s2 cells  (Thermo Fisher)


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    Structured Review

    Thermo Fisher s2 cells
    Western blot analysis of recombinant anosmin-1 in Drosophila <t>S2</t> cells ( a ) Transient expression of recombinant anosmin-1 was induced by 500 μM CuSO 4 in 10% serum-containing medium. Recombinant anosmin-1 proteins present in cell lysate (L), salt extract (S) and conditioned medium (M) were identified using an anti-His monoclonal antibody. The molecular-mass-markers are shown. Transiently transfected Drosophila S2 cells in serum-free medium culture were induced to express proteins in the absence ( b ) and presence ( c ) of 100 μg/ml HS. ( d ) PIWF1 and PIWF4 proteins generated from stable Drosophila S2 cell line in serum-free medium were analysed in presence (+) or absence (−) of HS in comparison with vector (control), GFP, mPIWF1 172 and mPIWF4 172 .
    S2 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Cross-talk of anosmin-1, the protein implicated in X-linked Kallmann's syndrome, with heparan sulphate and urokinase-type plasminogen activator"

    Article Title: Cross-talk of anosmin-1, the protein implicated in X-linked Kallmann's syndrome, with heparan sulphate and urokinase-type plasminogen activator

    Journal: Biochemical Journal

    doi: 10.1042/BJ20041078

    Western blot analysis of recombinant anosmin-1 in Drosophila S2 cells ( a ) Transient expression of recombinant anosmin-1 was induced by 500 μM CuSO 4 in 10% serum-containing medium. Recombinant anosmin-1 proteins present in cell lysate (L), salt extract (S) and conditioned medium (M) were identified using an anti-His monoclonal antibody. The molecular-mass-markers are shown. Transiently transfected Drosophila S2 cells in serum-free medium culture were induced to express proteins in the absence ( b ) and presence ( c ) of 100 μg/ml HS. ( d ) PIWF1 and PIWF4 proteins generated from stable Drosophila S2 cell line in serum-free medium were analysed in presence (+) or absence (−) of HS in comparison with vector (control), GFP, mPIWF1 172 and mPIWF4 172 .
    Figure Legend Snippet: Western blot analysis of recombinant anosmin-1 in Drosophila S2 cells ( a ) Transient expression of recombinant anosmin-1 was induced by 500 μM CuSO 4 in 10% serum-containing medium. Recombinant anosmin-1 proteins present in cell lysate (L), salt extract (S) and conditioned medium (M) were identified using an anti-His monoclonal antibody. The molecular-mass-markers are shown. Transiently transfected Drosophila S2 cells in serum-free medium culture were induced to express proteins in the absence ( b ) and presence ( c ) of 100 μg/ml HS. ( d ) PIWF1 and PIWF4 proteins generated from stable Drosophila S2 cell line in serum-free medium were analysed in presence (+) or absence (−) of HS in comparison with vector (control), GFP, mPIWF1 172 and mPIWF4 172 .

    Techniques Used: Western Blot, Recombinant, Expressing, Transfection, Generated, Plasmid Preparation

    2) Product Images from "Three microtubule severing enzymes contribute to the "Pacman-flux" machinery that moves chromosomes"

    Article Title: Three microtubule severing enzymes contribute to the "Pacman-flux" machinery that moves chromosomes

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200612011

    Dm-Kat60 stimulates Pacman, whereas Dm-Spastin and Dm-Fidgetin drive poleward flux. (A) Frames of time-lapse recordings of anaphase EGFP–α-tubulin expressing S2 cells in which chromosomes were Hoechst stained just before visualization, and bars were photobleached across each half spindle. Yellow arrowheads mark the initial position of bleach marks; yellow arrows and dots mark their current positions. Blue arrowheads mark the initial positions of the leading edge of a selected chromosome; blue arrows and dots mark the current positions of the chromosome. The control spindle displays both poleward flux and Pacman motility (manifested as chromosomes overtaking bleach marks), but the Dm-Kat60 RNAi spindle has visibly diminished Pacman motility, and the Dm-Spastin and Dm-Fidgetin spindles have diminished flux. (B) Representative plots of distance versus time of individual chromosomes measured from the RNAi-treated cells in A. Red lines represent flux, and blue lines represent Pacman motility (measured as the approach of chromosomes to bleach marks). (C) Mean flux, Pacman, and chromatid-to-pole velocities (+SD) for anaphase spindles. Statistical differences from the control are marked with asterisks. Numbers in parentheses are sample sizes.
    Figure Legend Snippet: Dm-Kat60 stimulates Pacman, whereas Dm-Spastin and Dm-Fidgetin drive poleward flux. (A) Frames of time-lapse recordings of anaphase EGFP–α-tubulin expressing S2 cells in which chromosomes were Hoechst stained just before visualization, and bars were photobleached across each half spindle. Yellow arrowheads mark the initial position of bleach marks; yellow arrows and dots mark their current positions. Blue arrowheads mark the initial positions of the leading edge of a selected chromosome; blue arrows and dots mark the current positions of the chromosome. The control spindle displays both poleward flux and Pacman motility (manifested as chromosomes overtaking bleach marks), but the Dm-Kat60 RNAi spindle has visibly diminished Pacman motility, and the Dm-Spastin and Dm-Fidgetin spindles have diminished flux. (B) Representative plots of distance versus time of individual chromosomes measured from the RNAi-treated cells in A. Red lines represent flux, and blue lines represent Pacman motility (measured as the approach of chromosomes to bleach marks). (C) Mean flux, Pacman, and chromatid-to-pole velocities (+SD) for anaphase spindles. Statistical differences from the control are marked with asterisks. Numbers in parentheses are sample sizes.

    Techniques Used: Expressing, Staining

    Dm-Kat60, Dm-Spastin, and Dm-Fidgetin are all required for proper chromosome segregation during anaphase A. (A) After RNAi, EGFP–α-tubulin expressing S2 cells were recorded from metaphase until late anaphase as single confocal sections. Chromosomes are visible as black dots at the ends of the prominent kinetochore fibers. In the last frames, yellow dots mark the positions of the chromosomes, and blue dots the centrosome position, in a half spindle. Numbers are elapsed time after the first frame. (B) Anaphase chromatid-to-pole velocities (+SD) after RNAi. *, P
    Figure Legend Snippet: Dm-Kat60, Dm-Spastin, and Dm-Fidgetin are all required for proper chromosome segregation during anaphase A. (A) After RNAi, EGFP–α-tubulin expressing S2 cells were recorded from metaphase until late anaphase as single confocal sections. Chromosomes are visible as black dots at the ends of the prominent kinetochore fibers. In the last frames, yellow dots mark the positions of the chromosomes, and blue dots the centrosome position, in a half spindle. Numbers are elapsed time after the first frame. (B) Anaphase chromatid-to-pole velocities (+SD) after RNAi. *, P

    Techniques Used: Expressing

    Dm-Spastin and Dm-Fidgetin stimulate the turnover of α-tubulin at MT ends and γ-tubulin at centrosomes. (A, top) FRAP of metaphase spindles of EGFP–α-tubulin expressing S2 cells after RNAi. One spindle pole region (MT minus ends) and one spindle equator region (MT plus ends) were photobleached, and their fluorescence recoveries were recorded. Images are frames from time-lapse recordings; the elapsed times after photobleaching are shown to the left. Fluorescent α-tubulin recovery after Dm-Kat60 RNAi is similar to control, whereas the recoveries after Dm-Spastin and Dm-Fidgetin RNAi are visibly slower. (middle) Mean fluorescence recovery half-times ( t 1/2 ) for MT minus ends (left). Asterisks indicate treatments with statistically slower α-tubulin turnover rates compared with control. Percentage of fluorescence recoveries are shown (right). Numbers within bars are sample sizes, and error bars are SD. (bottom) Mean t 1/2 (+SD) of MT plus ends. (B, top) The two intensely fluorescent centrosomes of γ-tubulin–EGFP expressing S2 cells were photobleached, and their fluorescent recoveries were recorded. Selected time-point images and their postbleach elapsed times are shown. (middle) Mean t 1/2 (+SD) of γ-tubulin turnover at centrosomes. (bottom) Percentage of fluorescence recoveries is shown.
    Figure Legend Snippet: Dm-Spastin and Dm-Fidgetin stimulate the turnover of α-tubulin at MT ends and γ-tubulin at centrosomes. (A, top) FRAP of metaphase spindles of EGFP–α-tubulin expressing S2 cells after RNAi. One spindle pole region (MT minus ends) and one spindle equator region (MT plus ends) were photobleached, and their fluorescence recoveries were recorded. Images are frames from time-lapse recordings; the elapsed times after photobleaching are shown to the left. Fluorescent α-tubulin recovery after Dm-Kat60 RNAi is similar to control, whereas the recoveries after Dm-Spastin and Dm-Fidgetin RNAi are visibly slower. (middle) Mean fluorescence recovery half-times ( t 1/2 ) for MT minus ends (left). Asterisks indicate treatments with statistically slower α-tubulin turnover rates compared with control. Percentage of fluorescence recoveries are shown (right). Numbers within bars are sample sizes, and error bars are SD. (bottom) Mean t 1/2 (+SD) of MT plus ends. (B, top) The two intensely fluorescent centrosomes of γ-tubulin–EGFP expressing S2 cells were photobleached, and their fluorescent recoveries were recorded. Selected time-point images and their postbleach elapsed times are shown. (middle) Mean t 1/2 (+SD) of γ-tubulin turnover at centrosomes. (bottom) Percentage of fluorescence recoveries is shown.

    Techniques Used: Expressing, Fluorescence

    Dm-Spastin and Dm-Fidgetin, but not Dm-Kat60, stimulate poleward flux in metaphase spindles.  (A) To visualize poleward flux, bars were photobleached across metaphase spindles of EGFP–α-tubulin expressing S2 cells, after RNAi treatment. Images are frames taken from time-lapse videos; the elapsed times (s) after photobleaching are indicated to the left or within the panels. Orange arrowheads mark the initial position of the photobleached bars, and blue arrowheads indicate their current positions. Kymographs were generated from the time-lapse recordings of these spindles to further illustrate the flux rates: a large angle between the tracks of bleached bar and pole indicates a relatively high flux rate (control and Dm-Kat60 RNAi), whereas more parallel tracks indicate decreased flux (Dm-Spastin and Dm-Fidgetin RNAi). t and d indicate the time and distance axes, respectively. (B) Mean flux rates (+SD) for metaphase cells after RNAi treatment. The flux rate after Dm-Kat60 RNAi is equivalent to control, whereas flux is significantly decreased after Dm-Spastin or Dm-Fidgetin RNAi. *, P
    Figure Legend Snippet: Dm-Spastin and Dm-Fidgetin, but not Dm-Kat60, stimulate poleward flux in metaphase spindles. (A) To visualize poleward flux, bars were photobleached across metaphase spindles of EGFP–α-tubulin expressing S2 cells, after RNAi treatment. Images are frames taken from time-lapse videos; the elapsed times (s) after photobleaching are indicated to the left or within the panels. Orange arrowheads mark the initial position of the photobleached bars, and blue arrowheads indicate their current positions. Kymographs were generated from the time-lapse recordings of these spindles to further illustrate the flux rates: a large angle between the tracks of bleached bar and pole indicates a relatively high flux rate (control and Dm-Kat60 RNAi), whereas more parallel tracks indicate decreased flux (Dm-Spastin and Dm-Fidgetin RNAi). t and d indicate the time and distance axes, respectively. (B) Mean flux rates (+SD) for metaphase cells after RNAi treatment. The flux rate after Dm-Kat60 RNAi is equivalent to control, whereas flux is significantly decreased after Dm-Spastin or Dm-Fidgetin RNAi. *, P

    Techniques Used: Expressing, Generated

    Overexpression of Dm-Kat60, Dm-Spastin, or Dm-Fidgetin eliminates MTs in interphase S2 cells. (A–C) mRFP–α-tubulin expressing S2 cells were transiently transfected with full-length AAA-EGFP constructs. AAA refers specifically to Dm-Kat60, Dm-Spastin, and Dm-Fidgetin. Fluorescent MTs are pseudocolored green; expressed AAA fusion protein is pseudocolored red. Overexpression of any one of the AAA constructs visibly decreases the MT polymer mass. Bars, 5 μm. (D) Mean fluorescence intensities (+SD) of mRFP– α-tubulin for live whole cells after transient transfection with the indicated full-length AAA-EGFP construct. Red bars are tubulin fluorescence intensities of cells that visibly expressed fluorescent AAA protein. Gray bars are intensities of neighboring cells not visibly expressing fluorescent AAA protein. *, P
    Figure Legend Snippet: Overexpression of Dm-Kat60, Dm-Spastin, or Dm-Fidgetin eliminates MTs in interphase S2 cells. (A–C) mRFP–α-tubulin expressing S2 cells were transiently transfected with full-length AAA-EGFP constructs. AAA refers specifically to Dm-Kat60, Dm-Spastin, and Dm-Fidgetin. Fluorescent MTs are pseudocolored green; expressed AAA fusion protein is pseudocolored red. Overexpression of any one of the AAA constructs visibly decreases the MT polymer mass. Bars, 5 μm. (D) Mean fluorescence intensities (+SD) of mRFP– α-tubulin for live whole cells after transient transfection with the indicated full-length AAA-EGFP construct. Red bars are tubulin fluorescence intensities of cells that visibly expressed fluorescent AAA protein. Gray bars are intensities of neighboring cells not visibly expressing fluorescent AAA protein. *, P

    Techniques Used: Over Expression, Expressing, Transfection, Construct, Fluorescence

    3) Product Images from "RNAi screen identifies a role for adaptor protein AP-3 in sorting to the regulated secretory pathway"

    Article Title: RNAi screen identifies a role for adaptor protein AP-3 in sorting to the regulated secretory pathway

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201006131

    S2 cell screen identifies genes that regulate the surface expression of dVMAT. (A) The flow chart illustrates the procedure used for screening. S2 cells transfected with GFP-/HA-dVMAT were treated twice with dsRNA over a 6-d period in a 96-well plate, incubated with external HA antibody conjugated to Alexa Fluor 647 for 2 h, washed, and the fluorescence of both GFP and Alexa Fluor 647 was measured at the level of individual cells by flow cytometry. Of 7,200 genes conserved from Drosophila to mammals, 18 positives (z score ≥ 3) were identified and retested using nonoverlapping dsRNA. Kolmogorov-Smirnov analysis of the cumulative frequency distribution reveals that 16 of 18 genes were again positive (P
    Figure Legend Snippet: S2 cell screen identifies genes that regulate the surface expression of dVMAT. (A) The flow chart illustrates the procedure used for screening. S2 cells transfected with GFP-/HA-dVMAT were treated twice with dsRNA over a 6-d period in a 96-well plate, incubated with external HA antibody conjugated to Alexa Fluor 647 for 2 h, washed, and the fluorescence of both GFP and Alexa Fluor 647 was measured at the level of individual cells by flow cytometry. Of 7,200 genes conserved from Drosophila to mammals, 18 positives (z score ≥ 3) were identified and retested using nonoverlapping dsRNA. Kolmogorov-Smirnov analysis of the cumulative frequency distribution reveals that 16 of 18 genes were again positive (P

    Techniques Used: Expressing, Flow Cytometry, Transfection, Incubation, Fluorescence, Cytometry

    Classification of genes identified in the screen by mechanism and effect on the regulated secretion of soluble cargo. S2 cells transfected with DE/AA GFP-/HA-dVMAT were treated with dsRNA-targeting genes identified in the screen, and the uptake of external HA antibody was measured as described in Fig. 1 . (left) Representative cumulative frequency distributions are shown for selected genes in each class. Class I genes increase, class II genes decrease, and class III genes have no effect on antibody uptake by DE/AA dVMAT. (middle) S2 cells expressing ANF-GFP were treated with dsRNA and basal (unstimulated) secretion of GFP fluorescence determined as in Fig. 1 C . Secretion was normalized to cellular ANF-GFP and expressed as a percentage of the fluorescence secreted by control cells. (right) S2 cells expressing ANF-GFP were treated with dsRNA and LPS-induced secretion measured as in Fig. 1 D . *, P
    Figure Legend Snippet: Classification of genes identified in the screen by mechanism and effect on the regulated secretion of soluble cargo. S2 cells transfected with DE/AA GFP-/HA-dVMAT were treated with dsRNA-targeting genes identified in the screen, and the uptake of external HA antibody was measured as described in Fig. 1 . (left) Representative cumulative frequency distributions are shown for selected genes in each class. Class I genes increase, class II genes decrease, and class III genes have no effect on antibody uptake by DE/AA dVMAT. (middle) S2 cells expressing ANF-GFP were treated with dsRNA and basal (unstimulated) secretion of GFP fluorescence determined as in Fig. 1 C . Secretion was normalized to cellular ANF-GFP and expressed as a percentage of the fluorescence secreted by control cells. (right) S2 cells expressing ANF-GFP were treated with dsRNA and LPS-induced secretion measured as in Fig. 1 D . *, P

    Techniques Used: Transfection, Expressing, Fluorescence

    S2 cells express an RSP. (A and B) S2 cells were transiently transfected with wt (black) or DE/AA (red) GFP- and HA-tagged dVMAT, incubated for 2 h at room temperature with external HA antibody conjugated to Alexa Fluor 647, washed, and the fluorescence of individual cells was determined by flow cytometry (A). (inset) The bar graph displays the mean ratio of surface/total fluorescence ( n > 1,800 cells). Kolmogorov-Smirnov analysis of the cumulative frequency distributions binned by surface/total dVMAT ratios (B) indicates a significant change in the DE/AA distribution relative to wt (***, P
    Figure Legend Snippet: S2 cells express an RSP. (A and B) S2 cells were transiently transfected with wt (black) or DE/AA (red) GFP- and HA-tagged dVMAT, incubated for 2 h at room temperature with external HA antibody conjugated to Alexa Fluor 647, washed, and the fluorescence of individual cells was determined by flow cytometry (A). (inset) The bar graph displays the mean ratio of surface/total fluorescence ( n > 1,800 cells). Kolmogorov-Smirnov analysis of the cumulative frequency distributions binned by surface/total dVMAT ratios (B) indicates a significant change in the DE/AA distribution relative to wt (***, P

    Techniques Used: Transfection, Incubation, Fluorescence, Flow Cytometry, Cytometry

    4) Product Images from "Enhancer Sequences Influence the Role of the Amino-Terminal Domain of Bicoid in Transcription"

    Article Title: Enhancer Sequences Influence the Role of the Amino-Terminal Domain of Bicoid in Transcription

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.23.13.4439-4448.2003

    The amino-terminal domain of Bcd plays different roles on hb and kni enhancer elements. (A) Schematic diagrams of the 250-bp hb enhancer element and the 64-bp kni enhancer element. The Bcd binding sites are represented by arrows. (B) CAT assay results of S2 cells transfected with hb-CAT and kni-CAT reporter plasmids (1 μg) and increasing amounts of an effector plasmid expressing Bcd. Activities obtained with 1 μg of transfected effector plasmid on each reporter were arbitrarily set to 100 (fold activation was 72 and 74 for hb-CAT and kni-CAT , respectively). (C and D) CAT assay activities (in logarithmic scale) for wild-type Bcd and Bcd(92-489) on hb-CAT (C) and kni-CAT (D) reporters at different concentrations. The activities of wild-type Bcd at 1 μg of transfected effector DNA on each reporters were set to 100. (E) Representative Western blot results detecting Bcd proteins in transfected cells. For the experiments shown in this figure, the amounts of the transfected effector plasmids were 0.01, 0.03, 0.1, 0.3, and 1 μg. Wt, wild type.
    Figure Legend Snippet: The amino-terminal domain of Bcd plays different roles on hb and kni enhancer elements. (A) Schematic diagrams of the 250-bp hb enhancer element and the 64-bp kni enhancer element. The Bcd binding sites are represented by arrows. (B) CAT assay results of S2 cells transfected with hb-CAT and kni-CAT reporter plasmids (1 μg) and increasing amounts of an effector plasmid expressing Bcd. Activities obtained with 1 μg of transfected effector plasmid on each reporter were arbitrarily set to 100 (fold activation was 72 and 74 for hb-CAT and kni-CAT , respectively). (C and D) CAT assay activities (in logarithmic scale) for wild-type Bcd and Bcd(92-489) on hb-CAT (C) and kni-CAT (D) reporters at different concentrations. The activities of wild-type Bcd at 1 μg of transfected effector DNA on each reporters were set to 100. (E) Representative Western blot results detecting Bcd proteins in transfected cells. For the experiments shown in this figure, the amounts of the transfected effector plasmids were 0.01, 0.03, 0.1, 0.3, and 1 μg. Wt, wild type.

    Techniques Used: Binding Assay, Transfection, Plasmid Preparation, Expressing, Activation Assay, Western Blot

    The self-inhibitory function of Bcd is differentially implemented on hb and kni enhancers. (A) CAT assay results from S2 cells transfected with 1 μg of hb-CAT or kni-CAT reporter plasmid and 1 μg of effector plasmid expressing wild-type Bcd (lane 1), Bcd(92-489) (lane 2), Bcd(A52-56) (lane 3), or Bcd(A57-61) (lane 4). The activities of wild-type Bcd on each reporter were set to 100. The data for the hb-CAT ). Wt, wild type.
    Figure Legend Snippet: The self-inhibitory function of Bcd is differentially implemented on hb and kni enhancers. (A) CAT assay results from S2 cells transfected with 1 μg of hb-CAT or kni-CAT reporter plasmid and 1 μg of effector plasmid expressing wild-type Bcd (lane 1), Bcd(92-489) (lane 2), Bcd(A52-56) (lane 3), or Bcd(A57-61) (lane 4). The activities of wild-type Bcd on each reporter were set to 100. The data for the hb-CAT ). Wt, wild type.

    Techniques Used: Transfection, Plasmid Preparation, Expressing

    5) Product Images from "Transcriptomic comparison of Drosophila snRNP biogenesis mutants reveals mutant-specific changes in pre-mRNA processing: implications for spinal muscular atrophy"

    Article Title: Transcriptomic comparison of Drosophila snRNP biogenesis mutants reveals mutant-specific changes in pre-mRNA processing: implications for spinal muscular atrophy

    Journal: RNA

    doi: 10.1261/rna.057208.116

    snRNP-specific protein knockdown in S2 cells. ( A ) qRT-PCR of untreated cells versus those treated with dsRNA for  Smn ,  Prp6 , or  Prp8  mRNAs. ( B ) Western blot of dUTPase levels in RNAi-treated cells. Anti-SMN and anti-α-tubulin verify SMN knockdown
    Figure Legend Snippet: snRNP-specific protein knockdown in S2 cells. ( A ) qRT-PCR of untreated cells versus those treated with dsRNA for Smn , Prp6 , or Prp8 mRNAs. ( B ) Western blot of dUTPase levels in RNAi-treated cells. Anti-SMN and anti-α-tubulin verify SMN knockdown

    Techniques Used: Quantitative RT-PCR, Western Blot

    6) Product Images from "B56-Associated Protein Phosphatase 2A Is Required For Survival and Protects from Apoptosis in Drosophila melanogaster"

    Article Title: B56-Associated Protein Phosphatase 2A Is Required For Survival and Protects from Apoptosis in Drosophila melanogaster

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.22.11.3674-3684.2002

    Apoptosis induced by loss of B56-regulated PP2A is accompanied by activation of DEVD-specific caspases and inhibited by RNAi of caspases Dredd, Dronc, and Drice. (A) Caspase activation following RNAi of B56-regulated PP2A. DEVD-specific caspase activity in the cell extracts was assayed 4 days after RNAi of the indicated genes. OD 405 , optical density at 405 nm. (B) Caspases Dredd, Dronc, and Drice are required for the cell death that follows RNAi of B56-targeted PP2A. Viable S2 cells were counted 4 days following RNAi of the indicated genes. (C to F) RNAi of dB56-1 and -2 (C) induces apoptotic morphology in S2 cells 4 days after application of the dsRNA, an effect blocked (D) by addition of dsRNA-targeting drice . Similar apoptotic changes were seen with RNAi of dIAP - 1 (E). Control cell morphology is shown in panel F.
    Figure Legend Snippet: Apoptosis induced by loss of B56-regulated PP2A is accompanied by activation of DEVD-specific caspases and inhibited by RNAi of caspases Dredd, Dronc, and Drice. (A) Caspase activation following RNAi of B56-regulated PP2A. DEVD-specific caspase activity in the cell extracts was assayed 4 days after RNAi of the indicated genes. OD 405 , optical density at 405 nm. (B) Caspases Dredd, Dronc, and Drice are required for the cell death that follows RNAi of B56-targeted PP2A. Viable S2 cells were counted 4 days following RNAi of the indicated genes. (C to F) RNAi of dB56-1 and -2 (C) induces apoptotic morphology in S2 cells 4 days after application of the dsRNA, an effect blocked (D) by addition of dsRNA-targeting drice . Similar apoptotic changes were seen with RNAi of dIAP - 1 (E). Control cell morphology is shown in panel F.

