File S1 - Mutating RBF Can Enhance Its Pro-Apoptotic Activity and Uncovers a New Role in Tissue Homeostasis

Figure S1 in File S1. RBF contains a consensus site of caspase cleavage. (A) RBF sequence was scanned for potential caspase cleavage site(s) using the CASVM web server (http://www.casbase.org/). This was done with the P14P10′ window (tetrapeptide cleavage sites with ten additional upstream and downstream flanking sequences) which have the highest accuracy. Only one predicted caspase cleavage site was found in RBF: TELD-253. (B) Amino acid sequences alignment of retinoblastoma protein homologs. Amino acid sequences of proteins from H. sapiens (top), C. elegans (middle), D. melanogaster (bottom) were aligned using the Clustal Omega program. Dashes represent gaps in the sequence. Amino acid sequences shown in boxes correspond to consensus caspase cleavage sites. (C) RBF and RBF cleaved forms analysed by Western Blot. Proteins extracts are made from S2 cells transfected with pActine Gal4 vector or pUAS RBFp76-HA (RBFp76-HA), pUAS RBF-HA (RBF-HA) or pUAS RBFD253A-HA (RBFD253A-HA) (Effecten kit, Quiagen). 2.106 cells were cryolysed in PBS pH 7.6 and homogenized in buffer containing 50 mM Tris-Cl pH = 7,4, 150 mM NaCl, 1% NP40, 1 mM DTT, AEBSFSC. Proteins were separated in 4–12% Bis-Tris polyacrylamide gels according to the manufacturer's instructions (BioRad) and transferred onto PVDF membrane (Millipore). Blots were incubated with mouse anti-HA (HA.11, Covance) and rabbit polyclonal anti-Actin (1∶500, Sigma). Arrow shows wholes RBF forms and dotted-line arrow shows RBFp76. Figure S2 in File S1. Quantification of RBF and RBFD253A protein rates and rbf mRNA. (A) RBF and RBFD253A protein rates detected by Western blot analysis. Protein extracts were prepared from embryos carrying the da-Gal4 driver to induce UAS-RBF and UAS-RBFD253A expressions ubiquitously. Three genotypes were tested: da-Gal4/+ (control), da-Gal4/UAS-RBF, UAS-RBFD253A/+; da-Gal4/+ at 25°C. Actin was used as a loading control, and an RBF antibody was used to detect RBF and RBFD253A (rabbit polyclonal anti-RBF,1∶500, Custom antibody, Proteogenix and rabbit polyclonal anti-Actin, 1∶500, Sigma). Immunoreactive bands were detected by Immobilon™ Western Chemiluminescent HRP Substrate (Millipore) with facilities of ChemiDoc MP System (BioRad). (B) Immunoreactive bands were quantified using the Quantity One software. Under these conditions, the level of RBF protein is significantly higher in embryos expressing UAS-RBF and UAS-RBFD253A than in control embryos (asterisk, ANOVA, p = 7.6E-3); furthermore, there is no significant difference between RBF and RBFD253A protein expression levels (ANOVA, p = 0.48). (C) Quantification of rbf mRNA by RT-qPCR in wing imaginal discs. Fifty wing imaginal discs per genotype were dissected on ice. Total RNAs were extracted from each sample using the RNeasy Mini kit (QIAGEN), RT was performed on each sample using random primer oligonucleotides (Invitrogen) with Recombinant Taq DNA Polymerase (Invitrogen). Real-time PCR analysis was performed using the C1000 Touch™ Thermal cycler (Biorad). Data are normalized against rp49 and correspond to the mean of three independent experiments. Error bars are the S.E.M. Asterisks indicate statistical significant difference between two genotypes (Student test, p<0,05). Figure S3 in File S1. RBFD253A is pro-apoptotic in the ZNC and induces more apoptosis than RBF in third instar larvae wing imaginal discs. (A, E) C96-Gal4 and vg-Gal4 expression patterns are visualized by UAS-mtGFP expression in third instar larvae wing imaginal discs. (B–D, F–H) Apoptotic cells are labeled with Acridine Orange in wing imaginal discs (2 min in 100 ng/ml AO, Molecular Probes); specific staining of apoptotic cells corresponds to bright white