEFH and SPP1 of the fastigial nucleus with Nissl counterstaining. Immunoreactivity against NEFH was greater in the large SPP1+ neurons in rFN (top) than in the small SPP1- neurons in cFN (e.g. asterisk) (bottom). The medium to large SPP1+ neurons (indicated with '+') that were scattered in cFN showed greater NEFH immunoreactivity than neighboring small neurons (bottom). ( G–I ) Linear positive correlation between expression levels (in qPCR Ct) of Nefl ( F ), Nefm ( G ) or Nefh ( H ) vs Scn4b , Scn8a , Kcnc1 , and Syt2 . Plotted are population averaged data for each cell type. Error bars represent SEM. Scale bar in C applies to B and C. Scale bar in F applies to both images in F.
    Figure Legend Snippet: Correlation of neurofilament gene expression with axonal caliber and the expression of ion channels and synaptic molecules. ( A–C ) High magnification analysis of fastigial axons that originate from rostral or caudal parts of the FN. As illustrated in ( A ), small, localized injections of AAV9.RFP to rostral and caudal parts of the FN anterogradely labeled subsets of fastigial axons, which were then imaged at the level of the superior cerebellar peduncle (scp). ( B ) Shows a case of rostral FN injection (n = 2 males), where majority of labeled axons had large axonal calibers. In contrast, ( C ) shows a case of caudal FN injection (n = 2 males), where the calibers of the labeled axons were significantly smaller than those in ( B ). Because the rostral and caudal parts of the FN are dominated by NEFH-rich and -poor neurons, respectively, these results are in accordance with a positive correlation between neurofilament expression level and axonal diameter previously reported ( Hoffman et al., 1987 ). ( D and E ) Expression levels (in qPCR Ct) of Nefm ( D ) or Nefh ( E ) in the molecularly defined cell types are plotted against cell body areas of corresponding cell types measured from Nissl images in combination with marker immunostaining (n = 210). Plots are color-coded for the cell-type as indicated in inset (F1, orange; F2, blue; F3, green; F4, purple; this color-code also applies to E and G-I of this Figure). Population averaged data for each cell type are plotted. Error bars represent SEM. ( F ) Double immunostaining for NEFH and SPP1 of the fastigial nucleus with Nissl counterstaining. Immunoreactivity against NEFH was greater in the large SPP1+ neurons in rFN (top) than in the small SPP1- neurons in cFN (e.g. asterisk) (bottom). The medium to large SPP1+ neurons (indicated with '+') that were scattered in cFN showed greater NEFH immunoreactivity than neighboring small neurons (bottom). ( G–I ) Linear positive correlation between expression levels (in qPCR Ct) of Nefl ( F ), Nefm ( G ) or Nefh ( H ) vs Scn4b , Scn8a , Kcnc1 , and Syt2 . Plotted are population averaged data for each cell type. Error bars represent SEM. Scale bar in C applies to B and C. Scale bar in F applies to both images in F.

    Techniques Used: Expressing, Labeling, Injection, Real-time Polymerase Chain Reaction, Marker, Immunostaining, Double Immunostaining