    Techniques Used: Activation Assay, Activity Assay

    RNAi-mediated knockdown suggests that PP2A is an obligate heterotrimer in S2 cells. Fifteen micrograms of each indicated dsRNA was added to S2 cells in six-well dishes as described (Materials and Methods), and the cognate proteins were examined by immunoblot analysis 96 h later. Dbt, the Drosophila CKIɛ homologue, was examined both as a control for equal protein loading and as a test of the specificity of RNAi. The dsRNA added is indicated along the top. −, no dsRNA added. The star indicates a cross-reacting band detected by the antibody directed against PR55. (A) RNAi of dPP2A A down-regulates A, C, and B subunits. (B) RNAi of dPP2A C down-regulates A, C, and B subunits. (C) RNAi of the four known B subunits in Drosophila down-regulates A and C subunits. (D) RNAi of dPP2A C has no effect on the mRNA levels of A and B subunits. Total RNA was extracted from S2 cells 4 days following RNAi of the indicated genes, followed by RT-PCR (RT) for A, C, B56-1, and Dbt. PCR products were separated by electrophoresis on 1% agarose and examined by ethidium bromide staining.
    Figure Legend Snippet: RNAi-mediated knockdown suggests that PP2A is an obligate heterotrimer in S2 cells. Fifteen micrograms of each indicated dsRNA was added to S2 cells in six-well dishes as described (Materials and Methods), and the cognate proteins were examined by immunoblot analysis 96 h later. Dbt, the Drosophila CKIɛ homologue, was examined both as a control for equal protein loading and as a test of the specificity of RNAi. The dsRNA added is indicated along the top. −, no dsRNA added. The star indicates a cross-reacting band detected by the antibody directed against PR55. (A) RNAi of dPP2A A down-regulates A, C, and B subunits. (B) RNAi of dPP2A C down-regulates A, C, and B subunits. (C) RNAi of the four known B subunits in Drosophila down-regulates A and C subunits. (D) RNAi of dPP2A C has no effect on the mRNA levels of A and B subunits. Total RNA was extracted from S2 cells 4 days following RNAi of the indicated genes, followed by RT-PCR (RT) for A, C, B56-1, and Dbt. PCR products were separated by electrophoresis on 1% agarose and examined by ethidium bromide staining.

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

    PP2A and its B56 regulatory subunits are required for S2 cell multiplication. Viable S2 cells per well (determined by trypan blue exclusion) were counted at days 1 to 4 after the addition of the indicated dsRNAs. The B56 subunits appear to be redundant for cell multiplication.
    Figure Legend Snippet: PP2A and its B56 regulatory subunits are required for S2 cell multiplication. Viable S2 cells per well (determined by trypan blue exclusion) were counted at days 1 to 4 after the addition of the indicated dsRNAs. The B56 subunits appear to be redundant for cell multiplication.

    Techniques Used:

    Loss of B56:PP2A induces apoptotic morphology in S2 cells. S2 cells were fixed and stained with DAPI 4 days after treatment. Nuclear morphology was examined after DAPI staining, and cell morphology was examined by phase-contrast microscopy. Cells were either mock treated (A) or treated with dsRNA from dPR55 (B), 40 μM actinomycin D for only 15 h (C), dsRNA from both B56-1 and B56-2 (dB56-1+2) (D), dPP2A A subunit (E), or dPP2A C subunit (F). PP2A A, C, and combined B56 subunits produced morphological changes similar to those induced by actinomycin D. No morphological changes were seen following RNAi of individual B56 subunits or of PR55 or PR72 either alone or combined (data not shown).
    Figure Legend Snippet: Loss of B56:PP2A induces apoptotic morphology in S2 cells. S2 cells were fixed and stained with DAPI 4 days after treatment. Nuclear morphology was examined after DAPI staining, and cell morphology was examined by phase-contrast microscopy. Cells were either mock treated (A) or treated with dsRNA from dPR55 (B), 40 μM actinomycin D for only 15 h (C), dsRNA from both B56-1 and B56-2 (dB56-1+2) (D), dPP2A A subunit (E), or dPP2A C subunit (F). PP2A A, C, and combined B56 subunits produced morphological changes similar to those induced by actinomycin D. No morphological changes were seen following RNAi of individual B56 subunits or of PR55 or PR72 either alone or combined (data not shown).

    Techniques Used: Staining, Microscopy, Produced

    B56-targeted PP2A acts functionally upstream of Dark/dApaf1, Reaper, and Hid, and dp53. (A) Loss of dark abolishes B56:PP2A-regulated activation of DEVD-dependent caspases. Caspase activity was determined 4 days following RNAi of the indicated genes. (B) RNAi of dPP2A C induces morphological changes characteristic of apoptosis. S2 cells were examined by phase-contrast microscopy 4 days following RNAi of dPP2A C. (C) Addition of dsRNA for dark blocks the morphology changes. Similar results were seen with dB56 subunits (data not shown). (D) Partial knockdown of upstream activators of apoptosis by RNAi in S2 cells. dsRNA derived from reaper , hid , grim , and dp53 was added to S2 cells as described earlier, and mRNA levels were assessed by RT-PCR 4 days later. RT-PCR (RT) against dbt was performed on each sample as a control for RNA recovery. (E) RNAi of rpr , hid , or dp53 but not of grim partially suppresses B56:PP2A-regulated activation of DEVD-specific caspases. OD 405 , optical density at 405 nm.
    Figure Legend Snippet: B56-targeted PP2A acts functionally upstream of Dark/dApaf1, Reaper, and Hid, and dp53. (A) Loss of dark abolishes B56:PP2A-regulated activation of DEVD-dependent caspases. Caspase activity was determined 4 days following RNAi of the indicated genes. (B) RNAi of dPP2A C induces morphological changes characteristic of apoptosis. S2 cells were examined by phase-contrast microscopy 4 days following RNAi of dPP2A C. (C) Addition of dsRNA for dark blocks the morphology changes. Similar results were seen with dB56 subunits (data not shown). (D) Partial knockdown of upstream activators of apoptosis by RNAi in S2 cells. dsRNA derived from reaper , hid , grim , and dp53 was added to S2 cells as described earlier, and mRNA levels were assessed by RT-PCR 4 days later. RT-PCR (RT) against dbt was performed on each sample as a control for RNA recovery. (E) RNAi of rpr , hid , or dp53 but not of grim partially suppresses B56:PP2A-regulated activation of DEVD-specific caspases. OD 405 , optical density at 405 nm.

    Techniques Used: Activation Assay, Activity Assay, Microscopy, Derivative Assay, Reverse Transcription Polymerase Chain Reaction

    7) Product Images from "N-linked glycosylation restricts the function of Short gastrulation to bind and shuttle BMPs"

    Article Title: N-linked glycosylation restricts the function of Short gastrulation to bind and shuttle BMPs

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.167338

    Drosophila sog  has three conserved glycosylation sites.  (A) Alignment of Sog sequences from  Drosophila sp . shows that three putative glycosylation sites are conserved (Arg residue in pink in  D. melanogaster ). A Tld/Tlr cleavage site sequence close to N1 is shown in red. (B) Sog protein scheme with the location of the four conserved CR domains, Tld/Tlr cleavage sites (red arrows) and putative glycosylation sites (pink). TM, transmembrane domain. (C-E) Analysis of Sog constructs expressed in S2 cells. (C) Glycosylation mutants produced by site-directed mutagenesis and their effect on Sog migration in SDS-PAGE. N23 and N123 migrate faster than wild-type Sog. All mutants and wild-type Sog bear a C-terminal V5/His tag. Treatment with Tunicamycin decreases wild-type Sog Mw and confirms that Sog is glycosylated. (D) SDS-PAGE for Sog protein in cells (C) and extracellular medium (M) shows that Sog is secreted and that only the N2 mutation slightly decreases Sog secretion. (E) ELISA for S2 cell-secreted Sog confirms the analysis in D.
    Figure Legend Snippet: Drosophila sog has three conserved glycosylation sites. (A) Alignment of Sog sequences from Drosophila sp . shows that three putative glycosylation sites are conserved (Arg residue in pink in D. melanogaster ). A Tld/Tlr cleavage site sequence close to N1 is shown in red. (B) Sog protein scheme with the location of the four conserved CR domains, Tld/Tlr cleavage sites (red arrows) and putative glycosylation sites (pink). TM, transmembrane domain. (C-E) Analysis of Sog constructs expressed in S2 cells. (C) Glycosylation mutants produced by site-directed mutagenesis and their effect on Sog migration in SDS-PAGE. N23 and N123 migrate faster than wild-type Sog. All mutants and wild-type Sog bear a C-terminal V5/His tag. Treatment with Tunicamycin decreases wild-type Sog Mw and confirms that Sog is glycosylated. (D) SDS-PAGE for Sog protein in cells (C) and extracellular medium (M) shows that Sog is secreted and that only the N2 mutation slightly decreases Sog secretion. (E) ELISA for S2 cell-secreted Sog confirms the analysis in D.

    Techniques Used: Sequencing, Construct, Produced, Mutagenesis, Migration, SDS Page, Enzyme-linked Immunosorbent Assay

    Loss of Sog glycosylation modifies Dpp binding. (A) S2 cells transfected with wild-type Sog-myc and different combinations of Tsg and BMP ligands. Co-immunoprecipitation (Co-IP) with cell supernatants shows that Sog binds only BMP heterodimers in the absence of Tsg, but binds Dpp homodimers in the presence of Tsg. (B) S2 cells transfected with wild-type Sog-myc or the N-terminal Supersog fragment reveal that only Supersog binds Dpp alone using cell pellets (Pellet). That Supersog interacts with Dpp in the extracellular space is confirmed by using cell supernatants (Sup). The presence of Sax or Tkv in lane 3 does not change this pattern. (C) S2 cells transfected with wild-type sog- V5 or sogN123 -V5, and dpp- HA. Co-IP for V5 suggests that binding of the N123 mutant to Dpp is weakly enhanced compared with wild-type Sog. (D) S2 cells transfected with wild-type sog (S) or single glycosylation mutants (N1, N2, N3) plus dpp -HA, or plus dpp- HA and tld -HA. Co-IP for V5. SogN1 binds weakly to Dpp alone, whereas other mutants and wild-type Sog do not. Arrows in D point to the different Sog fragments produced by the Tld metalloprotease and show that the cleavage pattern is similar among all constructs. Asterisks indicate nonspecific bands.
    Figure Legend Snippet: Loss of Sog glycosylation modifies Dpp binding. (A) S2 cells transfected with wild-type Sog-myc and different combinations of Tsg and BMP ligands. Co-immunoprecipitation (Co-IP) with cell supernatants shows that Sog binds only BMP heterodimers in the absence of Tsg, but binds Dpp homodimers in the presence of Tsg. (B) S2 cells transfected with wild-type Sog-myc or the N-terminal Supersog fragment reveal that only Supersog binds Dpp alone using cell pellets (Pellet). That Supersog interacts with Dpp in the extracellular space is confirmed by using cell supernatants (Sup). The presence of Sax or Tkv in lane 3 does not change this pattern. (C) S2 cells transfected with wild-type sog- V5 or sogN123 -V5, and dpp- HA. Co-IP for V5 suggests that binding of the N123 mutant to Dpp is weakly enhanced compared with wild-type Sog. (D) S2 cells transfected with wild-type sog (S) or single glycosylation mutants (N1, N2, N3) plus dpp -HA, or plus dpp- HA and tld -HA. Co-IP for V5. SogN1 binds weakly to Dpp alone, whereas other mutants and wild-type Sog do not. Arrows in D point to the different Sog fragments produced by the Tld metalloprotease and show that the cleavage pattern is similar among all constructs. Asterisks indicate nonspecific bands.

    Techniques Used: Binding Assay, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Mutagenesis, Produced, Construct

    Sog levels are controlled by glycan and receptor-based retrieval from the extracellular space. (A) S2 cells transfected with wild-type sog- myc construct and different combinations of constructs producing Tsg and BMP ligands. Cellular levels of Sog are detected with anti-myc antiserum. Sog amounts increase in the presence of Dpp plus Tsg. These cellular levels correspond to Sog retrieved from the medium, as equivalent intracellular levels are observed when medium (S) from construct-expressing cells is added to cells that do not express sog- myc . (B) Intracellular Sog levels retrieved from Sog-myc+Dpp-HA+Tsg-His medium decrease by knocking down the Dpp receptor gene tkv or αPS1 ( mew ), αPS2 ( if ) or βPS ( mys ) integrin expression. (C) S2 cells transfected with wild-type sog-V5 or sogN1-V5 , sogN2-V5 , sogN3-V5 or triple sogN123-V5 mutant constructs in the presence of constructs producing Tsg and Dpp. Sog retrieval decreases by mutating the N1 and N2 sites. (D) Predicted conformation of Sog protein with regions depicted for binding to established extracellular partners. Red arrows indicate Tld/Tlr cleavage sites; pink represents glycosylated residues; gray shaded and open circles indicate BMPs. (E-J) Medium from S2 cells transfected with dpp -HA and tsg -His plus wild-type sog-V5 , or sogN1-V5 , sogN2-V5 or sogN3-V5 was added to naive cells and immunolabeled with anti-V5 (red) and anti-Rab5 (green) (E-H′). (E′′) High magnification for wild-type Sog-V5 and Rab5 shows partial colocalization, indicative of Sog endocytosis. Arrows indicate V5 + Rab5 + punctae. Quantification of these cells (I) shows that loss of N1, N2 and N3 glycosylation sites decreases the number of cells with V5 + Rab5 + endocytic punctae. (J) In cells expressing dpp- HA , gbb- His and tsg- His plus wild-type (wt) sog-V5 , or sogN1-V5 , sogN2-V5 or sogN3-V5 , the number of cells with V5 + Rab5 + punctae is equivalent. ** P ≤0.01 (Student's t -test). Scale bars: 20 μm in E′; 10 μm in E″.
    Figure Legend Snippet: Sog levels are controlled by glycan and receptor-based retrieval from the extracellular space. (A) S2 cells transfected with wild-type sog- myc construct and different combinations of constructs producing Tsg and BMP ligands. Cellular levels of Sog are detected with anti-myc antiserum. Sog amounts increase in the presence of Dpp plus Tsg. These cellular levels correspond to Sog retrieved from the medium, as equivalent intracellular levels are observed when medium (S) from construct-expressing cells is added to cells that do not express sog- myc . (B) Intracellular Sog levels retrieved from Sog-myc+Dpp-HA+Tsg-His medium decrease by knocking down the Dpp receptor gene tkv or αPS1 ( mew ), αPS2 ( if ) or βPS ( mys ) integrin expression. (C) S2 cells transfected with wild-type sog-V5 or sogN1-V5 , sogN2-V5 , sogN3-V5 or triple sogN123-V5 mutant constructs in the presence of constructs producing Tsg and Dpp. Sog retrieval decreases by mutating the N1 and N2 sites. (D) Predicted conformation of Sog protein with regions depicted for binding to established extracellular partners. Red arrows indicate Tld/Tlr cleavage sites; pink represents glycosylated residues; gray shaded and open circles indicate BMPs. (E-J) Medium from S2 cells transfected with dpp -HA and tsg -His plus wild-type sog-V5 , or sogN1-V5 , sogN2-V5 or sogN3-V5 was added to naive cells and immunolabeled with anti-V5 (red) and anti-Rab5 (green) (E-H′). (E′′) High magnification for wild-type Sog-V5 and Rab5 shows partial colocalization, indicative of Sog endocytosis. Arrows indicate V5 + Rab5 + punctae. Quantification of these cells (I) shows that loss of N1, N2 and N3 glycosylation sites decreases the number of cells with V5 + Rab5 + endocytic punctae. (J) In cells expressing dpp- HA , gbb- His and tsg- His plus wild-type (wt) sog-V5 , or sogN1-V5 , sogN2-V5 or sogN3-V5 , the number of cells with V5 + Rab5 + punctae is equivalent. ** P ≤0.01 (Student's t -test). Scale bars: 20 μm in E′; 10 μm in E″.

    Techniques Used: Transfection, Construct, Expressing, Mutagenesis, Binding Assay, Immunolabeling

    8) Product Images from "The NDNF-like factor Nord is a Hedgehog-induced extracellular BMP modulator that regulates Drosophila wing patterning and growth"

    Article Title: The NDNF-like factor Nord is a Hedgehog-induced extracellular BMP modulator that regulates Drosophila wing patterning and growth

    Journal: bioRxiv

    doi: 10.1101/2021.09.06.459106

    Nord binds to Dpp and attenuates BMP signaling in vitro (A) Co-immunoprecipitation of Nord with the BMP ligands Dpp and Gbb. Medium from S2 cells transfected for expression of GFP-tagged Nord were mixed with medium from cells expressing Flag-tagged Dpp and HA-tagged Gbb alone or in combination, followed by incubation with anti-FLAG or anti-HA antibody coupled beads overnight at 4°C. Precipitated proteins were analyzed by Western blotting with indicated antibodies. Nord was immunoprecipitated with Dpp and, to a lesser extent, with Gbb. The amount of immunoprecipitated Nord was increased when Dpp and Gbb were co-transfected. (B) S2 cells were transfected for expression of Flag-tagged Dpp or Gbb with or without GFP-tagged Nord (source cell). Both cell lysate and conditioned medium from the source cells were collected and followed by Western blot analysis. Loading was controlled by probing the blot for tubulin. The amount of Dpp or Dpp-Gbb ligands released into the medium was reduced when Nord was co-expressed in the source cells. (C) Comparison of BMP signaling activities of conditioned media in a cell-based signaling assay. After incubating the conditioned media collected in (B) with S2 cells stably expressing the FLAG-Mad transgene (Mad-S2, responding cell) for 1 hour at room temperature, the responding cells were washed and lysed. The lysates were probe with anti-pMad and anti-FLAG antibodies to detect both the phosphorylated Mad and total Mad protein, respectively. (D) Quantification of the Western blot data in panel C: the phosphorylated Mad (anti-pMad) levels were measured and normalized to the total Mad (anti-FLAG) levels. (E) Quantification of relative ligand activity [(pMad/Mad)/BMPs] by normalizing the medium signaling activity pMad/Mad in panel C to the ligand amount in panel B. (F) Mad-S2 cells were treated with a recombinant Dpp peptide (rDpp) in the absence or presence of conditioned medium containing raising levels of Nord for 1 hour at room temperature, the responding cells were washed and lysed. The lysates were probe with anti-pMad and anti-FLAG antibodies to detect both the phosphorylated Mad and total Mad protein, respectively. (G) Quantification of the Western blot data in panel F: the phosphorylated Mad (anti-pMad) levels were measured and normalized to the total Mad (anti-FLAG) levels. (H) Mad-S2 cells were transiently transfected for expression of GFP or Nord-GFP. 48 hours after transfection, the cells were treated with recombinant Dpp peptides for 1 hour. Upon treatment, the cells were washed, fixed, and stained by anti-FLAG to detect total Mad and anti-pMad to detect phosphorylated Mad. The average pMad levels were measured and normalized to the total Mad levels, and then plotted against different Dpp concentrations (0 to 5 nM). Each point shows the mean ± SD, n > 10. au: arbitrary units. The unpaired two-tailed t-test was used for statistical analysis. ***P
    Figure Legend Snippet: Nord binds to Dpp and attenuates BMP signaling in vitro (A) Co-immunoprecipitation of Nord with the BMP ligands Dpp and Gbb. Medium from S2 cells transfected for expression of GFP-tagged Nord were mixed with medium from cells expressing Flag-tagged Dpp and HA-tagged Gbb alone or in combination, followed by incubation with anti-FLAG or anti-HA antibody coupled beads overnight at 4°C. Precipitated proteins were analyzed by Western blotting with indicated antibodies. Nord was immunoprecipitated with Dpp and, to a lesser extent, with Gbb. The amount of immunoprecipitated Nord was increased when Dpp and Gbb were co-transfected. (B) S2 cells were transfected for expression of Flag-tagged Dpp or Gbb with or without GFP-tagged Nord (source cell). Both cell lysate and conditioned medium from the source cells were collected and followed by Western blot analysis. Loading was controlled by probing the blot for tubulin. The amount of Dpp or Dpp-Gbb ligands released into the medium was reduced when Nord was co-expressed in the source cells. (C) Comparison of BMP signaling activities of conditioned media in a cell-based signaling assay. After incubating the conditioned media collected in (B) with S2 cells stably expressing the FLAG-Mad transgene (Mad-S2, responding cell) for 1 hour at room temperature, the responding cells were washed and lysed. The lysates were probe with anti-pMad and anti-FLAG antibodies to detect both the phosphorylated Mad and total Mad protein, respectively. (D) Quantification of the Western blot data in panel C: the phosphorylated Mad (anti-pMad) levels were measured and normalized to the total Mad (anti-FLAG) levels. (E) Quantification of relative ligand activity [(pMad/Mad)/BMPs] by normalizing the medium signaling activity pMad/Mad in panel C to the ligand amount in panel B. (F) Mad-S2 cells were treated with a recombinant Dpp peptide (rDpp) in the absence or presence of conditioned medium containing raising levels of Nord for 1 hour at room temperature, the responding cells were washed and lysed. The lysates were probe with anti-pMad and anti-FLAG antibodies to detect both the phosphorylated Mad and total Mad protein, respectively. (G) Quantification of the Western blot data in panel F: the phosphorylated Mad (anti-pMad) levels were measured and normalized to the total Mad (anti-FLAG) levels. (H) Mad-S2 cells were transiently transfected for expression of GFP or Nord-GFP. 48 hours after transfection, the cells were treated with recombinant Dpp peptides for 1 hour. Upon treatment, the cells were washed, fixed, and stained by anti-FLAG to detect total Mad and anti-pMad to detect phosphorylated Mad. The average pMad levels were measured and normalized to the total Mad levels, and then plotted against different Dpp concentrations (0 to 5 nM). Each point shows the mean ± SD, n > 10. au: arbitrary units. The unpaired two-tailed t-test was used for statistical analysis. ***P

    Techniques Used: In Vitro, Immunoprecipitation, Transfection, Expressing, Incubation, Western Blot, Stable Transfection, Activity Assay, Recombinant, Staining, Two Tailed Test

    9) Product Images from "Enhanced antimicrobial peptide-induced activity in the mollusc Toll-2 family through evolution via tandem Toll/interleukin-1 receptor"

    Article Title: Enhanced antimicrobial peptide-induced activity in the mollusc Toll-2 family through evolution via tandem Toll/interleukin-1 receptor

    Journal: Royal Society Open Science

    doi: 10.1098/rsos.160123

    Activation of shrimp  Pen4  antimicrobial peptide gene by over expression of the full length of  Hcu_Toll-I  and  Hcu_Toll-2-2  ( a ),  Hcu_Toll-2-2 -TIR-1,  Hcu_Toll-2-2 -TIR-2 and  Hcu_Toll-2-2 -TIR-1 + TIR-2 ( b ) in S2 cells. The detailed methods could be seen in Material and methods section. All data are representative of three independent experiments. The bars indicate the s.d. of the luciferase activity ( n  = 3).
    Figure Legend Snippet: Activation of shrimp Pen4 antimicrobial peptide gene by over expression of the full length of Hcu_Toll-I and Hcu_Toll-2-2 ( a ), Hcu_Toll-2-2 -TIR-1, Hcu_Toll-2-2 -TIR-2 and Hcu_Toll-2-2 -TIR-1 + TIR-2 ( b ) in S2 cells. The detailed methods could be seen in Material and methods section. All data are representative of three independent experiments. The bars indicate the s.d. of the luciferase activity ( n  = 3).

    Techniques Used: Activation Assay, Over Expression, Luciferase, Activity Assay

    10) Product Images from "Post-mitotic myotubes repurpose the cytokinesis machinery to effect cellular guidance"

    Article Title: Post-mitotic myotubes repurpose the cytokinesis machinery to effect cellular guidance

    Journal: bioRxiv

    doi: 10.1101/2020.06.16.155333

    related to Figure 4. (A) Coomassie stained control gel for GST immunoprecipitation experiment described in Figure 3 . Bsd-bound GST beads (lane 5) recovered a large number of unique bands compared to control GST beads (lane 4). (B) Co-immunoprecipitation screen. S2 cells were transfected with Bsd.Myc and either a control plasmid (lane 2) or a plasmid expressing a Flag-tagged ‘prey’ protein (lanes 3-8) identified by AP-MS. Polo showed the strongest interaction with Bsd among the proteins tested. Lane 1 is a positive control. (C) Muscle morphogenesis phenotypes in polo mutants. Stage 16 embryos labeled with Tropomyosin. DT1, LO1, and VL1 muscles are pseudocolored orange, green, and violet. The polo alleles showed stronger phenotypes showed stronger phenotypes in trans to Df(3L)BSC447 that uncovers polo , arguing the alleles are hypomorphic. Quantification of muscle phenotypes is as described in Fig. 1 .
    Figure Legend Snippet: related to Figure 4. (A) Coomassie stained control gel for GST immunoprecipitation experiment described in Figure 3 . Bsd-bound GST beads (lane 5) recovered a large number of unique bands compared to control GST beads (lane 4). (B) Co-immunoprecipitation screen. S2 cells were transfected with Bsd.Myc and either a control plasmid (lane 2) or a plasmid expressing a Flag-tagged ‘prey’ protein (lanes 3-8) identified by AP-MS. Polo showed the strongest interaction with Bsd among the proteins tested. Lane 1 is a positive control. (C) Muscle morphogenesis phenotypes in polo mutants. Stage 16 embryos labeled with Tropomyosin. DT1, LO1, and VL1 muscles are pseudocolored orange, green, and violet. The polo alleles showed stronger phenotypes showed stronger phenotypes in trans to Df(3L)BSC447 that uncovers polo , arguing the alleles are hypomorphic. Quantification of muscle phenotypes is as described in Fig. 1 .

