Slik maintains tissue homeostasis by preventing JNK-mediated apoptosis

Background The c-Jun N-terminal kinase (JNK) pathway is an evolutionarily conserved regulator of cell death, which is essential for coordinating tissue homeostasis. In this study, we have characterized the Drosophila Ste20-like kinase Slik as a novel modulator of JNK pathway-mediated apoptotic cell death. Results First, ectopic JNK signaling-triggered cell death is enhanced by slik depletion whereas suppressed by Slik overexpression. Second, loss of slik activates JNK signaling, which results in enhanced apoptosis and impaired tissue homeostasis. In addition, genetic epistasis analysis suggests that Slik acts upstream of or in parallel to Hep to regulate JNK-mediated apoptotic cell death. Moreover, Slik is necessary and sufficient for preventing physiologic JNK signaling-mediated cell death in development. Furthermore, introduction of STK10, the human ortholog of Slik, into Drosophila restores slik depletion-induced cell death and compromised tissue homeostasis. Lastly, knockdown of STK10 in human cancer cells also leads to JNK activation, which is cancelled by expression of Slik. Conclusions This study has uncovered an evolutionarily conserved role of Slik/STK10 in blocking JNK signaling, which is required for cell death inhibition and tissue homeostasis maintenance in development. Supplementary Information The online version contains supplementary material available at 10.1186/s13008-023-00097-4.

The Sterile-20 (Ste20) kinases have been implicated in numerous physiological processes including cell proliferation and survival [20], cytoskeletal dynamics [21,22], and ion transport [23].The Ste20-kinase family consists of a heterogeneous group of Ser/Thr kinases including two large families (PAK-and GCK-like) and ten divergent subfamilies [21,24].Drosophila Slik and its mammalian ortholog STK10 belong to the GCK-V subfamily [25], which has emerged as an evolutionarily conserved modulator of ezrin/radixin/moesin (ERM) family proteins [26,27].In Drosophila, Moesin and Merlin, two FERM domain proteins, are the best known substrates of Slik [28].While Slik-mediated phosphorylation activates Moesin to stabilize microtubule organization and epithelial integrity [29,30], phosphorylation by Slik inactivates Merlin to adjust cell proliferation [31,32].Most of the physiological activities are governed by signaling pathways, though Slik is known to participate in a variety of cellular processes, the connection between Slik and potential signaling pathway remains poorly understood.
Drosophila is one of the best model organisms to study genetics, with many components of signal pathways being identified in fruit flies.We have previously performed a genetic screen with ectopic JNK-triggered cell death, and have characterized multiple components and regulators of the JNK pathway in Drosophila [33,34].In the current study, we report that Slik is required for maintaining tissue homeostasis by preventing JNKmediated apoptotic cell death in development.Loss of slik leads to JNK activation and triggers JNK-mediated cell death.Genetic epistasis analysis suggests that Slik acts upstream of or in parallel to Hep to suppress JNK pathway.Finally, the human ortholog STK10 has retained Slik functions to modulate JNK activity when introduced into Drosophila or in human cells.Taken together, this study unravels a crucial role of Slik in maintaining tissue homeostasis and organ development by preventing JNKmediated apoptotic cell death.

