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    Echelon Biosciences p-3016
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    Echelon Biosciences dic16 pi3p echelon p
    (A) Schematic illustrating the putative enzymatic routes by which PI5P can be synthesised in Drosophila . The activities of enzymes labelled in blue are known in mammalian cells, the activity of enzymes labelled in red followed by “?” are still unknown in Drosophila , the activity of enzymes labelled in green are known in Drosophila . The activity of PI5P to PI(4,5)P 2 is boxed and is linked to cell size regulation in Drosophila . PI, phosphatidylinositol; <t>PI3P,</t> <t>phosphatidylinositol</t> <t>3-phosphate;</t> <t>PI(3,5)P</t> 2 , phosphatidylinositol 3,5 bisphosphate; PI5P, phosphatidylinositol 5-phosphate; PI(4,5)P 2 , phosphatidylinositol 4,5 bisphosphate. Kinase and phosphatase activities are denoted by black solid arrows and black dashed arrows, respectively. (B) Schematic illustrating the LC-MS/MS-based in vitro 5-kinase activity assay using S2R+ cells overexpressing dFab1 enzyme to convert synthetic PI or PI3P to PI5P and PI(3,5)P 2 , respectively. (C) (i) Immunoprecipitated protein levels were analysed by Western blotting with an anti-mCherry antibody. Control (IgG) was prepared without anti-mCherry. Input lane shows the correct size of dFab1 protein ∼230 kD. UTC, Untransfected control. (ii) In vitro kinase assay on synthetic PI and PI3P. Graph representing the kinase activity (%) as the normalised response ratio of “PI 5-kinase activity on PI” to “PI3P 5-kinase activity on PI3P” upon enzymatic activity of immunoprecipitated mCherry::dFab1 on the respective substrates. Response ratio of PI 5-kinase activity on PI is obtained from area under the curve of 17:0 14:1 PI5P (product)/17:0 14:1 PI (substrate), Response ratio of PI3P 5-kinase activity on PI3P is obtained from area under the curve of 17:0 20:4 PI(3,5)P 2 (product)/17:0 20:4 PI3P (substrate) and is represented as mean ± S.E.M. on addition of either negative control (no beads), control (mCherry beads) or dFab1 (mCherry::dFab1 beads). Number of immunoprecipitated samples = 2. (D) Schematic illustrating the LC-MS/MS-based in vitro PI(3,5)P 2 3-phosphatase activity assay using dsRNA-treated S2R+ cells as the enzyme source to convert synthetic PI(3,5)P 2 [d5-PI(3,5)P 2 to d5- 18 O- PIP 2 ] using a two-step reaction scheme. (E) In vitro phosphatase assay on synthetic PI(3,5)P 2 . Graph representing the 3-phosphatase activity (%) as the percent formation of d5- 18 O-PIP 2 formed from starting d5-PI(3,5)P 2 as mean ± S.E.M. on addition of either control (GFP ds RNA) or Mtm , CG3632 , CG3530 ds RNA-treated S2R+ cell lysates. One-way ANOVA with a post hoc Tukey’s test shows P -value = 0.003 between GFP and Mtm ds RNA-treated lysates, shows P -value = 0.63 between GFP and CG3632 ds RNA-treated lysate and shows P -value = 0.11 between GFP and CG3530 ds RNA-treated lysates.
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    1) Product Images from "PI3P-dependent regulation of cell size and autophagy by phosphatidylinositol 5-phosphate 4-kinase"

    Article Title: PI3P-dependent regulation of cell size and autophagy by phosphatidylinositol 5-phosphate 4-kinase

    Journal: Life Science Alliance

    doi: 10.26508/lsa.202301920

    (A) Schematic illustrating the putative enzymatic routes by which PI5P can be synthesised in Drosophila . The activities of enzymes labelled in blue are known in mammalian cells, the activity of enzymes labelled in red followed by “?” are still unknown in Drosophila , the activity of enzymes labelled in green are known in Drosophila . The activity of PI5P to PI(4,5)P 2 is boxed and is linked to cell size regulation in Drosophila . PI, phosphatidylinositol; PI3P, phosphatidylinositol 3-phosphate; PI(3,5)P 2 , phosphatidylinositol 3,5 bisphosphate; PI5P, phosphatidylinositol 5-phosphate; PI(4,5)P 2 , phosphatidylinositol 4,5 bisphosphate. Kinase and phosphatase activities are denoted by black solid arrows and black dashed arrows, respectively. (B) Schematic illustrating the LC-MS/MS-based in vitro 5-kinase activity assay using S2R+ cells overexpressing dFab1 enzyme to convert synthetic PI or PI3P to PI5P and PI(3,5)P 2 , respectively. (C) (i) Immunoprecipitated protein levels were analysed by Western blotting with an anti-mCherry antibody. Control (IgG) was prepared without anti-mCherry. Input lane shows the correct size of dFab1 protein ∼230 kD. UTC, Untransfected control. (ii) In vitro kinase assay on synthetic PI and PI3P. Graph representing the kinase activity (%) as the normalised response ratio of “PI 5-kinase activity on PI” to “PI3P 5-kinase activity on PI3P” upon enzymatic activity of immunoprecipitated mCherry::dFab1 on the respective substrates. Response ratio of PI 5-kinase activity on PI is obtained from area under the curve of 17:0 14:1 PI5P (product)/17:0 14:1 PI (substrate), Response ratio of PI3P 5-kinase activity on PI3P is obtained from area under the curve of 17:0 20:4 PI(3,5)P 2 (product)/17:0 20:4 PI3P (substrate) and is represented as mean ± S.E.M. on addition of either negative control (no beads), control (mCherry beads) or dFab1 (mCherry::dFab1 beads). Number of immunoprecipitated samples = 2. (D) Schematic illustrating the LC-MS/MS-based in vitro PI(3,5)P 2 3-phosphatase activity assay using dsRNA-treated S2R+ cells as the enzyme source to convert synthetic PI(3,5)P 2 [d5-PI(3,5)P 2 to d5- 18 O- PIP 2 ] using a two-step reaction scheme. (E) In vitro phosphatase assay on synthetic PI(3,5)P 2 . Graph representing the 3-phosphatase activity (%) as the percent formation of d5- 18 O-PIP 2 formed from starting d5-PI(3,5)P 2 as mean ± S.E.M. on addition of either control (GFP ds RNA) or Mtm , CG3632 , CG3530 ds RNA-treated S2R+ cell lysates. One-way ANOVA with a post hoc Tukey’s test shows P -value = 0.003 between GFP and Mtm ds RNA-treated lysates, shows P -value = 0.63 between GFP and CG3632 ds RNA-treated lysate and shows P -value = 0.11 between GFP and CG3530 ds RNA-treated lysates.
    Figure Legend Snippet: (A) Schematic illustrating the putative enzymatic routes by which PI5P can be synthesised in Drosophila . The activities of enzymes labelled in blue are known in mammalian cells, the activity of enzymes labelled in red followed by “?” are still unknown in Drosophila , the activity of enzymes labelled in green are known in Drosophila . The activity of PI5P to PI(4,5)P 2 is boxed and is linked to cell size regulation in Drosophila . PI, phosphatidylinositol; PI3P, phosphatidylinositol 3-phosphate; PI(3,5)P 2 , phosphatidylinositol 3,5 bisphosphate; PI5P, phosphatidylinositol 5-phosphate; PI(4,5)P 2 , phosphatidylinositol 4,5 bisphosphate. Kinase and phosphatase activities are denoted by black solid arrows and black dashed arrows, respectively. (B) Schematic illustrating the LC-MS/MS-based in vitro 5-kinase activity assay using S2R+ cells overexpressing dFab1 enzyme to convert synthetic PI or PI3P to PI5P and PI(3,5)P 2 , respectively. (C) (i) Immunoprecipitated protein levels were analysed by Western blotting with an anti-mCherry antibody. Control (IgG) was prepared without anti-mCherry. Input lane shows the correct size of dFab1 protein ∼230 kD. UTC, Untransfected control. (ii) In vitro kinase assay on synthetic PI and PI3P. Graph representing the kinase activity (%) as the normalised response ratio of “PI 5-kinase activity on PI” to “PI3P 5-kinase activity on PI3P” upon enzymatic activity of immunoprecipitated mCherry::dFab1 on the respective substrates. Response ratio of PI 5-kinase activity on PI is obtained from area under the curve of 17:0 14:1 PI5P (product)/17:0 14:1 PI (substrate), Response ratio of PI3P 5-kinase activity on PI3P is obtained from area under the curve of 17:0 20:4 PI(3,5)P 2 (product)/17:0 20:4 PI3P (substrate) and is represented as mean ± S.E.M. on addition of either negative control (no beads), control (mCherry beads) or dFab1 (mCherry::dFab1 beads). Number of immunoprecipitated samples = 2. (D) Schematic illustrating the LC-MS/MS-based in vitro PI(3,5)P 2 3-phosphatase activity assay using dsRNA-treated S2R+ cells as the enzyme source to convert synthetic PI(3,5)P 2 [d5-PI(3,5)P 2 to d5- 18 O- PIP 2 ] using a two-step reaction scheme. (E) In vitro phosphatase assay on synthetic PI(3,5)P 2 . Graph representing the 3-phosphatase activity (%) as the percent formation of d5- 18 O-PIP 2 formed from starting d5-PI(3,5)P 2 as mean ± S.E.M. on addition of either control (GFP ds RNA) or Mtm , CG3632 , CG3530 ds RNA-treated S2R+ cell lysates. One-way ANOVA with a post hoc Tukey’s test shows P -value = 0.003 between GFP and Mtm ds RNA-treated lysates, shows P -value = 0.63 between GFP and CG3632 ds RNA-treated lysate and shows P -value = 0.11 between GFP and CG3530 ds RNA-treated lysates.

    Techniques Used: Activity Assay, Liquid Chromatography with Mass Spectroscopy, In Vitro, Kinase Assay, Immunoprecipitation, Western Blot, Negative Control, Phosphatase Assay

    (A) In vitro phosphatase assay on synthetic PI3P. Graph representing the response ratio of 17:0 20:4 PI (product)/17:0 20:4 PI3P (substrate) formed as mean ± S.E.M. on addition of either control ( da/+ ) or da>Mtm WT ::GFP lysates. Lysate samples = 3, where each sample was made from five third instar wandering larvae. unpaired t test with Welch correction showed P -value = 0.007. (B) Extracted ion chromatogram (XIC) of deacylated PI3P or GroPI3P (glycerophosphoinositol 3-phosphate) peak at R t = 7.37 min, separated from deacylated PI4P or GroPI4P (glycerophosphoinositol 4-phosphate) peak at R t = 9.12 min obtained from injecting WT larval lipid extract (details of sample preparation is discussed in methods). (C) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of da/+; dPIP4K 29 (green) and da> Mtm WT ::GFP; dPIP4K 29 (magenta). Biological samples = 3, where each sample was made from three third instar wandering larvae. unpaired t test with Welch correction showed P -value = 0.07. (D) Graph representing normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of da/+ (green) and da> Mtm WT ::GFP (magenta). Biological samples = 3, where each sample was made from three third instar wandering larvae. Unpaired t test with Welch correction showed P -value = 0.13.
    Figure Legend Snippet: (A) In vitro phosphatase assay on synthetic PI3P. Graph representing the response ratio of 17:0 20:4 PI (product)/17:0 20:4 PI3P (substrate) formed as mean ± S.E.M. on addition of either control ( da/+ ) or da>Mtm WT ::GFP lysates. Lysate samples = 3, where each sample was made from five third instar wandering larvae. unpaired t test with Welch correction showed P -value = 0.007. (B) Extracted ion chromatogram (XIC) of deacylated PI3P or GroPI3P (glycerophosphoinositol 3-phosphate) peak at R t = 7.37 min, separated from deacylated PI4P or GroPI4P (glycerophosphoinositol 4-phosphate) peak at R t = 9.12 min obtained from injecting WT larval lipid extract (details of sample preparation is discussed in methods). (C) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of da/+; dPIP4K 29 (green) and da> Mtm WT ::GFP; dPIP4K 29 (magenta). Biological samples = 3, where each sample was made from three third instar wandering larvae. unpaired t test with Welch correction showed P -value = 0.07. (D) Graph representing normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of da/+ (green) and da> Mtm WT ::GFP (magenta). Biological samples = 3, where each sample was made from three third instar wandering larvae. Unpaired t test with Welch correction showed P -value = 0.13.

    Techniques Used: In Vitro, Phosphatase Assay, Sample Prep

    (A) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value as mean ± S.E.M. of WT (green) and dPIP4K 29 (magenta). Biological samples = 5, where each sample was made from five third instar wandering larvae. Unpaired t test with Welch correction showed P -value = 0.008. (B) Graph representing normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C/+ ; dPIP4K 29 (green) or Act5C> dPIP4K WT ::GFP; dPIP4K 29 (magenta). Biological samples = 5, where each sample was made from three third instar wandering larvae. unpaired t test with Welch correction showed P -value = 0.008. (C) Graph representing normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C/+ ; dPIP4K 29 (green) or Act5C> dPIP4K D271A; dPIP4K 29 (magenta). Biological samples = 6, where each sample was made from three third instar wandering larvae. Unpaired t test with Welch correction showed P -value = 0.818. (D) In vitro phosphatase assay on synthetic PI3P. Graph representing the response ratio of 17:0 20:4 PI (product)/17:0 20:4 PI3P (substrate) formed as mean ± S.E.M. on addition of either WT or dPIP4K 29 lysates for either a 5-min or a 15-min reaction. Lysate samples = 3, where each sample was made from five third instar wandering larvae. Multiple unpaired t test showed P -value = 0.26 for 5 min time point and P -value = 0.052. (E) qPCR measurements for mRNA levels of PI3K59F and PI3K68D from either WT (green) or dPIP4K 29 (magenta). unpaired t test showed P -value = 0.01 for PI3K59F and P -value = 0.58 for PI3K68D . qPCR measurements for mRNA levels of Mtm , CG3632, and CG3530 from either WT (green) or dPIP4K 29 (magenta). Unpaired t test showed P -value = 0.03 for Mtm , P -value = 0.23 for CG3632, and P -value = 0.0006 for CG3530 .
    Figure Legend Snippet: (A) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value as mean ± S.E.M. of WT (green) and dPIP4K 29 (magenta). Biological samples = 5, where each sample was made from five third instar wandering larvae. Unpaired t test with Welch correction showed P -value = 0.008. (B) Graph representing normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C/+ ; dPIP4K 29 (green) or Act5C> dPIP4K WT ::GFP; dPIP4K 29 (magenta). Biological samples = 5, where each sample was made from three third instar wandering larvae. unpaired t test with Welch correction showed P -value = 0.008. (C) Graph representing normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C/+ ; dPIP4K 29 (green) or Act5C> dPIP4K D271A; dPIP4K 29 (magenta). Biological samples = 6, where each sample was made from three third instar wandering larvae. Unpaired t test with Welch correction showed P -value = 0.818. (D) In vitro phosphatase assay on synthetic PI3P. Graph representing the response ratio of 17:0 20:4 PI (product)/17:0 20:4 PI3P (substrate) formed as mean ± S.E.M. on addition of either WT or dPIP4K 29 lysates for either a 5-min or a 15-min reaction. Lysate samples = 3, where each sample was made from five third instar wandering larvae. Multiple unpaired t test showed P -value = 0.26 for 5 min time point and P -value = 0.052. (E) qPCR measurements for mRNA levels of PI3K59F and PI3K68D from either WT (green) or dPIP4K 29 (magenta). unpaired t test showed P -value = 0.01 for PI3K59F and P -value = 0.58 for PI3K68D . qPCR measurements for mRNA levels of Mtm , CG3632, and CG3530 from either WT (green) or dPIP4K 29 (magenta). Unpaired t test showed P -value = 0.03 for Mtm , P -value = 0.23 for CG3632, and P -value = 0.0006 for CG3530 .

