Figure 3: Loser cells secrete TyrRS under serum deprivation and during cell competition. | Nature Communications

Figure 3: Loser cells secrete TyrRS under serum deprivation and during cell competition.

From: Active JNK-dependent secretion of Drosophila Tyrosyl-tRNA synthetase by loser cells recruits haemocytes during cell competition

Figure 3

(a) Western blot of cellular fraction and supernatant of Drosophila S2 cells transfected with TyrRS-HA-tagged (C terminus). Cells are cultured with (+) or without (−) serum, as indicated. Anti-HA is used to detect TyrRS-HA expression and anti-tubulin is used as a negative control. (b,c) Microscopy images of cultured Drosophila S2 cells with and without serum at 24 h. Sytox Green is used as a marker of cell death. Scale bar, 25 μm. (d) Quantifications of total number of cells and dead cells. (e, left panel) Western blot of haemolymph (see Supplementary Video 1) from larvae of the following genotypes: ywF; tub>dmyc>gal4, TyrRS-HA (TyrRS-HA) and yw (WT). Tubulin is used as a negative control to discard contamination from cellular fractions. (e, right panel) Western blot from homogenized larvae of the following genotypes: ywF; tub>dmyc>gal4, UAS-TyrRS-HA (TyrRS-HA); ywF; tub>dmyc>gal4, UAS-TyrRS-HA, UAS-TyrRS-RNAi (TyrRSRNAi) and yw (WT).Western blot of haemolymph from larvae of the following genotype: ywF; tub>dmyc>gal4, TyrRS-HA, BskDN (BskDN), tubulin is used as a loading control. (f) Western blot of the third instar larvae haemolymph 48 h after cell competition clones were induced. Genotype ywF; tub>dmyc>gal4, UAS-LacZ (control), UAS-RNAi TyrRS, UAS-p35, UAS-BskDN or UAS-RNAi Kish. TyrRS cleaved fragments are named according to the molecular weight 51, 46 and 41 kDa. Lipophorin was used as a loading control. (g) Schematic representation of TyrRS including the linker region, MiniTyr domain (41 kDa) that includes the Tyrosine tRNA ligase signature and the C-terminal EMAP domain (17 kDa) including the tRNA-binding domain. (h) Quantification of secreted TyrRS normalized by the loading control Lipophorin (Lip), this ratio from the control is considered as 100%. (i) Cleaved secreted TyrRS/total secreted TyrRS in haemolymph. Control samples are protein extracts from larvae in which cell competition has been induced and loser clones express UAS-LacZ, innocuous for the cell. T-test *P<0.05. s.d. is represented.

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