    Techniques Used: Staining, Immunoprecipitation, Transfection, Plasmid Preparation, Expressing, Positive Control, Labeling

    related to Figure 5. (A) Control phosphorylation assay. Lysates from S2 cells transfected with a control plasmid or a plasmid expressing Bsd.Myc were immunoblotted with anti-phospho-threonine (pThr). Whole lystates showed equivalent pThr levels. (B) in vitro phosphorylation assay. Polo was immunoprecipitated from S2 cell lysates and blotted with a phospho-threonine (pThr) antibody. Cells transfected with Bsd.I129A showed equivalent phosphorylated Polo as controls. (C) Stage 12 embryos with an endogenous GFP-tagged Polo ( Polo GFP ) that expressed rp298 > nLacZ were immunolabeled for GFP (green) and lacZ (red). Localization of total Polo protein was comparable between control and bsd 1 embryos. Graph shows Polo and nlacZ fluorescent intensity across myoblasts. (D) Early and late Stage 12 embryos immunolabeled for phospo-Polo (pPolo, green) and Mef2 (violet). pPolo localization in myonuclei increased during Stage 12 in control ( bsd 1 heterozygous) but not bsd 1 embryos. Control and bsd 1 embryos were labeled in the same preparation. (E) Immunoprecipitation of S2 cell lysates transfected with Bsd and Pav. Bsd did not physically interact with Pav.
    Figure Legend Snippet: related to Figure 5. (A) Control phosphorylation assay. Lysates from S2 cells transfected with a control plasmid or a plasmid expressing Bsd.Myc were immunoblotted with anti-phospho-threonine (pThr). Whole lystates showed equivalent pThr levels. (B) in vitro phosphorylation assay. Polo was immunoprecipitated from S2 cell lysates and blotted with a phospho-threonine (pThr) antibody. Cells transfected with Bsd.I129A showed equivalent phosphorylated Polo as controls. (C) Stage 12 embryos with an endogenous GFP-tagged Polo ( Polo GFP ) that expressed rp298 > nLacZ were immunolabeled for GFP (green) and lacZ (red). Localization of total Polo protein was comparable between control and bsd 1 embryos. Graph shows Polo and nlacZ fluorescent intensity across myoblasts. (D) Early and late Stage 12 embryos immunolabeled for phospo-Polo (pPolo, green) and Mef2 (violet). pPolo localization in myonuclei increased during Stage 12 in control ( bsd 1 heterozygous) but not bsd 1 embryos. Control and bsd 1 embryos were labeled in the same preparation. (E) Immunoprecipitation of S2 cell lysates transfected with Bsd and Pav. Bsd did not physically interact with Pav.

    Techniques Used: Phosphorylation Assay, Transfection, Plasmid Preparation, Expressing, In Vitro, Immunoprecipitation, Immunolabeling, Labeling

    Bsd activates Polo to regulate myotube guidance. (A)  in vitro  phosphorylation assays. Polo was immunoprecipitated from S2 cell lysates and blotted with a phospho-threonine (pThr) antibody. Cells transfected with Bsd showed more phosphorylated Polo than controls. (B) Polo localization in S2 cells. Bsd.Myc, Polo.Flag, and Polo.T182A.Flag were transfected into S2 cells; transgenic proteins were detected with anti-Myc (red, Bsd) and anti-Flag (green, Polo). Polo localized to discrete nuclear foci in a subset of control cells (middle column). The frequency of cells showing nuclear Polo foci was increased when Bsd was co-transfected with Polo (left column and graph). Cells transfected with Polo that lacked a phosphorylated threonine (T182A) showed no nuclear Polo foci (right column and graph). (C) Polo acts downstream of Bsd during myogenesis.  bsd 1  UAS.Polo.T182D  and  bsd 1  Mef2.Gal4 > Polo.T182D  embryos labeled for Tropomyosin. DT1, LO1, and VL1 muscles are pseudocolored orange, green, and violet. Expressing active (phosphomimetic) Polo T182D  in the mesoderm of  bsd 1  embryos suppressed the  bsd 1  myogenic phenotype. Quantification of myogenic phenotypes is as described in   Fig. 1 . (D)  in vivo  phosphorylation assays. Endogenous GFP-tagged Polo was immunoprecipitated from embryo lysates and blotted with pThr.  bsd 1  embryo lysates showed significantly less phosphorylated Polo than controls. (E) Stage 12 embryos immunolabeled for phosphorylated Polo (Polo pT182 , green) and Mef2 (violet). Polo pT182  localized to myonuclei in control embryos. (F) Polo pT182  levels in myonuclei were significantly reduced in  bsd 1  embryos. (G)  tum DH15  VL1, DT1, LO1, and VT1 muscle phenotypes. Stage 16 embryos labeled for  5053 > GFP  (green) and Tropomyosin (violet) or  slou > GFP  (green) and Tropomyosin (violet).  tum DH15  muscles made incorrect tendon attachments similar to  bsd 1  muscles (white arrowheads). (H) Histogram of VL1, DT1, LO1, and VT1 phenotypes (n≥54 per muscle). (ns) not significant, **(p
    Figure Legend Snippet: Bsd activates Polo to regulate myotube guidance. (A) in vitro phosphorylation assays. Polo was immunoprecipitated from S2 cell lysates and blotted with a phospho-threonine (pThr) antibody. Cells transfected with Bsd showed more phosphorylated Polo than controls. (B) Polo localization in S2 cells. Bsd.Myc, Polo.Flag, and Polo.T182A.Flag were transfected into S2 cells; transgenic proteins were detected with anti-Myc (red, Bsd) and anti-Flag (green, Polo). Polo localized to discrete nuclear foci in a subset of control cells (middle column). The frequency of cells showing nuclear Polo foci was increased when Bsd was co-transfected with Polo (left column and graph). Cells transfected with Polo that lacked a phosphorylated threonine (T182A) showed no nuclear Polo foci (right column and graph). (C) Polo acts downstream of Bsd during myogenesis. bsd 1 UAS.Polo.T182D and bsd 1 Mef2.Gal4 > Polo.T182D embryos labeled for Tropomyosin. DT1, LO1, and VL1 muscles are pseudocolored orange, green, and violet. Expressing active (phosphomimetic) Polo T182D in the mesoderm of bsd 1 embryos suppressed the bsd 1 myogenic phenotype. Quantification of myogenic phenotypes is as described in Fig. 1 . (D) in vivo phosphorylation assays. Endogenous GFP-tagged Polo was immunoprecipitated from embryo lysates and blotted with pThr. bsd 1 embryo lysates showed significantly less phosphorylated Polo than controls. (E) Stage 12 embryos immunolabeled for phosphorylated Polo (Polo pT182 , green) and Mef2 (violet). Polo pT182 localized to myonuclei in control embryos. (F) Polo pT182 levels in myonuclei were significantly reduced in bsd 1 embryos. (G) tum DH15 VL1, DT1, LO1, and VT1 muscle phenotypes. Stage 16 embryos labeled for 5053 > GFP (green) and Tropomyosin (violet) or slou > GFP (green) and Tropomyosin (violet). tum DH15 muscles made incorrect tendon attachments similar to bsd 1 muscles (white arrowheads). (H) Histogram of VL1, DT1, LO1, and VT1 phenotypes (n≥54 per muscle). (ns) not significant, **(p

    Techniques Used: In Vitro, Immunoprecipitation, Transfection, Transgenic Assay, Labeling, Expressing, In Vivo, Immunolabeling

    11) Product Images from "Identification of replication fork-associated proteins in Drosophila embryos and cultured cells using iPOND coupled to quantitative mass spectrometry"

    Article Title: Identification of replication fork-associated proteins in Drosophila embryos and cultured cells using iPOND coupled to quantitative mass spectrometry

    Journal: bioRxiv

    doi: 10.1101/2022.01.18.476773

    BRWD3 affects genome stability and replication fork progression.  (A)  RNAi-based depletion screen of candidate replication fork-associated proteins in S2 cells. Violin plots of the ɣ-H2Av intensity per nucleus normalized to total DNA content. Each distribution represents the signal intensities of 700 randomly selected cells from two biological replicates. **** p
    Figure Legend Snippet: BRWD3 affects genome stability and replication fork progression. (A) RNAi-based depletion screen of candidate replication fork-associated proteins in S2 cells. Violin plots of the ɣ-H2Av intensity per nucleus normalized to total DNA content. Each distribution represents the signal intensities of 700 randomly selected cells from two biological replicates. **** p

    Techniques Used:

    iPOND coupled to quantitative mass spectrometry in S2 cells.  (A)  A schematic of the labeling and mass spectrometry process for iPOND-TMT in Drosophila S2 cells.  (B)  Volcano plot visualizing those proteins identified as enriched or depleted in the pulse versus the chase cell culture samples. Enrichment on the X-axis (log2[pulse]-log 2 [chase]) on the X-axis and -log 10 (p-value) on the Y-axis.  (C)  The top 10 enriched biological processes of the proteins enriched in the pulse sample as determined by Gene Ontology (GO) analysis.  (D)  Network map of the proteins enriched in the pulse sample, clustered into groups of known interactors using the STRING database with no additional interactors added. For visualization, we included proteins with a corrected p value of
    Figure Legend Snippet: iPOND coupled to quantitative mass spectrometry in S2 cells. (A) A schematic of the labeling and mass spectrometry process for iPOND-TMT in Drosophila S2 cells. (B) Volcano plot visualizing those proteins identified as enriched or depleted in the pulse versus the chase cell culture samples. Enrichment on the X-axis (log2[pulse]-log 2 [chase]) on the X-axis and -log 10 (p-value) on the Y-axis. (C) The top 10 enriched biological processes of the proteins enriched in the pulse sample as determined by Gene Ontology (GO) analysis. (D) Network map of the proteins enriched in the pulse sample, clustered into groups of known interactors using the STRING database with no additional interactors added. For visualization, we included proteins with a corrected p value of

    Techniques Used: Mass Spectrometry, Labeling, Cell Culture, Significance Assay

    12) Product Images from "Self-oligomerization regulates stability of Survival Motor Neuron (SMN) protein isoforms by sequestering an SCFSlmb degron"

    Article Title: Self-oligomerization regulates stability of Survival Motor Neuron (SMN) protein isoforms by sequestering an SCFSlmb degron

    Journal: bioRxiv

    doi: 10.1101/078337

    A) The expression of endogenous SMN in S2 cells following transient transfection of either modified ‘vertebrate’ SMN constructs (vSMN and vSMN S201A ) or Drosophila SMN constructs (Smn G210V and Smn 10V-1A ) is unaffected, as compared to mock transfection. Protein is detected by anti-Flag antibody or anti-SMN antibody as indicated to the left of the blots. B) Transient transfections in S2 cells express Flag-SMN from the endogenous promoter. Protein levels of all transfected constructs are lower than endogenous SMN protein levels. Protein is detected by anti-Flag antibody or anti-SMN antibody as indicated to the left of the blots.
    Figure Legend Snippet: A) The expression of endogenous SMN in S2 cells following transient transfection of either modified ‘vertebrate’ SMN constructs (vSMN and vSMN S201A ) or Drosophila SMN constructs (Smn G210V and Smn 10V-1A ) is unaffected, as compared to mock transfection. Protein is detected by anti-Flag antibody or anti-SMN antibody as indicated to the left of the blots. B) Transient transfections in S2 cells express Flag-SMN from the endogenous promoter. Protein levels of all transfected constructs are lower than endogenous SMN protein levels. Protein is detected by anti-Flag antibody or anti-SMN antibody as indicated to the left of the blots.

    Techniques Used: Expressing, Transfection, Modification, Construct

    Identification and mutation of a putative Slmb/B-TrCP phospho-degron  A.  Identification of a conserved putative Slmb phospho-degron (DpSGXXpS/T motif variant) in the C-terminal self-oligomerization domain (YG Box) of SMN. The amino acid sequence of Smn from a variety of vertebrates is shown to illustrate conservation of this motif and rationale for the amino acid changes. Full-length human SMN is labeled as “Human” and the truncated isoform is labeled “hSMN∆7”. Endogenous  Drosophila melanogaster  Smn is labeled “Fruitfly”. To generate a more vertebrate-like SMN, key amino acids in Drosophila SMN were changed to amino acids conserved in vertebrates. Using this SMN backbone, a serine to alanine mutation was made in the putative degron in both full-length (vSMN S201A ) and truncated SMN∆7 (vSMN∆7A). An additional SMN construct that is the same length as SMN∆7, but has the amino acid sequence GLRQ (the next amino acids in the sequence) rather than EMLA (the amino acids introduced by mis-splicing of  SMN2 ) was made. The same serine to alanine mutation was made in this construct as well (MGLRQ* and MGLRQ* S201A ). Finally, to mimic a phosphorylated serine the full-length SMN construct (vSmn S201D ) and truncated SM N (vSMN∆7D) were made.  B.  Western blotting was used to determine protein levels of each of these SMN constructs, with expression driven by the endogenous promoter, in Drosophila S2 cells. Both the vSMN and vSMN∆7S proteins show increased levels when the serine is mutated to an alanine, indicating disruption of the normal degradation of SMN. Additionally, MGLRQ* protein is present at higher levels than is vSMN∆7S and protein levels do not change when the serine is mutated to an alanine. Normalized fold change as compared to vSmn levels is indicated at the bottom. *p
    Figure Legend Snippet: Identification and mutation of a putative Slmb/B-TrCP phospho-degron A. Identification of a conserved putative Slmb phospho-degron (DpSGXXpS/T motif variant) in the C-terminal self-oligomerization domain (YG Box) of SMN. The amino acid sequence of Smn from a variety of vertebrates is shown to illustrate conservation of this motif and rationale for the amino acid changes. Full-length human SMN is labeled as “Human” and the truncated isoform is labeled “hSMN∆7”. Endogenous Drosophila melanogaster Smn is labeled “Fruitfly”. To generate a more vertebrate-like SMN, key amino acids in Drosophila SMN were changed to amino acids conserved in vertebrates. Using this SMN backbone, a serine to alanine mutation was made in the putative degron in both full-length (vSMN S201A ) and truncated SMN∆7 (vSMN∆7A). An additional SMN construct that is the same length as SMN∆7, but has the amino acid sequence GLRQ (the next amino acids in the sequence) rather than EMLA (the amino acids introduced by mis-splicing of SMN2 ) was made. The same serine to alanine mutation was made in this construct as well (MGLRQ* and MGLRQ* S201A ). Finally, to mimic a phosphorylated serine the full-length SMN construct (vSmn S201D ) and truncated SM N (vSMN∆7D) were made. B. Western blotting was used to determine protein levels of each of these SMN constructs, with expression driven by the endogenous promoter, in Drosophila S2 cells. Both the vSMN and vSMN∆7S proteins show increased levels when the serine is mutated to an alanine, indicating disruption of the normal degradation of SMN. Additionally, MGLRQ* protein is present at higher levels than is vSMN∆7S and protein levels do not change when the serine is mutated to an alanine. Normalized fold change as compared to vSmn levels is indicated at the bottom. *p

    Techniques Used: Mutagenesis, Variant Assay, Sequencing, Labeling, Construct, Western Blot, Expressing

    Depletion of Slmb/B-TrCP results in an increase of SMN levels. A. Depletion of Slmb using 10 day (1 Od) treatment with dsRNA in Drosophila S2 cells resulted in modestly increased SMN levels. Following Slmb RNAi, full-length SMN levels were increased as compared to cells treated with control dsRNA against Gaussia Luciferase, which is not expressed in S2 cells. B. The effect of B-TrCP depletion on SMN levels in human cells was tested using siRNA that targets both B-TrCP1 and B-TrCP2 in HeLa cells. We detected a modest increase in levels of full-length endogenous SMN after B-TrCP RNAi but not control (scramble) RNAi. C. Drosophila S2 cells were treated with cycloheximide (CHX), an inhibitor of protein synthesis, following Slmb depletion following a 3d dsRNA treatment to test whether differences in protein levels would be exacerbated when the production of new protein was prevented. SMN protein levels were also directly targeted using dsRNA against Smn as a positive control for the RNAi treatment. As a negative control (Ctrl), dsRNA against oskar , which is not expressed in S2 cells, was used. Protein was collected at 0, 2, and 6 hours post CHX treatment. At 6 hours post-CHX treatment there is a modest increase in full-length SMN levels following Slmb RNAi as compared to the initial timepoint (0h) and as compared to control RNAi treatment.
    Figure Legend Snippet: Depletion of Slmb/B-TrCP results in an increase of SMN levels. A. Depletion of Slmb using 10 day (1 Od) treatment with dsRNA in Drosophila S2 cells resulted in modestly increased SMN levels. Following Slmb RNAi, full-length SMN levels were increased as compared to cells treated with control dsRNA against Gaussia Luciferase, which is not expressed in S2 cells. B. The effect of B-TrCP depletion on SMN levels in human cells was tested using siRNA that targets both B-TrCP1 and B-TrCP2 in HeLa cells. We detected a modest increase in levels of full-length endogenous SMN after B-TrCP RNAi but not control (scramble) RNAi. C. Drosophila S2 cells were treated with cycloheximide (CHX), an inhibitor of protein synthesis, following Slmb depletion following a 3d dsRNA treatment to test whether differences in protein levels would be exacerbated when the production of new protein was prevented. SMN protein levels were also directly targeted using dsRNA against Smn as a positive control for the RNAi treatment. As a negative control (Ctrl), dsRNA against oskar , which is not expressed in S2 cells, was used. Protein was collected at 0, 2, and 6 hours post CHX treatment. At 6 hours post-CHX treatment there is a modest increase in full-length SMN levels following Slmb RNAi as compared to the initial timepoint (0h) and as compared to control RNAi treatment.

    Techniques Used: Luciferase, Positive Control, Negative Control

    13) Product Images from "The Drosophila metallopeptidase superdeath decouples apoptosis from the activation of the ER stress response"

    Article Title: The Drosophila metallopeptidase superdeath decouples apoptosis from the activation of the ER stress response

    Journal: bioRxiv

    doi: 10.1101/620492

    Loss of superdeath reduces ER stress-associated apoptosis. Reducing expression of superdeath reduces apoptosis and degeneration in models of ER stress. A. Degeneration caused by overexpression of Rh1 G69D is partially rescued by RNAi-mediated knockdown of superdeath expression (15299 ± 1658 pixels, N = 10 in Rh1 G69D / superdeathi flies as compared to 11942 ± 473 pixels, N = 10 in Rh1 G69D /controls). B. Eye size also showed a small increase when the superdeath RNAi construct was expressed in a wild-type background (28867 ± 1566 pixels, N = 10) as compared to controls (25968 ± 1026 pixels, N = 10), but no qualitative differences were observed. C. Rh1 G69D / superdeathi eye imaginal discs display reduced apoptosis compared to Rh1 G69D /controls as measured by TUNEL staining. D. S2 cells treated with DsRNA against EGFP increased expression of the apoptotic gene rpr after 7.5 hours of DTT exposure (2.23 ± 0.33, N = 6) as compared to DMSO-treated control cells (1.00 ± 0.09, N = 6). Activation of rpr expression was significantly reduced in S2 cells that were treated with DsRNA against superdeath (1.47 ± 0.07, N = 6 in DTT-treated cells compared to 1.00 ± 0.11, N = 6 with DMSO). E. Activation of JNK signaling was reduced in Rh1 G69D / superdeathi eye imaginal discs compared to Rh1 G69D /controls as determined by expression of puc - LacZ . F. When quantified, LacZ levels were significantly lower in Rh1 G69D / superdeathi eye discs (0.367 ± 0.082, N = 3) as compared to Rh1 G69D /controls (1.00 ± 0.39, N = 4). Values are average ± SD. Scale bars = 0.1 mm. * P
    Figure Legend Snippet: Loss of superdeath reduces ER stress-associated apoptosis. Reducing expression of superdeath reduces apoptosis and degeneration in models of ER stress. A. Degeneration caused by overexpression of Rh1 G69D is partially rescued by RNAi-mediated knockdown of superdeath expression (15299 ± 1658 pixels, N = 10 in Rh1 G69D / superdeathi flies as compared to 11942 ± 473 pixels, N = 10 in Rh1 G69D /controls). B. Eye size also showed a small increase when the superdeath RNAi construct was expressed in a wild-type background (28867 ± 1566 pixels, N = 10) as compared to controls (25968 ± 1026 pixels, N = 10), but no qualitative differences were observed. C. Rh1 G69D / superdeathi eye imaginal discs display reduced apoptosis compared to Rh1 G69D /controls as measured by TUNEL staining. D. S2 cells treated with DsRNA against EGFP increased expression of the apoptotic gene rpr after 7.5 hours of DTT exposure (2.23 ± 0.33, N = 6) as compared to DMSO-treated control cells (1.00 ± 0.09, N = 6). Activation of rpr expression was significantly reduced in S2 cells that were treated with DsRNA against superdeath (1.47 ± 0.07, N = 6 in DTT-treated cells compared to 1.00 ± 0.11, N = 6 with DMSO). E. Activation of JNK signaling was reduced in Rh1 G69D / superdeathi eye imaginal discs compared to Rh1 G69D /controls as determined by expression of puc - LacZ . F. When quantified, LacZ levels were significantly lower in Rh1 G69D / superdeathi eye discs (0.367 ± 0.082, N = 3) as compared to Rh1 G69D /controls (1.00 ± 0.39, N = 4). Values are average ± SD. Scale bars = 0.1 mm. * P

    Techniques Used: Expressing, Over Expression, Construct, TUNEL Assay, Staining, Activation Assay

    Loss of superdeath does not alter IRE1 or PERK activation. Activation of the UPR is not altered by loss of superdeath in models of ER stress. A. Rh1 G69D / superdeathi eye discs do not display altered expression of Xbp1-EGFP or rhodopsin as compared to Rh1 G69D /controls. Eye discs were dissected from wandering L3 larvae expressing Rh1 G69D and UAS - Xbp1 - EGFP , stained for rhodopsin and GFP as an indicator of ER stress, and counterstained with 4’,6-diamidino-2-pheneylindole (DAPI). B. Loss of superdeath does not significantly alter Xbp1-EGFP expression (0.905 ± 0.057, N = 4) compared to Rh1 G69D /controls (1.00 ± 0.25, N = 4). C. Rhodopsin levels were also not significantly altered (0.899 ± 0.071, N = 4 relative to 1.00 ± 0.10, N = 4 in controls). D. DTT treatment increased Xbp1 splicing in S2 cells compared to control cells treated with DMSO. This increase was similar in S2 cells treated with DsRNA against either EGFP or superdeath . E. Rh1 G69D / superdeathi eye discs had similar levels of P-eif2α as compared to Rh1 G69D /controls. F. DTT treatment in S2 cells increased levels of P-eif2α compared to the control DMSO treatment. This increase was similar to cells treated with DsRNA against either EGFP or superdeath . Values are average ± SD. Scale bars = 0.1 mm.
    Figure Legend Snippet: Loss of superdeath does not alter IRE1 or PERK activation. Activation of the UPR is not altered by loss of superdeath in models of ER stress. A. Rh1 G69D / superdeathi eye discs do not display altered expression of Xbp1-EGFP or rhodopsin as compared to Rh1 G69D /controls. Eye discs were dissected from wandering L3 larvae expressing Rh1 G69D and UAS - Xbp1 - EGFP , stained for rhodopsin and GFP as an indicator of ER stress, and counterstained with 4’,6-diamidino-2-pheneylindole (DAPI). B. Loss of superdeath does not significantly alter Xbp1-EGFP expression (0.905 ± 0.057, N = 4) compared to Rh1 G69D /controls (1.00 ± 0.25, N = 4). C. Rhodopsin levels were also not significantly altered (0.899 ± 0.071, N = 4 relative to 1.00 ± 0.10, N = 4 in controls). D. DTT treatment increased Xbp1 splicing in S2 cells compared to control cells treated with DMSO. This increase was similar in S2 cells treated with DsRNA against either EGFP or superdeath . E. Rh1 G69D / superdeathi eye discs had similar levels of P-eif2α as compared to Rh1 G69D /controls. F. DTT treatment in S2 cells increased levels of P-eif2α compared to the control DMSO treatment. This increase was similar to cells treated with DsRNA against either EGFP or superdeath . Values are average ± SD. Scale bars = 0.1 mm.