    Modular circuit connections of excitatory fastigial projection neurons provide circuit substrates for coordinating five broad organismal functions. Schematics summarize cerebellar modular circuit connections that link distinct types of fastigial nucleus neurons with specific neurons in the inferior olive, cerebellar cortex, and downstream brain regions. Projection targets of each FN cell type are indicated in shaded colors. Specific functions associated with each collection of projection targets are indicated at the left; proposed broad organismal functions of each module are encircled above. To show the distribution of Purkinje cells associated with each module, a flatmap of the mouse cerebellar cortex with vermis lobules indicated numerically and Aldoc/zebrin stripes indicated in grey, with medial-lateral width expanded 5x for clarity, was redrawn from Fujita et al., 2014 . Inferior olive subnuclei of the caudal MAO are denoted a, b, c, d and beta. Circles around each fastigial nucleus cell type indicate relative size and parasagittal position. Abbreviations, 7N, facial nucleus; CL, centrolateral thalamic nucleus; Cr I, Crus I; Cr II, Crus II; INC, interstitial nucleus of Cajal; IRt, intermediate reticular nucleus; IVN, inferior vestibular nucleus; KF, Kölliker-Fuse nucleus; LDTg, laterodorsal tegmental nucleus; LPAG, lateral periaqueductal gray; LPGi, lateral paragigantocellular nucleus; LVN, lateral vestibular nucleus; MAO, medial accessory olive; MD, mediodorsal thalamic nucleus; MDJ, mesodiencephalic junction; MdV, medullary reticular nucleus, ventral; mRt, mesencephalic reticular formation; MVN, medial vestibular nucleus; NPH, nucleus prepositus hypoglossi; NI, nucleus incertus; NRG, nucleus reticularis gigantocellularis; PCRt, parvocellular reticular nucleus; PF, parafascicular thalamic nucleus; PMn, paramedian reticular nucleus; PnC, pontine reticular nucleus, caudal; PPRF, paramedian pontine reticular formation; PTg, pedunculotegmental nucleus; Sim, simplex lobule; SC, superior colliculus; SNc, substantia nigra, pars compacta; SubC, subcoeruleus nucleus; SuM, supramammillary region; VL, ventrolateral thalamic nucleus; vlPAG, ventrolateral periaqueductal gray; VM, ventromedial thalamic nucleus; ZI, zona incerta.
    Figure Legend Snippet: Modular circuit connections of excitatory fastigial projection neurons provide circuit substrates for coordinating five broad organismal functions. Schematics summarize cerebellar modular circuit connections that link distinct types of fastigial nucleus neurons with specific neurons in the inferior olive, cerebellar cortex, and downstream brain regions. Projection targets of each FN cell type are indicated in shaded colors. Specific functions associated with each collection of projection targets are indicated at the left; proposed broad organismal functions of each module are encircled above. To show the distribution of Purkinje cells associated with each module, a flatmap of the mouse cerebellar cortex with vermis lobules indicated numerically and Aldoc/zebrin stripes indicated in grey, with medial-lateral width expanded 5x for clarity, was redrawn from Fujita et al., 2014 . Inferior olive subnuclei of the caudal MAO are denoted a, b, c, d and beta. Circles around each fastigial nucleus cell type indicate relative size and parasagittal position. Abbreviations, 7N, facial nucleus; CL, centrolateral thalamic nucleus; Cr I, Crus I; Cr II, Crus II; INC, interstitial nucleus of Cajal; IRt, intermediate reticular nucleus; IVN, inferior vestibular nucleus; KF, Kölliker-Fuse nucleus; LDTg, laterodorsal tegmental nucleus; LPAG, lateral periaqueductal gray; LPGi, lateral paragigantocellular nucleus; LVN, lateral vestibular nucleus; MAO, medial accessory olive; MD, mediodorsal thalamic nucleus; MDJ, mesodiencephalic junction; MdV, medullary reticular nucleus, ventral; mRt, mesencephalic reticular formation; MVN, medial vestibular nucleus; NPH, nucleus prepositus hypoglossi; NI, nucleus incertus; NRG, nucleus reticularis gigantocellularis; PCRt, parvocellular reticular nucleus; PF, parafascicular thalamic nucleus; PMn, paramedian reticular nucleus; PnC, pontine reticular nucleus, caudal; PPRF, paramedian pontine reticular formation; PTg, pedunculotegmental nucleus; Sim, simplex lobule; SC, superior colliculus; SNc, substantia nigra, pars compacta; SubC, subcoeruleus nucleus; SuM, supramammillary region; VL, ventrolateral thalamic nucleus; vlPAG, ventrolateral periaqueductal gray; VM, ventromedial thalamic nucleus; ZI, zona incerta.

    Techniques Used:

