Journal: EMBO Reports

Article Title: Functional profiling and visualization of the sphingolipid metabolic network in vivo

doi: 10.1038/s44319-025-00632-0

Figure Lengend Snippet: ( A ) OlyA w expression and SMases RNAi knockdowns in neurons. UAS-OlyA w expression and SMases RNAi were driven by nSyb -GAL4 ( nSyb -GAL4> OlyA w + UAS-RNAi ). Confocal images were captured from the posterior view of the Calyx in the brain of young adult flies (1 week old). (Right) Quantitative analysis of OlyA w intensity in the cortical region. P values [dSMPD4-IR (BDSC 51682), P < 0.001; dSMPD4-IR (VDRC 110163), P = 0.03; nSMase-IR (BDSC 36759), P = 0.09; nSMase-IR (VDRC 107062), P = 0.25; aSMase-IR (VDRC 12227), P = 0.46] were calculated using one-way ANOVA with Dunnett’s multiple comparisons. Data are represented as mean ± SEM ( n ≥ 4, biological repeats). Data are representative of at least two independent experiments. ( B ) OlyA w expression in neurons and SMases RNAi knockdowns in glia. LexAop-OlyA w expression is driven by nSyb -LexA, while SMases RNAi were driven by repo -GAL4 ( nSyb -LexA> LexAop-OlyA w ; repo -GAL4 > UAS-RNAi ). Confocal images were captured from the posterior view of the Calyx in the adult brain of young adult flies (1 week old). (Right) Quantifications of OlyA w intensity in the cortical region. P values [dSMPD4-IR (BDSC 51682), P = 0.10; dSMPD4-IR (VDRC 110163), P = 0.49; nSMase-IR (BDSC 36759), P = 0.90; nSMase-IR (VDRC 107062), P = 0.41; aSMase-IR (VDRC 12227), P < 0.001] were calculated using one-way ANOVA with Tukey’s multiple comparisons. Data are represented as mean ± SEM ( n ≥ 4, biological repeats). Data are representative of at least two independent experiments. ( C ) Quantification of the fold change in total lipid levels of dSMPD4 KO and nSMase KO compared to w 1118 brains (day 3 females). P values ( nSMase KO vs. w 1118 : Total lipids, P = 0.973; PS, P = 0.955; PE, P = 0.995; PC, P = 0.294; CerPE, P < 0.001; Cer, P < 0.001. dSMPD4 KO vs. w 1118 : Total lipids, P = 0.946; PS, P = 0.988; PE, P = 0.938; PC, P = 0.779; CerPE, P = 0.101; Cer, P = 0.768.) were calculated using one-way ANOVA with Dunnett’s multiple comparisons. Data are represented as mean ± SEM ( n = 4, biological repeats). ( D ) Quantification of the fold change in CerPE of dSMPD4 -knockout compared to w 1118 brains (day 3 females). P values (d16:2/22:0, P = 0.345; d16:2/20:0, P = 0.832; d16:2/18:0, P = 0.428; d16:1/22:0, P = 0.842; d16:0/22:0, P = 0.122; d14:2/24:0, P = 0.932; d14:2/22:0, P = 0.227; d14:2/20:0, P = 0.096; d14:2/18:1, P = 0.005; d14:2/18:0, P = 0.027; d14:1/24:1, P = 0.171; d14:1/24:0, P = 0.766; d14:1/22:0, P = 0.379; d14:1/20:0, P = 0.024; d14:1/18:0, P = 0.001; d14:0/22:1, P = 0.152; d14:0/20:1, P = 0.061) were calculated using two-tailed unpaired Student’s t test. Data are represented as mean ± SEM ( n = 4, biological repeats). ( E ) Quantification of the fold change in CerPE of nSMase -knockout compared to w 1118 brains (day 3 females). P values (d16:2/22:0, P = 0.081; d16:2/20:0, P < 0.001; d16:2/18:0, P < 0.001; d16:1/22:0, P < 0.001; d16:1/20:0, P < 0.001; d16:1/18:0, P < 0.001; d14:2/24:0, P = 0.610; d14:2/22:0, P = 0.663; d14:2/20:0, P < 0.001; d14:2/18:1, P < 0.001; d14:2/18:0, P < 0.001; d14:1/24:0, P = 0.203; d14:1/22:0, P = 0.016; d14:1/20:0, P < 0.001; d14:1/18:0, P < 0.001; d14:1/16:0, P < 0.001; d14:0/22:1, P = 0.001; d14:0/20:1, P < 0.001; d14:0/18:1, P < 0.001) were calculated using two-tailed unpaired Student’s t-test. Data are represented as mean ± SEM ( n = 4, biological repeats). ( F ) Anti-HA and anti-Lamin co-staining of the adult brain from dSMPD4 -HG line. Lamin is used as a marker of the nuclear envelope (NE). ( G ) Quantification of dSMPD4-3XHA subcellular localization. Box plot shows the Pearson’s coefficient of anti-HA and organellar markers, including anti-Lamin (nuclear envelope), anti-ATP5A (mitochondria), anti-Na + -K + -ATPase (Plasma membrane), and anti-Cathepsin L (Lysosome). Box plots illustrate the data distribution. The center line within the box represents the median, which is the 50th percentile. The lower and upper bounds of the box correspond to the 25th and 75th percentiles, respectively. The ends of the whiskers indicate the minima and maxima. Data points represent biological repeats. P values (Nuclear envelope vs. Mitochondria, P < 0.001; Nuclear envelope vs. Plasma membrane, P < 0.001; Nuclear envelope vs. Lysosome, P < 0.001) were calculated using one-way ANOVA with Tukey’s multiple comparisons. Representative images of anti-HA and organellar markers co-stainings are presented in Appendix Fig. . ( H ) Morphology of the nuclear membrane (anti-Lamin, magenta) in adult brains (1 week old) of indicated genotypes. ( I ) Quantification of the percentage of nuclei with blebs. Data are represented as mean ± SEM ( n = 3, biological repeats). P values ( w 1118 vs. dSMPD4 KO , P < 0.001; w 1118 vs. dSMPD4 KO ; actin > dSMPD4-myc , P = 0.999; w 1118 vs. nSMase KO , P = 0.547) were calculated using one-way ANOVA with Dunnett’s multiple comparisons. Data are representative of at least two independent experiments. ( J ) Olfactory associative memory assay in dSMPD4 -knockout, nSMase -knockout, and w 1118 control flies (1-week-old). Data are represented as mean ± SEM. P values ( w 1118 vs. dSMPD4 KO , P = 0.018; w 1118 vs. nSMase KO , P = 0.915) were calculated using one-way ANOVA with Dunnett’s multiple comparisons. Data are representative of four independent experiments of biological repeats. .