    Techniques Used: Activation Assay, Expressing, Staining

    Specificity of V5 staining. The signal for Superdeath-V5 in Figure 6 is specific to expression of the V5 tag. A. S2 cells that were not transformed with the pMT-DEST48-Superdeath-V5 vector were stained for rabbit αV5 (green) and counterstained with DAPI. Only background staining and no punctate staining indicative of Superdeath is detectable. B. S2 cells that were not transformed with the pMT-DEST48-Superdeath-V5 vector were stained for mouse αV5 (green) and counterstained with DAPI. No punctate staining indicative of Superdeath is detectable. Scale bars = 0.01 mm.
    Figure Legend Snippet: Specificity of V5 staining. The signal for Superdeath-V5 in Figure 6 is specific to expression of the V5 tag. A. S2 cells that were not transformed with the pMT-DEST48-Superdeath-V5 vector were stained for rabbit αV5 (green) and counterstained with DAPI. Only background staining and no punctate staining indicative of Superdeath is detectable. B. S2 cells that were not transformed with the pMT-DEST48-Superdeath-V5 vector were stained for mouse αV5 (green) and counterstained with DAPI. No punctate staining indicative of Superdeath is detectable. Scale bars = 0.01 mm.

    Techniques Used: Staining, Expressing, Transformation Assay, Plasmid Preparation

    Superdeath is localized to the endoplasmic reticulum. Superdeath predominantly localizes to the ER membrane. A. Superdeath localizes to the ER. S2 cells expressing Superdeath-V5 were stained for V5 (green) and Calnexin 99A (red) and counterstained with DAPI. A’ and A” represent the highlighted panels from A . White arrows highlight select sites of V5 and Calnexin 99A overlap. B. Superdeath does not localize to the golgi. S2 cells expressing Superdeath-V5 were stained for V5 (green) and Golgin-84 (red) and counterstained with DAPI. B’ and B” represent the highlighted panels from B . White arrows indicate select sites of independent V5 staining or Golgin-84 staining. C. Superdeath does not primarily localize to the lysosome. S2 cells expressing Superdeath-V5 were stained for V5 (green) and Lamp1 (red) and counterstained with DAPI. C’ and C” represent the highlighted panels from C . White arrows indicate select sites of independent V5 staining or Lamp1 staining. Scale bars = 0.01 mm.
    Figure Legend Snippet: Superdeath is localized to the endoplasmic reticulum. Superdeath predominantly localizes to the ER membrane. A. Superdeath localizes to the ER. S2 cells expressing Superdeath-V5 were stained for V5 (green) and Calnexin 99A (red) and counterstained with DAPI. A’ and A” represent the highlighted panels from A . White arrows highlight select sites of V5 and Calnexin 99A overlap. B. Superdeath does not localize to the golgi. S2 cells expressing Superdeath-V5 were stained for V5 (green) and Golgin-84 (red) and counterstained with DAPI. B’ and B” represent the highlighted panels from B . White arrows indicate select sites of independent V5 staining or Golgin-84 staining. C. Superdeath does not primarily localize to the lysosome. S2 cells expressing Superdeath-V5 were stained for V5 (green) and Lamp1 (red) and counterstained with DAPI. C’ and C” represent the highlighted panels from C . White arrows indicate select sites of independent V5 staining or Lamp1 staining. Scale bars = 0.01 mm.

    Techniques Used: Expressing, Staining

    superdeath expression is efficiently reduced by both RNAi and DsRNA. The RNAi and DsRNA targeting superdeath in our models of ER stress significantly reduce superdeath expression. A. The Bloomington Drosophila Stock Center superdeath RNAi strain (42947) used in this study efficiently reduces expression of superdeath (27.8% of controls, N = 4). The RNAi construct was driven ubiquitously by Tubulin - GAL4 , and expression determined in wandering L3 larvae compared to controls expressing only Tubulin - GAL4 (N = 4). B. S2 Cells treated with DsRNA targeted against superdeath also had near complete reduction in superdeath transcript levels (10.2%, N = 6) as compared to control cells treated with DsRNA against EGFP (N = 6). Values are average ± SD. *** P
    Figure Legend Snippet: superdeath expression is efficiently reduced by both RNAi and DsRNA. The RNAi and DsRNA targeting superdeath in our models of ER stress significantly reduce superdeath expression. A. The Bloomington Drosophila Stock Center superdeath RNAi strain (42947) used in this study efficiently reduces expression of superdeath (27.8% of controls, N = 4). The RNAi construct was driven ubiquitously by Tubulin - GAL4 , and expression determined in wandering L3 larvae compared to controls expressing only Tubulin - GAL4 (N = 4). B. S2 Cells treated with DsRNA targeted against superdeath also had near complete reduction in superdeath transcript levels (10.2%, N = 6) as compared to control cells treated with DsRNA against EGFP (N = 6). Values are average ± SD. *** P

    Techniques Used: Expressing, Construct

    14) Product Images from "CBP / p300-mediated acetylation of histone H3 on lysine 56"

    Article Title: CBP / p300-mediated acetylation of histone H3 on lysine 56

    Journal: Nature

    doi: 10.1038/nature07861

    Drosophila CBP acetylates H3 K56, while Sir2 deacetylates H3 K56Ac in vivo a . Inhibition of CBP by curcumin lowers levels of H3 K56Ac in S2 cells. Duplicate analyses are shown. b . CBP acetylates H3 K56. c . CBP is required for the increase of H3 K56Ac on chromatin after DNA damage. d . Inhibition of NAD-dependent HDACs by nicotinamide increases K56Ac. e . Sir2 deacetylates H3 K56Ac.
    Figure Legend Snippet: Drosophila CBP acetylates H3 K56, while Sir2 deacetylates H3 K56Ac in vivo a . Inhibition of CBP by curcumin lowers levels of H3 K56Ac in S2 cells. Duplicate analyses are shown. b . CBP acetylates H3 K56. c . CBP is required for the increase of H3 K56Ac on chromatin after DNA damage. d . Inhibition of NAD-dependent HDACs by nicotinamide increases K56Ac. e . Sir2 deacetylates H3 K56Ac.

    Techniques Used: In Vivo, Inhibition

    15) Product Images from "Three-tier regulation of cell number plasticity by neurotrophins and Tolls in Drosophila"

    Article Title: Three-tier regulation of cell number plasticity by neurotrophins and Tolls in Drosophila

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201607098

    DNT1 and 2 are cleaved by conserved furin proteases. (A) The prodomain α-helix of Spz (boxes) required to activate Toll-1 is not conserved in DNT1 and 2. Yellow highlights indicate corresponding sequences that are not conserved. (B) The prodomains of DNT1 and 2 but not Spz have conserved furin sites. c.s., cleavage site. (C) Site-directed mutagenesis of furin sequences. Red letters mark amino acid substitutions. (D) Mutant DNT1-FL-HA and DNT2-FL-HA forms expressed in S2 cells and visualized in Western blots with anti-HA from lysate and secreted medium. Black arrows indicate normal forms, and red arrows indicate mutant products. (E) Anti-GFP Western blot upon overexpression of C-terminally tagged DNTs in the retina with GMR-GAL4 shows that furin cleavage occurs in vivo (black arrows: DNT1, blue arrows: DNT2). Molecular masses on the left of each blot are given in kilodaltons.
    Figure Legend Snippet: DNT1 and 2 are cleaved by conserved furin proteases. (A) The prodomain α-helix of Spz (boxes) required to activate Toll-1 is not conserved in DNT1 and 2. Yellow highlights indicate corresponding sequences that are not conserved. (B) The prodomains of DNT1 and 2 but not Spz have conserved furin sites. c.s., cleavage site. (C) Site-directed mutagenesis of furin sequences. Red letters mark amino acid substitutions. (D) Mutant DNT1-FL-HA and DNT2-FL-HA forms expressed in S2 cells and visualized in Western blots with anti-HA from lysate and secreted medium. Black arrows indicate normal forms, and red arrows indicate mutant products. (E) Anti-GFP Western blot upon overexpression of C-terminally tagged DNTs in the retina with GMR-GAL4 shows that furin cleavage occurs in vivo (black arrows: DNT1, blue arrows: DNT2). Molecular masses on the left of each blot are given in kilodaltons.

    Techniques Used: Mutagenesis, Western Blot, Over Expression, In Vivo

    16) Product Images from "Diverse Forms of RPS9 Splicing Are Part of an Evolving Autoregulatory Circuit"

    Article Title: Diverse Forms of RPS9 Splicing Are Part of an Evolving Autoregulatory Circuit

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1002620

    D. melanogaster RpS9 is autoregulated by alternative splicing coupled to NMD. A) Illustration of RpS9 mRNA isoforms assessed by PCR (the PTC+ isoform is indicated by a red octagon). Primers sets (arrows) were designed to amplify multiple or specific RpS9 mRNA isoforms (RT-PCR and qPCR primers, respectively). B) RT-PCR validation of the RpS9 PTC+ mRNA isoform degraded by NMD. C) Experimental design used to assess the affect of UPF1 knock-down on the abundance of RpS9 mRNA isoforms. D) RT-qPCR determination of RpS9 PTC+ mRNA isoform abundance (top panels) and total endogenous RpS9 mRNA abundance (bottom panels) in S2 cells transfected with a plasmid constitutively expressing an RpS9 cDNA (red circles) or an empty vector control (blue circles). The affect of UPF1 knock-down (via incubation with dsRNA) on each RpS9 mRNA isoform (right panels) is compared to a non-specific dsRNA control (left panels). RpS9 mRNA isoform abundance values were divided by GAPDH1 mRNA abundance values to obtain ratios internally controlled for variations in cDNA quantity. Log 2 transformed ratios for each of three biological replicates is shown as a point and the mean as a dash.
    Figure Legend Snippet: D. melanogaster RpS9 is autoregulated by alternative splicing coupled to NMD. A) Illustration of RpS9 mRNA isoforms assessed by PCR (the PTC+ isoform is indicated by a red octagon). Primers sets (arrows) were designed to amplify multiple or specific RpS9 mRNA isoforms (RT-PCR and qPCR primers, respectively). B) RT-PCR validation of the RpS9 PTC+ mRNA isoform degraded by NMD. C) Experimental design used to assess the affect of UPF1 knock-down on the abundance of RpS9 mRNA isoforms. D) RT-qPCR determination of RpS9 PTC+ mRNA isoform abundance (top panels) and total endogenous RpS9 mRNA abundance (bottom panels) in S2 cells transfected with a plasmid constitutively expressing an RpS9 cDNA (red circles) or an empty vector control (blue circles). The affect of UPF1 knock-down (via incubation with dsRNA) on each RpS9 mRNA isoform (right panels) is compared to a non-specific dsRNA control (left panels). RpS9 mRNA isoform abundance values were divided by GAPDH1 mRNA abundance values to obtain ratios internally controlled for variations in cDNA quantity. Log 2 transformed ratios for each of three biological replicates is shown as a point and the mean as a dash.

    Techniques Used: Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Transfection, Plasmid Preparation, Expressing, Incubation, Transformation Assay

    17) Product Images from "Drosophila ZDHHC8 palmitoylates scribble and Ras64B and controls growth and viability"

    Article Title: Drosophila ZDHHC8 palmitoylates scribble and Ras64B and controls growth and viability

    Journal: bioRxiv

    doi: 10.1101/365247

    dZDHHC8 resides at the Golgi (A) Antibody against dZDHHC8 specifically detects dZDHHC8 in immunofluorescent stainings of Drosophila S2 cells treated with control (luciferase) dsRNA as there is no staining upon dZDHHC8 knockdown (dsRNA anti dZDHHC8). (B) Endogenous dZDHHC8 (red) was co-stained with markers for different cellular compartments (green). Overexpressed organelle markers are Golgi-tethered fringe-myc, HA-Rab5 marking endosomes, GFP-KDEL marking endoplasmic reticulum and dLamp1-GFP marking lysosomes. DAPI was used to stain nuclei. dZDHHC8 co-localises with the Golgi-tethered marker.
    Figure Legend Snippet: dZDHHC8 resides at the Golgi (A) Antibody against dZDHHC8 specifically detects dZDHHC8 in immunofluorescent stainings of Drosophila S2 cells treated with control (luciferase) dsRNA as there is no staining upon dZDHHC8 knockdown (dsRNA anti dZDHHC8). (B) Endogenous dZDHHC8 (red) was co-stained with markers for different cellular compartments (green). Overexpressed organelle markers are Golgi-tethered fringe-myc, HA-Rab5 marking endosomes, GFP-KDEL marking endoplasmic reticulum and dLamp1-GFP marking lysosomes. DAPI was used to stain nuclei. dZDHHC8 co-localises with the Golgi-tethered marker.

    Techniques Used: Luciferase, Staining, Marker

    18) Product Images from "Regulation of Yki/Yap subcellular localization and Hpo signaling by a nuclear kinase PRP4K"

    Article Title: Regulation of Yki/Yap subcellular localization and Hpo signaling by a nuclear kinase PRP4K

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04090-2

    PRP4K inhibits Yki nuclear localization and activity by phosphorylating Yki on Ser111/250. a , b S2 cells treated with control or Wts dsRNA were transfected with the indicated constructs. Cell extracts were separated on phos-tag conjugated (as indicated) or regular SDS-PAGE and immunoblotted with the indicated antibodies. c , d Cell extracts from eye discs carrying control (ctrl) or PRP4K mutant clones were separated on phos-tag conjugated or regular SDS-PAGE and immunoblotted with the indicated antibodies. e Sequence alignment of wild type and mutated Wts phosphorylation sites included in the indicated GST-Yki fusion constructs. The phospho acceptor sites are highlighted in red. “X” in the consensus sequence denotes any amino acid. f Autoradiograph (top panel) of an in vitro kinase assay using GST-Yki fusion proteins containing the indicated Wts phosphorylation sites and immunopurified HA-PRP4K or HA-PRP4K KR in the presence of [γ- 32 p] ATP. Coomassie blue staining (middle) and western blot (bottom) show that equal amounts of GST fusion proteins and HA-PRP4K were used. g Immunostaining of S2 cells transfected with the indicated constructs. h Quantification of subcellular localization of Myc-tagged wild type and mutant Yki transfected into S2 cells with GFP, PRP4K-GFP, or PRP4K KR -GFP. C > N: higher Myc signal intensity in cytoplasm than in nucleus; C = N: equal Myc signal intensity in cytoplasm and nucleus; C
    Figure Legend Snippet: PRP4K inhibits Yki nuclear localization and activity by phosphorylating Yki on Ser111/250. a , b S2 cells treated with control or Wts dsRNA were transfected with the indicated constructs. Cell extracts were separated on phos-tag conjugated (as indicated) or regular SDS-PAGE and immunoblotted with the indicated antibodies. c , d Cell extracts from eye discs carrying control (ctrl) or PRP4K mutant clones were separated on phos-tag conjugated or regular SDS-PAGE and immunoblotted with the indicated antibodies. e Sequence alignment of wild type and mutated Wts phosphorylation sites included in the indicated GST-Yki fusion constructs. The phospho acceptor sites are highlighted in red. “X” in the consensus sequence denotes any amino acid. f Autoradiograph (top panel) of an in vitro kinase assay using GST-Yki fusion proteins containing the indicated Wts phosphorylation sites and immunopurified HA-PRP4K or HA-PRP4K KR in the presence of [γ- 32 p] ATP. Coomassie blue staining (middle) and western blot (bottom) show that equal amounts of GST fusion proteins and HA-PRP4K were used. g Immunostaining of S2 cells transfected with the indicated constructs. h Quantification of subcellular localization of Myc-tagged wild type and mutant Yki transfected into S2 cells with GFP, PRP4K-GFP, or PRP4K KR -GFP. C > N: higher Myc signal intensity in cytoplasm than in nucleus; C = N: equal Myc signal intensity in cytoplasm and nucleus; C

    Techniques Used: Activity Assay, Transfection, Construct, SDS Page, Mutagenesis, Clone Assay, Sequencing, Autoradiography, In Vitro, Kinase Assay, Staining, Western Blot, Immunostaining

    19) Product Images from "Regulation of Yki/Yap subcellular localization and Hpo signaling by a nuclear kinase PRP4K"

    Article Title: Regulation of Yki/Yap subcellular localization and Hpo signaling by a nuclear kinase PRP4K

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04090-2

    PRP4K inhibits Yki nuclear localization and activity by phosphorylating Yki on Ser111/250. a , b S2 cells treated with control or Wts dsRNA were transfected with the indicated constructs. Cell extracts were separated on phos-tag conjugated (as indicated) or regular SDS-PAGE and immunoblotted with the indicated antibodies. c , d Cell extracts from eye discs carrying control (ctrl) or PRP4K mutant clones were separated on phos-tag conjugated or regular SDS-PAGE and immunoblotted with the indicated antibodies. e Sequence alignment of wild type and mutated Wts phosphorylation sites included in the indicated GST-Yki fusion constructs. The phospho acceptor sites are highlighted in red. “X” in the consensus sequence denotes any amino acid. f Autoradiograph (top panel) of an in vitro kinase assay using GST-Yki fusion proteins containing the indicated Wts phosphorylation sites and immunopurified HA-PRP4K or HA-PRP4K KR in the presence of [γ- 32 p] ATP. Coomassie blue staining (middle) and western blot (bottom) show that equal amounts of GST fusion proteins and HA-PRP4K were used. g Immunostaining of S2 cells transfected with the indicated constructs. h Quantification of subcellular localization of Myc-tagged wild type and mutant Yki transfected into S2 cells with GFP, PRP4K-GFP, or PRP4K KR -GFP. C > N: higher Myc signal intensity in cytoplasm than in nucleus; C = N: equal Myc signal intensity in cytoplasm and nucleus; C
    Figure Legend Snippet: PRP4K inhibits Yki nuclear localization and activity by phosphorylating Yki on Ser111/250. a , b S2 cells treated with control or Wts dsRNA were transfected with the indicated constructs. Cell extracts were separated on phos-tag conjugated (as indicated) or regular SDS-PAGE and immunoblotted with the indicated antibodies. c , d Cell extracts from eye discs carrying control (ctrl) or PRP4K mutant clones were separated on phos-tag conjugated or regular SDS-PAGE and immunoblotted with the indicated antibodies. e Sequence alignment of wild type and mutated Wts phosphorylation sites included in the indicated GST-Yki fusion constructs. The phospho acceptor sites are highlighted in red. “X” in the consensus sequence denotes any amino acid. f Autoradiograph (top panel) of an in vitro kinase assay using GST-Yki fusion proteins containing the indicated Wts phosphorylation sites and immunopurified HA-PRP4K or HA-PRP4K KR in the presence of [γ- 32 p] ATP. Coomassie blue staining (middle) and western blot (bottom) show that equal amounts of GST fusion proteins and HA-PRP4K were used. g Immunostaining of S2 cells transfected with the indicated constructs. h Quantification of subcellular localization of Myc-tagged wild type and mutant Yki transfected into S2 cells with GFP, PRP4K-GFP, or PRP4K KR -GFP. C > N: higher Myc signal intensity in cytoplasm than in nucleus; C = N: equal Myc signal intensity in cytoplasm and nucleus; C

    Techniques Used: Activity Assay, Transfection, Construct, SDS Page, Mutagenesis, Clone Assay, Sequencing, Autoradiography, In Vitro, Kinase Assay, Staining, Western Blot, Immunostaining

    20) Product Images from "Reduced Stimulation of Recombinant DNA Polymerase ? and Mitochondrial DNA (mtDNA) Helicase by Variants of Mitochondrial Single-stranded DNA-binding Protein (mtSSB) Correlates with Defects in mtDNA Replication in Animal Cells *"

    Article Title: Reduced Stimulation of Recombinant DNA Polymerase ? and Mitochondrial DNA (mtDNA) Helicase by Variants of Mitochondrial Single-stranded DNA-binding Protein (mtSSB) Correlates with Defects in mtDNA Replication in Animal Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.289983

    Knockdown of Dm mtSSB endo and overexpression of Dm mtSSB variants in S2 cells causes mtDNA depletion under conditions of mitochondrial homeostasis. A , cells carrying pMt/ Dm mtSSBwt/Hy were cultured in the presence of dsRNA homologous to the 3′-UTR
    Figure Legend Snippet: Knockdown of Dm mtSSB endo and overexpression of Dm mtSSB variants in S2 cells causes mtDNA depletion under conditions of mitochondrial homeostasis. A , cells carrying pMt/ Dm mtSSBwt/Hy were cultured in the presence of dsRNA homologous to the 3′-UTR

    Techniques Used: Over Expression, Cell Culture

    Expression of  Dm mtSSB variants impedes recovery from mtDNA depletion in  Drosophila  S2 cells. A ,  left panel , 0.2 μg/ml of EtBr was applied for 3 days to the growth media of cells carrying the  Dm mtSSBwt gene, followed by a recovery time of 12 days
    Figure Legend Snippet: Expression of Dm mtSSB variants impedes recovery from mtDNA depletion in Drosophila S2 cells. A , left panel , 0.2 μg/ml of EtBr was applied for 3 days to the growth media of cells carrying the Dm mtSSBwt gene, followed by a recovery time of 12 days

    Techniques Used: Expressing

    21) Product Images from "Intronic gRNAs for the Construction of Minimal Gene Drive Systems"

    Article Title: Intronic gRNAs for the Construction of Minimal Gene Drive Systems

    Journal: Frontiers in Bioengineering and Biotechnology

    doi: 10.3389/fbioe.2022.857460

    Characterization of intron-encoded gRNAs in Drosophila S2 cells. (A) Assay consists of a GFP reporter split by the presence of an intron and the dCas9-VPR activator component. In the absence of a gRNA guiding the activator, the reporter exhibits low levels of eGFP fluorescence. Following the activation by the dCas9:gRNA complex at the 5′ QUAS motif, eGFP fluorescence is amplified. This requires both successful splicing and the gRNA expression from within the intron. Relative fluorescence is subsequently measured by flow cytometry, allowing each gRNA configuration to be compared to a negative control (no gRNA) and a positive control (gRNA provided separately). (B) Three intronic gRNA designs were tested with or without a U6 promoter, each with three previously characterized QUAS-targeting gRNA spacers, cloned at the multiple cloning site (MCS) near the intronic branch point, or directly adjacent to the polypyrimidine stretch toward the 3′ end of each intron. (C) Relative mean eGFP fluorescence and SD from triplicate transfections of each intronic gRNA variant, using either the ftz or minimal introns. The p values were calculated using one-way ANOVA and Tukey multiple comparisons of means (* p
    Figure Legend Snippet: Characterization of intron-encoded gRNAs in Drosophila S2 cells. (A) Assay consists of a GFP reporter split by the presence of an intron and the dCas9-VPR activator component. In the absence of a gRNA guiding the activator, the reporter exhibits low levels of eGFP fluorescence. Following the activation by the dCas9:gRNA complex at the 5′ QUAS motif, eGFP fluorescence is amplified. This requires both successful splicing and the gRNA expression from within the intron. Relative fluorescence is subsequently measured by flow cytometry, allowing each gRNA configuration to be compared to a negative control (no gRNA) and a positive control (gRNA provided separately). (B) Three intronic gRNA designs were tested with or without a U6 promoter, each with three previously characterized QUAS-targeting gRNA spacers, cloned at the multiple cloning site (MCS) near the intronic branch point, or directly adjacent to the polypyrimidine stretch toward the 3′ end of each intron. (C) Relative mean eGFP fluorescence and SD from triplicate transfections of each intronic gRNA variant, using either the ftz or minimal introns. The p values were calculated using one-way ANOVA and Tukey multiple comparisons of means (* p

    Techniques Used: Fluorescence, Activation Assay, Amplification, Expressing, Flow Cytometry, Negative Control, Positive Control, Clone Assay, Transfection, Variant Assay

    22) Product Images from "The Non-Receptor Protein Tyrosine Phosphatase PTPN6 Mediates a Positive Regulatory Approach From the Interferon Regulatory Factor to the JAK/STAT Pathway in Litopenaeus vannamei"

    Article Title: The Non-Receptor Protein Tyrosine Phosphatase PTPN6 Mediates a Positive Regulatory Approach From the Interferon Regulatory Factor to the JAK/STAT Pathway in Litopenaeus vannamei

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2022.913955

    Tissue distribution and subcellular localization of LvPTPN6. (A) Expression of LvPTPN6 in L. vannamei tissues detected by qRT-PCR with EF-1α as internal control. The expression level of LvPTPN6 in pyloric cecum was set as the baseline (1.0). Each bar represents the mean ± SD ( n = 4). (B) Subcellular localization of HA-tagged LvPTPN6 was detected by confocal laser scanning microscopy analysis in S2 cells. LvPTPN6 was stained with Alexa Fluor 488 (green), the cytomembranes were visualized by β-actin stain with Alexa Flour 594 (red), and the nuclei were stained with Hoechst 33342 (blue).
    Figure Legend Snippet: Tissue distribution and subcellular localization of LvPTPN6. (A) Expression of LvPTPN6 in L. vannamei tissues detected by qRT-PCR with EF-1α as internal control. The expression level of LvPTPN6 in pyloric cecum was set as the baseline (1.0). Each bar represents the mean ± SD ( n = 4). (B) Subcellular localization of HA-tagged LvPTPN6 was detected by confocal laser scanning microscopy analysis in S2 cells. LvPTPN6 was stained with Alexa Fluor 488 (green), the cytomembranes were visualized by β-actin stain with Alexa Flour 594 (red), and the nuclei were stained with Hoechst 33342 (blue).