    Techniques Used: In Vitro, Phosphatase Assay

    (A) Schematic illustrating the methodology to assay PI3P by a dFab1-mediated radioactivity-based mass assay. dFab1 is purified from S2R+ cells by immunoprecipitation and used to convert PI3P from total lipid extracts obtained from larvae in the presence of ϒ 32 P-ATP to radiolabelled PI(3,5)P 2 product which is finally analysed using TLC. A portion of the total lipid extract is used for organic phosphate assay to normalise for sample size. (B) (i) Autoradiograph of TLC ran with lipid samples from in vitro PI3P mass assay using WT and dPIP4K 29 lipid samples. The first two lanes from the left are obtained from mass assay reactions using synthetic PI3P standard without or with addition of dFab1 enzyme, respectively. The origin spot and PI(3,5)P 2 spots are marked. (B) (ii) Graph represents normalised PI3P levels. Briefly, the spot marked as PI(3,5)P 2 on TLC in (B) is obtained by converting PI3P in the samples using immunoprecipitated dFab1 in the presence of ϒ 32 P-ATP are quantified using image analysis and then normalised to organic phosphate value (indicated in blue embedded text under TLC) to obtain normalised PI3P levels. Biological samples = 3, where each sample was made from five third instar wandering larvae. Unpaired t test with Welch correction showed P -value = 0.036. (C) Illustration depicting an indirect model where the increased PI3P levels in dPIP4K 29 (denoted by a red cross on dPIP4K) can be explained by either an activation of PI 3-kinase activity (green arrow) or an inhibition of PI3P 3-phosphatase activity (red stubbed arrow).
    Figure Legend Snippet: (A) Schematic illustrating the methodology to assay PI3P by a dFab1-mediated radioactivity-based mass assay. dFab1 is purified from S2R+ cells by immunoprecipitation and used to convert PI3P from total lipid extracts obtained from larvae in the presence of ϒ 32 P-ATP to radiolabelled PI(3,5)P 2 product which is finally analysed using TLC. A portion of the total lipid extract is used for organic phosphate assay to normalise for sample size. (B) (i) Autoradiograph of TLC ran with lipid samples from in vitro PI3P mass assay using WT and dPIP4K 29 lipid samples. The first two lanes from the left are obtained from mass assay reactions using synthetic PI3P standard without or with addition of dFab1 enzyme, respectively. The origin spot and PI(3,5)P 2 spots are marked. (B) (ii) Graph represents normalised PI3P levels. Briefly, the spot marked as PI(3,5)P 2 on TLC in (B) is obtained by converting PI3P in the samples using immunoprecipitated dFab1 in the presence of ϒ 32 P-ATP are quantified using image analysis and then normalised to organic phosphate value (indicated in blue embedded text under TLC) to obtain normalised PI3P levels. Biological samples = 3, where each sample was made from five third instar wandering larvae. Unpaired t test with Welch correction showed P -value = 0.036. (C) Illustration depicting an indirect model where the increased PI3P levels in dPIP4K 29 (denoted by a red cross on dPIP4K) can be explained by either an activation of PI 3-kinase activity (green arrow) or an inhibition of PI3P 3-phosphatase activity (red stubbed arrow).

    Techniques Used: Radioactivity, Mass Assay, Purification, Immunoprecipitation, Autoradiography, In Vitro, Activation Assay, Activity Assay, Inhibition

    (A) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+; dPIP4K 29 (n = 9), AB1>PI3K59F RNAi; dPIP4K 29 (n = 9). Sample size is represented on individual bars. Unpaired t test with Welch correction showed P -value < 0.0001. (B) Graph representing normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C /+; dPIP4K 29 (green) or Act5C > PI3K59F RNAi; dPIP4K 29 (magenta). Biological samples = 5, where each sample was made from five third instar wandering larvae. Unpaired t test with Welch correction showed P -value = 0.011. (C) (i) Representative confocal z-projections depicting a subpopulation of early endosomal compartment using 2XFYVE-mCherry in the salivary glands from wandering third instar larvae of AB1> 2XFYVE::mCherry (Control) and AB1> 2XFYVE::mCherry; dPIP4K 29 . Scale bar indicated at 20 μm. (ii) Graph representing 2XFYVE punctae measurement in the salivary glands from wandering third instar larvae of AB1> 2XFYVE::mCherry (N = 8, n = 40) and AB1>2XFYVE::mCherry; dPIP4K 29 (N = 8, n = 40). Unpaired t test with Welch correction showed P -value = 0.4057. (D) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+; dPIP4K 29 (n = 8), AB1>dPIP4K 2XFYVE ; dPIP4K 29 (n = 8). Sample size is represented on individual bars. Unpaired t test with Welch correction showed P -value = 0.171. (E) qPCR measurements for mRNA levels of UVRAG from either da/+ (green) or da> UVRAG RNAi (magenta). Unpaired t test showed P -value = 0.0046. (n = 6, where each n is derived from five third instar wandering larvae). (F) Graph representing average cell size measurement as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+; dPIP4K 29 (n = 12) and AB1> UVRAG RNAi; dPIP4K 29 (n = 12). unpaired t test with Welch correction showed P -value = 0.4306. (G) (i) Representative confocal z-projections depicting 2XFYVE punctae using 2XFYVE::mCherry in the salivary glands from the genotypes a. AB1> 2XFYVE :: mCherry (Control) b. AB1> 2XFYVE::mCherry; UVRAG RNAi. (ii) Graph representing measurement of 2XFYVE punctae numbers per unit cell area of the salivary glands from wandering third instar larvae of AB1> 2XFYVE::mCherry (N = 3, n = 11), AB1> 2XFYVE::mCherry; UVRAG RNAi (N = 3,n = 11).
    Figure Legend Snippet: (A) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+; dPIP4K 29 (n = 9), AB1>PI3K59F RNAi; dPIP4K 29 (n = 9). Sample size is represented on individual bars. Unpaired t test with Welch correction showed P -value < 0.0001. (B) Graph representing normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C /+; dPIP4K 29 (green) or Act5C > PI3K59F RNAi; dPIP4K 29 (magenta). Biological samples = 5, where each sample was made from five third instar wandering larvae. Unpaired t test with Welch correction showed P -value = 0.011. (C) (i) Representative confocal z-projections depicting a subpopulation of early endosomal compartment using 2XFYVE-mCherry in the salivary glands from wandering third instar larvae of AB1> 2XFYVE::mCherry (Control) and AB1> 2XFYVE::mCherry; dPIP4K 29 . Scale bar indicated at 20 μm. (ii) Graph representing 2XFYVE punctae measurement in the salivary glands from wandering third instar larvae of AB1> 2XFYVE::mCherry (N = 8, n = 40) and AB1>2XFYVE::mCherry; dPIP4K 29 (N = 8, n = 40). Unpaired t test with Welch correction showed P -value = 0.4057. (D) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+; dPIP4K 29 (n = 8), AB1>dPIP4K 2XFYVE ; dPIP4K 29 (n = 8). Sample size is represented on individual bars. Unpaired t test with Welch correction showed P -value = 0.171. (E) qPCR measurements for mRNA levels of UVRAG from either da/+ (green) or da> UVRAG RNAi (magenta). Unpaired t test showed P -value = 0.0046. (n = 6, where each n is derived from five third instar wandering larvae). (F) Graph representing average cell size measurement as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+; dPIP4K 29 (n = 12) and AB1> UVRAG RNAi; dPIP4K 29 (n = 12). unpaired t test with Welch correction showed P -value = 0.4306. (G) (i) Representative confocal z-projections depicting 2XFYVE punctae using 2XFYVE::mCherry in the salivary glands from the genotypes a. AB1> 2XFYVE :: mCherry (Control) b. AB1> 2XFYVE::mCherry; UVRAG RNAi. (ii) Graph representing measurement of 2XFYVE punctae numbers per unit cell area of the salivary glands from wandering third instar larvae of AB1> 2XFYVE::mCherry (N = 3, n = 11), AB1> 2XFYVE::mCherry; UVRAG RNAi (N = 3,n = 11).

    Techniques Used: Derivative Assay

    (A) qPCR measurements for mRNA levels of Mtm , CG3632, and CG3530 from either control ( da /+, in green) or da > Mtm RNAi, in magenta. Multiple t test with post hoc Holm-Sidak’s test showed P -value < 0.0001 between da/+ and da> Mtm RNAi for Mtm and P -value = 0.35 between da/+ and da> Mtm RNAi for CG3632 , and P -value = 0.04 between da/+ and da> Mtm RNAi for CG3530 . (B) (i) Representative confocal images of salivary glands from the genotypes a. AB1/+ , b. AB1>Mtm RNAi. Cell body is marked magenta by BODIPY-conjugated lipid dye, and the nucleus is marked by DAPI shown in green. Scale bar indicated at 50 μm. (ii) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+ (n = 7), AB1> Mtm RNAi (n = 7). Sample size is represented on individual bars. Unpaired t test with Welch correction showed P -value = 0.0005. (C) Graph representing normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value as mean ± S.E.M. of da/+ (green) and da> Mtm RNAi (magenta). Biological samples = 4, where each sample was made from five third instar wandering larvae. Unpaired t test with Welch correction showed P -value = 0.07. (D) (i) Representative confocal z-projections depicting autophagosomal levels using Atg8a-mCherry in the salivary glands from the genotypes a. AB1>Atg8a::mCherry (control), b. AB1>Atg8a::mCherry; Mtm RNAi. Scale bar indicated at 20 μm. (ii) Graph representing Atg8a punctae measurement in the salivary glands from wandering third instar larvae of AB1>Atg8a::mCherry (N = 7, n = 40), AB1>Atg8a::mCherry; Mtm RNAi (N = 7, n = 40). Unpaired t test with Welch correction showed P -value < 0.0001. (E) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+ (n = 11), AB1>Mtm RNAi (n = 8), AB1>Mtm RNAi, Atg8a RNAi (n = 12). Sample size is represented on individual bars. One-way ANOVA with post hoc Tukey’s test showed P -value < 0.0001 between AB1/+ and AB1>Mtm RNAi and P -value = 0.0002 between AB1/+; dPIP4K 29 and AB1>Mtm RNAi, Atg8a RNAi.
    Figure Legend Snippet: (A) qPCR measurements for mRNA levels of Mtm , CG3632, and CG3530 from either control ( da /+, in green) or da > Mtm RNAi, in magenta. Multiple t test with post hoc Holm-Sidak’s test showed P -value < 0.0001 between da/+ and da> Mtm RNAi for Mtm and P -value = 0.35 between da/+ and da> Mtm RNAi for CG3632 , and P -value = 0.04 between da/+ and da> Mtm RNAi for CG3530 . (B) (i) Representative confocal images of salivary glands from the genotypes a. AB1/+ , b. AB1>Mtm RNAi. Cell body is marked magenta by BODIPY-conjugated lipid dye, and the nucleus is marked by DAPI shown in green. Scale bar indicated at 50 μm. (ii) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+ (n = 7), AB1> Mtm RNAi (n = 7). Sample size is represented on individual bars. Unpaired t test with Welch correction showed P -value = 0.0005. (C) Graph representing normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value as mean ± S.E.M. of da/+ (green) and da> Mtm RNAi (magenta). Biological samples = 4, where each sample was made from five third instar wandering larvae. Unpaired t test with Welch correction showed P -value = 0.07. (D) (i) Representative confocal z-projections depicting autophagosomal levels using Atg8a-mCherry in the salivary glands from the genotypes a. AB1>Atg8a::mCherry (control), b. AB1>Atg8a::mCherry; Mtm RNAi. Scale bar indicated at 20 μm. (ii) Graph representing Atg8a punctae measurement in the salivary glands from wandering third instar larvae of AB1>Atg8a::mCherry (N = 7, n = 40), AB1>Atg8a::mCherry; Mtm RNAi (N = 7, n = 40). Unpaired t test with Welch correction showed P -value < 0.0001. (E) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+ (n = 11), AB1>Mtm RNAi (n = 8), AB1>Mtm RNAi, Atg8a RNAi (n = 12). Sample size is represented on individual bars. One-way ANOVA with post hoc Tukey’s test showed P -value < 0.0001 between AB1/+ and AB1>Mtm RNAi and P -value = 0.0002 between AB1/+; dPIP4K 29 and AB1>Mtm RNAi, Atg8a RNAi.

    Techniques Used:

    Schematic showing the regulation of PI3P at an autophagic membrane downstream of Atg1. In this study, dPIP4K which metabolizes PI3P has been shown to genetically interact with Vps34 and Mtm, two established PI3P regulators. Atg8a-marked autophagosomes form, mature to autolysosomes, and function in maintaining normal cell size in WT cells. In dPIP4K 29 cells, PI3P levels increase, leading to an increase in Atg8a punctae by increased autophagy initiation, which in turn leads to decreased cell size phenotype.
    Figure Legend Snippet: Schematic showing the regulation of PI3P at an autophagic membrane downstream of Atg1. In this study, dPIP4K which metabolizes PI3P has been shown to genetically interact with Vps34 and Mtm, two established PI3P regulators. Atg8a-marked autophagosomes form, mature to autolysosomes, and function in maintaining normal cell size in WT cells. In dPIP4K 29 cells, PI3P levels increase, leading to an increase in Atg8a punctae by increased autophagy initiation, which in turn leads to decreased cell size phenotype.

    Techniques Used:

    Multiple reaction monitoring (MRM) values for commercial lipids used.
    Figure Legend Snippet: Multiple reaction monitoring (MRM) values for commercial lipids used.