Slik inhibits ectopic JNK-induced cell death in development
Given that JNK signaling is of vital importance in regulating cell death, to identify new factors that regulate JNKmediated cell death, we performed a genetic screen for dominant modifiers of GMR > Egr-induced cell death in the Drosophila eyes using the Bloomington Drosophila Stock Center Deficiency kit [5,35].We found that three overlapping deficiencies, Df(2R)BSC603, Df(2R)ED4065 and Df(2R)ED4071, enhanced the small eye phenotype caused by GMR > Egr, indicating a potential enhancer is located within the common region (Additional file 1: Fig. S1A-E).This was confirmed by subsequent experiments showing that either mutation or knockdown of slik, one of the genes in this region, significantly enhanced the GMR > Egr-induced small eye phenotype (Additional file 11 Fig.S1F, G).Furthermore, ectopic expression of a constitutive active form of Hep (Hep CA ) triggered strong cell death posterior to the morphogenetic furrow (MF) in the eye imaginal discs (Fig. 1F, G), indicated by Acridine Orange (AO) staining that detects dying cells [36], and produced a small eye phenotype in the adults (Fig. 1A,  B).We found that GMR > Hep CA -induced cell death and small eye phenotype were enhanced in heterozygous slik mutants, but were significantly suppressed by overexpression of Slik (Fig. 1C-E, H-J).
To investigate whether Slik inhibits JNK-mediated cell death in other tissues, we turned to the developing wing.Compared with the controls (Fig. 1K, P), expression of Hep along the anterior/posterior (A/P) compartment border driven by patched (ptc)-Gal4 [37] resulted in increased cell death in the wing imaginal discs (Fig. 1Q), and generated a loss of anterior cross vein (ACV) phenotype in the adult wings (Fig. 1L).Both phenotypes were considerably suppressed by ectopic expression of Slik, but remained unaffected by that of LacZ (Fig. 1M-O, R-T).Collectively, these results indicate that Slik negatively regulates ectopic JNK-induced cell death during eye and wing development.

Depletion of slik triggers apoptotic cell death in development
Given that Slik is an inhibitor of ectopic JNK-mediated cell death, we wonder whether slik is physiologically required for preventing cell death in development.To test this, we depleted slik with three independent RNA interference (RNAi), whose knockdown efficiencies were confirmed by the quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay (Fig. 2P).Compared with the GMR-Gal4 or ptc-Gal4 controls, cell death was significantly increased upon slik knockdown in the corresponding areas of 3rd instar larval eye (Fig. 2A-E) or wing (Fig. 2F-J) discs, suggesting that endogenous Slik inhibits cell death in normal development.
Apoptosis is the major form of cell death in Drosophila, which is mediated by the cleavage and activation of caspases [38,39].To check whether Slik regulates apoptotic cell death, we utilized an anticleaved Dcp-1 antibody that specifically recognizes the activated form of the effector caspase Dcp-1 [40].We found that knockdown of slik induced strong apoptosis in the wing discs (Fig. 2K-O).Consistently, slik depletion-induced cell death was efficiently blocked by expressing P35, a viral caspase inhibitor [41], but not a lacZ RNAi (Additional file 1: Fig. S2).Taken together, these data suggest that slik is physiologically required for preventing apoptotic cell death in normal development.

JNK signaling is required for loss of slik-induced apoptotic cell death
Given that slik depletion activates JNK and apoptosis, we hypothesized that loss of slik might promotes cell death through activating JNK signaling.In agreement with this speculation, we found that ptc-Gal4 (Fig. 4A) driven slik depletion-caused cell death (Fig. 4B) was soundly suppressed by knocking down bsk, which encodes the Drosophila JNK, or expressing a dominant negative form of Bsk (Bsk DN ) or Puc, an inhibitor of JNK [18,19] (Fig. 4H-K).Consistently, loss of slik-induced apoptosis, visualized by anti-cleaved Dcp-1 (cDcp-1) antibody staining, was remarkably inhibited by Bsk DN (Fig. 4L, M, P, Q).In conclusion, these results suggest that slik depletion triggers JNK-dependent apoptotic cell death.

Slik functions upstream of or in parallel to Hep
To understand how Slik regulates JNK signaling-mediated cell death, we performed genetic epistasis analysis between Slik and core components of the Egr-JNK pathway.We found that slik knockdown-induced cell death was significantly suppressed by depletion of hep, but not by that of egr, dTRAF2, dTAK1 or lacZ serving as a negative control (Fig. 4A-G, K).Consistently,

