thermomutatus chrysovirus 1  (Millipore)


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    Structured Review

    Millipore thermomutatus chrysovirus 1
    Alignment of amino acid sequences of the putative RdRP of A . <t>thermomutatus</t> <t>chrysovirus</t> 1 (AthCV1) with those of closely related chrysoviruses. The eight conserved RdRP motifs characteristic of dsRNA mycoviruses are referred to as M1-M8. Shading: black, 100% amino acid similarity; dark gray, 80–99% amino acid similarity; gray, 60–79% amino acid similarity; white, less than 60% amino acid similarity. Virus sequences are as follows: AGZ84312: RdRP aa sequence of Botryosphaeria dothidea chrysovirus 1 [ 48 ], BBC27878: RdRP aa sequence of Alternaria alternata chrysovirus 1 [ 49 ], AWC67507: RdRP aa sequence of AthCV1, ALO50149: RdRP aa sequence of Penicillium janczewskii chrysovirus 2 [ 50 ], AXP19674: RdRP aa sequence of Colletotrichum fructicola chrysovirus 1 [ 51 ], CBY84993: RdRP aa sequence of Tolypocladium cylindrosporum virus 2 [ 58 ], BAJ15133: RdRP aa sequence of Magnaporthe oryzae chrysovirus 1-A [ 59 ], BAO20927: RdRP aa sequence of Magnaporthe oryzae chrysovirus 1-B [ 60 ], ABX79996: RdRP aa sequence of Aspergillus mycovirus 1816 [ 61 ], ALO50142: RdRP aa sequence of Penicillium janczewskii chrysovirus 1 [ 50 ], AKP45145: RdRP aa sequence of Fusarium oxysporum f . sp . dianthi virus [ 62 ], ADU54123: RdRP aa sequence of Fusarium graminearum mycovirus-China 9 [ 63 ], ADW08802: RdRP aa sequence of Fusarium graminearum dsRNA mycovirus -2 [ 64 ], AAM68953: RdRP aa sequence of Helminthosporium victoriae 145S virus [ 65 ], CAG77602: RdRP aa sequence of Amasya cherry disease associated chrysovirus [ 66 ], ACT79255: RdRP aa sequence of Cryphonectria nitschkei chrysovirus 1[ 67 ], ADG21213: RdRP aa sequence of Verticillium dahliae chrysovirus 1 [ 68 ], ALD89090: RdRP aa sequence of Macrophomina phaseolina chrysovirus 1 [ 69 ], APR73428: RdRP aa sequence of Isaria javanica chrysovirus 1 [ 70 ], AAM95601: RdRP aa sequence of Penicillium chrysogenum virus [ 71 ], CAX48749: RdRP aa sequence of A . fumigatus chrysovirus [ 19 ], ABQ53134: RdRP aa sequence of Fusarium oxysporum chrysovirus 1 [ 72 ].
    Thermomutatus Chrysovirus 1, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 1249 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "A novel chrysovirus from a clinical isolate of Aspergillus thermomutatus affects sporulation"

    Article Title: A novel chrysovirus from a clinical isolate of Aspergillus thermomutatus affects sporulation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0209443

    Alignment of amino acid sequences of the putative RdRP of A . thermomutatus chrysovirus 1 (AthCV1) with those of closely related chrysoviruses. The eight conserved RdRP motifs characteristic of dsRNA mycoviruses are referred to as M1-M8. Shading: black, 100% amino acid similarity; dark gray, 80–99% amino acid similarity; gray, 60–79% amino acid similarity; white, less than 60% amino acid similarity. Virus sequences are as follows: AGZ84312: RdRP aa sequence of Botryosphaeria dothidea chrysovirus 1 [ 48 ], BBC27878: RdRP aa sequence of Alternaria alternata chrysovirus 1 [ 49 ], AWC67507: RdRP aa sequence of AthCV1, ALO50149: RdRP aa sequence of Penicillium janczewskii chrysovirus 2 [ 50 ], AXP19674: RdRP aa sequence of Colletotrichum fructicola chrysovirus 1 [ 51 ], CBY84993: RdRP aa sequence of Tolypocladium cylindrosporum virus 2 [ 58 ], BAJ15133: RdRP aa sequence of Magnaporthe oryzae chrysovirus 1-A [ 59 ], BAO20927: RdRP aa sequence of Magnaporthe oryzae chrysovirus 1-B [ 60 ], ABX79996: RdRP aa sequence of Aspergillus mycovirus 1816 [ 61 ], ALO50142: RdRP aa sequence of Penicillium janczewskii chrysovirus 1 [ 50 ], AKP45145: RdRP aa sequence of Fusarium oxysporum f . sp . dianthi virus [ 62 ], ADU54123: RdRP aa sequence of Fusarium graminearum mycovirus-China 9 [ 63 ], ADW08802: RdRP aa sequence of Fusarium graminearum dsRNA mycovirus -2 [ 64 ], AAM68953: RdRP aa sequence of Helminthosporium victoriae 145S virus [ 65 ], CAG77602: RdRP aa sequence of Amasya cherry disease associated chrysovirus [ 66 ], ACT79255: RdRP aa sequence of Cryphonectria nitschkei chrysovirus 1[ 67 ], ADG21213: RdRP aa sequence of Verticillium dahliae chrysovirus 1 [ 68 ], ALD89090: RdRP aa sequence of Macrophomina phaseolina chrysovirus 1 [ 69 ], APR73428: RdRP aa sequence of Isaria javanica chrysovirus 1 [ 70 ], AAM95601: RdRP aa sequence of Penicillium chrysogenum virus [ 71 ], CAX48749: RdRP aa sequence of A . fumigatus chrysovirus [ 19 ], ABQ53134: RdRP aa sequence of Fusarium oxysporum chrysovirus 1 [ 72 ].
    Figure Legend Snippet: Alignment of amino acid sequences of the putative RdRP of A . thermomutatus chrysovirus 1 (AthCV1) with those of closely related chrysoviruses. The eight conserved RdRP motifs characteristic of dsRNA mycoviruses are referred to as M1-M8. Shading: black, 100% amino acid similarity; dark gray, 80–99% amino acid similarity; gray, 60–79% amino acid similarity; white, less than 60% amino acid similarity. Virus sequences are as follows: AGZ84312: RdRP aa sequence of Botryosphaeria dothidea chrysovirus 1 [ 48 ], BBC27878: RdRP aa sequence of Alternaria alternata chrysovirus 1 [ 49 ], AWC67507: RdRP aa sequence of AthCV1, ALO50149: RdRP aa sequence of Penicillium janczewskii chrysovirus 2 [ 50 ], AXP19674: RdRP aa sequence of Colletotrichum fructicola chrysovirus 1 [ 51 ], CBY84993: RdRP aa sequence of Tolypocladium cylindrosporum virus 2 [ 58 ], BAJ15133: RdRP aa sequence of Magnaporthe oryzae chrysovirus 1-A [ 59 ], BAO20927: RdRP aa sequence of Magnaporthe oryzae chrysovirus 1-B [ 60 ], ABX79996: RdRP aa sequence of Aspergillus mycovirus 1816 [ 61 ], ALO50142: RdRP aa sequence of Penicillium janczewskii chrysovirus 1 [ 50 ], AKP45145: RdRP aa sequence of Fusarium oxysporum f . sp . dianthi virus [ 62 ], ADU54123: RdRP aa sequence of Fusarium graminearum mycovirus-China 9 [ 63 ], ADW08802: RdRP aa sequence of Fusarium graminearum dsRNA mycovirus -2 [ 64 ], AAM68953: RdRP aa sequence of Helminthosporium victoriae 145S virus [ 65 ], CAG77602: RdRP aa sequence of Amasya cherry disease associated chrysovirus [ 66 ], ACT79255: RdRP aa sequence of Cryphonectria nitschkei chrysovirus 1[ 67 ], ADG21213: RdRP aa sequence of Verticillium dahliae chrysovirus 1 [ 68 ], ALD89090: RdRP aa sequence of Macrophomina phaseolina chrysovirus 1 [ 69 ], APR73428: RdRP aa sequence of Isaria javanica chrysovirus 1 [ 70 ], AAM95601: RdRP aa sequence of Penicillium chrysogenum virus [ 71 ], CAX48749: RdRP aa sequence of A . fumigatus chrysovirus [ 19 ], ABQ53134: RdRP aa sequence of Fusarium oxysporum chrysovirus 1 [ 72 ].

    Techniques Used: Sequencing

    Phylogenetic analysis of A . thermomutatus chrysovirus 1 and its closely related members of the family Chrysoviridae and unclassified viruses based on amino acid sequences of their RdRPs. The amino acid sequences were aligned using the program MUSCLE [ 38 ]. A neighbour-joining phylogenetic tree [ 39 ] constructed from this alignment for distance calculated with a Poisson model and a gamma distribution of rates between sites using the program MEGA 6 [ 40 ]. Bootstrap values based on 1000 replicates. Cluster I contains viruses with 4 or 3 genome segments (classified members of the genus Chrysovirus are in bold font). Cluster II contains 4, 5 or 7 genome segments.
    Figure Legend Snippet: Phylogenetic analysis of A . thermomutatus chrysovirus 1 and its closely related members of the family Chrysoviridae and unclassified viruses based on amino acid sequences of their RdRPs. The amino acid sequences were aligned using the program MUSCLE [ 38 ]. A neighbour-joining phylogenetic tree [ 39 ] constructed from this alignment for distance calculated with a Poisson model and a gamma distribution of rates between sites using the program MEGA 6 [ 40 ]. Bootstrap values based on 1000 replicates. Cluster I contains viruses with 4 or 3 genome segments (classified members of the genus Chrysovirus are in bold font). Cluster II contains 4, 5 or 7 genome segments.

    Techniques Used: Construct

    2) Product Images from "PATZ1 is a target of miR-29b that is induced by Ha-Ras oncogene in rat thyroid cells"

    Article Title: PATZ1 is a target of miR-29b that is induced by Ha-Ras oncogene in rat thyroid cells

    Journal: Scientific Reports

    doi: 10.1038/srep25268

    miR-29b targeting of PATZ1 in rat thyroid cells. ( a ) Western blot using anti-PATZ1 on PC Cl3 total extracts previously transfected with synthetic miR-29b precursor or scramble oligonucleotide. Three major specific bands were observed (arrows). Vinculin was used for normalization. Densitometric analysis by Image J software was applied on the gel: Relative expression levels of PATZ1, compared to scramble-transfected control and normalized with respect to vinculin, are indicated on the bottom. Black lines delineate the boundary between not contiguous lanes of the same gel. ( b ) qRT-PCR on total RNA from PC Cl3 and FRTL-5 cells previously transfected with synthetic miR-29b precursor or scramble oligonucleotide. PATZ1 mRNA levels were normalized for endogenous G6PD levels. The mean ± SE of three independent experiments performed in duplicate for each cell line is reported. *P
    Figure Legend Snippet: miR-29b targeting of PATZ1 in rat thyroid cells. ( a ) Western blot using anti-PATZ1 on PC Cl3 total extracts previously transfected with synthetic miR-29b precursor or scramble oligonucleotide. Three major specific bands were observed (arrows). Vinculin was used for normalization. Densitometric analysis by Image J software was applied on the gel: Relative expression levels of PATZ1, compared to scramble-transfected control and normalized with respect to vinculin, are indicated on the bottom. Black lines delineate the boundary between not contiguous lanes of the same gel. ( b ) qRT-PCR on total RNA from PC Cl3 and FRTL-5 cells previously transfected with synthetic miR-29b precursor or scramble oligonucleotide. PATZ1 mRNA levels were normalized for endogenous G6PD levels. The mean ± SE of three independent experiments performed in duplicate for each cell line is reported. *P

    Techniques Used: Western Blot, Transfection, Software, Expressing, Quantitative RT-PCR

    Validation of PATZ1 as a target of miR-29b. ( a ) predicted miR-29b/ PATZ1 alignment, according to microRNA.org web system. mirSVR (cutoff 0.1 or lower) and PhastCons (cutoff 0.57 or higher) are downregulation and conservation scores, respectively. ( b ) Western blot using anti-PATZ1 on HEK293 total cellular extracts collected 72 h after transfection with increasing amount (50–100 nM) of synthetic miR-29b precursor or scramble (100 nM) oligonucleotide. Vinculin was used for normalization. Relative expression levels, compared to scramble-transfected control and normalized with respect to vinculin, are indicated on the bottom. Black lines delineate the boundary between not contiguous lanes of the same gel. ( c ) qRT-PCR on total RNA from HEK293 cells previously transfected with 100 nM synthetic miR-29b precursor or scramble oligonucleotide. PATZ1 mRNA levels were normalized for endogenous G6PD levels. The mean ± SE of four independent experiments performed in duplicate is reported. ( d ) Luciferase assay on HEK293 cells co-transfected with the Luc-PATZ1-3′UTR and pCMV renilla reporter vectors along with 100 nM synthetic miR-29b precursor or scramble oligonucleotide. Relative firefly luciferase activity levels were normalized for renilla luciferase activity and analysed relatively to scramble-transfected cells, which were set to 1. The mean ± SE of four independent experiments performed in duplicate is reported. ****P
    Figure Legend Snippet: Validation of PATZ1 as a target of miR-29b. ( a ) predicted miR-29b/ PATZ1 alignment, according to microRNA.org web system. mirSVR (cutoff 0.1 or lower) and PhastCons (cutoff 0.57 or higher) are downregulation and conservation scores, respectively. ( b ) Western blot using anti-PATZ1 on HEK293 total cellular extracts collected 72 h after transfection with increasing amount (50–100 nM) of synthetic miR-29b precursor or scramble (100 nM) oligonucleotide. Vinculin was used for normalization. Relative expression levels, compared to scramble-transfected control and normalized with respect to vinculin, are indicated on the bottom. Black lines delineate the boundary between not contiguous lanes of the same gel. ( c ) qRT-PCR on total RNA from HEK293 cells previously transfected with 100 nM synthetic miR-29b precursor or scramble oligonucleotide. PATZ1 mRNA levels were normalized for endogenous G6PD levels. The mean ± SE of four independent experiments performed in duplicate is reported. ( d ) Luciferase assay on HEK293 cells co-transfected with the Luc-PATZ1-3′UTR and pCMV renilla reporter vectors along with 100 nM synthetic miR-29b precursor or scramble oligonucleotide. Relative firefly luciferase activity levels were normalized for renilla luciferase activity and analysed relatively to scramble-transfected cells, which were set to 1. The mean ± SE of four independent experiments performed in duplicate is reported. ****P

    Techniques Used: Western Blot, Transfection, Expressing, Quantitative RT-PCR, Luciferase, Activity Assay

    3) Product Images from "Arabidopsis protein disulfide isomerase-8 is a type I endoplasmic reticulum transmembrane protein with thiol-disulfide oxidase activity"

    Article Title: Arabidopsis protein disulfide isomerase-8 is a type I endoplasmic reticulum transmembrane protein with thiol-disulfide oxidase activity

    Journal: BMC Plant Biology

    doi: 10.1186/s12870-016-0869-2

    Membrane orientation of PDI8. a Immunoblot analyses of proteins extracted from 35S pro :PDI8 plants. The 35S pro :PDI8 total protein homogenate was separated into soluble (sol) and microsomal membrane (mem) fractions by centrifugation. Immunoblots were incubated with affinity-purified anti-PDI8 antiserum (upper panel). A polyclonal goat anti-BiP antibody was used as a marker for microsomes (middle panel). The large subunit of Rubisco (LSU) was used as a marker for the soluble phase in an SDS-PAGE gel stained with Coomassie (lower panel). b Protease protection assays were performed with 35S pro :PDI8 microsomes. Samples were either treated (+) or not treated (-) with 50 μg/mL proteinase K (PK) and 0.1 % Triton X-100 (TX), and immunoblot analysis was performed using the anti-PDI8 antiserum. c Model of the PDI8 polypeptide oriented in the ER membrane
    Figure Legend Snippet: Membrane orientation of PDI8. a Immunoblot analyses of proteins extracted from 35S pro :PDI8 plants. The 35S pro :PDI8 total protein homogenate was separated into soluble (sol) and microsomal membrane (mem) fractions by centrifugation. Immunoblots were incubated with affinity-purified anti-PDI8 antiserum (upper panel). A polyclonal goat anti-BiP antibody was used as a marker for microsomes (middle panel). The large subunit of Rubisco (LSU) was used as a marker for the soluble phase in an SDS-PAGE gel stained with Coomassie (lower panel). b Protease protection assays were performed with 35S pro :PDI8 microsomes. Samples were either treated (+) or not treated (-) with 50 μg/mL proteinase K (PK) and 0.1 % Triton X-100 (TX), and immunoblot analysis was performed using the anti-PDI8 antiserum. c Model of the PDI8 polypeptide oriented in the ER membrane

    Techniques Used: Centrifugation, Western Blot, Incubation, Affinity Purification, Marker, SDS Page, Staining

    Alkaline phosphatase activity of E. coli dsbA − cells expressing the lumenal region of PDI8. PhoA activities were measured from cell lysates obtained from the dsbA + strain RI89 (wild-type; WT), the untransformed dsbA − strain RI90, and RI90 cells transformed with either the pFLAG-CTS empty vector (+EV) or the pFLAG-PDI8 abb’ construct (+PDI8). The values are averages of three independent trials, with error bars representing standard deviations
    Figure Legend Snippet: Alkaline phosphatase activity of E. coli dsbA − cells expressing the lumenal region of PDI8. PhoA activities were measured from cell lysates obtained from the dsbA + strain RI89 (wild-type; WT), the untransformed dsbA − strain RI90, and RI90 cells transformed with either the pFLAG-CTS empty vector (+EV) or the pFLAG-PDI8 abb’ construct (+PDI8). The values are averages of three independent trials, with error bars representing standard deviations

    Techniques Used: Activity Assay, Expressing, Transformation Assay, Plasmid Preparation, Construct

    Domain arrangement of PDI8. a The secondary structure of PDI8. Positions of α-helices (E) and β-strands (H) are based on prediction by SPIDER2. The thioredoxin-fold domains ( a , b and b’ ), and predicted signal peptide (SP) and TMD of PDI8 are boxed. Each thioredoxin fold consists of 5 β-strands and 4 α-helices (underlined), in the arrangement β 1 -α 1 -β 2 -α 2 -β 3 -α 3 -β 4 -β 5 -α 4 . b Comparison of the domain organizations of Homo sapiens TMX3 and Arabidopsis PDI8, showing the relative positions of the SP, TMD, and domains a , b and b’ . PDI8 abb’ and PDI8 bb’ represent truncated forms of PDI8 used in this study
    Figure Legend Snippet: Domain arrangement of PDI8. a The secondary structure of PDI8. Positions of α-helices (E) and β-strands (H) are based on prediction by SPIDER2. The thioredoxin-fold domains ( a , b and b’ ), and predicted signal peptide (SP) and TMD of PDI8 are boxed. Each thioredoxin fold consists of 5 β-strands and 4 α-helices (underlined), in the arrangement β 1 -α 1 -β 2 -α 2 -β 3 -α 3 -β 4 -β 5 -α 4 . b Comparison of the domain organizations of Homo sapiens TMX3 and Arabidopsis PDI8, showing the relative positions of the SP, TMD, and domains a , b and b’ . PDI8 abb’ and PDI8 bb’ represent truncated forms of PDI8 used in this study

    Techniques Used:

    Expression pattern of the PDI8 pro :GUS reporter construct in seedlings and flowering plants. GUS staining pattern of 7-day-old seedlings ( a ), with close-up images of a cotyledon stomata (so) and vasculature (v) ( b ), the root mature zone ( c ), and the root tip ( d ). GUS staining pattern of 14-day-old seedlings ( e ), with close-up images of an expanding leaf ( f ) and the shoot apex ( g ). In panel g , the emerging leaves were pulled back to expose the shoot apical meristem (sm). GUS staining pattern of 6-week-old plants in flowers ( h ), an expanding silique ( i ), and the inflorescence stem ( j ). In j , staining is shown at the junction between the stem (st) and the pedicel (pd) of a silique
    Figure Legend Snippet: Expression pattern of the PDI8 pro :GUS reporter construct in seedlings and flowering plants. GUS staining pattern of 7-day-old seedlings ( a ), with close-up images of a cotyledon stomata (so) and vasculature (v) ( b ), the root mature zone ( c ), and the root tip ( d ). GUS staining pattern of 14-day-old seedlings ( e ), with close-up images of an expanding leaf ( f ) and the shoot apex ( g ). In panel g , the emerging leaves were pulled back to expose the shoot apical meristem (sm). GUS staining pattern of 6-week-old plants in flowers ( h ), an expanding silique ( i ), and the inflorescence stem ( j ). In j , staining is shown at the junction between the stem (st) and the pedicel (pd) of a silique

    Techniques Used: Expressing, Construct, Staining

    Detection of native PDI8 specifically at the ER by immunoelectron microscopy. TEM analysis was performed on sections taken from the shoot apex ( a ), and the root apex ( b ), after primary labeling with rabbit anti-PDI8 antiserum and secondary labeling with 10 or 15 nm gold-conjugated goat anti-rabbit IgG antibodies (respectively). Labeling was detected at the endoplasmic reticulum (ER)
    Figure Legend Snippet: Detection of native PDI8 specifically at the ER by immunoelectron microscopy. TEM analysis was performed on sections taken from the shoot apex ( a ), and the root apex ( b ), after primary labeling with rabbit anti-PDI8 antiserum and secondary labeling with 10 or 15 nm gold-conjugated goat anti-rabbit IgG antibodies (respectively). Labeling was detected at the endoplasmic reticulum (ER)

    Techniques Used: Immuno-Electron Microscopy, Transmission Electron Microscopy, Labeling

    GFP fusions to PDI8 localize to the ER. a Position of GFP(S65T) within the fusions spGFP-PDI8 and PDI8-GFP-KKED. b Transient co-expression of the ER-mCherry marker with unfused GFP(S65T) (top row), the spGFP-PDI8 construct (middle row), or the PDI8-GFP-KKED construct (bottom row). GFP(S65T) signal is shown in column 1, mCherry signal in column 2, and a merge of both signal patterns in column 3. The scale bar in each panel represents 5 μm
    Figure Legend Snippet: GFP fusions to PDI8 localize to the ER. a Position of GFP(S65T) within the fusions spGFP-PDI8 and PDI8-GFP-KKED. b Transient co-expression of the ER-mCherry marker with unfused GFP(S65T) (top row), the spGFP-PDI8 construct (middle row), or the PDI8-GFP-KKED construct (bottom row). GFP(S65T) signal is shown in column 1, mCherry signal in column 2, and a merge of both signal patterns in column 3. The scale bar in each panel represents 5 μm

    Techniques Used: Expressing, Marker, Construct

    4) Product Images from "NLRP3 Inflammasome: Key Mediator of Neuroinflammation in Murine Japanese Encephalitis"

    Article Title: NLRP3 Inflammasome: Key Mediator of Neuroinflammation in Murine Japanese Encephalitis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0032270

    Generation of ROS is critical for caspase-1 activity and subsequent IL-1β and IL-18 maturation. BV-2 cells were incubated with 1 µM of DPI for inhibition of ROS generation. ( A ) Representative FACS plot showing intracellular ROS production, after 4 h of JEV infection and representation of the Mean Fluorescent Intensities (M.F.I) (right graph) in mock-infected control (C), JEV+DPI and JEV alone condition. ( B ) Caspase-1 activity measured after 6 h of JEV infection in presence or absence of DPI. Graph represents fold change in caspase-1 activity with respect to mock-infected control. ( C–D ) ELISA study showing the levels of mature IL-1β and IL-18 in JEV+DPI condition with respect to JEV alone infected sample. Graph represents cytokine levels in pg/ml. For Potassium efflux study, BV-2 cells were incubated with 50 mM KCl for 20 min in order to study the requirement of K + efflux for caspase-1 activity and its downstream effects. Data represent mean ± SEM from 3 independent experiments performed in duplicate. Statistical differences were evaluated using one way ANOVA with Bonferroni's post hoc test. *, **, Statistical difference in comparison to mock-infected control values (*p
    Figure Legend Snippet: Generation of ROS is critical for caspase-1 activity and subsequent IL-1β and IL-18 maturation. BV-2 cells were incubated with 1 µM of DPI for inhibition of ROS generation. ( A ) Representative FACS plot showing intracellular ROS production, after 4 h of JEV infection and representation of the Mean Fluorescent Intensities (M.F.I) (right graph) in mock-infected control (C), JEV+DPI and JEV alone condition. ( B ) Caspase-1 activity measured after 6 h of JEV infection in presence or absence of DPI. Graph represents fold change in caspase-1 activity with respect to mock-infected control. ( C–D ) ELISA study showing the levels of mature IL-1β and IL-18 in JEV+DPI condition with respect to JEV alone infected sample. Graph represents cytokine levels in pg/ml. For Potassium efflux study, BV-2 cells were incubated with 50 mM KCl for 20 min in order to study the requirement of K + efflux for caspase-1 activity and its downstream effects. Data represent mean ± SEM from 3 independent experiments performed in duplicate. Statistical differences were evaluated using one way ANOVA with Bonferroni's post hoc test. *, **, Statistical difference in comparison to mock-infected control values (*p

    Techniques Used: Activity Assay, Incubation, Inhibition, FACS, Infection, Enzyme-linked Immunosorbent Assay

    Schematic showing the signalling pathway leading to IL-1β and IL-18 production upon JEV infection in microglia. NLRP3 is the key PRR involved in the identification of JEV intracellularly in a microglia. The NLRP3 interacts with an adaptor molecule, ASC and recruits pro-caspase-1 forming a biochemical complex termed as inflammasome. In response to JEV, NLRP3 cleaves pro-caspase-1 into its active form, caspase-1. Caspase-1 then cleaves the inactive pro-forms of IL-1β and IL-18 to their mature forms which are then secreted out. ROS and K + efflux are the host derived danger signals that are critical for the formation of NLRP3 biochemical complex during JEV infection.
    Figure Legend Snippet: Schematic showing the signalling pathway leading to IL-1β and IL-18 production upon JEV infection in microglia. NLRP3 is the key PRR involved in the identification of JEV intracellularly in a microglia. The NLRP3 interacts with an adaptor molecule, ASC and recruits pro-caspase-1 forming a biochemical complex termed as inflammasome. In response to JEV, NLRP3 cleaves pro-caspase-1 into its active form, caspase-1. Caspase-1 then cleaves the inactive pro-forms of IL-1β and IL-18 to their mature forms which are then secreted out. ROS and K + efflux are the host derived danger signals that are critical for the formation of NLRP3 biochemical complex during JEV infection.