    Techniques Used:

    dic16 pi3p  (Echelon Biosciences)


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    Echelon Biosciences dic16 pi3p
    (A) Schematic illustrating the putative enzymatic routes by which PI5P can be synthesised in Drosophila . The activities of enzymes labelled in blue are known in mammalian cells, the activity of enzymes labelled in red followed by “?” are still unknown in Drosophila , the activity of enzymes labelled in green are known in Drosophila . The activity of PI5P to PI(4,5)P 2 is boxed and is linked to cell size regulation in Drosophila . PI, phosphatidylinositol; <t>PI3P,</t> <t>phosphatidylinositol</t> <t>3-phosphate;</t> <t>PI(3,5)P</t> 2 , phosphatidylinositol 3,5 bisphosphate; PI5P, phosphatidylinositol 5-phosphate; PI(4,5)P 2 , phosphatidylinositol 4,5 bisphosphate. Kinase and phosphatase activities are denoted by black solid arrows and black dashed arrows, respectively. (B) Schematic illustrating the LC-MS/MS-based in vitro 5-kinase activity assay using S2R+ cells overexpressing dFab1 enzyme to convert synthetic PI or PI3P to PI5P and PI(3,5)P 2 , respectively. (C) (i) Immunoprecipitated protein levels were analysed by Western blotting with an anti-mCherry antibody. Control (IgG) was prepared without anti-mCherry. Input lane shows the correct size of dFab1 protein ∼230 kD. UTC, Untransfected control. (ii) In vitro kinase assay on synthetic PI and PI3P. Graph representing the kinase activity (%) as the normalised response ratio of “PI 5-kinase activity on PI” to “PI3P 5-kinase activity on PI3P” upon enzymatic activity of immunoprecipitated mCherry::dFab1 on the respective substrates. Response ratio of PI 5-kinase activity on PI is obtained from area under the curve of 17:0 14:1 PI5P (product)/17:0 14:1 PI (substrate), Response ratio of PI3P 5-kinase activity on PI3P is obtained from area under the curve of 17:0 20:4 PI(3,5)P 2 (product)/17:0 20:4 PI3P (substrate) and is represented as mean ± S.E.M. on addition of either negative control (no beads), control (mCherry beads) or dFab1 (mCherry::dFab1 beads). Number of immunoprecipitated samples = 2. (D) Schematic illustrating the LC-MS/MS-based in vitro PI(3,5)P 2 3-phosphatase activity assay using dsRNA-treated S2R+ cells as the enzyme source to convert synthetic PI(3,5)P 2 [d5-PI(3,5)P 2 to d5- 18 O- PIP 2 ] using a two-step reaction scheme. (E) In vitro phosphatase assay on synthetic PI(3,5)P 2 . Graph representing the 3-phosphatase activity (%) as the percent formation of d5- 18 O-PIP 2 formed from starting d5-PI(3,5)P 2 as mean ± S.E.M. on addition of either control (GFP ds RNA) or Mtm , CG3632 , CG3530 ds RNA-treated S2R+ cell lysates. One-way ANOVA with a post hoc Tukey’s test shows P -value = 0.003 between GFP and Mtm ds RNA-treated lysates, shows P -value = 0.63 between GFP and CG3632 ds RNA-treated lysate and shows P -value = 0.11 between GFP and CG3530 ds RNA-treated lysates.
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    1) Product Images from "PI3P-dependent regulation of cell size and autophagy by phosphatidylinositol 5-phosphate 4-kinase"

    Article Title: PI3P-dependent regulation of cell size and autophagy by phosphatidylinositol 5-phosphate 4-kinase

    Journal: Life Science Alliance

    doi: 10.26508/lsa.202301920

    (A) Schematic illustrating the putative enzymatic routes by which PI5P can be synthesised in Drosophila . The activities of enzymes labelled in blue are known in mammalian cells, the activity of enzymes labelled in red followed by “?” are still unknown in Drosophila , the activity of enzymes labelled in green are known in Drosophila . The activity of PI5P to PI(4,5)P 2 is boxed and is linked to cell size regulation in Drosophila . PI, phosphatidylinositol; PI3P, phosphatidylinositol 3-phosphate; PI(3,5)P 2 , phosphatidylinositol 3,5 bisphosphate; PI5P, phosphatidylinositol 5-phosphate; PI(4,5)P 2 , phosphatidylinositol 4,5 bisphosphate. Kinase and phosphatase activities are denoted by black solid arrows and black dashed arrows, respectively. (B) Schematic illustrating the LC-MS/MS-based in vitro 5-kinase activity assay using S2R+ cells overexpressing dFab1 enzyme to convert synthetic PI or PI3P to PI5P and PI(3,5)P 2 , respectively. (C) (i) Immunoprecipitated protein levels were analysed by Western blotting with an anti-mCherry antibody. Control (IgG) was prepared without anti-mCherry. Input lane shows the correct size of dFab1 protein ∼230 kD. UTC, Untransfected control. (ii) In vitro kinase assay on synthetic PI and PI3P. Graph representing the kinase activity (%) as the normalised response ratio of “PI 5-kinase activity on PI” to “PI3P 5-kinase activity on PI3P” upon enzymatic activity of immunoprecipitated mCherry::dFab1 on the respective substrates. Response ratio of PI 5-kinase activity on PI is obtained from area under the curve of 17:0 14:1 PI5P (product)/17:0 14:1 PI (substrate), Response ratio of PI3P 5-kinase activity on PI3P is obtained from area under the curve of 17:0 20:4 PI(3,5)P 2 (product)/17:0 20:4 PI3P (substrate) and is represented as mean ± S.E.M. on addition of either negative control (no beads), control (mCherry beads) or dFab1 (mCherry::dFab1 beads). Number of immunoprecipitated samples = 2. (D) Schematic illustrating the LC-MS/MS-based in vitro PI(3,5)P 2 3-phosphatase activity assay using dsRNA-treated S2R+ cells as the enzyme source to convert synthetic PI(3,5)P 2 [d5-PI(3,5)P 2 to d5- 18 O- PIP 2 ] using a two-step reaction scheme. (E) In vitro phosphatase assay on synthetic PI(3,5)P 2 . Graph representing the 3-phosphatase activity (%) as the percent formation of d5- 18 O-PIP 2 formed from starting d5-PI(3,5)P 2 as mean ± S.E.M. on addition of either control (GFP ds RNA) or Mtm , CG3632 , CG3530 ds RNA-treated S2R+ cell lysates. One-way ANOVA with a post hoc Tukey’s test shows P -value = 0.003 between GFP and Mtm ds RNA-treated lysates, shows P -value = 0.63 between GFP and CG3632 ds RNA-treated lysate and shows P -value = 0.11 between GFP and CG3530 ds RNA-treated lysates.
    Figure Legend Snippet: (A) Schematic illustrating the putative enzymatic routes by which PI5P can be synthesised in Drosophila . The activities of enzymes labelled in blue are known in mammalian cells, the activity of enzymes labelled in red followed by “?” are still unknown in Drosophila , the activity of enzymes labelled in green are known in Drosophila . The activity of PI5P to PI(4,5)P 2 is boxed and is linked to cell size regulation in Drosophila . PI, phosphatidylinositol; PI3P, phosphatidylinositol 3-phosphate; PI(3,5)P 2 , phosphatidylinositol 3,5 bisphosphate; PI5P, phosphatidylinositol 5-phosphate; PI(4,5)P 2 , phosphatidylinositol 4,5 bisphosphate. Kinase and phosphatase activities are denoted by black solid arrows and black dashed arrows, respectively. (B) Schematic illustrating the LC-MS/MS-based in vitro 5-kinase activity assay using S2R+ cells overexpressing dFab1 enzyme to convert synthetic PI or PI3P to PI5P and PI(3,5)P 2 , respectively. (C) (i) Immunoprecipitated protein levels were analysed by Western blotting with an anti-mCherry antibody. Control (IgG) was prepared without anti-mCherry. Input lane shows the correct size of dFab1 protein ∼230 kD. UTC, Untransfected control. (ii) In vitro kinase assay on synthetic PI and PI3P. Graph representing the kinase activity (%) as the normalised response ratio of “PI 5-kinase activity on PI” to “PI3P 5-kinase activity on PI3P” upon enzymatic activity of immunoprecipitated mCherry::dFab1 on the respective substrates. Response ratio of PI 5-kinase activity on PI is obtained from area under the curve of 17:0 14:1 PI5P (product)/17:0 14:1 PI (substrate), Response ratio of PI3P 5-kinase activity on PI3P is obtained from area under the curve of 17:0 20:4 PI(3,5)P 2 (product)/17:0 20:4 PI3P (substrate) and is represented as mean ± S.E.M. on addition of either negative control (no beads), control (mCherry beads) or dFab1 (mCherry::dFab1 beads). Number of immunoprecipitated samples = 2. (D) Schematic illustrating the LC-MS/MS-based in vitro PI(3,5)P 2 3-phosphatase activity assay using dsRNA-treated S2R+ cells as the enzyme source to convert synthetic PI(3,5)P 2 [d5-PI(3,5)P 2 to d5- 18 O- PIP 2 ] using a two-step reaction scheme. (E) In vitro phosphatase assay on synthetic PI(3,5)P 2 . Graph representing the 3-phosphatase activity (%) as the percent formation of d5- 18 O-PIP 2 formed from starting d5-PI(3,5)P 2 as mean ± S.E.M. on addition of either control (GFP ds RNA) or Mtm , CG3632 , CG3530 ds RNA-treated S2R+ cell lysates. One-way ANOVA with a post hoc Tukey’s test shows P -value = 0.003 between GFP and Mtm ds RNA-treated lysates, shows P -value = 0.63 between GFP and CG3632 ds RNA-treated lysate and shows P -value = 0.11 between GFP and CG3530 ds RNA-treated lysates.

    Techniques Used: Activity Assay, Liquid Chromatography with Mass Spectroscopy, In Vitro, Kinase Assay, Immunoprecipitation, Western Blot, Negative Control, Phosphatase Assay

    (A) In vitro phosphatase assay on synthetic PI3P. Graph representing the response ratio of 17:0 20:4 PI (product)/17:0 20:4 PI3P (substrate) formed as mean ± S.E.M. on addition of either control ( da/+ ) or da>Mtm WT ::GFP lysates. Lysate samples = 3, where each sample was made from five third instar wandering larvae. unpaired t test with Welch correction showed P -value = 0.007. (B) Extracted ion chromatogram (XIC) of deacylated PI3P or GroPI3P (glycerophosphoinositol 3-phosphate) peak at R t = 7.37 min, separated from deacylated PI4P or GroPI4P (glycerophosphoinositol 4-phosphate) peak at R t = 9.12 min obtained from injecting WT larval lipid extract (details of sample preparation is discussed in methods). (C) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of da/+; dPIP4K 29 (green) and da> Mtm WT ::GFP; dPIP4K 29 (magenta). Biological samples = 3, where each sample was made from three third instar wandering larvae. unpaired t test with Welch correction showed P -value = 0.07. (D) Graph representing normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of da/+ (green) and da> Mtm WT ::GFP (magenta). Biological samples = 3, where each sample was made from three third instar wandering larvae. Unpaired t test with Welch correction showed P -value = 0.13.
    Figure Legend Snippet: (A) In vitro phosphatase assay on synthetic PI3P. Graph representing the response ratio of 17:0 20:4 PI (product)/17:0 20:4 PI3P (substrate) formed as mean ± S.E.M. on addition of either control ( da/+ ) or da>Mtm WT ::GFP lysates. Lysate samples = 3, where each sample was made from five third instar wandering larvae. unpaired t test with Welch correction showed P -value = 0.007. (B) Extracted ion chromatogram (XIC) of deacylated PI3P or GroPI3P (glycerophosphoinositol 3-phosphate) peak at R t = 7.37 min, separated from deacylated PI4P or GroPI4P (glycerophosphoinositol 4-phosphate) peak at R t = 9.12 min obtained from injecting WT larval lipid extract (details of sample preparation is discussed in methods). (C) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of da/+; dPIP4K 29 (green) and da> Mtm WT ::GFP; dPIP4K 29 (magenta). Biological samples = 3, where each sample was made from three third instar wandering larvae. unpaired t test with Welch correction showed P -value = 0.07. (D) Graph representing normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of da/+ (green) and da> Mtm WT ::GFP (magenta). Biological samples = 3, where each sample was made from three third instar wandering larvae. Unpaired t test with Welch correction showed P -value = 0.13.

    Techniques Used: In Vitro, Phosphatase Assay, Sample Prep

    (A) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value as mean ± S.E.M. of WT (green) and dPIP4K 29 (magenta). Biological samples = 5, where each sample was made from five third instar wandering larvae. Unpaired t test with Welch correction showed P -value = 0.008. (B) Graph representing normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C/+ ; dPIP4K 29 (green) or Act5C> dPIP4K WT ::GFP; dPIP4K 29 (magenta). Biological samples = 5, where each sample was made from three third instar wandering larvae. unpaired t test with Welch correction showed P -value = 0.008. (C) Graph representing normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C/+ ; dPIP4K 29 (green) or Act5C> dPIP4K D271A; dPIP4K 29 (magenta). Biological samples = 6, where each sample was made from three third instar wandering larvae. Unpaired t test with Welch correction showed P -value = 0.818. (D) In vitro phosphatase assay on synthetic PI3P. Graph representing the response ratio of 17:0 20:4 PI (product)/17:0 20:4 PI3P (substrate) formed as mean ± S.E.M. on addition of either WT or dPIP4K 29 lysates for either a 5-min or a 15-min reaction. Lysate samples = 3, where each sample was made from five third instar wandering larvae. Multiple unpaired t test showed P -value = 0.26 for 5 min time point and P -value = 0.052. (E) qPCR measurements for mRNA levels of PI3K59F and PI3K68D from either WT (green) or dPIP4K 29 (magenta). unpaired t test showed P -value = 0.01 for PI3K59F and P -value = 0.58 for PI3K68D . qPCR measurements for mRNA levels of Mtm , CG3632, and CG3530 from either WT (green) or dPIP4K 29 (magenta). Unpaired t test showed P -value = 0.03 for Mtm , P -value = 0.23 for CG3632, and P -value = 0.0006 for CG3530 .
    Figure Legend Snippet: (A) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value as mean ± S.E.M. of WT (green) and dPIP4K 29 (magenta). Biological samples = 5, where each sample was made from five third instar wandering larvae. Unpaired t test with Welch correction showed P -value = 0.008. (B) Graph representing normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C/+ ; dPIP4K 29 (green) or Act5C> dPIP4K WT ::GFP; dPIP4K 29 (magenta). Biological samples = 5, where each sample was made from three third instar wandering larvae. unpaired t test with Welch correction showed P -value = 0.008. (C) Graph representing normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C/+ ; dPIP4K 29 (green) or Act5C> dPIP4K D271A; dPIP4K 29 (magenta). Biological samples = 6, where each sample was made from three third instar wandering larvae. Unpaired t test with Welch correction showed P -value = 0.818. (D) In vitro phosphatase assay on synthetic PI3P. Graph representing the response ratio of 17:0 20:4 PI (product)/17:0 20:4 PI3P (substrate) formed as mean ± S.E.M. on addition of either WT or dPIP4K 29 lysates for either a 5-min or a 15-min reaction. Lysate samples = 3, where each sample was made from five third instar wandering larvae. Multiple unpaired t test showed P -value = 0.26 for 5 min time point and P -value = 0.052. (E) qPCR measurements for mRNA levels of PI3K59F and PI3K68D from either WT (green) or dPIP4K 29 (magenta). unpaired t test showed P -value = 0.01 for PI3K59F and P -value = 0.58 for PI3K68D . qPCR measurements for mRNA levels of Mtm , CG3632, and CG3530 from either WT (green) or dPIP4K 29 (magenta). Unpaired t test showed P -value = 0.03 for Mtm , P -value = 0.23 for CG3632, and P -value = 0.0006 for CG3530 .