Slik modulates JNK-dependent tissue homeostasis in normal development
Strict regulation of cell death, which contributes to the adjustment of cell number and elimination of unwanted cells, is vital for tissue homeostasis and organism fitness [49].To characterize the physiological function of  Furthermore, slik knockdown either in the posterior compartment by en-Gal4 or in the wing pouch by nub-Gal4 resulted in diminished sizes of corresponding areas, which were largely recovered by blocking Bsk activity (Fig. 5E-L).Moreover, we also generated slik mutant clones by FLP/FRT-mediated genetic mosaic technique [50], and observed that slik mutant clones (GFP − ) were significantly smaller than their simultaneously generated wild-type twin clones (2×GFP), while the control clones were almost the same size as their counterparts (Additional file 1: Fig. S3B-D).Together, these data suggest that slik is required to maintain JNK-dependent tissue homeostasis in normal development.

Human STK10 rescues slik depletion-induced developmental defects inDrosophila
Slik encodes a member of the Sterile-20 kinase family, sharing 46% similarity and 32% identity with its human ortholog STK10.To check whether STK10 has retained the developmental functions of Slik in Drosophila, we produced UAS-STK10 transgenic flies and checked whether expression of STK10 could rescue slik losscaused developmental defects.We noted that ectopic expression of Slik or STK10, verified by RT-qPCR (Additional file 1: Fig. S4), could largely rescue ptc > slik-IRinduced L3-L4 area reduction in adult wings (Fig. 6A-E) and robust cell death along A/P compartment boundary in larval wing discs (Fig. 6F-J).In addition, area reduction in the posterior compartment of en > slik-IR wing discs was efficiently rescued by expressing Slik or STK10 (Fig. 6K-O).Furthermore, MARCM analyses showed that the augmented apoptosis and diminished size of slik mutant clones (marked by GFP) were effectively mitigated upon the expression of Bsk DN , Slik, or STK10 (Additional file 1: Fig. S5).Thus, these data demonstrate that the developmental functions of Slik are evolutionarily conserved by STK10.

Slik function in the JNK pathway is retained by STK10
As the JNK pathway is evolutionarily conserved from fly to human, and STK10 is able to rescue slik loss-induced developmental defects, it is plausible that STK10 has retained the regulatory role of Slik in the JNK pathway.
Consistently, GMR > Egr-triggered cell death in the larval eye discs and the small eye phenotype in the adults were effectively restored by expressing STK10, while hep depletion was included as a positive control (Additional file 1: Fig. S6).Since Egr triggers both JNK-dependent and JNK-independent cell death in development [12,35,51], to determine whether STK10 modulates JNK-mediated cell death, we activated JNK signaling by overexpressing the JNK kinase Hep.We found that expression of STK10 markedly inhibited GMR > Hep CA -caused small eye in adults (Fig. 7A-D) and increased AO staining in larval eye discs (Fig. 7E-H), and ptc > Hep-induced lossof-ACV phenotype in adult wings (Fig. 7I-L) and cell death in larval wing discs (Fig. 7M-P).Hence, we conclude that STK10 could functionally substitute for Slik to regulate JNK-dependent cell death in Drosophila.

