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    QIAamp DNA Blood Mini Kit
    Description:
    For purification of up to 12 µg genomic mitochondrial or viral DNA from blood and related body fluids Kit contents Qiagen QIAamp DNA Blood Mini Kit 50 preps 1 to 200L Sample 50 to 200L Elution Volume Whole Blood Body Fluids Sample Spin Column Format Silica Technology Manual Processing 20 to 40 min Time Run 4 to 12g Yield For Purification of Up to 12g Genomic Mitochondrial or Viral DNA from Blood and Related Body Fluids Includes 50 QIAamp Mini Spin Columns Qiagen Protease Reagents Buffers 2mL Collection Tubes Benefits Rapid purification of high quality ready to use DNA No organic extraction or alcohol precipitation Consistent high yields Complete removal of contaminants and inhibitors for reliable results
    Catalog Number:
    51104
    Price:
    157
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    QIAamp DNA Blood Mini Kit
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    Structured Review

    Qiagen genomic dna
    QIAamp DNA Blood Mini Kit
    For purification of up to 12 µg genomic mitochondrial or viral DNA from blood and related body fluids Kit contents Qiagen QIAamp DNA Blood Mini Kit 50 preps 1 to 200L Sample 50 to 200L Elution Volume Whole Blood Body Fluids Sample Spin Column Format Silica Technology Manual Processing 20 to 40 min Time Run 4 to 12g Yield For Purification of Up to 12g Genomic Mitochondrial or Viral DNA from Blood and Related Body Fluids Includes 50 QIAamp Mini Spin Columns Qiagen Protease Reagents Buffers 2mL Collection Tubes Benefits Rapid purification of high quality ready to use DNA No organic extraction or alcohol precipitation Consistent high yields Complete removal of contaminants and inhibitors for reliable results
    https://www.bioz.com/result/genomic dna/product/Qiagen
    Average 99 stars, based on 10301 article reviews
    Price from $9.99 to $1999.99
    genomic dna - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) Product Images from "ASF1a inhibition induces p53-dependent growth arrest and senescence of cancer cells"

    Article Title: ASF1a inhibition induces p53-dependent growth arrest and senescence of cancer cells

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-019-1357-z

    Silencing ASF1a triggers DNA damage response. a Immunofluorescence staining of control (nc) and ASF1a knockdown (ASF1a si1/si2) groups of HepG2 and LNCaP cells. Nuclei were stained with DAPI (blue signals). γH2AX and 53BP1 were stained with specific antibodies (green and red signals, respectively; scale bar: 50 μm). Quantification is shown at the bottom (data are presented as the mean ± SD value of three independent experiments for HepG2 and LNCaP, respectively). Senescence-associated heterochromatin foci (SAHF) were not detected by using DAPI staining. DAPI, 4-6-diamidino-2-phenylindole dihydrochloride
    Figure Legend Snippet: Silencing ASF1a triggers DNA damage response. a Immunofluorescence staining of control (nc) and ASF1a knockdown (ASF1a si1/si2) groups of HepG2 and LNCaP cells. Nuclei were stained with DAPI (blue signals). γH2AX and 53BP1 were stained with specific antibodies (green and red signals, respectively; scale bar: 50 μm). Quantification is shown at the bottom (data are presented as the mean ± SD value of three independent experiments for HepG2 and LNCaP, respectively). Senescence-associated heterochromatin foci (SAHF) were not detected by using DAPI staining. DAPI, 4-6-diamidino-2-phenylindole dihydrochloride

    Techniques Used: Immunofluorescence, Staining

    2) Product Images from "Herpes Simplex Virus Type 2 Infection-Induced Expression of CXCR3 Ligands Promotes CD4+ T Cell Migration and Is Regulated by the Viral Immediate-Early Protein ICP4"

    Article Title: Herpes Simplex Virus Type 2 Infection-Induced Expression of CXCR3 Ligands Promotes CD4+ T Cell Migration and Is Regulated by the Viral Immediate-Early Protein ICP4

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02932

    HSV-2 ICP4 regulates the expression of CXCR3 ligands via the p38 MAPK signaling pathway. (A–C) HSV-2 regulates the expression of CXCL9 (A) , CXCL10 (B) , and CXCL11 (C) via p38/MAPK signaling pathway. ME180 cells in 24-well plates were co-transfected with 150 ng CXCR3 ligand reporter and 15 ng phRL-TK. At 4 h post-transfection, cells were infected with HSV-2 at an MOI of 1 and supplemented with inhibitor PD98059, SP600125, or SB203580. DLR assay was performed at 24 h post-transfection. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in mock-infected samples. (D–F) HSV-2 ICP4 regulates the expression of CXCL9 (D) , CXCL10 (E) , and CXCL11 (F) via p38/MAPK signaling pathway. ME180 cells in 24-well plates were co-transfected with 300 ng empty vector or ICP4 expression plasmid together with 150 ng CXCR3 ligand reporter and 15 ng phRL-TK. At 4 h post-transfection, cells were cultured in complete DMEM supplemented with inhibitor PD98059, SP600125, or SB203580. DLR assay was performed at 24 h post-transfection. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in cells transfected with empty vector. (G) ICP4 activates p38 MAPK signaling pathway. ME180 cells were transfected with 3 μg ICP4 expression plasmid. The protein level of p38, phospho-p38 (p-p38) or phospho-C/EBP-β (p-C/EBP-β) was detected by Western Blot. Data shown are mean ± S.D. of three independent experiments (A–F) . ns, not significant, *** p
    Figure Legend Snippet: HSV-2 ICP4 regulates the expression of CXCR3 ligands via the p38 MAPK signaling pathway. (A–C) HSV-2 regulates the expression of CXCL9 (A) , CXCL10 (B) , and CXCL11 (C) via p38/MAPK signaling pathway. ME180 cells in 24-well plates were co-transfected with 150 ng CXCR3 ligand reporter and 15 ng phRL-TK. At 4 h post-transfection, cells were infected with HSV-2 at an MOI of 1 and supplemented with inhibitor PD98059, SP600125, or SB203580. DLR assay was performed at 24 h post-transfection. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in mock-infected samples. (D–F) HSV-2 ICP4 regulates the expression of CXCL9 (D) , CXCL10 (E) , and CXCL11 (F) via p38/MAPK signaling pathway. ME180 cells in 24-well plates were co-transfected with 300 ng empty vector or ICP4 expression plasmid together with 150 ng CXCR3 ligand reporter and 15 ng phRL-TK. At 4 h post-transfection, cells were cultured in complete DMEM supplemented with inhibitor PD98059, SP600125, or SB203580. DLR assay was performed at 24 h post-transfection. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in cells transfected with empty vector. (G) ICP4 activates p38 MAPK signaling pathway. ME180 cells were transfected with 3 μg ICP4 expression plasmid. The protein level of p38, phospho-p38 (p-p38) or phospho-C/EBP-β (p-C/EBP-β) was detected by Western Blot. Data shown are mean ± S.D. of three independent experiments (A–F) . ns, not significant, *** p

    Techniques Used: Expressing, Transfection, Infection, Luciferase, Plasmid Preparation, Cell Culture, Western Blot

    HSV-2 infection induces the production of CXCR3 ligands in human cervical epithelial cells. (A) HSV-2 infection activates the promoters of human CXCR3 ligands. ME180 cells in 24-well plates were co-transfected with 150 ng CXCL9-Luc, CXCL10-Luc or CXCL11-Luc, and 15 ng internal control plasmid phRL-TK. At 4 h post-transfection, cells were infected with HSV-2 or ultraviolet-inactivated HSV-2 (UV-HSV-2) at an MOI of 1 for 24 h. DLR assay was performed. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in mock-infected samples. (B) HSV-2 infection induces the mRNA production of CXCR3 ligands. ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cells were harvested and total RNA was extracted. The expression of CXCR3 ligands and GAPDH was evaluated by relative real-time quantitative PCR. The Ct values of GAPDH among all groups were equable and not overloaded. mRNA copies of CXCR3 ligands were normalized using GAPDH and expressed as fold increase of the value for the mock-infected control. (C) HSV-2 infection induces the production of CXCR3 ligands. As depicted in (B) , cell supernatants were collected, and the protein level of CXCR3 ligands was measured by CBA. Data shown are mean ± S.D. of three independent experiments (A, B, and C). * p
    Figure Legend Snippet: HSV-2 infection induces the production of CXCR3 ligands in human cervical epithelial cells. (A) HSV-2 infection activates the promoters of human CXCR3 ligands. ME180 cells in 24-well plates were co-transfected with 150 ng CXCL9-Luc, CXCL10-Luc or CXCL11-Luc, and 15 ng internal control plasmid phRL-TK. At 4 h post-transfection, cells were infected with HSV-2 or ultraviolet-inactivated HSV-2 (UV-HSV-2) at an MOI of 1 for 24 h. DLR assay was performed. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in mock-infected samples. (B) HSV-2 infection induces the mRNA production of CXCR3 ligands. ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cells were harvested and total RNA was extracted. The expression of CXCR3 ligands and GAPDH was evaluated by relative real-time quantitative PCR. The Ct values of GAPDH among all groups were equable and not overloaded. mRNA copies of CXCR3 ligands were normalized using GAPDH and expressed as fold increase of the value for the mock-infected control. (C) HSV-2 infection induces the production of CXCR3 ligands. As depicted in (B) , cell supernatants were collected, and the protein level of CXCR3 ligands was measured by CBA. Data shown are mean ± S.D. of three independent experiments (A, B, and C). * p

    Techniques Used: Infection, Transfection, Plasmid Preparation, Luciferase, Expressing, Real-time Polymerase Chain Reaction, Crocin Bleaching Assay

    HSV-2 infection-induced CXCL9 plays a predominant role in mediating CD4 + T cell migration. (A) The concentrations of CXCR3 ligands in the supernatants of ME180 cells infected with HSV-2 or mock-infected with DMEM were detected by CBA. (B,C) CXCL9 induced by HSV-2 recruits the migration of PBMCs (B) and CD4 + T cells (C) . ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates in the absence or presence of anti-CXCL9, –CXCL10, or/and –CXCL11 neutralizing Ab or control Ab for 1 h. (D) Neutralization of CXCR3 reduces the migration of CD4 + T cells induced by HSV-2 infection. ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates. The activated CD4 + T cells were incubated with RPMI 1,640 medium containing anti-CXCR3 neutralizing Ab for 1 h and placed in the upper chamber. (E) Recombinant CXCL9 significantly induces the migration of CD4 + T cells. DMEM containing recombinant CXCL9, CXCL10, or CXCL11 (48 pg/mL, 55 pg/mL and 175 pg/mL, respectively; the lowest concentration induced by HSV-2 infection) was added to the lower chamber of transwell plates. (F,G) Recombinant CXCL10 or CXCL11 mediates the migration of CD4 + T cells in a dose-dependent manner. DMEM containing recombinant CXCL10 or CXCL11 was added to the lower chamber of transwell plates. CXCL10 or CXCL11 was started from 55 pg/mL and 175 pg/mL, respectively, at a concentration gradient of two times. The activated CD4 + T cells were placed in the upper chamber. After 2 h incubation, cells migrated to lower chambers were collected and counted using an automatic cell counter. Cells migration was expressed as percentage of input. Input cells in the upper chamber were 5 × 10 5 . Data shown are mean ± S.D. of three independent experiments (A–G) . ns, not significant, * p
    Figure Legend Snippet: HSV-2 infection-induced CXCL9 plays a predominant role in mediating CD4 + T cell migration. (A) The concentrations of CXCR3 ligands in the supernatants of ME180 cells infected with HSV-2 or mock-infected with DMEM were detected by CBA. (B,C) CXCL9 induced by HSV-2 recruits the migration of PBMCs (B) and CD4 + T cells (C) . ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates in the absence or presence of anti-CXCL9, –CXCL10, or/and –CXCL11 neutralizing Ab or control Ab for 1 h. (D) Neutralization of CXCR3 reduces the migration of CD4 + T cells induced by HSV-2 infection. ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates. The activated CD4 + T cells were incubated with RPMI 1,640 medium containing anti-CXCR3 neutralizing Ab for 1 h and placed in the upper chamber. (E) Recombinant CXCL9 significantly induces the migration of CD4 + T cells. DMEM containing recombinant CXCL9, CXCL10, or CXCL11 (48 pg/mL, 55 pg/mL and 175 pg/mL, respectively; the lowest concentration induced by HSV-2 infection) was added to the lower chamber of transwell plates. (F,G) Recombinant CXCL10 or CXCL11 mediates the migration of CD4 + T cells in a dose-dependent manner. DMEM containing recombinant CXCL10 or CXCL11 was added to the lower chamber of transwell plates. CXCL10 or CXCL11 was started from 55 pg/mL and 175 pg/mL, respectively, at a concentration gradient of two times. The activated CD4 + T cells were placed in the upper chamber. After 2 h incubation, cells migrated to lower chambers were collected and counted using an automatic cell counter. Cells migration was expressed as percentage of input. Input cells in the upper chamber were 5 × 10 5 . Data shown are mean ± S.D. of three independent experiments (A–G) . ns, not significant, * p

    Techniques Used: Infection, Migration, Crocin Bleaching Assay, Neutralization, Incubation, Recombinant, Concentration Assay

    HSV-2 ICP4 promotes the production of human CXCR3 ligands. (A) ICP4 induces the activation of CXCR3 ligand promoters. ME180 cells in 24-well plates were transfected with 300 ng expression plasmid of HSV-2 gene or empty vector together with 150 ng CXCR3 ligand reporter and 15 ng phRL-TK. At 24 h post-transfection, DLR assay was performed. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in cells transfected with empty vector. (B) The expression of HSV-2 genes was detected using anti-Flag Ab by Western Blot. ME180 cells were transfected with 3 μg HSV-2 gene expression plasmid for 24 h. The proteins were collected and detected using mouse anti-Flag Ab. (C) ICP4 induces the mRNA production of CXCR3 ligands. ME180 cells in 6-well plates were transfected with 3 μg ICP4 expression plasmid for 24 h. Cells were harvested and total RNA was extracted. The expression of CXCR3 ligands and GAPDH gene was evaluated by relative real-time quantitative PCR. The Ct values of GAPDH among all groups were equable and not overloaded. mRNA copies of CXCR3 ligands were normalized using GAPDH and expressed as fold increase of the value for the empty vector-transfected control. (D) ICP4 induces the production of CXCR3 ligands. As depicted in (C) , cell supernatants were collected, and the protein levels of CXCR3 ligands were measured by CBA. (E,F) CXCL9 induced by ICP4 recruits the migration of PBMCs (E) and CD4 + T cells (F) . ME180 cells in 6-well plates were transfected with 3 μg ICP4 expression plasmid for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates in the absence or presence of anti-CXCL9, –CXCL10, or/and –CXCL11 neutralizing Ab or control Ab for 1h. (G) Neutralization of CXCR3 reduces the migration of CD4 + T cells induced by ICP4. ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates. The activated CD4 + T cells were incubated with RPMI 1,640 medium containing anti-CXCR3 neutralizing Ab for 1 h and placed in the upper chamber. As depicted in Figure 3 , cells migrated to lower chambers were counted. Cells migration was expressed as percentage of input. One representative out of three independent experiments is shown (B) . Data shown are mean ± S.D. of three independent experiments (A,C–G) . ns, not significant, * p
    Figure Legend Snippet: HSV-2 ICP4 promotes the production of human CXCR3 ligands. (A) ICP4 induces the activation of CXCR3 ligand promoters. ME180 cells in 24-well plates were transfected with 300 ng expression plasmid of HSV-2 gene or empty vector together with 150 ng CXCR3 ligand reporter and 15 ng phRL-TK. At 24 h post-transfection, DLR assay was performed. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in cells transfected with empty vector. (B) The expression of HSV-2 genes was detected using anti-Flag Ab by Western Blot. ME180 cells were transfected with 3 μg HSV-2 gene expression plasmid for 24 h. The proteins were collected and detected using mouse anti-Flag Ab. (C) ICP4 induces the mRNA production of CXCR3 ligands. ME180 cells in 6-well plates were transfected with 3 μg ICP4 expression plasmid for 24 h. Cells were harvested and total RNA was extracted. The expression of CXCR3 ligands and GAPDH gene was evaluated by relative real-time quantitative PCR. The Ct values of GAPDH among all groups were equable and not overloaded. mRNA copies of CXCR3 ligands were normalized using GAPDH and expressed as fold increase of the value for the empty vector-transfected control. (D) ICP4 induces the production of CXCR3 ligands. As depicted in (C) , cell supernatants were collected, and the protein levels of CXCR3 ligands were measured by CBA. (E,F) CXCL9 induced by ICP4 recruits the migration of PBMCs (E) and CD4 + T cells (F) . ME180 cells in 6-well plates were transfected with 3 μg ICP4 expression plasmid for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates in the absence or presence of anti-CXCL9, –CXCL10, or/and –CXCL11 neutralizing Ab or control Ab for 1h. (G) Neutralization of CXCR3 reduces the migration of CD4 + T cells induced by ICP4. ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates. The activated CD4 + T cells were incubated with RPMI 1,640 medium containing anti-CXCR3 neutralizing Ab for 1 h and placed in the upper chamber. As depicted in Figure 3 , cells migrated to lower chambers were counted. Cells migration was expressed as percentage of input. One representative out of three independent experiments is shown (B) . Data shown are mean ± S.D. of three independent experiments (A,C–G) . ns, not significant, * p

    Techniques Used: Activation Assay, Transfection, Expressing, Plasmid Preparation, Luciferase, Western Blot, Real-time Polymerase Chain Reaction, Crocin Bleaching Assay, Migration, Neutralization, Infection, Incubation

    HSV-2 ICP4 binds to the promoters of CXCR3 ligands by interaction with TBP. (A) Schematic representation of the predicted NLSs of ICP4 amino acid (AA) sequence. (B) ICP4 is located in the nucleus. ME180 cells in 35-mm dishes with glass bottom were transfected with 2 μg empty vector or HA-tagged ICP4 expression plasmid for 24 h. Cells were stained with mouse anti-HA mAb, followed by Cy3-conjugated goat anti-mouse (red) as the secondary Ab. Cell nuclei (blue) were stained with DAPI. The images were obtained by fluorescence microscopy using 60 × objective. The scale bar indicates 21 μm. (C) The expression of ICP4 was stained using anti-HA mAb. (D) ICP4 binds to the promoters of CXCR3 ligands. ME180 cells were transfected with 3 μg empty vector or HA-tagged ICP4 expression plasmid for 24 h. Cells were lysed and subjected to ChIP assay using mouse anti-HA mAb, mouse anti-RNA polymerase II mAb (positive control) or mouse normal IgG (negative control) for immunoprecipitation. (E) ICP4 interacts with TBP. ME180 cells in 6-well plates were transfected with 3 μg empty vector or HA-tagged ICP4 expression plasmid for 24 h. Cells were lysed and subjected to co-immunoprecipitation (IP) using rabbit anti-HA or anti-TBP Ab. Rabbit normal IgG was used as a negative control. IP products and 5% input samples were examined using rabbit anti-HA and rabbit anti-TBP Abs by western blot. One representative out of three independent experiments is shown (B–E) .
    Figure Legend Snippet: HSV-2 ICP4 binds to the promoters of CXCR3 ligands by interaction with TBP. (A) Schematic representation of the predicted NLSs of ICP4 amino acid (AA) sequence. (B) ICP4 is located in the nucleus. ME180 cells in 35-mm dishes with glass bottom were transfected with 2 μg empty vector or HA-tagged ICP4 expression plasmid for 24 h. Cells were stained with mouse anti-HA mAb, followed by Cy3-conjugated goat anti-mouse (red) as the secondary Ab. Cell nuclei (blue) were stained with DAPI. The images were obtained by fluorescence microscopy using 60 × objective. The scale bar indicates 21 μm. (C) The expression of ICP4 was stained using anti-HA mAb. (D) ICP4 binds to the promoters of CXCR3 ligands. ME180 cells were transfected with 3 μg empty vector or HA-tagged ICP4 expression plasmid for 24 h. Cells were lysed and subjected to ChIP assay using mouse anti-HA mAb, mouse anti-RNA polymerase II mAb (positive control) or mouse normal IgG (negative control) for immunoprecipitation. (E) ICP4 interacts with TBP. ME180 cells in 6-well plates were transfected with 3 μg empty vector or HA-tagged ICP4 expression plasmid for 24 h. Cells were lysed and subjected to co-immunoprecipitation (IP) using rabbit anti-HA or anti-TBP Ab. Rabbit normal IgG was used as a negative control. IP products and 5% input samples were examined using rabbit anti-HA and rabbit anti-TBP Abs by western blot. One representative out of three independent experiments is shown (B–E) .

    Techniques Used: Sequencing, Transfection, Plasmid Preparation, Expressing, Staining, Fluorescence, Microscopy, Chromatin Immunoprecipitation, Positive Control, Negative Control, Immunoprecipitation, Western Blot

    Contribution of HSV-2 infection-induced CXCR3 ligands to CD4 + T cell infiltration into mouse vagina. Seven days prior to HSV-2 challenge, BALB/c mice were injected with progesterone in multiple sites. One day prior to HSV-2 challenge, CXCL9, CXCL10, and CXCL11 neutralizing antibodies were delivered to the vagina of mice, alone or in combination, while isotype matched control IgG was used as the control. Mice were then anesthetized with pentobarbital sodium and challenged intravaginally with 10 μL/ mouse HSV-2 at a concentration of 6 × 10 7 PFU/ml or mock- challenged. Vaginal lavage fluids and cervical-vaginal tissues were collected at day 7 after challenge. (A) HSV-2 infection induces the production of mouse CXCR3 ligands. The protein levels of CXCL9 and CXCL10 ligands in vaginal lavage fluids were measured by CBA, and the protein level of CXCL11 was detected by ELISA. (B) CXCL9 mediates the migration of CD4 + T cells to the vaginal foci of infected mice. CD4 + T cells in infection foci were detected using anti-CD4 Ab by IHC. The scale bar indicates 100 μm. Data shown are mean ± S.D. ( n = 5 mice/group) of three independent experiments (A) . *** p
    Figure Legend Snippet: Contribution of HSV-2 infection-induced CXCR3 ligands to CD4 + T cell infiltration into mouse vagina. Seven days prior to HSV-2 challenge, BALB/c mice were injected with progesterone in multiple sites. One day prior to HSV-2 challenge, CXCL9, CXCL10, and CXCL11 neutralizing antibodies were delivered to the vagina of mice, alone or in combination, while isotype matched control IgG was used as the control. Mice were then anesthetized with pentobarbital sodium and challenged intravaginally with 10 μL/ mouse HSV-2 at a concentration of 6 × 10 7 PFU/ml or mock- challenged. Vaginal lavage fluids and cervical-vaginal tissues were collected at day 7 after challenge. (A) HSV-2 infection induces the production of mouse CXCR3 ligands. The protein levels of CXCL9 and CXCL10 ligands in vaginal lavage fluids were measured by CBA, and the protein level of CXCL11 was detected by ELISA. (B) CXCL9 mediates the migration of CD4 + T cells to the vaginal foci of infected mice. CD4 + T cells in infection foci were detected using anti-CD4 Ab by IHC. The scale bar indicates 100 μm. Data shown are mean ± S.D. ( n = 5 mice/group) of three independent experiments (A) . *** p

    Techniques Used: Infection, Mouse Assay, Injection, Concentration Assay, Crocin Bleaching Assay, Enzyme-linked Immunosorbent Assay, Migration, Immunohistochemistry

    3) Product Images from "Epigenetic Modifications of the α-Synuclein Gene and Relative Protein Content Are Affected by Ageing and Physical Exercise in Blood from Healthy Subjects"

    Article Title: Epigenetic Modifications of the α-Synuclein Gene and Relative Protein Content Are Affected by Ageing and Physical Exercise in Blood from Healthy Subjects

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2018/3740345

    Correlation analyses between SNCA I1 relative DNA methylation levels, Dnmt1, total α -syn, and age. Correlation analysis between SNCA I1 relative DNA methylation and age (a) in the total population, (b) in the “95% CI α -syn population” (i.e., subjects characterized by RBC α -syn concentration of 76 ng/mg protein or lower), and (c) in the SED group. (d) Correlation analysis between Dnmt1 levels and age in the ATHL group. (e) Correlation analysis between total α -syn levels in RBCs and age in the ATHL group. The correlation between variables was determined by simple linear regression analysis. P and R 2 were reported in the respective panels.
    Figure Legend Snippet: Correlation analyses between SNCA I1 relative DNA methylation levels, Dnmt1, total α -syn, and age. Correlation analysis between SNCA I1 relative DNA methylation and age (a) in the total population, (b) in the “95% CI α -syn population” (i.e., subjects characterized by RBC α -syn concentration of 76 ng/mg protein or lower), and (c) in the SED group. (d) Correlation analysis between Dnmt1 levels and age in the ATHL group. (e) Correlation analysis between total α -syn levels in RBCs and age in the ATHL group. The correlation between variables was determined by simple linear regression analysis. P and R 2 were reported in the respective panels.

    Techniques Used: DNA Methylation Assay, Concentration Assay

    (a) SNCA I1 relative DNA methylation levels were evaluated by MRSE digestion of genomic DNA extracted from the blood cells of healthy subjects and followed by quantitative real-time PCR. The results were expressed as the percentage of methylation in young, older, ATHL, and SED subgroups. (b) Dnmt1 and (c) Dnmt3a levels were determined in the blood of young, older, ATHL, and SED subgroups. Differences between groups (i.e., young versus older and ATHL versus SED) were evaluated by one-way ANOVA followed by a Kruskal-Wallis post hoc test. P values were adjusted with Sidak's multiple comparison test. ∗ P
    Figure Legend Snippet: (a) SNCA I1 relative DNA methylation levels were evaluated by MRSE digestion of genomic DNA extracted from the blood cells of healthy subjects and followed by quantitative real-time PCR. The results were expressed as the percentage of methylation in young, older, ATHL, and SED subgroups. (b) Dnmt1 and (c) Dnmt3a levels were determined in the blood of young, older, ATHL, and SED subgroups. Differences between groups (i.e., young versus older and ATHL versus SED) were evaluated by one-way ANOVA followed by a Kruskal-Wallis post hoc test. P values were adjusted with Sidak's multiple comparison test. ∗ P

    Techniques Used: DNA Methylation Assay, Real-time Polymerase Chain Reaction, Methylation

    4) Product Images from "Host transcription factor Speckled 110 kDa (Sp110), a nuclear body protein, is hijacked by hepatitis B virus protein X for viral persistence"

    Article Title: Host transcription factor Speckled 110 kDa (Sp110), a nuclear body protein, is hijacked by hepatitis B virus protein X for viral persistence

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.796839

    Sp110 hyper-expression upon HBV infection. A, increased expression of the Sp110 protein in normal and HBV-infected patient serum (having a viral load > 10 5 copies/ml), quantified by ELISA, n = 15. B, immunohistochemistry staining of normal, NASH, and CHB liver tissues ( n = 10) with anti-Sp110 antibody and quantification of the positively stained area also show a similar increase. Scale bar, 100 μm. C, Southern blot with DNA extracted from HepG2 with or without 1.3-mer HBV plasmid transfection confirms that viral production and thus the effectiveness of the transfection. Relaxed circular double-stranded ( RC ), covalently closed circular DNA ( CCC ), and single-stranded ( SS ) are the various HBV DNA replicative intermediates shown. The agarose gel image shows comparable loading of total DNA. D and E, relative expression of Sp110 in HepG2 cells on 1.3-mer HBV transfection at mRNA level by qRT-PCR ( D ) and protein level by Western blotting and quantification ( E ) show a significant increase. F, Southern blot with the DNA isolated from the of HepG2.2.15 cells confirms the viral production. G and H, Sp110 mRNA ( G ) and protein expression quantified ( H ) in HepG2.2.15 cells show a similar increase. Data are represented as mean ± S.D. from at least three independent experiments. The statistical significance has been represented as follows: **, p
    Figure Legend Snippet: Sp110 hyper-expression upon HBV infection. A, increased expression of the Sp110 protein in normal and HBV-infected patient serum (having a viral load > 10 5 copies/ml), quantified by ELISA, n = 15. B, immunohistochemistry staining of normal, NASH, and CHB liver tissues ( n = 10) with anti-Sp110 antibody and quantification of the positively stained area also show a similar increase. Scale bar, 100 μm. C, Southern blot with DNA extracted from HepG2 with or without 1.3-mer HBV plasmid transfection confirms that viral production and thus the effectiveness of the transfection. Relaxed circular double-stranded ( RC ), covalently closed circular DNA ( CCC ), and single-stranded ( SS ) are the various HBV DNA replicative intermediates shown. The agarose gel image shows comparable loading of total DNA. D and E, relative expression of Sp110 in HepG2 cells on 1.3-mer HBV transfection at mRNA level by qRT-PCR ( D ) and protein level by Western blotting and quantification ( E ) show a significant increase. F, Southern blot with the DNA isolated from the of HepG2.2.15 cells confirms the viral production. G and H, Sp110 mRNA ( G ) and protein expression quantified ( H ) in HepG2.2.15 cells show a similar increase. Data are represented as mean ± S.D. from at least three independent experiments. The statistical significance has been represented as follows: **, p

    Techniques Used: Expressing, Infection, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Staining, Southern Blot, Plasmid Preparation, Transfection, Countercurrent Chromatography, Agarose Gel Electrophoresis, Quantitative RT-PCR, Western Blot, Isolation