    Techniques Used: In Vitro, Phosphatase Assay

    (A) Schematic illustrating the methodology to assay PI3P by a dFab1-mediated radioactivity-based mass assay. dFab1 is purified from S2R+ cells by immunoprecipitation and used to convert PI3P from total lipid extracts obtained from larvae in the presence of ϒ 32 P-ATP to radiolabelled PI(3,5)P 2 product which is finally analysed using TLC. A portion of the total lipid extract is used for organic phosphate assay to normalise for sample size. (B) (i) Autoradiograph of TLC ran with lipid samples from in vitro PI3P mass assay using WT and dPIP4K 29 lipid samples. The first two lanes from the left are obtained from mass assay reactions using synthetic PI3P standard without or with addition of dFab1 enzyme, respectively. The origin spot and PI(3,5)P 2 spots are marked. (B) (ii) Graph represents normalised PI3P levels. Briefly, the spot marked as PI(3,5)P 2 on TLC in (B) is obtained by converting PI3P in the samples using immunoprecipitated dFab1 in the presence of ϒ 32 P-ATP are quantified using image analysis and then normalised to organic phosphate value (indicated in blue embedded text under TLC) to obtain normalised PI3P levels. Biological samples = 3, where each sample was made from five third instar wandering larvae. Unpaired t test with Welch correction showed P -value = 0.036. (C) Illustration depicting an indirect model where the increased PI3P levels in dPIP4K 29 (denoted by a red cross on dPIP4K) can be explained by either an activation of PI 3-kinase activity (green arrow) or an inhibition of PI3P 3-phosphatase activity (red stubbed arrow).
    Figure Legend Snippet: (A) Schematic illustrating the methodology to assay PI3P by a dFab1-mediated radioactivity-based mass assay. dFab1 is purified from S2R+ cells by immunoprecipitation and used to convert PI3P from total lipid extracts obtained from larvae in the presence of ϒ 32 P-ATP to radiolabelled PI(3,5)P 2 product which is finally analysed using TLC. A portion of the total lipid extract is used for organic phosphate assay to normalise for sample size. (B) (i) Autoradiograph of TLC ran with lipid samples from in vitro PI3P mass assay using WT and dPIP4K 29 lipid samples. The first two lanes from the left are obtained from mass assay reactions using synthetic PI3P standard without or with addition of dFab1 enzyme, respectively. The origin spot and PI(3,5)P 2 spots are marked. (B) (ii) Graph represents normalised PI3P levels. Briefly, the spot marked as PI(3,5)P 2 on TLC in (B) is obtained by converting PI3P in the samples using immunoprecipitated dFab1 in the presence of ϒ 32 P-ATP are quantified using image analysis and then normalised to organic phosphate value (indicated in blue embedded text under TLC) to obtain normalised PI3P levels. Biological samples = 3, where each sample was made from five third instar wandering larvae. Unpaired t test with Welch correction showed P -value = 0.036. (C) Illustration depicting an indirect model where the increased PI3P levels in dPIP4K 29 (denoted by a red cross on dPIP4K) can be explained by either an activation of PI 3-kinase activity (green arrow) or an inhibition of PI3P 3-phosphatase activity (red stubbed arrow).

    Techniques Used: Radioactivity, Mass Assay, Purification, Immunoprecipitation, Autoradiography, In Vitro, Activation Assay, Activity Assay, Inhibition

    (A) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+; dPIP4K 29 (n = 9), AB1>PI3K59F RNAi; dPIP4K 29 (n = 9). Sample size is represented on individual bars. Unpaired t test with Welch correction showed P -value < 0.0001. (B) Graph representing normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C /+; dPIP4K 29 (green) or Act5C > PI3K59F RNAi; dPIP4K 29 (magenta). Biological samples = 5, where each sample was made from five third instar wandering larvae. Unpaired t test with Welch correction showed P -value = 0.011. (C) (i) Representative confocal z-projections depicting a subpopulation of early endosomal compartment using 2XFYVE-mCherry in the salivary glands from wandering third instar larvae of AB1> 2XFYVE::mCherry (Control) and AB1> 2XFYVE::mCherry; dPIP4K 29 . Scale bar indicated at 20 μm. (ii) Graph representing 2XFYVE punctae measurement in the salivary glands from wandering third instar larvae of AB1> 2XFYVE::mCherry (N = 8, n = 40) and AB1>2XFYVE::mCherry; dPIP4K 29 (N = 8, n = 40). Unpaired t test with Welch correction showed P -value = 0.4057. (D) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+; dPIP4K 29 (n = 8), AB1>dPIP4K 2XFYVE ; dPIP4K 29 (n = 8). Sample size is represented on individual bars. Unpaired t test with Welch correction showed P -value = 0.171. (E) qPCR measurements for mRNA levels of UVRAG from either da/+ (green) or da> UVRAG RNAi (magenta). Unpaired t test showed P -value = 0.0046. (n = 6, where each n is derived from five third instar wandering larvae). (F) Graph representing average cell size measurement as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+; dPIP4K 29 (n = 12) and AB1> UVRAG RNAi; dPIP4K 29 (n = 12). unpaired t test with Welch correction showed P -value = 0.4306. (G) (i) Representative confocal z-projections depicting 2XFYVE punctae using 2XFYVE::mCherry in the salivary glands from the genotypes a. AB1> 2XFYVE :: mCherry (Control) b. AB1> 2XFYVE::mCherry; UVRAG RNAi. (ii) Graph representing measurement of 2XFYVE punctae numbers per unit cell area of the salivary glands from wandering third instar larvae of AB1> 2XFYVE::mCherry (N = 3, n = 11), AB1> 2XFYVE::mCherry; UVRAG RNAi (N = 3,n = 11).
    Figure Legend Snippet: (A) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+; dPIP4K 29 (n = 9), AB1>PI3K59F RNAi; dPIP4K 29 (n = 9). Sample size is represented on individual bars. Unpaired t test with Welch correction showed P -value < 0.0001. (B) Graph representing normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C /+; dPIP4K 29 (green) or Act5C > PI3K59F RNAi; dPIP4K 29 (magenta). Biological samples = 5, where each sample was made from five third instar wandering larvae. Unpaired t test with Welch correction showed P -value = 0.011. (C) (i) Representative confocal z-projections depicting a subpopulation of early endosomal compartment using 2XFYVE-mCherry in the salivary glands from wandering third instar larvae of AB1> 2XFYVE::mCherry (Control) and AB1> 2XFYVE::mCherry; dPIP4K 29 . Scale bar indicated at 20 μm. (ii) Graph representing 2XFYVE punctae measurement in the salivary glands from wandering third instar larvae of AB1> 2XFYVE::mCherry (N = 8, n = 40) and AB1>2XFYVE::mCherry; dPIP4K 29 (N = 8, n = 40). Unpaired t test with Welch correction showed P -value = 0.4057. (D) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+; dPIP4K 29 (n = 8), AB1>dPIP4K 2XFYVE ; dPIP4K 29 (n = 8). Sample size is represented on individual bars. Unpaired t test with Welch correction showed P -value = 0.171. (E) qPCR measurements for mRNA levels of UVRAG from either da/+ (green) or da> UVRAG RNAi (magenta). Unpaired t test showed P -value = 0.0046. (n = 6, where each n is derived from five third instar wandering larvae). (F) Graph representing average cell size measurement as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+; dPIP4K 29 (n = 12) and AB1> UVRAG RNAi; dPIP4K 29 (n = 12). unpaired t test with Welch correction showed P -value = 0.4306. (G) (i) Representative confocal z-projections depicting 2XFYVE punctae using 2XFYVE::mCherry in the salivary glands from the genotypes a. AB1> 2XFYVE :: mCherry (Control) b. AB1> 2XFYVE::mCherry; UVRAG RNAi. (ii) Graph representing measurement of 2XFYVE punctae numbers per unit cell area of the salivary glands from wandering third instar larvae of AB1> 2XFYVE::mCherry (N = 3, n = 11), AB1> 2XFYVE::mCherry; UVRAG RNAi (N = 3,n = 11).

    Techniques Used: Derivative Assay

    (A) qPCR measurements for mRNA levels of Mtm , CG3632, and CG3530 from either control ( da /+, in green) or da > Mtm RNAi, in magenta. Multiple t test with post hoc Holm-Sidak’s test showed P -value < 0.0001 between da/+ and da> Mtm RNAi for Mtm and P -value = 0.35 between da/+ and da> Mtm RNAi for CG3632 , and P -value = 0.04 between da/+ and da> Mtm RNAi for CG3530 . (B) (i) Representative confocal images of salivary glands from the genotypes a. AB1/+ , b. AB1>Mtm RNAi. Cell body is marked magenta by BODIPY-conjugated lipid dye, and the nucleus is marked by DAPI shown in green. Scale bar indicated at 50 μm. (ii) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+ (n = 7), AB1> Mtm RNAi (n = 7). Sample size is represented on individual bars. Unpaired t test with Welch correction showed P -value = 0.0005. (C) Graph representing normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value as mean ± S.E.M. of da/+ (green) and da> Mtm RNAi (magenta). Biological samples = 4, where each sample was made from five third instar wandering larvae. Unpaired t test with Welch correction showed P -value = 0.07. (D) (i) Representative confocal z-projections depicting autophagosomal levels using Atg8a-mCherry in the salivary glands from the genotypes a. AB1>Atg8a::mCherry (control), b. AB1>Atg8a::mCherry; Mtm RNAi. Scale bar indicated at 20 μm. (ii) Graph representing Atg8a punctae measurement in the salivary glands from wandering third instar larvae of AB1>Atg8a::mCherry (N = 7, n = 40), AB1>Atg8a::mCherry; Mtm RNAi (N = 7, n = 40). Unpaired t test with Welch correction showed P -value < 0.0001. (E) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+ (n = 11), AB1>Mtm RNAi (n = 8), AB1>Mtm RNAi, Atg8a RNAi (n = 12). Sample size is represented on individual bars. One-way ANOVA with post hoc Tukey’s test showed P -value < 0.0001 between AB1/+ and AB1>Mtm RNAi and P -value = 0.0002 between AB1/+; dPIP4K 29 and AB1>Mtm RNAi, Atg8a RNAi.
    Figure Legend Snippet: (A) qPCR measurements for mRNA levels of Mtm , CG3632, and CG3530 from either control ( da /+, in green) or da > Mtm RNAi, in magenta. Multiple t test with post hoc Holm-Sidak’s test showed P -value < 0.0001 between da/+ and da> Mtm RNAi for Mtm and P -value = 0.35 between da/+ and da> Mtm RNAi for CG3632 , and P -value = 0.04 between da/+ and da> Mtm RNAi for CG3530 . (B) (i) Representative confocal images of salivary glands from the genotypes a. AB1/+ , b. AB1>Mtm RNAi. Cell body is marked magenta by BODIPY-conjugated lipid dye, and the nucleus is marked by DAPI shown in green. Scale bar indicated at 50 μm. (ii) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+ (n = 7), AB1> Mtm RNAi (n = 7). Sample size is represented on individual bars. Unpaired t test with Welch correction showed P -value = 0.0005. (C) Graph representing normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value as mean ± S.E.M. of da/+ (green) and da> Mtm RNAi (magenta). Biological samples = 4, where each sample was made from five third instar wandering larvae. Unpaired t test with Welch correction showed P -value = 0.07. (D) (i) Representative confocal z-projections depicting autophagosomal levels using Atg8a-mCherry in the salivary glands from the genotypes a. AB1>Atg8a::mCherry (control), b. AB1>Atg8a::mCherry; Mtm RNAi. Scale bar indicated at 20 μm. (ii) Graph representing Atg8a punctae measurement in the salivary glands from wandering third instar larvae of AB1>Atg8a::mCherry (N = 7, n = 40), AB1>Atg8a::mCherry; Mtm RNAi (N = 7, n = 40). Unpaired t test with Welch correction showed P -value < 0.0001. (E) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+ (n = 11), AB1>Mtm RNAi (n = 8), AB1>Mtm RNAi, Atg8a RNAi (n = 12). Sample size is represented on individual bars. One-way ANOVA with post hoc Tukey’s test showed P -value < 0.0001 between AB1/+ and AB1>Mtm RNAi and P -value = 0.0002 between AB1/+; dPIP4K 29 and AB1>Mtm RNAi, Atg8a RNAi.

    Techniques Used:

    Schematic showing the regulation of PI3P at an autophagic membrane downstream of Atg1. In this study, dPIP4K which metabolizes PI3P has been shown to genetically interact with Vps34 and Mtm, two established PI3P regulators. Atg8a-marked autophagosomes form, mature to autolysosomes, and function in maintaining normal cell size in WT cells. In dPIP4K 29 cells, PI3P levels increase, leading to an increase in Atg8a punctae by increased autophagy initiation, which in turn leads to decreased cell size phenotype.
    Figure Legend Snippet: Schematic showing the regulation of PI3P at an autophagic membrane downstream of Atg1. In this study, dPIP4K which metabolizes PI3P has been shown to genetically interact with Vps34 and Mtm, two established PI3P regulators. Atg8a-marked autophagosomes form, mature to autolysosomes, and function in maintaining normal cell size in WT cells. In dPIP4K 29 cells, PI3P levels increase, leading to an increase in Atg8a punctae by increased autophagy initiation, which in turn leads to decreased cell size phenotype.

    Techniques Used:

    Multiple reaction monitoring (MRM) values for commercial lipids used.
    Figure Legend Snippet: Multiple reaction monitoring (MRM) values for commercial lipids used.

    Techniques Used:

    p-3016  (Echelon Biosciences)


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    dic16 pi3p  (Echelon Biosciences)


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    Echelon Biosciences dic16 pi3p
    (A) Schematic illustrating the putative enzymatic routes by which PI5Pcan be synthesised in Drosophila . The activities of enzymes labelled in blue are known in mammalian cells, the activity of enzymes labelled in red followed by “?” are still unknown in Drosophila , the activity of enzymes labelled in green are known in Drosophila . The activity of PI5P to PI(4,5)P 2 is boxed and is linked to cell size regulation in Drosophila . PI: Phosphatidylinositol, <t>PI3P:</t> <t>Phosphatidylinositol</t> <t>3-phosphate,</t> <t>PI(3,5)P</t> 2 : Phosphatidylinositol 3,5 bisphosphate, PI5P: Phosphatidylinositol 5-phosphate, PI(4,5)P 2 : Phosphatidylinositol 4,5 bisphosphate. (B) Schematic illustrating the LC-MS/MS based in vitro 5-kinaseactivity assay using S2R+ cells over-expressing dFab1 enzyme to convert synthetic PI or PI3P to PI5P and PI(3,5)P 2 , respectively. (C) (i) Immunoprecipitated protein levels were analysed by Western blotting with an anti-mCherry antibody. Control (IgG) was prepared without anti-mCherry. Input lane shows the correct size of dFab1 protein ∼230 kDa. UTC: Untransfected control. (ii) In vitro kinase assay on synthetic PI and PI3P. Graph representing the Kinase activity (%) as the normalised response ratio of PI 5-kinase activity on PI” to PI3P 5-kinase activity on PI3P” upon enzymatic activity of immunoprecipitated mCherry::dFab1 on the respective substrates. Response ratio of PI 5-kinase activity on PI is obtained from area under the curve (AUC) of 17:0 14:1 PI5P (Product)/17:0 14:1 PI (Substrate), Response ratio of PI3P 5-kinaseactivity on PI3P isobtained from area under the curve (AUC) of 17:0 20:4 PI(3,5)P 2 (Product)/17:0 20:4 PI3P (Substrate) and is represented as mean ± S.E.M. on addition of either negative control (no beads), Control (mCherry beads) or dFab1 (mCherry::dFab1 beads). Number of immunoprecipitated samples= 2. (D) Schematic illustrating the LC-MS/MS based in vitro PI(3,5)P 2 3-phosphataseactivity assay using dsRNA treated S2R+ cells as enzyme source to convert synthetic PI(3,5)P 2 [d5-PI(3,5)P 2 to d5- 18 O-PIP 2 ] using a two-step reaction scheme. (E) In vitro phosphatase assay on synthetic PI(3,5)P 2 . Graph representing the 3-Phosphatase activity (%) as the percent formation of d5- 18 O-PIP 2 formed from starting d5-PI(3,5)P 2 as mean ± S.E.M. on addition of either control (GFPdsRNA) or Mtm, CG3632, CG3530 dsRNA treated S2R+ cell lysates. One way ANOVA with a post hoc Tukey’s test shows p value = 0.003 between GFP and Mtm ds RNA treated lysates, shows p value = 0.63 between GFP and CG3632 ds RNA treated lysate and shows p value= 0.11 between GFP and CG3530 dsRNA treated lysates.
    Dic16 Pi3p, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "PI3P dependent regulation of cell size by phosphatidylinositol 5-phosphate 4-kinase"