    Fastigial projection targets revealed with AAV- mediated anterograde tracing. ( A ) Schematic of AAV injection experiments to examine fastigial output projections. Vertical lines in a sagittal scheme indicates approximate rostrocaudal levels of images in C-E of this figure and in Figure 3 . ( B ) Representative coronal section images of injection sites. Two images are from the same case, showing that the labeled injection site (red) covered the entire fastigial nucleus with little spread to the neighboring anterior interpositus nucleus (indicated with '+'). ( C–E ) Fastigial axonal innervation (red) of reticular formation, midbrain, and thalamus are shown with Nissl counterstaining (blue). ( C ) Shows a labeled axonal bundle exiting the cerebellum from the contralateral cerebellar peduncle (asterisk) which then innervated the pontine reticular nucleus (PnC), intermediate reticular nucleus (IRt), and parvocellular reticular nucleus (PCRt). Labeled axonal innervation was also found at laterodorsal tegmental nucleus (LDTg) and a confined part of the facial nucleus (7N). ( D ) Shows significant axonal innervation of perioculomotor area including the interstitial nucleus of Cajal (INC) and sparse innervation of the anterior pretectal nucleus (APT), nucleus of posterior commissure (Pcom), and suprageniculate thalamic nucleus/posterior thalamic group (SG/Po). The labeled axonal terminals also reached the supramammillary region (SuM) and a medial part of the substantia nigra pars compacta (SNc). ( E ) Shows fastigial projections to the thalamic nuclei including ventromedial (VM), centrolateral (CL), and mediodorsal (MD) nuclei and to the zona incerta (ZI). The panel also shows labeled fastigial axons traversing the midline to project to the thalamic nuclei ipsilateral to the injection side (right in the image). ( F ) Fastigial projections to the bilateral cerebellar cortex with ipsilateral predominance. The image shows that the projections terminate as mossy fibers at or beneath the Purkinje cell layer (pcl), as described previously for nucleocortical projections from other cerebellar nuclei ( Houck and Person, 2015 ; Gao et al., 2016 ; Low et al., 2018 ). Dotted line indicates the midline. The result is in accordance with a previous study in cats and monkeys ( Tolbert et al., 1978 ). ( G ) Fastigial projections to the nucleus incertus (NI) and laterodorsal tegmental nucleus (LDTg). ( H ) Fastigial projections to the ventrolateral periaqueductal gray (vlPAG). ( I ) Fastigial projections to the interpeduncular nucleus. A double injection case is shown in which red and green AAVs were injected in the right and left FN, respectively. Resultant labeling showed sparse but consistent axonal projections to lateral interpeduncular nucleus (IPL). ( J ) Fastigial projections to the zona incerta (ZI). Labeled fastigial axons (red) within the Lhx6+ neuronal clusters (green, visualized with Lhx6 reporter mouse; n = 2 females), which demarcates the ZI, are shown. No clear fastigial projections to the lateral hypothalamus, which was identified with clustered orexinergic neurons that are visualized with immunostaining for orexin (blue). Scale bar in E applies to C-E. Abbreviations, 7N, facial nucleus; APT, anterior pretectal nucleus; CL, centrolateral thalamic nucleus; gcl, granule cell layer; IRt, intermediate reticular nucleus; INC, interstitial nucleus of Cajal; IPL, lateral interpeduncular nucleus; LDTg, laterodorsal tegmental nucleus; MD, mediodorsal thalamic nucleus; ml, molecular layer; NI, nucleus incertus; pcl, Purkinje cell layer; Pcom, nucleus of posterior commissure; PCRt, parvocellular reticular nucleus; PF, parafascicular thalamic nucleus; PnC, caudal pontine reticular nucleus; SG/Po, suprageniculate thalamic nucleus/posterior thalamic group; SNc, substantia nigra, pars compacta; SuM, supramammillary region; VM, ventromedial thalamic nucleus; vlPAG, ventrolateral periaqueductal gray; VTA, ventral tegmental area; wm, cerebellar white matter; ZI, zona incerta.
    Figure Legend Snippet: Fastigial projection targets revealed with AAV- mediated anterograde tracing. ( A ) Schematic of AAV injection experiments to examine fastigial output projections. Vertical lines in a sagittal scheme indicates approximate rostrocaudal levels of images in C-E of this figure and in Figure 3 . ( B ) Representative coronal section images of injection sites. Two images are from the same case, showing that the labeled injection site (red) covered the entire fastigial nucleus with little spread to the neighboring anterior interpositus nucleus (indicated with '+'). ( C–E ) Fastigial axonal innervation (red) of reticular formation, midbrain, and thalamus are shown with Nissl counterstaining (blue). ( C ) Shows a labeled axonal bundle exiting the cerebellum from the contralateral cerebellar peduncle (asterisk) which then innervated the pontine reticular nucleus (PnC), intermediate reticular nucleus (IRt), and parvocellular reticular nucleus (PCRt). Labeled axonal innervation was also found at laterodorsal tegmental nucleus (LDTg) and a confined part of the facial nucleus (7N). ( D ) Shows significant axonal innervation of perioculomotor area including the interstitial nucleus of Cajal (INC) and sparse innervation of the anterior pretectal nucleus (APT), nucleus of posterior commissure (Pcom), and suprageniculate thalamic nucleus/posterior thalamic group (SG/Po). The labeled axonal terminals also reached the supramammillary region (SuM) and a medial part of the substantia nigra pars compacta (SNc). ( E ) Shows fastigial projections to the thalamic nuclei including ventromedial (VM), centrolateral (CL), and mediodorsal (MD) nuclei and to the zona incerta (ZI). The panel also shows labeled fastigial axons traversing the midline to project to the thalamic nuclei ipsilateral to the injection side (right in the image). ( F ) Fastigial projections to the bilateral cerebellar cortex with ipsilateral predominance. The image shows that the projections terminate as mossy fibers at or beneath the Purkinje cell layer (pcl), as described previously for nucleocortical projections from other cerebellar nuclei ( Houck and Person, 2015 ; Gao et al., 2016 ; Low et al., 2018 ). Dotted line indicates the midline. The result is in accordance with a previous study in cats and monkeys ( Tolbert et al., 1978 ). ( G ) Fastigial projections to the nucleus incertus (NI) and laterodorsal tegmental nucleus (LDTg). ( H ) Fastigial projections to the ventrolateral periaqueductal gray (vlPAG). ( I ) Fastigial projections to the interpeduncular nucleus. A double injection case is shown in which red and green AAVs were injected in the right and left FN, respectively. Resultant labeling showed sparse but consistent axonal projections to lateral interpeduncular nucleus (IPL). ( J ) Fastigial projections to the zona incerta (ZI). Labeled fastigial axons (red) within the Lhx6+ neuronal clusters (green, visualized with Lhx6 reporter mouse; n = 2 females), which demarcates the ZI, are shown. No clear fastigial projections to the lateral hypothalamus, which was identified with clustered orexinergic neurons that are visualized with immunostaining for orexin (blue). Scale bar in E applies to C-E. Abbreviations, 7N, facial nucleus; APT, anterior pretectal nucleus; CL, centrolateral thalamic nucleus; gcl, granule cell layer; IRt, intermediate reticular nucleus; INC, interstitial nucleus of Cajal; IPL, lateral interpeduncular nucleus; LDTg, laterodorsal tegmental nucleus; MD, mediodorsal thalamic nucleus; ml, molecular layer; NI, nucleus incertus; pcl, Purkinje cell layer; Pcom, nucleus of posterior commissure; PCRt, parvocellular reticular nucleus; PF, parafascicular thalamic nucleus; PnC, caudal pontine reticular nucleus; SG/Po, suprageniculate thalamic nucleus/posterior thalamic group; SNc, substantia nigra, pars compacta; SuM, supramammillary region; VM, ventromedial thalamic nucleus; vlPAG, ventrolateral periaqueductal gray; VTA, ventral tegmental area; wm, cerebellar white matter; ZI, zona incerta.