    HBx shows cooperative association with Sp110 and drives it out of the PML-NB. A, co-immunofluorescence staining showing that Sp110 associates with HBx to exit the PML-NBs ( panel I ) shows HBx (Alexa 488) does not co-localize with PML-NBs, stained with its marker protein PML (Alexa 594). Panel II shows the usual PML-NB distribution of Sp110 (Alexa 594) altered upon 1.3-mer HBV transfection, and Sp110 co-localizes with HBx. Panel III shows Sp110 remains in its PML-NB distribution upon 1.3-mer HBV X-null transfection. B, panel I, co-immunoprecipitation with α-HA antibody in HA-HBx-transfected HepG2 cells, followed by immunoblotting with a α-Sp110 antibody, confirms the interaction between HBx and Sp110. Panel II, co-immunoprecipitation with α-FLAG antibody in HepG2 cells co-transfected with GFP-HBx and FLAG-Sp110c, followed by immunoblotting with α-GFP antibody, validated the interaction. C, schematic representation of the domain organization in Sp110. D, immunofluorescence staining of HBx-GFP and α-FLAG (Alexa 594) to show HBx co-localizes with both FLAG-Sp110c-full ( panel I ) and FLAG-Sp110-SPB ( panel II ) (Pearson's coefficient 0.60 ± 0.054 and 0.71 ± 0.082, respectively) and partially co-localizes with FLAG-Sp110-N-term ( panel III ) (Pearson's coefficient 0.38 ± 0.081). Scale bar, 10 μm. E, overexpression of FLAG-Sp110 and FLAG-Sp110-SPB or in HepG2.2.15 cells results in a significant increase in the HBV viral DNA load. FLAG-Sp110-N-term overexpression also increases the viral load but to a comparatively lesser extent. The experiments were independently repeated at least three times and for co-localization studies, n = 20. All the data are represented as a mean ± S.D. The statistical significance has been represented as follows: **, p
    Figure Legend Snippet: HBx shows cooperative association with Sp110 and drives it out of the PML-NB. A, co-immunofluorescence staining showing that Sp110 associates with HBx to exit the PML-NBs ( panel I ) shows HBx (Alexa 488) does not co-localize with PML-NBs, stained with its marker protein PML (Alexa 594). Panel II shows the usual PML-NB distribution of Sp110 (Alexa 594) altered upon 1.3-mer HBV transfection, and Sp110 co-localizes with HBx. Panel III shows Sp110 remains in its PML-NB distribution upon 1.3-mer HBV X-null transfection. B, panel I, co-immunoprecipitation with α-HA antibody in HA-HBx-transfected HepG2 cells, followed by immunoblotting with a α-Sp110 antibody, confirms the interaction between HBx and Sp110. Panel II, co-immunoprecipitation with α-FLAG antibody in HepG2 cells co-transfected with GFP-HBx and FLAG-Sp110c, followed by immunoblotting with α-GFP antibody, validated the interaction. C, schematic representation of the domain organization in Sp110. D, immunofluorescence staining of HBx-GFP and α-FLAG (Alexa 594) to show HBx co-localizes with both FLAG-Sp110c-full ( panel I ) and FLAG-Sp110-SPB ( panel II ) (Pearson's coefficient 0.60 ± 0.054 and 0.71 ± 0.082, respectively) and partially co-localizes with FLAG-Sp110-N-term ( panel III ) (Pearson's coefficient 0.38 ± 0.081). Scale bar, 10 μm. E, overexpression of FLAG-Sp110 and FLAG-Sp110-SPB or in HepG2.2.15 cells results in a significant increase in the HBV viral DNA load. FLAG-Sp110-N-term overexpression also increases the viral load but to a comparatively lesser extent. The experiments were independently repeated at least three times and for co-localization studies, n = 20. All the data are represented as a mean ± S.D. The statistical significance has been represented as follows: **, p

    Techniques Used: Immunofluorescence, Staining, Marker, Transfection, Immunoprecipitation, Over Expression

    5) Product Images from "miR-147b-mediated TCA cycle dysfunction and pseudohypoxia initiate drug tolerance to EGFR inhibitors in lung adenocarcinoma"

    Article Title: miR-147b-mediated TCA cycle dysfunction and pseudohypoxia initiate drug tolerance to EGFR inhibitors in lung adenocarcinoma

    Journal: Nature metabolism

    doi: 10.1038/s42255-019-0052-9

    miR-147b initiates drug-tolerance. a , A heat map showing top upregulated and downregulated miRNAs in two paired osimertinib-tolerant (OTR) and parental cells in PC9 and HCC827 by miRNA-seq analysis. b , qRT-PCR analysis of miR-147b expressions in parental, recovered, primary and secondary osimertinib-tolerant cells in PC9. The parental tumor cells treated with 160 nM EGFR-TKI osimertinib for 6 days enter a drug-tolerant state (primary tolerant cells) with a brief therapy withdrawal up to 18 days (recovered cells) followed by reinstatement of the 160 nM dose for 11 days (secondary tolerant cells). The relative miR-147b expression level in the parental cells were calibrated as 1. miR-423 was used as endogenous control. n=3 independent biological replicates. c -d, Osimertinib (c) and gefitinib (d) treatment response on scrambled control (Scr) and miR-147b-overexpressing cells (147b) in HCC827 for 3 days. n=3 independent biological replicates. e , Osimertinib (40 nM) and gefitinib (40 nM) treatment response on scrambled control and miR-147b-overexpressing cells in HCC827 by colony formation assay. 20, 40 and 80 cells were plated in 10-cm dish and the colonies were stained with Giemsa on day 10 and the total number of colonies were quantified. n=3 independent biological replicates. f , Osimertinib treatment response on H1975 cells with miR-147b knockdown (anti147b) and scrambled control (antictrl). The cell viability was measured on day 4. n=3 independent biological replicates. g , Osimertinib (160 nM) treatment response on H1975 cells with miR-147b knockdown. Left, the monolayer colonies were treated for 10 days and stained with Giemsa. Right, the 3D structures were treated for 14 days. -, vehicle; +, osimertinib. Scale bar, 1000 μm. n=3 independent biological replicates. Data are mean ± s.e.m. and were analysed with one-way ANOVA ( b ); unpaired two-tailed t -test with Holm-Sidak’s correction ( e,g ).
    Figure Legend Snippet: miR-147b initiates drug-tolerance. a , A heat map showing top upregulated and downregulated miRNAs in two paired osimertinib-tolerant (OTR) and parental cells in PC9 and HCC827 by miRNA-seq analysis. b , qRT-PCR analysis of miR-147b expressions in parental, recovered, primary and secondary osimertinib-tolerant cells in PC9. The parental tumor cells treated with 160 nM EGFR-TKI osimertinib for 6 days enter a drug-tolerant state (primary tolerant cells) with a brief therapy withdrawal up to 18 days (recovered cells) followed by reinstatement of the 160 nM dose for 11 days (secondary tolerant cells). The relative miR-147b expression level in the parental cells were calibrated as 1. miR-423 was used as endogenous control. n=3 independent biological replicates. c -d, Osimertinib (c) and gefitinib (d) treatment response on scrambled control (Scr) and miR-147b-overexpressing cells (147b) in HCC827 for 3 days. n=3 independent biological replicates. e , Osimertinib (40 nM) and gefitinib (40 nM) treatment response on scrambled control and miR-147b-overexpressing cells in HCC827 by colony formation assay. 20, 40 and 80 cells were plated in 10-cm dish and the colonies were stained with Giemsa on day 10 and the total number of colonies were quantified. n=3 independent biological replicates. f , Osimertinib treatment response on H1975 cells with miR-147b knockdown (anti147b) and scrambled control (antictrl). The cell viability was measured on day 4. n=3 independent biological replicates. g , Osimertinib (160 nM) treatment response on H1975 cells with miR-147b knockdown. Left, the monolayer colonies were treated for 10 days and stained with Giemsa. Right, the 3D structures were treated for 14 days. -, vehicle; +, osimertinib. Scale bar, 1000 μm. n=3 independent biological replicates. Data are mean ± s.e.m. and were analysed with one-way ANOVA ( b ); unpaired two-tailed t -test with Holm-Sidak’s correction ( e,g ).

    Techniques Used: Quantitative RT-PCR, Expressing, Colony Assay, Staining, Two Tailed Test

    NSCLC cells adopt a tolerance strategy against EGFR-TKIs. a, Representative phase contrast images of 3D structures from AALE cells cultured according to the protocol at top of the panel. Scale bar, 50 μm. Repeated six times with similar results. b , Top, the scenario of anti-EGFR tolerance and resistance in lung cancer. The tumor cells treated with the EGFR-TKI gefitinib or osimertinib enter a reversible drug-tolerant cycle (in green arrows, 1° Tolerant) with a brief therapy withdrawal (up to 21 days) followed by reinstatement of the 160 nM dose (2° Tolerant). Alternatively, the tumor cells treated continuously with gefitinib or osimertinib without therapy interruption undergo drug-tolerance briefly and go into a drug-resistance state in which cells do not respond to gefitinib (1° Resistant)/osimertinib (2° Resistant). Bottom, osimertinib treatment response on HCC827 3D structures. Representative images of Parental cells, 1° Tolerant cells (derived from the Parental cells treated with 160 nM osimertinib for 11 days), Recovered cells (derived from the 1° Tolerant cells with a therapy withdrawal up to 21 days) and 2° Tolerant cells (derived from the Recovered cells by reinstatement of the 160 nM dose for 11 days). Scale bar, 200 μm. Repeated six times with similar results. c, Representative phase contrast microscopy (left panel) and H E staining of HCC827 3D structures derived from parental (top) and osimertinib-tolerant (bottom) cells. Images in blue dotted squares (middle panel) were amplified (right panel) and shown. Scale bar, 50 μm. Repeated six times with similar results. d, qRT-PCR analysis of SFTPC , HOPX , ID2 and CEACAM5 expression in single cell clone HCC827-derived 3D structures in the presence of osimertinib. Single cell clone derived cells were plated with geltrex and treated with 100 nM osimertinib (tolerant) or vehicle (parental) for 24 days. Gene expression for surviving 3D structures were analyzed. n=3 independent biological replicates. e , Single-cell clonogenicity of PC9 cells treated with gefitinib. A single cell was sorted by FACS into a 96-well plate and treated with 0.1, 0.4, and 2 μM gefitinib or the vehicle for 14 days. The frequency of colony formation was calculated as a ratio of the total number of colonies to the total number of wells plated with a single cell in a 96-well plate. n=3 independent biological replicates. f, qRT-PCR analysis of top upregulated and downregulated genes in gefitinib-tolerant clones (n=2) compared with vehicle-treated parental single cell clone (n=1) in PC9. The gene expression in parental sensitive clone was calibrated as 1. ACTB was used as endogenous control. n=4 independent biological replicates. g, Whole transcriptome and gene ontology analysis of gefitinib-tolerant clones compared with the parental single cell clone in PC9. n=4 independent biological replicates. h, qRT-PCR analysis of genes in top regulated signaling pathways including Wnt planar cell polarity signaling, glutamine metabolic process, cellular response to hypoxia, and tricarboxylic acid cycle in gefitinib-tolerant clones compared with parental the single cell clone in PC9. The gene expression in parental sensitive clone was calibrated as 1. ACTB was used as endogenous control. n=4 independent biological replicates. Data are mean ± s.e.m. and were analysed with unpaired two-tailed t -test with Welch’s correction ( d ); modified one-tailed Fisher’s Exact test ( g).
    Figure Legend Snippet: NSCLC cells adopt a tolerance strategy against EGFR-TKIs. a, Representative phase contrast images of 3D structures from AALE cells cultured according to the protocol at top of the panel. Scale bar, 50 μm. Repeated six times with similar results. b , Top, the scenario of anti-EGFR tolerance and resistance in lung cancer. The tumor cells treated with the EGFR-TKI gefitinib or osimertinib enter a reversible drug-tolerant cycle (in green arrows, 1° Tolerant) with a brief therapy withdrawal (up to 21 days) followed by reinstatement of the 160 nM dose (2° Tolerant). Alternatively, the tumor cells treated continuously with gefitinib or osimertinib without therapy interruption undergo drug-tolerance briefly and go into a drug-resistance state in which cells do not respond to gefitinib (1° Resistant)/osimertinib (2° Resistant). Bottom, osimertinib treatment response on HCC827 3D structures. Representative images of Parental cells, 1° Tolerant cells (derived from the Parental cells treated with 160 nM osimertinib for 11 days), Recovered cells (derived from the 1° Tolerant cells with a therapy withdrawal up to 21 days) and 2° Tolerant cells (derived from the Recovered cells by reinstatement of the 160 nM dose for 11 days). Scale bar, 200 μm. Repeated six times with similar results. c, Representative phase contrast microscopy (left panel) and H E staining of HCC827 3D structures derived from parental (top) and osimertinib-tolerant (bottom) cells. Images in blue dotted squares (middle panel) were amplified (right panel) and shown. Scale bar, 50 μm. Repeated six times with similar results. d, qRT-PCR analysis of SFTPC , HOPX , ID2 and CEACAM5 expression in single cell clone HCC827-derived 3D structures in the presence of osimertinib. Single cell clone derived cells were plated with geltrex and treated with 100 nM osimertinib (tolerant) or vehicle (parental) for 24 days. Gene expression for surviving 3D structures were analyzed. n=3 independent biological replicates. e , Single-cell clonogenicity of PC9 cells treated with gefitinib. A single cell was sorted by FACS into a 96-well plate and treated with 0.1, 0.4, and 2 μM gefitinib or the vehicle for 14 days. The frequency of colony formation was calculated as a ratio of the total number of colonies to the total number of wells plated with a single cell in a 96-well plate. n=3 independent biological replicates. f, qRT-PCR analysis of top upregulated and downregulated genes in gefitinib-tolerant clones (n=2) compared with vehicle-treated parental single cell clone (n=1) in PC9. The gene expression in parental sensitive clone was calibrated as 1. ACTB was used as endogenous control. n=4 independent biological replicates. g, Whole transcriptome and gene ontology analysis of gefitinib-tolerant clones compared with the parental single cell clone in PC9. n=4 independent biological replicates. h, qRT-PCR analysis of genes in top regulated signaling pathways including Wnt planar cell polarity signaling, glutamine metabolic process, cellular response to hypoxia, and tricarboxylic acid cycle in gefitinib-tolerant clones compared with parental the single cell clone in PC9. The gene expression in parental sensitive clone was calibrated as 1. ACTB was used as endogenous control. n=4 independent biological replicates. Data are mean ± s.e.m. and were analysed with unpaired two-tailed t -test with Welch’s correction ( d ); modified one-tailed Fisher’s Exact test ( g).

    Techniques Used: Cell Culture, Derivative Assay, Microscopy, Staining, Amplification, Quantitative RT-PCR, Expressing, FACS, Clone Assay, Two Tailed Test, Modification, One-tailed Test

    6) Product Images from "miR-147b-mediated TCA cycle dysfunction and pseudohypoxia initiate drug tolerance to EGFR inhibitors in lung adenocarcinoma"

    Article Title: miR-147b-mediated TCA cycle dysfunction and pseudohypoxia initiate drug tolerance to EGFR inhibitors in lung adenocarcinoma

    Journal: Nature metabolism

    doi: 10.1038/s42255-019-0052-9

    miR-147b initiates drug-tolerance. a , A heat map showing top upregulated and downregulated miRNAs in two paired osimertinib-tolerant (OTR) and parental cells in PC9 and HCC827 by miRNA-seq analysis. b , qRT-PCR analysis of miR-147b expressions in parental, recovered, primary and secondary osimertinib-tolerant cells in PC9. The parental tumor cells treated with 160 nM EGFR-TKI osimertinib for 6 days enter a drug-tolerant state (primary tolerant cells) with a brief therapy withdrawal up to 18 days (recovered cells) followed by reinstatement of the 160 nM dose for 11 days (secondary tolerant cells). The relative miR-147b expression level in the parental cells were calibrated as 1. miR-423 was used as endogenous control. n=3 independent biological replicates. c -d, Osimertinib (c) and gefitinib (d) treatment response on scrambled control (Scr) and miR-147b-overexpressing cells (147b) in HCC827 for 3 days. n=3 independent biological replicates. e , Osimertinib (40 nM) and gefitinib (40 nM) treatment response on scrambled control and miR-147b-overexpressing cells in HCC827 by colony formation assay. 20, 40 and 80 cells were plated in 10-cm dish and the colonies were stained with Giemsa on day 10 and the total number of colonies were quantified. n=3 independent biological replicates. f , Osimertinib treatment response on H1975 cells with miR-147b knockdown (anti147b) and scrambled control (antictrl). The cell viability was measured on day 4. n=3 independent biological replicates. g , Osimertinib (160 nM) treatment response on H1975 cells with miR-147b knockdown. Left, the monolayer colonies were treated for 10 days and stained with Giemsa. Right, the 3D structures were treated for 14 days. -, vehicle; +, osimertinib. Scale bar, 1000 μm. n=3 independent biological replicates. Data are mean ± s.e.m. and were analysed with one-way ANOVA ( b ); unpaired two-tailed t -test with Holm-Sidak’s correction ( e,g ).
    Figure Legend Snippet: miR-147b initiates drug-tolerance. a , A heat map showing top upregulated and downregulated miRNAs in two paired osimertinib-tolerant (OTR) and parental cells in PC9 and HCC827 by miRNA-seq analysis. b , qRT-PCR analysis of miR-147b expressions in parental, recovered, primary and secondary osimertinib-tolerant cells in PC9. The parental tumor cells treated with 160 nM EGFR-TKI osimertinib for 6 days enter a drug-tolerant state (primary tolerant cells) with a brief therapy withdrawal up to 18 days (recovered cells) followed by reinstatement of the 160 nM dose for 11 days (secondary tolerant cells). The relative miR-147b expression level in the parental cells were calibrated as 1. miR-423 was used as endogenous control. n=3 independent biological replicates. c -d, Osimertinib (c) and gefitinib (d) treatment response on scrambled control (Scr) and miR-147b-overexpressing cells (147b) in HCC827 for 3 days. n=3 independent biological replicates. e , Osimertinib (40 nM) and gefitinib (40 nM) treatment response on scrambled control and miR-147b-overexpressing cells in HCC827 by colony formation assay. 20, 40 and 80 cells were plated in 10-cm dish and the colonies were stained with Giemsa on day 10 and the total number of colonies were quantified. n=3 independent biological replicates. f , Osimertinib treatment response on H1975 cells with miR-147b knockdown (anti147b) and scrambled control (antictrl). The cell viability was measured on day 4. n=3 independent biological replicates. g , Osimertinib (160 nM) treatment response on H1975 cells with miR-147b knockdown. Left, the monolayer colonies were treated for 10 days and stained with Giemsa. Right, the 3D structures were treated for 14 days. -, vehicle; +, osimertinib. Scale bar, 1000 μm. n=3 independent biological replicates. Data are mean ± s.e.m. and were analysed with one-way ANOVA ( b ); unpaired two-tailed t -test with Holm-Sidak’s correction ( e,g ).

    Techniques Used: Quantitative RT-PCR, Expressing, Colony Assay, Staining, Two Tailed Test

    NSCLC cells adopt a tolerance strategy against EGFR-TKIs. a, Representative phase contrast images of 3D structures from AALE cells cultured according to the protocol at top of the panel. Scale bar, 50 μm. Repeated six times with similar results. b , Top, the scenario of anti-EGFR tolerance and resistance in lung cancer. The tumor cells treated with the EGFR-TKI gefitinib or osimertinib enter a reversible drug-tolerant cycle (in green arrows, 1° Tolerant) with a brief therapy withdrawal (up to 21 days) followed by reinstatement of the 160 nM dose (2° Tolerant). Alternatively, the tumor cells treated continuously with gefitinib or osimertinib without therapy interruption undergo drug-tolerance briefly and go into a drug-resistance state in which cells do not respond to gefitinib (1° Resistant)/osimertinib (2° Resistant). Bottom, osimertinib treatment response on HCC827 3D structures. Representative images of Parental cells, 1° Tolerant cells (derived from the Parental cells treated with 160 nM osimertinib for 11 days), Recovered cells (derived from the 1° Tolerant cells with a therapy withdrawal up to 21 days) and 2° Tolerant cells (derived from the Recovered cells by reinstatement of the 160 nM dose for 11 days). Scale bar, 200 μm. Repeated six times with similar results. c, Representative phase contrast microscopy (left panel) and H E staining of HCC827 3D structures derived from parental (top) and osimertinib-tolerant (bottom) cells. Images in blue dotted squares (middle panel) were amplified (right panel) and shown. Scale bar, 50 μm. Repeated six times with similar results. d, qRT-PCR analysis of SFTPC , HOPX , ID2 and CEACAM5 expression in single cell clone HCC827-derived 3D structures in the presence of osimertinib. Single cell clone derived cells were plated with geltrex and treated with 100 nM osimertinib (tolerant) or vehicle (parental) for 24 days. Gene expression for surviving 3D structures were analyzed. n=3 independent biological replicates. e , Single-cell clonogenicity of PC9 cells treated with gefitinib. A single cell was sorted by FACS into a 96-well plate and treated with 0.1, 0.4, and 2 μM gefitinib or the vehicle for 14 days. The frequency of colony formation was calculated as a ratio of the total number of colonies to the total number of wells plated with a single cell in a 96-well plate. n=3 independent biological replicates. f, qRT-PCR analysis of top upregulated and downregulated genes in gefitinib-tolerant clones (n=2) compared with vehicle-treated parental single cell clone (n=1) in PC9. The gene expression in parental sensitive clone was calibrated as 1. ACTB was used as endogenous control. n=4 independent biological replicates. g, Whole transcriptome and gene ontology analysis of gefitinib-tolerant clones compared with the parental single cell clone in PC9. n=4 independent biological replicates. h, qRT-PCR analysis of genes in top regulated signaling pathways including Wnt planar cell polarity signaling, glutamine metabolic process, cellular response to hypoxia, and tricarboxylic acid cycle in gefitinib-tolerant clones compared with parental the single cell clone in PC9. The gene expression in parental sensitive clone was calibrated as 1. ACTB was used as endogenous control. n=4 independent biological replicates. Data are mean ± s.e.m. and were analysed with unpaired two-tailed t -test with Welch’s correction ( d ); modified one-tailed Fisher’s Exact test ( g).
    Figure Legend Snippet: NSCLC cells adopt a tolerance strategy against EGFR-TKIs. a, Representative phase contrast images of 3D structures from AALE cells cultured according to the protocol at top of the panel. Scale bar, 50 μm. Repeated six times with similar results. b , Top, the scenario of anti-EGFR tolerance and resistance in lung cancer. The tumor cells treated with the EGFR-TKI gefitinib or osimertinib enter a reversible drug-tolerant cycle (in green arrows, 1° Tolerant) with a brief therapy withdrawal (up to 21 days) followed by reinstatement of the 160 nM dose (2° Tolerant). Alternatively, the tumor cells treated continuously with gefitinib or osimertinib without therapy interruption undergo drug-tolerance briefly and go into a drug-resistance state in which cells do not respond to gefitinib (1° Resistant)/osimertinib (2° Resistant). Bottom, osimertinib treatment response on HCC827 3D structures. Representative images of Parental cells, 1° Tolerant cells (derived from the Parental cells treated with 160 nM osimertinib for 11 days), Recovered cells (derived from the 1° Tolerant cells with a therapy withdrawal up to 21 days) and 2° Tolerant cells (derived from the Recovered cells by reinstatement of the 160 nM dose for 11 days). Scale bar, 200 μm. Repeated six times with similar results. c, Representative phase contrast microscopy (left panel) and H E staining of HCC827 3D structures derived from parental (top) and osimertinib-tolerant (bottom) cells. Images in blue dotted squares (middle panel) were amplified (right panel) and shown. Scale bar, 50 μm. Repeated six times with similar results. d, qRT-PCR analysis of SFTPC , HOPX , ID2 and CEACAM5 expression in single cell clone HCC827-derived 3D structures in the presence of osimertinib. Single cell clone derived cells were plated with geltrex and treated with 100 nM osimertinib (tolerant) or vehicle (parental) for 24 days. Gene expression for surviving 3D structures were analyzed. n=3 independent biological replicates. e , Single-cell clonogenicity of PC9 cells treated with gefitinib. A single cell was sorted by FACS into a 96-well plate and treated with 0.1, 0.4, and 2 μM gefitinib or the vehicle for 14 days. The frequency of colony formation was calculated as a ratio of the total number of colonies to the total number of wells plated with a single cell in a 96-well plate. n=3 independent biological replicates. f, qRT-PCR analysis of top upregulated and downregulated genes in gefitinib-tolerant clones (n=2) compared with vehicle-treated parental single cell clone (n=1) in PC9. The gene expression in parental sensitive clone was calibrated as 1. ACTB was used as endogenous control. n=4 independent biological replicates. g, Whole transcriptome and gene ontology analysis of gefitinib-tolerant clones compared with the parental single cell clone in PC9. n=4 independent biological replicates. h, qRT-PCR analysis of genes in top regulated signaling pathways including Wnt planar cell polarity signaling, glutamine metabolic process, cellular response to hypoxia, and tricarboxylic acid cycle in gefitinib-tolerant clones compared with parental the single cell clone in PC9. The gene expression in parental sensitive clone was calibrated as 1. ACTB was used as endogenous control. n=4 independent biological replicates. Data are mean ± s.e.m. and were analysed with unpaired two-tailed t -test with Welch’s correction ( d ); modified one-tailed Fisher’s Exact test ( g).

    Techniques Used: Cell Culture, Derivative Assay, Microscopy, Staining, Amplification, Quantitative RT-PCR, Expressing, FACS, Clone Assay, Two Tailed Test, Modification, One-tailed Test

    miR-147b-VHL axis mediates drug-tolerance through impaired VHL activity. a , Left, gene candidates predicted for miR-147b by the TargetScan tool were shown in signaling pathways enriched for gefitinib-tolerance in PC9 single-cell clones in fig. 1f . Right, qRT-PCR analysis for the predicted gene candidates for miR-147b in H1975 cells with miR-147b knockdown compared with scrambled control. n=3 independent biological replicates. b , Left, computational prediction of RNA duplex formation between miR-147b and the 3’UTR (untranslated region) of VHL mRNA. Mutations generated within the 3’UTR for the luciferase assay are shown in red. Right, dual-luciferase reporter assay in miR-147b-overexpressing AALE cells. The Firefly luciferase and Renilla luciferase activities were measured 48 hours post co-transfection with miR-147b or control vector and wild-type (WT) or mutant (Mut) VHL 3’UTR. n=3 independent biological replicates. c , Western blot analysis and quantification of VHL in miR-147b-overexpressing AALE cells. β-Actin was used as loading control. n=3 independent biological replicates. d , qRT-PCR analysis for fold change of hypoxia gene expression in AALE cells with miR-147b overexpression relative to scrambled control (147b/Scr) and cells with co-overexpression of miR-147b and VHL relative to scrambled control (147b+VHL/Scr). ACTB was used as endogenous control. n=3 independent biological replicates. e , Fractional viability of HCC827 cells treated with vehicle, osimertinib (20 nM), miR-147b vector, VHL vector or combinations. The cell viability was measured on day 3. The relative cell viability treated with vehicle on day 3 was calibrated as 1. n=7 independent biological replicates. Data are mean ± s.e.m. and were analysed with unpaired two-tailed t -test ( a , b , c , d ); Kruskal-Wallis test (e).
    Figure Legend Snippet: miR-147b-VHL axis mediates drug-tolerance through impaired VHL activity. a , Left, gene candidates predicted for miR-147b by the TargetScan tool were shown in signaling pathways enriched for gefitinib-tolerance in PC9 single-cell clones in fig. 1f . Right, qRT-PCR analysis for the predicted gene candidates for miR-147b in H1975 cells with miR-147b knockdown compared with scrambled control. n=3 independent biological replicates. b , Left, computational prediction of RNA duplex formation between miR-147b and the 3’UTR (untranslated region) of VHL mRNA. Mutations generated within the 3’UTR for the luciferase assay are shown in red. Right, dual-luciferase reporter assay in miR-147b-overexpressing AALE cells. The Firefly luciferase and Renilla luciferase activities were measured 48 hours post co-transfection with miR-147b or control vector and wild-type (WT) or mutant (Mut) VHL 3’UTR. n=3 independent biological replicates. c , Western blot analysis and quantification of VHL in miR-147b-overexpressing AALE cells. β-Actin was used as loading control. n=3 independent biological replicates. d , qRT-PCR analysis for fold change of hypoxia gene expression in AALE cells with miR-147b overexpression relative to scrambled control (147b/Scr) and cells with co-overexpression of miR-147b and VHL relative to scrambled control (147b+VHL/Scr). ACTB was used as endogenous control. n=3 independent biological replicates. e , Fractional viability of HCC827 cells treated with vehicle, osimertinib (20 nM), miR-147b vector, VHL vector or combinations. The cell viability was measured on day 3. The relative cell viability treated with vehicle on day 3 was calibrated as 1. n=7 independent biological replicates. Data are mean ± s.e.m. and were analysed with unpaired two-tailed t -test ( a , b , c , d ); Kruskal-Wallis test (e).

    Techniques Used: Activity Assay, Clone Assay, Quantitative RT-PCR, Generated, Luciferase, Reporter Assay, Cotransfection, Plasmid Preparation, Mutagenesis, Western Blot, Expressing, Over Expression, Two Tailed Test

    7) Product Images from "Development of a Membrane-Based Method for Isolation of Genomic DNA from Human Blood"

    Article Title: Development of a Membrane-Based Method for Isolation of Genomic DNA from Human Blood

    Journal: Journal of Biomolecular Techniques : JBT

    doi: 10.7171/jbt.18-2902-001

    PCR of different HLA targets using gDNA prepared using the PES membrane or QIAamp kit. Buffy coat from the same blood sample was equally distributed and processed for gDNA extraction using either 0.22 µm PES membrane from EMD Millipore ( A ) or QIAamp DNA Blood Mini Kit from Qiagen ( B ). Different targets of HLA loci were PCR amplified, as described in Validation of the gDNA isolation method using the PES membrane filter . PCR amplicons were analyzed on 2.5% agarose gel electrophoresis. Representative of a minimum of 4 independent experiments. Lanes 1 and 2: HLA-A Exons 2 and 3; lanes 3 and 4: HLA-B Exons 2 and 3; lanes 5 and 6: HLA-C Exons 2 and 3; lanes 7 and 10: HLA-DPB1 Exons 3 and 2, respectively; lanes 8 and 11: HLA-DQB1 Exons 3 and 2, respectively; lanes 9 and 12: HLA-DRB1 Exons 3 and 2, respectively. M, DNA MW marker (250 bp–10 kb).
    Figure Legend Snippet: PCR of different HLA targets using gDNA prepared using the PES membrane or QIAamp kit. Buffy coat from the same blood sample was equally distributed and processed for gDNA extraction using either 0.22 µm PES membrane from EMD Millipore ( A ) or QIAamp DNA Blood Mini Kit from Qiagen ( B ). Different targets of HLA loci were PCR amplified, as described in Validation of the gDNA isolation method using the PES membrane filter . PCR amplicons were analyzed on 2.5% agarose gel electrophoresis. Representative of a minimum of 4 independent experiments. Lanes 1 and 2: HLA-A Exons 2 and 3; lanes 3 and 4: HLA-B Exons 2 and 3; lanes 5 and 6: HLA-C Exons 2 and 3; lanes 7 and 10: HLA-DPB1 Exons 3 and 2, respectively; lanes 8 and 11: HLA-DQB1 Exons 3 and 2, respectively; lanes 9 and 12: HLA-DRB1 Exons 3 and 2, respectively. M, DNA MW marker (250 bp–10 kb).