    Article Title: PI3P dependent regulation of cell size by phosphatidylinositol 5-phosphate 4-kinase

    Journal: bioRxiv

    doi: 10.1101/2022.06.18.496676

    (A) Schematic illustrating the putative enzymatic routes by which PI5Pcan be synthesised in Drosophila . The activities of enzymes labelled in blue are known in mammalian cells, the activity of enzymes labelled in red followed by “?” are still unknown in Drosophila , the activity of enzymes labelled in green are known in Drosophila . The activity of PI5P to PI(4,5)P 2 is boxed and is linked to cell size regulation in Drosophila . PI: Phosphatidylinositol, PI3P: Phosphatidylinositol 3-phosphate, PI(3,5)P 2 : Phosphatidylinositol 3,5 bisphosphate, PI5P: Phosphatidylinositol 5-phosphate, PI(4,5)P 2 : Phosphatidylinositol 4,5 bisphosphate. (B) Schematic illustrating the LC-MS/MS based in vitro 5-kinaseactivity assay using S2R+ cells over-expressing dFab1 enzyme to convert synthetic PI or PI3P to PI5P and PI(3,5)P 2 , respectively. (C) (i) Immunoprecipitated protein levels were analysed by Western blotting with an anti-mCherry antibody. Control (IgG) was prepared without anti-mCherry. Input lane shows the correct size of dFab1 protein ∼230 kDa. UTC: Untransfected control. (ii) In vitro kinase assay on synthetic PI and PI3P. Graph representing the Kinase activity (%) as the normalised response ratio of PI 5-kinase activity on PI” to PI3P 5-kinase activity on PI3P” upon enzymatic activity of immunoprecipitated mCherry::dFab1 on the respective substrates. Response ratio of PI 5-kinase activity on PI is obtained from area under the curve (AUC) of 17:0 14:1 PI5P (Product)/17:0 14:1 PI (Substrate), Response ratio of PI3P 5-kinaseactivity on PI3P isobtained from area under the curve (AUC) of 17:0 20:4 PI(3,5)P 2 (Product)/17:0 20:4 PI3P (Substrate) and is represented as mean ± S.E.M. on addition of either negative control (no beads), Control (mCherry beads) or dFab1 (mCherry::dFab1 beads). Number of immunoprecipitated samples= 2. (D) Schematic illustrating the LC-MS/MS based in vitro PI(3,5)P 2 3-phosphataseactivity assay using dsRNA treated S2R+ cells as enzyme source to convert synthetic PI(3,5)P 2 [d5-PI(3,5)P 2 to d5- 18 O-PIP 2 ] using a two-step reaction scheme. (E) In vitro phosphatase assay on synthetic PI(3,5)P 2 . Graph representing the 3-Phosphatase activity (%) as the percent formation of d5- 18 O-PIP 2 formed from starting d5-PI(3,5)P 2 as mean ± S.E.M. on addition of either control (GFPdsRNA) or Mtm, CG3632, CG3530 dsRNA treated S2R+ cell lysates. One way ANOVA with a post hoc Tukey’s test shows p value = 0.003 between GFP and Mtm ds RNA treated lysates, shows p value = 0.63 between GFP and CG3632 ds RNA treated lysate and shows p value= 0.11 between GFP and CG3530 dsRNA treated lysates.
    Figure Legend Snippet: (A) Schematic illustrating the putative enzymatic routes by which PI5Pcan be synthesised in Drosophila . The activities of enzymes labelled in blue are known in mammalian cells, the activity of enzymes labelled in red followed by “?” are still unknown in Drosophila , the activity of enzymes labelled in green are known in Drosophila . The activity of PI5P to PI(4,5)P 2 is boxed and is linked to cell size regulation in Drosophila . PI: Phosphatidylinositol, PI3P: Phosphatidylinositol 3-phosphate, PI(3,5)P 2 : Phosphatidylinositol 3,5 bisphosphate, PI5P: Phosphatidylinositol 5-phosphate, PI(4,5)P 2 : Phosphatidylinositol 4,5 bisphosphate. (B) Schematic illustrating the LC-MS/MS based in vitro 5-kinaseactivity assay using S2R+ cells over-expressing dFab1 enzyme to convert synthetic PI or PI3P to PI5P and PI(3,5)P 2 , respectively. (C) (i) Immunoprecipitated protein levels were analysed by Western blotting with an anti-mCherry antibody. Control (IgG) was prepared without anti-mCherry. Input lane shows the correct size of dFab1 protein ∼230 kDa. UTC: Untransfected control. (ii) In vitro kinase assay on synthetic PI and PI3P. Graph representing the Kinase activity (%) as the normalised response ratio of PI 5-kinase activity on PI” to PI3P 5-kinase activity on PI3P” upon enzymatic activity of immunoprecipitated mCherry::dFab1 on the respective substrates. Response ratio of PI 5-kinase activity on PI is obtained from area under the curve (AUC) of 17:0 14:1 PI5P (Product)/17:0 14:1 PI (Substrate), Response ratio of PI3P 5-kinaseactivity on PI3P isobtained from area under the curve (AUC) of 17:0 20:4 PI(3,5)P 2 (Product)/17:0 20:4 PI3P (Substrate) and is represented as mean ± S.E.M. on addition of either negative control (no beads), Control (mCherry beads) or dFab1 (mCherry::dFab1 beads). Number of immunoprecipitated samples= 2. (D) Schematic illustrating the LC-MS/MS based in vitro PI(3,5)P 2 3-phosphataseactivity assay using dsRNA treated S2R+ cells as enzyme source to convert synthetic PI(3,5)P 2 [d5-PI(3,5)P 2 to d5- 18 O-PIP 2 ] using a two-step reaction scheme. (E) In vitro phosphatase assay on synthetic PI(3,5)P 2 . Graph representing the 3-Phosphatase activity (%) as the percent formation of d5- 18 O-PIP 2 formed from starting d5-PI(3,5)P 2 as mean ± S.E.M. on addition of either control (GFPdsRNA) or Mtm, CG3632, CG3530 dsRNA treated S2R+ cell lysates. One way ANOVA with a post hoc Tukey’s test shows p value = 0.003 between GFP and Mtm ds RNA treated lysates, shows p value = 0.63 between GFP and CG3632 ds RNA treated lysate and shows p value= 0.11 between GFP and CG3530 dsRNA treated lysates.

    Techniques Used: Activity Assay, Liquid Chromatography with Mass Spectroscopy, In Vitro, Expressing, Immunoprecipitation, Western Blot, Kinase Assay, Negative Control, Phosphatase Assay

    (A) In vitro phosphatase assay on synthetic PI3P. Graph representing the response ratio of 17:0 20:4 PI (Product)/17:0 20:4 PI3P (Substrate) formed as mean ± S.E.M. on addition of either control (da/+) or da>Mtm WT GFP lysates. Lysate samples= 3, where each sample was made from fivethird instar wandering larvae. Strudent’s Unpaired t-test with Welch correction showed p value= 0.007. (B) Extracted ion chromatogram (XIC) of deacylated PI3P or GroPI3P (Glycerophosphoinositol 3-phosphate) peak at Rt = 7.37 min, separated from deacylated PI4P or GroPI4P (Glycerophosphoinositol 4-phosphate) peak at Rt = 9.12 min obtained from injecting wild type larval lipid extract (details of sample preparation is discussed in methods). (C) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of da/+; dPIP4K 29 (green) and da> Mtm WT GFP, dPIP4K 29 (majenta). Biological samples = 3, where each sample was made from three third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.07. (D) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of da/+ (green) andda> Mtm WT GFP, (majenta). Biological samples= 3, where each sample was made from three third instar wandering larvae.. Student’s unpaired t-test with Welch correction showed p value = 0.13.
    Figure Legend Snippet: (A) In vitro phosphatase assay on synthetic PI3P. Graph representing the response ratio of 17:0 20:4 PI (Product)/17:0 20:4 PI3P (Substrate) formed as mean ± S.E.M. on addition of either control (da/+) or da>Mtm WT GFP lysates. Lysate samples= 3, where each sample was made from fivethird instar wandering larvae. Strudent’s Unpaired t-test with Welch correction showed p value= 0.007. (B) Extracted ion chromatogram (XIC) of deacylated PI3P or GroPI3P (Glycerophosphoinositol 3-phosphate) peak at Rt = 7.37 min, separated from deacylated PI4P or GroPI4P (Glycerophosphoinositol 4-phosphate) peak at Rt = 9.12 min obtained from injecting wild type larval lipid extract (details of sample preparation is discussed in methods). (C) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of da/+; dPIP4K 29 (green) and da> Mtm WT GFP, dPIP4K 29 (majenta). Biological samples = 3, where each sample was made from three third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.07. (D) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of da/+ (green) andda> Mtm WT GFP, (majenta). Biological samples= 3, where each sample was made from three third instar wandering larvae.. Student’s unpaired t-test with Welch correction showed p value = 0.13.

    Techniques Used: In Vitro, Phosphatase Assay, Sample Prep

    (A) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value as mean ± S.E.M. of wild type (green) and dPIP4K 29 (majenta). Biological samples = 5, where each sample was made from five third instar wandering larvae. Unpaired t-test with Welch correction showed p value= 0.008. (B) Autoradiograph of TLC ran with lipid samples from in vitro PI3Pmassassay using wild type(WT) and dPIP4K 29 lipid samples. The first two lanes from the left are obtained from mass assay reactions using synthetic PI3P standard without or with addition of dFab1 enzyme, respectively. The origin spot and PI(3,5)P 2 spots are marked. (C) The graph represents normalised PI3P levels. Briefly, the spot marked asPI(3,5)P 2 on TLC in (B) is obtained by converting PI3P in the samples using immunoprecipitated dFab1 in presence of ϒ 32 P-ATP are quantified using image analysis and then normalised to organic phosphate value (indicated in blue embedded text under TLC) to obtain normalised PI3P levels. Biological samples = 3, where each sample was made from five third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.036. (D) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C/+; dPIP4K 29 (green), or Act5C> dPIP4K WT GFP, dPIP4K 29 (majenta). Biological samples= 5, where each sample was made from three third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.008. (E) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4Pnormalised to organicphosphatevalueof total lipid extractsasmean ± S.E.M. of Act5C/+; dPIP4K 29 (green) or Act5C> dPIP4K D271A , dPIP4K 29 (majenta). Biological samples = 6, where each sample was made from three third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.818. (F) In vitro phosphataseassay on synthetic PI3P. Graph representing the response ratio of 17:0 20:4 PI (Product)/17:0 20:4 PI3P (Substrate) formed asmean ± S.E.M. on addition of either wildtype (WT) or dPIP4K 29 lysatesfor either a 5 min or a 15 min reaction. Lysate samples= 3, where each sample was made from five third instar wandering larvae. Multiple unpaired t-test showed p value = 0.26 for 5 min time point and p value= 0.052. (G) qPCR measurements for mRNA levels of PI3K59F and PI3K68D from either Wild type (green) or dPIP4K 29 (majenta). Student’s unpaired t-test showed p value= 0.01 for PI3K59F and p value = 0.58 for PI3K68D . qPCR measurements for mRNA levels of Mtm, CG3632 and CG3530 from either Wild type (green) or dPIP4K 29 (majenta). Student’s unparied t-test showed p value = 0.03 for Mtm , p value= 0.23 for CG3632 and p value= 0.0006 for CG3530 .
    Figure Legend Snippet: (A) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value as mean ± S.E.M. of wild type (green) and dPIP4K 29 (majenta). Biological samples = 5, where each sample was made from five third instar wandering larvae. Unpaired t-test with Welch correction showed p value= 0.008. (B) Autoradiograph of TLC ran with lipid samples from in vitro PI3Pmassassay using wild type(WT) and dPIP4K 29 lipid samples. The first two lanes from the left are obtained from mass assay reactions using synthetic PI3P standard without or with addition of dFab1 enzyme, respectively. The origin spot and PI(3,5)P 2 spots are marked. (C) The graph represents normalised PI3P levels. Briefly, the spot marked asPI(3,5)P 2 on TLC in (B) is obtained by converting PI3P in the samples using immunoprecipitated dFab1 in presence of ϒ 32 P-ATP are quantified using image analysis and then normalised to organic phosphate value (indicated in blue embedded text under TLC) to obtain normalised PI3P levels. Biological samples = 3, where each sample was made from five third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.036. (D) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C/+; dPIP4K 29 (green), or Act5C> dPIP4K WT GFP, dPIP4K 29 (majenta). Biological samples= 5, where each sample was made from three third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.008. (E) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4Pnormalised to organicphosphatevalueof total lipid extractsasmean ± S.E.M. of Act5C/+; dPIP4K 29 (green) or Act5C> dPIP4K D271A , dPIP4K 29 (majenta). Biological samples = 6, where each sample was made from three third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.818. (F) In vitro phosphataseassay on synthetic PI3P. Graph representing the response ratio of 17:0 20:4 PI (Product)/17:0 20:4 PI3P (Substrate) formed asmean ± S.E.M. on addition of either wildtype (WT) or dPIP4K 29 lysatesfor either a 5 min or a 15 min reaction. Lysate samples= 3, where each sample was made from five third instar wandering larvae. Multiple unpaired t-test showed p value = 0.26 for 5 min time point and p value= 0.052. (G) qPCR measurements for mRNA levels of PI3K59F and PI3K68D from either Wild type (green) or dPIP4K 29 (majenta). Student’s unpaired t-test showed p value= 0.01 for PI3K59F and p value = 0.58 for PI3K68D . qPCR measurements for mRNA levels of Mtm, CG3632 and CG3530 from either Wild type (green) or dPIP4K 29 (majenta). Student’s unparied t-test showed p value = 0.03 for Mtm , p value= 0.23 for CG3632 and p value= 0.0006 for CG3530 .