    Techniques Used: Infection, Derivative Assay

    NLRP3 is critical for caspase-1 activity as well as IL-1β and IL-18 production. Transient knockdown of NLRP3 using 100 nM of NLRP3 SiRNA was carried out in BV-2 microglial cells. Scrambed RNA (ScRNA) was used as a transfection control. This was followed by virus infection with 5 MOI dose for 6 h. ( A ) NLRP3 mRNA levels were measured by qRT-PCR from total RNA isolated from JEV infected as well as uninfected BV-2 cells. The graph represent a fold change in NLRP3 mRNA with respect to mock-infected control normalised to 18 s rRNA internal control. ( B ) Caspase-1 activity was also measured in NLRP3 knockdown condition upon JEV infection. Graph represents fold change in caspase-1 activity in different conditions with respect to mock-infected control. ( C–D ) In order to measure IL-1β and IL-18 production, ELISA was then carried out in NLRP3 knockdown (SiRNA+JEV) condition with respect to JEV infected sample. Graph represents cytokine levels in pg/ml. Data represent mean ± SEM from 3 independent experiments performed in duplicate. Statistical differences were evaluated using one way ANOVA with Bonferroni's post hoc test. *, **, Statistical difference in comparison to mock-infected control values (* p
    Figure Legend Snippet: NLRP3 is critical for caspase-1 activity as well as IL-1β and IL-18 production. Transient knockdown of NLRP3 using 100 nM of NLRP3 SiRNA was carried out in BV-2 microglial cells. Scrambed RNA (ScRNA) was used as a transfection control. This was followed by virus infection with 5 MOI dose for 6 h. ( A ) NLRP3 mRNA levels were measured by qRT-PCR from total RNA isolated from JEV infected as well as uninfected BV-2 cells. The graph represent a fold change in NLRP3 mRNA with respect to mock-infected control normalised to 18 s rRNA internal control. ( B ) Caspase-1 activity was also measured in NLRP3 knockdown condition upon JEV infection. Graph represents fold change in caspase-1 activity in different conditions with respect to mock-infected control. ( C–D ) In order to measure IL-1β and IL-18 production, ELISA was then carried out in NLRP3 knockdown (SiRNA+JEV) condition with respect to JEV infected sample. Graph represents cytokine levels in pg/ml. Data represent mean ± SEM from 3 independent experiments performed in duplicate. Statistical differences were evaluated using one way ANOVA with Bonferroni's post hoc test. *, **, Statistical difference in comparison to mock-infected control values (* p

    Techniques Used: Activity Assay, Transfection, Infection, Quantitative RT-PCR, Isolation, Enzyme-linked Immunosorbent Assay

    Potassium efflux is required for caspase-1 activity and subsequent inflammation upon JEV infection. ( A ) Caspase-1 activity was measured in presence of KCl upon JEV infection. Graph represents caspase-1 activity in JEV infected cells incubated with KCl with respect to untreated JEV alone condition. ( B–C ) ELISA study showing the levels of IL-1β and IL-18 in KCl treated condition upon JEV infection over that of JEV alone condition. Graph represents IL-1β and IL-18 levels in pg/ml. Data represent mean ± SEM from 3 independent experiments performed in duplicate. Statistical differences were evaluated using one way ANOVA with Bonferroni's post hoc test. *, **, Statistical difference in comparison to mock-infected control values (*p
    Figure Legend Snippet: Potassium efflux is required for caspase-1 activity and subsequent inflammation upon JEV infection. ( A ) Caspase-1 activity was measured in presence of KCl upon JEV infection. Graph represents caspase-1 activity in JEV infected cells incubated with KCl with respect to untreated JEV alone condition. ( B–C ) ELISA study showing the levels of IL-1β and IL-18 in KCl treated condition upon JEV infection over that of JEV alone condition. Graph represents IL-1β and IL-18 levels in pg/ml. Data represent mean ± SEM from 3 independent experiments performed in duplicate. Statistical differences were evaluated using one way ANOVA with Bonferroni's post hoc test. *, **, Statistical difference in comparison to mock-infected control values (*p

    Techniques Used: Activity Assay, Infection, Incubation, Enzyme-linked Immunosorbent Assay

    Caspase-1 activity is required for the production of IL-1β and IL-18 during JEV infection. BV-2 cells were incubated with 5 µM YVAD for 30 min to inhibit caspase-1 activity followed by JEV infection. ( A–B ) ELISA for IL-1β and IL-18 was carried out in JEV infected BV-2 cells upon caspase-1 inhibition. The cytokine levels were then measured using ELISA and the values are represented in pg/ml. Data represent mean ± SEM from 3 independent experiments performed in duplicate. Statistical differences were evaluated using one way ANOVA with Bonferroni's post hoc test. **, Statistical difference in comparison to mock-infected control values (** p
    Figure Legend Snippet: Caspase-1 activity is required for the production of IL-1β and IL-18 during JEV infection. BV-2 cells were incubated with 5 µM YVAD for 30 min to inhibit caspase-1 activity followed by JEV infection. ( A–B ) ELISA for IL-1β and IL-18 was carried out in JEV infected BV-2 cells upon caspase-1 inhibition. The cytokine levels were then measured using ELISA and the values are represented in pg/ml. Data represent mean ± SEM from 3 independent experiments performed in duplicate. Statistical differences were evaluated using one way ANOVA with Bonferroni's post hoc test. **, Statistical difference in comparison to mock-infected control values (** p

    Techniques Used: Activity Assay, Infection, Incubation, Enzyme-linked Immunosorbent Assay, Inhibition

    JEV induces the production of IL-1β and IL-18 in vitro . BV-2 microglial cells were infected with 5 MOI of JEV for different time points. LPS+ATP condition was used as a positive control for qRT-PCR, ELISA and caspase-1 activity studies. ( A ) Cytokine analysis from mock-infected control and JEV infected BV-2 cells was carried out after 6 h of JEV infection using cytokine bead array. Graphs represent the levels of pro-inflammatory cytokines, TNF- α, CCL2 and IL-6 after JEV infection with respect to mock-infected control condition. ( B–C ) qRT-PCR studies on total RNA isolated from uninfected cells as well as on BV-2 cells infected with JEV for 3 h. IL-1β and IL-18 mRNA levels are represented in terms of fold change with respect to mock-infected control normalized to 18 s rRNA internal control. ( D–E ) Levels of mature IL-1β and IL-18 cytokines upon JEV infection with respect to mock-infected control condition were measured by ELISA. Graphs represent the fold change values in JEV infected cells with respect to mock-infected control condition. Data represent mean ± SEM from 3 independent experiments performed in duplicate. Statistical differences were evaluated using one way ANOVA with Bonferroni's post hoc test. *, **, Statistical differences in comparison to mock-infected control values (* p
    Figure Legend Snippet: JEV induces the production of IL-1β and IL-18 in vitro . BV-2 microglial cells were infected with 5 MOI of JEV for different time points. LPS+ATP condition was used as a positive control for qRT-PCR, ELISA and caspase-1 activity studies. ( A ) Cytokine analysis from mock-infected control and JEV infected BV-2 cells was carried out after 6 h of JEV infection using cytokine bead array. Graphs represent the levels of pro-inflammatory cytokines, TNF- α, CCL2 and IL-6 after JEV infection with respect to mock-infected control condition. ( B–C ) qRT-PCR studies on total RNA isolated from uninfected cells as well as on BV-2 cells infected with JEV for 3 h. IL-1β and IL-18 mRNA levels are represented in terms of fold change with respect to mock-infected control normalized to 18 s rRNA internal control. ( D–E ) Levels of mature IL-1β and IL-18 cytokines upon JEV infection with respect to mock-infected control condition were measured by ELISA. Graphs represent the fold change values in JEV infected cells with respect to mock-infected control condition. Data represent mean ± SEM from 3 independent experiments performed in duplicate. Statistical differences were evaluated using one way ANOVA with Bonferroni's post hoc test. *, **, Statistical differences in comparison to mock-infected control values (* p

    Techniques Used: In Vitro, Infection, Positive Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Activity Assay, Isolation

    JEV induces the production of IL-1β and IL-18 in vivo . Infection was carried out in BALB/c mice with 5×10 5 PFU of JEV Intravenously. Brain samples were isolated from mock-infected control (C) as well JEV infected mice after 3 d, 5 d and 7 d post infection. ( A–C ) Cytokine levels were estimated using CBA. The graphs represent the levels of pro-inflammatory cytokines, TNF-α, CCL2 and IL-6 in pg/ml from protein homogenates isolated from infected and mock-infected mice brain ( D–E ) qRT-PCR analysis was carried out from total RNA isolated from mock-infected and JEV infected mice brains on all the time points and levels of IL-1β as well as IL-18 mRNA were measured. Graphs represent fold change in mRNA values with respect to mock-infected control normalized to 18 s rRNA internal control. ( F–G ) ELISA study was carried out to measure the levels of mature IL-1β and IL-18 cytokine from JEV infected as well as uninfected brain samples. Graphs represent the cytokine levels in pg/ml in mock-infected control as well as infected brain samples. Data represent mean ± SEM of 5 animals in each group. Statistical differences were evaluated using one way ANOVA with Bonferroni's post hoc test. *, **, Statistical differences in comparison to mock-infected control values (* p
    Figure Legend Snippet: JEV induces the production of IL-1β and IL-18 in vivo . Infection was carried out in BALB/c mice with 5×10 5 PFU of JEV Intravenously. Brain samples were isolated from mock-infected control (C) as well JEV infected mice after 3 d, 5 d and 7 d post infection. ( A–C ) Cytokine levels were estimated using CBA. The graphs represent the levels of pro-inflammatory cytokines, TNF-α, CCL2 and IL-6 in pg/ml from protein homogenates isolated from infected and mock-infected mice brain ( D–E ) qRT-PCR analysis was carried out from total RNA isolated from mock-infected and JEV infected mice brains on all the time points and levels of IL-1β as well as IL-18 mRNA were measured. Graphs represent fold change in mRNA values with respect to mock-infected control normalized to 18 s rRNA internal control. ( F–G ) ELISA study was carried out to measure the levels of mature IL-1β and IL-18 cytokine from JEV infected as well as uninfected brain samples. Graphs represent the cytokine levels in pg/ml in mock-infected control as well as infected brain samples. Data represent mean ± SEM of 5 animals in each group. Statistical differences were evaluated using one way ANOVA with Bonferroni's post hoc test. *, **, Statistical differences in comparison to mock-infected control values (* p

    Techniques Used: In Vivo, Infection, Mouse Assay, Isolation, Crocin Bleaching Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    5) Product Images from "Protective functions of ZO-2/Tjp2 expressed in hepatocytes and cholangiocytes against liver injury and cholestasis"

    Article Title: Protective functions of ZO-2/Tjp2 expressed in hepatocytes and cholangiocytes against liver injury and cholestasis

    Journal: bioRxiv

    doi: 10.1101/841080

    Blood and liver biochemistry and liver histology of control and Tjp2 cKO mice show progressive cholestasis in Tjp2 cKO mice. ( A ) Liver to body ratio. ( B, C ) ALT and AST plasma levels as markers for liver injury. ( D ) Quantification of Sirius red staining indicative of fibrosis from images as shown in panel F. H E ( E ) and Sirius red ( F ) staining indicative of liver fibrosis. Plasma BA ( G ), liver BA ( H ) and AP levels ( I ). ( J ) Bile flow in livers of control and Tjp2 cKO mice. mRNA expression levels for BA synthesis genes ( K ), BA transporters ( L ) and detoxification enzymes ( M ) in the livers of control and Tjp2 cKO mice determined by qRT-PCR. Data are shown as mean ± SD, paired Student’s t-test p-value
    Figure Legend Snippet: Blood and liver biochemistry and liver histology of control and Tjp2 cKO mice show progressive cholestasis in Tjp2 cKO mice. ( A ) Liver to body ratio. ( B, C ) ALT and AST plasma levels as markers for liver injury. ( D ) Quantification of Sirius red staining indicative of fibrosis from images as shown in panel F. H E ( E ) and Sirius red ( F ) staining indicative of liver fibrosis. Plasma BA ( G ), liver BA ( H ) and AP levels ( I ). ( J ) Bile flow in livers of control and Tjp2 cKO mice. mRNA expression levels for BA synthesis genes ( K ), BA transporters ( L ) and detoxification enzymes ( M ) in the livers of control and Tjp2 cKO mice determined by qRT-PCR. Data are shown as mean ± SD, paired Student’s t-test p-value

    Techniques Used: Mouse Assay, AST Assay, Staining, Expressing, Quantitative RT-PCR

    Mice with a hepatocyte-restricted inactivation of Tjp2 are less susceptible to CA-diet induced liver injury as compared to those lacking Tjp2 in both hepatocytes and cholangiocytes. ( A ) LacZ staining showing specific activation of the LacZ reporter gene in hepatocytes and not in cholangiocytes, indicative of a restricted deletion of Tjp2 in hepatocytes of Tjp2 icKO HC mice. ( B ) Immunofluorescence microscopy of liver sections from Tjp2 icKO HC mice treated or not with tamoxifen and stained with antibodies against Tjp2 and CK19, confirming the absence of Tjp2 protein in hepatocytes but not in CK19 positive cholangiocytes. Nuclei are labelled with DAPI. ( C ) H E staining and quantification of necrotic area ( D ) showing reduced susceptibility of Tjp2 icKO HC mice to CA-diet induced injury. ( E ). Liver to body weight ratios of control and Tjp2 icKO HC mice fed 0.5% or 1% CA diet. Plasma ( F ) and liver ( G ) bile acid, ALT ( H ), AST ( I ), AP ( J ) and bilirubin ( K ) levels showing enhanced tolerance of Tjp2 icKO HC animals to CA-diet induced injury (compare to respective parameters in Fig. 3 for Tjp2 cKO mice). Data are shown as mean ± SD, paired Student’s t-test p-value
    Figure Legend Snippet: Mice with a hepatocyte-restricted inactivation of Tjp2 are less susceptible to CA-diet induced liver injury as compared to those lacking Tjp2 in both hepatocytes and cholangiocytes. ( A ) LacZ staining showing specific activation of the LacZ reporter gene in hepatocytes and not in cholangiocytes, indicative of a restricted deletion of Tjp2 in hepatocytes of Tjp2 icKO HC mice. ( B ) Immunofluorescence microscopy of liver sections from Tjp2 icKO HC mice treated or not with tamoxifen and stained with antibodies against Tjp2 and CK19, confirming the absence of Tjp2 protein in hepatocytes but not in CK19 positive cholangiocytes. Nuclei are labelled with DAPI. ( C ) H E staining and quantification of necrotic area ( D ) showing reduced susceptibility of Tjp2 icKO HC mice to CA-diet induced injury. ( E ). Liver to body weight ratios of control and Tjp2 icKO HC mice fed 0.5% or 1% CA diet. Plasma ( F ) and liver ( G ) bile acid, ALT ( H ), AST ( I ), AP ( J ) and bilirubin ( K ) levels showing enhanced tolerance of Tjp2 icKO HC animals to CA-diet induced injury (compare to respective parameters in Fig. 3 for Tjp2 cKO mice). Data are shown as mean ± SD, paired Student’s t-test p-value

    Techniques Used: Mouse Assay, Staining, Activation Assay, Immunofluorescence, Microscopy, AST Assay

    Tjp2 cKO mice are more susceptible to CA diet induced liver injury and cholestasis. ( A ) Plasma from control and Tjp2 cKO mice after a 7-day 0.5% CA diet. Bilirubin ( B ), AP ( C ), plasma BA ( D ), and liver BA ( E ) levels. ( F ) H E staining showing liver necrosis (arrows) in CA diet fed Tjp2 cKO mice. ( G ) Quantification of necrotic area. ( H ) Plasma AST levels. mRNA expression levels for detoxification enzymes ( I ), BA synthesis genes ( J ), BA transporters ( K ) in the livers of control and Tjp2 cKO mice fed with normal or 0.5% CA diet as determined by qRT-PCR. Data are shown as mean ± SD, paired Student’s t-test p-value
    Figure Legend Snippet: Tjp2 cKO mice are more susceptible to CA diet induced liver injury and cholestasis. ( A ) Plasma from control and Tjp2 cKO mice after a 7-day 0.5% CA diet. Bilirubin ( B ), AP ( C ), plasma BA ( D ), and liver BA ( E ) levels. ( F ) H E staining showing liver necrosis (arrows) in CA diet fed Tjp2 cKO mice. ( G ) Quantification of necrotic area. ( H ) Plasma AST levels. mRNA expression levels for detoxification enzymes ( I ), BA synthesis genes ( J ), BA transporters ( K ) in the livers of control and Tjp2 cKO mice fed with normal or 0.5% CA diet as determined by qRT-PCR. Data are shown as mean ± SD, paired Student’s t-test p-value

    Techniques Used: Mouse Assay, Staining, AST Assay, Expressing, Quantitative RT-PCR

    The CAR agonist TCPOBOP suppresses CA diet-induced necrotic injury in Tjp2 cKO mice.( A ) mRNA expression levels for selected detoxification enzymes and BA transporters in control and Tjp2 cKO mice after a 5-day CA diet with or without daily TCPOBPOP injections, monitored by qRT-PCR. ( B, C ) TCPOBOP H E staining and quantification of the necrotic area in livers of CA-diet fed control or Tjp2 cKO mice with or without TCPOBPOP administration. Plasma BA ( D ), liver BA ( E ), AST ( F ) and bilirubin ( G ) levels in CA-diet fed control or Tjp2 cKO mice with or without TCPOBOP treatment. Data are shown as mean ± SD, paired Student’s t-test p-value
    Figure Legend Snippet: The CAR agonist TCPOBOP suppresses CA diet-induced necrotic injury in Tjp2 cKO mice.( A ) mRNA expression levels for selected detoxification enzymes and BA transporters in control and Tjp2 cKO mice after a 5-day CA diet with or without daily TCPOBPOP injections, monitored by qRT-PCR. ( B, C ) TCPOBOP H E staining and quantification of the necrotic area in livers of CA-diet fed control or Tjp2 cKO mice with or without TCPOBPOP administration. Plasma BA ( D ), liver BA ( E ), AST ( F ) and bilirubin ( G ) levels in CA-diet fed control or Tjp2 cKO mice with or without TCPOBOP treatment. Data are shown as mean ± SD, paired Student’s t-test p-value

    Techniques Used: Mouse Assay, Expressing, Quantitative RT-PCR, Staining, AST Assay

    Conditional deletion of Tjp2 in the mouse liver and TJ structure. ( A ) Immunofluorescence microscopy of liver sections from control and Tjp2 cKO mice stained with antibodies against Tjp2, Tjp1 or VE-cadherin, showing absence of Tjp2 protein in both hepatocytes and bile duct cholangiocytes (thick arrows), but not in endothelial cells (thin arrows). Nuclei are labelled with DAPI. ( B ) Western blot analysis of whole liver lysates from a control and two independent Tjp2 cKO mice probed with antibodies to Tjp2, Tjp1 and GAPDH, confirming the absence of Tjp2 in the Tjp2 cKO liver, while Tjp1 protein levels are not affected. GAPDH was used as a control. ( C ) mRNA expression levels of Tjp2 and Tjp1 in whole liver lysates from P60 and P300 control and Tjp2 cKO mice determined by qRT-PCR. Residual Tjp2 mRNA levels likely reflect the presence of cell types other than hepatocytes and cholangiocytes, for example endothelial cells, which express Tjp2. ( D ) Western blot analysis of whole liver lysates from a control and two Tjp2 cKO mice at P60 probed with antibodies to Cldn1, Cldn3, and Ocln. GAPDH was used as a control. ( E ) mRNA expression levels of Cldn1, Cldn2 and Cldn3 in whole liver lysates from P60 control and Tjp2 cKO mice determined by qRT-PCR. Data in (D) and (E) are shown as mean ± SD, paired Student’s t-test p-value
    Figure Legend Snippet: Conditional deletion of Tjp2 in the mouse liver and TJ structure. ( A ) Immunofluorescence microscopy of liver sections from control and Tjp2 cKO mice stained with antibodies against Tjp2, Tjp1 or VE-cadherin, showing absence of Tjp2 protein in both hepatocytes and bile duct cholangiocytes (thick arrows), but not in endothelial cells (thin arrows). Nuclei are labelled with DAPI. ( B ) Western blot analysis of whole liver lysates from a control and two independent Tjp2 cKO mice probed with antibodies to Tjp2, Tjp1 and GAPDH, confirming the absence of Tjp2 in the Tjp2 cKO liver, while Tjp1 protein levels are not affected. GAPDH was used as a control. ( C ) mRNA expression levels of Tjp2 and Tjp1 in whole liver lysates from P60 and P300 control and Tjp2 cKO mice determined by qRT-PCR. Residual Tjp2 mRNA levels likely reflect the presence of cell types other than hepatocytes and cholangiocytes, for example endothelial cells, which express Tjp2. ( D ) Western blot analysis of whole liver lysates from a control and two Tjp2 cKO mice at P60 probed with antibodies to Cldn1, Cldn3, and Ocln. GAPDH was used as a control. ( E ) mRNA expression levels of Cldn1, Cldn2 and Cldn3 in whole liver lysates from P60 control and Tjp2 cKO mice determined by qRT-PCR. Data in (D) and (E) are shown as mean ± SD, paired Student’s t-test p-value

    Techniques Used: Immunofluorescence, Microscopy, Mouse Assay, Staining, Western Blot, Expressing, Quantitative RT-PCR

    Deletion of Tjp2 in cholangiocytes contributes to the susceptibility to CA-diet induced liver injury. ( A ) LacZ staining showing specific activation of the LacZ reporter gene in bile ducts and not in hepatocytes, indicative of a restricted deletion of Tjp2 in cholangiocytes of Tjp2 icKO CC mice. ( B ) Immunofluorescence microscopy of liver sections from Tjp2 icKO CC mice treated or not with tamoxifen and stained with antibodies to Tjp2 and CK19, confirming the absence of Tjp2 protein in CK19 positive cholangiocytes but not in hepatocytes. Nuclei are labelled with DAPI. ( C ) H E staining and quantification of necrotic area ( D ) of control and Tjp2 icKO CC mice fed 0.5% CA diet. Liver ( E ) and plasma ( F ) BA, ALT ( G ), AST ( H ), AP ( I ) and bilirubin ( J ), and gall bladder BA ( K ) levels in control and Tjp2 icKO CC mice fed 0.5% CA-diet. ( L ) Liver to body weight ratios. ( M ) mRNA expression levels for Abcb11 and Cyp2b10 in control, Tjp2 cKO, Tjp2 icKO HC and Tjp2 icKO CC mice fed with 0.5% CA diet monitored by qRT-PCR. Note the specific reduction of Abcb11 and Cyp2b10 expression levels only after deletion of Tjp2 in hepatocytes (e.g. in Tjp2 cKO and Tjp2 icKO HC livers). Data are shown as mean ± SD, paired Student’s t-test p-value
    Figure Legend Snippet: Deletion of Tjp2 in cholangiocytes contributes to the susceptibility to CA-diet induced liver injury. ( A ) LacZ staining showing specific activation of the LacZ reporter gene in bile ducts and not in hepatocytes, indicative of a restricted deletion of Tjp2 in cholangiocytes of Tjp2 icKO CC mice. ( B ) Immunofluorescence microscopy of liver sections from Tjp2 icKO CC mice treated or not with tamoxifen and stained with antibodies to Tjp2 and CK19, confirming the absence of Tjp2 protein in CK19 positive cholangiocytes but not in hepatocytes. Nuclei are labelled with DAPI. ( C ) H E staining and quantification of necrotic area ( D ) of control and Tjp2 icKO CC mice fed 0.5% CA diet. Liver ( E ) and plasma ( F ) BA, ALT ( G ), AST ( H ), AP ( I ) and bilirubin ( J ), and gall bladder BA ( K ) levels in control and Tjp2 icKO CC mice fed 0.5% CA-diet. ( L ) Liver to body weight ratios. ( M ) mRNA expression levels for Abcb11 and Cyp2b10 in control, Tjp2 cKO, Tjp2 icKO HC and Tjp2 icKO CC mice fed with 0.5% CA diet monitored by qRT-PCR. Note the specific reduction of Abcb11 and Cyp2b10 expression levels only after deletion of Tjp2 in hepatocytes (e.g. in Tjp2 cKO and Tjp2 icKO HC livers). Data are shown as mean ± SD, paired Student’s t-test p-value

    Techniques Used: Staining, Activation Assay, Mouse Assay, Immunofluorescence, Microscopy, AST Assay, Expressing, Quantitative RT-PCR

    6) Product Images from "PIAS-family proteins negatively regulate Glis3 transactivation function through SUMO modification in pancreatic β cells"

    Article Title: PIAS-family proteins negatively regulate Glis3 transactivation function through SUMO modification in pancreatic β cells

    Journal: Heliyon

    doi: 10.1016/j.heliyon.2018.e00709

    Glis3 is SUMOylated in BRIN BD11 cells under conditions of chronically elevated glucose. A. BRIN BD11 cells were grown in media containing 3 mM or 25 mM D-glucose. Media was changed after 24 h and after 48 h, total RNA was collected and the specified mRNA was measured by qRT-PCR analysis. A representative experiment is shown. Each bar represents relative mRNA levels normalized to 18s rRNA ± S.D. * indicates statistically different value compared to cells grown in 3 mM glucose p
    Figure Legend Snippet: Glis3 is SUMOylated in BRIN BD11 cells under conditions of chronically elevated glucose. A. BRIN BD11 cells were grown in media containing 3 mM or 25 mM D-glucose. Media was changed after 24 h and after 48 h, total RNA was collected and the specified mRNA was measured by qRT-PCR analysis. A representative experiment is shown. Each bar represents relative mRNA levels normalized to 18s rRNA ± S.D. * indicates statistically different value compared to cells grown in 3 mM glucose p

    Techniques Used: Quantitative RT-PCR

    SUMOylation of Glis3 decreases its transcriptional activity. A. BRIN BD11 cells were transfected with the indicated plasmids. After 48 h, total RNA was collected and the specified mRNA was measured by qRT-PCR analysis. A representative experiment is shown. Each bar represents relative mRNA levels normalized to 18s rRNA ± S.D. * indicates statistically different value compared to empty vector control p
    Figure Legend Snippet: SUMOylation of Glis3 decreases its transcriptional activity. A. BRIN BD11 cells were transfected with the indicated plasmids. After 48 h, total RNA was collected and the specified mRNA was measured by qRT-PCR analysis. A representative experiment is shown. Each bar represents relative mRNA levels normalized to 18s rRNA ± S.D. * indicates statistically different value compared to empty vector control p

    Techniques Used: Activity Assay, Transfection, Quantitative RT-PCR, Plasmid Preparation

    7) Product Images from "Kr?ppel-Like Factor 4, a Tumor Suppressor in Hepatocellular Carcinoma Cells Reverts Epithelial Mesenchymal Transition by Suppressing Slug Expression"

    Article Title: Kr?ppel-Like Factor 4, a Tumor Suppressor in Hepatocellular Carcinoma Cells Reverts Epithelial Mesenchymal Transition by Suppressing Slug Expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0043593

    Klf4 suppressed tumor growth and lung colonization. (A) Quantification of the weight of the tumor lesions in mice (n = 7) subcutaneously injected with MM189 PB-Klf4 or MM189 PB cells. Bar, SE. *, p
    Figure Legend Snippet: Klf4 suppressed tumor growth and lung colonization. (A) Quantification of the weight of the tumor lesions in mice (n = 7) subcutaneously injected with MM189 PB-Klf4 or MM189 PB cells. Bar, SE. *, p

    Techniques Used: Mouse Assay, Injection

    Ectopic Slug expression reversed Klf4-mediated phenotypes. (A) Slug protein level was detected in HCC cell lines, MM189 with only ectopic Klf4 (MM189 PB-Klf4/PB) and MM189 with both Klf4 and Slug expression (MM189 PB-Klf4/PB-Slug) by immunoblot assay. α-tubulin served as a loading control. (B) Observations of morphological change by the simultaneous ectopic expression of Slug and Klf4 in MM189 cells from epithelial- to mesenchymal-like shape under phase contrast microscopy with 200× magnification (upper panel). Cytoskelton F-actin proteins were stained with rodamine-phalloidin and viewed under fluorescence microscope with 630× magnification (lower panel, shown in grey mode). (C) Immunoblot analysis of mesenchymal and epithelial proteins in MM189 PB-Klf4/PB and MM189 PB-Slug/PB-Klf4 cells. α-tubulin served as a loading control. (D) Representative data shows the relative migration activity of MM189 cells expressing Klf4/Slug (MM189 PB-Klf4/PB-Slug) and its vector control (MM189 PB-Klf4/PB). The migrated cells were observed at magnification (100×) in the upper panel. The relative migration activity was defined by normalizing the mean of migrated cells/per field in MM189 PB-Klf4/PB-Slug cells to that in MM189 PB-Klf4/PB cells. Bar, SE. ***, p
    Figure Legend Snippet: Ectopic Slug expression reversed Klf4-mediated phenotypes. (A) Slug protein level was detected in HCC cell lines, MM189 with only ectopic Klf4 (MM189 PB-Klf4/PB) and MM189 with both Klf4 and Slug expression (MM189 PB-Klf4/PB-Slug) by immunoblot assay. α-tubulin served as a loading control. (B) Observations of morphological change by the simultaneous ectopic expression of Slug and Klf4 in MM189 cells from epithelial- to mesenchymal-like shape under phase contrast microscopy with 200× magnification (upper panel). Cytoskelton F-actin proteins were stained with rodamine-phalloidin and viewed under fluorescence microscope with 630× magnification (lower panel, shown in grey mode). (C) Immunoblot analysis of mesenchymal and epithelial proteins in MM189 PB-Klf4/PB and MM189 PB-Slug/PB-Klf4 cells. α-tubulin served as a loading control. (D) Representative data shows the relative migration activity of MM189 cells expressing Klf4/Slug (MM189 PB-Klf4/PB-Slug) and its vector control (MM189 PB-Klf4/PB). The migrated cells were observed at magnification (100×) in the upper panel. The relative migration activity was defined by normalizing the mean of migrated cells/per field in MM189 PB-Klf4/PB-Slug cells to that in MM189 PB-Klf4/PB cells. Bar, SE. ***, p

    Techniques Used: Expressing, Microscopy, Staining, Fluorescence, Migration, Activity Assay, Plasmid Preparation

    Klf4 bound and repressed the Slug promoter. (A) Schematic representation of Slug gene structure containing 1500 bp of promoter region ( Slug promoter) and SLUG luciferase construct ( SLUG -Luc). Grey ovals (Klf4) represented GC-boxes containing putative Klf4 binding sites predicted using MatInspector; black ovals (KLF4) represented putative KLF4 binding sites predicted using MatInspector. Black arrows depicted the location of the forward and reverse primers used for PCR amplification from immunoprecipitated DNA fragments. (B) SLUG promoter activity was reduced due to ectopic KLF4 expression in a dose-dependent manner. The SLUG -Luc reporter plasmid or pGL3-basic was co-transfected with Renilla-expressing control (pRL-TK) and KLF4-expression plasmids into 293T. The relative luciferase activity was defined as luciferase value, normalized to Renilla levels, was shown as –fold change over vector control. Bar, SE. *, p
    Figure Legend Snippet: Klf4 bound and repressed the Slug promoter. (A) Schematic representation of Slug gene structure containing 1500 bp of promoter region ( Slug promoter) and SLUG luciferase construct ( SLUG -Luc). Grey ovals (Klf4) represented GC-boxes containing putative Klf4 binding sites predicted using MatInspector; black ovals (KLF4) represented putative KLF4 binding sites predicted using MatInspector. Black arrows depicted the location of the forward and reverse primers used for PCR amplification from immunoprecipitated DNA fragments. (B) SLUG promoter activity was reduced due to ectopic KLF4 expression in a dose-dependent manner. The SLUG -Luc reporter plasmid or pGL3-basic was co-transfected with Renilla-expressing control (pRL-TK) and KLF4-expression plasmids into 293T. The relative luciferase activity was defined as luciferase value, normalized to Renilla levels, was shown as –fold change over vector control. Bar, SE. *, p

    Techniques Used: Luciferase, Construct, Binding Assay, Polymerase Chain Reaction, Amplification, Immunoprecipitation, Activity Assay, Expressing, Plasmid Preparation, Transfection

    Ectopic Klf4 expression inhibited colony formation, migration and invasion. (A) Klf4 and β-actin protein levels were detected in murine HCC cell lines, MM189 with ectopic Klf4 expression (MM189 PB-Klf4) and its corresponding control (MM189 PB) by immunoblot assay. β-actin served as a loading control. (B) Representative anchorage-independent growth activity for MM189 cells with ectopic Klf4 expression (MM189 PB-Klf4) and its corresponding control (MM189 PB). The colonies were observed at lower magnification (40×) in the left panel. The relative activity was determined by normalizing the mean number of colonies in MM189 PB-Klf4 cells to that in MM189 PB cells. Bar, SE. *, p
    Figure Legend Snippet: Ectopic Klf4 expression inhibited colony formation, migration and invasion. (A) Klf4 and β-actin protein levels were detected in murine HCC cell lines, MM189 with ectopic Klf4 expression (MM189 PB-Klf4) and its corresponding control (MM189 PB) by immunoblot assay. β-actin served as a loading control. (B) Representative anchorage-independent growth activity for MM189 cells with ectopic Klf4 expression (MM189 PB-Klf4) and its corresponding control (MM189 PB). The colonies were observed at lower magnification (40×) in the left panel. The relative activity was determined by normalizing the mean number of colonies in MM189 PB-Klf4 cells to that in MM189 PB cells. Bar, SE. *, p

    Techniques Used: Expressing, Migration, Activity Assay

    Down-regulation of KLF4 mRNA is frequently observed in HCC cell tissues. (A) Decreased KLF4 mRNA levels in HCC tissues (N = 225) in comparison with normal liver tissues (N = 220) [31] . Data were obtained from GEO/GSE14520 and statistics were calculated by unpaired t test. ***, p
    Figure Legend Snippet: Down-regulation of KLF4 mRNA is frequently observed in HCC cell tissues. (A) Decreased KLF4 mRNA levels in HCC tissues (N = 225) in comparison with normal liver tissues (N = 220) [31] . Data were obtained from GEO/GSE14520 and statistics were calculated by unpaired t test. ***, p

    Techniques Used:

    Klf4 promoted an epithelial phenotype in MM189 cells. (A) Ectopic Klf4 expression shifts cell morphology from a mesenchymal- to an epithelial phenotype. Phase contrast microscopy with 200× magnification (upper panel). Note the cobblestone appearance of the Klf4-expressing cells. Cytoskelton F-actin proteins were stained with rodamine-phalloidin and viewed under fluorescence microscope with 630× magnification (lower panel, shown in grey mode). (B) Immunoblot analysis of epithelial and mesenchymal proteins in MM189 PB and MM189 PB-Klf4 cells. BL185 cells and 3T3L1 cells served as positive controls for the expression of E-cadherin and Vimentin, respectively. α-tubulin served as a loading control. (C) Quantitative RT-PCR demonstrated the relative mRNA levels for E-cadherin (Cdh1) and epithelial mesenchymal transition (EMT)-related transcription factors in MM189 PB-Klf4 and MM189 PB cells. All amplifications were normalized to an endogenous β-actin control. For each gene, the relative expression of mRNA in MM189 PB-Klf4 cells was normalized to that in MM189 PB cells. Bar, SE. **, p
    Figure Legend Snippet: Klf4 promoted an epithelial phenotype in MM189 cells. (A) Ectopic Klf4 expression shifts cell morphology from a mesenchymal- to an epithelial phenotype. Phase contrast microscopy with 200× magnification (upper panel). Note the cobblestone appearance of the Klf4-expressing cells. Cytoskelton F-actin proteins were stained with rodamine-phalloidin and viewed under fluorescence microscope with 630× magnification (lower panel, shown in grey mode). (B) Immunoblot analysis of epithelial and mesenchymal proteins in MM189 PB and MM189 PB-Klf4 cells. BL185 cells and 3T3L1 cells served as positive controls for the expression of E-cadherin and Vimentin, respectively. α-tubulin served as a loading control. (C) Quantitative RT-PCR demonstrated the relative mRNA levels for E-cadherin (Cdh1) and epithelial mesenchymal transition (EMT)-related transcription factors in MM189 PB-Klf4 and MM189 PB cells. All amplifications were normalized to an endogenous β-actin control. For each gene, the relative expression of mRNA in MM189 PB-Klf4 cells was normalized to that in MM189 PB cells. Bar, SE. **, p

    Techniques Used: Expressing, Microscopy, Staining, Fluorescence, Quantitative RT-PCR

    8) Product Images from "Investigating the efficacy of monovalent and tetravalent dengue vaccine formulations against DENV-4 challenge in AG129 mice"

    Article Title: Investigating the efficacy of monovalent and tetravalent dengue vaccine formulations against DENV-4 challenge in AG129 mice

    Journal: Vaccine

    doi: 10.1016/j.vaccine.2014.08.087

    Viremia data following DENV-4 challenge of animals immunized with TDV-2, TDV-4 or TDV vaccines. Levels of DENV-4 RNA in the serum post-challenge were quantified by qRT-PCR using a Fam labeled 5 prime nuclease assay designed to target the 3 prime non-coding region of dengue virus. Limit of detection is 2.5 log 10 genomes/ml.
    Figure Legend Snippet: Viremia data following DENV-4 challenge of animals immunized with TDV-2, TDV-4 or TDV vaccines. Levels of DENV-4 RNA in the serum post-challenge were quantified by qRT-PCR using a Fam labeled 5 prime nuclease assay designed to target the 3 prime non-coding region of dengue virus. Limit of detection is 2.5 log 10 genomes/ml.