    Techniques Used: Polymerase Chain Reaction, Amplification, Isolation, Agarose Gel Electrophoresis, Marker

    Agarose gel electrophoresis of gDNA samples prepared using different PES membranes or the QIAamp commercial kit. A ) Buffy coat from the same blood sample was equally distributed and processed for gDNA extraction using either 0.22 µm PES membrane from EMD Millipore (lane 1) or QIAamp DNA Blood Mini Kit from Qiagen (lane 2). B ) Buffy coat from different samples was lysed independently, pooled together, and equally distributed and processed for gDNA extraction using 0.22 µm PES membrane from different suppliers (lane 1: EMD Millipore; lane 2: Pall; and lane 3: Sterlitech). C ) Buffy coat from the same sample was equally distributed and processed for gDNA extraction using either 0.22 µm (lane 1) or 0.45 µm (lane 2) PES membrane from EMD Millipore. M, DNA MW marker (75 bp–20 kb). Representative of a minimum of 4 independent experiments.
    Figure Legend Snippet: Agarose gel electrophoresis of gDNA samples prepared using different PES membranes or the QIAamp commercial kit. A ) Buffy coat from the same blood sample was equally distributed and processed for gDNA extraction using either 0.22 µm PES membrane from EMD Millipore (lane 1) or QIAamp DNA Blood Mini Kit from Qiagen (lane 2). B ) Buffy coat from different samples was lysed independently, pooled together, and equally distributed and processed for gDNA extraction using 0.22 µm PES membrane from different suppliers (lane 1: EMD Millipore; lane 2: Pall; and lane 3: Sterlitech). C ) Buffy coat from the same sample was equally distributed and processed for gDNA extraction using either 0.22 µm (lane 1) or 0.45 µm (lane 2) PES membrane from EMD Millipore. M, DNA MW marker (75 bp–20 kb). Representative of a minimum of 4 independent experiments.

    Techniques Used: Agarose Gel Electrophoresis, Marker

    Agarose gel electrophoresis of gDNA samples digested with either Hin dIII or Eco RI. DNAs digested with Hin dIII ( A – C ); Eco R1 digested gDNAs ( D – F ). A , D ) Buffy coat from the same sample was equally distributed and processed for gDNA extraction using either 0.22 µm PES membrane from EMD Millipore (lane 1) or QIAamp DNA Blood Mini Kit from Qiagen (lane 2). B , E ) Buffy coat from 2 different samples was lysed independently, pooled together, and equally distributed and processed for gDNA extraction using the 0.22 µm PES membrane from different suppliers (lane 1: EMD Millipore; lane 2: Pall; lane 3: Sterlitech). C , F ) Buffy coat from the same sample was equally distributed and processed for gDNA extraction using either the 0.22 µm (lane 1) or 0.45 µm (lane 2) PES membrane from EMD Millipore. M, DNA MW marker (75 bp–20 kb).
    Figure Legend Snippet: Agarose gel electrophoresis of gDNA samples digested with either Hin dIII or Eco RI. DNAs digested with Hin dIII ( A – C ); Eco R1 digested gDNAs ( D – F ). A , D ) Buffy coat from the same sample was equally distributed and processed for gDNA extraction using either 0.22 µm PES membrane from EMD Millipore (lane 1) or QIAamp DNA Blood Mini Kit from Qiagen (lane 2). B , E ) Buffy coat from 2 different samples was lysed independently, pooled together, and equally distributed and processed for gDNA extraction using the 0.22 µm PES membrane from different suppliers (lane 1: EMD Millipore; lane 2: Pall; lane 3: Sterlitech). C , F ) Buffy coat from the same sample was equally distributed and processed for gDNA extraction using either the 0.22 µm (lane 1) or 0.45 µm (lane 2) PES membrane from EMD Millipore. M, DNA MW marker (75 bp–20 kb).

    Techniques Used: Agarose Gel Electrophoresis, Marker

    8) Product Images from "Characterization of human bone morphogenetic protein gene variants for possible roles in congenital heart disease"

    Article Title: Characterization of human bone morphogenetic protein gene variants for possible roles in congenital heart disease

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2016.5428

    Schematic diagrams of rs1049007, rs235768 and rs17563 locations within the translated regions of the BMP 2 and 4 genes. BMP, bone mor phogenetic protein.
    Figure Legend Snippet: Schematic diagrams of rs1049007, rs235768 and rs17563 locations within the translated regions of the BMP 2 and 4 genes. BMP, bone mor phogenetic protein.

    Techniques Used:

    DNA sequence chromatograms of the rs1049007, rs235768 and rs17563 single nucleotide polymorphisms. (A) rs1049007; (B) rs235768; (C) rs17563. Arrows indicate the sites of variation.
    Figure Legend Snippet: DNA sequence chromatograms of the rs1049007, rs235768 and rs17563 single nucleotide polymorphisms. (A) rs1049007; (B) rs235768; (C) rs17563. Arrows indicate the sites of variation.

    Techniques Used: Sequencing

    9) Product Images from "Rearranged JC Virus Noncoding Control Regions Found in Progressive Multifocal Leukoencephalopathy Patient Samples Increase Virus Early Gene Expression and Replication Rate ▿Rearranged JC Virus Noncoding Control Regions Found in Progressive Multifocal Leukoencephalopathy Patient Samples Increase Virus Early Gene Expression and Replication Rate ▿ †"

    Article Title: Rearranged JC Virus Noncoding Control Regions Found in Progressive Multifocal Leukoencephalopathy Patient Samples Increase Virus Early Gene Expression and Replication Rate ▿Rearranged JC Virus Noncoding Control Regions Found in Progressive Multifocal Leukoencephalopathy Patient Samples Increase Virus Early Gene Expression and Replication Rate ▿ †

    Journal: Journal of Virology

    doi: 10.1128/JVI.00614-10

    Replication of rr -NCCR JCV in cell culture. (A) Time course for prototypes CSF (patient 8), CSF-1 (patient 1), Mad-4, and archetype urine (patient 1) in Hs683 cells. DNase-protected JCV DNA was quantified by qPCR from cell supernatants collected at the
    Figure Legend Snippet: Replication of rr -NCCR JCV in cell culture. (A) Time course for prototypes CSF (patient 8), CSF-1 (patient 1), Mad-4, and archetype urine (patient 1) in Hs683 cells. DNase-protected JCV DNA was quantified by qPCR from cell supernatants collected at the

    Techniques Used: Cell Culture, Real-time Polymerase Chain Reaction

    Replication of recombinant rr -NCCR JCV in COS-7 cells. (A) Time course for prototypes CSF (patient 8), CSF-1 (patient 1), Mad-4, and urine (patient 1). DNase-protected JCV DNA was quantified by qPCR from cell supernatants collected at the indicated times
    Figure Legend Snippet: Replication of recombinant rr -NCCR JCV in COS-7 cells. (A) Time course for prototypes CSF (patient 8), CSF-1 (patient 1), Mad-4, and urine (patient 1). DNase-protected JCV DNA was quantified by qPCR from cell supernatants collected at the indicated times

    Techniques Used: Recombinant, Real-time Polymerase Chain Reaction

    10) Product Images from "Adaptation of red blood cell lysis represents a fundamental breakthrough that improves the sensitivity of Salmonella detection in blood"

    Article Title: Adaptation of red blood cell lysis represents a fundamental breakthrough that improves the sensitivity of Salmonella detection in blood

    Journal: Journal of Applied Microbiology

    doi: 10.1111/jam.12769

    Real-time PCR detection of Salmonella DNA. Two millilitres of whole blood was lysed using erythrocyte lysis buffer and spiked with the shown CFU of Salmonella Typhi CVD 909. DNA was extracted using a QIAamp Mini blood kit and S. Typhi was detected by qPCR.
    Figure Legend Snippet: Real-time PCR detection of Salmonella DNA. Two millilitres of whole blood was lysed using erythrocyte lysis buffer and spiked with the shown CFU of Salmonella Typhi CVD 909. DNA was extracted using a QIAamp Mini blood kit and S. Typhi was detected by qPCR.

    Techniques Used: Real-time Polymerase Chain Reaction, Lysis

    11) Product Images from "Herpes Simplex Virus Type 2 Infection-Induced Expression of CXCR3 Ligands Promotes CD4+ T Cell Migration and Is Regulated by the Viral Immediate-Early Protein ICP4"

    Article Title: Herpes Simplex Virus Type 2 Infection-Induced Expression of CXCR3 Ligands Promotes CD4+ T Cell Migration and Is Regulated by the Viral Immediate-Early Protein ICP4

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02932

    HSV-2 ICP4 regulates the expression of CXCR3 ligands via the p38 MAPK signaling pathway. (A–C) HSV-2 regulates the expression of CXCL9 (A) , CXCL10 (B) , and CXCL11 (C) via p38/MAPK signaling pathway. ME180 cells in 24-well plates were co-transfected with 150 ng CXCR3 ligand reporter and 15 ng phRL-TK. At 4 h post-transfection, cells were infected with HSV-2 at an MOI of 1 and supplemented with inhibitor PD98059, SP600125, or SB203580. DLR assay was performed at 24 h post-transfection. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in mock-infected samples. (D–F) HSV-2 ICP4 regulates the expression of CXCL9 (D) , CXCL10 (E) , and CXCL11 (F) via p38/MAPK signaling pathway. ME180 cells in 24-well plates were co-transfected with 300 ng empty vector or ICP4 expression plasmid together with 150 ng CXCR3 ligand reporter and 15 ng phRL-TK. At 4 h post-transfection, cells were cultured in complete DMEM supplemented with inhibitor PD98059, SP600125, or SB203580. DLR assay was performed at 24 h post-transfection. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in cells transfected with empty vector. (G) ICP4 activates p38 MAPK signaling pathway. ME180 cells were transfected with 3 μg ICP4 expression plasmid. The protein level of p38, phospho-p38 (p-p38) or phospho-C/EBP-β (p-C/EBP-β) was detected by Western Blot. Data shown are mean ± S.D. of three independent experiments (A–F) . ns, not significant, *** p
    Figure Legend Snippet: HSV-2 ICP4 regulates the expression of CXCR3 ligands via the p38 MAPK signaling pathway. (A–C) HSV-2 regulates the expression of CXCL9 (A) , CXCL10 (B) , and CXCL11 (C) via p38/MAPK signaling pathway. ME180 cells in 24-well plates were co-transfected with 150 ng CXCR3 ligand reporter and 15 ng phRL-TK. At 4 h post-transfection, cells were infected with HSV-2 at an MOI of 1 and supplemented with inhibitor PD98059, SP600125, or SB203580. DLR assay was performed at 24 h post-transfection. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in mock-infected samples. (D–F) HSV-2 ICP4 regulates the expression of CXCL9 (D) , CXCL10 (E) , and CXCL11 (F) via p38/MAPK signaling pathway. ME180 cells in 24-well plates were co-transfected with 300 ng empty vector or ICP4 expression plasmid together with 150 ng CXCR3 ligand reporter and 15 ng phRL-TK. At 4 h post-transfection, cells were cultured in complete DMEM supplemented with inhibitor PD98059, SP600125, or SB203580. DLR assay was performed at 24 h post-transfection. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in cells transfected with empty vector. (G) ICP4 activates p38 MAPK signaling pathway. ME180 cells were transfected with 3 μg ICP4 expression plasmid. The protein level of p38, phospho-p38 (p-p38) or phospho-C/EBP-β (p-C/EBP-β) was detected by Western Blot. Data shown are mean ± S.D. of three independent experiments (A–F) . ns, not significant, *** p

    Techniques Used: Expressing, Transfection, Infection, Luciferase, Plasmid Preparation, Cell Culture, Western Blot

    HSV-2 infection induces the production of CXCR3 ligands in human cervical epithelial cells. (A) HSV-2 infection activates the promoters of human CXCR3 ligands. ME180 cells in 24-well plates were co-transfected with 150 ng CXCL9-Luc, CXCL10-Luc or CXCL11-Luc, and 15 ng internal control plasmid phRL-TK. At 4 h post-transfection, cells were infected with HSV-2 or ultraviolet-inactivated HSV-2 (UV-HSV-2) at an MOI of 1 for 24 h. DLR assay was performed. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in mock-infected samples. (B) HSV-2 infection induces the mRNA production of CXCR3 ligands. ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cells were harvested and total RNA was extracted. The expression of CXCR3 ligands and GAPDH was evaluated by relative real-time quantitative PCR. The Ct values of GAPDH among all groups were equable and not overloaded. mRNA copies of CXCR3 ligands were normalized using GAPDH and expressed as fold increase of the value for the mock-infected control. (C) HSV-2 infection induces the production of CXCR3 ligands. As depicted in (B) , cell supernatants were collected, and the protein level of CXCR3 ligands was measured by CBA. Data shown are mean ± S.D. of three independent experiments (A, B, and C). * p
    Figure Legend Snippet: HSV-2 infection induces the production of CXCR3 ligands in human cervical epithelial cells. (A) HSV-2 infection activates the promoters of human CXCR3 ligands. ME180 cells in 24-well plates were co-transfected with 150 ng CXCL9-Luc, CXCL10-Luc or CXCL11-Luc, and 15 ng internal control plasmid phRL-TK. At 4 h post-transfection, cells were infected with HSV-2 or ultraviolet-inactivated HSV-2 (UV-HSV-2) at an MOI of 1 for 24 h. DLR assay was performed. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in mock-infected samples. (B) HSV-2 infection induces the mRNA production of CXCR3 ligands. ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cells were harvested and total RNA was extracted. The expression of CXCR3 ligands and GAPDH was evaluated by relative real-time quantitative PCR. The Ct values of GAPDH among all groups were equable and not overloaded. mRNA copies of CXCR3 ligands were normalized using GAPDH and expressed as fold increase of the value for the mock-infected control. (C) HSV-2 infection induces the production of CXCR3 ligands. As depicted in (B) , cell supernatants were collected, and the protein level of CXCR3 ligands was measured by CBA. Data shown are mean ± S.D. of three independent experiments (A, B, and C). * p

    Techniques Used: Infection, Transfection, Plasmid Preparation, Luciferase, Expressing, Real-time Polymerase Chain Reaction, Crocin Bleaching Assay

    HSV-2 infection-induced CXCL9 plays a predominant role in mediating CD4 + T cell migration. (A) The concentrations of CXCR3 ligands in the supernatants of ME180 cells infected with HSV-2 or mock-infected with DMEM were detected by CBA. (B,C) CXCL9 induced by HSV-2 recruits the migration of PBMCs (B) and CD4 + T cells (C) . ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates in the absence or presence of anti-CXCL9, –CXCL10, or/and –CXCL11 neutralizing Ab or control Ab for 1 h. (D) Neutralization of CXCR3 reduces the migration of CD4 + T cells induced by HSV-2 infection. ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates. The activated CD4 + T cells were incubated with RPMI 1,640 medium containing anti-CXCR3 neutralizing Ab for 1 h and placed in the upper chamber. (E) Recombinant CXCL9 significantly induces the migration of CD4 + T cells. DMEM containing recombinant CXCL9, CXCL10, or CXCL11 (48 pg/mL, 55 pg/mL and 175 pg/mL, respectively; the lowest concentration induced by HSV-2 infection) was added to the lower chamber of transwell plates. (F,G) Recombinant CXCL10 or CXCL11 mediates the migration of CD4 + T cells in a dose-dependent manner. DMEM containing recombinant CXCL10 or CXCL11 was added to the lower chamber of transwell plates. CXCL10 or CXCL11 was started from 55 pg/mL and 175 pg/mL, respectively, at a concentration gradient of two times. The activated CD4 + T cells were placed in the upper chamber. After 2 h incubation, cells migrated to lower chambers were collected and counted using an automatic cell counter. Cells migration was expressed as percentage of input. Input cells in the upper chamber were 5 × 10 5 . Data shown are mean ± S.D. of three independent experiments (A–G) . ns, not significant, * p
    Figure Legend Snippet: HSV-2 infection-induced CXCL9 plays a predominant role in mediating CD4 + T cell migration. (A) The concentrations of CXCR3 ligands in the supernatants of ME180 cells infected with HSV-2 or mock-infected with DMEM were detected by CBA. (B,C) CXCL9 induced by HSV-2 recruits the migration of PBMCs (B) and CD4 + T cells (C) . ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates in the absence or presence of anti-CXCL9, –CXCL10, or/and –CXCL11 neutralizing Ab or control Ab for 1 h. (D) Neutralization of CXCR3 reduces the migration of CD4 + T cells induced by HSV-2 infection. ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates. The activated CD4 + T cells were incubated with RPMI 1,640 medium containing anti-CXCR3 neutralizing Ab for 1 h and placed in the upper chamber. (E) Recombinant CXCL9 significantly induces the migration of CD4 + T cells. DMEM containing recombinant CXCL9, CXCL10, or CXCL11 (48 pg/mL, 55 pg/mL and 175 pg/mL, respectively; the lowest concentration induced by HSV-2 infection) was added to the lower chamber of transwell plates. (F,G) Recombinant CXCL10 or CXCL11 mediates the migration of CD4 + T cells in a dose-dependent manner. DMEM containing recombinant CXCL10 or CXCL11 was added to the lower chamber of transwell plates. CXCL10 or CXCL11 was started from 55 pg/mL and 175 pg/mL, respectively, at a concentration gradient of two times. The activated CD4 + T cells were placed in the upper chamber. After 2 h incubation, cells migrated to lower chambers were collected and counted using an automatic cell counter. Cells migration was expressed as percentage of input. Input cells in the upper chamber were 5 × 10 5 . Data shown are mean ± S.D. of three independent experiments (A–G) . ns, not significant, * p

    Techniques Used: Infection, Migration, Crocin Bleaching Assay, Neutralization, Incubation, Recombinant, Concentration Assay

    HSV-2 ICP4 promotes the production of human CXCR3 ligands. (A) ICP4 induces the activation of CXCR3 ligand promoters. ME180 cells in 24-well plates were transfected with 300 ng expression plasmid of HSV-2 gene or empty vector together with 150 ng CXCR3 ligand reporter and 15 ng phRL-TK. At 24 h post-transfection, DLR assay was performed. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in cells transfected with empty vector. (B) The expression of HSV-2 genes was detected using anti-Flag Ab by Western Blot. ME180 cells were transfected with 3 μg HSV-2 gene expression plasmid for 24 h. The proteins were collected and detected using mouse anti-Flag Ab. (C) ICP4 induces the mRNA production of CXCR3 ligands. ME180 cells in 6-well plates were transfected with 3 μg ICP4 expression plasmid for 24 h. Cells were harvested and total RNA was extracted. The expression of CXCR3 ligands and GAPDH gene was evaluated by relative real-time quantitative PCR. The Ct values of GAPDH among all groups were equable and not overloaded. mRNA copies of CXCR3 ligands were normalized using GAPDH and expressed as fold increase of the value for the empty vector-transfected control. (D) ICP4 induces the production of CXCR3 ligands. As depicted in (C) , cell supernatants were collected, and the protein levels of CXCR3 ligands were measured by CBA. (E,F) CXCL9 induced by ICP4 recruits the migration of PBMCs (E) and CD4 + T cells (F) . ME180 cells in 6-well plates were transfected with 3 μg ICP4 expression plasmid for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates in the absence or presence of anti-CXCL9, –CXCL10, or/and –CXCL11 neutralizing Ab or control Ab for 1h. (G) Neutralization of CXCR3 reduces the migration of CD4 + T cells induced by ICP4. ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates. The activated CD4 + T cells were incubated with RPMI 1,640 medium containing anti-CXCR3 neutralizing Ab for 1 h and placed in the upper chamber. As depicted in Figure 3 , cells migrated to lower chambers were counted. Cells migration was expressed as percentage of input. One representative out of three independent experiments is shown (B) . Data shown are mean ± S.D. of three independent experiments (A,C–G) . ns, not significant, * p
    Figure Legend Snippet: HSV-2 ICP4 promotes the production of human CXCR3 ligands. (A) ICP4 induces the activation of CXCR3 ligand promoters. ME180 cells in 24-well plates were transfected with 300 ng expression plasmid of HSV-2 gene or empty vector together with 150 ng CXCR3 ligand reporter and 15 ng phRL-TK. At 24 h post-transfection, DLR assay was performed. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in cells transfected with empty vector. (B) The expression of HSV-2 genes was detected using anti-Flag Ab by Western Blot. ME180 cells were transfected with 3 μg HSV-2 gene expression plasmid for 24 h. The proteins were collected and detected using mouse anti-Flag Ab. (C) ICP4 induces the mRNA production of CXCR3 ligands. ME180 cells in 6-well plates were transfected with 3 μg ICP4 expression plasmid for 24 h. Cells were harvested and total RNA was extracted. The expression of CXCR3 ligands and GAPDH gene was evaluated by relative real-time quantitative PCR. The Ct values of GAPDH among all groups were equable and not overloaded. mRNA copies of CXCR3 ligands were normalized using GAPDH and expressed as fold increase of the value for the empty vector-transfected control. (D) ICP4 induces the production of CXCR3 ligands. As depicted in (C) , cell supernatants were collected, and the protein levels of CXCR3 ligands were measured by CBA. (E,F) CXCL9 induced by ICP4 recruits the migration of PBMCs (E) and CD4 + T cells (F) . ME180 cells in 6-well plates were transfected with 3 μg ICP4 expression plasmid for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates in the absence or presence of anti-CXCL9, –CXCL10, or/and –CXCL11 neutralizing Ab or control Ab for 1h. (G) Neutralization of CXCR3 reduces the migration of CD4 + T cells induced by ICP4. ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates. The activated CD4 + T cells were incubated with RPMI 1,640 medium containing anti-CXCR3 neutralizing Ab for 1 h and placed in the upper chamber. As depicted in Figure 3 , cells migrated to lower chambers were counted. Cells migration was expressed as percentage of input. One representative out of three independent experiments is shown (B) . Data shown are mean ± S.D. of three independent experiments (A,C–G) . ns, not significant, * p

    Techniques Used: Activation Assay, Transfection, Expressing, Plasmid Preparation, Luciferase, Western Blot, Real-time Polymerase Chain Reaction, Crocin Bleaching Assay, Migration, Neutralization, Infection, Incubation

    HSV-2 ICP4 binds to the promoters of CXCR3 ligands by interaction with TBP. (A) Schematic representation of the predicted NLSs of ICP4 amino acid (AA) sequence. (B) ICP4 is located in the nucleus. ME180 cells in 35-mm dishes with glass bottom were transfected with 2 μg empty vector or HA-tagged ICP4 expression plasmid for 24 h. Cells were stained with mouse anti-HA mAb, followed by Cy3-conjugated goat anti-mouse (red) as the secondary Ab. Cell nuclei (blue) were stained with DAPI. The images were obtained by fluorescence microscopy using 60 × objective. The scale bar indicates 21 μm. (C) The expression of ICP4 was stained using anti-HA mAb. (D) ICP4 binds to the promoters of CXCR3 ligands. ME180 cells were transfected with 3 μg empty vector or HA-tagged ICP4 expression plasmid for 24 h. Cells were lysed and subjected to ChIP assay using mouse anti-HA mAb, mouse anti-RNA polymerase II mAb (positive control) or mouse normal IgG (negative control) for immunoprecipitation. (E) ICP4 interacts with TBP. ME180 cells in 6-well plates were transfected with 3 μg empty vector or HA-tagged ICP4 expression plasmid for 24 h. Cells were lysed and subjected to co-immunoprecipitation (IP) using rabbit anti-HA or anti-TBP Ab. Rabbit normal IgG was used as a negative control. IP products and 5% input samples were examined using rabbit anti-HA and rabbit anti-TBP Abs by western blot. One representative out of three independent experiments is shown (B–E) .
    Figure Legend Snippet: HSV-2 ICP4 binds to the promoters of CXCR3 ligands by interaction with TBP. (A) Schematic representation of the predicted NLSs of ICP4 amino acid (AA) sequence. (B) ICP4 is located in the nucleus. ME180 cells in 35-mm dishes with glass bottom were transfected with 2 μg empty vector or HA-tagged ICP4 expression plasmid for 24 h. Cells were stained with mouse anti-HA mAb, followed by Cy3-conjugated goat anti-mouse (red) as the secondary Ab. Cell nuclei (blue) were stained with DAPI. The images were obtained by fluorescence microscopy using 60 × objective. The scale bar indicates 21 μm. (C) The expression of ICP4 was stained using anti-HA mAb. (D) ICP4 binds to the promoters of CXCR3 ligands. ME180 cells were transfected with 3 μg empty vector or HA-tagged ICP4 expression plasmid for 24 h. Cells were lysed and subjected to ChIP assay using mouse anti-HA mAb, mouse anti-RNA polymerase II mAb (positive control) or mouse normal IgG (negative control) for immunoprecipitation. (E) ICP4 interacts with TBP. ME180 cells in 6-well plates were transfected with 3 μg empty vector or HA-tagged ICP4 expression plasmid for 24 h. Cells were lysed and subjected to co-immunoprecipitation (IP) using rabbit anti-HA or anti-TBP Ab. Rabbit normal IgG was used as a negative control. IP products and 5% input samples were examined using rabbit anti-HA and rabbit anti-TBP Abs by western blot. One representative out of three independent experiments is shown (B–E) .

    Techniques Used: Sequencing, Transfection, Plasmid Preparation, Expressing, Staining, Fluorescence, Microscopy, Chromatin Immunoprecipitation, Positive Control, Negative Control, Immunoprecipitation, Western Blot

    Contribution of HSV-2 infection-induced CXCR3 ligands to CD4 + T cell infiltration into mouse vagina. Seven days prior to HSV-2 challenge, BALB/c mice were injected with progesterone in multiple sites. One day prior to HSV-2 challenge, CXCL9, CXCL10, and CXCL11 neutralizing antibodies were delivered to the vagina of mice, alone or in combination, while isotype matched control IgG was used as the control. Mice were then anesthetized with pentobarbital sodium and challenged intravaginally with 10 μL/ mouse HSV-2 at a concentration of 6 × 10 7 PFU/ml or mock- challenged. Vaginal lavage fluids and cervical-vaginal tissues were collected at day 7 after challenge. (A) HSV-2 infection induces the production of mouse CXCR3 ligands. The protein levels of CXCL9 and CXCL10 ligands in vaginal lavage fluids were measured by CBA, and the protein level of CXCL11 was detected by ELISA. (B) CXCL9 mediates the migration of CD4 + T cells to the vaginal foci of infected mice. CD4 + T cells in infection foci were detected using anti-CD4 Ab by IHC. The scale bar indicates 100 μm. Data shown are mean ± S.D. ( n = 5 mice/group) of three independent experiments (A) . *** p
    Figure Legend Snippet: Contribution of HSV-2 infection-induced CXCR3 ligands to CD4 + T cell infiltration into mouse vagina. Seven days prior to HSV-2 challenge, BALB/c mice were injected with progesterone in multiple sites. One day prior to HSV-2 challenge, CXCL9, CXCL10, and CXCL11 neutralizing antibodies were delivered to the vagina of mice, alone or in combination, while isotype matched control IgG was used as the control. Mice were then anesthetized with pentobarbital sodium and challenged intravaginally with 10 μL/ mouse HSV-2 at a concentration of 6 × 10 7 PFU/ml or mock- challenged. Vaginal lavage fluids and cervical-vaginal tissues were collected at day 7 after challenge. (A) HSV-2 infection induces the production of mouse CXCR3 ligands. The protein levels of CXCL9 and CXCL10 ligands in vaginal lavage fluids were measured by CBA, and the protein level of CXCL11 was detected by ELISA. (B) CXCL9 mediates the migration of CD4 + T cells to the vaginal foci of infected mice. CD4 + T cells in infection foci were detected using anti-CD4 Ab by IHC. The scale bar indicates 100 μm. Data shown are mean ± S.D. ( n = 5 mice/group) of three independent experiments (A) . *** p

    Techniques Used: Infection, Mouse Assay, Injection, Concentration Assay, Crocin Bleaching Assay, Enzyme-linked Immunosorbent Assay, Migration, Immunohistochemistry

    12) Product Images from "Complete, Long-Lasting Protection against Lethal Infectious Bursal Disease Virus Challenge by a Single Vaccination with an Avian Herpesvirus Vector Expressing VP2 Antigens"

    Article Title: Complete, Long-Lasting Protection against Lethal Infectious Bursal Disease Virus Challenge by a Single Vaccination with an Avian Herpesvirus Vector Expressing VP2 Antigens

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.11.5637-5645.2002

    Characterization of rHVTs. (A) Schematic representation of rHVT-pecVP2 (Pec) and rHVT-cmvVP2 (CMV). (B) Immunological staining of CEFs infected with either rHVT-pecVP2 or rHVT-cmvVP2 with anti-IBDV rabbit antiserum followed by horseradish peroxidase-labeled goat anti-rabbit immunoglobulin G. (C) CEFs infected with rHVT-cmvVP2 (CMV), rHVT-pecVP2 (Pec), HVT, or IBDV J1 strains were analyzed by immunoblotting for VP2 antigen expression with rabbit anti-IBDV antiserum. (D) PCR analysis was performed on cellular DNA of CEFs infected with rHVT-cmvVP2 (CMV), rHVT-pecVP2 (Pec), or HVT for the presence of the VP2 gene. Reverse transcription was performed on genomic double-stranded RNA of IBDV E/91 strain followed by PCR for amplification of VP2 gene.
    Figure Legend Snippet: Characterization of rHVTs. (A) Schematic representation of rHVT-pecVP2 (Pec) and rHVT-cmvVP2 (CMV). (B) Immunological staining of CEFs infected with either rHVT-pecVP2 or rHVT-cmvVP2 with anti-IBDV rabbit antiserum followed by horseradish peroxidase-labeled goat anti-rabbit immunoglobulin G. (C) CEFs infected with rHVT-cmvVP2 (CMV), rHVT-pecVP2 (Pec), HVT, or IBDV J1 strains were analyzed by immunoblotting for VP2 antigen expression with rabbit anti-IBDV antiserum. (D) PCR analysis was performed on cellular DNA of CEFs infected with rHVT-cmvVP2 (CMV), rHVT-pecVP2 (Pec), or HVT for the presence of the VP2 gene. Reverse transcription was performed on genomic double-stranded RNA of IBDV E/91 strain followed by PCR for amplification of VP2 gene.