Slik and STK10 modulates physiological JNK-mediated cell death
The above results suggest that Slik negatively regulates ectopic JNK-mediated cell death, triggered by overexpression of Egr or Hep, yet it remains unknown whether Slik modulates the physiological JNK signaling.It has been reported that disruption of cell polarity promotes JNK-mediated cell death in development [52,53].Consistently, knockdown of the cell polarity gene scrib along the A/P compartment boundary by ptc-Gal4 induced severe cell death in wing discs (Fig. 8F, G) and ACV loss in adult wings (Fig. 8A, B).These phenotypes, caused by the activation of endogenous JNK signaling [5], were effectively inhibited by expressing Slik or STK10 (Fig. 8C-E, H-J), while expression of Bsk DN and Puc served as the positive controls (Additional file 1: Fig. S7).Importantly, ptc > scrib-IR-increased cell death was potently aggravated in heterozygous slik mutants (Fig. 8K-M).Together, these results indicated that Slik modulates physiological JNK signaling-induced cell death in development.
Finally, to examine whether STK10 regulates JNK activity in human cells, we knockdown STK10 in SW480 human colon cancer cells by two independent siRNA, whose efficacies were verified by RT-qPCR (Additional file 1: Fig. S8).We found that p-JNK level was Comparable with the en-Gal4 control (K), slik knockdown-induced GFP area reduction in the posterior compartment L was restored by expressing Slik M or STK10 N. Statistical analysis of GFP + area/GFP − area ratio in K-N is shown (O, n = 10 for each genotype).One-way ANOVA with Bonferroni multiple-comparison test was used to compute P-values, ****P < 0.0001, n.s indicates not significant.See the supplementary material for detailed genotypes.Scale bar: 100 μm up-regulated whereas total JNK protein level remained unaffected upon STK10 knockdown (Fig. 8N).Intriguingly, STK10 depletion-augmented p-JNK level was effectively mitigated by the expression of Slik, but not its kinase-deficient variant (Additional file 1: Fig. S9).These observations provide further evidence supporting the notion that Slik/STK10 play a conserved role in regulating the JNK pathway across both Drosophila and human cells, and the kinase activity of Slik/STK10 likely being crucial for this function.

Discussion
Control of tissue growth requires coordinate actions of cell proliferation, growth and death [54,55].Cell death modulates a great deal of physiological processes, including regulation of cell number, sculpting of tissue structures, and elimination of abnormal or aged cells, which plays a fundamental role in tissue homeostasis and organ development [56,57].JNK pathway has been shown to play a critical role in tissue homeostasis by maintaining the proper balance of cell death and proliferation [58,59].In this study, we took advantage of the fly genetics and identified Slik, the Drosophila ortholog of mammalian STK10, as an essential regulator of JNK-mediated apoptosis.Consistent with our data, slik exhibits moderately high expression in 3rd instar larval imaginal discs [60].In particular, loss of slik activates JNK signaling and promotes JNK-dependent apoptotic cell death, while overexpression of Slik or its human ortholog STK10 significantly suppresses ectopic or physiologic JNK-induced cell death in development.Furthermore, our genetic epistasis analysis established that Slik acts upstream of or in parallel to Hep, to regulate the JNK signaling in cell death (Additional file 1: Fig. S10).Notably, knockdown of STK10 in human cells also results in JNK activation, suggest the inhibitory role of Slik/STK10 on JNK signaling has been evolutionarily conserved from fly to human.
Previous studies have revealed Moesin and Merlin as the main substrates of Slik.In addition, STRIPAK is shown to act as an upstream regulator that dephosphorylates Slik, which facilitates its association with the cortex [30].In this work, we characterized Slik as a negative regulator of JNK pathway, yet the direct target of Slik in JNK signaling remains to be identified.It is worth noting that a previous study has documented the involvement of Moesin in the regulation of JNK-dependent cell death via the Rho1-Slpr-dTAK1 axis [61].Notably, the inhibition of cell death induced by Moesin was observed upon the knockdown of dTAK1 [61], but no such effect was observed in the case of slik loss-triggered cell death (Fig. 4F, K).Therefore, it can be inferred that Slik may regulate JNK-mediated cell death through a distinct mechanism that is independent of Moesin.
As Slik encodes a kinase, Slik-mediated phosphorylation may suppress JNK activity via two possible mechanisms, activating a negative regulator or inactivating a positive regulator.It is well known that phosphorylation can either activate or inactivate a substrate protein.Intriguingly, Slik-mediated phosphorylation promotes the function of Moesin, but inhibits that of Merlin, which results in a coordinate regulation of proliferation and epithelial integrity [27,31].If Slik acts upstream of Hep, a possible mechanism is that Slik phosphorylates and activates an inhibitor of Hep, thus inhibits Hep-mediated JNK activation.The protein phosphatase PP2A has been shown to inactivate JNK signaling by dephosphorylating MKK7, the ortholog of Hep, in hepatocellular carcinoma (HCC) cells [61].Interestingly, Cka, a regulatory subunit of PP2A, acts upstream of Hep to inhibit JNK signaling during Drosophila spermatogenesis [62].Furthermore, a Cka-Slik physical interaction has been reported, which contributes to PP2A-mediated Slik dephosphorylation [30].Thus, it is likely that Slik, in a feed-back manner, phosphorylates and activates Cka, which dephosphorylates Hep and inhibits JNK signaling.If Slik acts in parallel to Hep, a possible target is Puc, a serine/threonine protein phosphatase for Bsk.Kinase-mediated phosphorylation has been reported to promote phosphatases activities.For instance, the extracellular signal-regulated kinase 2 (ERK2) activates MAP kinase phosphatase-3 (MKP-3) [63], and protein kinase A (PKA) activates protein phosphatase 2 A (PP2A) by phosphorylation [64].Of particular interest, mTORC2-induced phosphorylation on DUSP10, the human ortholog of Puc, blocks its proteasome-mediated degradation [65].Hence, it is plausible that Slik/STK10 may inhibit JNK signaling via phosphorylation and activation of Puc/DUSP10.Apart from this, while Slik regulates Moesin and Merlin through its kinase function, a kinase independent genetic interaction of Slik with Raf has also been described [32,66].