    Techniques Used: Autoradiography, In Vitro, Mass Assay, Immunoprecipitation

    (A) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvaeof AB1/+; dPIP4K 29 (n = 9), AB1>PI3K59F RNAi ; dPIP4K 29 (n = 9). Sample size is represented on individual bars. Student’s unpaired t-test with Welch correction showed p value<0.0001. (B) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4Pnormalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C/+; dPIP4K 29 (green), or Act5C> dPIP4K WT GFP, dPIP4K 29 (majenta). Biological samples= 5, where each sample was made from five third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.011. (C) (i) Representative confocal z-projections depicting a sub population of early endosomal compartment using 2xFYVE-mCherry in the salivary glands from wandering third instar larvaeof AB1> 2xFYVE-mCherry and AB1> 2xFYVE-mCherry ; dPIP4K 29 . Scalebar indicated at 20 μm. (ii) Graph representing 2xFYVE punctaemeasurement in the s alivary glands from wandering third instar larvae of AB1> 2xFYVEmCherry (N = 8, n=40) and AB1> 2xFYVEmCherry ; dPIP4K 29 (N =8, n = 40). Student’s unpaired t-test with Welch correction showed p value= 0.4057 (D) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvaeof AB1/+; dPIP4K 29 (n = 8), AB1>dPIP4K 2XFYVE ; dPIP4K 29 (n = 8). Sample size is represented on individual bars. Student’s unpaired t-test with Welch correction showed p value= 0.171. (E) (i) Representative confocal z-projections depicting autophagosomal levels using Atg8a-mCherry in the salivary glands from the wandering third instar larvae of AB1>ATG8a-mCherry and AB1>ATG8a-mCherry; dPIP4K 29 . Scale bar indicated at 20 μm. (ii) Graph representing Atg8a punctae measurement in the salivary glands from wandering third instar larvaeof AB1>ATG8a-mCherry (N = 10, n = 60) and AB1>ATG8a-mCherry; dPIP4K 29 (N = 10, n = 62). Student’s unpaired t-test with Welch correction showed p value<0.0001. (F) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvaeof AB1/+; dPIP4K 29 (n = 9), AB1>PI3K59F RNAi ; dPIP4K 29 (n = 9). Sample size is represented on individual bars. ired t-test with Welch correction showed p value<0.0001.
    Figure Legend Snippet: (A) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvaeof AB1/+; dPIP4K 29 (n = 9), AB1>PI3K59F RNAi ; dPIP4K 29 (n = 9). Sample size is represented on individual bars. Student’s unpaired t-test with Welch correction showed p value<0.0001. (B) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4Pnormalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C/+; dPIP4K 29 (green), or Act5C> dPIP4K WT GFP, dPIP4K 29 (majenta). Biological samples= 5, where each sample was made from five third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.011. (C) (i) Representative confocal z-projections depicting a sub population of early endosomal compartment using 2xFYVE-mCherry in the salivary glands from wandering third instar larvaeof AB1> 2xFYVE-mCherry and AB1> 2xFYVE-mCherry ; dPIP4K 29 . Scalebar indicated at 20 μm. (ii) Graph representing 2xFYVE punctaemeasurement in the s alivary glands from wandering third instar larvae of AB1> 2xFYVEmCherry (N = 8, n=40) and AB1> 2xFYVEmCherry ; dPIP4K 29 (N =8, n = 40). Student’s unpaired t-test with Welch correction showed p value= 0.4057 (D) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvaeof AB1/+; dPIP4K 29 (n = 8), AB1>dPIP4K 2XFYVE ; dPIP4K 29 (n = 8). Sample size is represented on individual bars. Student’s unpaired t-test with Welch correction showed p value= 0.171. (E) (i) Representative confocal z-projections depicting autophagosomal levels using Atg8a-mCherry in the salivary glands from the wandering third instar larvae of AB1>ATG8a-mCherry and AB1>ATG8a-mCherry; dPIP4K 29 . Scale bar indicated at 20 μm. (ii) Graph representing Atg8a punctae measurement in the salivary glands from wandering third instar larvaeof AB1>ATG8a-mCherry (N = 10, n = 60) and AB1>ATG8a-mCherry; dPIP4K 29 (N = 10, n = 62). Student’s unpaired t-test with Welch correction showed p value<0.0001. (F) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvaeof AB1/+; dPIP4K 29 (n = 9), AB1>PI3K59F RNAi ; dPIP4K 29 (n = 9). Sample size is represented on individual bars. ired t-test with Welch correction showed p value<0.0001.

    Techniques Used:

    (A) qPCR measurements for mRNA levels of mtm, CG3632 and CG3530 from either Control ( daGal4 /+, in green) or da > Mtm RNAi, in majenta. Multiple t-test with post hoc Holm-Sidak’s test showed p value< 0.0001 between daGal4 /+ and da > Mtm RNAi for Mtm and p value= 0.35 between daGal4 /+ and da > Mtm RNAi for CG3632 , and p value = 0.04 between daGal4 /+ and da > Mtm RNAi for CG3530 . (B) (i) Representative confocal images of salivary glands from the genotypes a. AB1Gal4/+, b. AB1>Mtm RNAi. Cell body is marked majenta by BODIPY conjugated lipid dye, nucleus is marked by DAPI shown in green. Scale bar indicated at 50 μm. (ii) Graph representing average cell size measurement (in μm 3 ) asmean ± S.E.M. of salivary glands from wandering third instar larvae of AB1Gal4/+ (n = 7), AB1> Mtm RNAi (n = 7). Sample size is represented on individual bars. Student’s unpaired t-test with Welch correction showed p value = 0.0005. (C) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value as mean ± S.E.M. of da /+ (green) and da> Mtm RNAi (majenta). Biological samples = 4, where each sample was made from five third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.07. (D) (i) Graph representing Atg8a punctae measurement in the salivary glands from wandering third instar larvaeof AB1>ATG8a-mCherry (N =7, n =40), AB1>ATG8a-mCherry; Mtm RNAi (N =7, n =40). Student’s unpaired t-test with Welch correction showed p value<0.0001. (ii) Representative confocal z-projections depicting autophagosomal levels using Atg8a-mCherry in the salivary glands from the genotypesa. AB1>ATG8a-mCherry, b. AB1>ATG8a-mCherry; Mtm RNAi. Scale bar indicated at 20 μm. (E) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+ (n = 11), AB1>Mtm RNAi (n = 8), AB1>Mtm RNAi, Atg8a RNAi (n = 12). Sample size is represented on individual bars. One way ANOVA with post hoc Tukey’s test showed p value < 0.0001 between AB1/+ and AB1>Mtm RNAi and p value = 0.0002 between AB1/+; dPIP4K 29 and AB1>Mtm RNAi, Atg8a RNAi.
    Figure Legend Snippet: (A) qPCR measurements for mRNA levels of mtm, CG3632 and CG3530 from either Control ( daGal4 /+, in green) or da > Mtm RNAi, in majenta. Multiple t-test with post hoc Holm-Sidak’s test showed p value< 0.0001 between daGal4 /+ and da > Mtm RNAi for Mtm and p value= 0.35 between daGal4 /+ and da > Mtm RNAi for CG3632 , and p value = 0.04 between daGal4 /+ and da > Mtm RNAi for CG3530 . (B) (i) Representative confocal images of salivary glands from the genotypes a. AB1Gal4/+, b. AB1>Mtm RNAi. Cell body is marked majenta by BODIPY conjugated lipid dye, nucleus is marked by DAPI shown in green. Scale bar indicated at 50 μm. (ii) Graph representing average cell size measurement (in μm 3 ) asmean ± S.E.M. of salivary glands from wandering third instar larvae of AB1Gal4/+ (n = 7), AB1> Mtm RNAi (n = 7). Sample size is represented on individual bars. Student’s unpaired t-test with Welch correction showed p value = 0.0005. (C) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value as mean ± S.E.M. of da /+ (green) and da> Mtm RNAi (majenta). Biological samples = 4, where each sample was made from five third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.07. (D) (i) Graph representing Atg8a punctae measurement in the salivary glands from wandering third instar larvaeof AB1>ATG8a-mCherry (N =7, n =40), AB1>ATG8a-mCherry; Mtm RNAi (N =7, n =40). Student’s unpaired t-test with Welch correction showed p value<0.0001. (ii) Representative confocal z-projections depicting autophagosomal levels using Atg8a-mCherry in the salivary glands from the genotypesa. AB1>ATG8a-mCherry, b. AB1>ATG8a-mCherry; Mtm RNAi. Scale bar indicated at 20 μm. (E) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+ (n = 11), AB1>Mtm RNAi (n = 8), AB1>Mtm RNAi, Atg8a RNAi (n = 12). Sample size is represented on individual bars. One way ANOVA with post hoc Tukey’s test showed p value < 0.0001 between AB1/+ and AB1>Mtm RNAi and p value = 0.0002 between AB1/+; dPIP4K 29 and AB1>Mtm RNAi, Atg8a RNAi.

    Techniques Used:

    dic16 pi3p echelon p  (Echelon Biosciences)


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    Echelon Biosciences dic16 pi3p echelon p
    (A) Schematic illustrating the putative enzymatic routes by which PI5Pcan be synthesised in Drosophila . The activities of enzymes labelled in blue are known in mammalian cells, the activity of enzymes labelled in red followed by “?” are still unknown in Drosophila , the activity of enzymes labelled in green are known in Drosophila . The activity of PI5P to PI(4,5)P 2 is boxed and is linked to cell size regulation in Drosophila . PI: Phosphatidylinositol, <t>PI3P:</t> <t>Phosphatidylinositol</t> <t>3-phosphate,</t> <t>PI(3,5)P</t> 2 : Phosphatidylinositol 3,5 bisphosphate, PI5P: Phosphatidylinositol 5-phosphate, PI(4,5)P 2 : Phosphatidylinositol 4,5 bisphosphate. (B) Schematic illustrating the LC-MS/MS based in vitro 5-kinaseactivity assay using S2R+ cells over-expressing dFab1 enzyme to convert synthetic PI or PI3P to PI5P and PI(3,5)P 2 , respectively. (C) (i) Immunoprecipitated protein levels were analysed by Western blotting with an anti-mCherry antibody. Control (IgG) was prepared without anti-mCherry. Input lane shows the correct size of dFab1 protein ∼230 kDa. UTC: Untransfected control. (ii) In vitro kinase assay on synthetic PI and PI3P. Graph representing the Kinase activity (%) as the normalised response ratio of PI 5-kinase activity on PI” to PI3P 5-kinase activity on PI3P” upon enzymatic activity of immunoprecipitated mCherry::dFab1 on the respective substrates. Response ratio of PI 5-kinase activity on PI is obtained from area under the curve (AUC) of 17:0 14:1 PI5P (Product)/17:0 14:1 PI (Substrate), Response ratio of PI3P 5-kinaseactivity on PI3P isobtained from area under the curve (AUC) of 17:0 20:4 PI(3,5)P 2 (Product)/17:0 20:4 PI3P (Substrate) and is represented as mean ± S.E.M. on addition of either negative control (no beads), Control (mCherry beads) or dFab1 (mCherry::dFab1 beads). Number of immunoprecipitated samples= 2. (D) Schematic illustrating the LC-MS/MS based in vitro PI(3,5)P 2 3-phosphataseactivity assay using dsRNA treated S2R+ cells as enzyme source to convert synthetic PI(3,5)P 2 [d5-PI(3,5)P 2 to d5- 18 O-PIP 2 ] using a two-step reaction scheme. (E) In vitro phosphatase assay on synthetic PI(3,5)P 2 . Graph representing the 3-Phosphatase activity (%) as the percent formation of d5- 18 O-PIP 2 formed from starting d5-PI(3,5)P 2 as mean ± S.E.M. on addition of either control (GFPdsRNA) or Mtm, CG3632, CG3530 dsRNA treated S2R+ cell lysates. One way ANOVA with a post hoc Tukey’s test shows p value = 0.003 between GFP and Mtm ds RNA treated lysates, shows p value = 0.63 between GFP and CG3632 ds RNA treated lysate and shows p value= 0.11 between GFP and CG3530 dsRNA treated lysates.
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    1) Product Images from "PI3P dependent regulation of cell size by phosphatidylinositol 5-phosphate 4-kinase"

    Article Title: PI3P dependent regulation of cell size by phosphatidylinositol 5-phosphate 4-kinase

    Journal: bioRxiv

    doi: 10.1101/2022.06.18.496676

    (A) Schematic illustrating the putative enzymatic routes by which PI5Pcan be synthesised in Drosophila . The activities of enzymes labelled in blue are known in mammalian cells, the activity of enzymes labelled in red followed by “?” are still unknown in Drosophila , the activity of enzymes labelled in green are known in Drosophila . The activity of PI5P to PI(4,5)P 2 is boxed and is linked to cell size regulation in Drosophila . PI: Phosphatidylinositol, PI3P: Phosphatidylinositol 3-phosphate, PI(3,5)P 2 : Phosphatidylinositol 3,5 bisphosphate, PI5P: Phosphatidylinositol 5-phosphate, PI(4,5)P 2 : Phosphatidylinositol 4,5 bisphosphate. (B) Schematic illustrating the LC-MS/MS based in vitro 5-kinaseactivity assay using S2R+ cells over-expressing dFab1 enzyme to convert synthetic PI or PI3P to PI5P and PI(3,5)P 2 , respectively. (C) (i) Immunoprecipitated protein levels were analysed by Western blotting with an anti-mCherry antibody. Control (IgG) was prepared without anti-mCherry. Input lane shows the correct size of dFab1 protein ∼230 kDa. UTC: Untransfected control. (ii) In vitro kinase assay on synthetic PI and PI3P. Graph representing the Kinase activity (%) as the normalised response ratio of PI 5-kinase activity on PI” to PI3P 5-kinase activity on PI3P” upon enzymatic activity of immunoprecipitated mCherry::dFab1 on the respective substrates. Response ratio of PI 5-kinase activity on PI is obtained from area under the curve (AUC) of 17:0 14:1 PI5P (Product)/17:0 14:1 PI (Substrate), Response ratio of PI3P 5-kinaseactivity on PI3P isobtained from area under the curve (AUC) of 17:0 20:4 PI(3,5)P 2 (Product)/17:0 20:4 PI3P (Substrate) and is represented as mean ± S.E.M. on addition of either negative control (no beads), Control (mCherry beads) or dFab1 (mCherry::dFab1 beads). Number of immunoprecipitated samples= 2. (D) Schematic illustrating the LC-MS/MS based in vitro PI(3,5)P 2 3-phosphataseactivity assay using dsRNA treated S2R+ cells as enzyme source to convert synthetic PI(3,5)P 2 [d5-PI(3,5)P 2 to d5- 18 O-PIP 2 ] using a two-step reaction scheme. (E) In vitro phosphatase assay on synthetic PI(3,5)P 2 . Graph representing the 3-Phosphatase activity (%) as the percent formation of d5- 18 O-PIP 2 formed from starting d5-PI(3,5)P 2 as mean ± S.E.M. on addition of either control (GFPdsRNA) or Mtm, CG3632, CG3530 dsRNA treated S2R+ cell lysates. One way ANOVA with a post hoc Tukey’s test shows p value = 0.003 between GFP and Mtm ds RNA treated lysates, shows p value = 0.63 between GFP and CG3632 ds RNA treated lysate and shows p value= 0.11 between GFP and CG3530 dsRNA treated lysates.
    Figure Legend Snippet: (A) Schematic illustrating the putative enzymatic routes by which PI5Pcan be synthesised in Drosophila . The activities of enzymes labelled in blue are known in mammalian cells, the activity of enzymes labelled in red followed by “?” are still unknown in Drosophila , the activity of enzymes labelled in green are known in Drosophila . The activity of PI5P to PI(4,5)P 2 is boxed and is linked to cell size regulation in Drosophila . PI: Phosphatidylinositol, PI3P: Phosphatidylinositol 3-phosphate, PI(3,5)P 2 : Phosphatidylinositol 3,5 bisphosphate, PI5P: Phosphatidylinositol 5-phosphate, PI(4,5)P 2 : Phosphatidylinositol 4,5 bisphosphate. (B) Schematic illustrating the LC-MS/MS based in vitro 5-kinaseactivity assay using S2R+ cells over-expressing dFab1 enzyme to convert synthetic PI or PI3P to PI5P and PI(3,5)P 2 , respectively. (C) (i) Immunoprecipitated protein levels were analysed by Western blotting with an anti-mCherry antibody. Control (IgG) was prepared without anti-mCherry. Input lane shows the correct size of dFab1 protein ∼230 kDa. UTC: Untransfected control. (ii) In vitro kinase assay on synthetic PI and PI3P. Graph representing the Kinase activity (%) as the normalised response ratio of PI 5-kinase activity on PI” to PI3P 5-kinase activity on PI3P” upon enzymatic activity of immunoprecipitated mCherry::dFab1 on the respective substrates. Response ratio of PI 5-kinase activity on PI is obtained from area under the curve (AUC) of 17:0 14:1 PI5P (Product)/17:0 14:1 PI (Substrate), Response ratio of PI3P 5-kinaseactivity on PI3P isobtained from area under the curve (AUC) of 17:0 20:4 PI(3,5)P 2 (Product)/17:0 20:4 PI3P (Substrate) and is represented as mean ± S.E.M. on addition of either negative control (no beads), Control (mCherry beads) or dFab1 (mCherry::dFab1 beads). Number of immunoprecipitated samples= 2. (D) Schematic illustrating the LC-MS/MS based in vitro PI(3,5)P 2 3-phosphataseactivity assay using dsRNA treated S2R+ cells as enzyme source to convert synthetic PI(3,5)P 2 [d5-PI(3,5)P 2 to d5- 18 O-PIP 2 ] using a two-step reaction scheme. (E) In vitro phosphatase assay on synthetic PI(3,5)P 2 . Graph representing the 3-Phosphatase activity (%) as the percent formation of d5- 18 O-PIP 2 formed from starting d5-PI(3,5)P 2 as mean ± S.E.M. on addition of either control (GFPdsRNA) or Mtm, CG3632, CG3530 dsRNA treated S2R+ cell lysates. One way ANOVA with a post hoc Tukey’s test shows p value = 0.003 between GFP and Mtm ds RNA treated lysates, shows p value = 0.63 between GFP and CG3632 ds RNA treated lysate and shows p value= 0.11 between GFP and CG3530 dsRNA treated lysates.