    Techniques Used: Anterograde Tracing, Injection, Labeling, Immunostaining

    Expression of marker candidate genes for fastigial cell types. ( A–I ) Expression of marker candidate genes for molecularly distinct fastigial neurons. The images are from the in situ hybridization database available at Allen Brain Atlas ( Lein et al., 2007 ). The experiment IDs are, 79556662 for Calb2 ( A ), 79568026 for Cdh8 ( B ), 297 for Crhr1 ( C ), 73636087 for Inhbb ( D ), 74512048 for Nefh ( E ), 72340134 for Rreb1 (F), 955 for Sema7a ( G ), 79908848 for Snca ( H ), and 70436740 for Spp1 ( I ). Each panel consists of two representative coronal sections showing caudal (top) and rostral (bottom) areas of the FN. Contour of the FN are drawn with dotted line in ( A ). Scale bar in A applies to A-I.
    Figure Legend Snippet: Expression of marker candidate genes for fastigial cell types. ( A–I ) Expression of marker candidate genes for molecularly distinct fastigial neurons. The images are from the in situ hybridization database available at Allen Brain Atlas ( Lein et al., 2007 ). The experiment IDs are, 79556662 for Calb2 ( A ), 79568026 for Cdh8 ( B ), 297 for Crhr1 ( C ), 73636087 for Inhbb ( D ), 74512048 for Nefh ( E ), 72340134 for Rreb1 (F), 955 for Sema7a ( G ), 79908848 for Snca ( H ), and 70436740 for Spp1 ( I ). Each panel consists of two representative coronal sections showing caudal (top) and rostral (bottom) areas of the FN. Contour of the FN are drawn with dotted line in ( A ). Scale bar in A applies to A-I.