    Techniques Used: Staining, Infection, Labeling, Expressing, Polymerase Chain Reaction, Amplification

    Protective efficacy of rHVTs against lethal IBDV challenge. (A) Chickens were vaccinated with each rHVT or conventional live vaccine IBDV-A and challenged with a vvIBDV at 4 weeks of age. One hundred percent (22 of 22), 58% (7 of 12), and 92% (11 of 12) of chickens vaccinated with rHVT-pecVP2, rHVT-cmvVP2, or IBDV-A, respectively, were protected against IBDV gross lesions. (B) Sera were tested for the presence of anti-VP2 antibodies detected by AGP tests. Ninety-six percent (21 of 22), 42% (5 of 12), and 67% (8 of 12) of chickens vaccinated with rHVT-pecVP2, rHVT-cmvVP2, or IBDV-A, respectively, had the AGP antibodies to the VP2 antigen.
    Figure Legend Snippet: Protective efficacy of rHVTs against lethal IBDV challenge. (A) Chickens were vaccinated with each rHVT or conventional live vaccine IBDV-A and challenged with a vvIBDV at 4 weeks of age. One hundred percent (22 of 22), 58% (7 of 12), and 92% (11 of 12) of chickens vaccinated with rHVT-pecVP2, rHVT-cmvVP2, or IBDV-A, respectively, were protected against IBDV gross lesions. (B) Sera were tested for the presence of anti-VP2 antibodies detected by AGP tests. Ninety-six percent (21 of 22), 42% (5 of 12), and 67% (8 of 12) of chickens vaccinated with rHVT-pecVP2, rHVT-cmvVP2, or IBDV-A, respectively, had the AGP antibodies to the VP2 antigen.

    Techniques Used:

    13) Product Images from "Herpes Simplex Virus Type 2 Infection-Induced Expression of CXCR3 Ligands Promotes CD4+ T Cell Migration and Is Regulated by the Viral Immediate-Early Protein ICP4"

    Article Title: Herpes Simplex Virus Type 2 Infection-Induced Expression of CXCR3 Ligands Promotes CD4+ T Cell Migration and Is Regulated by the Viral Immediate-Early Protein ICP4

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02932

    HSV-2 ICP4 regulates the expression of CXCR3 ligands via the p38 MAPK signaling pathway. (A–C) HSV-2 regulates the expression of CXCL9 (A) , CXCL10 (B) , and CXCL11 (C) via p38/MAPK signaling pathway. ME180 cells in 24-well plates were co-transfected with 150 ng CXCR3 ligand reporter and 15 ng phRL-TK. At 4 h post-transfection, cells were infected with HSV-2 at an MOI of 1 and supplemented with inhibitor PD98059, SP600125, or SB203580. DLR assay was performed at 24 h post-transfection. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in mock-infected samples. (D–F) HSV-2 ICP4 regulates the expression of CXCL9 (D) , CXCL10 (E) , and CXCL11 (F) via p38/MAPK signaling pathway. ME180 cells in 24-well plates were co-transfected with 300 ng empty vector or ICP4 expression plasmid together with 150 ng CXCR3 ligand reporter and 15 ng phRL-TK. At 4 h post-transfection, cells were cultured in complete DMEM supplemented with inhibitor PD98059, SP600125, or SB203580. DLR assay was performed at 24 h post-transfection. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in cells transfected with empty vector. (G) ICP4 activates p38 MAPK signaling pathway. ME180 cells were transfected with 3 μg ICP4 expression plasmid. The protein level of p38, phospho-p38 (p-p38) or phospho-C/EBP-β (p-C/EBP-β) was detected by Western Blot. Data shown are mean ± S.D. of three independent experiments (A–F) . ns, not significant, *** p
    Figure Legend Snippet: HSV-2 ICP4 regulates the expression of CXCR3 ligands via the p38 MAPK signaling pathway. (A–C) HSV-2 regulates the expression of CXCL9 (A) , CXCL10 (B) , and CXCL11 (C) via p38/MAPK signaling pathway. ME180 cells in 24-well plates were co-transfected with 150 ng CXCR3 ligand reporter and 15 ng phRL-TK. At 4 h post-transfection, cells were infected with HSV-2 at an MOI of 1 and supplemented with inhibitor PD98059, SP600125, or SB203580. DLR assay was performed at 24 h post-transfection. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in mock-infected samples. (D–F) HSV-2 ICP4 regulates the expression of CXCL9 (D) , CXCL10 (E) , and CXCL11 (F) via p38/MAPK signaling pathway. ME180 cells in 24-well plates were co-transfected with 300 ng empty vector or ICP4 expression plasmid together with 150 ng CXCR3 ligand reporter and 15 ng phRL-TK. At 4 h post-transfection, cells were cultured in complete DMEM supplemented with inhibitor PD98059, SP600125, or SB203580. DLR assay was performed at 24 h post-transfection. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in cells transfected with empty vector. (G) ICP4 activates p38 MAPK signaling pathway. ME180 cells were transfected with 3 μg ICP4 expression plasmid. The protein level of p38, phospho-p38 (p-p38) or phospho-C/EBP-β (p-C/EBP-β) was detected by Western Blot. Data shown are mean ± S.D. of three independent experiments (A–F) . ns, not significant, *** p

    Techniques Used: Expressing, Transfection, Infection, Luciferase, Plasmid Preparation, Cell Culture, Western Blot

    HSV-2 infection induces the production of CXCR3 ligands in human cervical epithelial cells. (A) HSV-2 infection activates the promoters of human CXCR3 ligands. ME180 cells in 24-well plates were co-transfected with 150 ng CXCL9-Luc, CXCL10-Luc or CXCL11-Luc, and 15 ng internal control plasmid phRL-TK. At 4 h post-transfection, cells were infected with HSV-2 or ultraviolet-inactivated HSV-2 (UV-HSV-2) at an MOI of 1 for 24 h. DLR assay was performed. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in mock-infected samples. (B) HSV-2 infection induces the mRNA production of CXCR3 ligands. ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cells were harvested and total RNA was extracted. The expression of CXCR3 ligands and GAPDH was evaluated by relative real-time quantitative PCR. The Ct values of GAPDH among all groups were equable and not overloaded. mRNA copies of CXCR3 ligands were normalized using GAPDH and expressed as fold increase of the value for the mock-infected control. (C) HSV-2 infection induces the production of CXCR3 ligands. As depicted in (B) , cell supernatants were collected, and the protein level of CXCR3 ligands was measured by CBA. Data shown are mean ± S.D. of three independent experiments (A, B, and C). * p
    Figure Legend Snippet: HSV-2 infection induces the production of CXCR3 ligands in human cervical epithelial cells. (A) HSV-2 infection activates the promoters of human CXCR3 ligands. ME180 cells in 24-well plates were co-transfected with 150 ng CXCL9-Luc, CXCL10-Luc or CXCL11-Luc, and 15 ng internal control plasmid phRL-TK. At 4 h post-transfection, cells were infected with HSV-2 or ultraviolet-inactivated HSV-2 (UV-HSV-2) at an MOI of 1 for 24 h. DLR assay was performed. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in mock-infected samples. (B) HSV-2 infection induces the mRNA production of CXCR3 ligands. ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cells were harvested and total RNA was extracted. The expression of CXCR3 ligands and GAPDH was evaluated by relative real-time quantitative PCR. The Ct values of GAPDH among all groups were equable and not overloaded. mRNA copies of CXCR3 ligands were normalized using GAPDH and expressed as fold increase of the value for the mock-infected control. (C) HSV-2 infection induces the production of CXCR3 ligands. As depicted in (B) , cell supernatants were collected, and the protein level of CXCR3 ligands was measured by CBA. Data shown are mean ± S.D. of three independent experiments (A, B, and C). * p

    Techniques Used: Infection, Transfection, Plasmid Preparation, Luciferase, Expressing, Real-time Polymerase Chain Reaction, Crocin Bleaching Assay

    HSV-2 infection-induced CXCL9 plays a predominant role in mediating CD4 + T cell migration. (A) The concentrations of CXCR3 ligands in the supernatants of ME180 cells infected with HSV-2 or mock-infected with DMEM were detected by CBA. (B,C) CXCL9 induced by HSV-2 recruits the migration of PBMCs (B) and CD4 + T cells (C) . ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates in the absence or presence of anti-CXCL9, –CXCL10, or/and –CXCL11 neutralizing Ab or control Ab for 1 h. (D) Neutralization of CXCR3 reduces the migration of CD4 + T cells induced by HSV-2 infection. ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates. The activated CD4 + T cells were incubated with RPMI 1,640 medium containing anti-CXCR3 neutralizing Ab for 1 h and placed in the upper chamber. (E) Recombinant CXCL9 significantly induces the migration of CD4 + T cells. DMEM containing recombinant CXCL9, CXCL10, or CXCL11 (48 pg/mL, 55 pg/mL and 175 pg/mL, respectively; the lowest concentration induced by HSV-2 infection) was added to the lower chamber of transwell plates. (F,G) Recombinant CXCL10 or CXCL11 mediates the migration of CD4 + T cells in a dose-dependent manner. DMEM containing recombinant CXCL10 or CXCL11 was added to the lower chamber of transwell plates. CXCL10 or CXCL11 was started from 55 pg/mL and 175 pg/mL, respectively, at a concentration gradient of two times. The activated CD4 + T cells were placed in the upper chamber. After 2 h incubation, cells migrated to lower chambers were collected and counted using an automatic cell counter. Cells migration was expressed as percentage of input. Input cells in the upper chamber were 5 × 10 5 . Data shown are mean ± S.D. of three independent experiments (A–G) . ns, not significant, * p
    Figure Legend Snippet: HSV-2 infection-induced CXCL9 plays a predominant role in mediating CD4 + T cell migration. (A) The concentrations of CXCR3 ligands in the supernatants of ME180 cells infected with HSV-2 or mock-infected with DMEM were detected by CBA. (B,C) CXCL9 induced by HSV-2 recruits the migration of PBMCs (B) and CD4 + T cells (C) . ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates in the absence or presence of anti-CXCL9, –CXCL10, or/and –CXCL11 neutralizing Ab or control Ab for 1 h. (D) Neutralization of CXCR3 reduces the migration of CD4 + T cells induced by HSV-2 infection. ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates. The activated CD4 + T cells were incubated with RPMI 1,640 medium containing anti-CXCR3 neutralizing Ab for 1 h and placed in the upper chamber. (E) Recombinant CXCL9 significantly induces the migration of CD4 + T cells. DMEM containing recombinant CXCL9, CXCL10, or CXCL11 (48 pg/mL, 55 pg/mL and 175 pg/mL, respectively; the lowest concentration induced by HSV-2 infection) was added to the lower chamber of transwell plates. (F,G) Recombinant CXCL10 or CXCL11 mediates the migration of CD4 + T cells in a dose-dependent manner. DMEM containing recombinant CXCL10 or CXCL11 was added to the lower chamber of transwell plates. CXCL10 or CXCL11 was started from 55 pg/mL and 175 pg/mL, respectively, at a concentration gradient of two times. The activated CD4 + T cells were placed in the upper chamber. After 2 h incubation, cells migrated to lower chambers were collected and counted using an automatic cell counter. Cells migration was expressed as percentage of input. Input cells in the upper chamber were 5 × 10 5 . Data shown are mean ± S.D. of three independent experiments (A–G) . ns, not significant, * p

    Techniques Used: Infection, Migration, Crocin Bleaching Assay, Neutralization, Incubation, Recombinant, Concentration Assay

    HSV-2 ICP4 binds to the promoters of CXCR3 ligands by interaction with TBP. (A) Schematic representation of the predicted NLSs of ICP4 amino acid (AA) sequence. (B) ICP4 is located in the nucleus. ME180 cells in 35-mm dishes with glass bottom were transfected with 2 μg empty vector or HA-tagged ICP4 expression plasmid for 24 h. Cells were stained with mouse anti-HA mAb, followed by Cy3-conjugated goat anti-mouse (red) as the secondary Ab. Cell nuclei (blue) were stained with DAPI. The images were obtained by fluorescence microscopy using 60 × objective. The scale bar indicates 21 μm. (C) The expression of ICP4 was stained using anti-HA mAb. (D) ICP4 binds to the promoters of CXCR3 ligands. ME180 cells were transfected with 3 μg empty vector or HA-tagged ICP4 expression plasmid for 24 h. Cells were lysed and subjected to ChIP assay using mouse anti-HA mAb, mouse anti-RNA polymerase II mAb (positive control) or mouse normal IgG (negative control) for immunoprecipitation. (E) ICP4 interacts with TBP. ME180 cells in 6-well plates were transfected with 3 μg empty vector or HA-tagged ICP4 expression plasmid for 24 h. Cells were lysed and subjected to co-immunoprecipitation (IP) using rabbit anti-HA or anti-TBP Ab. Rabbit normal IgG was used as a negative control. IP products and 5% input samples were examined using rabbit anti-HA and rabbit anti-TBP Abs by western blot. One representative out of three independent experiments is shown (B–E) .
    Figure Legend Snippet: HSV-2 ICP4 binds to the promoters of CXCR3 ligands by interaction with TBP. (A) Schematic representation of the predicted NLSs of ICP4 amino acid (AA) sequence. (B) ICP4 is located in the nucleus. ME180 cells in 35-mm dishes with glass bottom were transfected with 2 μg empty vector or HA-tagged ICP4 expression plasmid for 24 h. Cells were stained with mouse anti-HA mAb, followed by Cy3-conjugated goat anti-mouse (red) as the secondary Ab. Cell nuclei (blue) were stained with DAPI. The images were obtained by fluorescence microscopy using 60 × objective. The scale bar indicates 21 μm. (C) The expression of ICP4 was stained using anti-HA mAb. (D) ICP4 binds to the promoters of CXCR3 ligands. ME180 cells were transfected with 3 μg empty vector or HA-tagged ICP4 expression plasmid for 24 h. Cells were lysed and subjected to ChIP assay using mouse anti-HA mAb, mouse anti-RNA polymerase II mAb (positive control) or mouse normal IgG (negative control) for immunoprecipitation. (E) ICP4 interacts with TBP. ME180 cells in 6-well plates were transfected with 3 μg empty vector or HA-tagged ICP4 expression plasmid for 24 h. Cells were lysed and subjected to co-immunoprecipitation (IP) using rabbit anti-HA or anti-TBP Ab. Rabbit normal IgG was used as a negative control. IP products and 5% input samples were examined using rabbit anti-HA and rabbit anti-TBP Abs by western blot. One representative out of three independent experiments is shown (B–E) .

    Techniques Used: Sequencing, Transfection, Plasmid Preparation, Expressing, Staining, Fluorescence, Microscopy, Chromatin Immunoprecipitation, Positive Control, Negative Control, Immunoprecipitation, Western Blot

    Contribution of HSV-2 infection-induced CXCR3 ligands to CD4 + T cell infiltration into mouse vagina. Seven days prior to HSV-2 challenge, BALB/c mice were injected with progesterone in multiple sites. One day prior to HSV-2 challenge, CXCL9, CXCL10, and CXCL11 neutralizing antibodies were delivered to the vagina of mice, alone or in combination, while isotype matched control IgG was used as the control. Mice were then anesthetized with pentobarbital sodium and challenged intravaginally with 10 μL/ mouse HSV-2 at a concentration of 6 × 10 7 PFU/ml or mock- challenged. Vaginal lavage fluids and cervical-vaginal tissues were collected at day 7 after challenge. (A) HSV-2 infection induces the production of mouse CXCR3 ligands. The protein levels of CXCL9 and CXCL10 ligands in vaginal lavage fluids were measured by CBA, and the protein level of CXCL11 was detected by ELISA. (B) CXCL9 mediates the migration of CD4 + T cells to the vaginal foci of infected mice. CD4 + T cells in infection foci were detected using anti-CD4 Ab by IHC. The scale bar indicates 100 μm. Data shown are mean ± S.D. ( n = 5 mice/group) of three independent experiments (A) . *** p
    Figure Legend Snippet: Contribution of HSV-2 infection-induced CXCR3 ligands to CD4 + T cell infiltration into mouse vagina. Seven days prior to HSV-2 challenge, BALB/c mice were injected with progesterone in multiple sites. One day prior to HSV-2 challenge, CXCL9, CXCL10, and CXCL11 neutralizing antibodies were delivered to the vagina of mice, alone or in combination, while isotype matched control IgG was used as the control. Mice were then anesthetized with pentobarbital sodium and challenged intravaginally with 10 μL/ mouse HSV-2 at a concentration of 6 × 10 7 PFU/ml or mock- challenged. Vaginal lavage fluids and cervical-vaginal tissues were collected at day 7 after challenge. (A) HSV-2 infection induces the production of mouse CXCR3 ligands. The protein levels of CXCL9 and CXCL10 ligands in vaginal lavage fluids were measured by CBA, and the protein level of CXCL11 was detected by ELISA. (B) CXCL9 mediates the migration of CD4 + T cells to the vaginal foci of infected mice. CD4 + T cells in infection foci were detected using anti-CD4 Ab by IHC. The scale bar indicates 100 μm. Data shown are mean ± S.D. ( n = 5 mice/group) of three independent experiments (A) . *** p

    Techniques Used: Infection, Mouse Assay, Injection, Concentration Assay, Crocin Bleaching Assay, Enzyme-linked Immunosorbent Assay, Migration, Immunohistochemistry

    14) Product Images from "Isolation of Chlamydia Pneumoniae from Serum Samples of the Patients with Acute Coronary Syndrome"

    Article Title: Isolation of Chlamydia Pneumoniae from Serum Samples of the Patients with Acute Coronary Syndrome

    Journal: International Journal of Medical Sciences

    doi:

    Recovery of PCR product in DNA samples isolated from serum specimens using QIAamp DNA blood midi kit, protein A and phenol-chloroform extraction method. 1 - molecular size standards; 2, 6 and 10 - PCR-positive serum from patient M; 3, 7 and 11 - PCR-positive serum from patient P; 4, 8 and 12 - PCR negative serum from patient S; 5, 9 and 13 - extraction control; 14 - negative control; 15 - positive control.
    Figure Legend Snippet: Recovery of PCR product in DNA samples isolated from serum specimens using QIAamp DNA blood midi kit, protein A and phenol-chloroform extraction method. 1 - molecular size standards; 2, 6 and 10 - PCR-positive serum from patient M; 3, 7 and 11 - PCR-positive serum from patient P; 4, 8 and 12 - PCR negative serum from patient S; 5, 9 and 13 - extraction control; 14 - negative control; 15 - positive control.

    Techniques Used: Polymerase Chain Reaction, Isolation, Negative Control, Positive Control

    15) Product Images from "Detection of pathogenic microorganisms from bloodstream infection specimens using TaqMan array card technology"

    Article Title: Detection of pathogenic microorganisms from bloodstream infection specimens using TaqMan array card technology

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-31200-3

    TAC optimization. ( A ) 50% Tween-20/Triton X-100 and 0.1% saponin treatment on the yield of pathogen cells from whole blood specimens. 10 3 CFU S. aureus was mixed in 1 mL blood from healthy donors and then lysed by Tween-20/Triton X-100 or saponin. The colony numbers were determined by a plate count. ( B , C ). TNA extraction kit performance on blood culture specimens ( B ) and mock whole blood specimens ( C ). Kit 1 is the BiOstic bacteremia DNA isolation kit; Kit 2 is the QIAamp DNA Blood Mini Kit; Kit 3 is the QIAamp UCP Pathogen Mini Kit; Kit 4 is the TIANamp Blood DNA Kit; Kit 5 is the QIAamp cador Pathogen Mini Kit. M represents the benzyl alcohol-guanidine hydrochloride method. For blood culture specimens, three blood culture samples positive for S. aureus (G + ), E. coli (G − ), or C. albicans (fungi) were used. For whole blood specimens, three whole blood mock specimens spiked with 10 1 CFU/mL of S. aureus , E. coli , and C. albicans were used. ( D ) The effect of TNA combined with reverse transcription on amplifying efficiency. 10 1 and 10 2 CFU S. aureus (G + ), E. coli (G − ), or C. albicans (fungi) were used to make mock specimens. The cycle threshold (Ct) was compared with RT or without RT. ( E ) The effect of 0.1% blue dextran 2000 on the TAC assay. 10 6 CFU S. aureus (G + ), 10 7 CFU P. aeruginosa (G − ) and 10 4 CFU C. albicans (fungi) were used to make mock specimens. B+, TAC assay with blue dextran 2000; B−, TAC assay without blue dextran 2000.
    Figure Legend Snippet: TAC optimization. ( A ) 50% Tween-20/Triton X-100 and 0.1% saponin treatment on the yield of pathogen cells from whole blood specimens. 10 3 CFU S. aureus was mixed in 1 mL blood from healthy donors and then lysed by Tween-20/Triton X-100 or saponin. The colony numbers were determined by a plate count. ( B , C ). TNA extraction kit performance on blood culture specimens ( B ) and mock whole blood specimens ( C ). Kit 1 is the BiOstic bacteremia DNA isolation kit; Kit 2 is the QIAamp DNA Blood Mini Kit; Kit 3 is the QIAamp UCP Pathogen Mini Kit; Kit 4 is the TIANamp Blood DNA Kit; Kit 5 is the QIAamp cador Pathogen Mini Kit. M represents the benzyl alcohol-guanidine hydrochloride method. For blood culture specimens, three blood culture samples positive for S. aureus (G + ), E. coli (G − ), or C. albicans (fungi) were used. For whole blood specimens, three whole blood mock specimens spiked with 10 1 CFU/mL of S. aureus , E. coli , and C. albicans were used. ( D ) The effect of TNA combined with reverse transcription on amplifying efficiency. 10 1 and 10 2 CFU S. aureus (G + ), E. coli (G − ), or C. albicans (fungi) were used to make mock specimens. The cycle threshold (Ct) was compared with RT or without RT. ( E ) The effect of 0.1% blue dextran 2000 on the TAC assay. 10 6 CFU S. aureus (G + ), 10 7 CFU P. aeruginosa (G − ) and 10 4 CFU C. albicans (fungi) were used to make mock specimens. B+, TAC assay with blue dextran 2000; B−, TAC assay without blue dextran 2000.

    Techniques Used: DNA Extraction

    16) Product Images from "PCR-Based Method for Isolation and Detection of Chlamydia pneumoniae DNA in Cerebrospinal Fluids"

    Article Title: PCR-Based Method for Isolation and Detection of Chlamydia pneumoniae DNA in Cerebrospinal Fluids

    Journal: Clinical and Diagnostic Laboratory Immunology

    doi: 10.1128/CDLI.8.3.499-502.2001

    Optimization of annealing temperature and Mg 2+ concentration for PCR with primers for omp1 . The annealing temperature was optimized in a Mastercycler gradient (Eppendorf). The Mg concentration for PCR specific for omp1 was optimized with a PCR optimization kit (Invitrogen). The target DNA for PCR was extracted from C. pneumoniae with the QIAmp DNA Mini Kit.
    Figure Legend Snippet: Optimization of annealing temperature and Mg 2+ concentration for PCR with primers for omp1 . The annealing temperature was optimized in a Mastercycler gradient (Eppendorf). The Mg concentration for PCR specific for omp1 was optimized with a PCR optimization kit (Invitrogen). The target DNA for PCR was extracted from C. pneumoniae with the QIAmp DNA Mini Kit.

    Techniques Used: Concentration Assay, Polymerase Chain Reaction

    Detection sensitivity of PCR for omp1 versus the 16S rRNA gene. Two hundred microliters of the mock CSF containing a specific number of C. pneumoniae organisms (as indicated), was used for DNA extraction with the QIAmp DNA Mini Kit. A 2-μl portion of the 50-μl volume of DNA extracts was subjected to PCR. The optimized PCRs for omp1 and 16S rRNA gene were conducted (see Materials and Methods).
    Figure Legend Snippet: Detection sensitivity of PCR for omp1 versus the 16S rRNA gene. Two hundred microliters of the mock CSF containing a specific number of C. pneumoniae organisms (as indicated), was used for DNA extraction with the QIAmp DNA Mini Kit. A 2-μl portion of the 50-μl volume of DNA extracts was subjected to PCR. The optimized PCRs for omp1 and 16S rRNA gene were conducted (see Materials and Methods).

    Techniques Used: Polymerase Chain Reaction, DNA Extraction

    Comparison of extraction efficacy of C. pneumoniae DNA from mock CSFs using two DNA extraction kits. The mock CSFs (200 μl) spiked with serially diluted bacteria were extracted either with the QIAmp DNA Mini Kit with a bacterial DNA extraction protocol or with the QIAmp DNA Blood Mini Kit. Two microliters of the extracted DNA (50 μl) was subjected to PCR with primers for omp1 . Results are representative of three experiments.
    Figure Legend Snippet: Comparison of extraction efficacy of C. pneumoniae DNA from mock CSFs using two DNA extraction kits. The mock CSFs (200 μl) spiked with serially diluted bacteria were extracted either with the QIAmp DNA Mini Kit with a bacterial DNA extraction protocol or with the QIAmp DNA Blood Mini Kit. Two microliters of the extracted DNA (50 μl) was subjected to PCR with primers for omp1 . Results are representative of three experiments.

    Techniques Used: DNA Extraction, Polymerase Chain Reaction

    Detection of C. pneumoniae DNA by PCR in CSFs obtained from patients with MS. Two hundred microliters of CSFs obtained from MS patients was processed for extraction of DNA utilizing the QIAmp DNA Mini Kit, and 2 μl of the resulting 50-μl DNA solution was subjected to PCR with primers for either omp1 or the 16S rRNA gene. Data are representative of three PCR experiments. Boxed numbers indicate CSFs obtained from the same patient at different time points. Each number is the sample number. M, molecular marker; PC, positive control for PCR; NC, negative control for PCR; IC, negative control for DNA extraction.
    Figure Legend Snippet: Detection of C. pneumoniae DNA by PCR in CSFs obtained from patients with MS. Two hundred microliters of CSFs obtained from MS patients was processed for extraction of DNA utilizing the QIAmp DNA Mini Kit, and 2 μl of the resulting 50-μl DNA solution was subjected to PCR with primers for either omp1 or the 16S rRNA gene. Data are representative of three PCR experiments. Boxed numbers indicate CSFs obtained from the same patient at different time points. Each number is the sample number. M, molecular marker; PC, positive control for PCR; NC, negative control for PCR; IC, negative control for DNA extraction.

    Techniques Used: Polymerase Chain Reaction, Mass Spectrometry, Marker, Positive Control, Negative Control, DNA Extraction

    17) Product Images from "PCR-Based Method for Isolation and Detection of Chlamydia pneumoniae DNA in Cerebrospinal Fluids"

    Article Title: PCR-Based Method for Isolation and Detection of Chlamydia pneumoniae DNA in Cerebrospinal Fluids

    Journal: Clinical and Diagnostic Laboratory Immunology

    doi: 10.1128/CDLI.8.3.499-502.2001

    Optimization of annealing temperature and Mg 2+ concentration for PCR with primers for omp1 . The annealing temperature was optimized in a Mastercycler gradient (Eppendorf). The Mg concentration for PCR specific for omp1 was optimized with a PCR optimization kit (Invitrogen). The target DNA for PCR was extracted from C. pneumoniae with the QIAmp DNA Mini Kit.
    Figure Legend Snippet: Optimization of annealing temperature and Mg 2+ concentration for PCR with primers for omp1 . The annealing temperature was optimized in a Mastercycler gradient (Eppendorf). The Mg concentration for PCR specific for omp1 was optimized with a PCR optimization kit (Invitrogen). The target DNA for PCR was extracted from C. pneumoniae with the QIAmp DNA Mini Kit.

    Techniques Used: Concentration Assay, Polymerase Chain Reaction

    Detection sensitivity of PCR for omp1 versus the 16S rRNA gene. Two hundred microliters of the mock CSF containing a specific number of C. pneumoniae organisms (as indicated), was used for DNA extraction with the QIAmp DNA Mini Kit. A 2-μl portion of the 50-μl volume of DNA extracts was subjected to PCR. The optimized PCRs for omp1 and 16S rRNA gene were conducted (see Materials and Methods).
    Figure Legend Snippet: Detection sensitivity of PCR for omp1 versus the 16S rRNA gene. Two hundred microliters of the mock CSF containing a specific number of C. pneumoniae organisms (as indicated), was used for DNA extraction with the QIAmp DNA Mini Kit. A 2-μl portion of the 50-μl volume of DNA extracts was subjected to PCR. The optimized PCRs for omp1 and 16S rRNA gene were conducted (see Materials and Methods).

    Techniques Used: Polymerase Chain Reaction, DNA Extraction

    Comparison of extraction efficacy of C. pneumoniae DNA from mock CSFs using two DNA extraction kits. The mock CSFs (200 μl) spiked with serially diluted bacteria were extracted either with the QIAmp DNA Mini Kit with a bacterial DNA extraction protocol or with the QIAmp DNA Blood Mini Kit. Two microliters of the extracted DNA (50 μl) was subjected to PCR with primers for omp1 . Results are representative of three experiments.
    Figure Legend Snippet: Comparison of extraction efficacy of C. pneumoniae DNA from mock CSFs using two DNA extraction kits. The mock CSFs (200 μl) spiked with serially diluted bacteria were extracted either with the QIAmp DNA Mini Kit with a bacterial DNA extraction protocol or with the QIAmp DNA Blood Mini Kit. Two microliters of the extracted DNA (50 μl) was subjected to PCR with primers for omp1 . Results are representative of three experiments.

    Techniques Used: DNA Extraction, Polymerase Chain Reaction

    Detection of C. pneumoniae DNA by PCR in CSFs obtained from patients with MS. Two hundred microliters of CSFs obtained from MS patients was processed for extraction of DNA utilizing the QIAmp DNA Mini Kit, and 2 μl of the resulting 50-μl DNA solution was subjected to PCR with primers for either omp1 or the 16S rRNA gene. Data are representative of three PCR experiments. Boxed numbers indicate CSFs obtained from the same patient at different time points. Each number is the sample number. M, molecular marker; PC, positive control for PCR; NC, negative control for PCR; IC, negative control for DNA extraction.
    Figure Legend Snippet: Detection of C. pneumoniae DNA by PCR in CSFs obtained from patients with MS. Two hundred microliters of CSFs obtained from MS patients was processed for extraction of DNA utilizing the QIAmp DNA Mini Kit, and 2 μl of the resulting 50-μl DNA solution was subjected to PCR with primers for either omp1 or the 16S rRNA gene. Data are representative of three PCR experiments. Boxed numbers indicate CSFs obtained from the same patient at different time points. Each number is the sample number. M, molecular marker; PC, positive control for PCR; NC, negative control for PCR; IC, negative control for DNA extraction.