    Techniques Used: Quantitative RT-PCR, Labeling, Nuclease Assay

    9) Product Images from "Immunization with a MOMP-Based Vaccine Protects Mice against a Pulmonary Chlamydia Challenge and Identifies a Disconnection between Infection and Pathology"

    Article Title: Immunization with a MOMP-Based Vaccine Protects Mice against a Pulmonary Chlamydia Challenge and Identifies a Disconnection between Infection and Pathology

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0061962

    Percentage weight change of animals following IN challenge with C. muridarum . Percentage weight change was calculated by comparing the pre-infection body weight to daily p.i body weights. The figure depicts the effect of vaccination with MOMP and (A) CT/CpG- or (B) CTA1-DD-based vaccines on weight loss following IN challenge infection. Unimmunized (primary infection), live infection (secondary infection) and no infection controls are also included. (C) Area under the curve (AUC) analysis of percentage weight change was the total area, of both negative and positive peaks in weight change, over the 10 day course of infection in arbitrary units. Results are presented as the mean ± SD. Significant differences were determined using a one-way ANOVA with Tukey’s post-test by comparing the weight changes between groups at the same point in time. One P value, the most significant, is given for groups showing a significant change. Significance was set at P
    Figure Legend Snippet: Percentage weight change of animals following IN challenge with C. muridarum . Percentage weight change was calculated by comparing the pre-infection body weight to daily p.i body weights. The figure depicts the effect of vaccination with MOMP and (A) CT/CpG- or (B) CTA1-DD-based vaccines on weight loss following IN challenge infection. Unimmunized (primary infection), live infection (secondary infection) and no infection controls are also included. (C) Area under the curve (AUC) analysis of percentage weight change was the total area, of both negative and positive peaks in weight change, over the 10 day course of infection in arbitrary units. Results are presented as the mean ± SD. Significant differences were determined using a one-way ANOVA with Tukey’s post-test by comparing the weight changes between groups at the same point in time. One P value, the most significant, is given for groups showing a significant change. Significance was set at P

    Techniques Used: Infection

    10) Product Images from "Analysis of Erythrocyte Invasion Mechanisms of Plasmodium falciparum Clinical Isolates Across 3 Malaria-Endemic Areas in Ghana"

    Article Title: Analysis of Erythrocyte Invasion Mechanisms of Plasmodium falciparum Clinical Isolates Across 3 Malaria-Endemic Areas in Ghana

    Journal: The Journal of Infectious Diseases

    doi: 10.1093/infdis/jiv207

    Relative expression levels of genes encoding 8 invasion ligands in Plasmodium falciparum clinical isolates across 3 malaria-endemic areas of Ghana. Transcript levels of P. falciparum invasion ligand genes eba140, eba175, eba181, PfRh1, PfRh2a, PfRh2b, PfRh4 , and PfRh5 were determined by quantitative polymerase chain reaction analysis in 23 isolates from Accra (open circles), Navrongo (closed circles), and Kintampo (closed triangles). The relative abundance of each gene was expressed as a percentage of the total transcripts of all 8 genes. Transcript levels for individual isolates are shown by the symbols, and the dashes indicate the positions of the group means for the respective study sites.
    Figure Legend Snippet: Relative expression levels of genes encoding 8 invasion ligands in Plasmodium falciparum clinical isolates across 3 malaria-endemic areas of Ghana. Transcript levels of P. falciparum invasion ligand genes eba140, eba175, eba181, PfRh1, PfRh2a, PfRh2b, PfRh4 , and PfRh5 were determined by quantitative polymerase chain reaction analysis in 23 isolates from Accra (open circles), Navrongo (closed circles), and Kintampo (closed triangles). The relative abundance of each gene was expressed as a percentage of the total transcripts of all 8 genes. Transcript levels for individual isolates are shown by the symbols, and the dashes indicate the positions of the group means for the respective study sites.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    11) Product Images from "Pharmacological inhibition of focal segmental glomerulosclerosis‐related, gain of function mutants of TRPC6 channels by semi‐synthetic derivatives of larixol) Pharmacological inhibition of focal segmental glomerulosclerosis‐related, gain of function mutants of TRPC6 channels by semi‐synthetic derivatives of larixol"

    Article Title: Pharmacological inhibition of focal segmental glomerulosclerosis‐related, gain of function mutants of TRPC6 channels by semi‐synthetic derivatives of larixol) Pharmacological inhibition of focal segmental glomerulosclerosis‐related, gain of function mutants of TRPC6 channels by semi‐synthetic derivatives of larixol

    Journal: British Journal of Pharmacology

    doi: 10.1111/bph.13977

    TRPC6 mRNA and protein abundance in murine podocytes and glomeruli compared with PASMC. Quantitative RT‐PCR was performed on RNA isolated from rat (A, D, E) and mouse (B, C, F, G) podocytes or PASMC to detect total TRPC6, the TRPC6 transgene, podocin and α‐SMA transcripts. Panels A–C depict PCR products obtained from podocyte and PASMC cultures of different animals, and a water control with no RNA added to the RT‐PCR (H 2 O). Total (B) and transgenic (C) mouse TRPC6 expression was detected in wild‐type (WT) and in transgenic B6.Cg‐Tg(NPHS2‐Trpc6) F419Walz/J (TG) mice. (D–G) Quantitative PCR results from n = 6–11 experiments were normalized to 18s rRNA and to the relative expression of the investigated mRNA in podocytes from adult wild‐type animals. # P
    Figure Legend Snippet: TRPC6 mRNA and protein abundance in murine podocytes and glomeruli compared with PASMC. Quantitative RT‐PCR was performed on RNA isolated from rat (A, D, E) and mouse (B, C, F, G) podocytes or PASMC to detect total TRPC6, the TRPC6 transgene, podocin and α‐SMA transcripts. Panels A–C depict PCR products obtained from podocyte and PASMC cultures of different animals, and a water control with no RNA added to the RT‐PCR (H 2 O). Total (B) and transgenic (C) mouse TRPC6 expression was detected in wild‐type (WT) and in transgenic B6.Cg‐Tg(NPHS2‐Trpc6) F419Walz/J (TG) mice. (D–G) Quantitative PCR results from n = 6–11 experiments were normalized to 18s rRNA and to the relative expression of the investigated mRNA in podocytes from adult wild‐type animals. # P

    Techniques Used: Quantitative RT-PCR, Isolation, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Transgenic Assay, Expressing, Mouse Assay, Real-time Polymerase Chain Reaction

    Ca 2+ signals in acutely dissociated rat or mouse glomeruli are not significantly affected by LMA or LC. Glomeruli from 8‐week‐old Wistar rats (A, B) or from 4‐week‐old B6.Cg‐Tg(NPHS2‐Trpc6) F419Walz/J mice (C, D) were isolated by mechanical dissociation, collagenase treatment and fractional sieving. Glomeruli suspensions were loaded with fura‐2/AM washed and transferred to bath chambers for microfluorimetry (A–F) or loaded with fluo‐4/AM, washed and dispensed into microwell plates for imaging on a fluorescence plate imager (G). To elicit increases in [Ca 2+ ] i via stimulation of a PLC‐coupled GPCR, rat glomeruli were stimulated with 100 μM carbachol, followed by addition of 5 μM LMA or its solvent. (A) Representative background‐corrected ratio images of F 340 nm / F 380 nm before (basal) and 30 s after addition of 100 μM carbachol encoded with a rainbow pseudocolour scale ranging from 0.2 to 1.0. (B) Time course of F 340 nm / F 380 nm in regions of interest defined within single glomeruli (grey traces) and averaged signals (black trace) during stimulation with carbachol without (solvent; left panel) or with subsequent addition of 5 μM LMA (middle panel). The right panel displays the statistical analysis of fluorescence ratios taken at the indicated time points (1, 2, 3) in 5–6 independent experiments. (C–F) Similar experiments as in (A, B), but with glomeruli from TRPC6‐overexpressing mice either stimulated with 100 μM carbachol (C, D) or with 10 μM angiotensin II (E, F). Note that angiotensin II‐induced [Ca 2+ ] i responses were seen in some but not all glomeruli. (E) To level out inhomogenous responses, experiments were therefore repeated in fluo‐4‐loaded glomeruli suspensions measured in a microwell plate imager with and without preincubation with 5 μM LC (left panel). Statistical analysis of 16 measurements obtained in three independent experiments with glomeruli isolated from different animals. Signals with and without pretreatment with 5 μM LC did not reveal a significant LC‐sensitive component of angiotensin II‐induced [Ca 2+ ] i signals (right panel). White bars in (A, C, E) indicate 100 μm.
    Figure Legend Snippet: Ca 2+ signals in acutely dissociated rat or mouse glomeruli are not significantly affected by LMA or LC. Glomeruli from 8‐week‐old Wistar rats (A, B) or from 4‐week‐old B6.Cg‐Tg(NPHS2‐Trpc6) F419Walz/J mice (C, D) were isolated by mechanical dissociation, collagenase treatment and fractional sieving. Glomeruli suspensions were loaded with fura‐2/AM washed and transferred to bath chambers for microfluorimetry (A–F) or loaded with fluo‐4/AM, washed and dispensed into microwell plates for imaging on a fluorescence plate imager (G). To elicit increases in [Ca 2+ ] i via stimulation of a PLC‐coupled GPCR, rat glomeruli were stimulated with 100 μM carbachol, followed by addition of 5 μM LMA or its solvent. (A) Representative background‐corrected ratio images of F 340 nm / F 380 nm before (basal) and 30 s after addition of 100 μM carbachol encoded with a rainbow pseudocolour scale ranging from 0.2 to 1.0. (B) Time course of F 340 nm / F 380 nm in regions of interest defined within single glomeruli (grey traces) and averaged signals (black trace) during stimulation with carbachol without (solvent; left panel) or with subsequent addition of 5 μM LMA (middle panel). The right panel displays the statistical analysis of fluorescence ratios taken at the indicated time points (1, 2, 3) in 5–6 independent experiments. (C–F) Similar experiments as in (A, B), but with glomeruli from TRPC6‐overexpressing mice either stimulated with 100 μM carbachol (C, D) or with 10 μM angiotensin II (E, F). Note that angiotensin II‐induced [Ca 2+ ] i responses were seen in some but not all glomeruli. (E) To level out inhomogenous responses, experiments were therefore repeated in fluo‐4‐loaded glomeruli suspensions measured in a microwell plate imager with and without preincubation with 5 μM LC (left panel). Statistical analysis of 16 measurements obtained in three independent experiments with glomeruli isolated from different animals. Signals with and without pretreatment with 5 μM LC did not reveal a significant LC‐sensitive component of angiotensin II‐induced [Ca 2+ ] i signals (right panel). White bars in (A, C, E) indicate 100 μm.

    Techniques Used: Mouse Assay, Isolation, Imaging, Fluorescence, Planar Chromatography

    12) Product Images from "Intronic Deletions That Disrupt mRNA Splicing of the tva Receptor Gene Result in Decreased Susceptibility to Infection by Avian Sarcoma and Leukosis Virus Subgroup A"

    Article Title: Intronic Deletions That Disrupt mRNA Splicing of the tva Receptor Gene Result in Decreased Susceptibility to Infection by Avian Sarcoma and Leukosis Virus Subgroup A

    Journal: Journal of Virology

    doi: 10.1128/JVI.05771-11

    The tva r3 and tva r4 alleles in chicken line P and G. g. murghi contain deletions in the first intron. (A) PCR amplification of the whole tva coding sequence from G. g. murghi (GGM), L15, and line P CEFs. Two forms of normal tva transcripts in L15 420
    Figure Legend Snippet: The tva r3 and tva r4 alleles in chicken line P and G. g. murghi contain deletions in the first intron. (A) PCR amplification of the whole tva coding sequence from G. g. murghi (GGM), L15, and line P CEFs. Two forms of normal tva transcripts in L15 420

    Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing

    Time course of infection of line P (A) and G. g. murghi (B) CEFs with ASLVs. CEFs were infected at a multiplicity of infection of 10 with replication-competent ASLVs encoding the GFP reporter proteins RCASBP(A)GFP and RCASBP(B)GFP. The percentage of GFP-positive
    Figure Legend Snippet: Time course of infection of line P (A) and G. g. murghi (B) CEFs with ASLVs. CEFs were infected at a multiplicity of infection of 10 with replication-competent ASLVs encoding the GFP reporter proteins RCASBP(A)GFP and RCASBP(B)GFP. The percentage of GFP-positive

    Techniques Used: Infection

    13) Product Images from "Immunization with a MOMP-Based Vaccine Protects Mice against a Pulmonary Chlamydia Challenge and Identifies a Disconnection between Infection and Pathology"

    Article Title: Immunization with a MOMP-Based Vaccine Protects Mice against a Pulmonary Chlamydia Challenge and Identifies a Disconnection between Infection and Pathology

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0061962

    Chlamydial burden in lung tissue at day 10 p.i determined by omp A PCR. Genomic DNA (gDNA) was extracted from lung tissues collected at day 10 p.i. Chlamydial DNA was quantified from lung gDNA by omp A-specific qRT-PCR using a standard curve. The copies of C. muridarum DNA per µg of host gDNA for each vaccine were grouped with their respective route of immunization. Results are presented as the mean ± SD. Significant differences were determined using a one-way ANOVA with Tukey’s post-test. Significance was set at P
    Figure Legend Snippet: Chlamydial burden in lung tissue at day 10 p.i determined by omp A PCR. Genomic DNA (gDNA) was extracted from lung tissues collected at day 10 p.i. Chlamydial DNA was quantified from lung gDNA by omp A-specific qRT-PCR using a standard curve. The copies of C. muridarum DNA per µg of host gDNA for each vaccine were grouped with their respective route of immunization. Results are presented as the mean ± SD. Significant differences were determined using a one-way ANOVA with Tukey’s post-test. Significance was set at P

    Techniques Used: Polymerase Chain Reaction, Quantitative RT-PCR

    14) Product Images from "Isoflurane promotes glucose metabolism through up-regulation of miR-21 and suppresses mitochondrial oxidative phosphorylation in ovarian cancer cells"

    Article Title: Isoflurane promotes glucose metabolism through up-regulation of miR-21 and suppresses mitochondrial oxidative phosphorylation in ovarian cancer cells

    Journal: Bioscience Reports

    doi: 10.1042/BSR20170818

    Glycolytic enzymes were activated by isoflurane treatments SKOV3 and TOV21G cells were treated without or with isoflurane for 1 or 2 h, cells were then cultured in new medium for 24 h. ( A ) The protein expressions of GLUT1, HK2, PKM2, and LDHA were measured by Western blot. β-actin was used as a loading control. ( B ) The mRNA expressions of glycolytic enzymes were measured by qRT-PCR. ( C ) SKOV3 cells were treated without or with isoflurane for 2 h, cells were incubated in fresh medium for 24 h. Cells were fixed and stained with antibody against GLUT1. DAPI was used for the nuclei staining. ( D ) Quantitation of the membrane localization of GLUT1 based on the results of Figure 2 C using ImageJ software. All experiments were performed at least three times independently and the data shown are mean ± S.D.; *: P
    Figure Legend Snippet: Glycolytic enzymes were activated by isoflurane treatments SKOV3 and TOV21G cells were treated without or with isoflurane for 1 or 2 h, cells were then cultured in new medium for 24 h. ( A ) The protein expressions of GLUT1, HK2, PKM2, and LDHA were measured by Western blot. β-actin was used as a loading control. ( B ) The mRNA expressions of glycolytic enzymes were measured by qRT-PCR. ( C ) SKOV3 cells were treated without or with isoflurane for 2 h, cells were incubated in fresh medium for 24 h. Cells were fixed and stained with antibody against GLUT1. DAPI was used for the nuclei staining. ( D ) Quantitation of the membrane localization of GLUT1 based on the results of Figure 2 C using ImageJ software. All experiments were performed at least three times independently and the data shown are mean ± S.D.; *: P

    Techniques Used: Cell Culture, Western Blot, Quantitative RT-PCR, Incubation, Staining, Quantitation Assay, Software

    Isoflurane induces miR-21 expression and phosphorylation of AKT ( A ) SKOV3 and TOV21G cells were treated without or with isoflurane for 1 or 2 h, and the expressions of miR-21 were assessed by qRT-PCR. U6 was used as an internal control. ( B ) SKOV3 and TOV21G cells were treated with isoflurane for 1 or 2 h, the phosphorylation of AKT was measured by Western blot. β-actin was used as the loading control. ( C ) SKOV3 and TOV21G cells were transfected with control miRNAs or pre- miR-21 for 72 h. The expression of miR-21 was assessed by qRT-PCR. U6 was used as the internal control. ( D ) The proteins and ( E ) mRNAs of HK2, PKM2, and LDHA were measured in SKOV3 and TOV21G cells without or with miR-21 overexpression. ( F ) SKOV3 and TOV21G cells without or with isoflurane treatment were transfected with control antisense or anti- miR-21 for 72 h. The expression of miR-21 was assessed by qRT-PCR. U6 was used as an internal control. ( G ) The mRNAs of GLUT1, HK2, PKM2, and LDHA were measured by qRT-PCR. All experiments were performed at least three times independently and the data shown are mean ± S.D.; *: P
    Figure Legend Snippet: Isoflurane induces miR-21 expression and phosphorylation of AKT ( A ) SKOV3 and TOV21G cells were treated without or with isoflurane for 1 or 2 h, and the expressions of miR-21 were assessed by qRT-PCR. U6 was used as an internal control. ( B ) SKOV3 and TOV21G cells were treated with isoflurane for 1 or 2 h, the phosphorylation of AKT was measured by Western blot. β-actin was used as the loading control. ( C ) SKOV3 and TOV21G cells were transfected with control miRNAs or pre- miR-21 for 72 h. The expression of miR-21 was assessed by qRT-PCR. U6 was used as the internal control. ( D ) The proteins and ( E ) mRNAs of HK2, PKM2, and LDHA were measured in SKOV3 and TOV21G cells without or with miR-21 overexpression. ( F ) SKOV3 and TOV21G cells without or with isoflurane treatment were transfected with control antisense or anti- miR-21 for 72 h. The expression of miR-21 was assessed by qRT-PCR. U6 was used as an internal control. ( G ) The mRNAs of GLUT1, HK2, PKM2, and LDHA were measured by qRT-PCR. All experiments were performed at least three times independently and the data shown are mean ± S.D.; *: P

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Over Expression

    15) Product Images from "Heterologous DNA Uptake in Cultured Symbiodinium spp. Aided by Agrobacterium tumefaciens"

    Article Title: Heterologous DNA Uptake in Cultured Symbiodinium spp. Aided by Agrobacterium tumefaciens

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0132693

    GFP-fusion protein fluorescence from Symbiodinium spp. cells co-incubated with Agrobacterium tumefaciens harboring a gfp-AtRACK1C fusion. Cell cultures of S . KB8 (A, D), S . Mf11 (B, E), and S . kawagutii (E, F), after two weeks of selection with Basta. Cells were observed under phase contrast (A, B, and C), and under epifluorescence microscopy (D, E, and F). Bars equal 20 μm. Inset images show cells observed by CLSM.
    Figure Legend Snippet: GFP-fusion protein fluorescence from Symbiodinium spp. cells co-incubated with Agrobacterium tumefaciens harboring a gfp-AtRACK1C fusion. Cell cultures of S . KB8 (A, D), S . Mf11 (B, E), and S . kawagutii (E, F), after two weeks of selection with Basta. Cells were observed under phase contrast (A, B, and C), and under epifluorescence microscopy (D, E, and F). Bars equal 20 μm. Inset images show cells observed by CLSM.

    Techniques Used: Fluorescence, Incubation, Selection, Epifluorescence Microscopy, Confocal Laser Scanning Microscopy

    Genomic PCR of cells expressing the gfp - AtRACK1C construct. Amplification products from genomic DNA extracted from S . Mf11 (2, 3), S . KB8 (5, 6), and S . kawagutii (8, 9) co-incubated with A . tumefaciens harboring the gfp - AtRACK1C fusion. The gel shows the corresponding ~0.6 kbp and ~1 kbp fragments, respectively, obtained with AtRACK1C primers. DNA from control cells did not amplify any PCR product from S . Mf11, S . KB8, or S . kawagutii (lanes 1, 4 and 7, respectively). MWM shows the molecular standards. Lanes 1–6 and MWM are from an independent gel from that corresponding to lanes 7–9. The bands on the gel are shown in negative.
    Figure Legend Snippet: Genomic PCR of cells expressing the gfp - AtRACK1C construct. Amplification products from genomic DNA extracted from S . Mf11 (2, 3), S . KB8 (5, 6), and S . kawagutii (8, 9) co-incubated with A . tumefaciens harboring the gfp - AtRACK1C fusion. The gel shows the corresponding ~0.6 kbp and ~1 kbp fragments, respectively, obtained with AtRACK1C primers. DNA from control cells did not amplify any PCR product from S . Mf11, S . KB8, or S . kawagutii (lanes 1, 4 and 7, respectively). MWM shows the molecular standards. Lanes 1–6 and MWM are from an independent gel from that corresponding to lanes 7–9. The bands on the gel are shown in negative.

    Techniques Used: Polymerase Chain Reaction, Expressing, Construct, Amplification, Incubation

    GFP-fusion protein fluorescence from Symbiodinium spp. cells co-incubated with Agrobacterium tumefaciens harboring a gfp - FABD2 fusion. Cell cultures of S . Mf11 (A, B), and S . kawagutii (C, D) after 23 d of selection with Basta. Cells were observed under phase contrast (A and C) and under epifluorescence microscopy (B and D). Bars equal 10 μm for A and B, and 20 μm for C and D.
    Figure Legend Snippet: GFP-fusion protein fluorescence from Symbiodinium spp. cells co-incubated with Agrobacterium tumefaciens harboring a gfp - FABD2 fusion. Cell cultures of S . Mf11 (A, B), and S . kawagutii (C, D) after 23 d of selection with Basta. Cells were observed under phase contrast (A and C) and under epifluorescence microscopy (B and D). Bars equal 10 μm for A and B, and 20 μm for C and D.

    Techniques Used: Fluorescence, Incubation, Selection, Epifluorescence Microscopy

    Amplified products from reverse-transcribed cDNA obtained from Symbiodinium kawagutii cultures co-incubated with Agrobacterium tumefaciens harboring pCB302- gfp-AtRACK1C , pCB302- gfp-MBD , and pCB302- gfp-FABD2 . The reverse-transcribed cDNA from S . kawagutii cultures expressing the gfp -fusion constructs was used as template with the gfp primers for PCR amplification of the corresponding transcripts (see Materials and Methods ). Fragments of ~0.7 kbp corresponding to the gfp transcripts were obtained from cDNA of S . kawagutii co-incubated with A . tumefaciens harboring: gfp-AtRACK1C (lane 3), gfp-MBD (lane 5), or gfp-FABD2 (lane 6). No amplifications were obtained when template cDNA was used from the following negative S . kawagutti controls: no shaking/no Agrobacterium (lane 1); shaking/no Agrobacterium (lane 2); or Agrobacterium /no shaking ( gfp-AtRACK1C ) (lane 7). The presence of cDNA in preparations from control and A . tumefaciens co-incubated cells was confirmed by RT-PCR of the endogenous S . kawagutii RACK1 transcript (data not shown). Lane 4 shows the molecular standards. The bands on the gel are shown in negative.
    Figure Legend Snippet: Amplified products from reverse-transcribed cDNA obtained from Symbiodinium kawagutii cultures co-incubated with Agrobacterium tumefaciens harboring pCB302- gfp-AtRACK1C , pCB302- gfp-MBD , and pCB302- gfp-FABD2 . The reverse-transcribed cDNA from S . kawagutii cultures expressing the gfp -fusion constructs was used as template with the gfp primers for PCR amplification of the corresponding transcripts (see Materials and Methods ). Fragments of ~0.7 kbp corresponding to the gfp transcripts were obtained from cDNA of S . kawagutii co-incubated with A . tumefaciens harboring: gfp-AtRACK1C (lane 3), gfp-MBD (lane 5), or gfp-FABD2 (lane 6). No amplifications were obtained when template cDNA was used from the following negative S . kawagutti controls: no shaking/no Agrobacterium (lane 1); shaking/no Agrobacterium (lane 2); or Agrobacterium /no shaking ( gfp-AtRACK1C ) (lane 7). The presence of cDNA in preparations from control and A . tumefaciens co-incubated cells was confirmed by RT-PCR of the endogenous S . kawagutii RACK1 transcript (data not shown). Lane 4 shows the molecular standards. The bands on the gel are shown in negative.

    Techniques Used: Amplification, Incubation, Expressing, Construct, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

    GFP-fusion protein fluorescence from Symbiodinium kawagutii cells co-incubated with Agrobacterium tumefaciens harboring a gfp-AtRACK1C fusion. Distinctive fluorescence pattern between dead (asterisks), dying (arrows) or GFP-fusion protein expressing (arrowheads) Symbiodinium cells. The images were obtained under phase contrast (A) or epifluorecence microscopy (B) after 16 d on selection medium. Bars equal 20 μm.
    Figure Legend Snippet: GFP-fusion protein fluorescence from Symbiodinium kawagutii cells co-incubated with Agrobacterium tumefaciens harboring a gfp-AtRACK1C fusion. Distinctive fluorescence pattern between dead (asterisks), dying (arrows) or GFP-fusion protein expressing (arrowheads) Symbiodinium cells. The images were obtained under phase contrast (A) or epifluorecence microscopy (B) after 16 d on selection medium. Bars equal 20 μm.

    Techniques Used: Fluorescence, Incubation, Expressing, Microscopy, Selection

    Genomic PCR of cells expressing the gfp - MBD or gfp - FABD2 constructs. Amplification of a 717 bp fragment using gfp specific primers and genomic DNA template from: S . kawagutii co-incubated with A . tumefaciens harboring fusions of gfp-MBD (lane 1), or gfp-FABD2 (lane 4); S . Mf11 co-incubated with A . tumefaciens harboring fusions of gfp-MBD (lane 2), or gfp-FABD2 (lane 5); and S . KB8 co-incubated with A . tumefaciens harboring fusions of gfp-MBD (lane 3), or gfp-FABD2 (lane 6). Genomic DNA from control S . kawagutii , S . Mf11, or S . KB8 cells did not amplify any PCR product (lanes 7, 8 and 9, respectively). MWM shows the molecular standards. The bands on the gel are shown in negative.
    Figure Legend Snippet: Genomic PCR of cells expressing the gfp - MBD or gfp - FABD2 constructs. Amplification of a 717 bp fragment using gfp specific primers and genomic DNA template from: S . kawagutii co-incubated with A . tumefaciens harboring fusions of gfp-MBD (lane 1), or gfp-FABD2 (lane 4); S . Mf11 co-incubated with A . tumefaciens harboring fusions of gfp-MBD (lane 2), or gfp-FABD2 (lane 5); and S . KB8 co-incubated with A . tumefaciens harboring fusions of gfp-MBD (lane 3), or gfp-FABD2 (lane 6). Genomic DNA from control S . kawagutii , S . Mf11, or S . KB8 cells did not amplify any PCR product (lanes 7, 8 and 9, respectively). MWM shows the molecular standards. The bands on the gel are shown in negative.