    Techniques Used: Expressing, Marker, In Situ Hybridization

    Distribution of molecularly distinct fastigial neurons that were identified with immunostaining for SPP1, CALB, and SNCA. Individual neurons that were immunostained for 1) SPP1, 2) CALB2, 3) SNCA, or doubly immunostained for 4) SPP1 and SNCA, were mapped from serial sections (interval 120 µm). Mapping results from n = 3 male mice for each set were grouped into caudal, central, and rostral levels and superimposed onto drawings of the fastigial nucleus. Total numbers of neurons identified and mapped from three mice each are 922, 242, 334, and 88 for SPP1, CALB2, SNCA, and SPP1+SNCA, respectively.
    Figure Legend Snippet: Distribution of molecularly distinct fastigial neurons that were identified with immunostaining for SPP1, CALB, and SNCA. Individual neurons that were immunostained for 1) SPP1, 2) CALB2, 3) SNCA, or doubly immunostained for 4) SPP1 and SNCA, were mapped from serial sections (interval 120 µm). Mapping results from n = 3 male mice for each set were grouped into caudal, central, and rostral levels and superimposed onto drawings of the fastigial nucleus. Total numbers of neurons identified and mapped from three mice each are 922, 242, 334, and 88 for SPP1, CALB2, SNCA, and SPP1+SNCA, respectively.

    Techniques Used: Immunostaining, Mouse Assay

    37) Product Images from "Histone H2B as a functionally important plasminogen receptor on macrophages"

    Article Title: Histone H2B as a functionally important plasminogen receptor on macrophages

    Journal:

    doi: 10.1182/blood-2007-03-079392

    Colocalization of Plg-R with Plg on the surface of J774.1 cells. Localization of Plg with either H2B, α-enolase, annexin 2, or p11 on the cell surface of J774A.1 cells assessed by confocal microscopy. The cells were grown on coverslips, serum
    Figure Legend Snippet: Colocalization of Plg-R with Plg on the surface of J774.1 cells. Localization of Plg with either H2B, α-enolase, annexin 2, or p11 on the cell surface of J774A.1 cells assessed by confocal microscopy. The cells were grown on coverslips, serum

    Techniques Used: Confocal Microscopy

    Cell-surface expression of Plg-R on murine macrophage cell lines
    Figure Legend Snippet: Cell-surface expression of Plg-R on murine macrophage cell lines

    Techniques Used: Expressing

    Plg-R expression on mouse blood monocytes and TG-recruited peritoneal macrophages
    Figure Legend Snippet: Plg-R expression on mouse blood monocytes and TG-recruited peritoneal macrophages

    Techniques Used: Expressing

    38) 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

    39) 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:

    40) 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

    Related Articles

    Isolation:

    Article Title: Construction of Cardiac Tissue Rings Using a Magnetic Tissue Fabrication Technique
    Article Snippet: .. Primary Culture of Neonatal Rat Cardiomyocytes Primary neonatal rat cardiomyocytes were isolated using a neonatal cardiomyocyte isolation kit (Worthington Biochemical, Lakewood, NJ, USA) according to the published procedure [ ]. .. Briefly, ventricles from 2–4 day-old Sprague-Dawley rats (Japan SLC, Inc., Hamamatsu, Japan) were incubated for 16–20 h at 4 °C in Hank’s balanced salt solution containing trypsin (50–100 μg/mL), followed by digestion with collagenase (75 U/mL) for 40 min at 37 °C.

    Article Title: Mitoregulin: A lncRNA-Encoded Microprotein that Supports Mitochondrial Supercomplexes and Respiratory Efficiency
    Article Snippet: .. Neonatal rat cardiomyocytes (NRCMs) were isolated using the Worthington Neonatal Cardiomyocyte Isolation System (Worthington Biochemical Corporation, Cat. No ). ..