    Techniques Used: Polymerase Chain Reaction, Mass Spectrometry, Marker, Positive Control, Negative Control, DNA Extraction

    18) Product Images from "Adaptation of red blood cell lysis represents a fundamental breakthrough that improves the sensitivity of Salmonella detection in blood"

    Article Title: Adaptation of red blood cell lysis represents a fundamental breakthrough that improves the sensitivity of Salmonella detection in blood

    Journal: Journal of Applied Microbiology

    doi: 10.1111/jam.12769

    Real-time PCR detection of Salmonella DNA. Two millilitres of whole blood was lysed using erythrocyte lysis buffer and spiked with the shown CFU of Salmonella Typhi CVD 909. DNA was extracted using a QIAamp Mini blood kit and S. Typhi was detected by qPCR.
    Figure Legend Snippet: Real-time PCR detection of Salmonella DNA. Two millilitres of whole blood was lysed using erythrocyte lysis buffer and spiked with the shown CFU of Salmonella Typhi CVD 909. DNA was extracted using a QIAamp Mini blood kit and S. Typhi was detected by qPCR.

    Techniques Used: Real-time Polymerase Chain Reaction, Lysis

    19) Product Images from "CRISPR/Cas9 cleavage of viral DNA efficiently suppresses hepatitis B virus"

    Article Title: CRISPR/Cas9 cleavage of viral DNA efficiently suppresses hepatitis B virus

    Journal: Scientific Reports

    doi: 10.1038/srep10833

    Sustained expression of CRISPR machinery enables large reductions in HBV DNA and cccDNA. ( a ) Schematic of lentiviral vector and experimental strategy for sustained CRISPR expression. ( b - c ) CRISPR constructs targeting HBV cause progressive reduction in ( b ) cccDNA and ( c ) total HBV DNA levels dependent on successful targeting of viral DNA and Cas9 nuclease activity; data shown are from one representative experiment pooled across 3 separate HBV-targeting guides (sg6, sg17, sg21), and consistent across multiple independent transduction experiments. ( d ) Southern blot of HBV DNA forms using Hirt’s extraction (to deplete high-molecular weight DNA), shows HBV-targeted sgRNAs with nuclease-active Cas9 generate near-total reduction in cccDNA. ( e ) Surveyor assay to detect indel formation in total HBV DNA (top) and episomal HBV DNA, enriched by treatment with plasmid-safe DNase (bottom); lentiviral transduction enables high levels of cutting of HBV. Arrowheads depict surveyor digestion products. Expected PCR product sizes for sg6, sg17 and sg21 are respectively 599, 946 and 507 bp. Approximate sizes of surveyor digestion products for sg6, sg17 and sg21 are respectively: 429 + 170, 570 + 376, 275 + 232. ( f ) Immunofluorescent imaging of HBV Core protein demonstrates large reduction in Core staining upon targeting by sg17 specifically against the Core ORF. ( g - h ) Cas9/gRNA-transduced Hep-NTCP cells are cocultured with HepG2.2.15 cells to infect them with HBV followed by depletion of HepG2.2.15 cells using puromycin selection (Schematic in Fig S6 left). ( g ) From left to right, HBsAg secretion, cccDNA copies, levels of HBV 3.5kb RNA relative to 5 bp mismatch control, and titer of HBV DNA in culture medium show that Cas9/sg17 reduce HBV infection in de novo infection. 17M: 5 bp mismatch control. 17D: dead Cas9 with g17. Data shown are from one representative experiment, and consistent across experiments. ( h ) Surveyor assay performed on DNA untreated (left) or treated (right) with Plasmid-Safe DNase to remove non-episomal viral forms. Arrowheads indicate indel formation. ( b - c ) * p
    Figure Legend Snippet: Sustained expression of CRISPR machinery enables large reductions in HBV DNA and cccDNA. ( a ) Schematic of lentiviral vector and experimental strategy for sustained CRISPR expression. ( b - c ) CRISPR constructs targeting HBV cause progressive reduction in ( b ) cccDNA and ( c ) total HBV DNA levels dependent on successful targeting of viral DNA and Cas9 nuclease activity; data shown are from one representative experiment pooled across 3 separate HBV-targeting guides (sg6, sg17, sg21), and consistent across multiple independent transduction experiments. ( d ) Southern blot of HBV DNA forms using Hirt’s extraction (to deplete high-molecular weight DNA), shows HBV-targeted sgRNAs with nuclease-active Cas9 generate near-total reduction in cccDNA. ( e ) Surveyor assay to detect indel formation in total HBV DNA (top) and episomal HBV DNA, enriched by treatment with plasmid-safe DNase (bottom); lentiviral transduction enables high levels of cutting of HBV. Arrowheads depict surveyor digestion products. Expected PCR product sizes for sg6, sg17 and sg21 are respectively 599, 946 and 507 bp. Approximate sizes of surveyor digestion products for sg6, sg17 and sg21 are respectively: 429 + 170, 570 + 376, 275 + 232. ( f ) Immunofluorescent imaging of HBV Core protein demonstrates large reduction in Core staining upon targeting by sg17 specifically against the Core ORF. ( g - h ) Cas9/gRNA-transduced Hep-NTCP cells are cocultured with HepG2.2.15 cells to infect them with HBV followed by depletion of HepG2.2.15 cells using puromycin selection (Schematic in Fig S6 left). ( g ) From left to right, HBsAg secretion, cccDNA copies, levels of HBV 3.5kb RNA relative to 5 bp mismatch control, and titer of HBV DNA in culture medium show that Cas9/sg17 reduce HBV infection in de novo infection. 17M: 5 bp mismatch control. 17D: dead Cas9 with g17. Data shown are from one representative experiment, and consistent across experiments. ( h ) Surveyor assay performed on DNA untreated (left) or treated (right) with Plasmid-Safe DNase to remove non-episomal viral forms. Arrowheads indicate indel formation. ( b - c ) * p

    Techniques Used: Expressing, CRISPR, Plasmid Preparation, Construct, Activity Assay, Transduction, Southern Blot, Molecular Weight, Polymerase Chain Reaction, Imaging, Staining, Selection, Infection

    Transiently transfected CRISPR constructs exhibit anti-HBV activity. ( a ) Schematic of HBV life cycle and putative anti-HBV effect of CRISPR constructs; Cas9-mediated DSB formation should linearize the small, episomal cccDNA repeatedly, potentially leading to indel formation (generating less-fit viral mutants) or even degradation. ( b ) (left) HBV genome organization and location of target sequences for several tested guide RNA constructs. (right) Table of all possible CRISPR target sites in each HBV ORF, including number of possible target sites in conserved genomic regions. ( c ) Experimental schematic for ( d - e ): HepG2 cells are co-transfected with 1.3x WT HBV and sgRNA/Cas9-2A-mCherry construct, and ( d ) intracellular HBV pregenomic RNA and ( e ) secreted HBsAg are quantified after 72 hours. Data shown were generated in one representative experiment, with intracellular pgRNA harvested from one pellet and HBsAg collected from replicate wells per group; all data are consistent across three independent experiments. ( f ) Experimental schematic for ( g - h ): 1.3x WT HBV and sgRNA/Cas9-2A-mCherry are delivered to the livers of immunodeficient NRG mice via hydrodynamic injection, and ( g ) HBsAg and ( h ) secreted HBV titer are quantified in mouse blood at 2 and 4 days post injection. 21M: guide RNA with 5 bp mismatch from g21. Data shown are from one representative experiment, and consistent across multiple experiments. UT: ‘untargeted’ guide RNA (no target sequence in HBV genome). *p
    Figure Legend Snippet: Transiently transfected CRISPR constructs exhibit anti-HBV activity. ( a ) Schematic of HBV life cycle and putative anti-HBV effect of CRISPR constructs; Cas9-mediated DSB formation should linearize the small, episomal cccDNA repeatedly, potentially leading to indel formation (generating less-fit viral mutants) or even degradation. ( b ) (left) HBV genome organization and location of target sequences for several tested guide RNA constructs. (right) Table of all possible CRISPR target sites in each HBV ORF, including number of possible target sites in conserved genomic regions. ( c ) Experimental schematic for ( d - e ): HepG2 cells are co-transfected with 1.3x WT HBV and sgRNA/Cas9-2A-mCherry construct, and ( d ) intracellular HBV pregenomic RNA and ( e ) secreted HBsAg are quantified after 72 hours. Data shown were generated in one representative experiment, with intracellular pgRNA harvested from one pellet and HBsAg collected from replicate wells per group; all data are consistent across three independent experiments. ( f ) Experimental schematic for ( g - h ): 1.3x WT HBV and sgRNA/Cas9-2A-mCherry are delivered to the livers of immunodeficient NRG mice via hydrodynamic injection, and ( g ) HBsAg and ( h ) secreted HBV titer are quantified in mouse blood at 2 and 4 days post injection. 21M: guide RNA with 5 bp mismatch from g21. Data shown are from one representative experiment, and consistent across multiple experiments. UT: ‘untargeted’ guide RNA (no target sequence in HBV genome). *p

    Techniques Used: Transfection, CRISPR, Construct, Activity Assay, Generated, Mouse Assay, Injection, Sequencing

    20) Product Images from "miR-147b-mediated TCA cycle dysfunction and pseudohypoxia initiate drug tolerance to EGFR inhibitors in lung adenocarcinoma"

    Article Title: miR-147b-mediated TCA cycle dysfunction and pseudohypoxia initiate drug tolerance to EGFR inhibitors in lung adenocarcinoma

    Journal: Nature metabolism

    doi: 10.1038/s42255-019-0052-9

    miR-147b initiates drug-tolerance. a , A heat map showing top upregulated and downregulated miRNAs in two paired osimertinib-tolerant (OTR) and parental cells in PC9 and HCC827 by miRNA-seq analysis. b , qRT-PCR analysis of miR-147b expressions in parental, recovered, primary and secondary osimertinib-tolerant cells in PC9. The parental tumor cells treated with 160 nM EGFR-TKI osimertinib for 6 days enter a drug-tolerant state (primary tolerant cells) with a brief therapy withdrawal up to 18 days (recovered cells) followed by reinstatement of the 160 nM dose for 11 days (secondary tolerant cells). The relative miR-147b expression level in the parental cells were calibrated as 1. miR-423 was used as endogenous control. n=3 independent biological replicates. c -d, Osimertinib (c) and gefitinib (d) treatment response on scrambled control (Scr) and miR-147b-overexpressing cells (147b) in HCC827 for 3 days. n=3 independent biological replicates. e , Osimertinib (40 nM) and gefitinib (40 nM) treatment response on scrambled control and miR-147b-overexpressing cells in HCC827 by colony formation assay. 20, 40 and 80 cells were plated in 10-cm dish and the colonies were stained with Giemsa on day 10 and the total number of colonies were quantified. n=3 independent biological replicates. f , Osimertinib treatment response on H1975 cells with miR-147b knockdown (anti147b) and scrambled control (antictrl). The cell viability was measured on day 4. n=3 independent biological replicates. g , Osimertinib (160 nM) treatment response on H1975 cells with miR-147b knockdown. Left, the monolayer colonies were treated for 10 days and stained with Giemsa. Right, the 3D structures were treated for 14 days. -, vehicle; +, osimertinib. Scale bar, 1000 μm. n=3 independent biological replicates. Data are mean ± s.e.m. and were analysed with one-way ANOVA ( b ); unpaired two-tailed t -test with Holm-Sidak’s correction ( e,g ).
    Figure Legend Snippet: miR-147b initiates drug-tolerance. a , A heat map showing top upregulated and downregulated miRNAs in two paired osimertinib-tolerant (OTR) and parental cells in PC9 and HCC827 by miRNA-seq analysis. b , qRT-PCR analysis of miR-147b expressions in parental, recovered, primary and secondary osimertinib-tolerant cells in PC9. The parental tumor cells treated with 160 nM EGFR-TKI osimertinib for 6 days enter a drug-tolerant state (primary tolerant cells) with a brief therapy withdrawal up to 18 days (recovered cells) followed by reinstatement of the 160 nM dose for 11 days (secondary tolerant cells). The relative miR-147b expression level in the parental cells were calibrated as 1. miR-423 was used as endogenous control. n=3 independent biological replicates. c -d, Osimertinib (c) and gefitinib (d) treatment response on scrambled control (Scr) and miR-147b-overexpressing cells (147b) in HCC827 for 3 days. n=3 independent biological replicates. e , Osimertinib (40 nM) and gefitinib (40 nM) treatment response on scrambled control and miR-147b-overexpressing cells in HCC827 by colony formation assay. 20, 40 and 80 cells were plated in 10-cm dish and the colonies were stained with Giemsa on day 10 and the total number of colonies were quantified. n=3 independent biological replicates. f , Osimertinib treatment response on H1975 cells with miR-147b knockdown (anti147b) and scrambled control (antictrl). The cell viability was measured on day 4. n=3 independent biological replicates. g , Osimertinib (160 nM) treatment response on H1975 cells with miR-147b knockdown. Left, the monolayer colonies were treated for 10 days and stained with Giemsa. Right, the 3D structures were treated for 14 days. -, vehicle; +, osimertinib. Scale bar, 1000 μm. n=3 independent biological replicates. Data are mean ± s.e.m. and were analysed with one-way ANOVA ( b ); unpaired two-tailed t -test with Holm-Sidak’s correction ( e,g ).

    Techniques Used: Quantitative RT-PCR, Expressing, Colony Assay, Staining, Two Tailed Test

    NSCLC cells adopt a tolerance strategy against EGFR-TKIs. a, Representative phase contrast images of 3D structures from AALE cells cultured according to the protocol at top of the panel. Scale bar, 50 μm. Repeated six times with similar results. b , Top, the scenario of anti-EGFR tolerance and resistance in lung cancer. The tumor cells treated with the EGFR-TKI gefitinib or osimertinib enter a reversible drug-tolerant cycle (in green arrows, 1° Tolerant) with a brief therapy withdrawal (up to 21 days) followed by reinstatement of the 160 nM dose (2° Tolerant). Alternatively, the tumor cells treated continuously with gefitinib or osimertinib without therapy interruption undergo drug-tolerance briefly and go into a drug-resistance state in which cells do not respond to gefitinib (1° Resistant)/osimertinib (2° Resistant). Bottom, osimertinib treatment response on HCC827 3D structures. Representative images of Parental cells, 1° Tolerant cells (derived from the Parental cells treated with 160 nM osimertinib for 11 days), Recovered cells (derived from the 1° Tolerant cells with a therapy withdrawal up to 21 days) and 2° Tolerant cells (derived from the Recovered cells by reinstatement of the 160 nM dose for 11 days). Scale bar, 200 μm. Repeated six times with similar results. c, Representative phase contrast microscopy (left panel) and H E staining of HCC827 3D structures derived from parental (top) and osimertinib-tolerant (bottom) cells. Images in blue dotted squares (middle panel) were amplified (right panel) and shown. Scale bar, 50 μm. Repeated six times with similar results. d, qRT-PCR analysis of SFTPC , HOPX , ID2 and CEACAM5 expression in single cell clone HCC827-derived 3D structures in the presence of osimertinib. Single cell clone derived cells were plated with geltrex and treated with 100 nM osimertinib (tolerant) or vehicle (parental) for 24 days. Gene expression for surviving 3D structures were analyzed. n=3 independent biological replicates. e , Single-cell clonogenicity of PC9 cells treated with gefitinib. A single cell was sorted by FACS into a 96-well plate and treated with 0.1, 0.4, and 2 μM gefitinib or the vehicle for 14 days. The frequency of colony formation was calculated as a ratio of the total number of colonies to the total number of wells plated with a single cell in a 96-well plate. n=3 independent biological replicates. f, qRT-PCR analysis of top upregulated and downregulated genes in gefitinib-tolerant clones (n=2) compared with vehicle-treated parental single cell clone (n=1) in PC9. The gene expression in parental sensitive clone was calibrated as 1. ACTB was used as endogenous control. n=4 independent biological replicates. g, Whole transcriptome and gene ontology analysis of gefitinib-tolerant clones compared with the parental single cell clone in PC9. n=4 independent biological replicates. h, qRT-PCR analysis of genes in top regulated signaling pathways including Wnt planar cell polarity signaling, glutamine metabolic process, cellular response to hypoxia, and tricarboxylic acid cycle in gefitinib-tolerant clones compared with parental the single cell clone in PC9. The gene expression in parental sensitive clone was calibrated as 1. ACTB was used as endogenous control. n=4 independent biological replicates. Data are mean ± s.e.m. and were analysed with unpaired two-tailed t -test with Welch’s correction ( d ); modified one-tailed Fisher’s Exact test ( g).
    Figure Legend Snippet: NSCLC cells adopt a tolerance strategy against EGFR-TKIs. a, Representative phase contrast images of 3D structures from AALE cells cultured according to the protocol at top of the panel. Scale bar, 50 μm. Repeated six times with similar results. b , Top, the scenario of anti-EGFR tolerance and resistance in lung cancer. The tumor cells treated with the EGFR-TKI gefitinib or osimertinib enter a reversible drug-tolerant cycle (in green arrows, 1° Tolerant) with a brief therapy withdrawal (up to 21 days) followed by reinstatement of the 160 nM dose (2° Tolerant). Alternatively, the tumor cells treated continuously with gefitinib or osimertinib without therapy interruption undergo drug-tolerance briefly and go into a drug-resistance state in which cells do not respond to gefitinib (1° Resistant)/osimertinib (2° Resistant). Bottom, osimertinib treatment response on HCC827 3D structures. Representative images of Parental cells, 1° Tolerant cells (derived from the Parental cells treated with 160 nM osimertinib for 11 days), Recovered cells (derived from the 1° Tolerant cells with a therapy withdrawal up to 21 days) and 2° Tolerant cells (derived from the Recovered cells by reinstatement of the 160 nM dose for 11 days). Scale bar, 200 μm. Repeated six times with similar results. c, Representative phase contrast microscopy (left panel) and H E staining of HCC827 3D structures derived from parental (top) and osimertinib-tolerant (bottom) cells. Images in blue dotted squares (middle panel) were amplified (right panel) and shown. Scale bar, 50 μm. Repeated six times with similar results. d, qRT-PCR analysis of SFTPC , HOPX , ID2 and CEACAM5 expression in single cell clone HCC827-derived 3D structures in the presence of osimertinib. Single cell clone derived cells were plated with geltrex and treated with 100 nM osimertinib (tolerant) or vehicle (parental) for 24 days. Gene expression for surviving 3D structures were analyzed. n=3 independent biological replicates. e , Single-cell clonogenicity of PC9 cells treated with gefitinib. A single cell was sorted by FACS into a 96-well plate and treated with 0.1, 0.4, and 2 μM gefitinib or the vehicle for 14 days. The frequency of colony formation was calculated as a ratio of the total number of colonies to the total number of wells plated with a single cell in a 96-well plate. n=3 independent biological replicates. f, qRT-PCR analysis of top upregulated and downregulated genes in gefitinib-tolerant clones (n=2) compared with vehicle-treated parental single cell clone (n=1) in PC9. The gene expression in parental sensitive clone was calibrated as 1. ACTB was used as endogenous control. n=4 independent biological replicates. g, Whole transcriptome and gene ontology analysis of gefitinib-tolerant clones compared with the parental single cell clone in PC9. n=4 independent biological replicates. h, qRT-PCR analysis of genes in top regulated signaling pathways including Wnt planar cell polarity signaling, glutamine metabolic process, cellular response to hypoxia, and tricarboxylic acid cycle in gefitinib-tolerant clones compared with parental the single cell clone in PC9. The gene expression in parental sensitive clone was calibrated as 1. ACTB was used as endogenous control. n=4 independent biological replicates. Data are mean ± s.e.m. and were analysed with unpaired two-tailed t -test with Welch’s correction ( d ); modified one-tailed Fisher’s Exact test ( g).

    Techniques Used: Cell Culture, Derivative Assay, Microscopy, Staining, Amplification, Quantitative RT-PCR, Expressing, FACS, Clone Assay, Two Tailed Test, Modification, One-tailed Test

    miR-147b-VHL axis mediates drug-tolerance through impaired VHL activity. a , Left, gene candidates predicted for miR-147b by the TargetScan tool were shown in signaling pathways enriched for gefitinib-tolerance in PC9 single-cell clones in fig. 1f . Right, qRT-PCR analysis for the predicted gene candidates for miR-147b in H1975 cells with miR-147b knockdown compared with scrambled control. n=3 independent biological replicates. b , Left, computational prediction of RNA duplex formation between miR-147b and the 3’UTR (untranslated region) of VHL mRNA. Mutations generated within the 3’UTR for the luciferase assay are shown in red. Right, dual-luciferase reporter assay in miR-147b-overexpressing AALE cells. The Firefly luciferase and Renilla luciferase activities were measured 48 hours post co-transfection with miR-147b or control vector and wild-type (WT) or mutant (Mut) VHL 3’UTR. n=3 independent biological replicates. c , Western blot analysis and quantification of VHL in miR-147b-overexpressing AALE cells. β-Actin was used as loading control. n=3 independent biological replicates. d , qRT-PCR analysis for fold change of hypoxia gene expression in AALE cells with miR-147b overexpression relative to scrambled control (147b/Scr) and cells with co-overexpression of miR-147b and VHL relative to scrambled control (147b+VHL/Scr). ACTB was used as endogenous control. n=3 independent biological replicates. e , Fractional viability of HCC827 cells treated with vehicle, osimertinib (20 nM), miR-147b vector, VHL vector or combinations. The cell viability was measured on day 3. The relative cell viability treated with vehicle on day 3 was calibrated as 1. n=7 independent biological replicates. Data are mean ± s.e.m. and were analysed with unpaired two-tailed t -test ( a , b , c , d ); Kruskal-Wallis test (e).
    Figure Legend Snippet: miR-147b-VHL axis mediates drug-tolerance through impaired VHL activity. a , Left, gene candidates predicted for miR-147b by the TargetScan tool were shown in signaling pathways enriched for gefitinib-tolerance in PC9 single-cell clones in fig. 1f . Right, qRT-PCR analysis for the predicted gene candidates for miR-147b in H1975 cells with miR-147b knockdown compared with scrambled control. n=3 independent biological replicates. b , Left, computational prediction of RNA duplex formation between miR-147b and the 3’UTR (untranslated region) of VHL mRNA. Mutations generated within the 3’UTR for the luciferase assay are shown in red. Right, dual-luciferase reporter assay in miR-147b-overexpressing AALE cells. The Firefly luciferase and Renilla luciferase activities were measured 48 hours post co-transfection with miR-147b or control vector and wild-type (WT) or mutant (Mut) VHL 3’UTR. n=3 independent biological replicates. c , Western blot analysis and quantification of VHL in miR-147b-overexpressing AALE cells. β-Actin was used as loading control. n=3 independent biological replicates. d , qRT-PCR analysis for fold change of hypoxia gene expression in AALE cells with miR-147b overexpression relative to scrambled control (147b/Scr) and cells with co-overexpression of miR-147b and VHL relative to scrambled control (147b+VHL/Scr). ACTB was used as endogenous control. n=3 independent biological replicates. e , Fractional viability of HCC827 cells treated with vehicle, osimertinib (20 nM), miR-147b vector, VHL vector or combinations. The cell viability was measured on day 3. The relative cell viability treated with vehicle on day 3 was calibrated as 1. n=7 independent biological replicates. Data are mean ± s.e.m. and were analysed with unpaired two-tailed t -test ( a , b , c , d ); Kruskal-Wallis test (e).

    Techniques Used: Activity Assay, Clone Assay, Quantitative RT-PCR, Generated, Luciferase, Reporter Assay, Cotransfection, Plasmid Preparation, Mutagenesis, Western Blot, Expressing, Over Expression, Two Tailed Test

    21) Product Images from "PCR-Based Method for Isolation and Detection of Chlamydia pneumoniae DNA in Cerebrospinal Fluids"

    Article Title: PCR-Based Method for Isolation and Detection of Chlamydia pneumoniae DNA in Cerebrospinal Fluids

    Journal: Clinical and Diagnostic Laboratory Immunology

    doi: 10.1128/CDLI.8.3.499-502.2001

    Optimization of annealing temperature and Mg 2+ concentration for PCR with primers for omp1 . The annealing temperature was optimized in a Mastercycler gradient (Eppendorf). The Mg concentration for PCR specific for omp1 was optimized with a PCR optimization kit (Invitrogen). The target DNA for PCR was extracted from C. pneumoniae with the QIAmp DNA Mini Kit.
    Figure Legend Snippet: Optimization of annealing temperature and Mg 2+ concentration for PCR with primers for omp1 . The annealing temperature was optimized in a Mastercycler gradient (Eppendorf). The Mg concentration for PCR specific for omp1 was optimized with a PCR optimization kit (Invitrogen). The target DNA for PCR was extracted from C. pneumoniae with the QIAmp DNA Mini Kit.

    Techniques Used: Concentration Assay, Polymerase Chain Reaction

    Detection sensitivity of PCR for omp1 versus the 16S rRNA gene. Two hundred microliters of the mock CSF containing a specific number of C. pneumoniae organisms (as indicated), was used for DNA extraction with the QIAmp DNA Mini Kit. A 2-μl portion of the 50-μl volume of DNA extracts was subjected to PCR. The optimized PCRs for omp1 and 16S rRNA gene were conducted (see Materials and Methods).
    Figure Legend Snippet: Detection sensitivity of PCR for omp1 versus the 16S rRNA gene. Two hundred microliters of the mock CSF containing a specific number of C. pneumoniae organisms (as indicated), was used for DNA extraction with the QIAmp DNA Mini Kit. A 2-μl portion of the 50-μl volume of DNA extracts was subjected to PCR. The optimized PCRs for omp1 and 16S rRNA gene were conducted (see Materials and Methods).

    Techniques Used: Polymerase Chain Reaction, DNA Extraction

    Comparison of extraction efficacy of C. pneumoniae DNA from mock CSFs using two DNA extraction kits. The mock CSFs (200 μl) spiked with serially diluted bacteria were extracted either with the QIAmp DNA Mini Kit with a bacterial DNA extraction protocol or with the QIAmp DNA Blood Mini Kit. Two microliters of the extracted DNA (50 μl) was subjected to PCR with primers for omp1 . Results are representative of three experiments.
    Figure Legend Snippet: Comparison of extraction efficacy of C. pneumoniae DNA from mock CSFs using two DNA extraction kits. The mock CSFs (200 μl) spiked with serially diluted bacteria were extracted either with the QIAmp DNA Mini Kit with a bacterial DNA extraction protocol or with the QIAmp DNA Blood Mini Kit. Two microliters of the extracted DNA (50 μl) was subjected to PCR with primers for omp1 . Results are representative of three experiments.

    Techniques Used: DNA Extraction, Polymerase Chain Reaction

    Detection of C. pneumoniae DNA by PCR in CSFs obtained from patients with MS. Two hundred microliters of CSFs obtained from MS patients was processed for extraction of DNA utilizing the QIAmp DNA Mini Kit, and 2 μl of the resulting 50-μl DNA solution was subjected to PCR with primers for either omp1 or the 16S rRNA gene. Data are representative of three PCR experiments. Boxed numbers indicate CSFs obtained from the same patient at different time points. Each number is the sample number. M, molecular marker; PC, positive control for PCR; NC, negative control for PCR; IC, negative control for DNA extraction.
    Figure Legend Snippet: Detection of C. pneumoniae DNA by PCR in CSFs obtained from patients with MS. Two hundred microliters of CSFs obtained from MS patients was processed for extraction of DNA utilizing the QIAmp DNA Mini Kit, and 2 μl of the resulting 50-μl DNA solution was subjected to PCR with primers for either omp1 or the 16S rRNA gene. Data are representative of three PCR experiments. Boxed numbers indicate CSFs obtained from the same patient at different time points. Each number is the sample number. M, molecular marker; PC, positive control for PCR; NC, negative control for PCR; IC, negative control for DNA extraction.