    Techniques Used: Polymerase Chain Reaction, Expressing, Construct, Amplification, Incubation

    GFP-fusion protein fluorescence from Symbiodinium spp. cells co-incubated with Agrobacterium tumefaciens harboring a gfp - MBD fusion. Cell cultures of S . Mf11 (A, B), and S . kawagutii (C, D) after 20 d of selection with Basta. Cells were observed under phase contrast (A and C), and under epifluorescence microscopy (B and D). Bars equal 20 μm for A and B, and 25μm for C and D.
    Figure Legend Snippet: GFP-fusion protein fluorescence from Symbiodinium spp. cells co-incubated with Agrobacterium tumefaciens harboring a gfp - MBD fusion. Cell cultures of S . Mf11 (A, B), and S . kawagutii (C, D) after 20 d of selection with Basta. Cells were observed under phase contrast (A and C), and under epifluorescence microscopy (B and D). Bars equal 20 μm for A and B, and 25μm for C and D.

    Techniques Used: Fluorescence, Incubation, Selection, Epifluorescence Microscopy

    16) Product Images from "Preconditioning of Microglia by ?-Synuclein Strongly Affects the Response Induced by Toll-like Receptor (TLR) Stimulation"

    Article Title: Preconditioning of Microglia by ?-Synuclein Strongly Affects the Response Induced by Toll-like Receptor (TLR) Stimulation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0079160

    Comparison of TLR gene expression levels of Wt αSyn-primed vs. non-primed microgia, after TLR stimulation . After treating cells as before (see legend to Figure 1 ), cells were lysed and the total RNA was extracted. Relative TLR gene expression levels were then examined by qRT-PCR and the hprt gene was used as the internal control to calculate ∆Ct values. The ∆∆Ct values were calculated by subtracting ∆Ct values of non-primed samples upon TLR stimulation (‘TLR ligand’) from ∆Ct values of samples treated with αSyn-priming upon TLR stimulation (‘Wt+TLR ligand’), to give the fold-change in TLR expression in cells with ‘αSyn+TLR ligand’ relative to ‘TLR ligand’ treatments. In all cases the TLR gene analysed (indicated inside bars) corresponded to the TLR agonist used in that particular sample. Fold-changes represent the average of three independent experiments (N=3), each one performed with duplicate samples. Bars correspond to SEM. Statistically significant differences (* p
    Figure Legend Snippet: Comparison of TLR gene expression levels of Wt αSyn-primed vs. non-primed microgia, after TLR stimulation . After treating cells as before (see legend to Figure 1 ), cells were lysed and the total RNA was extracted. Relative TLR gene expression levels were then examined by qRT-PCR and the hprt gene was used as the internal control to calculate ∆Ct values. The ∆∆Ct values were calculated by subtracting ∆Ct values of non-primed samples upon TLR stimulation (‘TLR ligand’) from ∆Ct values of samples treated with αSyn-priming upon TLR stimulation (‘Wt+TLR ligand’), to give the fold-change in TLR expression in cells with ‘αSyn+TLR ligand’ relative to ‘TLR ligand’ treatments. In all cases the TLR gene analysed (indicated inside bars) corresponded to the TLR agonist used in that particular sample. Fold-changes represent the average of three independent experiments (N=3), each one performed with duplicate samples. Bars correspond to SEM. Statistically significant differences (* p

    Techniques Used: Expressing, Quantitative RT-PCR

    17) Product Images from "Microglia express ABI3 in the brains of Alzheimer's disease and Nasu-Hakola disease"

    Article Title: Microglia express ABI3 in the brains of Alzheimer's disease and Nasu-Hakola disease

    Journal: Intractable & Rare Diseases Research

    doi: 10.5582/irdr.2017.01073

    Quantitative RT-PCR analysis of ABI3 expression in HMO6 microglia in culture. HMO6 cells were exposed for 24 hours to 1 µg/ml lipopolysaccharide (LPS), recombinant human IFNγ, IL-4, IL-13 or TGFβ1 (50 ng/mL each), followed by extraction of total cellular RNA that is processed for qRT-PCR. The expression levels of ABI3 were standardized against the levels of G3PDH.
    Figure Legend Snippet: Quantitative RT-PCR analysis of ABI3 expression in HMO6 microglia in culture. HMO6 cells were exposed for 24 hours to 1 µg/ml lipopolysaccharide (LPS), recombinant human IFNγ, IL-4, IL-13 or TGFβ1 (50 ng/mL each), followed by extraction of total cellular RNA that is processed for qRT-PCR. The expression levels of ABI3 were standardized against the levels of G3PDH.

    Techniques Used: Quantitative RT-PCR, Expressing, Recombinant

    18) Product Images from "Analysis of Human Innate Immune Responses to PRINT Fabricated Nanoparticles with Cross Validation Using a Humanized Mouse Model"

    Article Title: Analysis of Human Innate Immune Responses to PRINT Fabricated Nanoparticles with Cross Validation Using a Humanized Mouse Model

    Journal: Nanomedicine : nanotechnology, biology, and medicine

    doi: 10.1016/j.nano.2014.11.010

    Undetectable cytokine responses and complement activation following treatment of primary hu-PBMC with HP 4 A-based 80×320 nm rods
    Figure Legend Snippet: Undetectable cytokine responses and complement activation following treatment of primary hu-PBMC with HP 4 A-based 80×320 nm rods

    Techniques Used: Activation Assay

    HP 4 A-based 80×320 nm rod uptake without inflammatory cytokine responses by THP-1 cells
    Figure Legend Snippet: HP 4 A-based 80×320 nm rod uptake without inflammatory cytokine responses by THP-1 cells

    Techniques Used:

    HP 4 A-based 80×320 nm rod uptake and inflammatory cytokine responses using a humanized mouse model
    Figure Legend Snippet: HP 4 A-based 80×320 nm rod uptake and inflammatory cytokine responses using a humanized mouse model

    Techniques Used:

    19) Product Images from "The repositioning of epigenetic probes/inhibitors identifies new anti-schistosomal lead compounds and chemotherapeutic targets"

    Article Title: The repositioning of epigenetic probes/inhibitors identifies new anti-schistosomal lead compounds and chemotherapeutic targets

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0007693

    Schistosome motility, egg production and H3K36me2 are regulated by Smp_000700. RNAi of adult schistosome pairs (21 worm pairs/biological replicate; n = 3 replicates) using siRNAs directed against smp_000700 and luc was performed as described in the Materials and Methods. (A) qRT-PCR analysis of smp_000700 RNA levels in si Luc vs si Smp_000700 treated worms at 48 h. (B) Quantification of adult schistosome worm motility at 168 h. (C) Enumeration of in vitro laid egg (IVLE) production at 168 h. (D) Detection of H3K36me2 in adult schistosome nuclear extracts at 168 h. Statistical significance is indicated (Student’s t test, two tailed, unequal variance). *** represents p
    Figure Legend Snippet: Schistosome motility, egg production and H3K36me2 are regulated by Smp_000700. RNAi of adult schistosome pairs (21 worm pairs/biological replicate; n = 3 replicates) using siRNAs directed against smp_000700 and luc was performed as described in the Materials and Methods. (A) qRT-PCR analysis of smp_000700 RNA levels in si Luc vs si Smp_000700 treated worms at 48 h. (B) Quantification of adult schistosome worm motility at 168 h. (C) Enumeration of in vitro laid egg (IVLE) production at 168 h. (D) Detection of H3K36me2 in adult schistosome nuclear extracts at 168 h. Statistical significance is indicated (Student’s t test, two tailed, unequal variance). *** represents p

    Techniques Used: Quantitative RT-PCR, In Vitro, Two Tailed Test

    20) Product Images from "SMOC Binds to Pro-EGF, but Does Not Induce Erk Phosphorylation via the EGFR"

    Article Title: SMOC Binds to Pro-EGF, but Does Not Induce Erk Phosphorylation via the EGFR

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0154294

    Induction of MAPK signaling by SMOC does not require the EGF receptor. Immunoblot analysis of serum-starved HEK293 (A, D) or 32D/EGFR (B, C) cell lysates following addition of X SMOC-1 (100μg/ml) or hSMOC-1 (50μg/ml). (A) Following addition of X SMOC-1 to HEK293 cells, phosphorylation of MEK1/2 and Erk was evident at four minutes. (B) Phosphorylation of Erk and EGFR (Y1172) in 32D/EGFR cells was weak in the presence of X SMOC-1 compared to hSMOC-1. (C) Phosphorylation of Erk by hSMOC-1 in 32D/EGFR cells was blocked by the small molecule EGFR inhibitor, Gefitinib (5μM). (D) Phosphorylation of Erk by SMOC in HEK293 cells does not require the EGFR; X SMOC-1 and hSMOC-1 continue to induce Erk phosphorylation in the presence of Gefitinib but, in the absence of Gefitinib, hSMOC-1 stimulated ERK phosphorylation more than X SMOC-1. Non-phosphorylated Erk and EGFR are shown as loading controls. The results presented are representative of experiments conducted at least three times.
    Figure Legend Snippet: Induction of MAPK signaling by SMOC does not require the EGF receptor. Immunoblot analysis of serum-starved HEK293 (A, D) or 32D/EGFR (B, C) cell lysates following addition of X SMOC-1 (100μg/ml) or hSMOC-1 (50μg/ml). (A) Following addition of X SMOC-1 to HEK293 cells, phosphorylation of MEK1/2 and Erk was evident at four minutes. (B) Phosphorylation of Erk and EGFR (Y1172) in 32D/EGFR cells was weak in the presence of X SMOC-1 compared to hSMOC-1. (C) Phosphorylation of Erk by hSMOC-1 in 32D/EGFR cells was blocked by the small molecule EGFR inhibitor, Gefitinib (5μM). (D) Phosphorylation of Erk by SMOC in HEK293 cells does not require the EGFR; X SMOC-1 and hSMOC-1 continue to induce Erk phosphorylation in the presence of Gefitinib but, in the absence of Gefitinib, hSMOC-1 stimulated ERK phosphorylation more than X SMOC-1. Non-phosphorylated Erk and EGFR are shown as loading controls. The results presented are representative of experiments conducted at least three times.

    Techniques Used:

    X SMOC-1 binds to pro-EGF and co-localizes with pro-EGF in vivo . (A) Immunoblot of pro-EGF following co-immunoprecipitation of pro-EGF with X SMOC-1, X SMOC-1 ΔEC, or X SMOC-1 EC in the presence of TBST/0.1% SDS; pro-EGF binds to X SMOC-1 and X SMOC-1 ΔEC, but not X SMOC-1EC. (B) RT-PCR analysis of HEK293 cells showing positive signal for pro-EGF(C) Representative confocal image showing co-localization of X SMOC-1 and pro-EGF (red fluorophore) on HEK293 cells using the PLA method. Nuclei are stained blue with DAPI. (D, E) Representative whole mount hybridization in situ images of Xenopus neurula embryos (stage 26) stained for X SMOC-1 (C) or pro-EGF (D). The locations of the eye (e) and pronephros (pn) are indicated.
    Figure Legend Snippet: X SMOC-1 binds to pro-EGF and co-localizes with pro-EGF in vivo . (A) Immunoblot of pro-EGF following co-immunoprecipitation of pro-EGF with X SMOC-1, X SMOC-1 ΔEC, or X SMOC-1 EC in the presence of TBST/0.1% SDS; pro-EGF binds to X SMOC-1 and X SMOC-1 ΔEC, but not X SMOC-1EC. (B) RT-PCR analysis of HEK293 cells showing positive signal for pro-EGF(C) Representative confocal image showing co-localization of X SMOC-1 and pro-EGF (red fluorophore) on HEK293 cells using the PLA method. Nuclei are stained blue with DAPI. (D, E) Representative whole mount hybridization in situ images of Xenopus neurula embryos (stage 26) stained for X SMOC-1 (C) or pro-EGF (D). The locations of the eye (e) and pronephros (pn) are indicated.

    Techniques Used: In Vivo, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Proximity Ligation Assay, Staining, Hybridization, In Situ

    Pro-EGF is present as an impurity in a commercial hSMOC-1 product. (A) Immunoblot of 5μg hSMOC-1 (R D systems #6074-SM) with an antibody to mature EGF (EMD Millipore #PC08). The band detected at approximately 160 kDa is consistent with the mass of pro-EGF. Mature EGF (50ng), detected at 5kDa, is shown as control. (B) Immuno-quantitation of pro-EGF in hSMOC-1. Fluorescence scan analysis of pro-EGF signals obtained following immunoblotting of hSMOC-1 (R D Systems) and recombinant pro-EGF (R D Systems #4289-EG) at the amounts shown. The left-hand Y axis displays the pro-EGF fluorescence signal intensity; the X axis displays the amounts of commercial hSMOC-1 analyzed (blue squares); the right hand Y axis displays the known amounts of recombinant pro-EGF analyzed (red squares). Note: Additional pro-EGF data points were used for the best fit analysis, but the graph was cropped to show the intersect point with hSMOC-1 more clearly. (C) Immunoblot of 32D/EGFR cell lysates showing increased phosphorylation of Erk following a six minute exposure to hSMOC-1 or hSMOC-1 immuno-depleted of pro-EGF (hSMOC-1 –proEGF). (D) Immunoblot of HEK293 cell lysates showing Erk phosphorylation following a six minute exposure to X SMOC-1, hSMOC-1, or hSMOC-1 –proEGF; the apparent higher potency of undepleted hSMOC-1 was presumably due to the pro-EGF impurity. The cell culture experiments were conducted in triplicate and the results presented are representative of those obtained.
    Figure Legend Snippet: Pro-EGF is present as an impurity in a commercial hSMOC-1 product. (A) Immunoblot of 5μg hSMOC-1 (R D systems #6074-SM) with an antibody to mature EGF (EMD Millipore #PC08). The band detected at approximately 160 kDa is consistent with the mass of pro-EGF. Mature EGF (50ng), detected at 5kDa, is shown as control. (B) Immuno-quantitation of pro-EGF in hSMOC-1. Fluorescence scan analysis of pro-EGF signals obtained following immunoblotting of hSMOC-1 (R D Systems) and recombinant pro-EGF (R D Systems #4289-EG) at the amounts shown. The left-hand Y axis displays the pro-EGF fluorescence signal intensity; the X axis displays the amounts of commercial hSMOC-1 analyzed (blue squares); the right hand Y axis displays the known amounts of recombinant pro-EGF analyzed (red squares). Note: Additional pro-EGF data points were used for the best fit analysis, but the graph was cropped to show the intersect point with hSMOC-1 more clearly. (C) Immunoblot of 32D/EGFR cell lysates showing increased phosphorylation of Erk following a six minute exposure to hSMOC-1 or hSMOC-1 immuno-depleted of pro-EGF (hSMOC-1 –proEGF). (D) Immunoblot of HEK293 cell lysates showing Erk phosphorylation following a six minute exposure to X SMOC-1, hSMOC-1, or hSMOC-1 –proEGF; the apparent higher potency of undepleted hSMOC-1 was presumably due to the pro-EGF impurity. The cell culture experiments were conducted in triplicate and the results presented are representative of those obtained.

    Techniques Used: Quantitation Assay, Fluorescence, Recombinant, Cell Culture

    21) Product Images from "WIPI3 and WIPI4 β-propellers are scaffolds for LKB1-AMPK-TSC signalling circuits in the control of autophagy"

    Article Title: WIPI3 and WIPI4 β-propellers are scaffolds for LKB1-AMPK-TSC signalling circuits in the control of autophagy

    Journal: Nature Communications

    doi: 10.1038/ncomms15637

    The WIPI protein interactome. Monoclonal U2OS cell lines stably expressing GFP-WIPI1, GFP-WIPI2B, GFP-WIPI2D, GFP-WIPI3, GFP-WIPI4 or GFP alone were analysed by anti-GFP immunoprecipitation followed by nano liquid chromatography (LC)-MS/MS analysis (for detailed information, see Materials and Methods). ( a ) GeneMANIA was used to visualize identified WIPI protein–protein interactions. Red lines represent interactions identified in this study, and grey lines represent previously reported and predicted interactions (GeneMANIA, see Materials and Methods). ATG proteins and autophagy regulators are indicated with red letters. Complete results can be found in Supplementary Data 2 . Interactions identified using GFP-WIPI2B and GFP-WIPI2D were combined and are presented as WIPI2 interactions. Proteins exclusively interacting with either of the two WIPI2 isoforms can be found in Supplementary Data 2 . ( b ) A sub-network of the WIPI protein interactome focusing on autophagy-related and autophagy-relevant proteins is shown. Red lines represent interactions identified in this study, with dotted red lines representing interactions with low peptide counts in our LC-MS/MS analysis. Grey lines represent previously reported and predicted interactions (GeneMANIA, see Materials and Methods). In addition, the reported interactions of FIP200-ATG16L, TSC1-FIP200 and AMPK-TSC2 that did not appear when using GeneMANIA are also shown in grey. Supplementary Data demonstrating WIPI1, WIPI2B, WIPI2D and WIPI4 co-immunoprecipitation with NudC ( Supplementary Fig. 4 ) is available.
    Figure Legend Snippet: The WIPI protein interactome. Monoclonal U2OS cell lines stably expressing GFP-WIPI1, GFP-WIPI2B, GFP-WIPI2D, GFP-WIPI3, GFP-WIPI4 or GFP alone were analysed by anti-GFP immunoprecipitation followed by nano liquid chromatography (LC)-MS/MS analysis (for detailed information, see Materials and Methods). ( a ) GeneMANIA was used to visualize identified WIPI protein–protein interactions. Red lines represent interactions identified in this study, and grey lines represent previously reported and predicted interactions (GeneMANIA, see Materials and Methods). ATG proteins and autophagy regulators are indicated with red letters. Complete results can be found in Supplementary Data 2 . Interactions identified using GFP-WIPI2B and GFP-WIPI2D were combined and are presented as WIPI2 interactions. Proteins exclusively interacting with either of the two WIPI2 isoforms can be found in Supplementary Data 2 . ( b ) A sub-network of the WIPI protein interactome focusing on autophagy-related and autophagy-relevant proteins is shown. Red lines represent interactions identified in this study, with dotted red lines representing interactions with low peptide counts in our LC-MS/MS analysis. Grey lines represent previously reported and predicted interactions (GeneMANIA, see Materials and Methods). In addition, the reported interactions of FIP200-ATG16L, TSC1-FIP200 and AMPK-TSC2 that did not appear when using GeneMANIA are also shown in grey. Supplementary Data demonstrating WIPI1, WIPI2B, WIPI2D and WIPI4 co-immunoprecipitation with NudC ( Supplementary Fig. 4 ) is available.

    Techniques Used: Stable Transfection, Expressing, Immunoprecipitation, Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Glucose-starvation-mediated AMPK activation regulates WIPI4. ( a ) A lentiviral-based shRNA screening approach targeting the human kinome used for the assessment of autophagy identified AMPK and the AMPK-related protein kinases NUAK2 and BRSK2, along with CaMKKα, as autophagy regulators (for details see Methods section and Supplementary Fig. 8 ). Reported (red) and predicted (yellow) proteins interactions, and pathway interactions (blue) are indicated (GeneMANIA). ( b ) Stable GFP-WIPI1 U2OS cells were transfected with siRNAs for AMPKα, AMPKγ, LKB1, CaMKKα, NUAK2 or BRSK2 and knock-down confirmed by immunoblotting (left panels) or quantitative RT–PCR (right panels). ( c ) Stable GFP-WIPI4 U2OS cells with siRNAs targeting AMPKα, AMPKγ, LKB1, CaMKKα, NUAK2 or BRSK2 were starved (S) for 3 h. Mean percentages of GFP-WIPI4-puncta-positive cells (300 cells per condition, n =3) are presented. ( d ) U2OS cells were fed (F) or starved (S) with or without glucose, glutamine or AICAR and immunoblotted using anti-AMPKα and anti-tubulin antibodies. Stable GFP-WIPI4 U2OS cells ( e ) or GFP-WIPI1 ( f ) were fed or treated with complete medium without FCS (−FCS), without glucose (−FCS −Gluc) or without glucose and glutamine (−FCS −Gluc −Glut). Mean percentages of GFP-WIPI4-puncta-positive cells (300 cells per condition, n =3) ( e ) and GFP-WIPI1-puncta-positive cells (up to 3,904 cells per condition, n =6) ( f ) are presented. ( g ) U2OS cells expressing GFP-WIPI4 WT or GFP-WIPI4 D113A mutant were fed or glucose-starved (−FCS −Gluc) for 3 h. Mean percentages of GFP-WIPI4-puncta-positive cells (300 cells per condition, n =3) are presented. ( h ) U2OS cells expressing GFP-WIPI4 WT or GFP-WIPI4 D113A mutant with siControl (siCtr) or siAMPKα were glucose-starved (−FCS −Gluc) for 3 h. Mean percentages of GFP-WIPI4-puncta-positive cells (500 cells per condition, n =5) are presented. ( i ) A predicted model for the differential contributions of human WIPI β-propeller proteins in autophagy. Statistics and source data can be found in Supplementary Data 1 . Mean±s.d.; heteroscedastic t -testing; P values: * P
    Figure Legend Snippet: Glucose-starvation-mediated AMPK activation regulates WIPI4. ( a ) A lentiviral-based shRNA screening approach targeting the human kinome used for the assessment of autophagy identified AMPK and the AMPK-related protein kinases NUAK2 and BRSK2, along with CaMKKα, as autophagy regulators (for details see Methods section and Supplementary Fig. 8 ). Reported (red) and predicted (yellow) proteins interactions, and pathway interactions (blue) are indicated (GeneMANIA). ( b ) Stable GFP-WIPI1 U2OS cells were transfected with siRNAs for AMPKα, AMPKγ, LKB1, CaMKKα, NUAK2 or BRSK2 and knock-down confirmed by immunoblotting (left panels) or quantitative RT–PCR (right panels). ( c ) Stable GFP-WIPI4 U2OS cells with siRNAs targeting AMPKα, AMPKγ, LKB1, CaMKKα, NUAK2 or BRSK2 were starved (S) for 3 h. Mean percentages of GFP-WIPI4-puncta-positive cells (300 cells per condition, n =3) are presented. ( d ) U2OS cells were fed (F) or starved (S) with or without glucose, glutamine or AICAR and immunoblotted using anti-AMPKα and anti-tubulin antibodies. Stable GFP-WIPI4 U2OS cells ( e ) or GFP-WIPI1 ( f ) were fed or treated with complete medium without FCS (−FCS), without glucose (−FCS −Gluc) or without glucose and glutamine (−FCS −Gluc −Glut). Mean percentages of GFP-WIPI4-puncta-positive cells (300 cells per condition, n =3) ( e ) and GFP-WIPI1-puncta-positive cells (up to 3,904 cells per condition, n =6) ( f ) are presented. ( g ) U2OS cells expressing GFP-WIPI4 WT or GFP-WIPI4 D113A mutant were fed or glucose-starved (−FCS −Gluc) for 3 h. Mean percentages of GFP-WIPI4-puncta-positive cells (300 cells per condition, n =3) are presented. ( h ) U2OS cells expressing GFP-WIPI4 WT or GFP-WIPI4 D113A mutant with siControl (siCtr) or siAMPKα were glucose-starved (−FCS −Gluc) for 3 h. Mean percentages of GFP-WIPI4-puncta-positive cells (500 cells per condition, n =5) are presented. ( i ) A predicted model for the differential contributions of human WIPI β-propeller proteins in autophagy. Statistics and source data can be found in Supplementary Data 1 . Mean±s.d.; heteroscedastic t -testing; P values: * P

    Techniques Used: Activation Assay, shRNA, Transfection, Quantitative RT-PCR, Expressing, Mutagenesis

    Differential contributions of WIPI members to the formation of functional autophagosomes. G361 cell lines stably expressing shRNAs targeting WIPI1 (shWIPI1), WIPI2 (shWIPI2), WIPI3 (shWIPI3), WIPI4 (shWIPI4) or non-targeting shRNA (shControl) were assessed by quantitative RT–PCR ( a ) or electron microscopy analysis upon starvation (3 h), scale bars: 500 nm ( b ). ( c ) Monoclonal U2OS cell lines stably expressing GFP-LC3 and shWIPI2 or shControl were assessed by quantitative RT–PCR (left panel). Automated high-throughput image acquisition (middle panel, upper row: dashed lines indicate cell boundaries; lower row: magnified sections) and analysis (right panel). The numbers of GFP-LC3 puncta in control (shRNA) or WIPI2-KD (shWIPI2) cells were calculated under fed (F) or starved (S) conditions with or without wortmannin (WM) or bafilomycin A1 (BafA1). The mean number of GFP-LC3 puncta per cell was calculated (up to 15,401 cells per condition, n =3). Scale bars: 20 μm. ( d ) U2OS cells stably expressing GFP-LC3 were transiently transfected (48 h) with control siRNA (siControl) or siRNAs targeting WIPI3 (siWIPI3) or WIPI4 (siWIPI4). Total RNA was extracted for quantitative RT–PCR (left panel: WIPI3, middle panel: WIPI4). In addition, fed cells (F) cells were treated with or without of BafA1 for automated high-throughput image analysis (right panel). Mean numbers of GFP-LC3 puncta per cell are presented (up to 2,680 cells per condition, n =3). ( e ) Endogenous WIPI2 puncta formation was examined (anti-WIPI2/IgG-Alexa Fluor 488 antibodies) by confocal LSM and ImageJ (least 15 cells, n =3) using stable G361 WIPI3-KD (shWIPI3), WIPI4-KD (shWIPI4) and control cells (shControl). ( f ) G361 cells were transiently transfected with control siRNA (siControl), siWIPI3, siWIPI4 or a siWIPI3/siWIPI4 combination and fed (F) or starved (S) for 3 h with or without BafA1. Cell lysates were analysed by anti-LC3 and anti-tubulin western blotting. The migrations of LC3-I and LC3-II are indicated (left panel). LC3-I (middle panel) and LC3-II (right panel) abundances were quantified and results achieved in fed conditions (F) are presented ( n =3). Supplementary Material is available: Supplementary Fig. 2 . Statistics and source data can be found in Supplementary Data 1 . Mean±s.d.; heteroscedastic t -testing; P values: * P
    Figure Legend Snippet: Differential contributions of WIPI members to the formation of functional autophagosomes. G361 cell lines stably expressing shRNAs targeting WIPI1 (shWIPI1), WIPI2 (shWIPI2), WIPI3 (shWIPI3), WIPI4 (shWIPI4) or non-targeting shRNA (shControl) were assessed by quantitative RT–PCR ( a ) or electron microscopy analysis upon starvation (3 h), scale bars: 500 nm ( b ). ( c ) Monoclonal U2OS cell lines stably expressing GFP-LC3 and shWIPI2 or shControl were assessed by quantitative RT–PCR (left panel). Automated high-throughput image acquisition (middle panel, upper row: dashed lines indicate cell boundaries; lower row: magnified sections) and analysis (right panel). The numbers of GFP-LC3 puncta in control (shRNA) or WIPI2-KD (shWIPI2) cells were calculated under fed (F) or starved (S) conditions with or without wortmannin (WM) or bafilomycin A1 (BafA1). The mean number of GFP-LC3 puncta per cell was calculated (up to 15,401 cells per condition, n =3). Scale bars: 20 μm. ( d ) U2OS cells stably expressing GFP-LC3 were transiently transfected (48 h) with control siRNA (siControl) or siRNAs targeting WIPI3 (siWIPI3) or WIPI4 (siWIPI4). Total RNA was extracted for quantitative RT–PCR (left panel: WIPI3, middle panel: WIPI4). In addition, fed cells (F) cells were treated with or without of BafA1 for automated high-throughput image analysis (right panel). Mean numbers of GFP-LC3 puncta per cell are presented (up to 2,680 cells per condition, n =3). ( e ) Endogenous WIPI2 puncta formation was examined (anti-WIPI2/IgG-Alexa Fluor 488 antibodies) by confocal LSM and ImageJ (least 15 cells, n =3) using stable G361 WIPI3-KD (shWIPI3), WIPI4-KD (shWIPI4) and control cells (shControl). ( f ) G361 cells were transiently transfected with control siRNA (siControl), siWIPI3, siWIPI4 or a siWIPI3/siWIPI4 combination and fed (F) or starved (S) for 3 h with or without BafA1. Cell lysates were analysed by anti-LC3 and anti-tubulin western blotting. The migrations of LC3-I and LC3-II are indicated (left panel). LC3-I (middle panel) and LC3-II (right panel) abundances were quantified and results achieved in fed conditions (F) are presented ( n =3). Supplementary Material is available: Supplementary Fig. 2 . Statistics and source data can be found in Supplementary Data 1 . Mean±s.d.; heteroscedastic t -testing; P values: * P

    Techniques Used: Functional Assay, Stable Transfection, Expressing, shRNA, Quantitative RT-PCR, Electron Microscopy, High Throughput Screening Assay, Transfection, Western Blot