    Techniques Used: Activity Assay, Liquid Chromatography with Mass Spectroscopy, In Vitro, Expressing, Immunoprecipitation, Western Blot, Kinase Assay, Negative Control, Phosphatase Assay

    (A) In vitro phosphatase assay on synthetic PI3P. Graph representing the response ratio of 17:0 20:4 PI (Product)/17:0 20:4 PI3P (Substrate) formed as mean ± S.E.M. on addition of either control (da/+) or da>Mtm WT GFP lysates. Lysate samples= 3, where each sample was made from fivethird instar wandering larvae. Strudent’s Unpaired t-test with Welch correction showed p value= 0.007. (B) Extracted ion chromatogram (XIC) of deacylated PI3P or GroPI3P (Glycerophosphoinositol 3-phosphate) peak at Rt = 7.37 min, separated from deacylated PI4P or GroPI4P (Glycerophosphoinositol 4-phosphate) peak at Rt = 9.12 min obtained from injecting wild type larval lipid extract (details of sample preparation is discussed in methods). (C) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of da/+; dPIP4K 29 (green) and da> Mtm WT GFP, dPIP4K 29 (majenta). Biological samples = 3, where each sample was made from three third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.07. (D) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of da/+ (green) andda> Mtm WT GFP, (majenta). Biological samples= 3, where each sample was made from three third instar wandering larvae.. Student’s unpaired t-test with Welch correction showed p value = 0.13.
    Figure Legend Snippet: (A) In vitro phosphatase assay on synthetic PI3P. Graph representing the response ratio of 17:0 20:4 PI (Product)/17:0 20:4 PI3P (Substrate) formed as mean ± S.E.M. on addition of either control (da/+) or da>Mtm WT GFP lysates. Lysate samples= 3, where each sample was made from fivethird instar wandering larvae. Strudent’s Unpaired t-test with Welch correction showed p value= 0.007. (B) Extracted ion chromatogram (XIC) of deacylated PI3P or GroPI3P (Glycerophosphoinositol 3-phosphate) peak at Rt = 7.37 min, separated from deacylated PI4P or GroPI4P (Glycerophosphoinositol 4-phosphate) peak at Rt = 9.12 min obtained from injecting wild type larval lipid extract (details of sample preparation is discussed in methods). (C) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of da/+; dPIP4K 29 (green) and da> Mtm WT GFP, dPIP4K 29 (majenta). Biological samples = 3, where each sample was made from three third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.07. (D) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of da/+ (green) andda> Mtm WT GFP, (majenta). Biological samples= 3, where each sample was made from three third instar wandering larvae.. Student’s unpaired t-test with Welch correction showed p value = 0.13.

    Techniques Used: In Vitro, Phosphatase Assay, Sample Prep

    (A) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value as mean ± S.E.M. of wild type (green) and dPIP4K 29 (majenta). Biological samples = 5, where each sample was made from five third instar wandering larvae. Unpaired t-test with Welch correction showed p value= 0.008. (B) Autoradiograph of TLC ran with lipid samples from in vitro PI3Pmassassay using wild type(WT) and dPIP4K 29 lipid samples. The first two lanes from the left are obtained from mass assay reactions using synthetic PI3P standard without or with addition of dFab1 enzyme, respectively. The origin spot and PI(3,5)P 2 spots are marked. (C) The graph represents normalised PI3P levels. Briefly, the spot marked asPI(3,5)P 2 on TLC in (B) is obtained by converting PI3P in the samples using immunoprecipitated dFab1 in presence of ϒ 32 P-ATP are quantified using image analysis and then normalised to organic phosphate value (indicated in blue embedded text under TLC) to obtain normalised PI3P levels. Biological samples = 3, where each sample was made from five third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.036. (D) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C/+; dPIP4K 29 (green), or Act5C> dPIP4K WT GFP, dPIP4K 29 (majenta). Biological samples= 5, where each sample was made from three third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.008. (E) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4Pnormalised to organicphosphatevalueof total lipid extractsasmean ± S.E.M. of Act5C/+; dPIP4K 29 (green) or Act5C> dPIP4K D271A , dPIP4K 29 (majenta). Biological samples = 6, where each sample was made from three third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.818. (F) In vitro phosphataseassay on synthetic PI3P. Graph representing the response ratio of 17:0 20:4 PI (Product)/17:0 20:4 PI3P (Substrate) formed asmean ± S.E.M. on addition of either wildtype (WT) or dPIP4K 29 lysatesfor either a 5 min or a 15 min reaction. Lysate samples= 3, where each sample was made from five third instar wandering larvae. Multiple unpaired t-test showed p value = 0.26 for 5 min time point and p value= 0.052. (G) qPCR measurements for mRNA levels of PI3K59F and PI3K68D from either Wild type (green) or dPIP4K 29 (majenta). Student’s unpaired t-test showed p value= 0.01 for PI3K59F and p value = 0.58 for PI3K68D . qPCR measurements for mRNA levels of Mtm, CG3632 and CG3530 from either Wild type (green) or dPIP4K 29 (majenta). Student’s unparied t-test showed p value = 0.03 for Mtm , p value= 0.23 for CG3632 and p value= 0.0006 for CG3530 .
    Figure Legend Snippet: (A) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value as mean ± S.E.M. of wild type (green) and dPIP4K 29 (majenta). Biological samples = 5, where each sample was made from five third instar wandering larvae. Unpaired t-test with Welch correction showed p value= 0.008. (B) Autoradiograph of TLC ran with lipid samples from in vitro PI3Pmassassay using wild type(WT) and dPIP4K 29 lipid samples. The first two lanes from the left are obtained from mass assay reactions using synthetic PI3P standard without or with addition of dFab1 enzyme, respectively. The origin spot and PI(3,5)P 2 spots are marked. (C) The graph represents normalised PI3P levels. Briefly, the spot marked asPI(3,5)P 2 on TLC in (B) is obtained by converting PI3P in the samples using immunoprecipitated dFab1 in presence of ϒ 32 P-ATP are quantified using image analysis and then normalised to organic phosphate value (indicated in blue embedded text under TLC) to obtain normalised PI3P levels. Biological samples = 3, where each sample was made from five third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.036. (D) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C/+; dPIP4K 29 (green), or Act5C> dPIP4K WT GFP, dPIP4K 29 (majenta). Biological samples= 5, where each sample was made from three third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.008. (E) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4Pnormalised to organicphosphatevalueof total lipid extractsasmean ± S.E.M. of Act5C/+; dPIP4K 29 (green) or Act5C> dPIP4K D271A , dPIP4K 29 (majenta). Biological samples = 6, where each sample was made from three third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.818. (F) In vitro phosphataseassay on synthetic PI3P. Graph representing the response ratio of 17:0 20:4 PI (Product)/17:0 20:4 PI3P (Substrate) formed asmean ± S.E.M. on addition of either wildtype (WT) or dPIP4K 29 lysatesfor either a 5 min or a 15 min reaction. Lysate samples= 3, where each sample was made from five third instar wandering larvae. Multiple unpaired t-test showed p value = 0.26 for 5 min time point and p value= 0.052. (G) qPCR measurements for mRNA levels of PI3K59F and PI3K68D from either Wild type (green) or dPIP4K 29 (majenta). Student’s unpaired t-test showed p value= 0.01 for PI3K59F and p value = 0.58 for PI3K68D . qPCR measurements for mRNA levels of Mtm, CG3632 and CG3530 from either Wild type (green) or dPIP4K 29 (majenta). Student’s unparied t-test showed p value = 0.03 for Mtm , p value= 0.23 for CG3632 and p value= 0.0006 for CG3530 .

    Techniques Used: Autoradiography, In Vitro, Mass Assay, Immunoprecipitation

    (A) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvaeof AB1/+; dPIP4K 29 (n = 9), AB1>PI3K59F RNAi ; dPIP4K 29 (n = 9). Sample size is represented on individual bars. Student’s unpaired t-test with Welch correction showed p value<0.0001. (B) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4Pnormalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C/+; dPIP4K 29 (green), or Act5C> dPIP4K WT GFP, dPIP4K 29 (majenta). Biological samples= 5, where each sample was made from five third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.011. (C) (i) Representative confocal z-projections depicting a sub population of early endosomal compartment using 2xFYVE-mCherry in the salivary glands from wandering third instar larvaeof AB1> 2xFYVE-mCherry and AB1> 2xFYVE-mCherry ; dPIP4K 29 . Scalebar indicated at 20 μm. (ii) Graph representing 2xFYVE punctaemeasurement in the s alivary glands from wandering third instar larvae of AB1> 2xFYVEmCherry (N = 8, n=40) and AB1> 2xFYVEmCherry ; dPIP4K 29 (N =8, n = 40). Student’s unpaired t-test with Welch correction showed p value= 0.4057 (D) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvaeof AB1/+; dPIP4K 29 (n = 8), AB1>dPIP4K 2XFYVE ; dPIP4K 29 (n = 8). Sample size is represented on individual bars. Student’s unpaired t-test with Welch correction showed p value= 0.171. (E) (i) Representative confocal z-projections depicting autophagosomal levels using Atg8a-mCherry in the salivary glands from the wandering third instar larvae of AB1>ATG8a-mCherry and AB1>ATG8a-mCherry; dPIP4K 29 . Scale bar indicated at 20 μm. (ii) Graph representing Atg8a punctae measurement in the salivary glands from wandering third instar larvaeof AB1>ATG8a-mCherry (N = 10, n = 60) and AB1>ATG8a-mCherry; dPIP4K 29 (N = 10, n = 62). Student’s unpaired t-test with Welch correction showed p value<0.0001. (F) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvaeof AB1/+; dPIP4K 29 (n = 9), AB1>PI3K59F RNAi ; dPIP4K 29 (n = 9). Sample size is represented on individual bars. ired t-test with Welch correction showed p value<0.0001.
    Figure Legend Snippet: (A) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvaeof AB1/+; dPIP4K 29 (n = 9), AB1>PI3K59F RNAi ; dPIP4K 29 (n = 9). Sample size is represented on individual bars. Student’s unpaired t-test with Welch correction showed p value<0.0001. (B) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4Pnormalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C/+; dPIP4K 29 (green), or Act5C> dPIP4K WT GFP, dPIP4K 29 (majenta). Biological samples= 5, where each sample was made from five third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.011. (C) (i) Representative confocal z-projections depicting a sub population of early endosomal compartment using 2xFYVE-mCherry in the salivary glands from wandering third instar larvaeof AB1> 2xFYVE-mCherry and AB1> 2xFYVE-mCherry ; dPIP4K 29 . Scalebar indicated at 20 μm. (ii) Graph representing 2xFYVE punctaemeasurement in the s alivary glands from wandering third instar larvae of AB1> 2xFYVEmCherry (N = 8, n=40) and AB1> 2xFYVEmCherry ; dPIP4K 29 (N =8, n = 40). Student’s unpaired t-test with Welch correction showed p value= 0.4057 (D) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvaeof AB1/+; dPIP4K 29 (n = 8), AB1>dPIP4K 2XFYVE ; dPIP4K 29 (n = 8). Sample size is represented on individual bars. Student’s unpaired t-test with Welch correction showed p value= 0.171. (E) (i) Representative confocal z-projections depicting autophagosomal levels using Atg8a-mCherry in the salivary glands from the wandering third instar larvae of AB1>ATG8a-mCherry and AB1>ATG8a-mCherry; dPIP4K 29 . Scale bar indicated at 20 μm. (ii) Graph representing Atg8a punctae measurement in the salivary glands from wandering third instar larvaeof AB1>ATG8a-mCherry (N = 10, n = 60) and AB1>ATG8a-mCherry; dPIP4K 29 (N = 10, n = 62). Student’s unpaired t-test with Welch correction showed p value<0.0001. (F) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvaeof AB1/+; dPIP4K 29 (n = 9), AB1>PI3K59F RNAi ; dPIP4K 29 (n = 9). Sample size is represented on individual bars. ired t-test with Welch correction showed p value<0.0001.