    Techniques Used: Polymerase Chain Reaction, Mass Spectrometry, Marker, Positive Control, Negative Control, DNA Extraction

    22) Product Images from "Central metabolism of functionally heterogeneous mesenchymal stromal cells"

    Article Title: Central metabolism of functionally heterogeneous mesenchymal stromal cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-51937-9

    Mitochondrial DNA copy number and methylation are molecular markers of LL- or SL-CBMSC identity. ( A ) LL-CBMSC showed high mtDNAcn compared to SL-CBMSC. Mean and standard deviation are represented; *p
    Figure Legend Snippet: Mitochondrial DNA copy number and methylation are molecular markers of LL- or SL-CBMSC identity. ( A ) LL-CBMSC showed high mtDNAcn compared to SL-CBMSC. Mean and standard deviation are represented; *p

    Techniques Used: Methylation, Standard Deviation

    SL-CBMSC as compared to LL-CBMSC show a decrease of mitochondrial OXPHOS expression and function associated to higher mitochondrial potential. Cells were cultured for 48 hours in standard growth condition and then analyzed. ( A ) LL-CBMSC showed higher mtDNAcn, assessed by qPCR. Mean and standard deviation are represented. ( B ) qPCR analysis of the expression levels of the indicated mRNA, codified by mitochondrial DNA (mtDNA) and nuclear DNA (nuDNA). Actin was used as housekeeping gene to normalize the expression levels. ( C ) Western blot analysis of the expression levels of the indicated mitochondrial proteins. Vinculin was used to normalize the expression levels. Blots shown are derived from multiple gels run under the same experimental conditions. The membrane was cut based on molecular weight. All full length blots are presented in supplementary Fig. 4A . ( D , E ) Basal ( D ) and ATP production-coupled ( E ) oxygen consumption rate (OCR). The latter was calculated subtracting the OCR upon oligomycin to the basal OCR. Data are represented as mean ± SEM of the different cell populations. For each cell population n ≥ 2 biological analyses were performed with n ≥ 5 replicates for each. ( F ) Representative confocal microscopic images after staining with TMRE (50 nM). Scale bar 20 µm. ( G - H ) Quantification of TMRE signal using confocal microscopy ( G ) and flow cytometry ( H ). Mean and standard deviation are represented. For panels B-H, ns, not statistically significant; *p
    Figure Legend Snippet: SL-CBMSC as compared to LL-CBMSC show a decrease of mitochondrial OXPHOS expression and function associated to higher mitochondrial potential. Cells were cultured for 48 hours in standard growth condition and then analyzed. ( A ) LL-CBMSC showed higher mtDNAcn, assessed by qPCR. Mean and standard deviation are represented. ( B ) qPCR analysis of the expression levels of the indicated mRNA, codified by mitochondrial DNA (mtDNA) and nuclear DNA (nuDNA). Actin was used as housekeeping gene to normalize the expression levels. ( C ) Western blot analysis of the expression levels of the indicated mitochondrial proteins. Vinculin was used to normalize the expression levels. Blots shown are derived from multiple gels run under the same experimental conditions. The membrane was cut based on molecular weight. All full length blots are presented in supplementary Fig. 4A . ( D , E ) Basal ( D ) and ATP production-coupled ( E ) oxygen consumption rate (OCR). The latter was calculated subtracting the OCR upon oligomycin to the basal OCR. Data are represented as mean ± SEM of the different cell populations. For each cell population n ≥ 2 biological analyses were performed with n ≥ 5 replicates for each. ( F ) Representative confocal microscopic images after staining with TMRE (50 nM). Scale bar 20 µm. ( G - H ) Quantification of TMRE signal using confocal microscopy ( G ) and flow cytometry ( H ). Mean and standard deviation are represented. For panels B-H, ns, not statistically significant; *p

    Techniques Used: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Standard Deviation, Western Blot, Derivative Assay, Molecular Weight, Staining, Confocal Microscopy, Flow Cytometry, Cytometry

    23) Product Images from "Towards standardisation of cell-free DNA measurement in plasma: controls for extraction efficiency, fragment size bias and quantification"

    Article Title: Towards standardisation of cell-free DNA measurement in plasma: controls for extraction efficiency, fragment size bias and quantification

    Journal: Analytical and Bioanalytical Chemistry

    doi: 10.1007/s00216-014-7835-3

    Assessment of cell-free DNA (cfDNA) yield using four extraction methods. The mean yield ± one standard deviation from replicate extractions using plasma pool A (i) (with or without ADH ) performed with the QIAamp circulating nucleic acid ( CNA ), NucleoSpin Plasma XS ( NS ) and FitAmp plasma/serum DNA isolation ( FA ) kits ( n = 10) and the QIAamp DNA blood mini ( DBM ) kit ( n = 9) is displayed relative to the mean yield of the DBM kit. The yield of cfDNA was quantified by quantitative PCR (qPCR) assays to TERT and ALUJ
    Figure Legend Snippet: Assessment of cell-free DNA (cfDNA) yield using four extraction methods. The mean yield ± one standard deviation from replicate extractions using plasma pool A (i) (with or without ADH ) performed with the QIAamp circulating nucleic acid ( CNA ), NucleoSpin Plasma XS ( NS ) and FitAmp plasma/serum DNA isolation ( FA ) kits ( n = 10) and the QIAamp DNA blood mini ( DBM ) kit ( n = 9) is displayed relative to the mean yield of the DBM kit. The yield of cfDNA was quantified by quantitative PCR (qPCR) assays to TERT and ALUJ

    Techniques Used: Standard Deviation, DNA Extraction, Real-time Polymerase Chain Reaction

    24) Product Images from "Unlabeled Oligonucleotides as Internal Temperature Controls for Genotyping by Amplicon Melting"

    Article Title: Unlabeled Oligonucleotides as Internal Temperature Controls for Genotyping by Amplicon Melting

    Journal: The Journal of molecular diagnostics : JMD

    doi: 10.2353/jmoldx.2007.060136

    Derivative melting plots of MTHFR samples extracted using different methods. Samples were extracted in parallel using the MagNA Pure LC system (thick black line), QIAamp DNA blood mini kit (thin black line), and the Puregene DNA blood kit (dotted black
    Figure Legend Snippet: Derivative melting plots of MTHFR samples extracted using different methods. Samples were extracted in parallel using the MagNA Pure LC system (thick black line), QIAamp DNA blood mini kit (thin black line), and the Puregene DNA blood kit (dotted black

    Techniques Used:

    25) Product Images from "Efficient Extraction of Viral DNA and Viral RNA by the Chemagic Viral DNA/RNA Kit Allows Sensitive Detection of Cytomegalovirus, Hepatitis B Virus, and Hepatitis G Virus by PCR"

    Article Title: Efficient Extraction of Viral DNA and Viral RNA by the Chemagic Viral DNA/RNA Kit Allows Sensitive Detection of Cytomegalovirus, Hepatitis B Virus, and Hepatitis G Virus by PCR

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.41.11.5273-5276.2003

    Amplification of a serial dilution of the EUROHEP standard with HBV-specific primers in a nested PCR following extraction with the Chemagic DNA/RNA kit and the QIAamp DNA Blood Mini kit. (A) Chemagen extracts. (B) QIAGEN extracts. Lane M shows a 100-bp ladder, and lane C shows results for the negative control. Lanes 1 through 10 show results for different viral loads expressed in numbers of copies per ml: 1, 4 × 10 5 ; 2, 4 × 10 4 ; 3, 4 × 10 3 ; 4, 4 × 10 2 ; 5, 2 × 10 2 ; 6, 1 × 10 2 ; 7, 8 × 10 1 ; 8, 4 × 10 1 ; 9, 2 × 10 1 ; 10, 1 × 10 1 .
    Figure Legend Snippet: Amplification of a serial dilution of the EUROHEP standard with HBV-specific primers in a nested PCR following extraction with the Chemagic DNA/RNA kit and the QIAamp DNA Blood Mini kit. (A) Chemagen extracts. (B) QIAGEN extracts. Lane M shows a 100-bp ladder, and lane C shows results for the negative control. Lanes 1 through 10 show results for different viral loads expressed in numbers of copies per ml: 1, 4 × 10 5 ; 2, 4 × 10 4 ; 3, 4 × 10 3 ; 4, 4 × 10 2 ; 5, 2 × 10 2 ; 6, 1 × 10 2 ; 7, 8 × 10 1 ; 8, 4 × 10 1 ; 9, 2 × 10 1 ; 10, 1 × 10 1 .

    Techniques Used: Amplification, Serial Dilution, Nested PCR, Negative Control

    Amplification of a serial dilution of the standard plasmid pCR-gpB by real-time PCR with CMV-specific primers (amplicon, 254 bp) on the LightCycler instrument (software, version 3.5; analysis method, fit points with manual noise band adjustment, hybridization probe format) following extraction with the Chemagic DNA/RNA kit and the QIAamp DNA Blood Mini kit. pCR-gpB concentrations (from left to right) were 2 × 10 5 , 2 × 10 4 , 2 × 10 3 , and 2 × 10 2 copies per ml of serum. Continuous line, Chemagen extracts; broken line, QIAGEN extracts. Cycle number, cycle number of the amplification reaction; F2/F1, quotient of fluorescence channel F2 (hybridization probe) and fluorescence channel F1 (fluorescein).
    Figure Legend Snippet: Amplification of a serial dilution of the standard plasmid pCR-gpB by real-time PCR with CMV-specific primers (amplicon, 254 bp) on the LightCycler instrument (software, version 3.5; analysis method, fit points with manual noise band adjustment, hybridization probe format) following extraction with the Chemagic DNA/RNA kit and the QIAamp DNA Blood Mini kit. pCR-gpB concentrations (from left to right) were 2 × 10 5 , 2 × 10 4 , 2 × 10 3 , and 2 × 10 2 copies per ml of serum. Continuous line, Chemagen extracts; broken line, QIAGEN extracts. Cycle number, cycle number of the amplification reaction; F2/F1, quotient of fluorescence channel F2 (hybridization probe) and fluorescence channel F1 (fluorescein).

    Techniques Used: Amplification, Serial Dilution, Plasmid Preparation, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Software, Hybridization, Fluorescence

    26) Product Images from "Gene expression profiling of hematologic malignant cell lines resistant to oncolytic virus treatment"

    Article Title: Gene expression profiling of hematologic malignant cell lines resistant to oncolytic virus treatment

    Journal: Oncotarget

    doi: 10.18632/oncotarget.13598

    Physical viral particle count by qPCR assay The number of physical DNA copies targeting E9L gene of vaccinia virus is plotted against time after viral infection on IM-9 and Ramos cell lines. The total DNA was isolated from infected cell harvest using QIAamp DNA blood mini kit. Data is shown on average of duplicate runs. JX-594:Pexa-Vec, WR: western reserve virus.
    Figure Legend Snippet: Physical viral particle count by qPCR assay The number of physical DNA copies targeting E9L gene of vaccinia virus is plotted against time after viral infection on IM-9 and Ramos cell lines. The total DNA was isolated from infected cell harvest using QIAamp DNA blood mini kit. Data is shown on average of duplicate runs. JX-594:Pexa-Vec, WR: western reserve virus.

    Techniques Used: Real-time Polymerase Chain Reaction, Infection, Isolation, Western Blot

    27) Product Images from "A Microfluidic Device with Integrated Sonication and Immunoprecipitation for Sensitive Epigenetic Assays"

    Article Title: A Microfluidic Device with Integrated Sonication and Immunoprecipitation for Sensitive Epigenetic Assays

    Journal: Analytical Chemistry

    doi: 10.1021/acs.analchem.5b04707

    Chromatin/gDNA fragmentation by on-chip sonication. Sonication was conducted under sine-wave 61 kHz AC with a sonication time of 3 s in each 10 s cycle. (a–c) Microscopic images of cross-linked GM12878 cells before and after sonication. Scale bar is 100 μm. (a) Before sonication. (b) Sonication for 6 cycles with V peak of 20 V. (c) Sonication for 12 cycles with V peak of 20 V. (d–f) Gel electrophoresis of DNA fragments after chromatin fragmentation (starting from 50,000 cross-linked cells in each experiment). (d) Chromatin fragmentation is stronger with crescent structures than without them in the microfluidic chamber under the same sonication conditions (30 cycles, V peak = 20 V). (e) Chromatin fragmentation increases with higher V peak . Thirty cycles were applied for each sample. (f) Chromatin fragmentation increases with higher number of sonication cycles (12–48 cycles). V peak was 20 V for all the samples. (g) gDNA fragmentation under various sonication conditions measured by gel electrophoresis ( V peak of 15–25 V, 12–24 cycles); 200 ng of genomic DNA was used in each experiment.
    Figure Legend Snippet: Chromatin/gDNA fragmentation by on-chip sonication. Sonication was conducted under sine-wave 61 kHz AC with a sonication time of 3 s in each 10 s cycle. (a–c) Microscopic images of cross-linked GM12878 cells before and after sonication. Scale bar is 100 μm. (a) Before sonication. (b) Sonication for 6 cycles with V peak of 20 V. (c) Sonication for 12 cycles with V peak of 20 V. (d–f) Gel electrophoresis of DNA fragments after chromatin fragmentation (starting from 50,000 cross-linked cells in each experiment). (d) Chromatin fragmentation is stronger with crescent structures than without them in the microfluidic chamber under the same sonication conditions (30 cycles, V peak = 20 V). (e) Chromatin fragmentation increases with higher V peak . Thirty cycles were applied for each sample. (f) Chromatin fragmentation increases with higher number of sonication cycles (12–48 cycles). V peak was 20 V for all the samples. (g) gDNA fragmentation under various sonication conditions measured by gel electrophoresis ( V peak of 15–25 V, 12–24 cycles); 200 ng of genomic DNA was used in each experiment.

    Techniques Used: Chromatin Immunoprecipitation, Sonication, Nucleic Acid Electrophoresis

    Microfluidic ChIP-qPCR and MeDIP-qPCR conducted using our device. (a) qPCR analysis of ChIP DNA at two known positive loci and two negative loci using cross-linked cells from 10,000 to 100 cells. (b) qPCR analysis of MeDIP DNA at two known positive loci and two negative loci using single-stranded gDNA ranging from 50 ng down to 500 pg.
    Figure Legend Snippet: Microfluidic ChIP-qPCR and MeDIP-qPCR conducted using our device. (a) qPCR analysis of ChIP DNA at two known positive loci and two negative loci using cross-linked cells from 10,000 to 100 cells. (b) qPCR analysis of MeDIP DNA at two known positive loci and two negative loci using single-stranded gDNA ranging from 50 ng down to 500 pg.

    Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Methylated DNA Immunoprecipitation

    28) Product Images from "Herpes Simplex Virus Type 2 Infection-Induced Expression of CXCR3 Ligands Promotes CD4+ T Cell Migration and Is Regulated by the Viral Immediate-Early Protein ICP4"

    Article Title: Herpes Simplex Virus Type 2 Infection-Induced Expression of CXCR3 Ligands Promotes CD4+ T Cell Migration and Is Regulated by the Viral Immediate-Early Protein ICP4

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02932

    HSV-2 ICP4 regulates the expression of CXCR3 ligands via the p38 MAPK signaling pathway. (A–C) HSV-2 regulates the expression of CXCL9 (A) , CXCL10 (B) , and CXCL11 (C) via p38/MAPK signaling pathway. ME180 cells in 24-well plates were co-transfected with 150 ng CXCR3 ligand reporter and 15 ng phRL-TK. At 4 h post-transfection, cells were infected with HSV-2 at an MOI of 1 and supplemented with inhibitor PD98059, SP600125, or SB203580. DLR assay was performed at 24 h post-transfection. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in mock-infected samples. (D–F) HSV-2 ICP4 regulates the expression of CXCL9 (D) , CXCL10 (E) , and CXCL11 (F) via p38/MAPK signaling pathway. ME180 cells in 24-well plates were co-transfected with 300 ng empty vector or ICP4 expression plasmid together with 150 ng CXCR3 ligand reporter and 15 ng phRL-TK. At 4 h post-transfection, cells were cultured in complete DMEM supplemented with inhibitor PD98059, SP600125, or SB203580. DLR assay was performed at 24 h post-transfection. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in cells transfected with empty vector. (G) ICP4 activates p38 MAPK signaling pathway. ME180 cells were transfected with 3 μg ICP4 expression plasmid. The protein level of p38, phospho-p38 (p-p38) or phospho-C/EBP-β (p-C/EBP-β) was detected by Western Blot. Data shown are mean ± S.D. of three independent experiments (A–F) . ns, not significant, *** p
    Figure Legend Snippet: HSV-2 ICP4 regulates the expression of CXCR3 ligands via the p38 MAPK signaling pathway. (A–C) HSV-2 regulates the expression of CXCL9 (A) , CXCL10 (B) , and CXCL11 (C) via p38/MAPK signaling pathway. ME180 cells in 24-well plates were co-transfected with 150 ng CXCR3 ligand reporter and 15 ng phRL-TK. At 4 h post-transfection, cells were infected with HSV-2 at an MOI of 1 and supplemented with inhibitor PD98059, SP600125, or SB203580. DLR assay was performed at 24 h post-transfection. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in mock-infected samples. (D–F) HSV-2 ICP4 regulates the expression of CXCL9 (D) , CXCL10 (E) , and CXCL11 (F) via p38/MAPK signaling pathway. ME180 cells in 24-well plates were co-transfected with 300 ng empty vector or ICP4 expression plasmid together with 150 ng CXCR3 ligand reporter and 15 ng phRL-TK. At 4 h post-transfection, cells were cultured in complete DMEM supplemented with inhibitor PD98059, SP600125, or SB203580. DLR assay was performed at 24 h post-transfection. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in cells transfected with empty vector. (G) ICP4 activates p38 MAPK signaling pathway. ME180 cells were transfected with 3 μg ICP4 expression plasmid. The protein level of p38, phospho-p38 (p-p38) or phospho-C/EBP-β (p-C/EBP-β) was detected by Western Blot. Data shown are mean ± S.D. of three independent experiments (A–F) . ns, not significant, *** p

    Techniques Used: Expressing, Transfection, Infection, Luciferase, Plasmid Preparation, Cell Culture, Western Blot

    HSV-2 infection induces the production of CXCR3 ligands in human cervical epithelial cells. (A) HSV-2 infection activates the promoters of human CXCR3 ligands. ME180 cells in 24-well plates were co-transfected with 150 ng CXCL9-Luc, CXCL10-Luc or CXCL11-Luc, and 15 ng internal control plasmid phRL-TK. At 4 h post-transfection, cells were infected with HSV-2 or ultraviolet-inactivated HSV-2 (UV-HSV-2) at an MOI of 1 for 24 h. DLR assay was performed. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in mock-infected samples. (B) HSV-2 infection induces the mRNA production of CXCR3 ligands. ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cells were harvested and total RNA was extracted. The expression of CXCR3 ligands and GAPDH was evaluated by relative real-time quantitative PCR. The Ct values of GAPDH among all groups were equable and not overloaded. mRNA copies of CXCR3 ligands were normalized using GAPDH and expressed as fold increase of the value for the mock-infected control. (C) HSV-2 infection induces the production of CXCR3 ligands. As depicted in (B) , cell supernatants were collected, and the protein level of CXCR3 ligands was measured by CBA. Data shown are mean ± S.D. of three independent experiments (A, B, and C). * p
    Figure Legend Snippet: HSV-2 infection induces the production of CXCR3 ligands in human cervical epithelial cells. (A) HSV-2 infection activates the promoters of human CXCR3 ligands. ME180 cells in 24-well plates were co-transfected with 150 ng CXCL9-Luc, CXCL10-Luc or CXCL11-Luc, and 15 ng internal control plasmid phRL-TK. At 4 h post-transfection, cells were infected with HSV-2 or ultraviolet-inactivated HSV-2 (UV-HSV-2) at an MOI of 1 for 24 h. DLR assay was performed. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in mock-infected samples. (B) HSV-2 infection induces the mRNA production of CXCR3 ligands. ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cells were harvested and total RNA was extracted. The expression of CXCR3 ligands and GAPDH was evaluated by relative real-time quantitative PCR. The Ct values of GAPDH among all groups were equable and not overloaded. mRNA copies of CXCR3 ligands were normalized using GAPDH and expressed as fold increase of the value for the mock-infected control. (C) HSV-2 infection induces the production of CXCR3 ligands. As depicted in (B) , cell supernatants were collected, and the protein level of CXCR3 ligands was measured by CBA. Data shown are mean ± S.D. of three independent experiments (A, B, and C). * p

    Techniques Used: Infection, Transfection, Plasmid Preparation, Luciferase, Expressing, Real-time Polymerase Chain Reaction, Crocin Bleaching Assay

    HSV-2 infection-induced CXCL9 plays a predominant role in mediating CD4 + T cell migration. (A) The concentrations of CXCR3 ligands in the supernatants of ME180 cells infected with HSV-2 or mock-infected with DMEM were detected by CBA. (B,C) CXCL9 induced by HSV-2 recruits the migration of PBMCs (B) and CD4 + T cells (C) . ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates in the absence or presence of anti-CXCL9, –CXCL10, or/and –CXCL11 neutralizing Ab or control Ab for 1 h. (D) Neutralization of CXCR3 reduces the migration of CD4 + T cells induced by HSV-2 infection. ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates. The activated CD4 + T cells were incubated with RPMI 1,640 medium containing anti-CXCR3 neutralizing Ab for 1 h and placed in the upper chamber. (E) Recombinant CXCL9 significantly induces the migration of CD4 + T cells. DMEM containing recombinant CXCL9, CXCL10, or CXCL11 (48 pg/mL, 55 pg/mL and 175 pg/mL, respectively; the lowest concentration induced by HSV-2 infection) was added to the lower chamber of transwell plates. (F,G) Recombinant CXCL10 or CXCL11 mediates the migration of CD4 + T cells in a dose-dependent manner. DMEM containing recombinant CXCL10 or CXCL11 was added to the lower chamber of transwell plates. CXCL10 or CXCL11 was started from 55 pg/mL and 175 pg/mL, respectively, at a concentration gradient of two times. The activated CD4 + T cells were placed in the upper chamber. After 2 h incubation, cells migrated to lower chambers were collected and counted using an automatic cell counter. Cells migration was expressed as percentage of input. Input cells in the upper chamber were 5 × 10 5 . Data shown are mean ± S.D. of three independent experiments (A–G) . ns, not significant, * p
    Figure Legend Snippet: HSV-2 infection-induced CXCL9 plays a predominant role in mediating CD4 + T cell migration. (A) The concentrations of CXCR3 ligands in the supernatants of ME180 cells infected with HSV-2 or mock-infected with DMEM were detected by CBA. (B,C) CXCL9 induced by HSV-2 recruits the migration of PBMCs (B) and CD4 + T cells (C) . ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates in the absence or presence of anti-CXCL9, –CXCL10, or/and –CXCL11 neutralizing Ab or control Ab for 1 h. (D) Neutralization of CXCR3 reduces the migration of CD4 + T cells induced by HSV-2 infection. ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates. The activated CD4 + T cells were incubated with RPMI 1,640 medium containing anti-CXCR3 neutralizing Ab for 1 h and placed in the upper chamber. (E) Recombinant CXCL9 significantly induces the migration of CD4 + T cells. DMEM containing recombinant CXCL9, CXCL10, or CXCL11 (48 pg/mL, 55 pg/mL and 175 pg/mL, respectively; the lowest concentration induced by HSV-2 infection) was added to the lower chamber of transwell plates. (F,G) Recombinant CXCL10 or CXCL11 mediates the migration of CD4 + T cells in a dose-dependent manner. DMEM containing recombinant CXCL10 or CXCL11 was added to the lower chamber of transwell plates. CXCL10 or CXCL11 was started from 55 pg/mL and 175 pg/mL, respectively, at a concentration gradient of two times. The activated CD4 + T cells were placed in the upper chamber. After 2 h incubation, cells migrated to lower chambers were collected and counted using an automatic cell counter. Cells migration was expressed as percentage of input. Input cells in the upper chamber were 5 × 10 5 . Data shown are mean ± S.D. of three independent experiments (A–G) . ns, not significant, * p

    Techniques Used: Infection, Migration, Crocin Bleaching Assay, Neutralization, Incubation, Recombinant, Concentration Assay

    HSV-2 ICP4 binds to the promoters of CXCR3 ligands by interaction with TBP. (A) Schematic representation of the predicted NLSs of ICP4 amino acid (AA) sequence. (B) ICP4 is located in the nucleus. ME180 cells in 35-mm dishes with glass bottom were transfected with 2 μg empty vector or HA-tagged ICP4 expression plasmid for 24 h. Cells were stained with mouse anti-HA mAb, followed by Cy3-conjugated goat anti-mouse (red) as the secondary Ab. Cell nuclei (blue) were stained with DAPI. The images were obtained by fluorescence microscopy using 60 × objective. The scale bar indicates 21 μm. (C) The expression of ICP4 was stained using anti-HA mAb. (D) ICP4 binds to the promoters of CXCR3 ligands. ME180 cells were transfected with 3 μg empty vector or HA-tagged ICP4 expression plasmid for 24 h. Cells were lysed and subjected to ChIP assay using mouse anti-HA mAb, mouse anti-RNA polymerase II mAb (positive control) or mouse normal IgG (negative control) for immunoprecipitation. (E) ICP4 interacts with TBP. ME180 cells in 6-well plates were transfected with 3 μg empty vector or HA-tagged ICP4 expression plasmid for 24 h. Cells were lysed and subjected to co-immunoprecipitation (IP) using rabbit anti-HA or anti-TBP Ab. Rabbit normal IgG was used as a negative control. IP products and 5% input samples were examined using rabbit anti-HA and rabbit anti-TBP Abs by western blot. One representative out of three independent experiments is shown (B–E) .
    Figure Legend Snippet: HSV-2 ICP4 binds to the promoters of CXCR3 ligands by interaction with TBP. (A) Schematic representation of the predicted NLSs of ICP4 amino acid (AA) sequence. (B) ICP4 is located in the nucleus. ME180 cells in 35-mm dishes with glass bottom were transfected with 2 μg empty vector or HA-tagged ICP4 expression plasmid for 24 h. Cells were stained with mouse anti-HA mAb, followed by Cy3-conjugated goat anti-mouse (red) as the secondary Ab. Cell nuclei (blue) were stained with DAPI. The images were obtained by fluorescence microscopy using 60 × objective. The scale bar indicates 21 μm. (C) The expression of ICP4 was stained using anti-HA mAb. (D) ICP4 binds to the promoters of CXCR3 ligands. ME180 cells were transfected with 3 μg empty vector or HA-tagged ICP4 expression plasmid for 24 h. Cells were lysed and subjected to ChIP assay using mouse anti-HA mAb, mouse anti-RNA polymerase II mAb (positive control) or mouse normal IgG (negative control) for immunoprecipitation. (E) ICP4 interacts with TBP. ME180 cells in 6-well plates were transfected with 3 μg empty vector or HA-tagged ICP4 expression plasmid for 24 h. Cells were lysed and subjected to co-immunoprecipitation (IP) using rabbit anti-HA or anti-TBP Ab. Rabbit normal IgG was used as a negative control. IP products and 5% input samples were examined using rabbit anti-HA and rabbit anti-TBP Abs by western blot. One representative out of three independent experiments is shown (B–E) .

    Techniques Used: Sequencing, Transfection, Plasmid Preparation, Expressing, Staining, Fluorescence, Microscopy, Chromatin Immunoprecipitation, Positive Control, Negative Control, Immunoprecipitation, Western Blot

    Contribution of HSV-2 infection-induced CXCR3 ligands to CD4 + T cell infiltration into mouse vagina. Seven days prior to HSV-2 challenge, BALB/c mice were injected with progesterone in multiple sites. One day prior to HSV-2 challenge, CXCL9, CXCL10, and CXCL11 neutralizing antibodies were delivered to the vagina of mice, alone or in combination, while isotype matched control IgG was used as the control. Mice were then anesthetized with pentobarbital sodium and challenged intravaginally with 10 μL/ mouse HSV-2 at a concentration of 6 × 10 7 PFU/ml or mock- challenged. Vaginal lavage fluids and cervical-vaginal tissues were collected at day 7 after challenge. (A) HSV-2 infection induces the production of mouse CXCR3 ligands. The protein levels of CXCL9 and CXCL10 ligands in vaginal lavage fluids were measured by CBA, and the protein level of CXCL11 was detected by ELISA. (B) CXCL9 mediates the migration of CD4 + T cells to the vaginal foci of infected mice. CD4 + T cells in infection foci were detected using anti-CD4 Ab by IHC. The scale bar indicates 100 μm. Data shown are mean ± S.D. ( n = 5 mice/group) of three independent experiments (A) . *** p
    Figure Legend Snippet: Contribution of HSV-2 infection-induced CXCR3 ligands to CD4 + T cell infiltration into mouse vagina. Seven days prior to HSV-2 challenge, BALB/c mice were injected with progesterone in multiple sites. One day prior to HSV-2 challenge, CXCL9, CXCL10, and CXCL11 neutralizing antibodies were delivered to the vagina of mice, alone or in combination, while isotype matched control IgG was used as the control. Mice were then anesthetized with pentobarbital sodium and challenged intravaginally with 10 μL/ mouse HSV-2 at a concentration of 6 × 10 7 PFU/ml or mock- challenged. Vaginal lavage fluids and cervical-vaginal tissues were collected at day 7 after challenge. (A) HSV-2 infection induces the production of mouse CXCR3 ligands. The protein levels of CXCL9 and CXCL10 ligands in vaginal lavage fluids were measured by CBA, and the protein level of CXCL11 was detected by ELISA. (B) CXCL9 mediates the migration of CD4 + T cells to the vaginal foci of infected mice. CD4 + T cells in infection foci were detected using anti-CD4 Ab by IHC. The scale bar indicates 100 μm. Data shown are mean ± S.D. ( n = 5 mice/group) of three independent experiments (A) . *** p

    Techniques Used: Infection, Mouse Assay, Injection, Concentration Assay, Crocin Bleaching Assay, Enzyme-linked Immunosorbent Assay, Migration, Immunohistochemistry

    29) Product Images from "ASF1a inhibition induces p53-dependent growth arrest and senescence of cancer cells"

    Article Title: ASF1a inhibition induces p53-dependent growth arrest and senescence of cancer cells

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-019-1357-z

    ASF1a knockdown leads to growth arrest and cellular senescence in wild-type p53-carrying HCC and PCa cells. a Western blot was performed for the validation of ASF1a siRNA efficiency in HepG2 and LNCaP cells. ASF1a si1 and ASF1a si2, two different ASF1A siRNAs, were used in this study. b The cell number of control (nc siRNA) and ASF1a knockdown (ASF1a si1/si2) groups in HepG2 and LNCaP cells 72 h after siRNA transfection. Cells in negative control groups were set as 100% (reference). The siASF1a-transfected groups showed a significant decrease in cell counts compared with control cells (data are presented as the mean ± SD of four independent experiments in HepG2 and three independent experiments in LNCaP, respectively; P values are shown in each panel). c Colony-formation assay of HepG2 and LNCaP cells. The clonogenic abilities of HepG2 and LNCaP cells were decreased upon ASF1a knockdown. Quantification is shown at the bottom (data are presented as the mean ± SD of three independent experiments in HepG2 and four independent experiments in LNCaP, respectively; P values are shown in each panel). Colonies in control groups were set as 100% (reference). d Cell cycles of HepG2 and LNCaP recorded by propidium iodide (PI) staining. Cell cycles in control groups are shown with red peaks and cell cycles in ASF1a knockdown groups are shown with blue peaks. Proportions of G0/G1, S, and G2/M phases are presented in pie charts (data presented are of three independent experiments in HepG2 and four independent experiments in LNCaP; P values are shown in each panel). e β-Gal staining of HepG2 and LNCaP cells. Senescent cells were stained with blue color (scale bar: 100 μm). f Quantification of β-gal staining-positive HepG2 and LNCaP cells (data are presented as the mean ± SD value of three independent experiments for HepG2 and LNCaP, respectively). HCC, hepatocellular carcinoma; PCa, prostate cancer
    Figure Legend Snippet: ASF1a knockdown leads to growth arrest and cellular senescence in wild-type p53-carrying HCC and PCa cells. a Western blot was performed for the validation of ASF1a siRNA efficiency in HepG2 and LNCaP cells. ASF1a si1 and ASF1a si2, two different ASF1A siRNAs, were used in this study. b The cell number of control (nc siRNA) and ASF1a knockdown (ASF1a si1/si2) groups in HepG2 and LNCaP cells 72 h after siRNA transfection. Cells in negative control groups were set as 100% (reference). The siASF1a-transfected groups showed a significant decrease in cell counts compared with control cells (data are presented as the mean ± SD of four independent experiments in HepG2 and three independent experiments in LNCaP, respectively; P values are shown in each panel). c Colony-formation assay of HepG2 and LNCaP cells. The clonogenic abilities of HepG2 and LNCaP cells were decreased upon ASF1a knockdown. Quantification is shown at the bottom (data are presented as the mean ± SD of three independent experiments in HepG2 and four independent experiments in LNCaP, respectively; P values are shown in each panel). Colonies in control groups were set as 100% (reference). d Cell cycles of HepG2 and LNCaP recorded by propidium iodide (PI) staining. Cell cycles in control groups are shown with red peaks and cell cycles in ASF1a knockdown groups are shown with blue peaks. Proportions of G0/G1, S, and G2/M phases are presented in pie charts (data presented are of three independent experiments in HepG2 and four independent experiments in LNCaP; P values are shown in each panel). e β-Gal staining of HepG2 and LNCaP cells. Senescent cells were stained with blue color (scale bar: 100 μm). f Quantification of β-gal staining-positive HepG2 and LNCaP cells (data are presented as the mean ± SD value of three independent experiments for HepG2 and LNCaP, respectively). HCC, hepatocellular carcinoma; PCa, prostate cancer

    Techniques Used: Western Blot, Transfection, Negative Control, Colony Assay, Staining

    30) Product Images from "Characterization of human bone morphogenetic protein gene variants for possible roles in congenital heart disease"

    Article Title: Characterization of human bone morphogenetic protein gene variants for possible roles in congenital heart disease

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2016.5428

    Schematic diagrams of rs1049007, rs235768 and rs17563 locations within the translated regions of the BMP 2 and 4 genes. BMP, bone mor phogenetic protein.
    Figure Legend Snippet: Schematic diagrams of rs1049007, rs235768 and rs17563 locations within the translated regions of the BMP 2 and 4 genes. BMP, bone mor phogenetic protein.