Conclusions
Loss of slik triggers JNK-mediated apoptosis and impaired tissue homeostasis.Slik is necessary and sufficient for preventing physiological JNK-mediated apoptosis.Human STK10 rescues slik loss-induced cell death and impaired tissue homeostasis.Knockdown of STK10 in human cancer cells also leads to JNK activation, which is cancelled by expressing Slik.Thus, the inhibitory role of Slik/STK10 on JNK signaling has been evolutionarily conserved from fly to human.

Heat shock induction of clones
Fluorescently labelled clones were produced in larval imaginal discs using the yw hs-Flp; act > CD2 > Gal4 UAS-GFP strain.Clones were induced by heat shock at 37 °C for 30 min, late third-instar larvae were dissected after recovering for 4 days (Fig. 5A-C).

RT-qPCR
For RNAi-knockdown efficiency experiments, hs-Gal4 driver was used.Animals were raised at 25 °C, heatshocked at 37 °C for 30 min, and recovered at 29 °C for 2 h before dissection.-D, H-I).The bottom panels show high magnification views of the boxed areas in upper panels (A-D).Importantly, ptc > scrib-IR-induced cell death (K) was enhanced in heterozygous slik mutants (L).Statistical analysis of the presence of ACV in adult wings (E, n = 15 for each genotype) and cell death number in wing discs (J and M, n = 10 for each genotype) were shown.(N) Immunoblot analysis of p-JNK and total JNK protein level in SW480 cells treated by nonspecific siRNA (siCtrl) or two independent siRNA targeting STK10 (referred to as siSTK10-1 and siSTK10-2).p-JNK protein level was increased upon STK10 knockdown, while total JNK protein level remained unchanged.One-way ANOVA with Bonferroni multiple-comparison test and unpaired two tailed t-test were used to compute P-values, ****p < 0.0001, ***p < 0.001, **p < 0.01, *P < 0.05.See the supplementary material for detailed genotypes.Scale bar: 100 μm in A-D (upper panels), F-I and K-L, 50 μm in A-D (lower panels) Eastep Super (Shanghai Promega) was used to isolate total RNA from third instar larvae of indicated genotypes, and RT-qPCR was performed using SYBR Green PCR Premix Kit (TaKaRa).Primers used were as follows:   • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year