    Techniques Used: Expressing, Quantitative RT-PCR, Confocal Laser Scanning Microscopy, Staining

    23) Product Images from "Atrophin controls developmental signaling pathways via interactions with Trithorax-like"

    Article Title: Atrophin controls developmental signaling pathways via interactions with Trithorax-like

    Journal: eLife

    doi: 10.7554/eLife.23084

    Principal component analysis of Atro peaks. ( A ) Score plot showing the first two components from principal component analysis using enrichment of all factors mapped by modENCODE (S2 cells) within Atro-binding regions. The Atro-binding regions are colored according to the three classes defined by hierarchical clustering. R2X values represent the fraction of the total variation explained by each component. ( B ) The mean scaled enrichment values for each factor withing the three classes. All factors with enrichment values below 0.4 in all classes were excluded. DOI: http://dx.doi.org/10.7554/eLife.23084.006
    Figure Legend Snippet: Principal component analysis of Atro peaks. ( A ) Score plot showing the first two components from principal component analysis using enrichment of all factors mapped by modENCODE (S2 cells) within Atro-binding regions. The Atro-binding regions are colored according to the three classes defined by hierarchical clustering. R2X values represent the fraction of the total variation explained by each component. ( B ) The mean scaled enrichment values for each factor withing the three classes. All factors with enrichment values below 0.4 in all classes were excluded. DOI: http://dx.doi.org/10.7554/eLife.23084.006

    Techniques Used: Binding Assay

    Trl knockdown decreases Atro protein levels, coimmunoprecipitation of Trl and Atro, and pausing indices of Trl and Atro bound genes. ( A ) Trl knockdown by RNAi causes Atro protein levels to decrease (arrow), while no RNAi or GFP RNAi treatments do not affect Atro protein levels. Lamin was used as a loading control. ( B ) shows the coimmunoprecipitation of Atro and Trl. Each lane is labeled with the antibody used for each IP. Blot was stained with Trl antibody. The red arrows mark the Trl bands. Trl is coimmunoprecipitated with Atro but not with IgG control. ( C ) The pausing index (amount of Pol II in the promoter-proximal region versus the gene body calculated from PRO-seq data ( Kwak et al., 2013 ) is higher on average for genes bound by Atro and Trl than for all other expressed genes in S2 cells. DOI: http://dx.doi.org/10.7554/eLife.23084.021
    Figure Legend Snippet: Trl knockdown decreases Atro protein levels, coimmunoprecipitation of Trl and Atro, and pausing indices of Trl and Atro bound genes. ( A ) Trl knockdown by RNAi causes Atro protein levels to decrease (arrow), while no RNAi or GFP RNAi treatments do not affect Atro protein levels. Lamin was used as a loading control. ( B ) shows the coimmunoprecipitation of Atro and Trl. Each lane is labeled with the antibody used for each IP. Blot was stained with Trl antibody. The red arrows mark the Trl bands. Trl is coimmunoprecipitated with Atro but not with IgG control. ( C ) The pausing index (amount of Pol II in the promoter-proximal region versus the gene body calculated from PRO-seq data ( Kwak et al., 2013 ) is higher on average for genes bound by Atro and Trl than for all other expressed genes in S2 cells. DOI: http://dx.doi.org/10.7554/eLife.23084.021

    Techniques Used: Labeling, Staining

    24) Product Images from "IPIP27 Coordinates PtdIns(4,5)P2 Homeostasis for Successful Cytokinesis"

    Article Title: IPIP27 Coordinates PtdIns(4,5)P2 Homeostasis for Successful Cytokinesis

    Journal: Current Biology

    doi: 10.1016/j.cub.2019.01.043

    dIPIP Depletion Gives Rise to PtdIns(4,5)P 2 - and Actin-Rich Vacuoles with Mislocalized Cytokinetic Machinery (A) Top: live stills of control, dIPIP, or dOCRL-depleted S2 cells stably expressing GFP-Tubby. Scale bar, 5 μm. Bottom: quantitation of PtdIns(4,5) 2 -rich vacuoles. Bars represent the mean of 3 experiments with > 100 cells per condition per experiment. Error bars indicate SEM and ∗∗ p
    Figure Legend Snippet: dIPIP Depletion Gives Rise to PtdIns(4,5)P 2 - and Actin-Rich Vacuoles with Mislocalized Cytokinetic Machinery (A) Top: live stills of control, dIPIP, or dOCRL-depleted S2 cells stably expressing GFP-Tubby. Scale bar, 5 μm. Bottom: quantitation of PtdIns(4,5) 2 -rich vacuoles. Bars represent the mean of 3 experiments with > 100 cells per condition per experiment. Error bars indicate SEM and ∗∗ p

    Techniques Used: Stable Transfection, Expressing, Quantitation Assay

    Depletion of SH3PX1 or Pacsin 2 Phenocopies IPIP Depletion in Drosophila or Human Cells, Respectively (A) Pull-down assay using GST-dIPIP or GST-dIPIP PxxP mutant ( 156 RR 157 > AA) and Drosophila S2 cell lysate, followed by western blotting of input (5%), unbound (5%), and bound fractions (50%). (B) Schematic of SH3PX1 and pacsin 2. (C) Top left: S2 cells expressing dIPIP-mRuby were fixed and labeled for SH3PX1. Top right: live still of S2 cells expressing GFP-dOCRL and RFP-SH3PX1. Bottom left: S2 cells transiently expressing GFP-dOCRL and dIPIP-mRuby and fixed and labeled for SH3PX1. Arrowheads indicate colocalization in puncta. Bottom right: colocalization analysis using Pearson’s correlation coefficient. Bars represent means ± SEM from 3 experiments with ∼30 cells per experiment Scale bar, 5 μm. (D) Western blot showing SH3PX1 depletion. The bar graph shows relative protein abundance. Values are means ± SEM of 3 independent experiments each done in triplicate. ∗∗∗∗ p
    Figure Legend Snippet: Depletion of SH3PX1 or Pacsin 2 Phenocopies IPIP Depletion in Drosophila or Human Cells, Respectively (A) Pull-down assay using GST-dIPIP or GST-dIPIP PxxP mutant ( 156 RR 157 > AA) and Drosophila S2 cell lysate, followed by western blotting of input (5%), unbound (5%), and bound fractions (50%). (B) Schematic of SH3PX1 and pacsin 2. (C) Top left: S2 cells expressing dIPIP-mRuby were fixed and labeled for SH3PX1. Top right: live still of S2 cells expressing GFP-dOCRL and RFP-SH3PX1. Bottom left: S2 cells transiently expressing GFP-dOCRL and dIPIP-mRuby and fixed and labeled for SH3PX1. Arrowheads indicate colocalization in puncta. Bottom right: colocalization analysis using Pearson’s correlation coefficient. Bars represent means ± SEM from 3 experiments with ∼30 cells per experiment Scale bar, 5 μm. (D) Western blot showing SH3PX1 depletion. The bar graph shows relative protein abundance. Values are means ± SEM of 3 independent experiments each done in triplicate. ∗∗∗∗ p

    Techniques Used: Pull Down Assay, Mutagenesis, Western Blot, Expressing, Labeling

    dIPIP Binding to dOCRL and SH3PX1 Is Required for Successful Cytokinesis (A) Quantification of multinucleation upon expression of Myc-tagged wild-type (WT) dIPIP or the dOCRL binding-deficient F H mutant (F267A) in dIPIP-depleted S2 cells. ∗∗∗ p
    Figure Legend Snippet: dIPIP Binding to dOCRL and SH3PX1 Is Required for Successful Cytokinesis (A) Quantification of multinucleation upon expression of Myc-tagged wild-type (WT) dIPIP or the dOCRL binding-deficient F H mutant (F267A) in dIPIP-depleted S2 cells. ∗∗∗ p

    Techniques Used: Binding Assay, Expressing, Mutagenesis

    dIPIP Interacts with dOCRL and Is Required for Cytokinesis (A) Schematic of human IPIP27A and IPIP27B and Drosophila dIPIP. (B) Pull-down using GST- dIPIP wild-type (WT) or F H mutant (F267A) and Drosophila S2 cell lysate. Input (5%), unbound (5%), and bound fractions (50%) were blotted. (C) Confocal microscopy of dIPIP-mRuby (red) co-expressed with GFP-dOCRL (green) in live S2 cells. Scale bar, 5 μm. (D) Western blot showing RNAi-mediated depletion of dIPIP (left) or dOCRL (right) in S2 cells. Bar graphs show relative protein abundance. Values are means ± SEM of 3 independent experiments each done in triplicate, ∗∗∗∗ p
    Figure Legend Snippet: dIPIP Interacts with dOCRL and Is Required for Cytokinesis (A) Schematic of human IPIP27A and IPIP27B and Drosophila dIPIP. (B) Pull-down using GST- dIPIP wild-type (WT) or F H mutant (F267A) and Drosophila S2 cell lysate. Input (5%), unbound (5%), and bound fractions (50%) were blotted. (C) Confocal microscopy of dIPIP-mRuby (red) co-expressed with GFP-dOCRL (green) in live S2 cells. Scale bar, 5 μm. (D) Western blot showing RNAi-mediated depletion of dIPIP (left) or dOCRL (right) in S2 cells. Bar graphs show relative protein abundance. Values are means ± SEM of 3 independent experiments each done in triplicate, ∗∗∗∗ p

    Techniques Used: Mutagenesis, Confocal Microscopy, Western Blot

    Cortical Instability and Cytokinesis Failure upon dIPIP or IPIP27A Depletion Is Due to Dysregulated PtdIns(4,5)P 2 Homeostasis (A) Top: live stills of S2 cells stably expressing GFP-Tubby depleted of dIPIP or dOCRL and treated with 50 μM inactive analog ( o -3M3FBS) or PLC activator ( m -3M3FBS). Bottom: quantification of PtdIns(4,5)P 2 -rich vacuoles. Bars represent the mean of 3 experiments with 250–400 cells analyzed per condition per experiment. ∗∗ p
    Figure Legend Snippet: Cortical Instability and Cytokinesis Failure upon dIPIP or IPIP27A Depletion Is Due to Dysregulated PtdIns(4,5)P 2 Homeostasis (A) Top: live stills of S2 cells stably expressing GFP-Tubby depleted of dIPIP or dOCRL and treated with 50 μM inactive analog ( o -3M3FBS) or PLC activator ( m -3M3FBS). Bottom: quantification of PtdIns(4,5)P 2 -rich vacuoles. Bars represent the mean of 3 experiments with 250–400 cells analyzed per condition per experiment. ∗∗ p

    Techniques Used: Stable Transfection, Expressing, Planar Chromatography

    25) Product Images from "Therapeutic Targeting of Follicular T Cells with Chimeric Antigen Receptor-Expressing Natural Killer Cells"

    Article Title: Therapeutic Targeting of Follicular T Cells with Chimeric Antigen Receptor-Expressing Natural Killer Cells

    Journal: Cell reports. Medicine

    doi: 10.1016/j.xcrm.2020.100003

    PD-L1 CAR NK Cell Responses to Plate- and Cell-Associated PD-1 (A–C) Degranulation (surface exposure of CD107a) of control (left) or CAR (right) NK-92 following a 4-h incubation in the presence of (A) anti-PD-L1 IgG (20 μg/mL, control: goat IgG) or rhPD-1-Fc (10 μg/mL, control:IgG-Fc), (B) rhPD-1-Fc (displayed dose, control: IgG-Fc), or (C) PD-1 + Drosophila S2 cells (control: S2 cells) at an effector:target (E:T) ratio of 1:5. PMA/ionomycin used as positive control in (A) and (C). (D) Fold degranulation (over control NK-92, dotted line) of CAR NK-92 in the presence of control, PD-1 low , or PD-1 high Drosophila S2 cells at 1:5 E:T for 4 h (n = 2–3). (E) CAR NK-92 degranulation in the presence of control or PD-1 + Raji cells for 4 h at 1:5 E:T (n = 4). (F) Control or PD-1 + Raji uptake of PI following 4-h co-culture with either control or CAR NK-92 at a 20:1 E:T (n = 2–3). (G) Percentage lysis of 51 Cr-labeled PD-1 + Raji cells after incubation with control or CAR NK-92 for 4 h at various E:T ratios. Error bars in (B) and (G) denote standard deviations. Representative data from 1 of 2 (B and D–G) or 3 (A and C) experimental replicates are shown. Data analyzed via (D and F) 1-way ANOVA with multiple comparisons or (E) Student’s t test. See also Figures S1 – S3 .
    Figure Legend Snippet: PD-L1 CAR NK Cell Responses to Plate- and Cell-Associated PD-1 (A–C) Degranulation (surface exposure of CD107a) of control (left) or CAR (right) NK-92 following a 4-h incubation in the presence of (A) anti-PD-L1 IgG (20 μg/mL, control: goat IgG) or rhPD-1-Fc (10 μg/mL, control:IgG-Fc), (B) rhPD-1-Fc (displayed dose, control: IgG-Fc), or (C) PD-1 + Drosophila S2 cells (control: S2 cells) at an effector:target (E:T) ratio of 1:5. PMA/ionomycin used as positive control in (A) and (C). (D) Fold degranulation (over control NK-92, dotted line) of CAR NK-92 in the presence of control, PD-1 low , or PD-1 high Drosophila S2 cells at 1:5 E:T for 4 h (n = 2–3). (E) CAR NK-92 degranulation in the presence of control or PD-1 + Raji cells for 4 h at 1:5 E:T (n = 4). (F) Control or PD-1 + Raji uptake of PI following 4-h co-culture with either control or CAR NK-92 at a 20:1 E:T (n = 2–3). (G) Percentage lysis of 51 Cr-labeled PD-1 + Raji cells after incubation with control or CAR NK-92 for 4 h at various E:T ratios. Error bars in (B) and (G) denote standard deviations. Representative data from 1 of 2 (B and D–G) or 3 (A and C) experimental replicates are shown. Data analyzed via (D and F) 1-way ANOVA with multiple comparisons or (E) Student’s t test. See also Figures S1 – S3 .

    Techniques Used: Incubation, Positive Control, Co-Culture Assay, Lysis, Labeling

    26) Product Images from "Requirement for a Nuclear Function of ?-Catenin in Wnt Signaling"

    Article Title: Requirement for a Nuclear Function of ?-Catenin in Wnt Signaling

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.23.23.8462-8470.2003

    Requirement of endogenous β-catenin for the stimulatory effect of membrane-tethered β-catenin/plakoglobin on luciferase reporters. (A) Effect of β-catenin-specific siRNA on the expression of endogenous β-catenin. 293 cells or NIH 3T3 cells were treated with control (c) and human or mouse-specific β-catenin (hβ or mβ) siRNA, and the expression of endogenous β-catenin was determined by immunoblotting with anti-β-catenin antibodies. Equal loading was confirmed by Western blotting with anti-α-tubulin antibodies. (B) Requirement of endogenous β-catenin for the signaling activity of membrane-tethered plakoglobin (CnxPkg) in 293 cells. 293 cells were transfected with control (▪) or human β-catenin-specific ( ) siRNA, together with TOP-FLASH and the indicated expression constructs, and the luciferase activities were determined 48 h after transfection. (C) Requirement of endogenous β-catenin for the signaling activity of membrane-tethered β-catenin in NIH 3T3 cells. To achieve high sensitivity in NIH 3T3 cells, β-catenin was cotransfected with a mouse LEF-1 expression plasmid and a LEF-1 luciferase reporter that contains multiple LEF-1 binding sites. NIH 3T3 cells were transfected with control (▪) or mouse β-catenin-specific ( ) siRNA, together with the indicated expression constructs, and assayed for luciferase activity. Amino acid residues 675 to 781 of β-catenin were deleted from Cnxβ to form CnxβΔC. (D) Requirement of endogenous armadillo for the signaling activity of membrane-tethered β-catenin in Drosophila S2 cells. S2 cells were grown in six-well plates and treated with control (▪) or armadillo-specific ( ) dsRNA at a concentration of 15 μg/well. Cells were then transfected with LEF-luciferase and LEF-1 expression constructs with the indicated plasmids that encode various forms of plakoglobin and β-catenin. Amino acid residues 677 to 781 and 583 to 781 of β-catenin were removed from Cnxβ to form CnxβΔC1 and CnxβΔC2. (E) Requirement of endogenous β-catenin for membrane-tethered plakoglobin-induced DKK1 expression. 293 cells were transfected with control siRNA (▪) or human β-catenin-specific siRNA ( ) with the indicated expression plasmids. The expression of DKK1 gene was determined by quantitative real-time PCR after reverse transcription. vec, Vector.
    Figure Legend Snippet: Requirement of endogenous β-catenin for the stimulatory effect of membrane-tethered β-catenin/plakoglobin on luciferase reporters. (A) Effect of β-catenin-specific siRNA on the expression of endogenous β-catenin. 293 cells or NIH 3T3 cells were treated with control (c) and human or mouse-specific β-catenin (hβ or mβ) siRNA, and the expression of endogenous β-catenin was determined by immunoblotting with anti-β-catenin antibodies. Equal loading was confirmed by Western blotting with anti-α-tubulin antibodies. (B) Requirement of endogenous β-catenin for the signaling activity of membrane-tethered plakoglobin (CnxPkg) in 293 cells. 293 cells were transfected with control (▪) or human β-catenin-specific ( ) siRNA, together with TOP-FLASH and the indicated expression constructs, and the luciferase activities were determined 48 h after transfection. (C) Requirement of endogenous β-catenin for the signaling activity of membrane-tethered β-catenin in NIH 3T3 cells. To achieve high sensitivity in NIH 3T3 cells, β-catenin was cotransfected with a mouse LEF-1 expression plasmid and a LEF-1 luciferase reporter that contains multiple LEF-1 binding sites. NIH 3T3 cells were transfected with control (▪) or mouse β-catenin-specific ( ) siRNA, together with the indicated expression constructs, and assayed for luciferase activity. Amino acid residues 675 to 781 of β-catenin were deleted from Cnxβ to form CnxβΔC. (D) Requirement of endogenous armadillo for the signaling activity of membrane-tethered β-catenin in Drosophila S2 cells. S2 cells were grown in six-well plates and treated with control (▪) or armadillo-specific ( ) dsRNA at a concentration of 15 μg/well. Cells were then transfected with LEF-luciferase and LEF-1 expression constructs with the indicated plasmids that encode various forms of plakoglobin and β-catenin. Amino acid residues 677 to 781 and 583 to 781 of β-catenin were removed from Cnxβ to form CnxβΔC1 and CnxβΔC2. (E) Requirement of endogenous β-catenin for membrane-tethered plakoglobin-induced DKK1 expression. 293 cells were transfected with control siRNA (▪) or human β-catenin-specific siRNA ( ) with the indicated expression plasmids. The expression of DKK1 gene was determined by quantitative real-time PCR after reverse transcription. vec, Vector.

    Techniques Used: Luciferase, Expressing, Western Blot, Activity Assay, Transfection, Construct, Plasmid Preparation, Binding Assay, Concentration Assay, Real-time Polymerase Chain Reaction

    27) Product Images from "Heterochromatin Protein 1 (HP1a) Positively Regulates Euchromatic Gene Expression through RNA Transcript Association and Interaction with hnRNPs in Drosophila"

    Article Title: Heterochromatin Protein 1 (HP1a) Positively Regulates Euchromatic Gene Expression through RNA Transcript Association and Interaction with hnRNPs in Drosophila

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1000670

    The genes corresponding to transcript targets of HP1a in S2 cells show a good overlap with HP1a binding sites on polytene chromosomes and appear down-regulated in both S2 cells lacking HP1a and HP1a mutant larvae. (A) Localization of HP1a binding sites and the genes corresponding to the HP1a target transcripts along polytene chromosomes of Drosophila wild type larvae. Blue bars represent sites where the HP1a target genes overlap with HP1a immunosignals; orange bars indicate the localization of HP1a target genes that do not overlap with HP1a immunosignals. (B) Quantitative RT–PCR analysis of the expression, in S2 cells treated with dsRNA of HP1a, of a sub-set of target genes that overlap with HP1a on polytene chromosomes whose position is indicated in (A) by blue bars marked with asterisks and (C) a sub-set of HP1a target genes that do not overlap with HP1a on polytene chromosomes whose position is indicated in (A) by orange bars marked with asterisks. (D) Quantitative RT-PCR analysis of the expression, in S2 cells treated with dsRNA of HP1a, of a sub-set of genes that were not found among the HP1a target genes in S2 cells and do not co-map with any of the HP1a immunosignals along the polytene chromosomes. (E) Quantitative RT-PCR analysis of the expression, in wild type and HP1a mutant larvae, of the same sub-set of target genes reported in Figure 2B . (F) Quantitative RT-PCR analysis of the expression, in wild type and HP1a mutant larvae, of the same sub-set of target genes reported in Figure 2C . (G) Quantitative RT-PCR analysis of the expression, in wild type and HP1a mutant larvae, of the same sub-set of non target genes reported in Figure 2D .
    Figure Legend Snippet: The genes corresponding to transcript targets of HP1a in S2 cells show a good overlap with HP1a binding sites on polytene chromosomes and appear down-regulated in both S2 cells lacking HP1a and HP1a mutant larvae. (A) Localization of HP1a binding sites and the genes corresponding to the HP1a target transcripts along polytene chromosomes of Drosophila wild type larvae. Blue bars represent sites where the HP1a target genes overlap with HP1a immunosignals; orange bars indicate the localization of HP1a target genes that do not overlap with HP1a immunosignals. (B) Quantitative RT–PCR analysis of the expression, in S2 cells treated with dsRNA of HP1a, of a sub-set of target genes that overlap with HP1a on polytene chromosomes whose position is indicated in (A) by blue bars marked with asterisks and (C) a sub-set of HP1a target genes that do not overlap with HP1a on polytene chromosomes whose position is indicated in (A) by orange bars marked with asterisks. (D) Quantitative RT-PCR analysis of the expression, in S2 cells treated with dsRNA of HP1a, of a sub-set of genes that were not found among the HP1a target genes in S2 cells and do not co-map with any of the HP1a immunosignals along the polytene chromosomes. (E) Quantitative RT-PCR analysis of the expression, in wild type and HP1a mutant larvae, of the same sub-set of target genes reported in Figure 2B . (F) Quantitative RT-PCR analysis of the expression, in wild type and HP1a mutant larvae, of the same sub-set of target genes reported in Figure 2C . (G) Quantitative RT-PCR analysis of the expression, in wild type and HP1a mutant larvae, of the same sub-set of non target genes reported in Figure 2D .

    Techniques Used: Binding Assay, Mutagenesis, Quantitative RT-PCR, Expressing

    Genes corresponding to transcript targets of HP1a in S2 cells appear down-regulated also in DDP1 and PEP mutants larvae. (A) Quantitative RT–PCR analysis of the expression in wild type Ore-R and DDP1 mutant larvae showing a significant decrease in the amount of the transcripts corresponding to all genes. (B) Quantitative RT–PCR analysis in wild type Ore-R and PEP mutant larvae. In this case a significant decrease in the amount of the transcripts is evident for four of the six analyzed genes.
    Figure Legend Snippet: Genes corresponding to transcript targets of HP1a in S2 cells appear down-regulated also in DDP1 and PEP mutants larvae. (A) Quantitative RT–PCR analysis of the expression in wild type Ore-R and DDP1 mutant larvae showing a significant decrease in the amount of the transcripts corresponding to all genes. (B) Quantitative RT–PCR analysis in wild type Ore-R and PEP mutant larvae. In this case a significant decrease in the amount of the transcripts is evident for four of the six analyzed genes.