    Techniques Used:

    31) Product Images from "Mammalian cell surface display for monoclonal antibody-based FACS selection of viral envelope proteins"

    Article Title: Mammalian cell surface display for monoclonal antibody-based FACS selection of viral envelope proteins

    Journal: mAbs

    doi: 10.1080/19420862.2017.1364824

    TaqMan® Copy Number assay of stable cell lines. Stable cell lines were generated using either separate transfection of each of the pQL13-based Env/V3 chimeras or a mixture of all 5 Env/V3 chimeras for transfection (HEK293 cell library). Relative copy numbers of integrated pQL13 plasmids were explored using a TaqMan Copy Number Assay on 4 individual samples of genomic DNA of each cell line and probing for eGFP in relation to the human telomerase reverse transcriptase (TERT) genes, resulting in an integration rate of 1 for each human cell line (differences between the 6 stable cell lines statistically n.s., p > 0.05).
    Figure Legend Snippet: TaqMan® Copy Number assay of stable cell lines. Stable cell lines were generated using either separate transfection of each of the pQL13-based Env/V3 chimeras or a mixture of all 5 Env/V3 chimeras for transfection (HEK293 cell library). Relative copy numbers of integrated pQL13 plasmids were explored using a TaqMan Copy Number Assay on 4 individual samples of genomic DNA of each cell line and probing for eGFP in relation to the human telomerase reverse transcriptase (TERT) genes, resulting in an integration rate of 1 for each human cell line (differences between the 6 stable cell lines statistically n.s., p > 0.05).

    Techniques Used: TaqMan Copy Number Assay, Stable Transfection, Generated, Transfection

    32) Product Images from "miR-147b-mediated TCA cycle dysfunction and pseudohypoxia initiate drug tolerance to EGFR inhibitors in lung adenocarcinoma"

    Article Title: miR-147b-mediated TCA cycle dysfunction and pseudohypoxia initiate drug tolerance to EGFR inhibitors in lung adenocarcinoma

    Journal: Nature metabolism

    doi: 10.1038/s42255-019-0052-9

    miR-147b initiates drug-tolerance. a , A heat map showing top upregulated and downregulated miRNAs in two paired osimertinib-tolerant (OTR) and parental cells in PC9 and HCC827 by miRNA-seq analysis. b , qRT-PCR analysis of miR-147b expressions in parental, recovered, primary and secondary osimertinib-tolerant cells in PC9. The parental tumor cells treated with 160 nM EGFR-TKI osimertinib for 6 days enter a drug-tolerant state (primary tolerant cells) with a brief therapy withdrawal up to 18 days (recovered cells) followed by reinstatement of the 160 nM dose for 11 days (secondary tolerant cells). The relative miR-147b expression level in the parental cells were calibrated as 1. miR-423 was used as endogenous control. n=3 independent biological replicates. c -d, Osimertinib (c) and gefitinib (d) treatment response on scrambled control (Scr) and miR-147b-overexpressing cells (147b) in HCC827 for 3 days. n=3 independent biological replicates. e , Osimertinib (40 nM) and gefitinib (40 nM) treatment response on scrambled control and miR-147b-overexpressing cells in HCC827 by colony formation assay. 20, 40 and 80 cells were plated in 10-cm dish and the colonies were stained with Giemsa on day 10 and the total number of colonies were quantified. n=3 independent biological replicates. f , Osimertinib treatment response on H1975 cells with miR-147b knockdown (anti147b) and scrambled control (antictrl). The cell viability was measured on day 4. n=3 independent biological replicates. g , Osimertinib (160 nM) treatment response on H1975 cells with miR-147b knockdown. Left, the monolayer colonies were treated for 10 days and stained with Giemsa. Right, the 3D structures were treated for 14 days. -, vehicle; +, osimertinib. Scale bar, 1000 μm. n=3 independent biological replicates. Data are mean ± s.e.m. and were analysed with one-way ANOVA ( b ); unpaired two-tailed t -test with Holm-Sidak’s correction ( e,g ).
    Figure Legend Snippet: miR-147b initiates drug-tolerance. a , A heat map showing top upregulated and downregulated miRNAs in two paired osimertinib-tolerant (OTR) and parental cells in PC9 and HCC827 by miRNA-seq analysis. b , qRT-PCR analysis of miR-147b expressions in parental, recovered, primary and secondary osimertinib-tolerant cells in PC9. The parental tumor cells treated with 160 nM EGFR-TKI osimertinib for 6 days enter a drug-tolerant state (primary tolerant cells) with a brief therapy withdrawal up to 18 days (recovered cells) followed by reinstatement of the 160 nM dose for 11 days (secondary tolerant cells). The relative miR-147b expression level in the parental cells were calibrated as 1. miR-423 was used as endogenous control. n=3 independent biological replicates. c -d, Osimertinib (c) and gefitinib (d) treatment response on scrambled control (Scr) and miR-147b-overexpressing cells (147b) in HCC827 for 3 days. n=3 independent biological replicates. e , Osimertinib (40 nM) and gefitinib (40 nM) treatment response on scrambled control and miR-147b-overexpressing cells in HCC827 by colony formation assay. 20, 40 and 80 cells were plated in 10-cm dish and the colonies were stained with Giemsa on day 10 and the total number of colonies were quantified. n=3 independent biological replicates. f , Osimertinib treatment response on H1975 cells with miR-147b knockdown (anti147b) and scrambled control (antictrl). The cell viability was measured on day 4. n=3 independent biological replicates. g , Osimertinib (160 nM) treatment response on H1975 cells with miR-147b knockdown. Left, the monolayer colonies were treated for 10 days and stained with Giemsa. Right, the 3D structures were treated for 14 days. -, vehicle; +, osimertinib. Scale bar, 1000 μm. n=3 independent biological replicates. Data are mean ± s.e.m. and were analysed with one-way ANOVA ( b ); unpaired two-tailed t -test with Holm-Sidak’s correction ( e,g ).

    Techniques Used: Quantitative RT-PCR, Expressing, Colony Assay, Staining, Two Tailed Test

    NSCLC cells adopt a tolerance strategy against EGFR-TKIs. a, Representative phase contrast images of 3D structures from AALE cells cultured according to the protocol at top of the panel. Scale bar, 50 μm. Repeated six times with similar results. b , Top, the scenario of anti-EGFR tolerance and resistance in lung cancer. The tumor cells treated with the EGFR-TKI gefitinib or osimertinib enter a reversible drug-tolerant cycle (in green arrows, 1° Tolerant) with a brief therapy withdrawal (up to 21 days) followed by reinstatement of the 160 nM dose (2° Tolerant). Alternatively, the tumor cells treated continuously with gefitinib or osimertinib without therapy interruption undergo drug-tolerance briefly and go into a drug-resistance state in which cells do not respond to gefitinib (1° Resistant)/osimertinib (2° Resistant). Bottom, osimertinib treatment response on HCC827 3D structures. Representative images of Parental cells, 1° Tolerant cells (derived from the Parental cells treated with 160 nM osimertinib for 11 days), Recovered cells (derived from the 1° Tolerant cells with a therapy withdrawal up to 21 days) and 2° Tolerant cells (derived from the Recovered cells by reinstatement of the 160 nM dose for 11 days). Scale bar, 200 μm. Repeated six times with similar results. c, Representative phase contrast microscopy (left panel) and H E staining of HCC827 3D structures derived from parental (top) and osimertinib-tolerant (bottom) cells. Images in blue dotted squares (middle panel) were amplified (right panel) and shown. Scale bar, 50 μm. Repeated six times with similar results. d, qRT-PCR analysis of SFTPC , HOPX , ID2 and CEACAM5 expression in single cell clone HCC827-derived 3D structures in the presence of osimertinib. Single cell clone derived cells were plated with geltrex and treated with 100 nM osimertinib (tolerant) or vehicle (parental) for 24 days. Gene expression for surviving 3D structures were analyzed. n=3 independent biological replicates. e , Single-cell clonogenicity of PC9 cells treated with gefitinib. A single cell was sorted by FACS into a 96-well plate and treated with 0.1, 0.4, and 2 μM gefitinib or the vehicle for 14 days. The frequency of colony formation was calculated as a ratio of the total number of colonies to the total number of wells plated with a single cell in a 96-well plate. n=3 independent biological replicates. f, qRT-PCR analysis of top upregulated and downregulated genes in gefitinib-tolerant clones (n=2) compared with vehicle-treated parental single cell clone (n=1) in PC9. The gene expression in parental sensitive clone was calibrated as 1. ACTB was used as endogenous control. n=4 independent biological replicates. g, Whole transcriptome and gene ontology analysis of gefitinib-tolerant clones compared with the parental single cell clone in PC9. n=4 independent biological replicates. h, qRT-PCR analysis of genes in top regulated signaling pathways including Wnt planar cell polarity signaling, glutamine metabolic process, cellular response to hypoxia, and tricarboxylic acid cycle in gefitinib-tolerant clones compared with parental the single cell clone in PC9. The gene expression in parental sensitive clone was calibrated as 1. ACTB was used as endogenous control. n=4 independent biological replicates. Data are mean ± s.e.m. and were analysed with unpaired two-tailed t -test with Welch’s correction ( d ); modified one-tailed Fisher’s Exact test ( g).
    Figure Legend Snippet: NSCLC cells adopt a tolerance strategy against EGFR-TKIs. a, Representative phase contrast images of 3D structures from AALE cells cultured according to the protocol at top of the panel. Scale bar, 50 μm. Repeated six times with similar results. b , Top, the scenario of anti-EGFR tolerance and resistance in lung cancer. The tumor cells treated with the EGFR-TKI gefitinib or osimertinib enter a reversible drug-tolerant cycle (in green arrows, 1° Tolerant) with a brief therapy withdrawal (up to 21 days) followed by reinstatement of the 160 nM dose (2° Tolerant). Alternatively, the tumor cells treated continuously with gefitinib or osimertinib without therapy interruption undergo drug-tolerance briefly and go into a drug-resistance state in which cells do not respond to gefitinib (1° Resistant)/osimertinib (2° Resistant). Bottom, osimertinib treatment response on HCC827 3D structures. Representative images of Parental cells, 1° Tolerant cells (derived from the Parental cells treated with 160 nM osimertinib for 11 days), Recovered cells (derived from the 1° Tolerant cells with a therapy withdrawal up to 21 days) and 2° Tolerant cells (derived from the Recovered cells by reinstatement of the 160 nM dose for 11 days). Scale bar, 200 μm. Repeated six times with similar results. c, Representative phase contrast microscopy (left panel) and H E staining of HCC827 3D structures derived from parental (top) and osimertinib-tolerant (bottom) cells. Images in blue dotted squares (middle panel) were amplified (right panel) and shown. Scale bar, 50 μm. Repeated six times with similar results. d, qRT-PCR analysis of SFTPC , HOPX , ID2 and CEACAM5 expression in single cell clone HCC827-derived 3D structures in the presence of osimertinib. Single cell clone derived cells were plated with geltrex and treated with 100 nM osimertinib (tolerant) or vehicle (parental) for 24 days. Gene expression for surviving 3D structures were analyzed. n=3 independent biological replicates. e , Single-cell clonogenicity of PC9 cells treated with gefitinib. A single cell was sorted by FACS into a 96-well plate and treated with 0.1, 0.4, and 2 μM gefitinib or the vehicle for 14 days. The frequency of colony formation was calculated as a ratio of the total number of colonies to the total number of wells plated with a single cell in a 96-well plate. n=3 independent biological replicates. f, qRT-PCR analysis of top upregulated and downregulated genes in gefitinib-tolerant clones (n=2) compared with vehicle-treated parental single cell clone (n=1) in PC9. The gene expression in parental sensitive clone was calibrated as 1. ACTB was used as endogenous control. n=4 independent biological replicates. g, Whole transcriptome and gene ontology analysis of gefitinib-tolerant clones compared with the parental single cell clone in PC9. n=4 independent biological replicates. h, qRT-PCR analysis of genes in top regulated signaling pathways including Wnt planar cell polarity signaling, glutamine metabolic process, cellular response to hypoxia, and tricarboxylic acid cycle in gefitinib-tolerant clones compared with parental the single cell clone in PC9. The gene expression in parental sensitive clone was calibrated as 1. ACTB was used as endogenous control. n=4 independent biological replicates. Data are mean ± s.e.m. and were analysed with unpaired two-tailed t -test with Welch’s correction ( d ); modified one-tailed Fisher’s Exact test ( g).

    Techniques Used: Cell Culture, Derivative Assay, Microscopy, Staining, Amplification, Quantitative RT-PCR, Expressing, FACS, Clone Assay, Two Tailed Test, Modification, One-tailed Test

    33) Product Images from "Comparison of QIAsymphony Automated and QIAamp Manual DNA Extraction Systems for Measuring Epstein-Barr Virus DNA Load in Whole Blood Using Real-Time PCR"

    Article Title: Comparison of QIAsymphony Automated and QIAamp Manual DNA Extraction Systems for Measuring Epstein-Barr Virus DNA Load in Whole Blood Using Real-Time PCR

    Journal: The Journal of Molecular Diagnostics : JMD

    doi: 10.1016/j.jmoldx.2011.07.006

    The proportion of positive EBV PCR reactions for blood samples with various EBV DNA load values. Solid symbols, QIAsymphony-extracted samples; open symbols, QIAamp-extracted samples. The proportion values were obtained from extraction of six replicates
    Figure Legend Snippet: The proportion of positive EBV PCR reactions for blood samples with various EBV DNA load values. Solid symbols, QIAsymphony-extracted samples; open symbols, QIAamp-extracted samples. The proportion values were obtained from extraction of six replicates

    Techniques Used: Polymerase Chain Reaction

    34) Product Images from "Comparative analysis of four methods to extract DNA from paraffin-embedded tissues: effect on downstream molecular applications"

    Article Title: Comparative analysis of four methods to extract DNA from paraffin-embedded tissues: effect on downstream molecular applications

    Journal: BMC Research Notes

    doi: 10.1186/1756-0500-3-239

    Internal control amplification . Mean Ct value of PhHV PCR of the 4 materials (A, B, C and D) for the different extraction methods. The +/ and -/ indicate the use of proteinase K digestion or no digestion, respectively. The different extraction methods are indicated by: heat-treatment =/-, QIAamp DNA extraction =/Q, EasyMAG DNA extraction =/EM, Gentra DNA extraction =/G.
    Figure Legend Snippet: Internal control amplification . Mean Ct value of PhHV PCR of the 4 materials (A, B, C and D) for the different extraction methods. The +/ and -/ indicate the use of proteinase K digestion or no digestion, respectively. The different extraction methods are indicated by: heat-treatment =/-, QIAamp DNA extraction =/Q, EasyMAG DNA extraction =/EM, Gentra DNA extraction =/G.

    Techniques Used: Amplification, Polymerase Chain Reaction, DNA Extraction

    Assessment of maximum amplicon length: PCR product yield . Mean relative yield (%) of 200, 400 and 600 bp PCR products of 4 materials (A, B, C and D). The prot. K + QIAamp extraction's yield was set at 100%. The +/ and -/ indicate the use of proteinase K digestion or no digestion, respectively. The different extraction methods are indicated by: heat-treatment =/-, QIAamp DNA extraction =/Q, EasyMAG DNA extraction =/EM, Gentra DNA extraction =/G.
    Figure Legend Snippet: Assessment of maximum amplicon length: PCR product yield . Mean relative yield (%) of 200, 400 and 600 bp PCR products of 4 materials (A, B, C and D). The prot. K + QIAamp extraction's yield was set at 100%. The +/ and -/ indicate the use of proteinase K digestion or no digestion, respectively. The different extraction methods are indicated by: heat-treatment =/-, QIAamp DNA extraction =/Q, EasyMAG DNA extraction =/EM, Gentra DNA extraction =/G.

    Techniques Used: Amplification, Polymerase Chain Reaction, DNA Extraction

    SNP analysis using real time PCR . A . Two representative component plots generated after proteinase K treatment followed by EasyMAG extraction from colon tissue B and real time amplification using SNP assay rs1350138. The light grey line indicates allele 1 (VIC label), the dark grey line indicates allele 2 (FAM label). B . Mean Ct value of SNP rs2043731 and rs1350138 of the 4 materials (A, B, C and D) in JBZ 4× mastermix or ABI 2× mastermix for the different extraction methods. The +/ and -/ indicate the use of proteinase K digestion or no digestion, respectively. The different extraction methods are indicated by: heat-treatment =/-, QIAamp DNA extraction =/Q, EasyMAG DNA extraction =/EM, Gentra DNA extraction =/G.
    Figure Legend Snippet: SNP analysis using real time PCR . A . Two representative component plots generated after proteinase K treatment followed by EasyMAG extraction from colon tissue B and real time amplification using SNP assay rs1350138. The light grey line indicates allele 1 (VIC label), the dark grey line indicates allele 2 (FAM label). B . Mean Ct value of SNP rs2043731 and rs1350138 of the 4 materials (A, B, C and D) in JBZ 4× mastermix or ABI 2× mastermix for the different extraction methods. The +/ and -/ indicate the use of proteinase K digestion or no digestion, respectively. The different extraction methods are indicated by: heat-treatment =/-, QIAamp DNA extraction =/Q, EasyMAG DNA extraction =/EM, Gentra DNA extraction =/G.

    Techniques Used: Real-time Polymerase Chain Reaction, Generated, Amplification, DNA Extraction

    Assessment of maximum amplicon length: visualisation on gel . Gel image of 200-400-600 bp multiplex PCR-products of representative tissue B. The +/ and -/ indicate the use of proteinase K digestion or no digestion, respectively. The different extraction methods are indicated by: heat-treatment =/-, QIAamp DNA extraction =/Q, EasyMAG DNA extraction =/EM, Gentra DNA extraction =/G. Neg = ultrapure water in PCR, Pos = QIAamp extracted DNA from EDTA-blood.
    Figure Legend Snippet: Assessment of maximum amplicon length: visualisation on gel . Gel image of 200-400-600 bp multiplex PCR-products of representative tissue B. The +/ and -/ indicate the use of proteinase K digestion or no digestion, respectively. The different extraction methods are indicated by: heat-treatment =/-, QIAamp DNA extraction =/Q, EasyMAG DNA extraction =/EM, Gentra DNA extraction =/G. Neg = ultrapure water in PCR, Pos = QIAamp extracted DNA from EDTA-blood.

    Techniques Used: Amplification, Multiplex Assay, Polymerase Chain Reaction, DNA Extraction

    35) Product Images from "Isolation of Chlamydia Pneumoniae from Serum Samples of the Patients with Acute Coronary Syndrome"

    Article Title: Isolation of Chlamydia Pneumoniae from Serum Samples of the Patients with Acute Coronary Syndrome

    Journal: International Journal of Medical Sciences

    doi:

    Recovery of PCR product in DNA samples isolated from serum specimens using QIAamp DNA blood midi kit, protein A and phenol-chloroform extraction method. 1 - molecular size standards; 2, 6 and 10 - PCR-positive serum from patient M; 3, 7 and 11 - PCR-positive serum from patient P; 4, 8 and 12 - PCR negative serum from patient S; 5, 9 and 13 - extraction control; 14 - negative control; 15 - positive control.
    Figure Legend Snippet: Recovery of PCR product in DNA samples isolated from serum specimens using QIAamp DNA blood midi kit, protein A and phenol-chloroform extraction method. 1 - molecular size standards; 2, 6 and 10 - PCR-positive serum from patient M; 3, 7 and 11 - PCR-positive serum from patient P; 4, 8 and 12 - PCR negative serum from patient S; 5, 9 and 13 - extraction control; 14 - negative control; 15 - positive control.

    Techniques Used: Polymerase Chain Reaction, Isolation, Negative Control, Positive Control

    36) Product Images from "The Most Favourable Procedure for the Isolation of Cell-Free DNA from the Plasma of Iso-Immunized RHD-Negative Pregnant Women"

    Article Title: The Most Favourable Procedure for the Isolation of Cell-Free DNA from the Plasma of Iso-Immunized RHD-Negative Pregnant Women

    Journal: Journal of Circulating Biomarkers

    doi: 10.5772/62113

    Bar diagram showing a comparison of DNA concentrations isolated from the maternal plasma of iso-immunized women using different isolation procedures. (n=30). (*=50 μL QIAamp method) (#= 30 μL QIAamp method) (α= 10 μL QIAamp method) (β= 3 μL of QIAamp method) (ψ=50 μL CM1); **p
    Figure Legend Snippet: Bar diagram showing a comparison of DNA concentrations isolated from the maternal plasma of iso-immunized women using different isolation procedures. (n=30). (*=50 μL QIAamp method) (#= 30 μL QIAamp method) (α= 10 μL QIAamp method) (β= 3 μL of QIAamp method) (ψ=50 μL CM1); **p

    Techniques Used: Isolation

    Bar diagram showing a comparison of the quality of DNA isolated from the maternal plasma of iso-immunized women using different isolation procedures. (n=30). (*=50 μL QIAamp method) (#= 30 μL QIAamp method) (α= 10 μL QIAamp method) (β= 3 μL of QIAamp method) (ψ=50 μL CM1); **p
    Figure Legend Snippet: Bar diagram showing a comparison of the quality of DNA isolated from the maternal plasma of iso-immunized women using different isolation procedures. (n=30). (*=50 μL QIAamp method) (#= 30 μL QIAamp method) (α= 10 μL QIAamp method) (β= 3 μL of QIAamp method) (ψ=50 μL CM1); **p

    Techniques Used: Isolation

    37) Product Images from "PCR-Based Method for Isolation and Detection of Chlamydia pneumoniae DNA in Cerebrospinal Fluids"

    Article Title: PCR-Based Method for Isolation and Detection of Chlamydia pneumoniae DNA in Cerebrospinal Fluids

    Journal: Clinical and Diagnostic Laboratory Immunology

    doi: 10.1128/CDLI.8.3.499-502.2001

    Optimization of annealing temperature and Mg 2+ concentration for PCR with primers for omp1 . The annealing temperature was optimized in a Mastercycler gradient (Eppendorf). The Mg concentration for PCR specific for omp1 was optimized with a PCR optimization kit (Invitrogen). The target DNA for PCR was extracted from C. pneumoniae with the QIAmp DNA Mini Kit.
    Figure Legend Snippet: Optimization of annealing temperature and Mg 2+ concentration for PCR with primers for omp1 . The annealing temperature was optimized in a Mastercycler gradient (Eppendorf). The Mg concentration for PCR specific for omp1 was optimized with a PCR optimization kit (Invitrogen). The target DNA for PCR was extracted from C. pneumoniae with the QIAmp DNA Mini Kit.

    Techniques Used: Concentration Assay, Polymerase Chain Reaction

    Detection sensitivity of PCR for omp1 versus the 16S rRNA gene. Two hundred microliters of the mock CSF containing a specific number of C. pneumoniae organisms (as indicated), was used for DNA extraction with the QIAmp DNA Mini Kit. A 2-μl portion of the 50-μl volume of DNA extracts was subjected to PCR. The optimized PCRs for omp1 and 16S rRNA gene were conducted (see Materials and Methods).
    Figure Legend Snippet: Detection sensitivity of PCR for omp1 versus the 16S rRNA gene. Two hundred microliters of the mock CSF containing a specific number of C. pneumoniae organisms (as indicated), was used for DNA extraction with the QIAmp DNA Mini Kit. A 2-μl portion of the 50-μl volume of DNA extracts was subjected to PCR. The optimized PCRs for omp1 and 16S rRNA gene were conducted (see Materials and Methods).

    Techniques Used: Polymerase Chain Reaction, DNA Extraction

    Comparison of extraction efficacy of C. pneumoniae DNA from mock CSFs using two DNA extraction kits. The mock CSFs (200 μl) spiked with serially diluted bacteria were extracted either with the QIAmp DNA Mini Kit with a bacterial DNA extraction protocol or with the QIAmp DNA Blood Mini Kit. Two microliters of the extracted DNA (50 μl) was subjected to PCR with primers for omp1 . Results are representative of three experiments.
    Figure Legend Snippet: Comparison of extraction efficacy of C. pneumoniae DNA from mock CSFs using two DNA extraction kits. The mock CSFs (200 μl) spiked with serially diluted bacteria were extracted either with the QIAmp DNA Mini Kit with a bacterial DNA extraction protocol or with the QIAmp DNA Blood Mini Kit. Two microliters of the extracted DNA (50 μl) was subjected to PCR with primers for omp1 . Results are representative of three experiments.

    Techniques Used: DNA Extraction, Polymerase Chain Reaction

    Detection of C. pneumoniae DNA by PCR in CSFs obtained from patients with MS. Two hundred microliters of CSFs obtained from MS patients was processed for extraction of DNA utilizing the QIAmp DNA Mini Kit, and 2 μl of the resulting 50-μl DNA solution was subjected to PCR with primers for either omp1 or the 16S rRNA gene. Data are representative of three PCR experiments. Boxed numbers indicate CSFs obtained from the same patient at different time points. Each number is the sample number. M, molecular marker; PC, positive control for PCR; NC, negative control for PCR; IC, negative control for DNA extraction.
    Figure Legend Snippet: Detection of C. pneumoniae DNA by PCR in CSFs obtained from patients with MS. Two hundred microliters of CSFs obtained from MS patients was processed for extraction of DNA utilizing the QIAmp DNA Mini Kit, and 2 μl of the resulting 50-μl DNA solution was subjected to PCR with primers for either omp1 or the 16S rRNA gene. Data are representative of three PCR experiments. Boxed numbers indicate CSFs obtained from the same patient at different time points. Each number is the sample number. M, molecular marker; PC, positive control for PCR; NC, negative control for PCR; IC, negative control for DNA extraction.

    Techniques Used: Polymerase Chain Reaction, Mass Spectrometry, Marker, Positive Control, Negative Control, DNA Extraction

    38) Product Images from "Characterization of human bone morphogenetic protein gene variants for possible roles in congenital heart disease"

    Article Title: Characterization of human bone morphogenetic protein gene variants for possible roles in congenital heart disease

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2016.5428

    DNA sequence chromatograms of the rs1049007, rs235768 and rs17563 single nucleotide polymorphisms. (A) rs1049007; (B) rs235768; (C) rs17563. Arrows indicate the sites of variation.
    Figure Legend Snippet: DNA sequence chromatograms of the rs1049007, rs235768 and rs17563 single nucleotide polymorphisms. (A) rs1049007; (B) rs235768; (C) rs17563. Arrows indicate the sites of variation.