    WIPI3 interacts with the TSC complex. ( a ) Stable GFP-WIPI1, GFP-WIPI2B, GFP-WIPI3 or GFP-WIPI4 U2OS cells were starved (3 h), and subjected to anti-GFP immunoprecipitation and anti-TSC2, anti-phospho-TSC2 (S1387), anti-TSC1 or anti-GFP immunoblotting. ( b ) U2OS cells were analysed by anti-TSC1 immunoprecipitation (TSC1-IP), anti-TSC1, anti-TSC2 and anti-WIPI1 (right panel), anti-WIPI2 (right panel), anti-WIPI3 (left panel) or anti-WIPI4 (right panel) immunoblotting. ( c ) ATG5 WT or KO MEFs expressing GFP-WIPI3 (W3) or GFP were subjected to anti-GFP immunoprecipitation and anti-TSC1, anti-TSC2 and anti-GFP immunoblotting. Conditions (3 h): fed (F) or starved (S). ( d ) Stable GFP-WIPI3 or GFP U2OS cells were analysed by anti-GFP immunoprecipitation and anti-TSC2 and anti-GFP immunoblotting. Conditions (15 min. to 3 h): fed (F), starved (S), starved with LY294002 (S+LY). ( e ) Myc-tagged TSC1 fragments (TSC1-M, TSC1-C; full length: TSC-FL) were expressed in stable GFP-WIPI3 U2OS cells and subjected to anti-GFP immunoprecipitation and anti-GFP or anti-myc immunoblotting. ( f ) Lysosomal fractions (no. 1–7; total protein control: TP) from stable GFP-WIPI3 U2OS cells (BafA1, 3 h) were immunoblotted using anti-GFP, anti-LAMP2, anti-TSC2, anti-WIPI2 or anti-Rab4 antibodies. ( g ) Stable GFP-WIPI3 U2OS cells were starved (3 h) and immunostained with anti-TSC2/IgG-Alexa Fluor 546 and anti-LAMP2/IgG-Alexa Fluor 633 antibodies for confocal LSM. ( h ) Stable GFP-WIPI3 U2OS cells were immunostained with anti-Lamp2/IgG-Alexa Fluor 633 and anti-WIPI2/IgG-Alexa Fluor 546 for confocal LSM. Conditions (3 h): fed (F), starved (S). ( i ) Stable GFP-WIPI3 U2OS cells with siControl or siTSC2 were fed (F) or starved (S) with or without BafA1 (3 h). Upper panel: anti-TSC2 immunoblotting, lower panel: GFP-WIPI3 puncta assessment (up to 493 cells per condition, n =4). ( j ) U2OS cells were subjected to immunoblotting using anti-phospho-mTOR (S2481), anti-mTOR, anti-phospho-ULK1 (S757), anti-ULK1 and anti-tubulin antibodies. Treatments: fed (F, 4 h), starved (S, 4 h), starved (3 h) and fed (1 h) (S→F). ( k ) Stable GFP-WIPI3 U2OS cells were immunostained with anti-mTOR/IgG-Alexa Fluor 546 and anti-LAMP2/IgG-Alexa Fluor 633 antibodies. Treatments: starved (S, 2 h), starved (2 h) and fed (1 h) (S→F). Co-localizations are indicated with arrows ( g , h , k ). Supplementary Material is available: Supplementary Fig. 5 . Statistics and source data: Supplementary Data 1 . Mean±s.d.; heteroscedastic t -testing; P values: * P
    Figure Legend Snippet: WIPI3 interacts with the TSC complex. ( a ) Stable GFP-WIPI1, GFP-WIPI2B, GFP-WIPI3 or GFP-WIPI4 U2OS cells were starved (3 h), and subjected to anti-GFP immunoprecipitation and anti-TSC2, anti-phospho-TSC2 (S1387), anti-TSC1 or anti-GFP immunoblotting. ( b ) U2OS cells were analysed by anti-TSC1 immunoprecipitation (TSC1-IP), anti-TSC1, anti-TSC2 and anti-WIPI1 (right panel), anti-WIPI2 (right panel), anti-WIPI3 (left panel) or anti-WIPI4 (right panel) immunoblotting. ( c ) ATG5 WT or KO MEFs expressing GFP-WIPI3 (W3) or GFP were subjected to anti-GFP immunoprecipitation and anti-TSC1, anti-TSC2 and anti-GFP immunoblotting. Conditions (3 h): fed (F) or starved (S). ( d ) Stable GFP-WIPI3 or GFP U2OS cells were analysed by anti-GFP immunoprecipitation and anti-TSC2 and anti-GFP immunoblotting. Conditions (15 min. to 3 h): fed (F), starved (S), starved with LY294002 (S+LY). ( e ) Myc-tagged TSC1 fragments (TSC1-M, TSC1-C; full length: TSC-FL) were expressed in stable GFP-WIPI3 U2OS cells and subjected to anti-GFP immunoprecipitation and anti-GFP or anti-myc immunoblotting. ( f ) Lysosomal fractions (no. 1–7; total protein control: TP) from stable GFP-WIPI3 U2OS cells (BafA1, 3 h) were immunoblotted using anti-GFP, anti-LAMP2, anti-TSC2, anti-WIPI2 or anti-Rab4 antibodies. ( g ) Stable GFP-WIPI3 U2OS cells were starved (3 h) and immunostained with anti-TSC2/IgG-Alexa Fluor 546 and anti-LAMP2/IgG-Alexa Fluor 633 antibodies for confocal LSM. ( h ) Stable GFP-WIPI3 U2OS cells were immunostained with anti-Lamp2/IgG-Alexa Fluor 633 and anti-WIPI2/IgG-Alexa Fluor 546 for confocal LSM. Conditions (3 h): fed (F), starved (S). ( i ) Stable GFP-WIPI3 U2OS cells with siControl or siTSC2 were fed (F) or starved (S) with or without BafA1 (3 h). Upper panel: anti-TSC2 immunoblotting, lower panel: GFP-WIPI3 puncta assessment (up to 493 cells per condition, n =4). ( j ) U2OS cells were subjected to immunoblotting using anti-phospho-mTOR (S2481), anti-mTOR, anti-phospho-ULK1 (S757), anti-ULK1 and anti-tubulin antibodies. Treatments: fed (F, 4 h), starved (S, 4 h), starved (3 h) and fed (1 h) (S→F). ( k ) Stable GFP-WIPI3 U2OS cells were immunostained with anti-mTOR/IgG-Alexa Fluor 546 and anti-LAMP2/IgG-Alexa Fluor 633 antibodies. Treatments: starved (S, 2 h), starved (2 h) and fed (1 h) (S→F). Co-localizations are indicated with arrows ( g , h , k ). Supplementary Material is available: Supplementary Fig. 5 . Statistics and source data: Supplementary Data 1 . Mean±s.d.; heteroscedastic t -testing; P values: * P

    Techniques Used: Immunoprecipitation, Expressing

    WIPI1 assists WIPI2 in recruiting ATG16L for LC3 lipidation. ( a ) U2OS cells transiently expressing GFP-WIPI1, GFP-WIPI2B, GFP-WIPI2D, GFP-WIPI3, GFP-WIPI4 or GFP alone were starved for 3 h and analysed by anti-GFP immunoprecipitation followed by anti-GFP or anti-ATG16L immunoblotting. The asterisk in the right panel (GFP-IP) represents a nonspecific band. Endogenous ATG16L isoforms co-precipitated with GFP-WIPI1, GFP-WIPI2B and GFP-WIPI2D as indicated in the right panel. ( b , c ) G361 cells stably expressing shRNA targeting WIPI2 ( b , shWIPI2), WIPI1 ( c , shWIPI1) or the non-targeting control (shControl) were fed (F) or starved (S) for 3 h, and endogenous ATG16L was detected using anti-ATG16L/IgG-Alexa Fluor 488 antibodies and fluorescence microscopy. Mean percentages of ATG16L-puncta-positive cells (up to 369 individual cells per condition, n =3) are presented. ( d ) Monoclonal U2OS cells stably expressing GFP-WIPI1 and shWIPI2 or shControl were starved (S) for 3 h, and the percentage of cells displaying an accumulation of perinuclear GFP-WIPI1 was calculated using fluorescence microscopy. Mean values (400 cells per condition, n =4) are presented. ( e , f ) G361 cells stably expressing shRNAs targeting WIPI1 ( e , shWIPI1) or WIPI2 ( f , shWIPI2) were fed (F) or starved (S) for 3 h and immunostained using anti-WIPI4/IgG-Alexa Fluor 488 antibodies. Mean percentages of WIPI4-puncta-positive cells (up to 340 cells per condition, n =3) are presented. ( g ) G361 cells stably expressing shRNAs targeting WIPI3 (shWIPI3), WIPI4 (shWIPI4) or the non-targeting shRNA control (shControl) were fed (F) or starved (S) for 3 h and immunostained using anti-ATG16L/IgG-Alexa Fluor 488 antibodies for analysis by fluorescence microscopy (left panel). Mean percentages of ATG16L-puncta-positive cells (up to 387 cells per condition, n =3) are presented (right panel). ( h , i ) ATG5 wild-type (WT) or knockout (KO) mouse embryonic fibroblasts (MEFs) transiently expressing GFP-WIPI1, GFP-WIPI2B, GFP-WIPI3 or GFP-WIPI4 were analysed by confocal LSM, and images from ATG5 WT MEFs ( h , upper panel) were processed using Volocity to generate 3D-reconstruction fly-through movies ( Supplementary Movies 5–8 ), from which still images are presented ( h , lower panel). Mean percentages of GFP-WIPI puncta-positive cells (300 cells, n =3). Mean±s.d.; heteroscedastic t -testing; P values: * P
    Figure Legend Snippet: WIPI1 assists WIPI2 in recruiting ATG16L for LC3 lipidation. ( a ) U2OS cells transiently expressing GFP-WIPI1, GFP-WIPI2B, GFP-WIPI2D, GFP-WIPI3, GFP-WIPI4 or GFP alone were starved for 3 h and analysed by anti-GFP immunoprecipitation followed by anti-GFP or anti-ATG16L immunoblotting. The asterisk in the right panel (GFP-IP) represents a nonspecific band. Endogenous ATG16L isoforms co-precipitated with GFP-WIPI1, GFP-WIPI2B and GFP-WIPI2D as indicated in the right panel. ( b , c ) G361 cells stably expressing shRNA targeting WIPI2 ( b , shWIPI2), WIPI1 ( c , shWIPI1) or the non-targeting control (shControl) were fed (F) or starved (S) for 3 h, and endogenous ATG16L was detected using anti-ATG16L/IgG-Alexa Fluor 488 antibodies and fluorescence microscopy. Mean percentages of ATG16L-puncta-positive cells (up to 369 individual cells per condition, n =3) are presented. ( d ) Monoclonal U2OS cells stably expressing GFP-WIPI1 and shWIPI2 or shControl were starved (S) for 3 h, and the percentage of cells displaying an accumulation of perinuclear GFP-WIPI1 was calculated using fluorescence microscopy. Mean values (400 cells per condition, n =4) are presented. ( e , f ) G361 cells stably expressing shRNAs targeting WIPI1 ( e , shWIPI1) or WIPI2 ( f , shWIPI2) were fed (F) or starved (S) for 3 h and immunostained using anti-WIPI4/IgG-Alexa Fluor 488 antibodies. Mean percentages of WIPI4-puncta-positive cells (up to 340 cells per condition, n =3) are presented. ( g ) G361 cells stably expressing shRNAs targeting WIPI3 (shWIPI3), WIPI4 (shWIPI4) or the non-targeting shRNA control (shControl) were fed (F) or starved (S) for 3 h and immunostained using anti-ATG16L/IgG-Alexa Fluor 488 antibodies for analysis by fluorescence microscopy (left panel). Mean percentages of ATG16L-puncta-positive cells (up to 387 cells per condition, n =3) are presented (right panel). ( h , i ) ATG5 wild-type (WT) or knockout (KO) mouse embryonic fibroblasts (MEFs) transiently expressing GFP-WIPI1, GFP-WIPI2B, GFP-WIPI3 or GFP-WIPI4 were analysed by confocal LSM, and images from ATG5 WT MEFs ( h , upper panel) were processed using Volocity to generate 3D-reconstruction fly-through movies ( Supplementary Movies 5–8 ), from which still images are presented ( h , lower panel). Mean percentages of GFP-WIPI puncta-positive cells (300 cells, n =3). Mean±s.d.; heteroscedastic t -testing; P values: * P

    Techniques Used: Expressing, Immunoprecipitation, Stable Transfection, shRNA, Fluorescence, Microscopy, Knock-Out

    All WIPI members fold into seven-bladed β-propeller proteins that bind PtdIns3P and co-localize at nascent autophagosomes. ( a ) Structural homology modelling using HHpred. Propeller blades are indicated (1 to 7), and unstructured sequences are omitted. ( b ) Protein-phospholipid binding overlay assays using G361 cell extracts followed by the detection of endogenous WIPI1, WIPI2 or WIPI4 or using a monoclonal U2OS cell line stably expressing GFP-WIPI3 followed by anti-GFP enhanced chemiluminescence (ECL) detection (left panels). Monoclonal U2OS cell lines stably expressing GFP-WIPI1, GFP-WIPI2B or GFP-WIPI3 or transiently expressing GFP-WIPI4 were starved for 3 h with nutrient-free medium. Images were acquired by confocal LSM and processed using Volocity to generate 3D-reconstruction fly-through movies ( Supplementary Movies 1–4 ). Representative still images are presented (right panels). Scale bars: 3 μm. ( c ) U2OS cells stably expressing GFP-WIPI1, GFP-WIPI2B or GFP-WIPI3 or transiently expressing GFP-WIPI4 were fed (F) or starved (S) for 3 h with or without bafilomycin A1 (BafA1) or LY294002 (LY). The percentage GFP-WIPI puncta cells were calculated (up to 429 cells per condition, n =3). ( d ) U2OS cells stably expressing GFP-WIPI1, GFP-WIPI2B or GFP-WIPI3 or transiently expressing GFP-WIPI4 were starved (3 h), and endogenous ATG12, LC3, p62 (anti-ATG12, anti-LC3, anti-p62 and IgG-Alexa Fluor 546 antibodies) or transiently expressed myc-tagged ATG14 or DFCP1 detected (anti-myc/IgG-Alexa Fluor 546 antibodies). Merged confocal LSM images are presented (left panels, dashed lines: cell boundaries; right panels: magnified subsections). Scale bars: 20 μm. ( e ) Confocal LSM examinations of starved (3 h) U2OS cells stably expressing GFP-WIPI1 and immunostained with anti-WIPI2/IgG-Alexa Fluor 546 and anti-WIPI4/IgG-Alexa Fluor 633 antibodies. Intensity profiles of co-localizations (peaks 1 and 2, upper panel) are displayed, along with the corresponding magnified image sections. Scale bar: 2.5 μm. ( f ) U2OS cells stably expressing GFP-WIPI3 and transiently expressing myc-tagged WIPI1 or WIPI2B were starved (3 h) and immunostained using anti-myc or anti-WIPI4 and IgG-Alexa Fluor 546 antibodies. Scale bar: 2.5 μm. Arrows in magnified image sections indicate co-localization events ( d – f ). Supplementary Material is available: Supplementary Fig. 1 , Supplementary Note . Statistics and source data can be found in Supplementary Data 1 . Mean±s.d.; heteroscedastic t -testing; P values: * P
    Figure Legend Snippet: All WIPI members fold into seven-bladed β-propeller proteins that bind PtdIns3P and co-localize at nascent autophagosomes. ( a ) Structural homology modelling using HHpred. Propeller blades are indicated (1 to 7), and unstructured sequences are omitted. ( b ) Protein-phospholipid binding overlay assays using G361 cell extracts followed by the detection of endogenous WIPI1, WIPI2 or WIPI4 or using a monoclonal U2OS cell line stably expressing GFP-WIPI3 followed by anti-GFP enhanced chemiluminescence (ECL) detection (left panels). Monoclonal U2OS cell lines stably expressing GFP-WIPI1, GFP-WIPI2B or GFP-WIPI3 or transiently expressing GFP-WIPI4 were starved for 3 h with nutrient-free medium. Images were acquired by confocal LSM and processed using Volocity to generate 3D-reconstruction fly-through movies ( Supplementary Movies 1–4 ). Representative still images are presented (right panels). Scale bars: 3 μm. ( c ) U2OS cells stably expressing GFP-WIPI1, GFP-WIPI2B or GFP-WIPI3 or transiently expressing GFP-WIPI4 were fed (F) or starved (S) for 3 h with or without bafilomycin A1 (BafA1) or LY294002 (LY). The percentage GFP-WIPI puncta cells were calculated (up to 429 cells per condition, n =3). ( d ) U2OS cells stably expressing GFP-WIPI1, GFP-WIPI2B or GFP-WIPI3 or transiently expressing GFP-WIPI4 were starved (3 h), and endogenous ATG12, LC3, p62 (anti-ATG12, anti-LC3, anti-p62 and IgG-Alexa Fluor 546 antibodies) or transiently expressed myc-tagged ATG14 or DFCP1 detected (anti-myc/IgG-Alexa Fluor 546 antibodies). Merged confocal LSM images are presented (left panels, dashed lines: cell boundaries; right panels: magnified subsections). Scale bars: 20 μm. ( e ) Confocal LSM examinations of starved (3 h) U2OS cells stably expressing GFP-WIPI1 and immunostained with anti-WIPI2/IgG-Alexa Fluor 546 and anti-WIPI4/IgG-Alexa Fluor 633 antibodies. Intensity profiles of co-localizations (peaks 1 and 2, upper panel) are displayed, along with the corresponding magnified image sections. Scale bar: 2.5 μm. ( f ) U2OS cells stably expressing GFP-WIPI3 and transiently expressing myc-tagged WIPI1 or WIPI2B were starved (3 h) and immunostained using anti-myc or anti-WIPI4 and IgG-Alexa Fluor 546 antibodies. Scale bar: 2.5 μm. Arrows in magnified image sections indicate co-localization events ( d – f ). Supplementary Material is available: Supplementary Fig. 1 , Supplementary Note . Statistics and source data can be found in Supplementary Data 1 . Mean±s.d.; heteroscedastic t -testing; P values: * P

    Techniques Used: Binding Assay, Stable Transfection, Expressing

    WIPI4 interacts with ATG2, AMPK and ULK1. ( a ) U2OS cells transiently expressing GFP-tagged WIPI proteins and myc-ATG2A were starved and analysed by anti-myc immunoprecipitation and anti-myc and anti-GFP immunoblotting. ( b ) U2OS cells expressing myc-ATG2A and GFP, GFP-WIPI4 WT or GFP-WIPI4 mutants (N15A, Q16A, D17A, N15A/D17A) were analysed by anti-GFP immunoprecipitation, and anti-myc and anti-GFP immunoblotting. Amino acids in WIPI4 conferring ATG2 binding are highlighted in red (upper panel). ( c ) Stable GFP-WIPI1 U2OS cells with shRNAs targeting WIPI4 and ATG2 (shATG2/shWIPI4) or with shControl were analysed by automated imaging. Aberrant GFP-WIPI1 accumulations are indicated. ( d , e ) Stable GFP-WIPI1 or GFP-LC3 U2OS cells were transfected with siATG2 or siControl (fed conditions, F) and images of GFP-WIPI1 cells are shown ( d ). ( e ) GFP-WIPI1 (left, middle panel) and GFP-LC3 puncta (right panel) formation was assessed using up to 13,614 GFP-WIPI1 U2OS cells ( n =3) or up to 9,457 GFP-LC3 U2OS cells ( n =3) per condition. ( f ) Stable GFP-WIPI2B, GFP-WIPI4 or GFP U2OS cells expressing myc-ATG2A were fed (F) or starved (S) and analysed by anti-GFP immunoprecipitation and anti-GFP or anti-AMPKα immunoblotting ( n =2). ( g ) Stable GFP-WIPI4 or GFP U2OS cells expressing myc-ATG2A were fed (F) or starved (S) for 3 h and analysed by anti-GFP immunoprecipitation and immunoblotting (anti-GFP, anti-myc or anti-Ulk1 antibodies) ( n =3 with duplicates). ( h ) Stable GFP-WIPI4 or GFP U2OS cells expressing myc-ATG2A were analysed by anti-GFP immunoprecipitation and anti-GFP, anti-myc, anti-Ulk1 or anti-AMPKα immunoblotting. Conditions (3 h): fed (F), starved (S), starved with AICAR ( n =3 with duplicates). ( i ) The abundance of AMPK co-purifying with GFP-WIPI4 upon AICAR treatment was quantified ( n =3). ( j ) Stable GFP-WIPI4 cells expressing myc-ATG2A were treated (3 h) with complete medium (without FCS) lacking glucose (Gluc) or glucose/glutamine (Gluc/Glut), and analysed by anti-GFP immunoprecipitation and anti-myc, anti-AMPKα and anti-GFP immunoblotting. ( k ) U2OS cells expressing GFP or GFP-WIPI4 with myc-tagged AMPKα1, α2, β1 or γ1 were analysed by anti-GFP immunoprecipitation and anti-myc and anti-GFP immunoblotting. Additional Supplementary Material is available: Supplementary Fig. 7 . Statistics and source data: Supplementary Data 1 . Mean±s.d.; heteroscedastic t -testing; P values: * P
    Figure Legend Snippet: WIPI4 interacts with ATG2, AMPK and ULK1. ( a ) U2OS cells transiently expressing GFP-tagged WIPI proteins and myc-ATG2A were starved and analysed by anti-myc immunoprecipitation and anti-myc and anti-GFP immunoblotting. ( b ) U2OS cells expressing myc-ATG2A and GFP, GFP-WIPI4 WT or GFP-WIPI4 mutants (N15A, Q16A, D17A, N15A/D17A) were analysed by anti-GFP immunoprecipitation, and anti-myc and anti-GFP immunoblotting. Amino acids in WIPI4 conferring ATG2 binding are highlighted in red (upper panel). ( c ) Stable GFP-WIPI1 U2OS cells with shRNAs targeting WIPI4 and ATG2 (shATG2/shWIPI4) or with shControl were analysed by automated imaging. Aberrant GFP-WIPI1 accumulations are indicated. ( d , e ) Stable GFP-WIPI1 or GFP-LC3 U2OS cells were transfected with siATG2 or siControl (fed conditions, F) and images of GFP-WIPI1 cells are shown ( d ). ( e ) GFP-WIPI1 (left, middle panel) and GFP-LC3 puncta (right panel) formation was assessed using up to 13,614 GFP-WIPI1 U2OS cells ( n =3) or up to 9,457 GFP-LC3 U2OS cells ( n =3) per condition. ( f ) Stable GFP-WIPI2B, GFP-WIPI4 or GFP U2OS cells expressing myc-ATG2A were fed (F) or starved (S) and analysed by anti-GFP immunoprecipitation and anti-GFP or anti-AMPKα immunoblotting ( n =2). ( g ) Stable GFP-WIPI4 or GFP U2OS cells expressing myc-ATG2A were fed (F) or starved (S) for 3 h and analysed by anti-GFP immunoprecipitation and immunoblotting (anti-GFP, anti-myc or anti-Ulk1 antibodies) ( n =3 with duplicates). ( h ) Stable GFP-WIPI4 or GFP U2OS cells expressing myc-ATG2A were analysed by anti-GFP immunoprecipitation and anti-GFP, anti-myc, anti-Ulk1 or anti-AMPKα immunoblotting. Conditions (3 h): fed (F), starved (S), starved with AICAR ( n =3 with duplicates). ( i ) The abundance of AMPK co-purifying with GFP-WIPI4 upon AICAR treatment was quantified ( n =3). ( j ) Stable GFP-WIPI4 cells expressing myc-ATG2A were treated (3 h) with complete medium (without FCS) lacking glucose (Gluc) or glucose/glutamine (Gluc/Glut), and analysed by anti-GFP immunoprecipitation and anti-myc, anti-AMPKα and anti-GFP immunoblotting. ( k ) U2OS cells expressing GFP or GFP-WIPI4 with myc-tagged AMPKα1, α2, β1 or γ1 were analysed by anti-GFP immunoprecipitation and anti-myc and anti-GFP immunoblotting. Additional Supplementary Material is available: Supplementary Fig. 7 . Statistics and source data: Supplementary Data 1 . Mean±s.d.; heteroscedastic t -testing; P values: * P

    Techniques Used: Expressing, Immunoprecipitation, Binding Assay, Imaging, Transfection

    22) Product Images from "Lycorine Downregulates HMGB1 to Inhibit Autophagy and Enhances Bortezomib Activity in Multiple Myeloma"

    Article Title: Lycorine Downregulates HMGB1 to Inhibit Autophagy and Enhances Bortezomib Activity in Multiple Myeloma

    Journal: Theranostics

    doi: 10.7150/thno.15584

    A schematic illustration of proposed mechanism for anti-myeloma activity of lycorine. HMGB1 acts as an important mediator of autophagy. It can directly bind with Beclin-1 or activate MEK-ERK to free Beclin-1 from Bcl-2 and initiate autophagy which facilitates cell survival and confers resistance. Lycorine is suggested to mediate HMGB1 degradation through proteasome pathway, inhibits MEK-ERK activation and thereby increase Bcl-2-Beclin-1 interaction. As a result causes inhibition of pro-survival autophagy and leads to cell death.
    Figure Legend Snippet: A schematic illustration of proposed mechanism for anti-myeloma activity of lycorine. HMGB1 acts as an important mediator of autophagy. It can directly bind with Beclin-1 or activate MEK-ERK to free Beclin-1 from Bcl-2 and initiate autophagy which facilitates cell survival and confers resistance. Lycorine is suggested to mediate HMGB1 degradation through proteasome pathway, inhibits MEK-ERK activation and thereby increase Bcl-2-Beclin-1 interaction. As a result causes inhibition of pro-survival autophagy and leads to cell death.

    Techniques Used: Activity Assay, Activation Assay, Inhibition

    HMGB1 acts as the key mediator of lycorine induced autophagy inhibition. (A) Samples were prepared and separated by 2D gel electrophoresis to identify differentially expressed protein after lycorine treatment (5µM). The gel was stained with Coomassie blue and photographed. Section of the photograph is shown (top). The spot indicated by the red arrow in the top right panel corresponding to the most differentially expressed protein comparing the control (top left panel). HMGB1 was identified by the MASCOT search engine (bottom panel). The MASCOT search engine was used to assess the data from LC-MS/MS to identify the proteins from the UniProt protein database. HMGB1 had the highest hit score (549) among all possible hits contained in the database that matched the peptides from the sample. (B) ARH-77, ANBL6, ARP-1 and MM.1S cells were treated with indicated concentration of lycorine for 24 h and the expression of HMGB1 was analyzed in the cell extract by Western blotting. GAPDH was used as a loading control. The intensity of HMGB1 was determined by densitometry using ImageJ software and normalized with loading control (HMGB1/GAPDH). (C) ARP-1 cells were transfected with blank pCMV (Mock) or HMGB1-pCMV (ovHMGB1) vectors and cultured with or without lycorine for 24 h followed by cell viability analysis using a CCK-8 kit. Data are presented from three independent experiments. (D) ARP-1 cells transfected with blank or HMGB1 vectors were treated with or without lycorine and Western blotting was used to investigate the change in autophagy. GAPDH was used as a loading control. (E) ARP-1 cells were transfected with HMGB1 shRNA or scramble shRNA and cultured in the presence or absence of lycorine for 24 h and cell viability was measure using a CCK-8 kit. Data presented are mean±SD from three independent experiments. (F) Scramble or HMGB1 shRNA transfected ARP-1 cells were cultured for 24 h in the presence or absence of lycorine, the whole cell lysate was prepared and subjected to immunoblotting to detect autophagy. GAPDH was used as an internal control.
    Figure Legend Snippet: HMGB1 acts as the key mediator of lycorine induced autophagy inhibition. (A) Samples were prepared and separated by 2D gel electrophoresis to identify differentially expressed protein after lycorine treatment (5µM). The gel was stained with Coomassie blue and photographed. Section of the photograph is shown (top). The spot indicated by the red arrow in the top right panel corresponding to the most differentially expressed protein comparing the control (top left panel). HMGB1 was identified by the MASCOT search engine (bottom panel). The MASCOT search engine was used to assess the data from LC-MS/MS to identify the proteins from the UniProt protein database. HMGB1 had the highest hit score (549) among all possible hits contained in the database that matched the peptides from the sample. (B) ARH-77, ANBL6, ARP-1 and MM.1S cells were treated with indicated concentration of lycorine for 24 h and the expression of HMGB1 was analyzed in the cell extract by Western blotting. GAPDH was used as a loading control. The intensity of HMGB1 was determined by densitometry using ImageJ software and normalized with loading control (HMGB1/GAPDH). (C) ARP-1 cells were transfected with blank pCMV (Mock) or HMGB1-pCMV (ovHMGB1) vectors and cultured with or without lycorine for 24 h followed by cell viability analysis using a CCK-8 kit. Data are presented from three independent experiments. (D) ARP-1 cells transfected with blank or HMGB1 vectors were treated with or without lycorine and Western blotting was used to investigate the change in autophagy. GAPDH was used as a loading control. (E) ARP-1 cells were transfected with HMGB1 shRNA or scramble shRNA and cultured in the presence or absence of lycorine for 24 h and cell viability was measure using a CCK-8 kit. Data presented are mean±SD from three independent experiments. (F) Scramble or HMGB1 shRNA transfected ARP-1 cells were cultured for 24 h in the presence or absence of lycorine, the whole cell lysate was prepared and subjected to immunoblotting to detect autophagy. GAPDH was used as an internal control.