    Techniques Used: Quantitative RT-PCR, Expressing, Mutagenesis

    HP1a seems to be involved in forming a complex with hnRNP proteins. (A) Western blot analysis with anti-FLAG antibody of immunopurified fractions obtained from nuclear extracts and nuclear lysates of S2 cells tranfected with FLAG-tagged HP1a (IP HP1 ) or FLAG-tagged GFP (IP GFP ). Aliquots corresponding to 3×10 6 cells processed for immunopurification are loaded on each lane. Molecular weight markers, expressed in kDalton, are reported on the left. (B) Western blot analysis with anti-PEP, anti-DDP1 and anti-HRB87F antibodies of immunopurified fractions obtained from nuclear extracts and nuclear lysates of S2 cells tranfected with FLAG-tagged HP1a (IP HP1 ) or FLAG-tagged GFP (IP GFP ). Aliquots corresponding to nuclear extracts and lysates obtained from 3×10 5 cells before immunopurification (lanes In) are compared with aliquots corresponding to 3×10 6 cells processed for immunopurification (lanes IP HP1 and IP GFP ). Molecular weight markers, expressed in kDalton, are reported on the left. Bands marked with asterisks correspond to mouse IgGs present in the sample and revealed by secondary antibodies. (C) Western blot analysis with anti-FLAG, anti-HRB87F, anti-DDP1 and anti-PEP antibodies of immunopurified fractions obtained from nuclear lysates of S2 cells tranfected with FLAG-tagged HP1a resolved by native-PAGE before (lanes −) and after (lanes +) RNAse treatment. Aliquots corresponding to 3×10 6 cells processed for immunopurification are loaded on each lane. Complexes with low-mobility (** and *) and a fast mobility form of HP1a (°) are indicated.
    Figure Legend Snippet: HP1a seems to be involved in forming a complex with hnRNP proteins. (A) Western blot analysis with anti-FLAG antibody of immunopurified fractions obtained from nuclear extracts and nuclear lysates of S2 cells tranfected with FLAG-tagged HP1a (IP HP1 ) or FLAG-tagged GFP (IP GFP ). Aliquots corresponding to 3×10 6 cells processed for immunopurification are loaded on each lane. Molecular weight markers, expressed in kDalton, are reported on the left. (B) Western blot analysis with anti-PEP, anti-DDP1 and anti-HRB87F antibodies of immunopurified fractions obtained from nuclear extracts and nuclear lysates of S2 cells tranfected with FLAG-tagged HP1a (IP HP1 ) or FLAG-tagged GFP (IP GFP ). Aliquots corresponding to nuclear extracts and lysates obtained from 3×10 5 cells before immunopurification (lanes In) are compared with aliquots corresponding to 3×10 6 cells processed for immunopurification (lanes IP HP1 and IP GFP ). Molecular weight markers, expressed in kDalton, are reported on the left. Bands marked with asterisks correspond to mouse IgGs present in the sample and revealed by secondary antibodies. (C) Western blot analysis with anti-FLAG, anti-HRB87F, anti-DDP1 and anti-PEP antibodies of immunopurified fractions obtained from nuclear lysates of S2 cells tranfected with FLAG-tagged HP1a resolved by native-PAGE before (lanes −) and after (lanes +) RNAse treatment. Aliquots corresponding to 3×10 6 cells processed for immunopurification are loaded on each lane. Complexes with low-mobility (** and *) and a fast mobility form of HP1a (°) are indicated.

    Techniques Used: Western Blot, Immu-Puri, Molecular Weight, Clear Native PAGE

    The HP1a target transcripts are less stable in cells lacking HP1a. Quantitative RT–PCR analysis of three HP1a target transcripts at different times after blockage of transcription by actinomicyn D treatment. The blue lines and the red lines respectively indicate the transcripts amount in S2 control cells and S2 cells interfered with HP1a dsRNA. Target transcripts correspond to genes: (A) CG5389, (B) CG6779, (C) CG7975. Note that at 120 minutes, the HP1a target transcripts are not detectable in HP1a depleted cells while in control cells their amount is only slightly decreased.
    Figure Legend Snippet: The HP1a target transcripts are less stable in cells lacking HP1a. Quantitative RT–PCR analysis of three HP1a target transcripts at different times after blockage of transcription by actinomicyn D treatment. The blue lines and the red lines respectively indicate the transcripts amount in S2 control cells and S2 cells interfered with HP1a dsRNA. Target transcripts correspond to genes: (A) CG5389, (B) CG6779, (C) CG7975. Note that at 120 minutes, the HP1a target transcripts are not detectable in HP1a depleted cells while in control cells their amount is only slightly decreased.

    Techniques Used: Quantitative RT-PCR

    HP1a can directly bind RNA in vitro and in vivo and interacts with Pol II. (A) Immunolocalization of HP1a and active Pol II on polytene chromosomes of D. melanogaster . Signals produced by the two antibodies show an extensive colocalization. (B) Coimmunoprecipitation of HP1a and Pol II by an anti-HP1a antibody. To test the specificity of HP1a with Pol II interaction, we also probed with an antibody against α-actin. (C) UV crosslinking after incubation of four different concentrations of HP1a with Hsp70 RNA in vitro . Note that radioactive HP1a bands with molecular weights corresponding to an HP1a dimer and monomer are present in the third and fourth lanes. (D) Primer extension of RNA immunoprecipitated from S2 cells with CIA9 antibody. Two signals are present only in HP1a immunoprecipitates of heat-shocked cells (HS). (E) Electromobility shift assay. Left, a diagram of the HP1a fragments used in the gel shift assay. Right, the results of EMSA of radiolabelled HSP70 RNA using the different HP1a fragments (50 ng). The absence of shift (lane d) using the HP1a fragment lacking the chromo-domain strongly suggests that this part of the protein is responsible for the binding of HP1a to the RNA transcripts.
    Figure Legend Snippet: HP1a can directly bind RNA in vitro and in vivo and interacts with Pol II. (A) Immunolocalization of HP1a and active Pol II on polytene chromosomes of D. melanogaster . Signals produced by the two antibodies show an extensive colocalization. (B) Coimmunoprecipitation of HP1a and Pol II by an anti-HP1a antibody. To test the specificity of HP1a with Pol II interaction, we also probed with an antibody against α-actin. (C) UV crosslinking after incubation of four different concentrations of HP1a with Hsp70 RNA in vitro . Note that radioactive HP1a bands with molecular weights corresponding to an HP1a dimer and monomer are present in the third and fourth lanes. (D) Primer extension of RNA immunoprecipitated from S2 cells with CIA9 antibody. Two signals are present only in HP1a immunoprecipitates of heat-shocked cells (HS). (E) Electromobility shift assay. Left, a diagram of the HP1a fragments used in the gel shift assay. Right, the results of EMSA of radiolabelled HSP70 RNA using the different HP1a fragments (50 ng). The absence of shift (lane d) using the HP1a fragment lacking the chromo-domain strongly suggests that this part of the protein is responsible for the binding of HP1a to the RNA transcripts.

    Techniques Used: In Vitro, In Vivo, Produced, Incubation, Immunoprecipitation, Electro Mobility Shift Assay, Electrophoretic Mobility Shift Assay, Binding Assay

    28) Product Images from "A Cell Adhesion-Based Reconstitution Method for Studying Cell Polarity"

    Article Title: A Cell Adhesion-Based Reconstitution Method for Studying Cell Polarity

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2020.598492

    Ed-induced polarity as a minimal reconstitution system to model Drosophila neural stem cell spindle orientation. (A) Drosophila neural stem cells (neuroblasts) establish apical-basal polarity in the early stages of mitosis. Both apical and basal polarity complexes (blue and red, respectively) consist of numerous components connected by a complex network of protein-protein interactions and regulatory relationships. At metaphase, the mitotic spindle aligns along this polarity axis through the activity of the apical Pins/Mud/Dlg spindle orientation complex (green). Mitosis proceeds through an asymmetric cell division that is essential for generating differentiated progeny (via the ganglion mother cell, GMC) while also maintaining the stem cell pool through self-renewal. (B) Illustration of how the Ed-induced polarity assay can model Pins-mediated spindle orientation in a minimal reconstituted system (i.e., “X” would represent Pins in this case). Isolated S2 cells initially express an Ed:GFP-X recombinant protein uniformly around the entire cell membrane. Shaking causes collisions that generate cell adhesions wherein cortical Ed:GFP-X proteins concentrate at sites of cell-cell contact within small clusters, the simplest of which is two adhered cells as shown. As cells enter and proceed through mitosis, spindle orientation can be measured relative to the Ed:GFP-X induced crescent similar to how one would with the native Pins crescent in the neuroblast. Note the simplification of this S2 cell system as compared with the complex environment established natively within NBs.
    Figure Legend Snippet: Ed-induced polarity as a minimal reconstitution system to model Drosophila neural stem cell spindle orientation. (A) Drosophila neural stem cells (neuroblasts) establish apical-basal polarity in the early stages of mitosis. Both apical and basal polarity complexes (blue and red, respectively) consist of numerous components connected by a complex network of protein-protein interactions and regulatory relationships. At metaphase, the mitotic spindle aligns along this polarity axis through the activity of the apical Pins/Mud/Dlg spindle orientation complex (green). Mitosis proceeds through an asymmetric cell division that is essential for generating differentiated progeny (via the ganglion mother cell, GMC) while also maintaining the stem cell pool through self-renewal. (B) Illustration of how the Ed-induced polarity assay can model Pins-mediated spindle orientation in a minimal reconstituted system (i.e., “X” would represent Pins in this case). Isolated S2 cells initially express an Ed:GFP-X recombinant protein uniformly around the entire cell membrane. Shaking causes collisions that generate cell adhesions wherein cortical Ed:GFP-X proteins concentrate at sites of cell-cell contact within small clusters, the simplest of which is two adhered cells as shown. As cells enter and proceed through mitosis, spindle orientation can be measured relative to the Ed:GFP-X induced crescent similar to how one would with the native Pins crescent in the neuroblast. Note the simplification of this S2 cell system as compared with the complex environment established natively within NBs.

    Techniques Used: Activity Assay, Isolation, Recombinant

    29) Product Images from "A mosquito 2-Cys peroxiredoxin protects against nitrosative and oxidative stresses associated with malaria parasite infection"

    Article Title: A mosquito 2-Cys peroxiredoxin protects against nitrosative and oxidative stresses associated with malaria parasite infection

    Journal:

    doi: 10.1016/j.freeradbiomed.2005.10.059

    (A) Anti-DmPrx-4783 antiserum recognizes DmPrx-4783 and AsPrx-4783. Crude protein lysates (10 μg) from D. melanogaster S2 cells or from A. stephensi MSQ43 cells were subjected to 12% SDS–PAGE under reducing conditions (DTT), transferred
    Figure Legend Snippet: (A) Anti-DmPrx-4783 antiserum recognizes DmPrx-4783 and AsPrx-4783. Crude protein lysates (10 μg) from D. melanogaster S2 cells or from A. stephensi MSQ43 cells were subjected to 12% SDS–PAGE under reducing conditions (DTT), transferred

    Techniques Used: SDS Page

    Overexpressed AsPrx-4783 protects transfected D. melanogaster S2 cells from RNOS-specific cytotoxicity. D. melanogaster S2 cells were mock transfected (control cells) or transfected with the pTLP55 plasmid encoding AsPrx-4783 (PRX cells). Protection due
    Figure Legend Snippet: Overexpressed AsPrx-4783 protects transfected D. melanogaster S2 cells from RNOS-specific cytotoxicity. D. melanogaster S2 cells were mock transfected (control cells) or transfected with the pTLP55 plasmid encoding AsPrx-4783 (PRX cells). Protection due

    Techniques Used: Transfection, Plasmid Preparation

    30) Product Images from "CK2 Inhibits TIMELESS Nuclear Export and Modulates CLOCK Transcriptional Activity to Regulate Circadian Rhythms"

    Article Title: CK2 Inhibits TIMELESS Nuclear Export and Modulates CLOCK Transcriptional Activity to Regulate Circadian Rhythms

    Journal: Current biology : CB

    doi: 10.1016/j.cub.2020.10.061

    TIM(S1404) Phosphorylation Promotes TIM Nuclear Retention by Compromising TIM-XPO1 Interaction (A) S1404 is located next to a putative TIM NES : L1394-V1403. S1404 is underlined and shown in gray. Classical NES sequence motif is previously investigated. 58 Φ is hydrophobic amino acid (in gray): Leu; Val; Ile; Phe; or Met. X is any amino acid. (B) Western blots showing reciprocal coimmunoprecipitations (coIPs) to examine the interactions of TIM(WT) or TIM(S1404A) to XPO1 in Drosophila S2 cells expressing pAc- xpo1 -3XFLAG-6XHIS and pAc- per -V5 in the presence or absence of pAc-HA plasmids expressing tim variants. Protein extracts were directly analyzed by immunoblotting (α-V5 for PER) or immunoprecipitated with α-HA or α-FLAG resins to detect baits and interactors. (C and D) Bar graphs displaying quantification of reciprocal coIPs. Values for binding are normalized to amount of bait detected in the IPs and expressed as relative signal intensity (high value = 1). Error bars indicate ± SEM (n = 4); ***p
    Figure Legend Snippet: TIM(S1404) Phosphorylation Promotes TIM Nuclear Retention by Compromising TIM-XPO1 Interaction (A) S1404 is located next to a putative TIM NES : L1394-V1403. S1404 is underlined and shown in gray. Classical NES sequence motif is previously investigated. 58 Φ is hydrophobic amino acid (in gray): Leu; Val; Ile; Phe; or Met. X is any amino acid. (B) Western blots showing reciprocal coimmunoprecipitations (coIPs) to examine the interactions of TIM(WT) or TIM(S1404A) to XPO1 in Drosophila S2 cells expressing pAc- xpo1 -3XFLAG-6XHIS and pAc- per -V5 in the presence or absence of pAc-HA plasmids expressing tim variants. Protein extracts were directly analyzed by immunoblotting (α-V5 for PER) or immunoprecipitated with α-HA or α-FLAG resins to detect baits and interactors. (C and D) Bar graphs displaying quantification of reciprocal coIPs. Values for binding are normalized to amount of bait detected in the IPs and expressed as relative signal intensity (high value = 1). Error bars indicate ± SEM (n = 4); ***p

    Techniques Used: Sequencing, Western Blot, Expressing, Immunoprecipitation, Binding Assay

    CK2 Phosphorylates TIM(S1404) (A) CK2 consensus motifs generated by KinasePhos 2.0. S1404 corresponds to phosphoserine at amino acid position 0 (Support vector machine score = 0.9581). (B) Drosophila S2 cells were transfected with pAc- tim (WT)-HA or pAc- tim (S1404A)-HA and co-transfected with an empty plasmid (pMT-V5-His), pMT- ck2α -V5, or pMT- ck2α (M161K E165D)-V5, referred to as ck2α ( tik ). Protein extracts were incubated with α-HA resin. Total TIM isoforms, TIM(pS1404), and CK2α protein levels were analyzed by western blotting with indicated antibodies. (C) Bar graph showing relative TIM pS1404 levels in (B) normalized to total TIM isoforms. Error bars indicate ± SEM (n = 2); **p
    Figure Legend Snippet: CK2 Phosphorylates TIM(S1404) (A) CK2 consensus motifs generated by KinasePhos 2.0. S1404 corresponds to phosphoserine at amino acid position 0 (Support vector machine score = 0.9581). (B) Drosophila S2 cells were transfected with pAc- tim (WT)-HA or pAc- tim (S1404A)-HA and co-transfected with an empty plasmid (pMT-V5-His), pMT- ck2α -V5, or pMT- ck2α (M161K E165D)-V5, referred to as ck2α ( tik ). Protein extracts were incubated with α-HA resin. Total TIM isoforms, TIM(pS1404), and CK2α protein levels were analyzed by western blotting with indicated antibodies. (C) Bar graph showing relative TIM pS1404 levels in (B) normalized to total TIM isoforms. Error bars indicate ± SEM (n = 2); **p

    Techniques Used: Generated, Plasmid Preparation, Transfection, Incubation, Western Blot

    31) Product Images from "A Novel Pathway of Cell Death in Response to Cytosolic DNA in Drosophila Cells"

    Article Title: A Novel Pathway of Cell Death in Response to Cytosolic DNA in Drosophila Cells

    Journal: Journal of Innate Immunity

    doi: 10.1159/000368276

    Transfected DNA rapidly kills Drosophila S2 cells. The viability of S2 cells was measured by MTT cleavage at 1 h after electroporation, unless otherwise stated. a DNA-dependent cell death of S2 cells after electroporation with 10 µg of CT DNA. The bars represent data from 40 experiments (mean ± SEM) normalised to the no-DNA sample for each experiment. **** p
    Figure Legend Snippet: Transfected DNA rapidly kills Drosophila S2 cells. The viability of S2 cells was measured by MTT cleavage at 1 h after electroporation, unless otherwise stated. a DNA-dependent cell death of S2 cells after electroporation with 10 µg of CT DNA. The bars represent data from 40 experiments (mean ± SEM) normalised to the no-DNA sample for each experiment. **** p

    Techniques Used: Transfection, MTT Assay, Electroporation

    Response of S2 cells and BMMs to different types of DNA. a Cytotoxic effect of DNA from different sources on S2 cells; 10 µg of CT DNA or DNA from salmon sperm, S2 cells, E. coli and pBS plasmid were used in electroporations. Data are from 2 experiments (mean ± range). b Cytotoxic effect of DNA on S2 cells is not dependent on methylation. Cells were electroporated with 10 µg of CT DNA or pBS DNA, either methylated or unmethylated on CpG sequences. Each dot represents a separate electroporated sample, with different symbols denoting 2 different experiments. Response of BMM ( c ) and S2 cells ( d ) to boiled DNA; 10 µg of CT DNA was left intact or boiled for 10 min, transferred to ice and immediately used for electroporation. The bars represent data from 3 ( c ) and 5 ( d ) experiments (mean ± SEM), normalised to the no-DNA sample (* p = 0.029). e Cytotoxic effect of synthetic ssDNA and dsDNA in BMM; 5 µg of CT DNA or synthetic DNA [poly(dA), poly(dT) or poly(dA:dT)] or 1 µg of poly(dA:dT) was electroporated. f Cytotoxic effect of synthetic DNAs in S2 cells, as per panel e. Each dot represents a separate electroporated sample, with the different symbols denoting the 3 different experiments (n = 3, ## p = 0.054). g Absence of cytotoxic effect of poly(dC) on S2 cells; 5 µg of CT DNA or synthetic DNA was used for electroporation. Each dot represents a separate electroporated sample, with the different symbols denoting the different experiments. h Toxic effect of synthetic mixed-base DNA with or without thymidine [poly(dN) and poly(dGCA)] on S2 cells; 10 μg of DNA was used for electroporation. Data are from 5 experiments [CT DNA and poly(dN), mean ± SEM; ** p = 0.0085], or 2 experiments [poly(dGCA), mean ± range].
    Figure Legend Snippet: Response of S2 cells and BMMs to different types of DNA. a Cytotoxic effect of DNA from different sources on S2 cells; 10 µg of CT DNA or DNA from salmon sperm, S2 cells, E. coli and pBS plasmid were used in electroporations. Data are from 2 experiments (mean ± range). b Cytotoxic effect of DNA on S2 cells is not dependent on methylation. Cells were electroporated with 10 µg of CT DNA or pBS DNA, either methylated or unmethylated on CpG sequences. Each dot represents a separate electroporated sample, with different symbols denoting 2 different experiments. Response of BMM ( c ) and S2 cells ( d ) to boiled DNA; 10 µg of CT DNA was left intact or boiled for 10 min, transferred to ice and immediately used for electroporation. The bars represent data from 3 ( c ) and 5 ( d ) experiments (mean ± SEM), normalised to the no-DNA sample (* p = 0.029). e Cytotoxic effect of synthetic ssDNA and dsDNA in BMM; 5 µg of CT DNA or synthetic DNA [poly(dA), poly(dT) or poly(dA:dT)] or 1 µg of poly(dA:dT) was electroporated. f Cytotoxic effect of synthetic DNAs in S2 cells, as per panel e. Each dot represents a separate electroporated sample, with the different symbols denoting the 3 different experiments (n = 3, ## p = 0.054). g Absence of cytotoxic effect of poly(dC) on S2 cells; 5 µg of CT DNA or synthetic DNA was used for electroporation. Each dot represents a separate electroporated sample, with the different symbols denoting the different experiments. h Toxic effect of synthetic mixed-base DNA with or without thymidine [poly(dN) and poly(dGCA)] on S2 cells; 10 μg of DNA was used for electroporation. Data are from 5 experiments [CT DNA and poly(dN), mean ± SEM; ** p = 0.0085], or 2 experiments [poly(dGCA), mean ± range].

    Techniques Used: Plasmid Preparation, Methylation, Electroporation

    Cyclic dinucleotides have no toxic effect on S2 cells, and are not likely to be second messengers in the cell death response to DNA. a Electroporation of S2 cells with CT DNA and cyclic dinucleotides. Cells were electroporated with 10 µg of CT DNA or 20 µg of cyclic di-GMP or cyclic di-AMP, and MTT cleavage was measured 1 h after treatment. Results show the mean and range of duplicate electroporations. b Transfection of S2 cells with CT DNA and cyclic dinucleotides using Lipofectamine 2000; 2 µg of CT DNA and 4 µg of cyclic dinucleotides were transfected into S2 cells complexed with 4 µl of Lipofectamine 2000, and MTT cleavage was measured 18 h after transfection. The bars show data from 4 experiments (mean ± SEM). c Induction of IFN-β mRNA in BMM either untreated (no ZAP) or 2 h after electroporation with either no addition, CT DNA or various amounts of cyclic di-GMP. The real time PCR results shown are mean and range of duplicate assays of IFN-β mRNA relative to HPRT.
    Figure Legend Snippet: Cyclic dinucleotides have no toxic effect on S2 cells, and are not likely to be second messengers in the cell death response to DNA. a Electroporation of S2 cells with CT DNA and cyclic dinucleotides. Cells were electroporated with 10 µg of CT DNA or 20 µg of cyclic di-GMP or cyclic di-AMP, and MTT cleavage was measured 1 h after treatment. Results show the mean and range of duplicate electroporations. b Transfection of S2 cells with CT DNA and cyclic dinucleotides using Lipofectamine 2000; 2 µg of CT DNA and 4 µg of cyclic dinucleotides were transfected into S2 cells complexed with 4 µl of Lipofectamine 2000, and MTT cleavage was measured 18 h after transfection. The bars show data from 4 experiments (mean ± SEM). c Induction of IFN-β mRNA in BMM either untreated (no ZAP) or 2 h after electroporation with either no addition, CT DNA or various amounts of cyclic di-GMP. The real time PCR results shown are mean and range of duplicate assays of IFN-β mRNA relative to HPRT.