    Techniques Used: Sequencing

    39) Product Images from "ASF1a inhibition induces p53-dependent growth arrest and senescence of cancer cells"

    Article Title: ASF1a inhibition induces p53-dependent growth arrest and senescence of cancer cells

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-019-1357-z

    Silencing ASF1a triggers DNA damage response. a Immunofluorescence staining of control (nc) and ASF1a knockdown (ASF1a si1/si2) groups of HepG2 and LNCaP cells. Nuclei were stained with DAPI (blue signals). γH2AX and 53BP1 were stained with specific antibodies (green and red signals, respectively; scale bar: 50 μm). Quantification is shown at the bottom (data are presented as the mean ± SD value of three independent experiments for HepG2 and LNCaP, respectively). Senescence-associated heterochromatin foci (SAHF) were not detected by using DAPI staining. DAPI, 4-6-diamidino-2-phenylindole dihydrochloride
    Figure Legend Snippet: Silencing ASF1a triggers DNA damage response. a Immunofluorescence staining of control (nc) and ASF1a knockdown (ASF1a si1/si2) groups of HepG2 and LNCaP cells. Nuclei were stained with DAPI (blue signals). γH2AX and 53BP1 were stained with specific antibodies (green and red signals, respectively; scale bar: 50 μm). Quantification is shown at the bottom (data are presented as the mean ± SD value of three independent experiments for HepG2 and LNCaP, respectively). Senescence-associated heterochromatin foci (SAHF) were not detected by using DAPI staining. DAPI, 4-6-diamidino-2-phenylindole dihydrochloride

    Techniques Used: Immunofluorescence, Staining

    ASF1a knockdown leads to growth arrest and cellular senescence in wild-type p53-carrying HCC and PCa cells. a Western blot was performed for the validation of ASF1a siRNA efficiency in HepG2 and LNCaP cells. ASF1a si1 and ASF1a si2, two different ASF1A siRNAs, were used in this study. b The cell number of control (nc siRNA) and ASF1a knockdown (ASF1a si1/si2) groups in HepG2 and LNCaP cells 72 h after siRNA transfection. Cells in negative control groups were set as 100% (reference). The siASF1a-transfected groups showed a significant decrease in cell counts compared with control cells (data are presented as the mean ± SD of four independent experiments in HepG2 and three independent experiments in LNCaP, respectively; P values are shown in each panel). c Colony-formation assay of HepG2 and LNCaP cells. The clonogenic abilities of HepG2 and LNCaP cells were decreased upon ASF1a knockdown. Quantification is shown at the bottom (data are presented as the mean ± SD of three independent experiments in HepG2 and four independent experiments in LNCaP, respectively; P values are shown in each panel). Colonies in control groups were set as 100% (reference). d Cell cycles of HepG2 and LNCaP recorded by propidium iodide (PI) staining. Cell cycles in control groups are shown with red peaks and cell cycles in ASF1a knockdown groups are shown with blue peaks. Proportions of G0/G1, S, and G2/M phases are presented in pie charts (data presented are of three independent experiments in HepG2 and four independent experiments in LNCaP; P values are shown in each panel). e β-Gal staining of HepG2 and LNCaP cells. Senescent cells were stained with blue color (scale bar: 100 μm). f Quantification of β-gal staining-positive HepG2 and LNCaP cells (data are presented as the mean ± SD value of three independent experiments for HepG2 and LNCaP, respectively). HCC, hepatocellular carcinoma; PCa, prostate cancer
    Figure Legend Snippet: ASF1a knockdown leads to growth arrest and cellular senescence in wild-type p53-carrying HCC and PCa cells. a Western blot was performed for the validation of ASF1a siRNA efficiency in HepG2 and LNCaP cells. ASF1a si1 and ASF1a si2, two different ASF1A siRNAs, were used in this study. b The cell number of control (nc siRNA) and ASF1a knockdown (ASF1a si1/si2) groups in HepG2 and LNCaP cells 72 h after siRNA transfection. Cells in negative control groups were set as 100% (reference). The siASF1a-transfected groups showed a significant decrease in cell counts compared with control cells (data are presented as the mean ± SD of four independent experiments in HepG2 and three independent experiments in LNCaP, respectively; P values are shown in each panel). c Colony-formation assay of HepG2 and LNCaP cells. The clonogenic abilities of HepG2 and LNCaP cells were decreased upon ASF1a knockdown. Quantification is shown at the bottom (data are presented as the mean ± SD of three independent experiments in HepG2 and four independent experiments in LNCaP, respectively; P values are shown in each panel). Colonies in control groups were set as 100% (reference). d Cell cycles of HepG2 and LNCaP recorded by propidium iodide (PI) staining. Cell cycles in control groups are shown with red peaks and cell cycles in ASF1a knockdown groups are shown with blue peaks. Proportions of G0/G1, S, and G2/M phases are presented in pie charts (data presented are of three independent experiments in HepG2 and four independent experiments in LNCaP; P values are shown in each panel). e β-Gal staining of HepG2 and LNCaP cells. Senescent cells were stained with blue color (scale bar: 100 μm). f Quantification of β-gal staining-positive HepG2 and LNCaP cells (data are presented as the mean ± SD value of three independent experiments for HepG2 and LNCaP, respectively). HCC, hepatocellular carcinoma; PCa, prostate cancer

    Techniques Used: Western Blot, Transfection, Negative Control, Colony Assay, Staining

    Cellular senescence occurring in ASF1a-inhibited cells is rescued by p21 cip1 or p53 loss. a β-Gal staining in HepG2 and LNCaP cells. Senescent cells are stained with blue color (scale bar: 100 μm). b – e Quantifications of figure ( a ) (data are presented as the mean ± SD of three independent experiments for HepG2 and LNCaP, respectively; P values are shown in each panel)
    Figure Legend Snippet: Cellular senescence occurring in ASF1a-inhibited cells is rescued by p21 cip1 or p53 loss. a β-Gal staining in HepG2 and LNCaP cells. Senescent cells are stained with blue color (scale bar: 100 μm). b – e Quantifications of figure ( a ) (data are presented as the mean ± SD of three independent experiments for HepG2 and LNCaP, respectively; P values are shown in each panel)

    Techniques Used: Staining

    Cellular senescence is induced via the p53/p21 cip1 pathway in ASF1a-depleted cells. a The schematic expression showing that RB/p16 ink4 and p53/p21 cip1 cascades are the two major pathways involved in cellular senescence. b A screen of four cellular senescence-associated genes in HepG2 and LNCaP cells. p16 ink4 , p21 cip1 , p27 kip1 , and TERT mRNA expression in control (nc) and ASF1a knockdown (ASF1a si1) groups is shown in the bar charts (data are presented as the mean ± SD; P values are shown in each panel). c Loss of ASF1a increased p21 cip1 mRNA expression in HepG2, LNCaP, AGS, and MCF-7 cells (data are presented as the mean ± SD, at least three independent experiments for each cell line, respectively; P values are shown in each panel). d Loss of ASF1a increased p21 cip1 and p53 protein expression in HepG2 and LNCaP cells. Quantification is shown at the bottom. e Loss of ASF1a increased p21 cip1 and p53 protein expression in AGS and MCF-7 cells. Quantification is shown at the bottom. f The schematic of p21 cip1 (CDKN1A) promoter and luciferase activity. An increased luciferase activity was observed upon ASF1a knockdown in both HepG2 and LNCaP cells (data are presented as the mean ± SD of three independent experiments for HepG2 and LNCaP, respectively; P values are shown in each panel). The landscape figure was drawn using the GPS 3.0 tool ( http://gps.biocuckoo.org ). g Knockdown of p53 compromises ASF1a depletion-induced p21 cip1 accumulation, as assessed using immunoblotting. Quantification is shown at the bottom
    Figure Legend Snippet: Cellular senescence is induced via the p53/p21 cip1 pathway in ASF1a-depleted cells. a The schematic expression showing that RB/p16 ink4 and p53/p21 cip1 cascades are the two major pathways involved in cellular senescence. b A screen of four cellular senescence-associated genes in HepG2 and LNCaP cells. p16 ink4 , p21 cip1 , p27 kip1 , and TERT mRNA expression in control (nc) and ASF1a knockdown (ASF1a si1) groups is shown in the bar charts (data are presented as the mean ± SD; P values are shown in each panel). c Loss of ASF1a increased p21 cip1 mRNA expression in HepG2, LNCaP, AGS, and MCF-7 cells (data are presented as the mean ± SD, at least three independent experiments for each cell line, respectively; P values are shown in each panel). d Loss of ASF1a increased p21 cip1 and p53 protein expression in HepG2 and LNCaP cells. Quantification is shown at the bottom. e Loss of ASF1a increased p21 cip1 and p53 protein expression in AGS and MCF-7 cells. Quantification is shown at the bottom. f The schematic of p21 cip1 (CDKN1A) promoter and luciferase activity. An increased luciferase activity was observed upon ASF1a knockdown in both HepG2 and LNCaP cells (data are presented as the mean ± SD of three independent experiments for HepG2 and LNCaP, respectively; P values are shown in each panel). The landscape figure was drawn using the GPS 3.0 tool ( http://gps.biocuckoo.org ). g Knockdown of p53 compromises ASF1a depletion-induced p21 cip1 accumulation, as assessed using immunoblotting. Quantification is shown at the bottom

    Techniques Used: Expressing, Luciferase, Activity Assay

    40) Product Images from "ASF1a inhibition induces p53-dependent growth arrest and senescence of cancer cells"

    Article Title: ASF1a inhibition induces p53-dependent growth arrest and senescence of cancer cells

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-019-1357-z

    Silencing ASF1a triggers DNA damage response. a Immunofluorescence staining of control (nc) and ASF1a knockdown (ASF1a si1/si2) groups of HepG2 and LNCaP cells. Nuclei were stained with DAPI (blue signals). γH2AX and 53BP1 were stained with specific antibodies (green and red signals, respectively; scale bar: 50 μm). Quantification is shown at the bottom (data are presented as the mean ± SD value of three independent experiments for HepG2 and LNCaP, respectively). Senescence-associated heterochromatin foci (SAHF) were not detected by using DAPI staining. DAPI, 4-6-diamidino-2-phenylindole dihydrochloride
    Figure Legend Snippet: Silencing ASF1a triggers DNA damage response. a Immunofluorescence staining of control (nc) and ASF1a knockdown (ASF1a si1/si2) groups of HepG2 and LNCaP cells. Nuclei were stained with DAPI (blue signals). γH2AX and 53BP1 were stained with specific antibodies (green and red signals, respectively; scale bar: 50 μm). Quantification is shown at the bottom (data are presented as the mean ± SD value of three independent experiments for HepG2 and LNCaP, respectively). Senescence-associated heterochromatin foci (SAHF) were not detected by using DAPI staining. DAPI, 4-6-diamidino-2-phenylindole dihydrochloride

    Techniques Used: Immunofluorescence, Staining

    ASF1a knockdown leads to growth arrest and cellular senescence in wild-type p53-carrying HCC and PCa cells. a Western blot was performed for the validation of ASF1a siRNA efficiency in HepG2 and LNCaP cells. ASF1a si1 and ASF1a si2, two different ASF1A siRNAs, were used in this study. b The cell number of control (nc siRNA) and ASF1a knockdown (ASF1a si1/si2) groups in HepG2 and LNCaP cells 72 h after siRNA transfection. Cells in negative control groups were set as 100% (reference). The siASF1a-transfected groups showed a significant decrease in cell counts compared with control cells (data are presented as the mean ± SD of four independent experiments in HepG2 and three independent experiments in LNCaP, respectively; P values are shown in each panel). c Colony-formation assay of HepG2 and LNCaP cells. The clonogenic abilities of HepG2 and LNCaP cells were decreased upon ASF1a knockdown. Quantification is shown at the bottom (data are presented as the mean ± SD of three independent experiments in HepG2 and four independent experiments in LNCaP, respectively; P values are shown in each panel). Colonies in control groups were set as 100% (reference). d Cell cycles of HepG2 and LNCaP recorded by propidium iodide (PI) staining. Cell cycles in control groups are shown with red peaks and cell cycles in ASF1a knockdown groups are shown with blue peaks. Proportions of G0/G1, S, and G2/M phases are presented in pie charts (data presented are of three independent experiments in HepG2 and four independent experiments in LNCaP; P values are shown in each panel). e β-Gal staining of HepG2 and LNCaP cells. Senescent cells were stained with blue color (scale bar: 100 μm). f Quantification of β-gal staining-positive HepG2 and LNCaP cells (data are presented as the mean ± SD value of three independent experiments for HepG2 and LNCaP, respectively). HCC, hepatocellular carcinoma; PCa, prostate cancer
    Figure Legend Snippet: ASF1a knockdown leads to growth arrest and cellular senescence in wild-type p53-carrying HCC and PCa cells. a Western blot was performed for the validation of ASF1a siRNA efficiency in HepG2 and LNCaP cells. ASF1a si1 and ASF1a si2, two different ASF1A siRNAs, were used in this study. b The cell number of control (nc siRNA) and ASF1a knockdown (ASF1a si1/si2) groups in HepG2 and LNCaP cells 72 h after siRNA transfection. Cells in negative control groups were set as 100% (reference). The siASF1a-transfected groups showed a significant decrease in cell counts compared with control cells (data are presented as the mean ± SD of four independent experiments in HepG2 and three independent experiments in LNCaP, respectively; P values are shown in each panel). c Colony-formation assay of HepG2 and LNCaP cells. The clonogenic abilities of HepG2 and LNCaP cells were decreased upon ASF1a knockdown. Quantification is shown at the bottom (data are presented as the mean ± SD of three independent experiments in HepG2 and four independent experiments in LNCaP, respectively; P values are shown in each panel). Colonies in control groups were set as 100% (reference). d Cell cycles of HepG2 and LNCaP recorded by propidium iodide (PI) staining. Cell cycles in control groups are shown with red peaks and cell cycles in ASF1a knockdown groups are shown with blue peaks. Proportions of G0/G1, S, and G2/M phases are presented in pie charts (data presented are of three independent experiments in HepG2 and four independent experiments in LNCaP; P values are shown in each panel). e β-Gal staining of HepG2 and LNCaP cells. Senescent cells were stained with blue color (scale bar: 100 μm). f Quantification of β-gal staining-positive HepG2 and LNCaP cells (data are presented as the mean ± SD value of three independent experiments for HepG2 and LNCaP, respectively). HCC, hepatocellular carcinoma; PCa, prostate cancer

    Techniques Used: Western Blot, Transfection, Negative Control, Colony Assay, Staining

    Cellular senescence occurring in ASF1a-inhibited cells is rescued by p21 cip1 or p53 loss. a β-Gal staining in HepG2 and LNCaP cells. Senescent cells are stained with blue color (scale bar: 100 μm). b – e Quantifications of figure ( a ) (data are presented as the mean ± SD of three independent experiments for HepG2 and LNCaP, respectively; P values are shown in each panel)
    Figure Legend Snippet: Cellular senescence occurring in ASF1a-inhibited cells is rescued by p21 cip1 or p53 loss. a β-Gal staining in HepG2 and LNCaP cells. Senescent cells are stained with blue color (scale bar: 100 μm). b – e Quantifications of figure ( a ) (data are presented as the mean ± SD of three independent experiments for HepG2 and LNCaP, respectively; P values are shown in each panel)

    Techniques Used: Staining

    Cellular senescence is induced via the p53/p21 cip1 pathway in ASF1a-depleted cells. a The schematic expression showing that RB/p16 ink4 and p53/p21 cip1 cascades are the two major pathways involved in cellular senescence. b A screen of four cellular senescence-associated genes in HepG2 and LNCaP cells. p16 ink4 , p21 cip1 , p27 kip1 , and TERT mRNA expression in control (nc) and ASF1a knockdown (ASF1a si1) groups is shown in the bar charts (data are presented as the mean ± SD; P values are shown in each panel). c Loss of ASF1a increased p21 cip1 mRNA expression in HepG2, LNCaP, AGS, and MCF-7 cells (data are presented as the mean ± SD, at least three independent experiments for each cell line, respectively; P values are shown in each panel). d Loss of ASF1a increased p21 cip1 and p53 protein expression in HepG2 and LNCaP cells. Quantification is shown at the bottom. e Loss of ASF1a increased p21 cip1 and p53 protein expression in AGS and MCF-7 cells. Quantification is shown at the bottom. f The schematic of p21 cip1 (CDKN1A) promoter and luciferase activity. An increased luciferase activity was observed upon ASF1a knockdown in both HepG2 and LNCaP cells (data are presented as the mean ± SD of three independent experiments for HepG2 and LNCaP, respectively; P values are shown in each panel). The landscape figure was drawn using the GPS 3.0 tool ( http://gps.biocuckoo.org ). g Knockdown of p53 compromises ASF1a depletion-induced p21 cip1 accumulation, as assessed using immunoblotting. Quantification is shown at the bottom
    Figure Legend Snippet: Cellular senescence is induced via the p53/p21 cip1 pathway in ASF1a-depleted cells. a The schematic expression showing that RB/p16 ink4 and p53/p21 cip1 cascades are the two major pathways involved in cellular senescence. b A screen of four cellular senescence-associated genes in HepG2 and LNCaP cells. p16 ink4 , p21 cip1 , p27 kip1 , and TERT mRNA expression in control (nc) and ASF1a knockdown (ASF1a si1) groups is shown in the bar charts (data are presented as the mean ± SD; P values are shown in each panel). c Loss of ASF1a increased p21 cip1 mRNA expression in HepG2, LNCaP, AGS, and MCF-7 cells (data are presented as the mean ± SD, at least three independent experiments for each cell line, respectively; P values are shown in each panel). d Loss of ASF1a increased p21 cip1 and p53 protein expression in HepG2 and LNCaP cells. Quantification is shown at the bottom. e Loss of ASF1a increased p21 cip1 and p53 protein expression in AGS and MCF-7 cells. Quantification is shown at the bottom. f The schematic of p21 cip1 (CDKN1A) promoter and luciferase activity. An increased luciferase activity was observed upon ASF1a knockdown in both HepG2 and LNCaP cells (data are presented as the mean ± SD of three independent experiments for HepG2 and LNCaP, respectively; P values are shown in each panel). The landscape figure was drawn using the GPS 3.0 tool ( http://gps.biocuckoo.org ). g Knockdown of p53 compromises ASF1a depletion-induced p21 cip1 accumulation, as assessed using immunoblotting. Quantification is shown at the bottom

    Techniques Used: Expressing, Luciferase, Activity Assay

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    Isolation:

    Article Title: PCR-Based Method for Isolation and Detection of Chlamydia pneumoniae DNA in Cerebrospinal Fluids
    Article Snippet: .. In contrast, both Boman et al. ( ) and Layh-Schmitt et al. ( ) utilized a commercial kit (QIAmp Blood Mini Kit) for isolation of C. pneumoniae DNA, but they failed to detect bacterial DNA in CSFs of MS patients or found only a low positive rate. .. As shown in this study, the QIAmp Blood Mini Kit is not designed for isolation of bacterial DNA and was less effective for extracting bacterial DNA from CSFs, particularly when specimens contained few bacteria.

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    Infection:

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    other:

    Article Title: PCR-Based Method for Isolation and Detection of Chlamydia pneumoniae DNA in Cerebrospinal Fluids
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    Mass Spectrometry:

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    Binding Assay:

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    Qiagen qiaamp dna blood mini kit
    PCR of different HLA targets using gDNA prepared using the PES membrane or <t>QIAamp</t> kit. Buffy coat from the same blood sample was equally distributed and processed for gDNA extraction using either 0.22 µm PES membrane from EMD Millipore ( A ) or QIAamp <t>DNA</t> Blood Mini Kit from Qiagen ( B ). Different targets of HLA loci were PCR amplified, as described in Validation of the gDNA isolation method using the PES membrane filter . PCR amplicons were analyzed on 2.5% agarose gel electrophoresis. Representative of a minimum of 4 independent experiments. Lanes 1 and 2: HLA-A Exons 2 and 3; lanes 3 and 4: HLA-B Exons 2 and 3; lanes 5 and 6: HLA-C Exons 2 and 3; lanes 7 and 10: HLA-DPB1 Exons 3 and 2, respectively; lanes 8 and 11: HLA-DQB1 Exons 3 and 2, respectively; lanes 9 and 12: HLA-DRB1 Exons 3 and 2, respectively. M, DNA MW marker (250 bp–10 kb).
    Qiaamp Dna Blood Mini Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 1873 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PCR of different HLA targets using gDNA prepared using the PES membrane or QIAamp kit. Buffy coat from the same blood sample was equally distributed and processed for gDNA extraction using either 0.22 µm PES membrane from EMD Millipore ( A ) or QIAamp DNA Blood Mini Kit from Qiagen ( B ). Different targets of HLA loci were PCR amplified, as described in Validation of the gDNA isolation method using the PES membrane filter . PCR amplicons were analyzed on 2.5% agarose gel electrophoresis. Representative of a minimum of 4 independent experiments. Lanes 1 and 2: HLA-A Exons 2 and 3; lanes 3 and 4: HLA-B Exons 2 and 3; lanes 5 and 6: HLA-C Exons 2 and 3; lanes 7 and 10: HLA-DPB1 Exons 3 and 2, respectively; lanes 8 and 11: HLA-DQB1 Exons 3 and 2, respectively; lanes 9 and 12: HLA-DRB1 Exons 3 and 2, respectively. M, DNA MW marker (250 bp–10 kb).

    Journal: Journal of Biomolecular Techniques : JBT

    Article Title: Development of a Membrane-Based Method for Isolation of Genomic DNA from Human Blood

    doi: 10.7171/jbt.18-2902-001

    Figure Lengend Snippet: PCR of different HLA targets using gDNA prepared using the PES membrane or QIAamp kit. Buffy coat from the same blood sample was equally distributed and processed for gDNA extraction using either 0.22 µm PES membrane from EMD Millipore ( A ) or QIAamp DNA Blood Mini Kit from Qiagen ( B ). Different targets of HLA loci were PCR amplified, as described in Validation of the gDNA isolation method using the PES membrane filter . PCR amplicons were analyzed on 2.5% agarose gel electrophoresis. Representative of a minimum of 4 independent experiments. Lanes 1 and 2: HLA-A Exons 2 and 3; lanes 3 and 4: HLA-B Exons 2 and 3; lanes 5 and 6: HLA-C Exons 2 and 3; lanes 7 and 10: HLA-DPB1 Exons 3 and 2, respectively; lanes 8 and 11: HLA-DQB1 Exons 3 and 2, respectively; lanes 9 and 12: HLA-DRB1 Exons 3 and 2, respectively. M, DNA MW marker (250 bp–10 kb).

    Article Snippet: Among the various commonly available commercial kits for gDNA isolation, the kit from Qiagen has been shown to have columns with superior binding capability., Hence, the QIAamp DNA Blood Mini Kit (Qiagen) was chosen for comparison of the efficiency of the gDNA isolation with the described method here.

    Techniques: Polymerase Chain Reaction, Amplification, Isolation, Agarose Gel Electrophoresis, Marker

    Agarose gel electrophoresis of gDNA samples prepared using different PES membranes or the QIAamp commercial kit. A ) Buffy coat from the same blood sample was equally distributed and processed for gDNA extraction using either 0.22 µm PES membrane from EMD Millipore (lane 1) or QIAamp DNA Blood Mini Kit from Qiagen (lane 2). B ) Buffy coat from different samples was lysed independently, pooled together, and equally distributed and processed for gDNA extraction using 0.22 µm PES membrane from different suppliers (lane 1: EMD Millipore; lane 2: Pall; and lane 3: Sterlitech). C ) Buffy coat from the same sample was equally distributed and processed for gDNA extraction using either 0.22 µm (lane 1) or 0.45 µm (lane 2) PES membrane from EMD Millipore. M, DNA MW marker (75 bp–20 kb). Representative of a minimum of 4 independent experiments.

    Journal: Journal of Biomolecular Techniques : JBT

    Article Title: Development of a Membrane-Based Method for Isolation of Genomic DNA from Human Blood

    doi: 10.7171/jbt.18-2902-001

    Figure Lengend Snippet: Agarose gel electrophoresis of gDNA samples prepared using different PES membranes or the QIAamp commercial kit. A ) Buffy coat from the same blood sample was equally distributed and processed for gDNA extraction using either 0.22 µm PES membrane from EMD Millipore (lane 1) or QIAamp DNA Blood Mini Kit from Qiagen (lane 2). B ) Buffy coat from different samples was lysed independently, pooled together, and equally distributed and processed for gDNA extraction using 0.22 µm PES membrane from different suppliers (lane 1: EMD Millipore; lane 2: Pall; and lane 3: Sterlitech). C ) Buffy coat from the same sample was equally distributed and processed for gDNA extraction using either 0.22 µm (lane 1) or 0.45 µm (lane 2) PES membrane from EMD Millipore. M, DNA MW marker (75 bp–20 kb). Representative of a minimum of 4 independent experiments.

    Article Snippet: Among the various commonly available commercial kits for gDNA isolation, the kit from Qiagen has been shown to have columns with superior binding capability., Hence, the QIAamp DNA Blood Mini Kit (Qiagen) was chosen for comparison of the efficiency of the gDNA isolation with the described method here.

    Techniques: Agarose Gel Electrophoresis, Marker

    Agarose gel electrophoresis of gDNA samples digested with either Hin dIII or Eco RI. DNAs digested with Hin dIII ( A – C ); Eco R1 digested gDNAs ( D – F ). A , D ) Buffy coat from the same sample was equally distributed and processed for gDNA extraction using either 0.22 µm PES membrane from EMD Millipore (lane 1) or QIAamp DNA Blood Mini Kit from Qiagen (lane 2). B , E ) Buffy coat from 2 different samples was lysed independently, pooled together, and equally distributed and processed for gDNA extraction using the 0.22 µm PES membrane from different suppliers (lane 1: EMD Millipore; lane 2: Pall; lane 3: Sterlitech). C , F ) Buffy coat from the same sample was equally distributed and processed for gDNA extraction using either the 0.22 µm (lane 1) or 0.45 µm (lane 2) PES membrane from EMD Millipore. M, DNA MW marker (75 bp–20 kb).

    Journal: Journal of Biomolecular Techniques : JBT

    Article Title: Development of a Membrane-Based Method for Isolation of Genomic DNA from Human Blood

    doi: 10.7171/jbt.18-2902-001

    Figure Lengend Snippet: Agarose gel electrophoresis of gDNA samples digested with either Hin dIII or Eco RI. DNAs digested with Hin dIII ( A – C ); Eco R1 digested gDNAs ( D – F ). A , D ) Buffy coat from the same sample was equally distributed and processed for gDNA extraction using either 0.22 µm PES membrane from EMD Millipore (lane 1) or QIAamp DNA Blood Mini Kit from Qiagen (lane 2). B , E ) Buffy coat from 2 different samples was lysed independently, pooled together, and equally distributed and processed for gDNA extraction using the 0.22 µm PES membrane from different suppliers (lane 1: EMD Millipore; lane 2: Pall; lane 3: Sterlitech). C , F ) Buffy coat from the same sample was equally distributed and processed for gDNA extraction using either the 0.22 µm (lane 1) or 0.45 µm (lane 2) PES membrane from EMD Millipore. M, DNA MW marker (75 bp–20 kb).

    Article Snippet: Among the various commonly available commercial kits for gDNA isolation, the kit from Qiagen has been shown to have columns with superior binding capability., Hence, the QIAamp DNA Blood Mini Kit (Qiagen) was chosen for comparison of the efficiency of the gDNA isolation with the described method here.

    Techniques: Agarose Gel Electrophoresis, Marker

    Real-time PCR detection of Salmonella DNA. Two millilitres of whole blood was lysed using erythrocyte lysis buffer and spiked with the shown CFU of Salmonella Typhi CVD 909. DNA was extracted using a QIAamp Mini blood kit and S. Typhi was detected by qPCR.

    Journal: Journal of Applied Microbiology

    Article Title: Adaptation of red blood cell lysis represents a fundamental breakthrough that improves the sensitivity of Salmonella detection in blood

    doi: 10.1111/jam.12769

    Figure Lengend Snippet: Real-time PCR detection of Salmonella DNA. Two millilitres of whole blood was lysed using erythrocyte lysis buffer and spiked with the shown CFU of Salmonella Typhi CVD 909. DNA was extracted using a QIAamp Mini blood kit and S. Typhi was detected by qPCR.

    Article Snippet: The white blood cell pellets were resuspended in 200 μ l PBS and subsequently spiked with 4·4 × 103 CFU, 4·4 × 104 CFU, 4·4 × 105 CFU or 4·4 × 106 CFU of CVD 909 and DNA extraction was performed using the QIAamp DNA Blood Mini Kit (Qiagen) according to the manufacturer's instructions.

    Techniques: Real-time Polymerase Chain Reaction, Lysis

    Recovery of PCR product in DNA samples isolated from serum specimens using QIAamp DNA blood midi kit, protein A and phenol-chloroform extraction method. 1 - molecular size standards; 2, 6 and 10 - PCR-positive serum from patient M; 3, 7 and 11 - PCR-positive serum from patient P; 4, 8 and 12 - PCR negative serum from patient S; 5, 9 and 13 - extraction control; 14 - negative control; 15 - positive control.

    Journal: International Journal of Medical Sciences

    Article Title: Isolation of Chlamydia Pneumoniae from Serum Samples of the Patients with Acute Coronary Syndrome

    doi:

    Figure Lengend Snippet: Recovery of PCR product in DNA samples isolated from serum specimens using QIAamp DNA blood midi kit, protein A and phenol-chloroform extraction method. 1 - molecular size standards; 2, 6 and 10 - PCR-positive serum from patient M; 3, 7 and 11 - PCR-positive serum from patient P; 4, 8 and 12 - PCR negative serum from patient S; 5, 9 and 13 - extraction control; 14 - negative control; 15 - positive control.

    Article Snippet: DNA isolation from infected HL cells Cells were harvested from 24 well plates and resuspended in 200 µl of lysis buffer and DNA was extracted using QIAamp DNA Blood Mini Kit (QIAGEN INC., Valencia, Calif.) according to the manual.

    Techniques: Polymerase Chain Reaction, Isolation, Negative Control, Positive Control

    TAC optimization. ( A ) 50% Tween-20/Triton X-100 and 0.1% saponin treatment on the yield of pathogen cells from whole blood specimens. 10 3 CFU S. aureus was mixed in 1 mL blood from healthy donors and then lysed by Tween-20/Triton X-100 or saponin. The colony numbers were determined by a plate count. ( B , C ). TNA extraction kit performance on blood culture specimens ( B ) and mock whole blood specimens ( C ). Kit 1 is the BiOstic bacteremia DNA isolation kit; Kit 2 is the QIAamp DNA Blood Mini Kit; Kit 3 is the QIAamp UCP Pathogen Mini Kit; Kit 4 is the TIANamp Blood DNA Kit; Kit 5 is the QIAamp cador Pathogen Mini Kit. M represents the benzyl alcohol-guanidine hydrochloride method. For blood culture specimens, three blood culture samples positive for S. aureus (G + ), E. coli (G − ), or C. albicans (fungi) were used. For whole blood specimens, three whole blood mock specimens spiked with 10 1 CFU/mL of S. aureus , E. coli , and C. albicans were used. ( D ) The effect of TNA combined with reverse transcription on amplifying efficiency. 10 1 and 10 2 CFU S. aureus (G + ), E. coli (G − ), or C. albicans (fungi) were used to make mock specimens. The cycle threshold (Ct) was compared with RT or without RT. ( E ) The effect of 0.1% blue dextran 2000 on the TAC assay. 10 6 CFU S. aureus (G + ), 10 7 CFU P. aeruginosa (G − ) and 10 4 CFU C. albicans (fungi) were used to make mock specimens. B+, TAC assay with blue dextran 2000; B−, TAC assay without blue dextran 2000.

    Journal: Scientific Reports

    Article Title: Detection of pathogenic microorganisms from bloodstream infection specimens using TaqMan array card technology

    doi: 10.1038/s41598-018-31200-3

    Figure Lengend Snippet: TAC optimization. ( A ) 50% Tween-20/Triton X-100 and 0.1% saponin treatment on the yield of pathogen cells from whole blood specimens. 10 3 CFU S. aureus was mixed in 1 mL blood from healthy donors and then lysed by Tween-20/Triton X-100 or saponin. The colony numbers were determined by a plate count. ( B , C ). TNA extraction kit performance on blood culture specimens ( B ) and mock whole blood specimens ( C ). Kit 1 is the BiOstic bacteremia DNA isolation kit; Kit 2 is the QIAamp DNA Blood Mini Kit; Kit 3 is the QIAamp UCP Pathogen Mini Kit; Kit 4 is the TIANamp Blood DNA Kit; Kit 5 is the QIAamp cador Pathogen Mini Kit. M represents the benzyl alcohol-guanidine hydrochloride method. For blood culture specimens, three blood culture samples positive for S. aureus (G + ), E. coli (G − ), or C. albicans (fungi) were used. For whole blood specimens, three whole blood mock specimens spiked with 10 1 CFU/mL of S. aureus , E. coli , and C. albicans were used. ( D ) The effect of TNA combined with reverse transcription on amplifying efficiency. 10 1 and 10 2 CFU S. aureus (G + ), E. coli (G − ), or C. albicans (fungi) were used to make mock specimens. The cycle threshold (Ct) was compared with RT or without RT. ( E ) The effect of 0.1% blue dextran 2000 on the TAC assay. 10 6 CFU S. aureus (G + ), 10 7 CFU P. aeruginosa (G − ) and 10 4 CFU C. albicans (fungi) were used to make mock specimens. B+, TAC assay with blue dextran 2000; B−, TAC assay without blue dextran 2000.

    Article Snippet: These kits included the BiOstic bacteremia DNA isolation kit (MOBIO, Carlsbad, CA, USA), QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany), QIAamp UCP Pathogen Mini Kit (Qiagen, Hilden, Germany), TIANamp Blood DNA Kit (Tiangen, Beijing, China) and QIAamp cador Pathogen Mini Kit (Qiagen, Hilden, Germany).

    Techniques: DNA Extraction