Additional file 1 of Slik maintains tissue homeostasis by preventing JNK-mediated apoptosis
Creators: name: Li, Chenglin
name: Zhu, Xiaojie
name: Sun, Xinyue
name: Guo, Xiaowei
name: Li, Wenzhe
name: Chen, Ping
name: Shidlovskii, Yulii V.
name: Zhou, Qian
name: Xue, Lei
Publisher: name: figshare
Year: 2023
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer
subject: Mental Health
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Virology

description: Additional file 1: Figure S1. A genetic screen for modifiers of GMR&gt;Egr-induced eye-ablation phenotype. A A schematic depiction of the three deficiencies Df(2R)BSC603, Df(2R)ED4065, and Df(2R)ED4071. B–G Light micrographs of adult eyes. GMR&gt;Egr-induced small eye phenotype (B) was obviously enhanced by deficiency Df(2R)BSC603 (C), Df(2R)ED4065 (D) or Df(2R)ED4071 (E), or by slikKG04837(F) or slik RNAi (G). Scale bar: 100 µm. Figure S2. Depletion of slik causes apoptotic cell death. Fluorescence micrographs of third instar larval wing discs are shown (A–D). Compared with the control (A), knockdown of slik driven by ptc-Gal4 led to massive cell death along the A/P compartment boundary (B), which was completely impeded by expression of P35 (D), but not that of lacZ-IR (C). Statistical analysis of AO positive cell number in wing discs (E, n=10 for each genotype) is shown. One-way ANOVA with Bonferroni multiple comparison test was used to compute P-values, ****P &lt; 0.0001; ns, no significant difference. Scale bar: 40 µm. Figure S3. Slik is required for maintaining tissue homeostasis in development. A Statistical analysis of clone numbers in Fig. 5A-C are shown (n=10 for each group). B–C Fluorescence micrographs of third instar larval wing discs with twin clones (marked with 2ÍGFP or the absence of GFP) are shown. Compared with the control (B–B’), slik mutant clone (black) was obviously smaller than its wild-type twin clone (2ÍGFP) (C–C’). Statistical analysis of GFP-area/2ÍGFP area ratio (D, n=10 for each genotype) is shown. Unpaired two tailed t-test was used to compute P-values,****p &lt; 0.0001. Scale bar: 25 µm. Figure S4. The efficacies of Slik and STK10 overexpression. mRNA level of slik (A) or STK10 (B) was measured by quantitative RT-PCR (n=2). Unpaired two tailed t-test was used to compute P-values, ****p &lt; 0.0001, **p &lt; 0.01. Figure S5. STK10 suppresses JNK-mediated apoptosis in slik mutant clones. A–E Fluorescence micrographs of third instar larval wing discs with MARCM clones (marked with GFP) are shown. Compared with the control (A-A’’), apoptosis was triggered in slik mutant clone (B-B’’), which was suppressed by the expression of Slik (C-C’’), STK10 (D-D’’) or BskDN (E-E’’). Scale bar: 25 µm. Figure S6. Human STK10 suppresses Egr-triggered cell death in eye development. Light micrographs of adult eyes (A–D) and fluorescence micrographs of third instar larval eye discs (F–I) are shown. Compared with the controls (A, F), GMR&gt;Egr-induced small eye phenotype (B) and massive cell death in eye discs (G) were obviously suppressed by expressing STK10 (C, H), or hep-IR (D, I) serving as a positive control. Statistical analysis of adult eye size (E, n=10 for each genotype) and AO positive cell numbers in eye discs (J, n=10 for each genotype) are shown. One-way ANOVA with Bonferroni multiple-comparison test was used to compute P-values, ****p &lt; 0.0001, ***p&lt; 0.001, **p &lt; 0.01. Scale bar: 100 μm. Figure S7. scrib depletion-induced cell death depends on JNK pathway. Light micrographs of adult wings (A–C) and fluorescence micrographs of third instar larval wing discs (E–G) are shown. ptc&gt;scrib-IR-triggered loss-of-ACV phenotype in adult wings (A) and cell death in larval wing discs (E) were suppressed by expression of BskDN (B, F) or Puc (C, G). Statistical analysis of the presence of ACV in adult wings (D, n=15 for each genotype) and cell death number in wing discs (H, n≥10 for each genotype) are shown. One-way ANOVA with Bonferroni multiple-comparison test was used to compute P-values, ****p &lt; 0.0001, **p &lt; 0.01. Scale bar: 100 µm in A–C (upper panels), 50 µm in A–C (lower panels) and E–G.Figure S8. The knock-down efficacies of STK10 RNAi. Validation of two independent siRNA-STK10 used in current study. SW480 cells treated with siRNA for 72 hours were subjected to RT-qPCR (n=3). One-way ANOVA with Bonferroni multiple-comparison test was used to compute P-values, ****p &lt; 0.0001. Figure S9. Kinase activity is necessary for Slik/STK10 to regulate JNK pathway. Immunoblot analysis of p-JNK and total JNK protein level in SW480 cells. Compared with the control (lane 1), STK10 knockdown increased p-JNK level (lane 2), which was effectively suppressed by expressing Slik (lane 3), but not a truncated Slik with kinase domain deletion (lane 4). One-way ANOVA with Bonferroni multiple-comparison test was used to compute P-values, ****p&lt; 0.0001, ***p &lt; 0.001. Figure S10. Schematic summary of Slik in regulating JNK pathway. Slik inhibits JNK-mediated cell death. Genetic epistasis analysis suggests that Slik acts upstream of or in parallel to Hep to impede JNK pathway.