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Fig. 1
Fig. 1 Slik inhibits ectopic Hep-induced cell death.Light micrographs of Drosophila adult eyes A-D and fluorescence micrographs of third instar larval eye discs F-I are shown.Compared with the GMR-Gal4 controls (A, F, GMR > Hep CA -induced small eye phenotype B and cell death in eye discs G were partially inhibited by overexpression of Slik C, H, whereas enhanced in heterozygous slik mutants D, I. Statistical analysis of the adult eye sizes (E, n = 10 for each genotype) and AO-positive cell numbers in eye discs (J, n = 10 for each genotype) are shown.Light micrographs of Drosophila adult wings K-N and fluorescence micrographs of third instar larval wing discs P-S are shown.Compared with the ptc-Gal4 controls K, P, ectopic expression of Hep driven by ptc-Gal4 generated a loss-of-ACV phenotype in adults L and induced cell death in larval wing discs (Q), which were strongly blocked by expressing Slik (N, S), but not LacZ (M, R).The lower panels show high magnification view of the boxed areas in upper panels K-N).Quantification of the presence of ACV in adult wings (O, n = 15 for each genotype) and cell death in wing discs (T, n = 10 for each genotype) are shown.One-way ANOVA was used to compute P-values, ****p < 0.0001, ***p < 0.001, **p < 0.01, *P < 0.05, n.s indicates not significant.See the supplementary material for detailed genotypes.Scale bar:100 μm in A-D, F-I, K-N (upper panels) and P-S, 50 μm in K-N (lower panels)

Fig. 2
Fig. 2 Depletion of slik triggers apoptotic cell death.Fluorescence micrographs of third instar larval eye discs A-D are shown.Compared with the GMR-Gal4 controls (A), downregulation of slik with three independent RNA interference (RNAi) triggered extensive cell death posterior to the MF (B-D).Fluorescence micrographs of third instar larval wing discs F-I, K-N are shown.Compared with the ptc-Gal4 controls (F, K), depletion of slik induced massive cell death (G-I) and apoptosis L-N in wing discs.Quantification of cell death in eye discs (E, n = 12; n = 9; n = 10; n = 9) and statistical analysis of AO positive cell number (J, n = 10 for each genotype) and cleaved Dcp-1 (cDcp-1) activity (O, n = 10 for each genotype) in wing discs are shown.P The knock-down efficacies of slik RNAi lines.Expression of three independent slik RNAi significantly reduces the level of slik mRNA, as measured by quantitative RT-PCR.One-way ANOVA with Bonferroni multiple-comparison test was used to compute P-values, ****P < 0.0001, ***P < 0.001.See the supplementary material for detailed genotypes.Scale bar: 50 μm

Fig. 3
Fig. 3 Loss of slik promotes JNK pathway activation in vivo.Merged fluorescence micrographs of Drosophila third instar larval wing discs are shown A-H.The red channels detected only RFP A'-D' or pJNK signal E'-H' .Compared with the controls A, E, expressing three independent slik RNAi notably elevated the expression of TRE-RFP B-D and JNK phosphorylation (F-H) along the A/P compartment boundary.See the supplementary material for detailed genotype.Scale bar: 50 μm

Fig. 4
Fig. 4 Slik acts upstream of or in parallel to Hep to adjust JNK-dependent apoptotic cell death.Fluorescence micrographs of third instar larval wing discs are shown (A-J).Compared with the control (A), RNAi-mediated down-regulation of slik along the A/P compartment boundary by ptc-Gal4 induced obvious cell death (B), which was suppressed by depletion of hep (G) or bsk (H), or expressing a dominant-negative form of Bsk (I) or Puc (J), but remained unaffected by expressing RNAi of lacZ (C), egr (D), dTRAF2 (E) or dTAK1 (F).Merged fluorescence micrographs of third instar larval wing discs are shown (L-P).Compared with the control (L), loss of slik-induced apoptosis M was blocked by expressing hep RNAi O or Bsk DN (P), but not by egr RNAi (N).Statistical analysis of the AO positive cell numbers (K, n = 10 for each genotype) and cleaved Dcp-1 (cDcp-1) activity (Q, n = 10 for each genotype) in wing discs are shown.One-way ANOVA with Bonferroni multiple-comparison test was used to compute P-values, ****P < 0.0001, n.s indicates not significant.See the supplementary material for detailed genotypes.Scale bar: 50 μm