patches. (B, F) C96-Gal4/+ and vg-Gal4/+ control discs have few apoptotic cells. (C) C96-Gal4/UAS-RBF wing discs are similar to control. (D) Some apoptotic cells are observed within the C96-Gal4 expression domain in UAS-RBFD253A/X; C96-Gal4/+ discs (white arrows). (G, H) Apoptotic cells are observed within the vg-Gal4 expression domain in vg-Gal4/+; UAS-RBF/+ and UAS-RBFD253A/X; vg-Gal4/+ wing discs (white arrows). (G) The white arrowhead indicates a zone at the center of the pouch where cells are not AO-labeled in vg-Gal4/+; UAS-RBF/+ wing discs. All discs are shown with posterior to the top. Discs were mounted in AO and observed with a conventional Leica DMRHC research microscope using the L5 filter to detect AO fluorescence. Figure S4 in File S1. RBFD253A expression at the antero-posterior boundary of wing imaginal discs induces more apoptosis than RBF, but adult phenotypes are similar. (A–C) Distances between veins 3 and 4 (dv3-v4) were measured at the posterior third of the wings using the Adobe Photoshop CS3 software, as indicated by the black lines. 20 wings were measured to estimate the average distance between veins 3 and 4 (dv3-v4±s.e.m) for each genotype: (A) ptc-Gal4/+ control wings, (B) ptc-Gal4/UAS-RBF flies, (C) UAS-RBFD253A; ptc-Gal4 flies. UAS-RBF as well as UAS-RBFD253A expression under the control of ptc-Gal4 brings veins 3 and 4 closer. (D) ptc-Gal4 expression pattern is visualized by UAS-mtGFP expression in third instar larvae wing imaginal discs. Apoptotic cells are labeled with TUNEL (E–G) or Acridine Orange (H–J) in the wing imaginal discs; specific staining of apoptotic cells corresponds to bright white patches. (E, H) Few apoptotic cells are observed in ptc-Gal4/+ control discs. (F, G, I, J) In ptc-Gal4/UAS-RBF and UAS-RBFD253A/+; ptc-Gal4/+ discs, apoptotic cells are observed within the ptc-Gal4 expression domain (white arrows). More apoptotic cells are observed in UAS-RBFD253A/+; ptc-Gal4/+ discs. All discs are shown posterior to the top. Discs were observed with a conventional Leica DMRHC research microscope using the L5 filter to detect AO fluorescence and using the N2.1 filter to detect TUNEL. Figure S5 in File S1. RBFD253A expression at the antero-posterior boundary of wing discs alters the Wg pattern. (A–C) anti-Wg (green) and anti-RBF (red) staining with enlarged views of boxed areas. (A) Wg pattern in control ptc-Gal4/+ wing disc. (B) ptc-Gal4/UAS-RBF discs have the same Wg pattern than control discs. (C) In UAS-RBFD253A/X; ptc-Gal4/+ discs, the Wg pattern is altered (white arrowhead) when compared to control discs. More wg expressing cells adjacent to the RBFD253A expression domain in the posterior compartment are observed. All discs are shown with posterior to the top. Discs were observed with a conventional Leica DMRHC research microscope using the L5 filter to detect Wg-associated fluorescence and using the N2.1 filter to detect RBF-associated fluorescence. Figure S6 in File S1. Expression of RBFD253A and co-expression of RBF and p35 lead to different Wg patterns. (A–C) RBF immuno-staining (red). (D–F) anti-Wg immuno-staining (green). (D) Control Wg pattern in UAS-p35/X; en-Gal4/+ wing disc. (E) In UAS-p35/X; en-Gal4/+; UAS-RBF/+ wing discs, the Wg pattern is altered when compared to control discs: ectopic patches are observed in the posterior compartment (arrows and box b). (F) In UAS-RBFD253A/X; en-Gal4/+ wing discs, the Wg pattern is altered when compared to the control, and an enlargement of this pattern is observed in the posterior compartment (box a). All discs are shown with posterior to the top. Discs were observed with a conventional Leica DMRHC research microscope using the L5 filter to detect Wg and the N2.1 filter to detect RBF associated fluorescence.
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