Article Snippet: Following ISTD were added: Glucosyl-beta-Ceramide d18:1/8:0 (Avanti #860540), CerPE d18:1/24:0 (Avanti #860067) and EquisplashTM LIPIDOMIX® Quantitative Mass Spec Internal Standard (Avanti #330731).

Techniques: Expressing, Knock-Out, Two Tailed Test, Staining, Marker, Clinical Proteomics, Membrane, Control

Journal: EMBO Reports

Article Title: Functional profiling and visualization of the sphingolipid metabolic network in vivo

doi: 10.1038/s44319-025-00632-0

Figure Lengend Snippet: ( A ) OlyA w expression and the aSMases RNAi knockdown in glia. UAS-OlyA w expression and aSMases RNAi were driven by repo -GAL4 ( repo -GAL4 > UAS-OlyA w +aSMase-RNAi ). Confocal images were captured from the posterior view of the Calyx in the adult brain of young adult flies (1 week old). (Right) Quantifications of OlyA w intensity in the cortical region. The P value ( P = 0.003) was calculated using two-tailed unpaired Student’s t test. Data are represented as mean ± SEM ( n ≥ 7). Data are representative of 4 independent experiments. ( B ) Quantification of the total CerPE levels in control ( repo -GAL4/+) and aSMase knockdown ( repo -GAL4/UAS-aSMase-RNAi) flies (day 10 female). The P value ( P = 0.01) were calculated using two-tailed unpaired Student’s t test. Data are represented as mean ± SEM (n > 3). Data are representative of 3 independent experiments. ( C ) Ref(2)P/p62 immunostainings in the optic lobe of adult brains from control or aSMase conditional knockout flies (1-week-old). Cell type-specific aSMase knockout was achieved by using small guide RNA targeting aSMase and Cas9 driven by neuronal ( nSyb -GAL4) or glial ( repo -GAL4) drivers. Control: UAS-Cas9 /+; UAS-aSMase-sgRNA /+; neuronal cKO: nSyb > Cas9+aSMase-sgRNA; glial cKO: repo > Cas9+aSMase-sgRNA . ( D ) Quantification of Ref(2)P/p62 puncta counts. Data are represented as mean ± SEM ( n ≥ 6). P values [Control vs. aSMase cKO (neuron), P = 0.910; Control vs. aSMase cKO (glia), P = 0.012] were calculated using one-way ANOVA with Tukey’s multiple comparisons. Data are representative of >5 independent experiments. ( E ) Climbing assay of control or aSMase conditional knockout flies (1-week-old). Cell type-specific aSMase knockout was achieved by using small guide RNA targeting aSMase and Cas9 driven by neuronal ( nSyb -GAL4) or glial ( repo -GAL4) drivers. Data are represented as mean ± SEM ( n = 5). P values [Control vs. aSMase cKO (neuron), P = 0.109; Control vs. aSMase cKO (glia), P < 0.001] were calculated using one-way ANOVA with Tukey’s multiple comparisons. Data are representative of 5 independent experiments. Control: UAS-Cas9 /+; UAS-aSMase-sgRNA /+; neuronal cKO: nSyb > Cas9+aSMase-sgRNA; glial cKO: repo > Cas9+aSMase-sgRNA .

Article Snippet: Following ISTD were added: Glucosyl-beta-Ceramide d18:1/8:0 (Avanti #860540), CerPE d18:1/24:0 (Avanti #860067) and EquisplashTM LIPIDOMIX® Quantitative Mass Spec Internal Standard (Avanti #330731).

Techniques: Expressing, Knockdown, Two Tailed Test, Control, Knock-Out, Climbing Assay