    Techniques Used: Electroporation, MTT Assay, Transfection, Real-time Polymerase Chain Reaction

    32) Product Images from "Abelson kinase’s intrinsically disordered region plays essential roles in protein function and protein stability"

    Article Title: Abelson kinase’s intrinsically disordered region plays essential roles in protein function and protein stability

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/s12964-020-00703-w

    Abl∆IDR protein accumulates at much higher levels than wildtype Abl. a Immunoblot of 0–6 h embryonic extracts, blotted with antibody to GFP to detect our transgenic proteins. Tubulin serves as a loading control. Despite the fact that both transgenes are driven by the same endogenous abl promotor and the transgenes are at the same chromosomal location, Abl∆IDR protein accumulates at much higher levels than wildtype Abl. b Quantification of mean protein levels from four immunoblots, normalized to both wildtype Abl:GFP and using the loading controls. Colored dots indicate values of the individual blots (Values: 8.2, 11.4, 12.3, and 15.2, Mean: 11.7; Red dot indicates blot shown in a ). Error bar = standard error of the mean. c Immunoblot of extracts of Drosophila S2 cells expressing transgenes encoding wildtype Abl:GFP or Abl∆IDR (at similar transfection efficiencies (see Methods)), both under control of the metallothionine promotor, blotted with antibody to GFP to detect our transgenic proteins. d Representative images of transfected S2 cells stained to visualize F-actin and our transgenic Abl proteins. Wildtype Abl:GFP is enriched in the lamellipodium (arrowhead; highlighted by F-actin) and excluded from nuclei (arrow), while Abl∆IDR:GFP is not enriched in the lamellipodium or excluded from nuclei. Cells expressing Abl∆CR1:GFP (bottom row) resemble those expressing Abl:GFP (enriched in the lamellipodium (arrowhead), excluded from nuclei (arrow)). Scale Bar = 10 µm
    Figure Legend Snippet: Abl∆IDR protein accumulates at much higher levels than wildtype Abl. a Immunoblot of 0–6 h embryonic extracts, blotted with antibody to GFP to detect our transgenic proteins. Tubulin serves as a loading control. Despite the fact that both transgenes are driven by the same endogenous abl promotor and the transgenes are at the same chromosomal location, Abl∆IDR protein accumulates at much higher levels than wildtype Abl. b Quantification of mean protein levels from four immunoblots, normalized to both wildtype Abl:GFP and using the loading controls. Colored dots indicate values of the individual blots (Values: 8.2, 11.4, 12.3, and 15.2, Mean: 11.7; Red dot indicates blot shown in a ). Error bar = standard error of the mean. c Immunoblot of extracts of Drosophila S2 cells expressing transgenes encoding wildtype Abl:GFP or Abl∆IDR (at similar transfection efficiencies (see Methods)), both under control of the metallothionine promotor, blotted with antibody to GFP to detect our transgenic proteins. d Representative images of transfected S2 cells stained to visualize F-actin and our transgenic Abl proteins. Wildtype Abl:GFP is enriched in the lamellipodium (arrowhead; highlighted by F-actin) and excluded from nuclei (arrow), while Abl∆IDR:GFP is not enriched in the lamellipodium or excluded from nuclei. Cells expressing Abl∆CR1:GFP (bottom row) resemble those expressing Abl:GFP (enriched in the lamellipodium (arrowhead), excluded from nuclei (arrow)). Scale Bar = 10 µm

    Techniques Used: Transgenic Assay, Western Blot, Expressing, Transfection, Staining

    33) Product Images from "Syntaxin 5 Is Required for Copper Homeostasis in Drosophila and Mammals"

    Article Title: Syntaxin 5 Is Required for Copper Homeostasis in Drosophila and Mammals

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0014303

    Syx5 suppression in Drosophila S2 cells decreases copper accumulation. Metal accumulation was measured by ICP-AES in control (black) and Syx5 (grey) RNAi suppression cells grown in basal media (A). Syx5 gene expression was suppressed to 19–36% of wild-type levels. Values are mean with s.e.m of eight replicates over two experiments, normalized against control cells. Mean copper accumulation during a 24 h exposure to 2 µM Cu was measured using 64 Cu in S2 cell lines stably over-expressing Ctr1A , Ctr1B or an empty vector control and normalized to total cellular protein (B). Error bars are s.e.m. from nine replicates over three experiments. Syx5 gene expression levels relative to wild-type were 18–41% (control cells), 25-41% (Ctr1A) and 23-31% (Ctr1B). Copper accumulation is reduced by Syx5 suppression (A) even when Ctr1A or Ctr1B is over-expressed (B): suppression of Syx5 reduces copper levels to 50–70% of wild-type in all cell lines. *Significant difference between control and Syx5 suppression cells, determined by an independent samples T-Test (P
    Figure Legend Snippet: Syx5 suppression in Drosophila S2 cells decreases copper accumulation. Metal accumulation was measured by ICP-AES in control (black) and Syx5 (grey) RNAi suppression cells grown in basal media (A). Syx5 gene expression was suppressed to 19–36% of wild-type levels. Values are mean with s.e.m of eight replicates over two experiments, normalized against control cells. Mean copper accumulation during a 24 h exposure to 2 µM Cu was measured using 64 Cu in S2 cell lines stably over-expressing Ctr1A , Ctr1B or an empty vector control and normalized to total cellular protein (B). Error bars are s.e.m. from nine replicates over three experiments. Syx5 gene expression levels relative to wild-type were 18–41% (control cells), 25-41% (Ctr1A) and 23-31% (Ctr1B). Copper accumulation is reduced by Syx5 suppression (A) even when Ctr1A or Ctr1B is over-expressed (B): suppression of Syx5 reduces copper levels to 50–70% of wild-type in all cell lines. *Significant difference between control and Syx5 suppression cells, determined by an independent samples T-Test (P

    Techniques Used: Expressing, Stable Transfection, Plasmid Preparation

    34) Product Images from "The H3K4 Demethylase Lid Associates with and Inhibits Histone Deacetylase Rpd3 ▿"

    Article Title: The H3K4 Demethylase Lid Associates with and Inhibits Histone Deacetylase Rpd3 ▿

    Journal:

    doi: 10.1128/MCB.01643-08

    Lid inhibits the HDAC activity of Rpd3 in vivo. (A) Identification of Rpd3 targets in S2 cells. S2 cells were treated with 100 ng/ml TSA for 48 h to repress deacetylation by Rpd3. RNA was isolated from dimethyl sulfoxide-treated control cells (ctrl) or
    Figure Legend Snippet: Lid inhibits the HDAC activity of Rpd3 in vivo. (A) Identification of Rpd3 targets in S2 cells. S2 cells were treated with 100 ng/ml TSA for 48 h to repress deacetylation by Rpd3. RNA was isolated from dimethyl sulfoxide-treated control cells (ctrl) or

    Techniques Used: Activity Assay, In Vivo, Isolation

    35) Product Images from "Loss of the spectraplakin gene Short stop induces a DNA damage response in Drosophila epithelia"

    Article Title: Loss of the spectraplakin gene Short stop induces a DNA damage response in Drosophila epithelia

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-77159-y

    Knockdown of GADD45 exacerbates chromosome segregation defects in Drosophila S2 cells with concomitant Shot knockdown. ( A – D ) Drosophila S2 cells treated without (Control) or with RNAi targeting shot and gadd45 were paraformaldehyde fixed and stained for phosphohistone-H3 (PH3, green) and a-Tubulin (red). Yellow arrows indicate lagging or bridged chromosomes in anaphase (ana) and telophase (telo), respectively. Images are representative of at least 30 cells. ( E ) Graph depicts percentage of cells with normal or Bridged/Lagging chromosomes for indicated treatment conditions. *p
    Figure Legend Snippet: Knockdown of GADD45 exacerbates chromosome segregation defects in Drosophila S2 cells with concomitant Shot knockdown. ( A – D ) Drosophila S2 cells treated without (Control) or with RNAi targeting shot and gadd45 were paraformaldehyde fixed and stained for phosphohistone-H3 (PH3, green) and a-Tubulin (red). Yellow arrows indicate lagging or bridged chromosomes in anaphase (ana) and telophase (telo), respectively. Images are representative of at least 30 cells. ( E ) Graph depicts percentage of cells with normal or Bridged/Lagging chromosomes for indicated treatment conditions. *p

    Techniques Used: Staining

    GADD45 and Ask1 are required for cell cycle arrest in Drosophila S2 cells following Shot knockdown. ( A – C ) Drosophila S2 cells stably expressing GFP:CID and mCherry: α-Tubulin were treated without (Control) or with RNAi targeting shot and gadd45 . Cells were imaged from prior to nuclear envelope breakdown (NEBD) through anaphase onset. Images are representative of at least 10 cells. Timestamps are relative to NEBD. ( D ) Plots show timing from NEBD to anaphase onset for each recorded cell for the indicated treatment condition. Cells not progressing into anaphase after 3 h were considered to have undergone mitotic arrest (“Arrest”; indicated by percentages listed at top). *p
    Figure Legend Snippet: GADD45 and Ask1 are required for cell cycle arrest in Drosophila S2 cells following Shot knockdown. ( A – C ) Drosophila S2 cells stably expressing GFP:CID and mCherry: α-Tubulin were treated without (Control) or with RNAi targeting shot and gadd45 . Cells were imaged from prior to nuclear envelope breakdown (NEBD) through anaphase onset. Images are representative of at least 10 cells. Timestamps are relative to NEBD. ( D ) Plots show timing from NEBD to anaphase onset for each recorded cell for the indicated treatment condition. Cells not progressing into anaphase after 3 h were considered to have undergone mitotic arrest (“Arrest”; indicated by percentages listed at top). *p

    Techniques Used: Stable Transfection, Expressing

    36) Product Images from "PERIOD phosphoclusters control temperature compensation of the Drosophila circadian clock"

    Article Title: PERIOD phosphoclusters control temperature compensation of the Drosophila circadian clock

    Journal: bioRxiv

    doi: 10.1101/2021.12.23.474078

    S47A degradation kinetics is excessively temperature-compensated A) Representative western blots probing cell extracts from S2 cells expressing DBT and either wild-type (WT) PER, S47A or S45A. Cells were collected at the indicated time points after cycloheximide addition at the indicated temperatures. Top panel shows PER immunoblotting, bottom panel shows Ponceau S staining, used as loading control and for normalization. B-E) Western Blot quantifications. All experiments were performed with three independent replicates. *p
    Figure Legend Snippet: S47A degradation kinetics is excessively temperature-compensated A) Representative western blots probing cell extracts from S2 cells expressing DBT and either wild-type (WT) PER, S47A or S45A. Cells were collected at the indicated time points after cycloheximide addition at the indicated temperatures. Top panel shows PER immunoblotting, bottom panel shows Ponceau S staining, used as loading control and for normalization. B-E) Western Blot quantifications. All experiments were performed with three independent replicates. *p

    Techniques Used: Western Blot, Expressing, Staining

    S47A phosphorylation kinetics is slowed down at cold temperature Representative western blots probing cell extracts from S2 cells expressing DBT and either wild-type (WT) PER, S47A or S45A. Cells were incubated at the indicated temperatures, and collected at the indicated time points after DBT induction. Top panel shows PER immunoblotting, bottom panel shows Ponceau S staining, used as loading control and for normalization. B, C and D) Western Blot quantifications. PER signal shown at time point 0 is used as a reference to classify PER isoforms as hypo-phosphorylated. Graphs representing the relative amount of hypo-phosphorylated PER at 18, 25 and 29 0 C for the respective genotypes at different time points after DBT induction. S47A shows increased hypo-phosphorylated PER 6h after DBT induction.*p
    Figure Legend Snippet: S47A phosphorylation kinetics is slowed down at cold temperature Representative western blots probing cell extracts from S2 cells expressing DBT and either wild-type (WT) PER, S47A or S45A. Cells were incubated at the indicated temperatures, and collected at the indicated time points after DBT induction. Top panel shows PER immunoblotting, bottom panel shows Ponceau S staining, used as loading control and for normalization. B, C and D) Western Blot quantifications. PER signal shown at time point 0 is used as a reference to classify PER isoforms as hypo-phosphorylated. Graphs representing the relative amount of hypo-phosphorylated PER at 18, 25 and 29 0 C for the respective genotypes at different time points after DBT induction. S47A shows increased hypo-phosphorylated PER 6h after DBT induction.*p

    Techniques Used: Western Blot, Expressing, Incubation, Staining

    37) Product Images from "Egalitarian binding partners, Dynein Light Chain and Bicaudal-D, act sequentially to link mRNA to the Dynein motor"

    Article Title: Egalitarian binding partners, Dynein Light Chain and Bicaudal-D, act sequentially to link mRNA to the Dynein motor

    Journal: bioRxiv

    doi: 10.1101/550277

    (A) A co-immunoprecipitation was set up using S2 cell lysates from strains expressing the indicated constructs. The lysates were incubated with RFP-Trap beads. After binding and wash steps, the bound proteins were eluted and analyzed by western blotting using the indicated antibodies. Wild-type Egl was able to co-precipitate BicD and Dlc. Egl_4e-RFP was able to co-precipitate Dlc but not BicD. Egl_2pt-RFP was deficient for binding both Dlc and BicD. ( B) A co-immunoprecipitation experiment was set up using S2 cells expressing the indicated constructs. The lysates were incubated with GFP-trap beads. The bound proteins were analyzed using the indicated antibodies. (C) Ovaries from flies expressing either a control shRNA ( eb1 shRNA) or shRNA against dlc were fixed and processed using TRITC-Phalloidin and DAPI. The driver used for this experiment is restricted to the germline and is turned on early-stage egg chambers ( Sanghavi et al., 2016 ).
    Figure Legend Snippet: (A) A co-immunoprecipitation was set up using S2 cell lysates from strains expressing the indicated constructs. The lysates were incubated with RFP-Trap beads. After binding and wash steps, the bound proteins were eluted and analyzed by western blotting using the indicated antibodies. Wild-type Egl was able to co-precipitate BicD and Dlc. Egl_4e-RFP was able to co-precipitate Dlc but not BicD. Egl_2pt-RFP was deficient for binding both Dlc and BicD. ( B) A co-immunoprecipitation experiment was set up using S2 cells expressing the indicated constructs. The lysates were incubated with GFP-trap beads. The bound proteins were analyzed using the indicated antibodies. (C) Ovaries from flies expressing either a control shRNA ( eb1 shRNA) or shRNA against dlc were fixed and processed using TRITC-Phalloidin and DAPI. The driver used for this experiment is restricted to the germline and is turned on early-stage egg chambers ( Sanghavi et al., 2016 ).

    Techniques Used: Immunoprecipitation, Expressing, Construct, Incubation, Binding Assay, Western Blot, shRNA

    Dlc is required for Egl dimerization. (A) Ovarian lysates were prepared from strains expressing Khc-RFP, Egl_wt-RFP, Egl_2pt-RFP or Egl_4e-RFP. The strains also expressed endogenous Egl. The RFP tagged proteins were immunoprecipitated using RFP-trap beads and the co-precipitating proteins were analyzed by western blotting using the indicated antibodies. The total fraction is shown. Egl_wt-RFP and Egl_4e-RFP were able to co-precipitate endogenous Egl, whereas Khc-RFP and Egl_2pt-RFP were not. (B) A co-precipitation experiment was carried out using S2 cells transfected with the following constructs; lane 1 (GFP and Egl_wt-FLAG); lane 2 (Egl_wt-GFP and Egl_wt-FLAG), lane 3 (GFP and Egl_2pt-FLAG) and lane 4 (Egl_2pt-GFP and Egl_2pt-FLAG). The lysates were precipitated using GFP-Trap beads and the co-precipitated proteins were analyzed using the indicated antibodies. The total fraction is shown. Egl_2pt is dimerization defective. (C) A similar experiment was set up as in panel B using the following constructs; lane 1 (GFP), lane 2 (Egl_wt-GFP) and lane 3 (Egl_delRBD-GFP, an Egl construct lacking the RNA binding domain). Bound and total fractions are shown. Deletion of the RNA binding domain compromises the Egl-BicD interaction but not the Egl-Dlc interaction. (D) ILS or its anti-sense ( ILS-AS ) were bound to beads. Ovarian lysates were prepared from strains expressing a control shRNA (against the white gene) or an shRNA against dlc . These lysates were incubated with RNA-bound beads. After incubation, the bound proteins were eluted and analyzed by \ blotting using the indicated antibodies. The total fraction is also shown. Depletion of Dlc compromises the ability of Egl and BicD to associate with ILS . (E) The same lysates were used in a co-immunoprecipitation experiment using either an antibody against GST (lane 1) or BicD (lane 2 and 3). The bound proteins were analyzed using the indicated antibodies. Bound and total fractions are shown. Depletion of Dlc reduces the Egl-BicD interaction.
    Figure Legend Snippet: Dlc is required for Egl dimerization. (A) Ovarian lysates were prepared from strains expressing Khc-RFP, Egl_wt-RFP, Egl_2pt-RFP or Egl_4e-RFP. The strains also expressed endogenous Egl. The RFP tagged proteins were immunoprecipitated using RFP-trap beads and the co-precipitating proteins were analyzed by western blotting using the indicated antibodies. The total fraction is shown. Egl_wt-RFP and Egl_4e-RFP were able to co-precipitate endogenous Egl, whereas Khc-RFP and Egl_2pt-RFP were not. (B) A co-precipitation experiment was carried out using S2 cells transfected with the following constructs; lane 1 (GFP and Egl_wt-FLAG); lane 2 (Egl_wt-GFP and Egl_wt-FLAG), lane 3 (GFP and Egl_2pt-FLAG) and lane 4 (Egl_2pt-GFP and Egl_2pt-FLAG). The lysates were precipitated using GFP-Trap beads and the co-precipitated proteins were analyzed using the indicated antibodies. The total fraction is shown. Egl_2pt is dimerization defective. (C) A similar experiment was set up as in panel B using the following constructs; lane 1 (GFP), lane 2 (Egl_wt-GFP) and lane 3 (Egl_delRBD-GFP, an Egl construct lacking the RNA binding domain). Bound and total fractions are shown. Deletion of the RNA binding domain compromises the Egl-BicD interaction but not the Egl-Dlc interaction. (D) ILS or its anti-sense ( ILS-AS ) were bound to beads. Ovarian lysates were prepared from strains expressing a control shRNA (against the white gene) or an shRNA against dlc . These lysates were incubated with RNA-bound beads. After incubation, the bound proteins were eluted and analyzed by \ blotting using the indicated antibodies. The total fraction is also shown. Depletion of Dlc compromises the ability of Egl and BicD to associate with ILS . (E) The same lysates were used in a co-immunoprecipitation experiment using either an antibody against GST (lane 1) or BicD (lane 2 and 3). The bound proteins were analyzed using the indicated antibodies. Bound and total fractions are shown. Depletion of Dlc reduces the Egl-BicD interaction.

    Techniques Used: Expressing, Immunoprecipitation, Western Blot, Transfection, Construct, RNA Binding Assay, shRNA, Incubation

    Artificial dimerization of Egl_2pt restores RNA and BicD binding. (A) A co-precipitation experiment was carried out using S2 cells transfected with the following; lane 1 (Egl_2pt-GFP and Egl_2pt-FLAG), lane 2 (GFP and Egl_2pt-Zip-FLAG), and lane 3 (Egl_2pt-Zip-GFP and Egl_2pt-Zip-FLAG). Zip refers to a leucine zipper motif. The lysates were incubated with GFP-Trap beads. The co-precipitating proteins were analyzed using the indicated antibodies. Insertion of the leucine zipper restores Egl_2pt dimerization. (B) A similar experiment was set up using the following constructs; lane 1 (GFP), lane 2 (Egl_wt-GFP), lane 3 (Egl_2pt-GFP) and lane 4 (Egl_2pt-Zip-GFP). After incubation with GFP-trap beads, the co-precipitating proteins were analyzed by western blotting. Egl_2pt-Zip-GFP is able to associate with BicD. *a degradation product is consistently seen in the Egl_2pt-Zip-GFP lane. (C) ILS was bound to beads and incubated with S2 cell lysates expressing the indicated constructs. (D) The TLS localization element, ILS or its anti-sense ( ILS-AS ) were bound to beads as indicated. The RNA-bound beads were incubated with S2 cells lysates expressing the indicated constructs. The bound proteins were analyzed by blotting. Although Egl_2pt is compromised for RNA binding, artificial dimerization (Egl_2pt-Zip) restores RNA binding.
    Figure Legend Snippet: Artificial dimerization of Egl_2pt restores RNA and BicD binding. (A) A co-precipitation experiment was carried out using S2 cells transfected with the following; lane 1 (Egl_2pt-GFP and Egl_2pt-FLAG), lane 2 (GFP and Egl_2pt-Zip-FLAG), and lane 3 (Egl_2pt-Zip-GFP and Egl_2pt-Zip-FLAG). Zip refers to a leucine zipper motif. The lysates were incubated with GFP-Trap beads. The co-precipitating proteins were analyzed using the indicated antibodies. Insertion of the leucine zipper restores Egl_2pt dimerization. (B) A similar experiment was set up using the following constructs; lane 1 (GFP), lane 2 (Egl_wt-GFP), lane 3 (Egl_2pt-GFP) and lane 4 (Egl_2pt-Zip-GFP). After incubation with GFP-trap beads, the co-precipitating proteins were analyzed by western blotting. Egl_2pt-Zip-GFP is able to associate with BicD. *a degradation product is consistently seen in the Egl_2pt-Zip-GFP lane. (C) ILS was bound to beads and incubated with S2 cell lysates expressing the indicated constructs. (D) The TLS localization element, ILS or its anti-sense ( ILS-AS ) were bound to beads as indicated. The RNA-bound beads were incubated with S2 cells lysates expressing the indicated constructs. The bound proteins were analyzed by blotting. Although Egl_2pt is compromised for RNA binding, artificial dimerization (Egl_2pt-Zip) restores RNA binding.

    Techniques Used: Binding Assay, Transfection, Incubation, Construct, Western Blot, Expressing, RNA Binding Assay

    38) Product Images from "N-linked glycosylation of the antagonist Short gastrulation increases the functional complexity of BMP signals"

    Article Title: N-linked glycosylation of the antagonist Short gastrulation increases the functional complexity of BMP signals

    Journal: bioRxiv

    doi: 10.1101/316448

    Loss of Sog glycosylation modifies Dpp binding. A) S2 cells transfected with wild type Sog-myc and different combinations of Tsg and BMP ligands. Co-IP with cell supernatants shows that Sog binds only BMP heterodimers in the absence of Tsg, but binds Dpp homodimers in the presence of Tsg. B) S2 cells transfected with wild type Sog-myc or the N-terminal Supersog fragment reveal that Supersog binds Dpp alone, either using cell pellets or supernatants. C) S2 cells transfected with wild type sog- V5 or sogN123 -V5, and dpp- HA. Co-IP for V5 shows that the N123 mutant binds more Dpp than wild type Sog. D) S2 cells transfected with wild type sog (S) or single glycosylation mutants (N1, N2, N3) plus dpp -HA or dpp- HA and tld -HA. Co-IP for V5. SogN1 binds Dpp alone, while other mutants and wild type Sog do not. Arrows point to the different Sog fragments produced by the Tld metalloprotease and show that the cleavage pattern is similar among all constructs.
    Figure Legend Snippet: Loss of Sog glycosylation modifies Dpp binding. A) S2 cells transfected with wild type Sog-myc and different combinations of Tsg and BMP ligands. Co-IP with cell supernatants shows that Sog binds only BMP heterodimers in the absence of Tsg, but binds Dpp homodimers in the presence of Tsg. B) S2 cells transfected with wild type Sog-myc or the N-terminal Supersog fragment reveal that Supersog binds Dpp alone, either using cell pellets or supernatants. C) S2 cells transfected with wild type sog- V5 or sogN123 -V5, and dpp- HA. Co-IP for V5 shows that the N123 mutant binds more Dpp than wild type Sog. D) S2 cells transfected with wild type sog (S) or single glycosylation mutants (N1, N2, N3) plus dpp -HA or dpp- HA and tld -HA. Co-IP for V5. SogN1 binds Dpp alone, while other mutants and wild type Sog do not. Arrows point to the different Sog fragments produced by the Tld metalloprotease and show that the cleavage pattern is similar among all constructs.

    Techniques Used: Binding Assay, Transfection, Co-Immunoprecipitation Assay, Mutagenesis, Produced, Construct

    Drosophila sog  has three conserved glycosylation sites. A) Alignment of Sog sequences from  Drosophila sp . shows that three putative glycosylation sites are conserved (Arg residue in pink in  D. melanogaster ). In red a Tld/Tlr cleavage site sequence close to N1 is shown. B) Sog protein scheme with the location of the four conserved Cystein Rich (CR) domains, Tld/Tlr cleavage sites (red arrows) and putative glycosylation sites (pink). C-E) Analysis of Sog constructs expressed in S2 cells. C) Glycosylation mutants produced by site-directed mutagenesis and their effect on Sog migration in SDS-PAGE. N23 and N123 migrate faster that wild type Sog. All mutants and wild type Sog bear a C-terminal V5/His tag. Treatment with Tunicamycin decreases wild type Sog Mw and confirms that Sog is glycosylated. D) SDS-PAGE for Sog protein in cells (C) and extracellular medium (M) shows that Sog is secreted and that only the N2 mutation slightly decreases Sog secretion. E) Elisa for S2 cell secreted Sog confirms the analysis in D.
    Figure Legend Snippet: Drosophila sog has three conserved glycosylation sites. A) Alignment of Sog sequences from Drosophila sp . shows that three putative glycosylation sites are conserved (Arg residue in pink in D. melanogaster ). In red a Tld/Tlr cleavage site sequence close to N1 is shown. B) Sog protein scheme with the location of the four conserved Cystein Rich (CR) domains, Tld/Tlr cleavage sites (red arrows) and putative glycosylation sites (pink). C-E) Analysis of Sog constructs expressed in S2 cells. C) Glycosylation mutants produced by site-directed mutagenesis and their effect on Sog migration in SDS-PAGE. N23 and N123 migrate faster that wild type Sog. All mutants and wild type Sog bear a C-terminal V5/His tag. Treatment with Tunicamycin decreases wild type Sog Mw and confirms that Sog is glycosylated. D) SDS-PAGE for Sog protein in cells (C) and extracellular medium (M) shows that Sog is secreted and that only the N2 mutation slightly decreases Sog secretion. E) Elisa for S2 cell secreted Sog confirms the analysis in D.