Fig. 5
Fig. 5 slik is physiologically required for proper tissue homeostasis in development.Merged fluorescence micrographs of Drosophila third instar larval wing discs are shown (A-C, E-G, I-K).From the Flp-out clones (marked by GFP), compared with the control (A), slik knockdown clones (GFP + ) displayed decreased clonal size (B), which was rescued by expressing Bsk DN (C).Statistical analysis of clone size/total size ratio in A-C are shown (D, n = 10 for each group).Besides, compared with the en-Gal4 (E) and nub-Gal4 controls (I), slik depletion-evoked decreased GFP area in the posterior compartment F or the whole wing pouch J was restored by Bsk DN (G, K).Statistical analysis of GFP + area/GFP − area ratio in E-G (H, n = 10 for each group) and I-K (L, n = 11; n = 8; n = 7) are shown.One-way ANOVA with Bonferroni multiple-comparison test was used to compute P-values, ****P < 0.0001, ***p < 0.001, *P < 0.05, n.s indicates not significant.See the supplementary material for detailed genotypes.Scale bar: 100 μm

(
See figure on next page.)Fig. 6 Human STK10 reliefs slik depletion-caused cell death.Light micrographs of adult wings (A-D) and fluorescence micrographs of third instar larval wing discs F-I, K-N are shown.Compared with the controls (A, F), loss of slik driven by ptc-Gal4 resulted in reduced L3-L4 area in adult wings B and intense AO staining in wing discs (G), which were effectively rescued by expressing Slik (C, H) or STK10 (D, I).Statistical analysis of the adult wing L3-L4 size/total size ratio (E, n = 10 for each genotype) and cell death number (J, n = 10 for each genotype) in wing discs are shown.

Fig. 7 Fig. 7 (
Fig. 7 Human STK10 inhibits JNK signaling in Drosophila.Light micrographs of Drosophila adult eyes (A-C) and wings (I-K), and fluorescence micrographs of third instar larval eye (E-G) and wing discs (M-O) are shown.Compared with the controls (A, E), GMR > Hep CA -evoked small eye phenotype (B) and cell death in eye discs (F) were inhibited by expressing STK10 (C, G).Statistical analysis of the adult eye sizes (D, n = 10 for each genotype) and AO-positive cell numbers in eye discs (H, n = 10 for each genotype) are shown.Consistently, compared with the ptc-Gal4 controls (I, M), ectopic Hep induced a loss-of-ACV phenotype in adult wings (J) and robust cell death in wing discs (N), both of which were suppressed by expressing STK10 (K, O).The lower panels show high magnification view of the boxed areas in upper panels (I-K).The presence of ACV in adult wings (L, n = 15 for each genotype) and AO positive cell numbers in wing discs (P, n = 10 for each genotype) were quantified.One-way ANOVA with Bonferroni multiple-comparison test was used to compute P-values, ****p < 0.0001, ***p < 0.001.See the supplementary material for detailed genotypes.Scale bar: 50 μm in A-C, E-G, I-K (lower panels) and M-O, 100 μm in I-K (upper panels) (See figure on next page.)

(
See figure on next page.)Fig. 8 Slik/STK10 prevents physiological JNK-mediated cell death.Light micrographs of Drosophila adult wings (A-D) and fluorescence micrographs of third instar larval wing discs (F-I, K-L) are shown.RNAi-mediated down-regulation of scrib along the A/P compartment boundary by ptc-Gal4 triggered the loss-of-ACV phenotype in adult wings (B), which was resulted from cell death in larval wing discs (G).Both phenotypes were inhibited by expressing Slik or STK10 (C rp49 FP: CCA CCA GTC GGA TCG ATA TGC.rp49 RP: CTC TTG AGA ACG CAG GCG ACC.slik FP: TAG GAC AGC AGC AAT GAG CTGG.slik RP: TTC ACG TAG CTC CTG CTT ACGG.

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