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: Nature

Article Title: Inhibition of LTβR-signalling activates Wnt-induced regeneration in lung

doi: 10.1038/s41586-020-2882-8

Figure Lengend Snippet: a-b, Representative images of immunohistochemical analysis for RelA ( a ) and RelB ( b ) (brown signal, indicated by arrows, haematoxylin counter stained, scale bar 50μm, zoomed area 25μm) in lung core biopsy sections from healthy (n=3) and COPD patients (n=4), with the quantification of RelA and RelB positive alveolar epithelial nuclei shown as mean ± SD. c-e , Gene set enrichment analysis (GSEA) of the LTβR signalling, NF-κB signalling (gene lists from IPA software, Qiagen), TNFR-mediated signalling (GO:0033209), positive regulation of I-kappaB kinase NF-kappaB signalling (GO:0043123) and NIK NF-kappaB signalling (GO:0038061) pathways in publically available array data from lung tissue (GSE47460-GPL14550) of healthy (n=91) v COPD patients (n=145) ( c ), from lung tissue (GSE37768) of healthy (n=9) v COPD patients (n=18) ( d ) and from peripheral blood mononuclear cells (GSE56768) of healthy (n=5) v COPD patients (n=49) ( e ). f , mRNA expression levels of Lta , Ltbr , Tnfsf14 ( Light ), Tnf , Ccl2 and Cxcl13 determined by qPCR in whole lung from B6 mice exposed to filtered air (FA, n=6) or cigarette smoke (CS, n=8) for 6m, individual mice shown. g , GSEA of the pathways described in (c-e) in the publically available array data (GSE52509) of lungs from our mice exposed to filtere air (FA, n=3) and cigarette smoke (CS, n=6) for 4 and 6m. h , Western blot analysis for RelB, p100 and p52 in total lung homogenate from the mice described in (f). Quantification relative to vinculin of individual mice shown (n=3). For gel source data see . i , Schematic representation of the LTβR-Ig treatment protocol. j , Representative low and high magnification overlay images of Multiplex immunofluorescence staining to identify CD4 (Red), CD8 (Green), B220 (Turquoise) and DAPI (blue) counterstained lung sections (Scale bars 100μm, n=4) from B6 mice exposed to CS for 6m, plus LTβR-Ig fusion protein or control Ig (80 μg i.p., weekly) therapeutically from 4 to 6m, and analysed at 6m. k , mRNA expression levels of Cxcl13 and Ccl19 determined by qPCR in whole lung from B6 mice exposed to FA or CS for 4 and 6m, plus LTβR-Ig fusion or control Ig (80 μg i.p., weekly) prophylactically from 2 to 4m and analysed at 4m, and therapeutically from 4 to 6m and analysed at 6m (n=4 mice/group, repeated twice, pooled data shown). P values indicated, Mann-Whitney one-sided test (a-b), unpaired two-tailed Student’s t test (f, h), one-way ANOVA multiple comparisons Bonferroni test (k).

Article Snippet: 24 h later, cells were stimulated for 6 and 24 h with an agonistic antibody to LTβR [2 μg/ml] (5G11, Cat. No. HM1079, Hycult Biotech), recombinant murine TNF-α [1 ng/ml] (Cat. No. 315–01A, PeproTech) or a combination thereof.

Techniques: Immunohistochemical staining, Staining, Software, Expressing, Western Blot, Multiplex Assay, Immunofluorescence, Control, MANN-WHITNEY, Two Tailed Test

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