    Techniques Used: Inhibition, Two-Dimensional Gel Electrophoresis, Electrophoresis, Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Concentration Assay, Expressing, Western Blot, Software, Transfection, Cell Culture, CCK-8 Assay, shRNA

    HMGB1 is overexpressed in MM and contributes to MM cell survival. (A) The expression of HMGB1 from microarray analysis of samples obtained from healthy donors and from MGUS, SMM and MM patients. Data presented as the mean±SD. (B) Kaplan-Meier analyses of overall survival (OS). Publicly available microarray data sets GSE 5900 and GSE 2658 were downloaded and data were reproduced to estimate the OS. (C) Expression of HMGB1 mRNA in primary human BM CD138 + cell. Primary human CD138 + cells were isolated from the BM aspirates of normal subjects (n=9) and patient with myeloma (n=19). GAPDH normalized HMGB1 mRNA level was analyzed by quantitative RT-PCR. (D) HMGB1 expression in myeloma cell lines and B-cell was measured by Western blotting. GAPDH was used as a loading control. Densitometry analysis of HMGB1 intensity was performed using ImageJ software, normalized with loading control (HMGB1/GAPDH) and plotted on a bar diagram. (E) A CCK-8 assay was used to check cell viability after ARP-1 cells were transiently transfected with HMGB1 shRNA or scramble shRNA for various times. Data are presented as the mean±SD (n=3, ** p
    Figure Legend Snippet: HMGB1 is overexpressed in MM and contributes to MM cell survival. (A) The expression of HMGB1 from microarray analysis of samples obtained from healthy donors and from MGUS, SMM and MM patients. Data presented as the mean±SD. (B) Kaplan-Meier analyses of overall survival (OS). Publicly available microarray data sets GSE 5900 and GSE 2658 were downloaded and data were reproduced to estimate the OS. (C) Expression of HMGB1 mRNA in primary human BM CD138 + cell. Primary human CD138 + cells were isolated from the BM aspirates of normal subjects (n=9) and patient with myeloma (n=19). GAPDH normalized HMGB1 mRNA level was analyzed by quantitative RT-PCR. (D) HMGB1 expression in myeloma cell lines and B-cell was measured by Western blotting. GAPDH was used as a loading control. Densitometry analysis of HMGB1 intensity was performed using ImageJ software, normalized with loading control (HMGB1/GAPDH) and plotted on a bar diagram. (E) A CCK-8 assay was used to check cell viability after ARP-1 cells were transiently transfected with HMGB1 shRNA or scramble shRNA for various times. Data are presented as the mean±SD (n=3, ** p

    Techniques Used: Expressing, Microarray, Isolation, Quantitative RT-PCR, Western Blot, Software, CCK-8 Assay, Transfection, shRNA

    Lycorine mediated proteasomal degradation of HMGB1 inhibits the dissociation of Bcl-2 from Beclin-1. (A) Release of HMGB1 upon lycorine treatment was analyzed. The amount of HMGB1 was checked in the culture medium of ANBL6 and ARP-1 cells after incubation with or without lycorine for 12 and 24 h by Western blotting. ( B ) Subcellular localization of HMGB1 was observed under confocal microscope. ANBL6 and ARP-1 cells were treated with or without lycorine for 24 h and then immunostained with HMGB1-specific antibody/Cy3 secondary antibody (shown in red). Nuclei were stained with DAPI (blue). Images were acquired digitally by FV1000-X81 confocal microscope (Olympus, Japan) with 60× magnification. (C) Subcellular localization of HMGB1 was checked by Western blotting. Cytoplasmic and nuclear extracts of the harvested ANBL6 and ARP-1 cells after 24 h treatment with increasing doses of lycorine were prepared and subjected to Western blotting for the detection of HMGB1. Antibodies against α-tubulin and RCC1 were used to determine the purity of the cytoplasmic and nuclear fractions, respectively. (D) Total RNA was prepared from ANBL6 and ARP-1 cells 24 h after lycorine treatment and the level of HMGB1 mRNA was measured by quantitative RT-PCR. (E) Western blotting analysis of total cell lysate prepared from cells treated with 200 μg/ml CHX in the presence or absence of lycorine for the indicated time. The signal intensity from HMGB1 blot was normalized to GAPDH and plotted against the CHX incubation time. (F) Cells were incubated for 18 h with or without lycorine, followed by 6 h with 10 or 15 µM MG-132, and the lysate was used to detect HMGB1. GAPDH was used as a loading control. Densitometry analysis of HMGB1 intensity was performed using ImageJ software and normalized with loading control (HMGB1/GAPDH). (G) Quantitative Co-IP was adopted to investigate the interaction of HMGB1 with ubiquitin. Co-IP was performed using an anti-HMGB1 antibody in lysate from cells treated with or without lycorine and subjected for Western blotting. The blot was then probed with an anti-ubiquitin antibody. (H) Effect of different concentration of lycorine on MEK-ERK pathway. Total protein lysates were analyzed by immunoblotting using the indicated antibodies. Levels of total MEK, ERK 1/2 and Bcl2 were normalized for equal loading to detect their activated phosphorylation state. (I) Association of Bcl-2 with Beclin-1 was analyzed by Quantitative Co-IP. After immunoprecipitation using Beclin-1 antibody, sample was blotted and probed with Bcl-2 antibody.
    Figure Legend Snippet: Lycorine mediated proteasomal degradation of HMGB1 inhibits the dissociation of Bcl-2 from Beclin-1. (A) Release of HMGB1 upon lycorine treatment was analyzed. The amount of HMGB1 was checked in the culture medium of ANBL6 and ARP-1 cells after incubation with or without lycorine for 12 and 24 h by Western blotting. ( B ) Subcellular localization of HMGB1 was observed under confocal microscope. ANBL6 and ARP-1 cells were treated with or without lycorine for 24 h and then immunostained with HMGB1-specific antibody/Cy3 secondary antibody (shown in red). Nuclei were stained with DAPI (blue). Images were acquired digitally by FV1000-X81 confocal microscope (Olympus, Japan) with 60× magnification. (C) Subcellular localization of HMGB1 was checked by Western blotting. Cytoplasmic and nuclear extracts of the harvested ANBL6 and ARP-1 cells after 24 h treatment with increasing doses of lycorine were prepared and subjected to Western blotting for the detection of HMGB1. Antibodies against α-tubulin and RCC1 were used to determine the purity of the cytoplasmic and nuclear fractions, respectively. (D) Total RNA was prepared from ANBL6 and ARP-1 cells 24 h after lycorine treatment and the level of HMGB1 mRNA was measured by quantitative RT-PCR. (E) Western blotting analysis of total cell lysate prepared from cells treated with 200 μg/ml CHX in the presence or absence of lycorine for the indicated time. The signal intensity from HMGB1 blot was normalized to GAPDH and plotted against the CHX incubation time. (F) Cells were incubated for 18 h with or without lycorine, followed by 6 h with 10 or 15 µM MG-132, and the lysate was used to detect HMGB1. GAPDH was used as a loading control. Densitometry analysis of HMGB1 intensity was performed using ImageJ software and normalized with loading control (HMGB1/GAPDH). (G) Quantitative Co-IP was adopted to investigate the interaction of HMGB1 with ubiquitin. Co-IP was performed using an anti-HMGB1 antibody in lysate from cells treated with or without lycorine and subjected for Western blotting. The blot was then probed with an anti-ubiquitin antibody. (H) Effect of different concentration of lycorine on MEK-ERK pathway. Total protein lysates were analyzed by immunoblotting using the indicated antibodies. Levels of total MEK, ERK 1/2 and Bcl2 were normalized for equal loading to detect their activated phosphorylation state. (I) Association of Bcl-2 with Beclin-1 was analyzed by Quantitative Co-IP. After immunoprecipitation using Beclin-1 antibody, sample was blotted and probed with Bcl-2 antibody.

    Techniques Used: Incubation, Western Blot, Microscopy, Staining, Quantitative RT-PCR, Software, Co-Immunoprecipitation Assay, Concentration Assay, Immunoprecipitation

    Anti-MM activity of lycorine in MM xenograft mouse models. (A) Changes in tumor volume. Caliper measurement of the tumor diameters were performed very 4 days to estimate the tumor volume using the following formula (a×b 2 )/2, where 'a' and 'b' respectively are the longest and shortest perpendicular diameters of tumor. Data are presented as the mean±SD from five mice. (B) Representative tumor bearing mouse from each treatment group before dissection (top). The mice were euthanized at the treatment end point, tumors were removed and photographed (bottom). (C) During the treatment period, mouse body weight was measured every 4 days. Data are shown as mean±SD. (D) Histochemical examinations of mouse tissue section. Tissue sections from the tumor were fixed and stained with HE and TUNEL to examine the tumor cell morphology. The magnifications are 40×. (E) Western blotting analysis of the expression of HMGB1, Beclin-1 and LC3B in lysate extracted from tumor tissues. GAPDH was used as a loading control.
    Figure Legend Snippet: Anti-MM activity of lycorine in MM xenograft mouse models. (A) Changes in tumor volume. Caliper measurement of the tumor diameters were performed very 4 days to estimate the tumor volume using the following formula (a×b 2 )/2, where 'a' and 'b' respectively are the longest and shortest perpendicular diameters of tumor. Data are presented as the mean±SD from five mice. (B) Representative tumor bearing mouse from each treatment group before dissection (top). The mice were euthanized at the treatment end point, tumors were removed and photographed (bottom). (C) During the treatment period, mouse body weight was measured every 4 days. Data are shown as mean±SD. (D) Histochemical examinations of mouse tissue section. Tissue sections from the tumor were fixed and stained with HE and TUNEL to examine the tumor cell morphology. The magnifications are 40×. (E) Western blotting analysis of the expression of HMGB1, Beclin-1 and LC3B in lysate extracted from tumor tissues. GAPDH was used as a loading control.

    Techniques Used: Activity Assay, Mouse Assay, Dissection, Staining, TUNEL Assay, Western Blot, Expressing

    23) Product Images from "HEPARIN-BINDING EGF-LIKE GROWTH FACTOR IS A POTENT DILATOR OF TERMINAL MESENTERIC ARTERIOLES"

    Article Title: HEPARIN-BINDING EGF-LIKE GROWTH FACTOR IS A POTENT DILATOR OF TERMINAL MESENTERIC ARTERIOLES

    Journal: Microvascular research

    doi: 10.1016/j.mvr.2009.04.006

    Effect of HB-EGF on intracellular calcium levels in HIMEC. The fluorescent ratio (405 nm/485 nm) was used as an indicator of the relative concentration of intracellular calcium. Data are expressed as the ratio of 405/485 nm over time. The time of addition
    Figure Legend Snippet: Effect of HB-EGF on intracellular calcium levels in HIMEC. The fluorescent ratio (405 nm/485 nm) was used as an indicator of the relative concentration of intracellular calcium. Data are expressed as the ratio of 405/485 nm over time. The time of addition

    Techniques Used: Concentration Assay

    ET B receptor mRNA expression and protein detection in HIMEC. A. Effect of HB-EGF and 2-APB on ET B mRNA expression in HIMEC. ET B mRNA was quantified using real-time RT-PCR after treatment of the cells as indicated. ET B mRNA expression ± SEM is
    Figure Legend Snippet: ET B receptor mRNA expression and protein detection in HIMEC. A. Effect of HB-EGF and 2-APB on ET B mRNA expression in HIMEC. ET B mRNA was quantified using real-time RT-PCR after treatment of the cells as indicated. ET B mRNA expression ± SEM is

    Techniques Used: Expressing, Quantitative RT-PCR

    24) Product Images from "ZNF503/Zpo2 drives aggressive breast cancer progression by down-regulation of GATA3 expression"

    Article Title: ZNF503/Zpo2 drives aggressive breast cancer progression by down-regulation of GATA3 expression

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

    doi: 10.1073/pnas.1701690114

    ChIP analysis indicating that ZPO2 and ZBTB32 occupy the Gata3 promoter. EpH4.9 cells were grown in 150-mm culture dishes and cotransfected with full-length Zpo2 and Myc-tagged ZBTB32 (generous gifts from Dr. I.-Cheng Ho, Harvard Medical School) constructs via FuGENE6 transfection reagent (Promega). ChIP analysis was performed using the ChIP-IT High-Sensitivity Kit (Active Motif) in accordance with the manufacturer’s protocol. qPCR for ChIP samples was performed using Gata3 promoter sequence primers listed in the text. ChIP analysis indicated that both ZPO2 and ZBTB32 occupy the promoter sequence of GATA3.
    Figure Legend Snippet: ChIP analysis indicating that ZPO2 and ZBTB32 occupy the Gata3 promoter. EpH4.9 cells were grown in 150-mm culture dishes and cotransfected with full-length Zpo2 and Myc-tagged ZBTB32 (generous gifts from Dr. I.-Cheng Ho, Harvard Medical School) constructs via FuGENE6 transfection reagent (Promega). ChIP analysis was performed using the ChIP-IT High-Sensitivity Kit (Active Motif) in accordance with the manufacturer’s protocol. qPCR for ChIP samples was performed using Gata3 promoter sequence primers listed in the text. ChIP analysis indicated that both ZPO2 and ZBTB32 occupy the promoter sequence of GATA3.

    Techniques Used: Chromatin Immunoprecipitation, Construct, Transfection, Real-time Polymerase Chain Reaction, Sequencing

    Zpo1 -overexpressing EpH4.9 cells were placed in 3D Matrigel cultures. Zpo1 induced an aggressive phenotype. Zpo1-overexpressing cells infiltrated through the Matrigel matrix. Knockdown of Zbtb32 did not alter Zpo1 -induced cellular invasion. (Scale bar: 150 µm.)
    Figure Legend Snippet: Zpo1 -overexpressing EpH4.9 cells were placed in 3D Matrigel cultures. Zpo1 induced an aggressive phenotype. Zpo1-overexpressing cells infiltrated through the Matrigel matrix. Knockdown of Zbtb32 did not alter Zpo1 -induced cellular invasion. (Scale bar: 150 µm.)

    Techniques Used:

    Co-IP experiment indicating endogenous ZPO2 and ZBTB32 interaction in EpH4.9 cells. For each experiment, 10 mg of total protein was pulled down via anti-ZPO2 or anti-ZBTB32 antibody. The samples were examined via Western blot analysis using anti-ZPO2 or anti-ZBTB32 antibody. Bidirectional co-IP indicates that ZPO2 and ZBTB32 form a complex in EpH4.9 mammary cells.
    Figure Legend Snippet: Co-IP experiment indicating endogenous ZPO2 and ZBTB32 interaction in EpH4.9 cells. For each experiment, 10 mg of total protein was pulled down via anti-ZPO2 or anti-ZBTB32 antibody. The samples were examined via Western blot analysis using anti-ZPO2 or anti-ZBTB32 antibody. Bidirectional co-IP indicates that ZPO2 and ZBTB32 form a complex in EpH4.9 mammary cells.

    Techniques Used: Co-Immunoprecipitation Assay, Western Blot

    Analysis of Zpo2 and ZBTB32 interaction. ( A ) Co-IP experiment indicating Zpo2 and ZBTB32 interaction. EpH4.9 cells were cotransfected with V5-tagged Zpo2 and Myc-tagged Zbtb32 constructs. Pull-down was performed with control IgG, anti-Myc (ZBTB32), or anti-V5 tag (ZPO2) antibodies. Western blot analysis for the presence of Zpo2 or ZBTB32 was performed with anti-V5 tag or anti-Myc antibodies, respectively. ( B ) ChIP analysis indicating the presence of Zpo2 and ZBTB32 on the Gata3 promoter. qRT-PCR analysis was performed using primers specific to the Gata3 promoter. n = 4. ( C ) qRT-PCR analysis for Gata3 expression in EpH4.9 control or EpH4.9 Zpo2 -overexpressing cells in the presence or absence of ZBTB32. Inhibition of Zbtb32 restored Gata3 levels. ( D ) 3D Matrigel culture assay of control or Zpo2 -overexpressing EpH4.9 cells. Inhibition of Zbtb32 interferes with cellular invasion mediated by Zpo2. (Scale bar: 150 µm.)
    Figure Legend Snippet: Analysis of Zpo2 and ZBTB32 interaction. ( A ) Co-IP experiment indicating Zpo2 and ZBTB32 interaction. EpH4.9 cells were cotransfected with V5-tagged Zpo2 and Myc-tagged Zbtb32 constructs. Pull-down was performed with control IgG, anti-Myc (ZBTB32), or anti-V5 tag (ZPO2) antibodies. Western blot analysis for the presence of Zpo2 or ZBTB32 was performed with anti-V5 tag or anti-Myc antibodies, respectively. ( B ) ChIP analysis indicating the presence of Zpo2 and ZBTB32 on the Gata3 promoter. qRT-PCR analysis was performed using primers specific to the Gata3 promoter. n = 4. ( C ) qRT-PCR analysis for Gata3 expression in EpH4.9 control or EpH4.9 Zpo2 -overexpressing cells in the presence or absence of ZBTB32. Inhibition of Zbtb32 restored Gata3 levels. ( D ) 3D Matrigel culture assay of control or Zpo2 -overexpressing EpH4.9 cells. Inhibition of Zbtb32 interferes with cellular invasion mediated by Zpo2. (Scale bar: 150 µm.)

    Techniques Used: Co-Immunoprecipitation Assay, Construct, Western Blot, Chromatin Immunoprecipitation, Quantitative RT-PCR, Expressing, Inhibition

    25) Product Images from "Early alteration of epigenetic-related transcription in Huntington’s disease mouse models"

    Article Title: Early alteration of epigenetic-related transcription in Huntington’s disease mouse models

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-28185-4

    Profiling of histone posttranslational modifications in the early hippocampal HD transcriptome. ( a ) Distribution across the juvenile R6/1 transcriptome of the genes associated with reported histone modifications (panH2Bac, H3K9/14ac, H4K12ac, H3K4me3 and H3K27me in 3–5-month-old mice 46 ) at TSS (overlapping with the transcription start sites or comprising the whole gene). ( b ) From a previous study reporting changes in 10-week-old N171-82Q hippocampi 26 , we grouped three types of genes: those showing both gene downregulation and histone H3 deacetylation at the TSS (Expr H3ac), those showing only downregulation (Only Expr), and those showing only deacetylation (Only H3ac). On the left, fold change in differential expression was averaged within these subsets and represented as the mean ± s.d. for the indicated HD models and ages. * P
    Figure Legend Snippet: Profiling of histone posttranslational modifications in the early hippocampal HD transcriptome. ( a ) Distribution across the juvenile R6/1 transcriptome of the genes associated with reported histone modifications (panH2Bac, H3K9/14ac, H4K12ac, H3K4me3 and H3K27me in 3–5-month-old mice 46 ) at TSS (overlapping with the transcription start sites or comprising the whole gene). ( b ) From a previous study reporting changes in 10-week-old N171-82Q hippocampi 26 , we grouped three types of genes: those showing both gene downregulation and histone H3 deacetylation at the TSS (Expr H3ac), those showing only downregulation (Only Expr), and those showing only deacetylation (Only H3ac). On the left, fold change in differential expression was averaged within these subsets and represented as the mean ± s.d. for the indicated HD models and ages. * P

    Techniques Used: Mouse Assay, Expressing

    26) Product Images from "A Role of Central NELL2 in the Regulation of Feeding Behavior in Rats"

    Article Title: A Role of Central NELL2 in the Regulation of Feeding Behavior in Rats

    Journal: Molecules and Cells

    doi: 10.14348/molcells.2017.2278

    NELL2 is expressed in both POMC and NPY neuronal populations in the ARC without affecting their gene expression (A) A cytoplasmic distribution of POMC-ir developed by a green fluorescence was found mostly in the ventromedial part of the ARC through the rostral-to-caudal axis. (B, E) NELL2-ir was developed by a red fluorescence. (C) All POMC immuno-positive cells were colocalized with the NELL2-ir. (D) NPY-ir developed by a green fluorescence showed a clear cytoplasmic expression along the rostral-to-caudal axis. (F) All NPY-ir was found within the NELL2 immuno-positive cells in this area. Single NELL2-ir was also found in the experiment. Scale bar = 50 μm. (G, H) NPY and POMC transcripts in the MBH were measured by a relative RT-PCR in the NELL2 AS ODN (AS, n = 4) and NELL2 SCR ODN groups (SCR, n = 5), and then compared between the groups. The transcriptional level of NPY (G) and POMC (H) was not different between groups.
    Figure Legend Snippet: NELL2 is expressed in both POMC and NPY neuronal populations in the ARC without affecting their gene expression (A) A cytoplasmic distribution of POMC-ir developed by a green fluorescence was found mostly in the ventromedial part of the ARC through the rostral-to-caudal axis. (B, E) NELL2-ir was developed by a red fluorescence. (C) All POMC immuno-positive cells were colocalized with the NELL2-ir. (D) NPY-ir developed by a green fluorescence showed a clear cytoplasmic expression along the rostral-to-caudal axis. (F) All NPY-ir was found within the NELL2 immuno-positive cells in this area. Single NELL2-ir was also found in the experiment. Scale bar = 50 μm. (G, H) NPY and POMC transcripts in the MBH were measured by a relative RT-PCR in the NELL2 AS ODN (AS, n = 4) and NELL2 SCR ODN groups (SCR, n = 5), and then compared between the groups. The transcriptional level of NPY (G) and POMC (H) was not different between groups.

    Techniques Used: Expressing, Fluorescence, Reverse Transcription Polymerase Chain Reaction

    Fasting promoted NELL2 synthesis in the hypothalamus RT-PCR was performed using the hypothalamic total RNA to detect a transcriptional level of NELL2 from control fed (n = 4) and 48-h fasted animals (n = 4). The PCR products for NELL2 were normalized by GAPDH. Hypothalamic NELL2 synthesis was significantly increased in the fasted animals compared with that in control (P
    Figure Legend Snippet: Fasting promoted NELL2 synthesis in the hypothalamus RT-PCR was performed using the hypothalamic total RNA to detect a transcriptional level of NELL2 from control fed (n = 4) and 48-h fasted animals (n = 4). The PCR products for NELL2 were normalized by GAPDH. Hypothalamic NELL2 synthesis was significantly increased in the fasted animals compared with that in control (P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    27) Product Images from "1-(2-Hydroxy-5-methylphenyl)-3-phenyl-1,3-propanedione Induces G1 Cell Cycle Arrest and Autophagy in HeLa Cervical Cancer Cells"

    Article Title: 1-(2-Hydroxy-5-methylphenyl)-3-phenyl-1,3-propanedione Induces G1 Cell Cycle Arrest and Autophagy in HeLa Cervical Cancer Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms17081274

    Effects of HMDB on the expression of G1-related cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors (CKIs). ( a ) Relative protein expression levels of cyclin D1/D3/E, and CDK4/6/2 expressed in the G1 phase; ( b ) the total and phosphorylated forms of retinoblastoma (Rb) with specific antibodies for each; and ( c ) the change in the protein expression levels of CKIs (p15, p16, p21, and p27). HeLa cells were exposed to 40 μM HMDB for the indicated times. Then, cellular extracts were harvested and the protein levels were visualized by Western blotting using antibodies against G1 cell cycle regulators as indicated. The β-actin acts as an internal control for evaluating protein loading; and ( d ) the changes in mRNA expression levels of CKIs, including p15, p16, p21, and p27, by HMDB. The relative amounts of target mRNA, collected from HMDB-treated HeLa cells, were determined by qRT-PCR for the indicated time. All of the results that come from independent experiments three times are expressed as mean ± SE. The relative amounts of protein levels on the Western blots were quantitated with a computerized densitometer (ImageQuant LAS4000 Digital System, GE Healthcare, Uppsala, Sweden) compared to the control group. Values were statistically significant for * p
    Figure Legend Snippet: Effects of HMDB on the expression of G1-related cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors (CKIs). ( a ) Relative protein expression levels of cyclin D1/D3/E, and CDK4/6/2 expressed in the G1 phase; ( b ) the total and phosphorylated forms of retinoblastoma (Rb) with specific antibodies for each; and ( c ) the change in the protein expression levels of CKIs (p15, p16, p21, and p27). HeLa cells were exposed to 40 μM HMDB for the indicated times. Then, cellular extracts were harvested and the protein levels were visualized by Western blotting using antibodies against G1 cell cycle regulators as indicated. The β-actin acts as an internal control for evaluating protein loading; and ( d ) the changes in mRNA expression levels of CKIs, including p15, p16, p21, and p27, by HMDB. The relative amounts of target mRNA, collected from HMDB-treated HeLa cells, were determined by qRT-PCR for the indicated time. All of the results that come from independent experiments three times are expressed as mean ± SE. The relative amounts of protein levels on the Western blots were quantitated with a computerized densitometer (ImageQuant LAS4000 Digital System, GE Healthcare, Uppsala, Sweden) compared to the control group. Values were statistically significant for * p

    Techniques Used: Expressing, Western Blot, Quantitative RT-PCR

    HMDB induced autophagy and apoptosis in HeLa cells. ( a ) HeLa cells were treated with 40 μM HMDB for the indicated times, fixed and incubated with rabbit anti-LC3-II primary antibody. After incubation with Alexa Fluor 488 phalloidin (green) for conjugated anti-rabbit secondary antibodies, immune-labeled cells were monitored by microscopy; ( b ) HeLa cells were treated with 40 μM HMDB and then the protein expression levels of LC3-II, Beclin-1, and p62 were determined by Western blotting for the indicated times; ( c ) the cells were treated with 40 μM HMDB for different times. Cells were harvested and lysed for the detection of the indicated protein expression by Western blotting; ( d ) the cells were treated with 40 μM HMDB for the indicated times, and then the apoptotic cells were examined by TUNEL assay; and ( e ) the apoptosis-related proteins, cleaved caspase-3, and poly (ADP-ribose) polymerase (PARP), were assessed using Western blotting. The densities of the band on the Western blots from three independent experiments were calculated using a computerized densitometer (ImageQuant LAS4000 Digital System, GE Healthcare, Uppsala, Sweden). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI).
    Figure Legend Snippet: HMDB induced autophagy and apoptosis in HeLa cells. ( a ) HeLa cells were treated with 40 μM HMDB for the indicated times, fixed and incubated with rabbit anti-LC3-II primary antibody. After incubation with Alexa Fluor 488 phalloidin (green) for conjugated anti-rabbit secondary antibodies, immune-labeled cells were monitored by microscopy; ( b ) HeLa cells were treated with 40 μM HMDB and then the protein expression levels of LC3-II, Beclin-1, and p62 were determined by Western blotting for the indicated times; ( c ) the cells were treated with 40 μM HMDB for different times. Cells were harvested and lysed for the detection of the indicated protein expression by Western blotting; ( d ) the cells were treated with 40 μM HMDB for the indicated times, and then the apoptotic cells were examined by TUNEL assay; and ( e ) the apoptosis-related proteins, cleaved caspase-3, and poly (ADP-ribose) polymerase (PARP), were assessed using Western blotting. The densities of the band on the Western blots from three independent experiments were calculated using a computerized densitometer (ImageQuant LAS4000 Digital System, GE Healthcare, Uppsala, Sweden). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI).

    Techniques Used: Incubation, Labeling, Microscopy, Expressing, Western Blot, TUNEL Assay, Staining

    HMDB increased the number of massive vacuoles with acid content and the accumulation of autolysosomes in HeLa cells. ( a ) The cells were treated with 40 μM HMDB for the indicated times. Morphological changes and representative photographs of HeLa cells after neutral red staining in response to HMDB were observed by light contrast microscopy; ( b ) microphotograph of cells stained with monodansylcadaverine (MDC) and acridine orange (AO). Scale bar, 50 µm.
    Figure Legend Snippet: HMDB increased the number of massive vacuoles with acid content and the accumulation of autolysosomes in HeLa cells. ( a ) The cells were treated with 40 μM HMDB for the indicated times. Morphological changes and representative photographs of HeLa cells after neutral red staining in response to HMDB were observed by light contrast microscopy; ( b ) microphotograph of cells stained with monodansylcadaverine (MDC) and acridine orange (AO). Scale bar, 50 µm.

    Techniques Used: Staining, Microscopy

    HMDB inhibited proliferation of HeLa cells via inducing the G1 cell cycle arrest. ( a ) The chemical structure of HMDB; and ( b ) the effect of HMDB on cell viability of HeLa cells. Cells were treated with a variety of dosages of HMDB for 0–24 h or ( c ) with 40 µM HMDB for different time periods. Cell survival was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and trypan blue exclusion assays, respectively. The protein levels of PCNA were determined by Western blotting; and ( d ) a histogram of the cell cycle distribution. HeLa cells were treated with 40 μM HMDB for 0, 6, 12, and 24 h. Cell distribution at G1, S and G2/M phase was determined using flow cytometry. All of the data resulted from repeating independent experiments three times and results are expressed as mean ± SE. Values were statistically significant (versus HMDB treatment) for * p
    Figure Legend Snippet: HMDB inhibited proliferation of HeLa cells via inducing the G1 cell cycle arrest. ( a ) The chemical structure of HMDB; and ( b ) the effect of HMDB on cell viability of HeLa cells. Cells were treated with a variety of dosages of HMDB for 0–24 h or ( c ) with 40 µM HMDB for different time periods. Cell survival was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and trypan blue exclusion assays, respectively. The protein levels of PCNA were determined by Western blotting; and ( d ) a histogram of the cell cycle distribution. HeLa cells were treated with 40 μM HMDB for 0, 6, 12, and 24 h. Cell distribution at G1, S and G2/M phase was determined using flow cytometry. All of the data resulted from repeating independent experiments three times and results are expressed as mean ± SE. Values were statistically significant (versus HMDB treatment) for * p

    Techniques Used: MTT Assay, Western Blot, Flow Cytometry, Cytometry

    The proposed signal pathway activated by HMDB in HeLa cervical cancer cells. Red arrows represent increased protein expression by HMDB, while black arrows indicate a decrease.
    Figure Legend Snippet: The proposed signal pathway activated by HMDB in HeLa cervical cancer cells. Red arrows represent increased protein expression by HMDB, while black arrows indicate a decrease.

    Techniques Used: Expressing

    Modulation of class III PI-3K and AMPK/Akt/mTOR signaling was linked to HMDB-induced cell cycle arrest and autophagy in HeLa cells. ( a ) The cells were pretreated with 1 mM autophagy inhibitor, 3-methyladenine, followed by 40 μM HMDB treatment for 24 h. The expression of the indicated proteins was determined by Western blotting; ( b ) HeLa cells were incubated in the presence of 40 μM HMDB for various time points. Cell extracts were harvested for determining the indicated protein expression by Western blotting. The densities of the band on the Western blots from three independent experiments were calculated using a computerized densitometer (ImageQuant LAS4000 Digital System).
    Figure Legend Snippet: Modulation of class III PI-3K and AMPK/Akt/mTOR signaling was linked to HMDB-induced cell cycle arrest and autophagy in HeLa cells. ( a ) The cells were pretreated with 1 mM autophagy inhibitor, 3-methyladenine, followed by 40 μM HMDB treatment for 24 h. The expression of the indicated proteins was determined by Western blotting; ( b ) HeLa cells were incubated in the presence of 40 μM HMDB for various time points. Cell extracts were harvested for determining the indicated protein expression by Western blotting. The densities of the band on the Western blots from three independent experiments were calculated using a computerized densitometer (ImageQuant LAS4000 Digital System).

    Techniques Used: Expressing, Western Blot, Incubation

    28) Product Images from "Potential role of an antimicrobial peptide, KLK in inhibiting lipopolysaccharide-induced macrophage inflammation"

    Article Title: Potential role of an antimicrobial peptide, KLK in inhibiting lipopolysaccharide-induced macrophage inflammation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0183852

    Effects of KLK peptide on iNOS, COX-2, IL-1β and TNF-α mRNA expression in LPS-stimulated RAW 264.7 macrophages. Cells were stimulated with LPS (1 μg/mL) in the presence or absence of different concentrations of KLK peptide for 18 h. Total mRNA was isolated and the mRNA expression of iNOS, COX-2, IL-1β and TNF-α was examined by RT-PCR. Data are expressed as means ± SD of three independent experiments. ##, P
    Figure Legend Snippet: Effects of KLK peptide on iNOS, COX-2, IL-1β and TNF-α mRNA expression in LPS-stimulated RAW 264.7 macrophages. Cells were stimulated with LPS (1 μg/mL) in the presence or absence of different concentrations of KLK peptide for 18 h. Total mRNA was isolated and the mRNA expression of iNOS, COX-2, IL-1β and TNF-α was examined by RT-PCR. Data are expressed as means ± SD of three independent experiments. ##, P

    Techniques Used: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction

    29) Product Images from "Exoribonuclease-Resistant RNAs Exist within both Coding and Noncoding Subgenomic RNAs"

    Article Title: Exoribonuclease-Resistant RNAs Exist within both Coding and Noncoding Subgenomic RNAs

    Journal: mBio

    doi: 10.1128/mBio.02461-18

    Widespread occurrence of Xrn1-resistant RNAs among plant viruses. (A) Consensus sequence and secondary structure of xrRNA D based on a comparative sequence alignment of 47 sequences of viruses belonging to the Tombusviridae and Luteoviridae families (shown in Fig. S1 in the supplemental material). Y = pyrimidine; R = purine. Non-Watson-Crick base pairs are shown using the Leontis-Westhof nomenclature ( 49 ). The numbering is that of the crystal structure of the SCNMV xrRNA ( 26 ). (B) Phylogenetic relationship between various plant viruses, based on the RNA-dependent RNA polymerase amino acid sequence ( 31 ). The viruses and corresponding genera in which we identified xrRNA D structures are marked by a star. Numbers at the nodes refer to bootstrap values as percentages obtained from 2,000 replications, shown only for branches supported by more than 40% of the data. Branch lengths are proportional to the number of changes. Further analysis will likely reveal xrRNA D elements in more of these viruses with additional sequence and structural variation.
    Figure Legend Snippet: Widespread occurrence of Xrn1-resistant RNAs among plant viruses. (A) Consensus sequence and secondary structure of xrRNA D based on a comparative sequence alignment of 47 sequences of viruses belonging to the Tombusviridae and Luteoviridae families (shown in Fig. S1 in the supplemental material). Y = pyrimidine; R = purine. Non-Watson-Crick base pairs are shown using the Leontis-Westhof nomenclature ( 49 ). The numbering is that of the crystal structure of the SCNMV xrRNA ( 26 ). (B) Phylogenetic relationship between various plant viruses, based on the RNA-dependent RNA polymerase amino acid sequence ( 31 ). The viruses and corresponding genera in which we identified xrRNA D structures are marked by a star. Numbers at the nodes refer to bootstrap values as percentages obtained from 2,000 replications, shown only for branches supported by more than 40% of the data. Branch lengths are proportional to the number of changes. Further analysis will likely reveal xrRNA D elements in more of these viruses with additional sequence and structural variation.

    Techniques Used: Sequencing

    Biochemical characterization of representative plant virus xrRNA D elements. (A) In vitro Xrn1 resistance assay of xrRNA D from various plant RNA viruses ( Table 1 ). The xrRNA from RCNMV was included as a positive control. Arrows indicate the size of full-length RNAs and Xrn1-resistant degradation products. (B) Classification of viruses used in the experiments represented in panel A ( Table 1 ). (C to E) In vitro Xrn1 resistance assay of wild-type (WT) and pseudoknot (PK) mutant versions of MCMV (C), PLRV (D), and HuPLV1 (E) xrRNAs. (F to H) Reverse transcription (RT) mapping of the Xrn1 stop site. Data represent distributions of RT products of Xrn1-resistant fragments of MCMV (F), PLRV (G), and HuPLV1 (H) degradation fragments. Experimentally validated stop sites are indicated on the secondary structure diagrams for all tested xrRNA D shown in Fig. S2 .
    Figure Legend Snippet: Biochemical characterization of representative plant virus xrRNA D elements. (A) In vitro Xrn1 resistance assay of xrRNA D from various plant RNA viruses ( Table 1 ). The xrRNA from RCNMV was included as a positive control. Arrows indicate the size of full-length RNAs and Xrn1-resistant degradation products. (B) Classification of viruses used in the experiments represented in panel A ( Table 1 ). (C to E) In vitro Xrn1 resistance assay of wild-type (WT) and pseudoknot (PK) mutant versions of MCMV (C), PLRV (D), and HuPLV1 (E) xrRNAs. (F to H) Reverse transcription (RT) mapping of the Xrn1 stop site. Data represent distributions of RT products of Xrn1-resistant fragments of MCMV (F), PLRV (G), and HuPLV1 (H) degradation fragments. Experimentally validated stop sites are indicated on the secondary structure diagrams for all tested xrRNA D shown in Fig. S2 .

    Techniques Used: In Vitro, Positive Control, Mutagenesis

    An expanding repertoire of structured RNAs for blocking exoribonuclease degradation. (Top) xrRNAs adopt a three-dimensional structure that blocks the progression of 5′-to-3′ exoribonucleases such as Xrn1 (gray). In the case of flaviviruses and dianthoviruses, xrRNAs are in the 3′UTR, resulting in accumulating noncoding sgRNAs. (Middle) Secondary structure diagrams of the two classes of xrRNAs from flaviviruses (xrRNA F1 and xrRNA F2 ) ( 15 , 22 , 23 ) and of xrRNA D from dianthoviruses ( 26 ). Secondary structure features are labeled, and nucleotides involved in tertiary interactions are shown in colors connected by dashed lines (pseudoknot shown in blue). Experimentally determined Xrn1 stop sites are indicated. (Bottom) The boxes below each secondary structure contain diagrams reflecting the currently available three-dimensional structures ( 24 – 26 ). The A8-G33 pair is highlighted in the open state of the Sweet clover necrotic mosaic virus (SCNMV) xrRNA (far left).
    Figure Legend Snippet: An expanding repertoire of structured RNAs for blocking exoribonuclease degradation. (Top) xrRNAs adopt a three-dimensional structure that blocks the progression of 5′-to-3′ exoribonucleases such as Xrn1 (gray). In the case of flaviviruses and dianthoviruses, xrRNAs are in the 3′UTR, resulting in accumulating noncoding sgRNAs. (Middle) Secondary structure diagrams of the two classes of xrRNAs from flaviviruses (xrRNA F1 and xrRNA F2 ) ( 15 , 22 , 23 ) and of xrRNA D from dianthoviruses ( 26 ). Secondary structure features are labeled, and nucleotides involved in tertiary interactions are shown in colors connected by dashed lines (pseudoknot shown in blue). Experimentally determined Xrn1 stop sites are indicated. (Bottom) The boxes below each secondary structure contain diagrams reflecting the currently available three-dimensional structures ( 24 – 26 ). The A8-G33 pair is highlighted in the open state of the Sweet clover necrotic mosaic virus (SCNMV) xrRNA (far left).

    Techniques Used: Blocking Assay, Labeling

    30) Product Images from "Polony analysis of gene expression in ES cells and blastocysts"

    Article Title: Polony analysis of gene expression in ES cells and blastocysts

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm1076

    Flow chart of typical experiment. Cells (either 1000 ES cells, single blastocysts or multiple blastocysts) were delivered to a lysis-binding solution containing oligo(dT) 25 Dynabeads®. After cell lysis, mRNA was captured by hybridization with poly(A) tails on the beads and mRNA was reverse-transcribed into cDNA. cDNA was added to non-polymerized polyacrylamide gel mix containing PCR components and deposited in an oval well on a microscope slide. After polymerization of the gel, slides were thermocycled so that cDNA templates gave rise to polonies. Polonies were visualized by hybridization with a labeled gene-specific probe.
    Figure Legend Snippet: Flow chart of typical experiment. Cells (either 1000 ES cells, single blastocysts or multiple blastocysts) were delivered to a lysis-binding solution containing oligo(dT) 25 Dynabeads®. After cell lysis, mRNA was captured by hybridization with poly(A) tails on the beads and mRNA was reverse-transcribed into cDNA. cDNA was added to non-polymerized polyacrylamide gel mix containing PCR components and deposited in an oval well on a microscope slide. After polymerization of the gel, slides were thermocycled so that cDNA templates gave rise to polonies. Polonies were visualized by hybridization with a labeled gene-specific probe.

    Techniques Used: Flow Cytometry, Lysis, Binding Assay, Hybridization, Polymerase Chain Reaction, Microscopy, Labeling

    Polonies and competitive PCR for three genes. ( A ) Polonies per slide and per ES cell equivalent for Oct3, Nanog and Rex1. The average number of polonies and standard deviation of three replicate slides containing 10.4 ES cell equivalents are shown. Calculated number of polonies per cell for each set of slides is indicated. ( B ) Competitive PCR for three genes. DNA competitors with 50-bp deletions were generated for Oct3, Nanog and Rex1. Competitive PCR reactions with the indicated number ES cell cDNA equivalents and varying amount of competitor are shown.
    Figure Legend Snippet: Polonies and competitive PCR for three genes. ( A ) Polonies per slide and per ES cell equivalent for Oct3, Nanog and Rex1. The average number of polonies and standard deviation of three replicate slides containing 10.4 ES cell equivalents are shown. Calculated number of polonies per cell for each set of slides is indicated. ( B ) Competitive PCR for three genes. DNA competitors with 50-bp deletions were generated for Oct3, Nanog and Rex1. Competitive PCR reactions with the indicated number ES cell cDNA equivalents and varying amount of competitor are shown.

    Techniques Used: Polymerase Chain Reaction, Standard Deviation, Generated

    31) Product Images from "Regulation of transferrin receptor-1 mRNA by the interplay between IRE-binding proteins and miR-7/miR-141 in the 3′-IRE stem–loops"

    Article Title: Regulation of transferrin receptor-1 mRNA by the interplay between IRE-binding proteins and miR-7/miR-141 in the 3′-IRE stem–loops

    Journal: RNA

    doi: 10.1261/rna.063941.117

    Blocking endogenous miR-7-5p and miR-141-3p increases TfR1 mRNA. ( A ) EGFR1 mRNA expression (normalized to HPRT1 expression) was measured in SW480 cells transfected with a combination of 20 pmol miR control (miR-Con) or miR-7-5p (miR-7) and 100 pmol antagomiR control (iCon) or antagomiR for miR-7-5p (i7). Means ± SE from three independent experiments are shown. SW480 ( B , C ) or NIH3T3 ( D ) cells were transfected with 200 pmol iCon or 100 pmol each antagomiR for miR-7-5p, miR-141-3p (i7/141 in B from Dharmacon, in C from Qiagen). After 48 h, cells were treated with 250 µM FAC for 2 h for SW480 or 1.5 h for NIH3T3 cells, and TfR1 or EGFR1 mRNA expression was measured by qPCR and normalized to HPRT1 expression. Means ± SD from three to five independent experiments are shown. ( E ) SW480 cells were transfected with a combination of 40 pmol siRNA control (siCon) or siIRP1 and 2 (siIRPs) and 100 pmol of iCon or i7/141. TfR1 mRNA was measured and normalized by HPRT1 mRNA expression. Means ± SD from five independent experiments are shown. ( F ) SW480 cells were treated with 0 or 250 µM FAC for 24 h, then mature miR-7-5p and miR-141-3p expressions were measured. Means ± SE from three independent experiments are shown.
    Figure Legend Snippet: Blocking endogenous miR-7-5p and miR-141-3p increases TfR1 mRNA. ( A ) EGFR1 mRNA expression (normalized to HPRT1 expression) was measured in SW480 cells transfected with a combination of 20 pmol miR control (miR-Con) or miR-7-5p (miR-7) and 100 pmol antagomiR control (iCon) or antagomiR for miR-7-5p (i7). Means ± SE from three independent experiments are shown. SW480 ( B , C ) or NIH3T3 ( D ) cells were transfected with 200 pmol iCon or 100 pmol each antagomiR for miR-7-5p, miR-141-3p (i7/141 in B from Dharmacon, in C from Qiagen). After 48 h, cells were treated with 250 µM FAC for 2 h for SW480 or 1.5 h for NIH3T3 cells, and TfR1 or EGFR1 mRNA expression was measured by qPCR and normalized to HPRT1 expression. Means ± SD from three to five independent experiments are shown. ( E ) SW480 cells were transfected with a combination of 40 pmol siRNA control (siCon) or siIRP1 and 2 (siIRPs) and 100 pmol of iCon or i7/141. TfR1 mRNA was measured and normalized by HPRT1 mRNA expression. Means ± SD from five independent experiments are shown. ( F ) SW480 cells were treated with 0 or 250 µM FAC for 24 h, then mature miR-7-5p and miR-141-3p expressions were measured. Means ± SE from three independent experiments are shown.

    Techniques Used: Blocking Assay, Expressing, Transfection, Real-time Polymerase Chain Reaction

    32) Product Images from "USP15 targets ALK3/BMPR1A for deubiquitylation to enhance bone morphogenetic protein signalling"

    Article Title: USP15 targets ALK3/BMPR1A for deubiquitylation to enhance bone morphogenetic protein signalling

    Journal: Open Biology

    doi: 10.1098/rsob.140065

    ALK3 undergoes proteasomal degradation. ( a ) HEK293 cells transfected with untagged ALK3 were treated with or without 20 μM cycloheximide for 24 h prior to lysis. Cells were treated with DMSO control, 100 nM bafilomycin A1 (to inhibit vacuolar-type H+ ATPase) or 10 μM bortezomib (to inhibit the proteasome) for 3 h prior to lysis. Extracts were resolved by SDS-PAGE and subjected to immunoblotting with the indicated antibodies. ( b ) HEK293 cells were transiently transfected with siFoxO4 or siUSP15-3 . Cells were serum-starved overnight, treated with or without 10 μM bortezomib for 3 h and then stimulated with or without 6.25 ng ml −1 BMP for 1 h prior to lysis. Extracts were resolved by SDS-PAGE and subjected to immunoblotting with antibodies against endogenous USP15, pSMAD1 and total SMAD1. ( c ) HEK293 cells were transiently transfected with siFoxO4 (–), siUSP15-3 or siSMAD6 as indicated. Twenty-four hours post siRNA transfection, cells were serum-starved overnight and stimulated with or without 6.25 ng ml −1 BMP for 1 h prior to lysis. Extracts were resolved by SDS-PAGE and subjected to immunoblotting with antibodies against pSMAD1, total SMAD1, USP15 and GAPDH. The SMAD6 knockdown was confirmed by qRT-PCR (electronic supplementary material, figure S7).
    Figure Legend Snippet: ALK3 undergoes proteasomal degradation. ( a ) HEK293 cells transfected with untagged ALK3 were treated with or without 20 μM cycloheximide for 24 h prior to lysis. Cells were treated with DMSO control, 100 nM bafilomycin A1 (to inhibit vacuolar-type H+ ATPase) or 10 μM bortezomib (to inhibit the proteasome) for 3 h prior to lysis. Extracts were resolved by SDS-PAGE and subjected to immunoblotting with the indicated antibodies. ( b ) HEK293 cells were transiently transfected with siFoxO4 or siUSP15-3 . Cells were serum-starved overnight, treated with or without 10 μM bortezomib for 3 h and then stimulated with or without 6.25 ng ml −1 BMP for 1 h prior to lysis. Extracts were resolved by SDS-PAGE and subjected to immunoblotting with antibodies against endogenous USP15, pSMAD1 and total SMAD1. ( c ) HEK293 cells were transiently transfected with siFoxO4 (–), siUSP15-3 or siSMAD6 as indicated. Twenty-four hours post siRNA transfection, cells were serum-starved overnight and stimulated with or without 6.25 ng ml −1 BMP for 1 h prior to lysis. Extracts were resolved by SDS-PAGE and subjected to immunoblotting with antibodies against pSMAD1, total SMAD1, USP15 and GAPDH. The SMAD6 knockdown was confirmed by qRT-PCR (electronic supplementary material, figure S7).

    Techniques Used: Transfection, Lysis, SDS Page, Quantitative RT-PCR

    USP15 impacts osteoblastic differentiation in C2C12 myoblasts and modulates BMP signalling in Xenopus embryogenesis. ( a ) Mouse myoblast cell line C2C12 were transfected with siRNAs targeting mouse FoxO4 or USP15. Cells were serum-starved overnight and treated with or without BMP for 1 h prior to lysis. Extracts were resolved by SDS-PAGE and immunoblotted with antibodies against USP15, pSMAD1, total SMAD1 and GAPDH. ( b ) C2C12 cells transfected with mouse siFoxO4 or mouse siUSP15 were grown for up to 4 days in the presence of BMP. Cells were lysed and the alkaline phosphatase activity measured using a fluorescence plate reader. Data are represented as mean of three biological replicates and error bars indicate s.d. Representative extracts were resolved by SDS-PAGE and subjected to immunoblotting with antibodies against USP15 and GAPDH. ( c ) Xenopus embryos were injected with 80 ng of either xUSP15- (xUSP15-MO) or control-MO morpholinos at the one-cell stage and then collected at the indicated stages. Lysates were resolved by SDS-PAGE and immunoblotted with antibodies against pSMAD1 and α-tubulin. ( d ) qRT-PCR analysis of xVENT1 mRNA expression. Embryos were injected with 80 ng of either USP15-MO or control-MO at the one-cell stage and then animal caps were cut at stage 8.5. The animal caps were collected at the equivalent embryo stage of 10.5 and processed for qRT-PCR.
    Figure Legend Snippet: USP15 impacts osteoblastic differentiation in C2C12 myoblasts and modulates BMP signalling in Xenopus embryogenesis. ( a ) Mouse myoblast cell line C2C12 were transfected with siRNAs targeting mouse FoxO4 or USP15. Cells were serum-starved overnight and treated with or without BMP for 1 h prior to lysis. Extracts were resolved by SDS-PAGE and immunoblotted with antibodies against USP15, pSMAD1, total SMAD1 and GAPDH. ( b ) C2C12 cells transfected with mouse siFoxO4 or mouse siUSP15 were grown for up to 4 days in the presence of BMP. Cells were lysed and the alkaline phosphatase activity measured using a fluorescence plate reader. Data are represented as mean of three biological replicates and error bars indicate s.d. Representative extracts were resolved by SDS-PAGE and subjected to immunoblotting with antibodies against USP15 and GAPDH. ( c ) Xenopus embryos were injected with 80 ng of either xUSP15- (xUSP15-MO) or control-MO morpholinos at the one-cell stage and then collected at the indicated stages. Lysates were resolved by SDS-PAGE and immunoblotted with antibodies against pSMAD1 and α-tubulin. ( d ) qRT-PCR analysis of xVENT1 mRNA expression. Embryos were injected with 80 ng of either USP15-MO or control-MO at the one-cell stage and then animal caps were cut at stage 8.5. The animal caps were collected at the equivalent embryo stage of 10.5 and processed for qRT-PCR.

    Techniques Used: Transfection, Lysis, SDS Page, Activity Assay, Fluorescence, Injection, Quantitative RT-PCR, Expressing

    Depletion of USP15 inhibits BMP signalling. ( a ) HEK293 cells were transiently transfected with three individual siRNAs targeting USP15, serum-starved overnight and stimulated with 6.25 ng ml −1 BMP for 1 h prior to lysis. Extracts were resolved by SDS-PAGE and subjected to immunoblotting with antibodies against endogenous USP15, pSMAD1, SMAD1 and GAPDH. ( b ) As in ( a ), except that siUSP15-3 was used to knockdown endogenous USP15 expression in HeLa cells. ( c ) As in ( b ) except that U2OS cells were used. ( d ) HEK293 cells were transiently transfected with siUSP15-3. Cells were serum-starved overnight and stimulated with 6.25 ng ml −1 BMP for 1 h. Cells were then washed and harvested 2 h later. The expression of USP15 and the BMP-target gene ID1 were assessed by qRT-PCR. Results are average of six biological replicates. The error bars indicate s.d. ( e ) As in ( d ), except that HEK293 cells were transfected with siUSP11. The expression of USP11 and ID1 were assessed by qRT-PCR. Results are average of three biological replicates. The error bars indicate s.d.
    Figure Legend Snippet: Depletion of USP15 inhibits BMP signalling. ( a ) HEK293 cells were transiently transfected with three individual siRNAs targeting USP15, serum-starved overnight and stimulated with 6.25 ng ml −1 BMP for 1 h prior to lysis. Extracts were resolved by SDS-PAGE and subjected to immunoblotting with antibodies against endogenous USP15, pSMAD1, SMAD1 and GAPDH. ( b ) As in ( a ), except that siUSP15-3 was used to knockdown endogenous USP15 expression in HeLa cells. ( c ) As in ( b ) except that U2OS cells were used. ( d ) HEK293 cells were transiently transfected with siUSP15-3. Cells were serum-starved overnight and stimulated with 6.25 ng ml −1 BMP for 1 h. Cells were then washed and harvested 2 h later. The expression of USP15 and the BMP-target gene ID1 were assessed by qRT-PCR. Results are average of six biological replicates. The error bars indicate s.d. ( e ) As in ( d ), except that HEK293 cells were transfected with siUSP11. The expression of USP11 and ID1 were assessed by qRT-PCR. Results are average of three biological replicates. The error bars indicate s.d.

    Techniques Used: Transfection, Lysis, SDS Page, Expressing, Quantitative RT-PCR

    33) Product Images from "Negative Feedback and Transcriptional Overshooting in a Regulatory Network for Horizontal Gene Transfer"

    Article Title: Negative Feedback and Transcriptional Overshooting in a Regulatory Network for Horizontal Gene Transfer

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1004171

    Transcriptional units in plasmid R388. ( A ) Statistics of the 66 primer pairs used to measure transcriptional levels in plasmid R388 (Upper right) Histogram showing the efficiency (calculated as indicated in materials and methods ) of the primer pairs. (Upper right) Histogram showing the Ct obtained in qPCR amplifications from plasmid cDNA. (Lower left) Histogram showing the Ct obtained in qPCR amplifications from purified plasmid DNA. (Lower right) Scatter plot showing the relationship between the promoter activity (obtained from figure 1 , in GFP/OD unit on the y axis) and the mRNA levels measured by RT-qPCR (ΔCt, x axis). ( B ) Transcriptional landscape of plasmid R388. The graph shows the relative abundance of mRNA, indicated as ΔCt = CtcDNA-CtDNA) along the plasmid genome. Each unit in the y axis corresponds to a 2 fold increase in mRNA. Peaks correspond to highly transcribed regions and valleys correspond to non-transcribed regions. The highlighted blue lines indicate the overlapping of the transcriptional units and the plasmid promoters identified in figure 1 .
    Figure Legend Snippet: Transcriptional units in plasmid R388. ( A ) Statistics of the 66 primer pairs used to measure transcriptional levels in plasmid R388 (Upper right) Histogram showing the efficiency (calculated as indicated in materials and methods ) of the primer pairs. (Upper right) Histogram showing the Ct obtained in qPCR amplifications from plasmid cDNA. (Lower left) Histogram showing the Ct obtained in qPCR amplifications from purified plasmid DNA. (Lower right) Scatter plot showing the relationship between the promoter activity (obtained from figure 1 , in GFP/OD unit on the y axis) and the mRNA levels measured by RT-qPCR (ΔCt, x axis). ( B ) Transcriptional landscape of plasmid R388. The graph shows the relative abundance of mRNA, indicated as ΔCt = CtcDNA-CtDNA) along the plasmid genome. Each unit in the y axis corresponds to a 2 fold increase in mRNA. Peaks correspond to highly transcribed regions and valleys correspond to non-transcribed regions. The highlighted blue lines indicate the overlapping of the transcriptional units and the plasmid promoters identified in figure 1 .

    Techniques Used: Plasmid Preparation, Real-time Polymerase Chain Reaction, Purification, Activity Assay, Quantitative RT-PCR

    34) Product Images from "CHD4 Has Oncogenic Functions in Initiating and Maintaining Epigenetic Suppression of Multiple Tumor Suppressor Genes"

    Article Title: CHD4 Has Oncogenic Functions in Initiating and Maintaining Epigenetic Suppression of Multiple Tumor Suppressor Genes

    Journal: Cancer cell

    doi: 10.1016/j.ccell.2017.04.005

    The Recruitment of CHD4 to Oxidative DNA Damage Sites Depends on OGG1 (A) CoIPs of lysates from SW480 cells untreated or treated with 2 mM H 2 O 2 for 30 min were performed with the indicated antibodies. (B) Purified OGG1 and Flag-CHD4 were incubated with antibodies against Flag or OGG1 in IP buffer. The immunoprecipitated samples were detected by western blot analyses using the antibodies indicated. (C) After SW480 OGG1 KO cells were transfected with pCMV-Taq or pCMV-OGG1 for 48 hr, the cells were untreated or treated with 2mMH 2 O 2 for 30 min. Whole-cell extracts and the tight chromatin fractions were analyzed by immunoblotting with the indicated antibodies. (D) Whole-cell extracts and the tight chromatin fractions from SW480 CHD4 KD cells untreated (Un) or treated with 2 mM H 2 O 2 for 30 min were analyzed by immunoblotting as in (C). (E) Purified OGG1 and Flag-CHD4 were incubated with antibodies against Flag or OGG1 in IP buffer with or without 8-OHdG oligonucleotide. The immuno-precipitated samples were detected by western blot analyses using the antibodies indicated. (F) Biotin labeled 8-OHdG oligonucleotide incubated with OGG1 and Flag-CHD4 was pulled down using streptavidin beads. Bound proteins were eluted and analyzed by immunoblotting with the indicated antibodies. (G) SW480 OGG1 KO cells were untreated or treated with 2mMH 2 O 2 for 30 min followed by ChIP for control IgG, 8-OHdG, and CHD4 at the promoter CpG islands of eight representative genes and analyzed by real-time RT-PCR. Data are represented as mean ± SEM for triplicate experiments. (H) Cells were untreated or treated with 2 mM H 2 O 2 for 30 min. Sequential ChIP analyses were performed to test the co-occupancy of CHD4 and 8-OHdG at the promoter CpG islands of eight TSGs. Data are represented as mean ± SEM for triplicate experiments. (I) Cells were untreated or treated with 2mMH 2 O 2 for 30 min, and nascent RNA was labeled concurrently. Real-time RT-PCR data are presented as mean ± SEM of the treated over untreated values for triplicate experiments. (J) Sequential ChIP analyses were performed to test the co-occupancy of CHD4 and 8-OHdG or epigenetic silencing proteins at the promoter CpG islands of eight representative TSGs in fresh frozen human CRC tissues (n = 20) and normal colon epithelial tissues (n = 6). Data are represented as mean ± SEM. .
    Figure Legend Snippet: The Recruitment of CHD4 to Oxidative DNA Damage Sites Depends on OGG1 (A) CoIPs of lysates from SW480 cells untreated or treated with 2 mM H 2 O 2 for 30 min were performed with the indicated antibodies. (B) Purified OGG1 and Flag-CHD4 were incubated with antibodies against Flag or OGG1 in IP buffer. The immunoprecipitated samples were detected by western blot analyses using the antibodies indicated. (C) After SW480 OGG1 KO cells were transfected with pCMV-Taq or pCMV-OGG1 for 48 hr, the cells were untreated or treated with 2mMH 2 O 2 for 30 min. Whole-cell extracts and the tight chromatin fractions were analyzed by immunoblotting with the indicated antibodies. (D) Whole-cell extracts and the tight chromatin fractions from SW480 CHD4 KD cells untreated (Un) or treated with 2 mM H 2 O 2 for 30 min were analyzed by immunoblotting as in (C). (E) Purified OGG1 and Flag-CHD4 were incubated with antibodies against Flag or OGG1 in IP buffer with or without 8-OHdG oligonucleotide. The immuno-precipitated samples were detected by western blot analyses using the antibodies indicated. (F) Biotin labeled 8-OHdG oligonucleotide incubated with OGG1 and Flag-CHD4 was pulled down using streptavidin beads. Bound proteins were eluted and analyzed by immunoblotting with the indicated antibodies. (G) SW480 OGG1 KO cells were untreated or treated with 2mMH 2 O 2 for 30 min followed by ChIP for control IgG, 8-OHdG, and CHD4 at the promoter CpG islands of eight representative genes and analyzed by real-time RT-PCR. Data are represented as mean ± SEM for triplicate experiments. (H) Cells were untreated or treated with 2 mM H 2 O 2 for 30 min. Sequential ChIP analyses were performed to test the co-occupancy of CHD4 and 8-OHdG at the promoter CpG islands of eight TSGs. Data are represented as mean ± SEM for triplicate experiments. (I) Cells were untreated or treated with 2mMH 2 O 2 for 30 min, and nascent RNA was labeled concurrently. Real-time RT-PCR data are presented as mean ± SEM of the treated over untreated values for triplicate experiments. (J) Sequential ChIP analyses were performed to test the co-occupancy of CHD4 and 8-OHdG or epigenetic silencing proteins at the promoter CpG islands of eight representative TSGs in fresh frozen human CRC tissues (n = 20) and normal colon epithelial tissues (n = 6). Data are represented as mean ± SEM. .

