EMBO/SourceData huggingface dataset:
Figure 4A. Immunoblotting for pro-caspase-3 (pro-CASP3) and active caspase-3 (CASP3) in GICs treated with prazosin (PRZ) or vehicle (V) demonstrating that prazosin activates caspase-3. ZVAD, a caspase inhibitor, prevents prazosin-induced caspase-3 activation. kDa: kilodaltons.B. Immunoblotting for pro-caspase-9 (pro-CASP9) and active caspase-9 (CASP9) in GICs treated with prazosin (PRZ) or vehicle (V) demonstrating that prazosin does not activate caspase-9.C. Viability analysis of GICs treated with prazosin for 24h in the presence or absence of ZVAD, a caspase inhibitor. ZVAD counteracts prazosin-induced GIC death. *P=0.0286, n=4, two-sided Mann-Whitney U-test.D. Prazosin induces glioblastoma cell apoptosis in vivo. GFP+ and GFP- cells were analyzed from tumors in vehicle or prazosin-treated mice. Annexin V / DAPI staining was used to identify apoptotic cells by FACS. Tumors were initiated by GBM44 GICs implantation.E. Dose-response curve of prazosin on GIC survival (24h treatment). EC50 was determined with curve fit using nonlinear regression.F. Viability analysis of GICs treated with prazosin for 24h in the presence or absence of cirazoline, a subtype agonist of the α-ARs. Cirazoline did not alter GIC survival and counteracted only poorly prazosin-induced GIC death. *P<0.05, n=4, two-sided Mann-Whitney U-test.G. Membrane binding assays demonstrating the absence of prazosin binding sites in GIC membrane preparations. Positive control shows that prazosin binds to membrane preparations of yeast expressing 1-AR. PRZ: prazosin. CRZ: cirazoline.H. Immunoblotting for phosphorylated ERK1/2 (P-ERK1/2) and total ERK1/2 following prazosin treatment for 30 min. Prazosin induces ERK1/2 phosphorylation in GICs.I. SRE-luciferase reporter activity analysis of GICs treated with prazosin in absence or presence of U0126 (10 μM), an inhibitor of the ERK-activating kinase MEK. U0126 prevented prazosin-induced SRE-luciferase activation in GICs.J. Viability analysis of GICs treated with prazosin for 24 h in the presence or absence of U0126 (10 μM), an inhibitor of the ERK-activating kinase MEK. U0126 did not counteract prazosin-induced GIC death. Blockade of prazosin-induced ERK1/2 phosphorylation by U0126 was confirmed by immunoblotting (insert). PRZ: prazosin.
Figure 5A. PKCδ expression analysis in two patient-derived GICs and NSCs by immunoblotting. GICs express higher levels of PKCδ than NSCs. kDa: kilodaltons.B. PKCδ by immunostaining (green). Nuclei were stained with DAPI (blue). Enhanced PKCδ punctate nuclear expression in prazosin-treated (PRZ, 5µM) GICs, as compared to vehicle-treated GICs. PRZ: prazosin. Scale bar: 10 µm.C. Total PKCδ (PKCδ 78kDa) and cleaved PKCδ (PKCδ 41kDa) expression analysis in GICs by immunoblotting. Prazosin (PRZ) induces PKCδ cleavage into a 41kDa active fragment. 2 µM rottlerin (Rott), a PKCδ inhibitor, inhibits prazosin-induced PKCδ cleavage. V: vehicle. kDa: kilodaltons.D. Pro-caspase-3 (Pro-CASP3) and activated caspase-3 (CASP3) expression analysis in GICs by immunoblotting. 