    Techniques Used: Sequencing, Construct, Produced, Mutagenesis, Migration, SDS Page, Enzyme-linked Immunosorbent Assay

    Sog levels are controlled by glycan and receptor-based retrieval from the extracellular space. A) S2 cells transfected with wild type sog-myc construct and different combinations of constructs producing Tsg and BMP ligands. Cellular amounts of Sog are detected with anti-myc antiserum. Sog amounts increase in the presence of Dpp plus Tsg. These cellular levels correspond to Sog retrieved from the medium, since equivalent intracellular amounts are observed when medium (S) from construct-expressing cells is added to cells that do not express sog-myc. B) Intracellular Sog levels retrieved from Sog-myc+Dpp-HA+Tsg-His medium decrease by knocking down the Dpp receptor tkv or αPS1 ( mew ), αPS2 ( if ) or βPS ( mys ) integrin expression. C) S2 cells transfected with wild type sog-V5 or sogN1-V5 , sogN2-V5 , sogN3-V5 or tripple sogN123-V5 mutant constructs in the presence of constructs producing Tsg and Dpp. Sog retrieval decreases by mutating the N1 and N2 sites. D) Predicted conformation of Sog protein with regions depicted for binding to established extracellular partners. E-J) Medium from S2 cells transfected with dpp-HA and tsg-His plus wild type sog-V5 or sogN1-V5 , sogN2-V5 , or sogN3-V5 , was added to naive cells and imunolabelled with anti-V5 (red) and anti-Rab5 (green, E’-H). E’’) High magnification for wild type Sog-V5 and Rab5 shows partial co-localization, indicative of Sog endocytosis. These cells were quantified in (I), showing that loss of N1, N2 and N3 glycosylation sites decreases the number of cells with V5+Rab5+ endocytic punctae. J) In cells expressing dpp-HA, gbb-His and tsg-His plus wild type sog-V5 or sogN1-V5 , sogN2-V5 , or sogN3-V5, the number of cells with V5+Rab5+ punctae is equivalent. Statistically significant differences based on Student’s t-test, **P≤0.01.
    Figure Legend Snippet: Sog levels are controlled by glycan and receptor-based retrieval from the extracellular space. A) S2 cells transfected with wild type sog-myc construct and different combinations of constructs producing Tsg and BMP ligands. Cellular amounts of Sog are detected with anti-myc antiserum. Sog amounts increase in the presence of Dpp plus Tsg. These cellular levels correspond to Sog retrieved from the medium, since equivalent intracellular amounts are observed when medium (S) from construct-expressing cells is added to cells that do not express sog-myc. B) Intracellular Sog levels retrieved from Sog-myc+Dpp-HA+Tsg-His medium decrease by knocking down the Dpp receptor tkv or αPS1 ( mew ), αPS2 ( if ) or βPS ( mys ) integrin expression. C) S2 cells transfected with wild type sog-V5 or sogN1-V5 , sogN2-V5 , sogN3-V5 or tripple sogN123-V5 mutant constructs in the presence of constructs producing Tsg and Dpp. Sog retrieval decreases by mutating the N1 and N2 sites. D) Predicted conformation of Sog protein with regions depicted for binding to established extracellular partners. E-J) Medium from S2 cells transfected with dpp-HA and tsg-His plus wild type sog-V5 or sogN1-V5 , sogN2-V5 , or sogN3-V5 , was added to naive cells and imunolabelled with anti-V5 (red) and anti-Rab5 (green, E’-H). E’’) High magnification for wild type Sog-V5 and Rab5 shows partial co-localization, indicative of Sog endocytosis. These cells were quantified in (I), showing that loss of N1, N2 and N3 glycosylation sites decreases the number of cells with V5+Rab5+ endocytic punctae. J) In cells expressing dpp-HA, gbb-His and tsg-His plus wild type sog-V5 or sogN1-V5 , sogN2-V5 , or sogN3-V5, the number of cells with V5+Rab5+ punctae is equivalent. Statistically significant differences based on Student’s t-test, **P≤0.01.

    Techniques Used: Transfection, Construct, Expressing, Mutagenesis, Binding Assay

    39) Product Images from "TBP-Related Factor 2 as a Trigger for Robertsonian Translocations and Speciation"

    Article Title: TBP-Related Factor 2 as a Trigger for Robertsonian Translocations and Speciation

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21228871

    Analysis of Trf2 and D1 genes mRNA levels in salivary glands of the third instar larvae, adult flies and S2 cells. The relative expression levels of Trf2 and D1 genes were examined using qRT-PCR as described in materials and methods. ( A ) Expression levels of Trf2 and D1 genes detected in salivary glands of: control UAS-Tris/+ larvae and after Trf2 knock-down by ddRNAi ( sgs > Tris ). ( B ) Expression levels of Trf2 and D1 genes in lawc p1 /EF520 adult flies compared to control EF520/+ flies. ( C ) Expression levels of Trf2 and D1 genes in lawc p1 /Df(1)RA2 adult flies compared to control Df(1)RA2/+ flies; ( D ) expression levels of Trf2 and D1 genes in the S2 cells after RNAi-mediated silencing of Trf2 (S2-TRFi) compared to control (S2 cells treated with dsRNA corresponding to eGFP gene). Data are presented as the mean ± SD of three independent experiments. * p
    Figure Legend Snippet: Analysis of Trf2 and D1 genes mRNA levels in salivary glands of the third instar larvae, adult flies and S2 cells. The relative expression levels of Trf2 and D1 genes were examined using qRT-PCR as described in materials and methods. ( A ) Expression levels of Trf2 and D1 genes detected in salivary glands of: control UAS-Tris/+ larvae and after Trf2 knock-down by ddRNAi ( sgs > Tris ). ( B ) Expression levels of Trf2 and D1 genes in lawc p1 /EF520 adult flies compared to control EF520/+ flies. ( C ) Expression levels of Trf2 and D1 genes in lawc p1 /Df(1)RA2 adult flies compared to control Df(1)RA2/+ flies; ( D ) expression levels of Trf2 and D1 genes in the S2 cells after RNAi-mediated silencing of Trf2 (S2-TRFi) compared to control (S2 cells treated with dsRNA corresponding to eGFP gene). Data are presented as the mean ± SD of three independent experiments. * p

    Techniques Used: Expressing, Quantitative RT-PCR

    RNAi-mediated depletion of Trf2 leads to micronuclei formation and disturbs nuclear envelope. Confocal immunofluorescence images of Drosophila S2 cells ( A ) before and ( B ) after RNAi-mediated silencing of Trf2 . Cells were stained for SytoxGreen to highlight DNA (green) and anti-Lamin Dm to visualized nuclear envelope (cian). The third column represents merged images. Arrowheads pointe to micronucleus, arrows indicate Lamin negative nuclear envelope. ( C ) Quantification of cells with micronuclei from control (Contr, n = 350) and Trf2 dsRNA transfected cells ( Trf2i , n = 229) from three independent experiments. A p -value from Student’s t -test was 0.0047. Error bars: SD.
    Figure Legend Snippet: RNAi-mediated depletion of Trf2 leads to micronuclei formation and disturbs nuclear envelope. Confocal immunofluorescence images of Drosophila S2 cells ( A ) before and ( B ) after RNAi-mediated silencing of Trf2 . Cells were stained for SytoxGreen to highlight DNA (green) and anti-Lamin Dm to visualized nuclear envelope (cian). The third column represents merged images. Arrowheads pointe to micronucleus, arrows indicate Lamin negative nuclear envelope. ( C ) Quantification of cells with micronuclei from control (Contr, n = 350) and Trf2 dsRNA transfected cells ( Trf2i , n = 229) from three independent experiments. A p -value from Student’s t -test was 0.0047. Error bars: SD.

    Techniques Used: Immunofluorescence, Staining, Transfection

    40) Product Images from "miR-71 and miR-263 Jointly Regulate Target Genes Chitin synthase and Chitinase to Control Locust Molting"

    Article Title: miR-71 and miR-263 Jointly Regulate Target Genes Chitin synthase and Chitinase to Control Locust Molting

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1006257

    Interactions between miRNAs (miR-71 and miR-263) and their targets CHS1 and CHT10 in locusts. (A, B) Luciferase reporter assays were analyzed in S2 cells co-transfected with miR-71 or miR-263 overexpression vectors and psi-CHECK2 vectors containing wild-type (WT) or mutant (MT, the 8 nt of the region that corresponds to the miRNA seed mutated) target gene sequences of CHS1 (A) and CHT10 (B) ( n = 6). (C, D) RIP was performed with an anti-Ago-1 antibody. qPCR analysis was performed to amplify the CHS1 and CHT10 mRNAs from the Ago-1 immunoprecipitates from extracts of integument tissue treated with agomir-71 or agomir-263 48 h later compared with the agomir controls (agomir-NC). The data for the luciferase activities and qPCR analyses are presented as means ± SEM ( n = 6). * p
    Figure Legend Snippet: Interactions between miRNAs (miR-71 and miR-263) and their targets CHS1 and CHT10 in locusts. (A, B) Luciferase reporter assays were analyzed in S2 cells co-transfected with miR-71 or miR-263 overexpression vectors and psi-CHECK2 vectors containing wild-type (WT) or mutant (MT, the 8 nt of the region that corresponds to the miRNA seed mutated) target gene sequences of CHS1 (A) and CHT10 (B) ( n = 6). (C, D) RIP was performed with an anti-Ago-1 antibody. qPCR analysis was performed to amplify the CHS1 and CHT10 mRNAs from the Ago-1 immunoprecipitates from extracts of integument tissue treated with agomir-71 or agomir-263 48 h later compared with the agomir controls (agomir-NC). The data for the luciferase activities and qPCR analyses are presented as means ± SEM ( n = 6). * p

    Techniques Used: Luciferase, Transfection, Over Expression, Mutagenesis, Real-time Polymerase Chain Reaction

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    Thermo Fisher dcv infected drosophila s2 cells
    The heat shock response is dynamic and requires viral replication in Drosophila S2 cells. Expression of the genes encoding the heat shock proteins Hsp70, Hsp23, Hsp26, and the Heat shock transcription factor (Hsf) was monitored at the indicated time points by RT-qPCR after infection with ( a ) <t>DCV,</t> ( b ) CrPV or ( c ) IIV-6 (MOI = 10). ( d ) <t>S2</t> cells were inoculated with UV-inactivated viruses and gene expression was measured at 24, 16 or 48 hpi with DCV, CrPV and IIV-6, respectively. Expression of the gene of interest was normalized to the housekeeping gene Ribosomal Protein 49 and expressed as fold change relative to mock infection. Data are mean and s.d. of three independent infections. Student’s t-tests were used to compare virus-infected samples to mock infections (* P
    Dcv Infected Drosophila S2 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher mouse mc1r
    (CKPV) 2 induces macrophage M1 to M2 polarization. Cytokine release profile (IL-1β, IL-6 and IL-10) and arginase activity after indicated treatment/s in primary cultured macrophages transfected with or without <t>MC1R</t> RNAi. LPS:5 ng/ml, IFN-γ:10 ng/ml, α-MSH: 10 µM, and (CKPV) 2 (0.1, 1 and 5 µM) (B–E). The supernatant of the above macrophages were collected and added to L929 cells, after 20 hours, cell viability was measured by MTT assay, the inhibitory rate was calculated by 100%-cell viability OD of treatment group/cell viability OD of untreated control group (A). * p
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    The heat shock response is dynamic and requires viral replication in Drosophila S2 cells. Expression of the genes encoding the heat shock proteins Hsp70, Hsp23, Hsp26, and the Heat shock transcription factor (Hsf) was monitored at the indicated time points by RT-qPCR after infection with ( a ) DCV, ( b ) CrPV or ( c ) IIV-6 (MOI = 10). ( d ) S2 cells were inoculated with UV-inactivated viruses and gene expression was measured at 24, 16 or 48 hpi with DCV, CrPV and IIV-6, respectively. Expression of the gene of interest was normalized to the housekeeping gene Ribosomal Protein 49 and expressed as fold change relative to mock infection. Data are mean and s.d. of three independent infections. Student’s t-tests were used to compare virus-infected samples to mock infections (* P

    Journal: Scientific Reports

    Article Title: The heat shock response restricts virus infection in Drosophila

    doi: 10.1038/srep12758

    Figure Lengend Snippet: The heat shock response is dynamic and requires viral replication in Drosophila S2 cells. Expression of the genes encoding the heat shock proteins Hsp70, Hsp23, Hsp26, and the Heat shock transcription factor (Hsf) was monitored at the indicated time points by RT-qPCR after infection with ( a ) DCV, ( b ) CrPV or ( c ) IIV-6 (MOI = 10). ( d ) S2 cells were inoculated with UV-inactivated viruses and gene expression was measured at 24, 16 or 48 hpi with DCV, CrPV and IIV-6, respectively. Expression of the gene of interest was normalized to the housekeeping gene Ribosomal Protein 49 and expressed as fold change relative to mock infection. Data are mean and s.d. of three independent infections. Student’s t-tests were used to compare virus-infected samples to mock infections (* P

    Article Snippet: The heat shock response is induced in DCV-infected Drosophila S2 cells To identify novel factors or processes involved in antiviral defence in Drosophila , we generated transcriptional profiles of DCV-infected Drosophila S2 cells at 8 and 24 hours post-infection (hpi) using Affymetrix GeneChip microarrays ( ).

    Techniques: Expressing, Quantitative RT-PCR, Infection

    Microarray analysis of DCV-infected Drosophila S2 cells. ( a ) Overview of the experimental workflow. S2 cells were infected with DCV (MOI = 10) or mock-infected with Schneider’s medium, and RNA was extracted at 8 and 24 hours post-infection (hpi) for microarray analyses. Figure drawn by S.H. Merkling. ( b ) Number of differentially expressed genes at 8 and 24 hpi (fold change ≥2 relative to mock infection). ( c ) Venn diagram representing the overlap between differentially induced genes after DCV infection at 8 and 24 hpi. ( d , e ) Gene ontology (GO) analysis of the genes that are upregulated ≥2-fold at ( d ) 8 hpi and ( e ) 24 hpi. All significantly enriched level 4 GO terms are shown ( P

    Journal: Scientific Reports

    Article Title: The heat shock response restricts virus infection in Drosophila

    doi: 10.1038/srep12758

    Figure Lengend Snippet: Microarray analysis of DCV-infected Drosophila S2 cells. ( a ) Overview of the experimental workflow. S2 cells were infected with DCV (MOI = 10) or mock-infected with Schneider’s medium, and RNA was extracted at 8 and 24 hours post-infection (hpi) for microarray analyses. Figure drawn by S.H. Merkling. ( b ) Number of differentially expressed genes at 8 and 24 hpi (fold change ≥2 relative to mock infection). ( c ) Venn diagram representing the overlap between differentially induced genes after DCV infection at 8 and 24 hpi. ( d , e ) Gene ontology (GO) analysis of the genes that are upregulated ≥2-fold at ( d ) 8 hpi and ( e ) 24 hpi. All significantly enriched level 4 GO terms are shown ( P

    Article Snippet: The heat shock response is induced in DCV-infected Drosophila S2 cells To identify novel factors or processes involved in antiviral defence in Drosophila , we generated transcriptional profiles of DCV-infected Drosophila S2 cells at 8 and 24 hours post-infection (hpi) using Affymetrix GeneChip microarrays ( ).

    Techniques: Microarray, Infection

    Binding of Upd to Dally. ( A ) Upd-HA was expressed in S2 cells with or without a secreted form of Dally-Myc. Fractions from the cell pellet (c) and supernatant (s) were probed with anti-HA antibody. ( B ) Upd-HA was expressed in S2 cells with or without

    Journal: Development (Cambridge, England)

    Article Title: Glypicans regulate JAK/STAT signaling and distribution of the Unpaired morphogen

    doi: 10.1242/dev.078055

    Figure Lengend Snippet: Binding of Upd to Dally. ( A ) Upd-HA was expressed in S2 cells with or without a secreted form of Dally-Myc. Fractions from the cell pellet (c) and supernatant (s) were probed with anti-HA antibody. ( B ) Upd-HA was expressed in S2 cells with or without

    Article Snippet: Drosophila S2 cells were grown in Schneider's medium (Invitrogen) supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin.

    Techniques: Binding Assay

    Dose-Dependent Inhibition of Imd Signaling by ThT in Cells and in Flies (A and B) S2* cells were treated with ThT before triggering the activation of the Imd pathway with DAP-type PGN (A), or the Toll pathway with recombinant cleaved Spätzle, Spz-C106 (B). Transcript levels of the target genes for the Imd and Toll pathways, Diptericin and Drosomycin , respectively, were measured by qRT-PCR and normalized to Rp49 expression. Graph shows individual data points from 3 or 4 independent experiments, the line indicating the mean. (C and D) ThT suppresses Diptericin induction in flies. w 1118 male and female flies were co-injected with vehicle (5% DMSO in sterile PBS), and 1 mM ThT ± 0.5 mg/mL PGN (C) or 40 μM TCT (D), and harvested 1 hr after injection. Diptericin transcript levels were measured by qRT-PCR and normalized to Rp49 values. Data shown are individual data points of six (C) and five (D) biological replicates, the bar indicating the mean. (E) Inhibition of the Imd pathway activation by ThT is dose dependent. Male flies were injected with vehicle (5% DMSO in sterile PBS), 0.2 mM, 0.5 mM, or 1 mM ThT ± 0.5 mg/mL PGN and harvested 1 hr after injection. Diptericin transcript levels were measured by qPCR and normalized to Rp49 values. Data shown are individual data points of five biological replicates, the bar indicating the mean. .

    Journal: Immunity

    Article Title: Peptidoglycan-Sensing Receptors Trigger the Formation of Functional Amyloids of the Adaptor Protein Imd to Initiate Drosophila NF-κB Signaling

    doi: 10.1016/j.immuni.2017.09.011

    Figure Lengend Snippet: Dose-Dependent Inhibition of Imd Signaling by ThT in Cells and in Flies (A and B) S2* cells were treated with ThT before triggering the activation of the Imd pathway with DAP-type PGN (A), or the Toll pathway with recombinant cleaved Spätzle, Spz-C106 (B). Transcript levels of the target genes for the Imd and Toll pathways, Diptericin and Drosomycin , respectively, were measured by qRT-PCR and normalized to Rp49 expression. Graph shows individual data points from 3 or 4 independent experiments, the line indicating the mean. (C and D) ThT suppresses Diptericin induction in flies. w 1118 male and female flies were co-injected with vehicle (5% DMSO in sterile PBS), and 1 mM ThT ± 0.5 mg/mL PGN (C) or 40 μM TCT (D), and harvested 1 hr after injection. Diptericin transcript levels were measured by qRT-PCR and normalized to Rp49 values. Data shown are individual data points of six (C) and five (D) biological replicates, the bar indicating the mean. (E) Inhibition of the Imd pathway activation by ThT is dose dependent. Male flies were injected with vehicle (5% DMSO in sterile PBS), 0.2 mM, 0.5 mM, or 1 mM ThT ± 0.5 mg/mL PGN and harvested 1 hr after injection. Diptericin transcript levels were measured by qPCR and normalized to Rp49 values. Data shown are individual data points of five biological replicates, the bar indicating the mean. .

    Article Snippet: Drosophila S2* cells were cultured in Schneider’s Drosophila medium (GIBCO) supplemented with 10% FBS (not heat-inactivated), 1% Glutamax (GIBCO), and 0.2% PenStrep (GIBCO) at 27°C.

    Techniques: Inhibition, Radial Immuno Diffusion, Activation Assay, Recombinant, Quantitative RT-PCR, Expressing, Injection, Real-time Polymerase Chain Reaction

    PGRP-LC, PGRP-LE, and Imd Form Amyloidal Aggregates in S2* Cells (A) SDD-AGE profiles of S2* cell lysates expressing WT or mutant forms of PGRP-LE and Imd. MCMV protein M45(1-277) was used as positive control and Kenny and M45(1-277) IQIG/AAAA mutant as negative controls. (B) S2* cells were transiently transfected with wild-type mCherry-tagged PGRP-LCx, PGRP-LE, or Imd, or respective cRHIM deletion mutants, and amyloidal protein aggregates were visualized by ThT fluorescence. Scale bar: 10 μm. (C) Quantification of ThT fluorescence in cells expressing mCherry-tagged PGRP-LCx, PGRP-LE, and Imd, wild-type, and cRHIM deletion mutants. Data shown are mean ± SEM of at least 15 cells pooled from three independent experiments. (D) Quantification of ThT fluorescence in S2* cells expressing single alanine substitution mutants of mCherry-PGRP-LCx. Columns represent the mean ± SEM of at least 20 cells pooled from three independent experiments. ****p

    Journal: Immunity

    Article Title: Peptidoglycan-Sensing Receptors Trigger the Formation of Functional Amyloids of the Adaptor Protein Imd to Initiate Drosophila NF-κB Signaling

    doi: 10.1016/j.immuni.2017.09.011

    Figure Lengend Snippet: PGRP-LC, PGRP-LE, and Imd Form Amyloidal Aggregates in S2* Cells (A) SDD-AGE profiles of S2* cell lysates expressing WT or mutant forms of PGRP-LE and Imd. MCMV protein M45(1-277) was used as positive control and Kenny and M45(1-277) IQIG/AAAA mutant as negative controls. (B) S2* cells were transiently transfected with wild-type mCherry-tagged PGRP-LCx, PGRP-LE, or Imd, or respective cRHIM deletion mutants, and amyloidal protein aggregates were visualized by ThT fluorescence. Scale bar: 10 μm. (C) Quantification of ThT fluorescence in cells expressing mCherry-tagged PGRP-LCx, PGRP-LE, and Imd, wild-type, and cRHIM deletion mutants. Data shown are mean ± SEM of at least 15 cells pooled from three independent experiments. (D) Quantification of ThT fluorescence in S2* cells expressing single alanine substitution mutants of mCherry-PGRP-LCx. Columns represent the mean ± SEM of at least 20 cells pooled from three independent experiments. ****p

    Article Snippet: Drosophila S2* cells were cultured in Schneider’s Drosophila medium (GIBCO) supplemented with 10% FBS (not heat-inactivated), 1% Glutamax (GIBCO), and 0.2% PenStrep (GIBCO) at 27°C.

    Techniques: Radial Immuno Diffusion, Expressing, Mutagenesis, Positive Control, Transfection, Fluorescence

    (CKPV) 2 induces macrophage M1 to M2 polarization. Cytokine release profile (IL-1β, IL-6 and IL-10) and arginase activity after indicated treatment/s in primary cultured macrophages transfected with or without MC1R RNAi. LPS:5 ng/ml, IFN-γ:10 ng/ml, α-MSH: 10 µM, and (CKPV) 2 (0.1, 1 and 5 µM) (B–E). The supernatant of the above macrophages were collected and added to L929 cells, after 20 hours, cell viability was measured by MTT assay, the inhibitory rate was calculated by 100%-cell viability OD of treatment group/cell viability OD of untreated control group (A). * p

    Journal: PLoS ONE

    Article Title: The Synthetic Melanocortin (CKPV)2 Exerts Anti-Fungal and Anti-Inflammatory Effects against Candida albicans Vaginitis via Inducing Macrophage M2 Polarization

    doi: 10.1371/journal.pone.0056004

    Figure Lengend Snippet: (CKPV) 2 induces macrophage M1 to M2 polarization. Cytokine release profile (IL-1β, IL-6 and IL-10) and arginase activity after indicated treatment/s in primary cultured macrophages transfected with or without MC1R RNAi. LPS:5 ng/ml, IFN-γ:10 ng/ml, α-MSH: 10 µM, and (CKPV) 2 (0.1, 1 and 5 µM) (B–E). The supernatant of the above macrophages were collected and added to L929 cells, after 20 hours, cell viability was measured by MTT assay, the inhibitory rate was calculated by 100%-cell viability OD of treatment group/cell viability OD of untreated control group (A). * p

    Article Snippet: MC1R RNA Interference (RNAi) The chemically synthesized MC1R siRNA (small interfering RNA) duplexes ( ) against mouse MC1R (s1, s2 and s3) were purchased from Ambion (GenePharm Co. Ltd. Shanghai, China).

    Techniques: Activity Assay, Cell Culture, Transfection, MTT Assay

    (CKPV) 2 promotes cAMP production via MC1R. (A) Upper panel: the effects of MC1R siRNA (S1, S2 and S3, see sequence on Tab. 1) on MC1R mRNA level in primary cultured macrophages. Lower panel: MC1R siRNA knockdown almost blocked (CKPV) 2 -induced cAMP production in macrophages. (B) Upper panel, RT-PCR results confirms (CKPV) 2 mRNA expression in MC1R cDNA-transfected COS-7 cells (COS-7/MC1R) or nonsense-cDNA-transfected control cells (COS-7). Lower panel: (CKPV) 2 -induced cAMP production in negative control-cDNA-transfected (NC) or MC1R cDNA-transfected COS-7 cells. * p

    Journal: PLoS ONE

    Article Title: The Synthetic Melanocortin (CKPV)2 Exerts Anti-Fungal and Anti-Inflammatory Effects against Candida albicans Vaginitis via Inducing Macrophage M2 Polarization

    doi: 10.1371/journal.pone.0056004

    Figure Lengend Snippet: (CKPV) 2 promotes cAMP production via MC1R. (A) Upper panel: the effects of MC1R siRNA (S1, S2 and S3, see sequence on Tab. 1) on MC1R mRNA level in primary cultured macrophages. Lower panel: MC1R siRNA knockdown almost blocked (CKPV) 2 -induced cAMP production in macrophages. (B) Upper panel, RT-PCR results confirms (CKPV) 2 mRNA expression in MC1R cDNA-transfected COS-7 cells (COS-7/MC1R) or nonsense-cDNA-transfected control cells (COS-7). Lower panel: (CKPV) 2 -induced cAMP production in negative control-cDNA-transfected (NC) or MC1R cDNA-transfected COS-7 cells. * p

    Article Snippet: MC1R RNA Interference (RNAi) The chemically synthesized MC1R siRNA (small interfering RNA) duplexes ( ) against mouse MC1R (s1, s2 and s3) were purchased from Ambion (GenePharm Co. Ltd. Shanghai, China).

    Techniques: Sequencing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Negative Control