    Techniques Used: Purification, Incubation, Immunoprecipitation, Western Blot, Transfection, Labeling, Chromatin Immunoprecipitation, Quantitative RT-PCR

    35) Product Images from "Rab40C is a novel Varp-binding protein that promotes proteasomal degradation of Varp in melanocytes"

    Article Title: Rab40C is a novel Varp-binding protein that promotes proteasomal degradation of Varp in melanocytes

    Journal: Biology Open

    doi: 10.1242/bio.201411114

    Effect of knockdown of Rab40C in melanocytes on the level of Varp expression and Tyrp1 expression. (A) Knockdown efficiency of Rab40C siRNAs as revealed by semi-quantitative RT-PCR analysis. Both Rab40C siRNAs (st1 and st2) dramatically suppressed the level of Rab40C mRNA expression (top panel) without affecting the level of Varp mRNA expression (middle panel). GAPDH mRNA expression (bottom panel) is shown as a control to ensure that equivalent amounts of first-strand cDNA were used for the RT-PCR analysis. The size of the molecular mass markers (bp, base pair) is shown on the left side of the panel. (B) Knockdown of Rab40C increased the amount of Varp protein in melanocytes. Cell lysates of B16-F1 cells that had been transfected with Rab40C siRNA (st1 or st2) and pEF-T7-Varp were subjected to SDS-PAGE followed by immunoblotting with the antibodies indicated. Note that the Rab40C-knockdown cells contained a greater amount of T7-Varp protein than the control cells (upper panel) even though their Varp mRNA level was unaltered (see A, middle panel). (C) Typical images of Rab40C-knockdown melanocytes (Tyrp1 images and their corresponding bright-field images). Melan-a cells were transfected with Rab40C siRNAs and then immunostained with anti-Tyrp1 mouse monoclonal antibody (upper panels). Note that Rab40C knockdown caused a dramatic reduction in Tyrp1 signals. Rab40C-knockdown cells are outlined with a broken line. Scale bars, 20 µm. (D) Quantification of the Tyrp1 signals shown in C. The bars represent the means and S.E. of data (30 cells for each siRNA) from one representative experiment. * p
    Figure Legend Snippet: Effect of knockdown of Rab40C in melanocytes on the level of Varp expression and Tyrp1 expression. (A) Knockdown efficiency of Rab40C siRNAs as revealed by semi-quantitative RT-PCR analysis. Both Rab40C siRNAs (st1 and st2) dramatically suppressed the level of Rab40C mRNA expression (top panel) without affecting the level of Varp mRNA expression (middle panel). GAPDH mRNA expression (bottom panel) is shown as a control to ensure that equivalent amounts of first-strand cDNA were used for the RT-PCR analysis. The size of the molecular mass markers (bp, base pair) is shown on the left side of the panel. (B) Knockdown of Rab40C increased the amount of Varp protein in melanocytes. Cell lysates of B16-F1 cells that had been transfected with Rab40C siRNA (st1 or st2) and pEF-T7-Varp were subjected to SDS-PAGE followed by immunoblotting with the antibodies indicated. Note that the Rab40C-knockdown cells contained a greater amount of T7-Varp protein than the control cells (upper panel) even though their Varp mRNA level was unaltered (see A, middle panel). (C) Typical images of Rab40C-knockdown melanocytes (Tyrp1 images and their corresponding bright-field images). Melan-a cells were transfected with Rab40C siRNAs and then immunostained with anti-Tyrp1 mouse monoclonal antibody (upper panels). Note that Rab40C knockdown caused a dramatic reduction in Tyrp1 signals. Rab40C-knockdown cells are outlined with a broken line. Scale bars, 20 µm. (D) Quantification of the Tyrp1 signals shown in C. The bars represent the means and S.E. of data (30 cells for each siRNA) from one representative experiment. * p

    Techniques Used: Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Transfection, SDS Page

    36) Product Images from "USP15 targets ALK3/BMPR1A for deubiquitylation to enhance bone morphogenetic protein signalling"

    Article Title: USP15 targets ALK3/BMPR1A for deubiquitylation to enhance bone morphogenetic protein signalling

    Journal: Open Biology

    doi: 10.1098/rsob.140065

    ALK3 undergoes proteasomal degradation. ( a ) HEK293 cells transfected with untagged ALK3 were treated with or without 20 μM cycloheximide for 24 h prior to lysis. Cells were treated with DMSO control, 100 nM bafilomycin A1 (to inhibit vacuolar-type H+ ATPase) or 10 μM bortezomib (to inhibit the proteasome) for 3 h prior to lysis. Extracts were resolved by SDS-PAGE and subjected to immunoblotting with the indicated antibodies. ( b ) HEK293 cells were transiently transfected with siFoxO4 or siUSP15-3 . Cells were serum-starved overnight, treated with or without 10 μM bortezomib for 3 h and then stimulated with or without 6.25 ng ml −1 BMP for 1 h prior to lysis. Extracts were resolved by SDS-PAGE and subjected to immunoblotting with antibodies against endogenous USP15, pSMAD1 and total SMAD1. ( c ) HEK293 cells were transiently transfected with siFoxO4 (–), siUSP15-3 or siSMAD6 as indicated. Twenty-four hours post siRNA transfection, cells were serum-starved overnight and stimulated with or without 6.25 ng ml −1 BMP for 1 h prior to lysis. Extracts were resolved by SDS-PAGE and subjected to immunoblotting with antibodies against pSMAD1, total SMAD1, USP15 and GAPDH. The SMAD6 knockdown was confirmed by qRT-PCR (electronic supplementary material, figure S7).
    Figure Legend Snippet: ALK3 undergoes proteasomal degradation. ( a ) HEK293 cells transfected with untagged ALK3 were treated with or without 20 μM cycloheximide for 24 h prior to lysis. Cells were treated with DMSO control, 100 nM bafilomycin A1 (to inhibit vacuolar-type H+ ATPase) or 10 μM bortezomib (to inhibit the proteasome) for 3 h prior to lysis. Extracts were resolved by SDS-PAGE and subjected to immunoblotting with the indicated antibodies. ( b ) HEK293 cells were transiently transfected with siFoxO4 or siUSP15-3 . Cells were serum-starved overnight, treated with or without 10 μM bortezomib for 3 h and then stimulated with or without 6.25 ng ml −1 BMP for 1 h prior to lysis. Extracts were resolved by SDS-PAGE and subjected to immunoblotting with antibodies against endogenous USP15, pSMAD1 and total SMAD1. ( c ) HEK293 cells were transiently transfected with siFoxO4 (–), siUSP15-3 or siSMAD6 as indicated. Twenty-four hours post siRNA transfection, cells were serum-starved overnight and stimulated with or without 6.25 ng ml −1 BMP for 1 h prior to lysis. Extracts were resolved by SDS-PAGE and subjected to immunoblotting with antibodies against pSMAD1, total SMAD1, USP15 and GAPDH. The SMAD6 knockdown was confirmed by qRT-PCR (electronic supplementary material, figure S7).

    Techniques Used: Transfection, Lysis, SDS Page, Quantitative RT-PCR

    37) Product Images from "Promoter-Associated RNAs Regulate HSPC152 Gene Expression in Malignant Melanoma"

    Article Title: Promoter-Associated RNAs Regulate HSPC152 Gene Expression in Malignant Melanoma

    Journal: Non-Coding RNA

    doi: 10.3390/ncrna2030007

    The 014mel cells were transfected with 75 nM of siRNA-targeting paHSPC152 or with 75 nM of scrambled siRNA (untreated). At 72 h post transfection, RNA was extracted and subjected to qRT-PCR with specific primers to HSPC152 mRNA. HSPC152 was normalized relative to the expression of RPLP0 RNA. The graph represents the mean results of at least three different experiments. * p value
    Figure Legend Snippet: The 014mel cells were transfected with 75 nM of siRNA-targeting paHSPC152 or with 75 nM of scrambled siRNA (untreated). At 72 h post transfection, RNA was extracted and subjected to qRT-PCR with specific primers to HSPC152 mRNA. HSPC152 was normalized relative to the expression of RPLP0 RNA. The graph represents the mean results of at least three different experiments. * p value

    Techniques Used: Transfection, Quantitative RT-PCR, Expressing

    38) Product Images from "Convergent Evolution of a New Arsenic Binding Site in the ArsR/SmtB Family of Metalloregulators"

    Article Title: Convergent Evolution of a New Arsenic Binding Site in the ArsR/SmtB Family of Metalloregulators

    Journal: The Journal of biological chemistry

    doi: 10.1074/jbc.M706565200

    DNA footprinting Top , DNase I footprinting was performed as described under “Materials and Methods” using light-sabre green fluorescently labeled double-stranded DNA. The sizes of major DNA fragments in nucleotides are shown at the top of the peaks. The sequence protected by AfArsR is shown between the arrows , with the positions of −60, −73, and −86 nucleotides relative to the start of the arsB gene indicated. Bottom , the sequence of the double-stranded DNA is shown, with the region protected by AfArsR boxed . The start sites for the arsR and arsB genes are indicated, with the −10, −60, −73, and −86 nucleotide positions relative to the start of arsB indicated.
    Figure Legend Snippet: DNA footprinting Top , DNase I footprinting was performed as described under “Materials and Methods” using light-sabre green fluorescently labeled double-stranded DNA. The sizes of major DNA fragments in nucleotides are shown at the top of the peaks. The sequence protected by AfArsR is shown between the arrows , with the positions of −60, −73, and −86 nucleotides relative to the start of the arsB gene indicated. Bottom , the sequence of the double-stranded DNA is shown, with the region protected by AfArsR boxed . The start sites for the arsR and arsB genes are indicated, with the −10, −60, −73, and −86 nucleotide positions relative to the start of arsB indicated.

    Techniques Used: DNA Footprinting, Footprinting, Labeling, Sequencing

    39) Product Images from "Chitinases Are Negative Regulators of Francisella novicida Biofilms"

    Article Title: Chitinases Are Negative Regulators of Francisella novicida Biofilms

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0093119

    Effect of chitinase inhibitors SAN and DEQ on antibacterial and antibiofilm activity. ( A, B ) Susceptibility of Fn WT and chi mutants to SAN ( A ) and DEQ ( B ). Survival percentage of bacteria was calculated by OD 600 measurements after 24 h incubation with various concentrations of SAN and DEQ in TSBC. The EC 50 s (μM) were determined by GraphPad software as indicated in the bottom table. ( C ) Effect of chitinase inhibitors SAN and DEQ on biofilm formation. Biofilm formation (CV570/OD600) was calculated by normalization with bacterial growth in each concentration of inhibitors. *P
    Figure Legend Snippet: Effect of chitinase inhibitors SAN and DEQ on antibacterial and antibiofilm activity. ( A, B ) Susceptibility of Fn WT and chi mutants to SAN ( A ) and DEQ ( B ). Survival percentage of bacteria was calculated by OD 600 measurements after 24 h incubation with various concentrations of SAN and DEQ in TSBC. The EC 50 s (μM) were determined by GraphPad software as indicated in the bottom table. ( C ) Effect of chitinase inhibitors SAN and DEQ on biofilm formation. Biofilm formation (CV570/OD600) was calculated by normalization with bacterial growth in each concentration of inhibitors. *P

    Techniques Used: Activity Assay, Incubation, Software, Concentration Assay

    Chitinase alters drug susceptibility of Fn biofilms. ( A, C ) Effect of chitinase on drug susceptibility of biofilms pre-formed in the TC plates to ( A ) gentamicin (Gm) and ( C ) ciprofloxacin (Cipro). ( B, D ) Effect of chitinase on drug susceptibility of biofilms pre-formed in the amine plates to ( B ) gentamicin and ( D ) ciprofloxacin. ( E ) Susceptibility of chitinase-pretreated biofilms to gentamicin. Biofilms were formed on Amine plates in the presence of chitinase (0, 0.2 and 2 μg/ml) for 24 h then Gm (2 μg/ml) was added to the biofilms for 24 h. The remaining bacteria were calculated by the relative bacteria to no Gm-treated control in each concentration of chitinase. *P
    Figure Legend Snippet: Chitinase alters drug susceptibility of Fn biofilms. ( A, C ) Effect of chitinase on drug susceptibility of biofilms pre-formed in the TC plates to ( A ) gentamicin (Gm) and ( C ) ciprofloxacin (Cipro). ( B, D ) Effect of chitinase on drug susceptibility of biofilms pre-formed in the amine plates to ( B ) gentamicin and ( D ) ciprofloxacin. ( E ) Susceptibility of chitinase-pretreated biofilms to gentamicin. Biofilms were formed on Amine plates in the presence of chitinase (0, 0.2 and 2 μg/ml) for 24 h then Gm (2 μg/ml) was added to the biofilms for 24 h. The remaining bacteria were calculated by the relative bacteria to no Gm-treated control in each concentration of chitinase. *P

    Techniques Used: Concentration Assay

    Fn chitinase affects the biophysical properties of the biofilm. Fn was grown to mid-log phase prior to the analyses. ( A ) The relative hydrophobicity of WT and chi mutants assayed by phenyl-sepharose column chromatography (HIC) and microbial adhesion to the nonpolar solvent hexadecane. *P
    Figure Legend Snippet: Fn chitinase affects the biophysical properties of the biofilm. Fn was grown to mid-log phase prior to the analyses. ( A ) The relative hydrophobicity of WT and chi mutants assayed by phenyl-sepharose column chromatography (HIC) and microbial adhesion to the nonpolar solvent hexadecane. *P

    Techniques Used: Column Chromatography, Hydrophobic Interaction Chromatography

    Fn chitinase affects biofilm formation in different surface charged microplates. ( A ) Biofilm formation based on CV staining (CV570) of cells adherent to negatively (TC), positively (Amine), neutral (PS) and positively/negatively (Primaria) charged 96-well plates, normalized by bacterial growth (OD600) expressed as CV570/OD600. ( B ) Attachment was assessed by CV staining 1 h post-inoculation of stationary-phase cultures (OD = 1.0). Initial attachment of Fn WT was very low to the TC and Primaria plates, but high to the amine and PS. *P
    Figure Legend Snippet: Fn chitinase affects biofilm formation in different surface charged microplates. ( A ) Biofilm formation based on CV staining (CV570) of cells adherent to negatively (TC), positively (Amine), neutral (PS) and positively/negatively (Primaria) charged 96-well plates, normalized by bacterial growth (OD600) expressed as CV570/OD600. ( B ) Attachment was assessed by CV staining 1 h post-inoculation of stationary-phase cultures (OD = 1.0). Initial attachment of Fn WT was very low to the TC and Primaria plates, but high to the amine and PS. *P

    Techniques Used: Staining

    Enzymatic activity of chitinase is required for regulation of Fn biofilm formation. ( A ) Effect of exogenously added chitinase on biofilm formation in the negatively-charged TC plates. EC 50 s of exogenous chitinase to WT, chiA and chiB mutants were determined to be 0.65, 0.18, and 0.21 μg/ml, respectively (n = 6). ( B ) Effect of exogenous chitinase on biofilm formation in the positively-charged amine plates. EC 50 s of chitinase to WT, chiA and chiB mutants were determined to be 87.46, 0.17, and 0.15 μg/ml, respectively (n = 6). ( C ) Detachment of Fn biofilms after exposure to proteinase K, chitinase and DNase I (50 μg/ml) in the TC plates. Untreated control CV 570 values were 0.149±0.032, 0.588±0.012, and 0.585±0.017 for Fn WT, chiA and chiB mutants, respectively. *P
    Figure Legend Snippet: Enzymatic activity of chitinase is required for regulation of Fn biofilm formation. ( A ) Effect of exogenously added chitinase on biofilm formation in the negatively-charged TC plates. EC 50 s of exogenous chitinase to WT, chiA and chiB mutants were determined to be 0.65, 0.18, and 0.21 μg/ml, respectively (n = 6). ( B ) Effect of exogenous chitinase on biofilm formation in the positively-charged amine plates. EC 50 s of chitinase to WT, chiA and chiB mutants were determined to be 87.46, 0.17, and 0.15 μg/ml, respectively (n = 6). ( C ) Detachment of Fn biofilms after exposure to proteinase K, chitinase and DNase I (50 μg/ml) in the TC plates. Untreated control CV 570 values were 0.149±0.032, 0.588±0.012, and 0.585±0.017 for Fn WT, chiA and chiB mutants, respectively. *P

    Techniques Used: Activity Assay

    40) Product Images from "Kindlin-2 suppresses transcription factor GATA4 through interaction with SUV39H1 to attenuate hypertrophy"

    Article Title: Kindlin-2 suppresses transcription factor GATA4 through interaction with SUV39H1 to attenuate hypertrophy

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-019-2121-0

    Cardiac-specific Kindlin-2 knockout leads to hypertrophy. a Strategy for generating myocardial conditional knockout Kindlin-2 mice. The targeting construct was designed to insert loxP sites flanking exons 5 and 6 of Kindlin-2 gene. Kindlin-2 floxed mice were then crossed with mice expressing recombinant α- MHC-Cre mice to produce Kindlin-2 myocardial conditional knockout (cKO) mice. b PCR analysis of extracted genomic DNA from tail clippings. c – e Western blot, RT-qPCR and immunohistochemical staining showed the level of Kindlin-2 in heart of wild-type or cKO mice (Scale bar = 50 µm). f Representative macroscopic observations of hearts (upper panel). HE staining of whole-heart sections (lower panel) (Scale bar = 1000 µm). g Ratio of heart weight to body weight or heart weight to tibia length was determined in saline or ISO-treated mice. Means ± S.D. * p
    Figure Legend Snippet: Cardiac-specific Kindlin-2 knockout leads to hypertrophy. a Strategy for generating myocardial conditional knockout Kindlin-2 mice. The targeting construct was designed to insert loxP sites flanking exons 5 and 6 of Kindlin-2 gene. Kindlin-2 floxed mice were then crossed with mice expressing recombinant α- MHC-Cre mice to produce Kindlin-2 myocardial conditional knockout (cKO) mice. b PCR analysis of extracted genomic DNA from tail clippings. c – e Western blot, RT-qPCR and immunohistochemical staining showed the level of Kindlin-2 in heart of wild-type or cKO mice (Scale bar = 50 µm). f Representative macroscopic observations of hearts (upper panel). HE staining of whole-heart sections (lower panel) (Scale bar = 1000 µm). g Ratio of heart weight to body weight or heart weight to tibia length was determined in saline or ISO-treated mice. Means ± S.D. * p

    Techniques Used: Knock-Out, Mouse Assay, Construct, Expressing, Recombinant, Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR, Immunohistochemistry, Staining

    Activation of GATA4 mediates cardiac hypertrophy in Kindlin-2 cKO mice. a Wild-type Kindlin-2 or cKO mice were injected with saline or ISO. Total protein was extracted from heart tissue for western blot (left panel). Protein bands on the left were scanned and relative band intensities were normalized (right panel). b Immunohistochemical staining showed GATA4 level (Scale bar = 50 µm). c Lysates from heart tissue of wild-type mice were prepared for Co-IP. d Lysates were extracted from heart tissue of wild-type and Kindlin-2 cKO mice for ChIP assays. Means ± S.D. * P
    Figure Legend Snippet: Activation of GATA4 mediates cardiac hypertrophy in Kindlin-2 cKO mice. a Wild-type Kindlin-2 or cKO mice were injected with saline or ISO. Total protein was extracted from heart tissue for western blot (left panel). Protein bands on the left were scanned and relative band intensities were normalized (right panel). b Immunohistochemical staining showed GATA4 level (Scale bar = 50 µm). c Lysates from heart tissue of wild-type mice were prepared for Co-IP. d Lysates were extracted from heart tissue of wild-type and Kindlin-2 cKO mice for ChIP assays. Means ± S.D. * P

    Techniques Used: Activation Assay, Mouse Assay, Injection, Western Blot, Immunohistochemistry, Staining, Co-Immunoprecipitation Assay, Chromatin Immunoprecipitation

    A working model for the role of Kindlin-2 in suppressing GATA4.
    Figure Legend Snippet: A working model for the role of Kindlin-2 in suppressing GATA4.

    Techniques Used:

    Kindlin-2 suppresses expression of GATA4. a GO analysis of differentially expressed genes in RNAseq. b 11 differentially expressed genes (log2 Fold > 1) in cardiovascular disease-related genes. c Control or Kindlin-2 siRNA was transfected into primary neonate rat cardiomyocytes for 48 h, followed western blot. d Protein bands were scanned and relative band intensities were normalized to each GAPDH band. The column diagrams represent average relative band intensity with standard error from three independent experiments. e Cardiomyocytes were infected by Kindlin-2 or control adenovirus, followed western blot. f Relative band intensities were analyzed. g Control or Kindlin-2 siRNA was transfected into cardiomyocytes and RT-qPCR detected Kindlin-2 and GATA4 mRNA level. h Adenovirus carrying Kindlin-2 or control was transfected into cardiomyocytes, followed RT-qPCR. i Schematic diagrams of the regions for ChIP assay. j Lysates were extracted from mouse cardiac tissues for ChIP assays using anti-Kindlin-2 antibody. Q-PCR assay was then performed to quantify ChIP-enriched DNA using the three primers.
    Figure Legend Snippet: Kindlin-2 suppresses expression of GATA4. a GO analysis of differentially expressed genes in RNAseq. b 11 differentially expressed genes (log2 Fold > 1) in cardiovascular disease-related genes. c Control or Kindlin-2 siRNA was transfected into primary neonate rat cardiomyocytes for 48 h, followed western blot. d Protein bands were scanned and relative band intensities were normalized to each GAPDH band. The column diagrams represent average relative band intensity with standard error from three independent experiments. e Cardiomyocytes were infected by Kindlin-2 or control adenovirus, followed western blot. f Relative band intensities were analyzed. g Control or Kindlin-2 siRNA was transfected into cardiomyocytes and RT-qPCR detected Kindlin-2 and GATA4 mRNA level. h Adenovirus carrying Kindlin-2 or control was transfected into cardiomyocytes, followed RT-qPCR. i Schematic diagrams of the regions for ChIP assay. j Lysates were extracted from mouse cardiac tissues for ChIP assays using anti-Kindlin-2 antibody. Q-PCR assay was then performed to quantify ChIP-enriched DNA using the three primers.

    Techniques Used: Expressing, Transfection, Western Blot, Infection, Quantitative RT-PCR, Chromatin Immunoprecipitation, Polymerase Chain Reaction

    SUV39H1 mediated suppression of Kindlin-2 on GATA4. a Flag or Flag-SUV39H1 was transfected into HEK293A cells and GATA4 were measured by Western blot. b Relative band intensities of western blot were analyzed. c Flag or Flag-SUV39H1 was transfected into HEK293A cells and GATA4 were measured by RT-qPCR. d HEK293A cells were treated with the SUV39H1 inhibitor Chaetomin at various doses for 48 h, and GATA4 was measured by western blot. e Relative band intensities of western blot were analyzed. f HEK293A cells were treated with Chaetomin and GATA4 was detected by RT-PCR. g Flag-SUV39H1 was transfected into HEK293A cells and lysates were then extracted for ChIP assays. Means ± S.D. ** indicates p
    Figure Legend Snippet: SUV39H1 mediated suppression of Kindlin-2 on GATA4. a Flag or Flag-SUV39H1 was transfected into HEK293A cells and GATA4 were measured by Western blot. b Relative band intensities of western blot were analyzed. c Flag or Flag-SUV39H1 was transfected into HEK293A cells and GATA4 were measured by RT-qPCR. d HEK293A cells were treated with the SUV39H1 inhibitor Chaetomin at various doses for 48 h, and GATA4 was measured by western blot. e Relative band intensities of western blot were analyzed. f HEK293A cells were treated with Chaetomin and GATA4 was detected by RT-PCR. g Flag-SUV39H1 was transfected into HEK293A cells and lysates were then extracted for ChIP assays. Means ± S.D. ** indicates p

    Techniques Used: Transfection, Western Blot, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Chromatin Immunoprecipitation

    Kindlin-2 prevents cardiomyocytes from hypertrophy through suppressing GATA4. a–c Cardiomyocytes were infected with Kindlin-2 or control adenovirus plus ISO treatment (5 µm, 24 h). The efficacy of Kindlin-2 overexpression was detected by Western blot a . Representative images of cardiomyocytes stained with anti-α-actinin2 (Alexa Fluor 568) b . Cell surface areas were measured c . d – f Kindlin-2 siRNA was transfected into primary neonate rat cardiomyocytes plus ISO treatment with or without knockdown of GATA4 and GATA6 alone or in combination. The efficacy of Kindlin-2 siRNA was detected by Western blotting d . Representative images of cardiomyocytes stained with anti-α-actinin2 e . Cell surface areas were measured f . g Cardiomyocytes were infected with Kindlin-2 or control adenovirus plus ISO, followed by Western blot (left panel). Relative band intensities were analyzed (right panel). h Cardiomyocytes were transfected with control or Kindlin-2 siRNA plus ISO treatment, followed Western blot (left panel). Relative band intensities were analyzed (right panel).
    Figure Legend Snippet: Kindlin-2 prevents cardiomyocytes from hypertrophy through suppressing GATA4. a–c Cardiomyocytes were infected with Kindlin-2 or control adenovirus plus ISO treatment (5 µm, 24 h). The efficacy of Kindlin-2 overexpression was detected by Western blot a . Representative images of cardiomyocytes stained with anti-α-actinin2 (Alexa Fluor 568) b . Cell surface areas were measured c . d – f Kindlin-2 siRNA was transfected into primary neonate rat cardiomyocytes plus ISO treatment with or without knockdown of GATA4 and GATA6 alone or in combination. The efficacy of Kindlin-2 siRNA was detected by Western blotting d . Representative images of cardiomyocytes stained with anti-α-actinin2 e . Cell surface areas were measured f . g Cardiomyocytes were infected with Kindlin-2 or control adenovirus plus ISO, followed by Western blot (left panel). Relative band intensities were analyzed (right panel). h Cardiomyocytes were transfected with control or Kindlin-2 siRNA plus ISO treatment, followed Western blot (left panel). Relative band intensities were analyzed (right panel).

    Techniques Used: Infection, Over Expression, Western Blot, Staining, Transfection

    Kindlin-2 interacts with SUV39H1. a HEK293A cells were treated by Chaetomin and lysates were then extracted for ChIP assays. b Flag-SUV39H1 was transfected into HEK293A cells and lysates were then extracted for ChIP assays. c Lysates from HEK293A cells were prepared and then anti-Kindlin-2 antibody was used for Co-IP. d Purified GST or GST-SUV39H1 was incubated with total lysates prepared from HEK293A cells for the GST-pull down assays. e Immunofluorescent staining of anti-Kindlin-2 (Alexa Fluor 488, green), anti-SUV39H1 (Alexa Fluor 568, red), and nuclei (DAPI, blue) was performed using primary neonate rat cardiomyocytes. Images were captured with a confocal microscope (Scale bar = 20 µm). Pearson’s coefficient was shown in the histogram.
    Figure Legend Snippet: Kindlin-2 interacts with SUV39H1. a HEK293A cells were treated by Chaetomin and lysates were then extracted for ChIP assays. b Flag-SUV39H1 was transfected into HEK293A cells and lysates were then extracted for ChIP assays. c Lysates from HEK293A cells were prepared and then anti-Kindlin-2 antibody was used for Co-IP. d Purified GST or GST-SUV39H1 was incubated with total lysates prepared from HEK293A cells for the GST-pull down assays. e Immunofluorescent staining of anti-Kindlin-2 (Alexa Fluor 488, green), anti-SUV39H1 (Alexa Fluor 568, red), and nuclei (DAPI, blue) was performed using primary neonate rat cardiomyocytes. Images were captured with a confocal microscope (Scale bar = 20 µm). Pearson’s coefficient was shown in the histogram.

    Techniques Used: Chromatin Immunoprecipitation, Transfection, Co-Immunoprecipitation Assay, Purification, Incubation, Staining, Microscopy

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    Modification:

    Article Title: Mutated NPM1 in combination with overexpression of Meis1 or Hoxa9 is not sufficient to induce acute myeloid leukemia
    Article Snippet: .. In brief, murine BM cells harvested from C57BL/6 J mice 4 days after 150 µg/g 5-fluorouracil (5-FU, Accord Healthcare AB, Solna, Sweden) treatment were transduced with NPMc+ and neo or YFP viruses (NPMc+ cells), Meis1 and GFP viruses (Meis1 cells), Hoxa9 and GFP viruses (Hoxa9 cells), Meis1 and NPMc+ viruses (Meis1-NPMc+ cells) or Hoxa9 and NPMc+ viruses (Hoxa9-NPMc+ cells) and cultured in Dulbecco modified Eagle medium (DMEM with high glucose, D6429, Sigma-Aldrich Sweden AB, Stockholm, Sweden) supplemented with 15 % fetal bovine serum (6250, StemCell Technologies Inc., Vancouver, Canada), 2 mM l -glutamine (G7513-100 ml, Sigma-Aldrich Sweden AB, Stockholm, Sweden), 1 % Penicillin and streptomycin (P4333-100 ml, Sigma-Aldrich Sweden AB, Stockholm, Sweden), 10 ng/ml human interleukin-6 (2506, StemCell Technologies SARL, Grenoble, France), 6 ng/ml murine interleukin-3 (2733, StemCell Technologies SARL, Grenoble, France), and 50 ng/ml murine stem cell factor (2931 StemCell Technologies SARL, Grenoble, France) (complete medium). .. Transduction was achieved by co-culturing 5-FU treated murine BM cells with irradiated (2 × 25 Gray) GP + E86 viral cells in complete medium supplemented with 5 µg/ml protamine sulfate (P-4020, Sigma-Aldrich Sweden AB, Stockholm, Sweden).

    Staining:

    Article Title: Drosophila cyclin D/Cdk4 regulates mitochondrial biogenesis and aging and sensitizes animals to hypoxic stress
    Article Snippet: .. 96AED En-Gal4 , UAS-GFP/UAS-X animals were dissected in PBS, incubated at RT with stained with 30 µM DHE (Sigma Aldrich D7008) solution for 10–15 min. ..

    Purification:

    Article Title: N-glycosylation converts non-glycoproteins into mannose receptor ligands and reveals antigen-specific T cell responses in vivo
    Article Snippet: .. For purification of NST-GFP, NST-GFP-S8L and QST-GFP, proteins were additionally purified by size exclusion chromatography (Sephacryl S200 HR, Sigma, Taufkirchen, Germany). .. Expression and purification of NST-GFP and QST-GFP from HEK293T cells NST-GFP and QST-GFP were cloned behind the IL-2 secretion sequence of pIgFuse, thereby removing the IgG sequence, and transfected into HEK293T cells.

    Plasmid Preparation:

    Article Title: Simple rules for passive diffusion through the nuclear pore complex
    Article Snippet: .. These GFP-xPrA genes were subcloned either into the yeast constitutive expression plasmid pYX242 (EMD Millipore, discontinued) or as 6xHIS fusion genes into the bacterial expression plasmid pET28a (EMD Millipore). .. GFP-1PrG and -2PrG constructs were made similarly, as yeast and bacterial expression constructs, by fusing to GFP either the C2 domain (for GFP-1PrG) or both the C2 and C3 domains (for GFP-2PrG) from Streptococcus protein G ( ).

    Article Title: Venezuelan Equine Encephalitis Virus Capsid Protein Forms a Tetrameric Complex with CRM1 and Importin ?/? That Obstructs Nuclear Pore Complex Function ▿
    Article Snippet: .. H68-GFP, H60-GFP, H68AA1-GFP, and H68AA2-GFP were expressed as N-terminal 6×His-tagged proteins from the pTriEx1 plasmid in Escherichia coli strain Turner(DE3)(pLysS) according to the manufacturer's instructions (Novagen). .. Cells were harvested by centrifugation and lysed in buffer containing 100 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 1 mg/ml of lysozyme.

    Article Title: The Effect of Transient Local Anti-inflammatory Treatment on the Survival of Pig Retinal Progenitor Cell Allotransplants
    Article Snippet: .. Dual-label IHC was performed using a cocktail of anti-GFP (Abcam, ab13970 1:500) to detect GFP pRPCs and anti-Ki-67 (Vector, VP-K452 1:100; proliferation), anti-recoverin (Millipore, AB5585 1:500; cones), or mouse anti-rhodopsin (Millipore, MABN15 1:500; rods) antibodies. .. For infiltrating leukocytes/macrophages, we used anti-CD45 antibody (Abcam, 1:200).

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