2 µM rottlerin (Rott) inhibits prazosin (PRZ)-induced caspase-3 activation. V: vehicle. kDa: kilodaltons.E. Inhibition of PKCδ by rottlerin prevents, in a concentration-dependent manner, prazosin-induced GIC death. Viability analysis of GICs treated with prazosin for 72 h in the presence or absence of rottlerin. *P=0.0286, n=4, two-sided Mann-Whitney U-test.F. Inhibition of PKCδ by the specific anti-PKCδ RACK peptide δV1.1 (10 μM) counteracts prazosin-induced GIC death. Viability analysis of GICs treated with prazosin for 72 h in the presence or absence of δV1.1. *P < 0.005 for Prazosin 1 and 5 µM and P<0.001 for Prazosin 10 µM by two-tailed unpaired Student's t-test, n=4.G. Decreased PKCδ expression using shRNA counteracts prazosin-induced GIC death. Viability analysis of GICs transduced with scrambled or PKCδ shRNA and treated with prazosin for 72 h. *P < 0.005 by two-tailed unpaired Student's t-test, n=4.H. Prazosin inhibits AKT phosphorylation in GICs. LY294002 (LY, 30 µM), an inhibitor of the PI3K/AKT pathway, was used as positive control. Terazosin, which does not affect GIC viability, does not alter AKT phosphorylation (see the corresponding cell viability counting in Fig EV4C). Phosphorylated AKT (P-AKT) and total AKT (AKT) expression analysis in GICs by immunoblotting. V: vehicle. kDa: kilodaltons.I. Prazosin (10 µM) does not inhibit AKT phosphorylation in NSCs. Analysis by immunoblotting. V: vehicle. kDa: kilodaltons.J. β-catenin expression, a downstream target of AKT, is decreased in prazosin-treated GICs, as opposed to NSCs. Analysis by immunoblotting. V: vehicle. kDa: kilodaltons.K. L. Inhibition of PKCδ using rottlerin (Rott, 2 µM) or δV1.1 (10 µM) counteracts prazosin inhibition of AKT phosphorylation. Analysis by immunoblotting. V: vehicle. kDa: kilodaltons.
Figure 6B. Bioluminescent in vivo images of tumors in mice grafted with GICs expressing either scrambled or PKCδ shRNA. All mice were treated with prazosin (PRZ) for 45 days.C. Quantification of the bioluminescent signals showing that decreased expression of PKC counteracts prazosin inhibition of tumor growth. Fold change in total flux represents the ratio: total flux after treatment/total flux prior treatment. *P=0.0007, n=8, two-sided Mann-Whitney U-test.
Figure 1Morphology of four-week-old soil-grown plants of Col-0, snc1, and two independent transgenic lines of SNIPER1 OE into snc1 background. OE stands for overexpression of SNIPER1. Scale bar = 1 cm. SNIPER1 gene expression in the indicated plants as determined by RT-PCR. Error bars represent mean ± SD (n = 3). Two independent experiments were carried out with similar results. Quantification of H.a. Noco2 sporulation in the indicated genotypes 7 days post inoculation (dpi) with 105 spores per ml water. Error bars represent mean ± SD (n=4). Three independent experiments were carried out with similar results. Growth of P.s.m. ES4326 on four-week-old leaves of the indicated genotypes at 0 and 3 dpi with bacterial inoculum of OD600 = 0.001. Error bars represent mean ± SD (n=4 for day 0; n=5 for day 3). Three independent experiments were carried out with similar results.Morphology of four-week-old soil-grown plants of two independent transgenic lines of SNIPER1 OE in Col-0 background. Scale bar = 1 cm. No difference in growth and development was observed. SNIPER1 gene expression in the indicated plants as determined by RT-PCR. Error bars represent mean ± SD (n = 3). Two independent experiments were carried out with similar results. Quantification of H.a. Noco2 sporulation in the indicated genotypes 7 days post inoculation (dpi) with 105 spores per ml water. Error bars represent mean ± SD (n=4). Three independent experiments were carried out with similar results. Growth of P.s.t. DC3000 on four-week-old leaves of the indicated genotypes at 0 and 3 dpi with bacterial inoculum of OD600 = 0.001. Error bars represent mean ± SD (n=4 for day 0; n=5 for day 3). Three independent experiments were carried out with similar results.