    Article Title: Single Cell Transcriptomics Reconstructs Fate Conversion from Fibroblast to Cardiomyocyte
    Article Snippet: .. Neonatal CMs were isolated using the neonatal cardiomyocytes isolation system (Worthington Biochemical Corporation) except that all enzymes were used at a ¼ of the recommended concentration to increase cell viability. .. After a 1.5 hour pre-plating on uncoated surface to remove attached nonmyocytes, the unattached CMs were collected in TRIzol ( > 80% viability by Trypan blue staining).

    Article Title: ADAP1 limits neonatal cardiomyocyte hypertrophy by reducing integrin cell surface expression
    Article Snippet: .. RNVC were extracted from the hearts of Sprague Dawley rat pups (strain code 001; Charles River) 1–3 days after birth using the Neonatal Cardiomyocyte Isolation System (Worthington Biochemical Corporation). .. The isolated ventricles were incubated in calcium/magnesium-free Hank’s Balanced Salt Solution containing trypsin (50 µg/mL) in vented-cap tubes with slow agitation at 4 °C for 18 h. The trypsin was then inhibited by adding Soybean Trypsin Inhibitor (200 µg/mL), and the ventricles were further digested with collagenase (100 units/mL) at 37 °C for 30 min.

    Article Title: Pro-survival function of MEF2 in cardiomyocytes is enhanced by β-blockers
    Article Snippet: .. Cell culture Primary neonatal rat cardiomyocytes were prepared from 1- to 3-day old Sprague Dawley rats using the Neonatal Cardiomyocyte Isolation System (Worthington Biochemical Corp, Lakewood, NJ, USA). .. Briefly, whole hearts were dissociated with trypsin (Promega, Madison, WI, USA) and collagenase (Worthington Biochemical Corp).

    Article Title: Docosahexaenoic acid inhibits protein kinase C translocation/activation and cardiac hypertrophy in rat cardiomyocytes
    Article Snippet: .. Isolation of cardiomyocytes Neonatal cardiomyocytes were obtained using an isolation system from Worthington Biochemical Corporation. ..

    Article Title: Muscle ring finger protein-1 inhibits PKC? activation and prevents cardiomyocyte hypertrophy
    Article Snippet: .. NRVM were isolated using the neonatal cardiomyocyte isolation kit (Worthington) and were plated on laminin. ..

    Article Title: Pivotal role of cardiac lineage protein-1 (CLP-1) in transcriptional elongation factor P-TEFb complex formation in cardiac hypertrophy
    Article Snippet: .. Cardiac cells of 2–4-day-old WKY rats were isolated using a neonatal cardiomyocyte isolation system (Worthington Biochemical Corp, Lakewood, New Jersey, USA). .. After pre-plating to reduce contaminating non-muscle cells, cardiomyocytes were plated on Bioflex collagen 6-well plates (Flexcell, McKeesport, Pennsylvania, USA) and cultured in DMEM/F-12 (1:1) media supplemented with 10% fetal bovine serum for approximately 1.5 days prior to switching to serum-free medium.

    Cell Culture:

    Article Title: Pro-survival function of MEF2 in cardiomyocytes is enhanced by β-blockers
    Article Snippet: .. Cell culture Primary neonatal rat cardiomyocytes were prepared from 1- to 3-day old Sprague Dawley rats using the Neonatal Cardiomyocyte Isolation System (Worthington Biochemical Corp, Lakewood, NJ, USA). .. Briefly, whole hearts were dissociated with trypsin (Promega, Madison, WI, USA) and collagenase (Worthington Biochemical Corp).

    Concentration Assay:

    Article Title: Single Cell Transcriptomics Reconstructs Fate Conversion from Fibroblast to Cardiomyocyte
    Article Snippet: .. Neonatal CMs were isolated using the neonatal cardiomyocytes isolation system (Worthington Biochemical Corporation) except that all enzymes were used at a ¼ of the recommended concentration to increase cell viability. .. After a 1.5 hour pre-plating on uncoated surface to remove attached nonmyocytes, the unattached CMs were collected in TRIzol ( > 80% viability by Trypan blue staining).

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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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    Worthington Biochemical ca free krebs solution
    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
    Ca Free Krebs Solution, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Worthington Biochemical chrestensen ca
    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
    Chrestensen Ca, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 89/100, based on 1 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

    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: