Journal: bioRxiv

Article Title: The deubiquitylating enzyme Fat Facets modulates Fat signalling and regulates tissue growth

doi: 10.1101/2024.01.27.577535

Figure Lengend Snippet: (A) Schematic representation of the Faf constructs used in this study. Numbers denote amino acid position. USP DUB domain is represented in green. (B) Faf binds to Ft. HA-tagged Ft ICD was co-expressed with FLAG-tagged GFP or Faf LD (B) in Drosophila S2 cells. Cells were lysed and lysates were subjected to co-immunoprecipitation using FLAG agarose beads. Lysates were analysed by immunoblot using the indicated antibodies for detection of protein expression and co-purification. Tubulin (Tub) was used as loading control. (C, D) Faf regulates Ft protein levels in Drosophila S2 cells. (C) HA-tagged Ft ICD was expressed in S2 cells in the presence or absence of Faf LD . 48h after transfection, cells were lysed and lysates were analysed by immunoblot using the indicated antibodies. Ø represents expression of empty vector. (D) S2 cells were treated with the indicated dsRNAs 24h before co-transfection with the indicated constructs. Ft ICD protein levels were analysed by Western blotting with the indicated antibodies 48h after cell transfection. GFP and Tubulin (Tub) were used as transfection and loading control, respectively. (E-J) Faf regulates Ft protein levels in vivo . Shown are XY confocal micrographs of third instar wing imaginal discs expressing the indicated constructs under the control of hh-Gal4 , showing Ft antibody staining (E-J and red in E’’-J’’), Arm antibody staining (E’-J’ and cyan in E’’-J’’), and direct fluorescence from GFP (green in E’’-J’’ merged images). Compared to the controls (E, lacZ RNAi ), faf depletion (F) and faf over-expression (G) resulted in a decrease or increase in Ft protein levels, respectively. Shown are also ft RNAi (H) and ft over-expression (I) controls to validate antibody specificity. Depletion of faf in the presence of ft over-expression resulted in a mild decrease in Ft protein levels (J). Ventral is up in XY sections, whilst GFP marks the hh-Gal4 -expressing posterior compartment (right). Dashed white line depicts boundary between anterior and posterior compartments. Scale bar represents 50 μm.

Article Snippet: Work involved the use of the Drosophila cell line Schneider S2 (RRID:CVCL_Z232), obtained from the ATCC and screened for mycoplasma presence, showing no contamination.

Techniques: Construct, Immunoprecipitation, Western Blot, Expressing, Copurification, Control, Transfection, Plasmid Preparation, Cotransfection, In Vivo, Staining, Fluorescence, Over Expression

Journal: bioRxiv

Article Title: The deubiquitylating enzyme Fat Facets modulates Fat signalling and regulates tissue growth

doi: 10.1101/2024.01.27.577535

Figure Lengend Snippet: (A-C) Faf regulates Ft protein levels in vivo in a DUB-dependent manner. Shown are XY confocal micrographs of third instar wing imaginal discs expressing the indicated constructs under the control of hh-Gal4 , showing Ft antibody staining (A-C and red in A’’-C’’), Arm antibody staining (A’-C’ and cyan in A’’-C’’), and direct fluorescence from GFP (green in A’’-C’’ merged images). Compared to the controls (A, lacZ RNAi ), over-expression of WT faf (B), but not of catalytically inactive faf ( faf CD , C), resulted in an increase in Ft protein levels. Ventral is up in XY sections, whilst GFP marks the hh-Gal4 -expressing posterior compartment (right). Dashed white line depicts boundary between anterior and posterior compartments. Scale bar represents 50 μm. (D-F) Quantification of in vivo Arm and Ft protein levels. Shown are the posterior/anterior (P/A) ratios for Fat protein levels (D), Arm protein levels (E), as well as the normalised Ft protein levels (F, normalised to the respective Arm levels). Data are shown as average ± standard deviation, with all data points represented. n=7, 12, 23 and 18 for lacZ RNAi , faf , faf WT and faf CD , respectively. Significance was assessed using a one-way ANOVA comparing all genotypes to the respective control ( hh>lacZ RNAi ), with Dunnett’s multiple comparisons test. **, p<0.01; ***, p<0.001. (G-J) Regulation of Ft protein levels in Drosophila S2 cells is mediated by the DUB activity of Faf. (G) HA-tagged Ft ICD was co-expressed with and V5-tagged Faf (WT or a catalytically inactive version, CD) in Drosophila S2 cells. Cells were lysed and lysates were analysed by immunoblot using the indicated antibodies for detection of protein expression. Tubulin was used as loading control. (H) Quantification of effect of Faf catalytic activity Ft protein levels in S2 cells. Shown are the relative Ft protein levels (fold change relative to controls, which were set to 1 in cells transfected with empty plasmid (ø); Ft protein levels were normalised to their respective Tubulin control) quantified from Western blot experiments where Faf was over-expressed. Data are represented as average ± standard deviation, with all data points represented. n=3 independent experiments. Significance was assessed by a one-way ANOVA comparing all samples to the respective control (ø), with Tukey’s multiple comparisons test or an unpaired t-test. *, p<0.05 **, p<0.01; ***, p<0.001. (I) Inhibition of DUB activity affects Ft protein stability. S2 cells were treated with 5 μM of WP1130 for 6 h before cell lysis. Protein levels were analysed by Western blotting using the indicated antibodies after cell lysis. FLAG-tagged GFP and Tubulin (Tub) were used as transfection and loading controls, respectively. (J) Quantification of effect of DUB inhibition on Ft protein levels in S2 cells. Significance was assessed by an unpaired t-test. *, p<0.05.

Article Snippet: Work involved the use of the Drosophila cell line Schneider S2 (RRID:CVCL_Z232), obtained from the ATCC and screened for mycoplasma presence, showing no contamination.

Techniques: In Vivo, Expressing, Construct, Control, Staining, Fluorescence, Over Expression, Standard Deviation, Activity Assay, Western Blot, Transfection, Plasmid Preparation, Inhibition, Lysis

Journal: STAR Protocols

Article Title: A computational pipeline to visualize DNA-protein binding states using dSMF data

doi: 10.1016/j.xpro.2022.101299

Figure Lengend Snippet:

Article Snippet: STARR-seq summits from Drosophila S2 cells , ( ) , https://data.starklab.org/publications/yanez-cuna_genomeRes_2014/S2_peakSummits.txt.

Techniques: Methylation Sequencing, Software

Journal: Molecular Cell

Article Title: BAP1 enhances Polycomb repression by counteracting widespread H2AK119ub1 deposition and chromatin condensation

doi: 10.1016/j.molcel.2021.06.020

Figure Lengend Snippet:

Article Snippet: Drosophila: S2 cell line , ATCC , ATCC CRL-1963.

Techniques: Control, Virus, Recombinant, Transfection, Purification, Extraction, Sequencing, Imaging, Plasmid Preparation, Software

Journal: Cell reports

Article Title: RNA polymerase II transcription initiation in holo-TFIID-depleted mouse embryonic stem cells

doi: 10.1016/j.celrep.2024.114791

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: S2 Drosophila cells , IGBMC PluriCell East platform , N/A.

Techniques: Recombinant, Staining, Protease Inhibitor, Random Hexamer, Imaging, SYBR Green Assay, RNA Extraction, Western Blot, Reverse Transcription, Sample Prep, Control, Cell Counting, Software