Figure 2Morphology of four-week-old soil-grown plants of Col-0, chs1-2 (A), chs2-1 (B) and two independent transgenic lines of SNIPER1 OE into chs1-2 (A) and chs2-1 (B) backgrounds. Plants were grown at 16 °C under long day conditions. Scale bar = 1 cm.Morphology of four-week-old soil-grown plants of Col-0, chs3-2D (C), snc2-1D eds5 npr1 (D) and two independent transgenic lines of SNIPER1 OE into chs3-2D (C) and snc2-1D eds5 npr1 (D) backgrounds. Scale bar = 1 cm.
Figure 3Morphology of four-week-old soil-grown plants of Col-0, mekk1-5 ndr1, and two independent transgenic lines of SNIPER1 OE into mekk1-5 ndr1 background. Scale bar = 1 cm. The mekk1 ndr1 double mutant was used instead of the mekk1-5 single mutant is to overcome the sterility. SNIPER1 gene expression in the indicated plants as determined by RT-PCR. Error bars represent mean ± SD (n = 3). Two independent experiments were carried out with similar results. Quantification of H.a. Noco2 sporulation in the indicated genotypes 7 days post inoculation (dpi) with 105 spores per ml water. Error bars represent mean ± SD (n=4). Three independent experiments were carried out with similar results. Growth of P.s.m. ES4326 on four-week-old leaves of the indicated genotypes at 0 and 3 dpi with bacterial inoculum of OD600 = 0.001. mn stands for mekk1-5 ndr1 double mutant. Error bars represent mean ± SD (n=4). Three independent experiments were carried out with similar results.In vitro ubiquitination assay using E. coli-expressed SNIPER1-myc (E3), AtUBC8 (E2), AtUBA1 (E1) and/or HIS-FLAG-Ubiquitin. The molecular mass markers are indicated on the left (kDa). Asterisk indicates SNIPER1-myc. Arrow points to His-FLAG-Ub and straight lines on the right indicate self-ubiquitinated SNIPER1.
Figure 6Morphology of four-week-old soil-grown plants of Col-0 and two independent transgenic lines of 35S-SNIPER1H129Y. Scale bar = 1 cm.PR1 and PR2 gene expression in the indicated plants as determined by RT-PCR. Error bars represent mean ± SD (n = 3). Two independent experiments were carried out with similar results.Morphology of four-week-old soil-grown plants of Col-0, sniper1-1, sniper2-1, eds1-2, SNIPER1H129Y, SNIPER1H129Y eds1-2, sniper1-1 sniper2-1 eds1-2 and sniper1-c1 sniper2-1 eds1-2. sniper1-1 is an exonic T-DNA insertional mutant. sniper1-c1 is a SNIPER1 knockout mutant containing 807bp deletion that was generated by CRISPR-Cas9 system. Scale bar = 1 cm.Quantification of H.a. Noco2 sporulation in the indicated plants 7 days post inoculation (dpi) with 105 spores per ml water. Error bars represent mean ± SD (n=4). Two independent experiments were carried out with similar results.Immunoblot of RPS4HA (E), SNC1 (F), RPS2HA (G), and RPM1myc (H) in SNIPER1H129Y background. Equal loading is shown by Ponceau S staining of a non-specific band. The numbers below represent the normalized ratio between the intensity of the protein band and the Ponceau S band ± SD (n=3). Molecular mass marker in kilo Daltons is indicated on the left. Three independent experiments were carried out with similar results.
Figure 7Immunoprecipitation and biotinylation of SUMM2NB-FLAG (A), RPP4NB-FLAG (B), or CHS1NB-FLAG (C) by SNIPER1 H129Y-HATurboID in N. benthamiana. Two biological repeats were carried out with similar results.Interaction of SNIPER1 H129Y-CLuc and SUMM2NB-NLuc (D), RPP4NB-NLuc (E) or CHS1NB-NLuc (F) as tested by split-luciferase complementary assay in N. benthamiana. Three biological repeats with four technical repeats each were carried out with similar results.
Figure 1(G) Reduced NMDA/AMPA ratio at SC-CA1 synapses of PtenΔC/ΔC mice (P17-21). The NMDAR component was obtained 60 ms after stimulation. (ratio, n = 11 cells from 8 mice for WT and 8 (8) for ΔC; decay, n = 12 cells from 8 mice for WT and 11 (8) for ΔC, **P < 0.01, ns, not significant, Mann-Whitney U test). The error bars represent SEM. (H) Reduced amplitude but not frequency of NMDA -mEPSCs at PtenΔC/ΔC CA1 pyramidal neurons. (n = 17 cells from 4 mice for WT and 18 (4) for ΔC, **P < 0.01, ns, not significant, Mann-Whitney U test, Student's t-test). The error bars represent SEM. (I) Reduced NMDA/AMPA ratio at synapses in layer II/III pyramidal neurons in the prelimbic region of the mPFC of PtenΔC/ΔC mice (P20-21). The NMDAR component was obtained 60 ms after stimulation. (n = 9 cells from 3 mice for WT and 10 (3) for ΔC, *P < 0.05, **P < 0.01, Student's t-test, Mann-Whitney U test). The error bars represent SEM. Data information: (C-F) Results from the last 10-minute recordings are shown in scatter plots.
Figure 2A) A volcano plot showing DEGs (adjusted p value < 0.1) derived from RNA-Seq results for PtenΔC/ΔC and WT mice (P21). For the calculation of adjusted p value, we have employed the R package DESeq2 where the P‐values obtained by the Wald test are corrected for multiple testing using the Benjamini and Hochberg method. (n = 3 mice for WT and ΔC). See also Dataset EV1 for the full RNA-Seq results.C) Immunoblot validation of the increase in SLC6A20 protein levels in hippocampal lysates of PtenΔC/ΔC mice (P21). We denoted the antibody as 'SLC6A20' here, not 'SLC6A20A', because this antibody was generated using a synthetic peptide sequence (YNEPSNNCQKHAI) that is commonly present in SLC6A20A and SLC6A20B, being a pan-SLC6A20 antibody (ThermoFisher). (n = 3 mice for WT and ∆C, *P < 0.05, Student's t-test). The error bars represent SEM.(D,E) Increased phosphorylation of β-catenin at Ser-675 but not at Ser-552 in the nucleus-enriched P1 fraction but not in whole-brain lysates of PtenΔC/ΔC mice (3 months). Note that total levels of β-catenin were not changed in the P1 fraction or whole-brain lysates. (n = 4 mice for WT-WB/P1 and ΔC-WB/P1, *P < 0.05, ns, not significant, Student's t-test). The error bars represent SEM.
Figure 3(B,C) Decreased extracellular levels of proline and glycine in the brains of PtenΔC/ΔC mice (2-4 month), as determined by microdialysis in the hippocampus (see Materials and Methods for details). (proline, n = 13 mice for WT and 8 for ΔC, **P < 0.01, Student's t-test; glycine, n = 9 for WT and 5 for ∆C, **P < 0.01, Student's t-test, Mann-Whitney U test). The error bars represent SEM.
Figure 4(B) Normalization of NMDAR-dependent HFS-LTP at synapses of PtenΔC/ΔC mice (4-5 weeks) by DCS treatment (20 μM), as shown by fEPSP slopes. (n = 15 slices from 9 mice for WT-V/vehicle, 11 (5) for WT-D/DCS, 16 (10) for ΔC-V, 13 (7) for ΔC-D, **P < 0.01, ns, not significant, two-way ANOVA with Tukey's test). The grey traces represent the baseline fEPSP prior to LTP induction. The error bars represent SEM.(F) Normalization of NMDAR-dependent HFS-LTP at synapses of PtenΔC/ΔC mice (4-5 weeks) by sarcosine treatment (750 µM), as shown by fEPSP slopes. Note that the data for WT-V and ΔC-V are identical to those shown in Fig. 4B because the whole experiments were performed together; we generated independent figures for DCS and sarcosine results for the clear presentation of the data. (n = 15 slices from 9 mice for WT-V/vehicle, 9 (4) for WT-Sarc/Sarcosine, 16 (10) for ΔC-V/Vehicle, 18 (4) for ΔC-S, *P < 0.05, ***P < 0.001, ns, not significant, two-way ANOVA with Tukey's test). The grey traces represent the baseline fEPSP prior to LTP induction. The error bars represent SEM.
Figure 5(D) PtenΔC/ΔC mice (2-4 months) treated with Slc6a20a-ASO display partially normalized levels of whole-brain glycine, as shown by the lack of difference between saline-treated WT and ASO-treated PtenΔC/ΔC mice. Note that the glycine levels observed here are ~4 times lower than those measured in Fig. 3A, which could be attributable to different mouse ages (P21 in Fig. 3A vs. 2-4 months in Fig. 5D), absence and presence of ASO injection, or lot-to-lot variation of the ELISA kits. (n = 8 mice for WT-saline, 5 for WT-ASO, 7 for ΔC-saline, and 8 for ΔC-ASO, **P < 0.01, ns, not significant, two-way ANOVA with Tukey's test). The error bars represent SEM.(E) PtenΔC/ΔC mice (P20-37) treated with Slc6a20a-ASO display an NMDA/AMPA ratio at hippocampal SC-CA1 synapses that is comparable to that in ASO-treated WT mice. (n = 11 neurons from 5 mice for WT-ASO, 10 (4) for ΔC-ASO, ns, not significant, Mann-Whitney's U test). The error bars represent SEM.(G) PtenΔC/ΔC mice (2-4 months) treated with Slc6a20a-ASO display normal levels of climbing in the Laboras test. n = 6 mice for WT-saline, 7 for WT-ASO, 7 for ΔC-saline, and 8 for ΔC-ASO, *P < 0.05, **P <0.01, ns, not significant, two-way ANOVA with Bonferroni's test. The error bars represent SEM.
Figure 6(D) Increased extracellular levels of glycine and proline in the brain of Slc6a20a+/- and Slc6a20a-/- mice, as shown by microdialysis analyses. Note that glycine levels are increased in both Slc6a20a+/- and Slc6a20a-/- mice, whereas proline levels are increased only in Slc6a20a-/- mice. (glycine, n = 19 mice for WT, 11 for HT, 8 for KO, **P < 0.01, Mann-Whitney U test; proline, n = 15 mice for WT, 11 for HT, 14 for KO, ***P < 0.001, ns, not significant, Mann-Whitney U test). The error bars represent SEM.
Figure 7(B) Glycine/proline-induced currents for mouse SLC6A20A are dependent on sodium chloride, as shown by the suppression of the currents when sodium chloride is replaced with choline chloride or sodium gluconate. Each experimental condition was sequentially applied to HEK293T cells, as shown in Appendix Fig. S7B. The indicated currents are average values from an ensemble of multiple (~20) cells in a single well. (glycine, n = 21 cells for untransfected/blank, 17 for human SLC6A20-V1, 25 for human SLC6A20-V2, 33 for mSLC6A20A, 28 for mSLC6A20B ***P < 0.001 (relative to buffer not containing glycine), two-way ANOVA with Bonferroni's test; proline, n = 17 cells for untransfected, 23 for human SLC6A20-V1, 47 for SLC6A20-V2, 31 for mSLC6A20A, and 31 for mSLC6A20B, ***P < 0.001, two-way ANOVA with Bonferroni's test). The error bars represent SEM.
Figure 1(A-C) Immunoblots for indicated proteins in BAT, extensor digitorum longus (EDL), epididymal white adipose tissue (eWAT) and heart from 10‐ to 12‐month (mo)‐old control (Con) and knock out (KO) mice. Arrows depict LC3‐I and II.(D) ATG5 and ATG7 mRNA levels in indicated tissues (n=4)(E) immunoblots for indicated proteins in spleen, liver, lung, kidney, mediobasal hypothalamus (MBH), inguinal white adipose tissue (ingWAT) and perinephric fat (pnWAT) from 10‐ to 12‐mo‐old Con and KO mice.(F) Body weights (wt) of chow diet (RD)‐fed (n=6-29), and (G) high‐fat diet (HFD)‐fed Con and KO mice at indicated ages (n=4-17).(H) Total body fat and lean mass of 4-7 mo RD‐fed Con and KO mice (n=8-12).(I) BAT wt (n=4-7), (J) gastrocnemius (GA) wt (n=5-7), (K) soleus wt (n=4-7), (L) eWAT wt (n=5-7) and (M) visceral (Visc) and subcutaneous (Subc) body fat distribution in 10‐mo‐old Con and KO mice (n=4). Values are mean±s.e., *P0.05, **P0.01, ***P0.001.
Figure 2(A-B) mRNA for indicated genes in BAT (n=4)(c) mRNA for the indicated genes in epididymal white adipose tissue (eWAT) (n=4)(D) immunoblots for indicated proteins in BAT from 10‐month (mo)‐old chow diet (RD)‐fed control (Con) and knock out (KO) mice. Arrows depict protein isoforms.(E) immunoblots for indicated proteins in eWAT from 10‐month (mo)‐old chow diet (RD)‐fed control (Con) and knock out (KO) mice. Arrows depict protein isoforms.(F) Electron micrographs ( × 10,000 magnification) of BAT depicting mitochondria from 4‐mo‐old Con and KO mice. m, mitochondria; LD, lipid droplet; n, nucleus.(G) mRNA levels (n=4) in BAT from 10‐mo‐old Con and KO mice(H) hematoxylin and eosin (HE) and (I) Sirius Red stains in BAT from 10‐mo‐old Con and KO mice (n=3-4). Average adipocyte and LD size, and LD number in BAT are shown. Scale bar, 50 μm.(J) BAT mRNA levels from 4‐mo‐old RD‐fed Con and KO mice cold‐challenged for 75 min (n=3). Values are mean±s.e. ***P0.001.
Figure 3(A) Body temperature before (Basal) and during cold exposure (n=7-8) in 10‐month (mo)‐old chow diet (RD)‐fed control (Con) and knock out (KO) mice(B) Oxygen consumption (VO2), (C) carbon dioxide production (VCO2) in 10‐month (mo)‐old chow diet (RD)‐fed control (Con) and knock out (KO) mice (n=4).(D) energy expenditure in 10‐month (mo)‐old chow diet (RD)‐fed control (Con) and knock out (KO) mice (n=4)(E) VO2, (F) VCO2 and (G) energy expenditure in 10‐mo‐old high‐fat diet (HFD)‐fed Con and KO mice (n=3-5).(H) Ambulation (AMB) in 10‐mo‐old RD‐fed (n=4) and HFD‐fed Con and KO mice (n=3-5).(I) Hematoxylin and eosin (HE)‐stained epididymal white adipose tissue (eWAT) from 6‐mo‐old RD‐fed Con and KO mice. Quantification for cell size is shown (n=3). Scale bar, 50 μm.(J) Adβ3 mRNA in eWAT from 10‐mo‐old RD‐fed Con and KO mice (n=4), and in (K) inguinal (ingWAT) from 4‐mo‐oldcold‐challenged RD‐fed Con and KO mice (n=3).(L) Serum‐free fatty acid (FFA) and glycerol (Gly) from 4‐ to 6‐mo‐old Con and KO mice untreated (basal) or treated with isoproterenol (Iso) i.p. for 20 min (n=4-6), and (M) from 4‐mo‐old RD‐fed cold‐challenged (75 min) mice (n=3-4). Values are mean±s.e. *P0.05, **P0.01, ***P0.001.
Figure 4(A,B) Tmem26 and tbx1 mRNA levels in epididymal eWAT, and (C,D) ingWAT from Atg7Flox/Floxmice (4 months (mo)) injected in BAT with empty (Con AdV) or Cre‐expressing (Cre AdV) adenoviruses and cold‐exposed or not (basal) for 75 min (n=4-5).(E,F) hematoxylin and eosin (HE)‐stained ingWAT section from Atg7Flox/Flox mice (4 months (mo)) injected in BAT with empty (Con AdV) or Cre‐expressing (Cre AdV) adenoviruses and cold‐exposed or not (basal) for 75 min (n=4-5).(G) mRNA for indicated genes in ingWAT from 4‐mo‐old cold‐exposed Con and KO mice (n=4).(H,I) mRNA levels in eWAT from 10‐mo‐old Con and KO mice (n=4).(J) β‐oxidation in eWAT and ingWAT from 4‐mo‐old Con and KO mice (n=4).(K) mRNA levels in soleus from 4‐mo‐old cold‐exposed Con and KO mice (n=3-6).(M) BAT from 4‐mo‐old Con and KO mice (n=4). Values are mean±s.e. *P0.05, **P0.01, ***P0.001.
Figure 5(A) Blood glucose in 6‐ to 10‐month (mo)‐old mice (n=12-15), and (B) serum insulin levels in 10‐mo‐old fed control (Con) and KO mice (n=6).(C) Glucose tolerance tests in 10‐mo‐old chow diet (RD)‐fed (n=5), and (D) in 10‐ to 12‐mo‐old high‐fat diet (HFD)‐fed Con and KO mice (n=4-9).(E) Insulin tolerance test in 10‐mo‐old RD‐fed Con and KO mice (n=5).(F) Immunoblots for indicated proteins in skeletal muscle from 10‐mo‐old Con and KO mice.(G) Hematoxylin and eosin (HE)‐stained gastrocnemius (GA) sections from 6‐mo‐old Con and KO mice. Myofiber cross‐sectional area (μm2) is shown (n=3).(H) Expression of MuRF‐1 and Atrogin‐1 in GA and TA from 10‐mo‐old Con and KO mice (n=4)(I) myogenic genes in GA from 10‐mo‐old Con and KO mice (n=6).(J) Immunoblots for indicated proteins in tissues from 4‐mo‐old Con and KO mice (n=4).(K) mRNA for IRS1, (L) IRS2 in epididymal white adipose tissue (eWAT) and GA from 10‐mo‐old Con and KO mice (n=6).
Figure 1(A) Cellomics images (top) of GFP-LC3‐HEK cells in full medium (FM) or after 2 h in starvation medium (ES) with leupeptin (EL). Scale bars=40 μm. Optimised automated analysis (bottom left) programme detecting hoechst‐labelled nuclei (purple line), cell outline (red line) and identifies GFP-LC3 spots (red spots) in FM and EL. Enlarged GFP-LC3 images in FM and EL (bottom right). Scale bars=10 μm.(B) SCPO, STIPO and STAPO after indicated siRNA treatment of GFP-LC3‐HEK cells incubated as in (A). Error bars represent s.e.m. Significance was determined using a two‐tailed paired t‐test compared with RISCfree (RF) in EL: SCPO siULK1, ***P=0.0002; siATG7, **P=0.0078; siNRBP2, **P=0.0088. STIPO siULK1, ***P0.0001; siATG7, **P=0.0060; siNRBP2, **P=0.0041. STAPO siULK1, ***P0.0001; siATG7, **P=0.0037; siNRBP2, **P=0.0028. The experiments were performed: RISCfree (n=5), siULK1 (n=5), siATG7 (n=3), siNRBP2 (n=3).(D) Scatter plot of median SCPO Z‐scores from siRNA pools in the primary screen that met the minimum object count (OC) cutoff. Dashed lines represent approximate cutoff for 500 best increasers and decreasers. RP of known autophagy genes (ATG4C, ATG14, RB1CC1, ATG9A, ATG7) are given in Supplementary Table S2; AMBRA1 (FLJ20294), TBC1D25 (OATL1) and SH3GLB1 (BIF‐1) in Supplementary Table S1; LAMTOR2 has a RP=708.7 and RUBICON has a RP=1126.