    Techniques Used:

    (A) qPCR measurements for mRNA levels of mtm, CG3632 and CG3530 from either Control ( daGal4 /+, in green) or da > Mtm RNAi, in majenta. Multiple t-test with post hoc Holm-Sidak’s test showed p value< 0.0001 between daGal4 /+ and da > Mtm RNAi for Mtm and p value= 0.35 between daGal4 /+ and da > Mtm RNAi for CG3632 , and p value = 0.04 between daGal4 /+ and da > Mtm RNAi for CG3530 . (B) (i) Representative confocal images of salivary glands from the genotypes a. AB1Gal4/+, b. AB1>Mtm RNAi. Cell body is marked majenta by BODIPY conjugated lipid dye, nucleus is marked by DAPI shown in green. Scale bar indicated at 50 μm. (ii) Graph representing average cell size measurement (in μm 3 ) asmean ± S.E.M. of salivary glands from wandering third instar larvae of AB1Gal4/+ (n = 7), AB1> Mtm RNAi (n = 7). Sample size is represented on individual bars. Student’s unpaired t-test with Welch correction showed p value = 0.0005. (C) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value as mean ± S.E.M. of da /+ (green) and da> Mtm RNAi (majenta). Biological samples = 4, where each sample was made from five third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.07. (D) (i) Graph representing Atg8a punctae measurement in the salivary glands from wandering third instar larvaeof AB1>ATG8a-mCherry (N =7, n =40), AB1>ATG8a-mCherry; Mtm RNAi (N =7, n =40). Student’s unpaired t-test with Welch correction showed p value<0.0001. (ii) Representative confocal z-projections depicting autophagosomal levels using Atg8a-mCherry in the salivary glands from the genotypesa. AB1>ATG8a-mCherry, b. AB1>ATG8a-mCherry; Mtm RNAi. Scale bar indicated at 20 μm. (E) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+ (n = 11), AB1>Mtm RNAi (n = 8), AB1>Mtm RNAi, Atg8a RNAi (n = 12). Sample size is represented on individual bars. One way ANOVA with post hoc Tukey’s test showed p value < 0.0001 between AB1/+ and AB1>Mtm RNAi and p value = 0.0002 between AB1/+; dPIP4K 29 and AB1>Mtm RNAi, Atg8a RNAi.
    Figure Legend Snippet: (A) qPCR measurements for mRNA levels of mtm, CG3632 and CG3530 from either Control ( daGal4 /+, in green) or da > Mtm RNAi, in majenta. Multiple t-test with post hoc Holm-Sidak’s test showed p value< 0.0001 between daGal4 /+ and da > Mtm RNAi for Mtm and p value= 0.35 between daGal4 /+ and da > Mtm RNAi for CG3632 , and p value = 0.04 between daGal4 /+ and da > Mtm RNAi for CG3530 . (B) (i) Representative confocal images of salivary glands from the genotypes a. AB1Gal4/+, b. AB1>Mtm RNAi. Cell body is marked majenta by BODIPY conjugated lipid dye, nucleus is marked by DAPI shown in green. Scale bar indicated at 50 μm. (ii) Graph representing average cell size measurement (in μm 3 ) asmean ± S.E.M. of salivary glands from wandering third instar larvae of AB1Gal4/+ (n = 7), AB1> Mtm RNAi (n = 7). Sample size is represented on individual bars. Student’s unpaired t-test with Welch correction showed p value = 0.0005. (C) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value as mean ± S.E.M. of da /+ (green) and da> Mtm RNAi (majenta). Biological samples = 4, where each sample was made from five third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.07. (D) (i) Graph representing Atg8a punctae measurement in the salivary glands from wandering third instar larvaeof AB1>ATG8a-mCherry (N =7, n =40), AB1>ATG8a-mCherry; Mtm RNAi (N =7, n =40). Student’s unpaired t-test with Welch correction showed p value<0.0001. (ii) Representative confocal z-projections depicting autophagosomal levels using Atg8a-mCherry in the salivary glands from the genotypesa. AB1>ATG8a-mCherry, b. AB1>ATG8a-mCherry; Mtm RNAi. Scale bar indicated at 20 μm. (E) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+ (n = 11), AB1>Mtm RNAi (n = 8), AB1>Mtm RNAi, Atg8a RNAi (n = 12). Sample size is represented on individual bars. One way ANOVA with post hoc Tukey’s test showed p value < 0.0001 between AB1/+ and AB1>Mtm RNAi and p value = 0.0002 between AB1/+; dPIP4K 29 and AB1>Mtm RNAi, Atg8a RNAi.

    Techniques Used:

    p-3016  (Echelon Biosciences)


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    Echelon Biosciences dic16 pi3p
    (A) Schematic illustrating the putative enzymatic routes by which PI5P can be synthesised in Drosophila . The activities of enzymes labelled in blue are known in mammalian cells, the activity of enzymes labelled in red followed by “?” are still unknown in Drosophila , the activity of enzymes labelled in green are known in Drosophila . The activity of PI5P to PI(4,5)P 2 is boxed and is linked to cell size regulation in Drosophila . PI, phosphatidylinositol; <t>PI3P,</t> <t>phosphatidylinositol</t> <t>3-phosphate;</t> <t>PI(3,5)P</t> 2 , phosphatidylinositol 3,5 bisphosphate; PI5P, phosphatidylinositol 5-phosphate; PI(4,5)P 2 , phosphatidylinositol 4,5 bisphosphate. Kinase and phosphatase activities are denoted by black solid arrows and black dashed arrows, respectively. (B) Schematic illustrating the LC-MS/MS-based in vitro 5-kinase activity assay using S2R+ cells overexpressing dFab1 enzyme to convert synthetic PI or PI3P to PI5P and PI(3,5)P 2 , respectively. (C) (i) Immunoprecipitated protein levels were analysed by Western blotting with an anti-mCherry antibody. Control (IgG) was prepared without anti-mCherry. Input lane shows the correct size of dFab1 protein ∼230 kD. UTC, Untransfected control. (ii) In vitro kinase assay on synthetic PI and PI3P. Graph representing the kinase activity (%) as the normalised response ratio of “PI 5-kinase activity on PI” to “PI3P 5-kinase activity on PI3P” upon enzymatic activity of immunoprecipitated mCherry::dFab1 on the respective substrates. Response ratio of PI 5-kinase activity on PI is obtained from area under the curve of 17:0 14:1 PI5P (product)/17:0 14:1 PI (substrate), Response ratio of PI3P 5-kinase activity on PI3P is obtained from area under the curve of 17:0 20:4 PI(3,5)P 2 (product)/17:0 20:4 PI3P (substrate) and is represented as mean ± S.E.M. on addition of either negative control (no beads), control (mCherry beads) or dFab1 (mCherry::dFab1 beads). Number of immunoprecipitated samples = 2. (D) Schematic illustrating the LC-MS/MS-based in vitro PI(3,5)P 2 3-phosphatase activity assay using dsRNA-treated S2R+ cells as the enzyme source to convert synthetic PI(3,5)P 2 [d5-PI(3,5)P 2 to d5- 18 O- PIP 2 ] using a two-step reaction scheme. (E) In vitro phosphatase assay on synthetic PI(3,5)P 2 . Graph representing the 3-phosphatase activity (%) as the percent formation of d5- 18 O-PIP 2 formed from starting d5-PI(3,5)P 2 as mean ± S.E.M. on addition of either control (GFP ds RNA) or Mtm , CG3632 , CG3530 ds RNA-treated S2R+ cell lysates. One-way ANOVA with a post hoc Tukey’s test shows P -value = 0.003 between GFP and Mtm ds RNA-treated lysates, shows P -value = 0.63 between GFP and CG3632 ds RNA-treated lysate and shows P -value = 0.11 between GFP and CG3530 ds RNA-treated lysates.
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    (A) Schematic illustrating the putative enzymatic routes by which PI5P can be synthesised in Drosophila . The activities of enzymes labelled in blue are known in mammalian cells, the activity of enzymes labelled in red followed by “?” are still unknown in Drosophila , the activity of enzymes labelled in green are known in Drosophila . The activity of PI5P to PI(4,5)P 2 is boxed and is linked to cell size regulation in Drosophila . PI, phosphatidylinositol; PI3P, phosphatidylinositol 3-phosphate; PI(3,5)P 2 , phosphatidylinositol 3,5 bisphosphate; PI5P, phosphatidylinositol 5-phosphate; PI(4,5)P 2 , phosphatidylinositol 4,5 bisphosphate. Kinase and phosphatase activities are denoted by black solid arrows and black dashed arrows, respectively. (B) Schematic illustrating the LC-MS/MS-based in vitro 5-kinase activity assay using S2R+ cells overexpressing dFab1 enzyme to convert synthetic PI or PI3P to PI5P and PI(3,5)P 2 , respectively. (C) (i) Immunoprecipitated protein levels were analysed by Western blotting with an anti-mCherry antibody. Control (IgG) was prepared without anti-mCherry. Input lane shows the correct size of dFab1 protein ∼230 kD. UTC, Untransfected control. (ii) In vitro kinase assay on synthetic PI and PI3P. Graph representing the kinase activity (%) as the normalised response ratio of “PI 5-kinase activity on PI” to “PI3P 5-kinase activity on PI3P” upon enzymatic activity of immunoprecipitated mCherry::dFab1 on the respective substrates. Response ratio of PI 5-kinase activity on PI is obtained from area under the curve of 17:0 14:1 PI5P (product)/17:0 14:1 PI (substrate), Response ratio of PI3P 5-kinase activity on PI3P is obtained from area under the curve of 17:0 20:4 PI(3,5)P 2 (product)/17:0 20:4 PI3P (substrate) and is represented as mean ± S.E.M. on addition of either negative control (no beads), control (mCherry beads) or dFab1 (mCherry::dFab1 beads). Number of immunoprecipitated samples = 2. (D) Schematic illustrating the LC-MS/MS-based in vitro PI(3,5)P 2 3-phosphatase activity assay using dsRNA-treated S2R+ cells as the enzyme source to convert synthetic PI(3,5)P 2 [d5-PI(3,5)P 2 to d5- 18 O- PIP 2 ] using a two-step reaction scheme. (E) In vitro phosphatase assay on synthetic PI(3,5)P 2 . Graph representing the 3-phosphatase activity (%) as the percent formation of d5- 18 O-PIP 2 formed from starting d5-PI(3,5)P 2 as mean ± S.E.M. on addition of either control (GFP ds RNA) or Mtm , CG3632 , CG3530 ds RNA-treated S2R+ cell lysates. One-way ANOVA with a post hoc Tukey’s test shows P -value = 0.003 between GFP and Mtm ds RNA-treated lysates, shows P -value = 0.63 between GFP and CG3632 ds RNA-treated lysate and shows P -value = 0.11 between GFP and CG3530 ds RNA-treated lysates.

    Journal: Life Science Alliance

    Article Title: PI3P-dependent regulation of cell size and autophagy by phosphatidylinositol 5-phosphate 4-kinase

    doi: 10.26508/lsa.202301920

    Figure Lengend Snippet: (A) Schematic illustrating the putative enzymatic routes by which PI5P can be synthesised in Drosophila . The activities of enzymes labelled in blue are known in mammalian cells, the activity of enzymes labelled in red followed by “?” are still unknown in Drosophila , the activity of enzymes labelled in green are known in Drosophila . The activity of PI5P to PI(4,5)P 2 is boxed and is linked to cell size regulation in Drosophila . PI, phosphatidylinositol; PI3P, phosphatidylinositol 3-phosphate; PI(3,5)P 2 , phosphatidylinositol 3,5 bisphosphate; PI5P, phosphatidylinositol 5-phosphate; PI(4,5)P 2 , phosphatidylinositol 4,5 bisphosphate. Kinase and phosphatase activities are denoted by black solid arrows and black dashed arrows, respectively. (B) Schematic illustrating the LC-MS/MS-based in vitro 5-kinase activity assay using S2R+ cells overexpressing dFab1 enzyme to convert synthetic PI or PI3P to PI5P and PI(3,5)P 2 , respectively. (C) (i) Immunoprecipitated protein levels were analysed by Western blotting with an anti-mCherry antibody. Control (IgG) was prepared without anti-mCherry. Input lane shows the correct size of dFab1 protein ∼230 kD. UTC, Untransfected control. (ii) In vitro kinase assay on synthetic PI and PI3P. Graph representing the kinase activity (%) as the normalised response ratio of “PI 5-kinase activity on PI” to “PI3P 5-kinase activity on PI3P” upon enzymatic activity of immunoprecipitated mCherry::dFab1 on the respective substrates. Response ratio of PI 5-kinase activity on PI is obtained from area under the curve of 17:0 14:1 PI5P (product)/17:0 14:1 PI (substrate), Response ratio of PI3P 5-kinase activity on PI3P is obtained from area under the curve of 17:0 20:4 PI(3,5)P 2 (product)/17:0 20:4 PI3P (substrate) and is represented as mean ± S.E.M. on addition of either negative control (no beads), control (mCherry beads) or dFab1 (mCherry::dFab1 beads). Number of immunoprecipitated samples = 2. (D) Schematic illustrating the LC-MS/MS-based in vitro PI(3,5)P 2 3-phosphatase activity assay using dsRNA-treated S2R+ cells as the enzyme source to convert synthetic PI(3,5)P 2 [d5-PI(3,5)P 2 to d5- 18 O- PIP 2 ] using a two-step reaction scheme. (E) In vitro phosphatase assay on synthetic PI(3,5)P 2 . Graph representing the 3-phosphatase activity (%) as the percent formation of d5- 18 O-PIP 2 formed from starting d5-PI(3,5)P 2 as mean ± S.E.M. on addition of either control (GFP ds RNA) or Mtm , CG3632 , CG3530 ds RNA-treated S2R+ cell lysates. One-way ANOVA with a post hoc Tukey’s test shows P -value = 0.003 between GFP and Mtm ds RNA-treated lysates, shows P -value = 0.63 between GFP and CG3632 ds RNA-treated lysate and shows P -value = 0.11 between GFP and CG3530 ds RNA-treated lysates.

    Article Snippet: diC16-PI3P (Echelon) was mixed with 20 μM phosphatidylserine (PS) (#P5660; Sigma-Aldrich) and dried in a centrifugal vacuum concentrator.

    Techniques: Activity Assay, Liquid Chromatography with Mass Spectroscopy, In Vitro, Kinase Assay, Immunoprecipitation, Western Blot, Negative Control, Phosphatase Assay

    (A) In vitro phosphatase assay on synthetic PI3P. Graph representing the response ratio of 17:0 20:4 PI (product)/17:0 20:4 PI3P (substrate) formed as mean ± S.E.M. on addition of either control ( da/+ ) or da>Mtm WT ::GFP lysates. Lysate samples = 3, where each sample was made from five third instar wandering larvae. unpaired t test with Welch correction showed P -value = 0.007. (B) Extracted ion chromatogram (XIC) of deacylated PI3P or GroPI3P (glycerophosphoinositol 3-phosphate) peak at R t = 7.37 min, separated from deacylated PI4P or GroPI4P (glycerophosphoinositol 4-phosphate) peak at R t = 9.12 min obtained from injecting WT larval lipid extract (details of sample preparation is discussed in methods). (C) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of da/+; dPIP4K 29 (green) and da> Mtm WT ::GFP; dPIP4K 29 (magenta). Biological samples = 3, where each sample was made from three third instar wandering larvae. unpaired t test with Welch correction showed P -value = 0.07. (D) Graph representing normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of da/+ (green) and da> Mtm WT ::GFP (magenta). Biological samples = 3, where each sample was made from three third instar wandering larvae. Unpaired t test with Welch correction showed P -value = 0.13.

    Journal: Life Science Alliance

    Article Title: PI3P-dependent regulation of cell size and autophagy by phosphatidylinositol 5-phosphate 4-kinase

    doi: 10.26508/lsa.202301920

    Figure Lengend Snippet: (A) In vitro phosphatase assay on synthetic PI3P. Graph representing the response ratio of 17:0 20:4 PI (product)/17:0 20:4 PI3P (substrate) formed as mean ± S.E.M. on addition of either control ( da/+ ) or da>Mtm WT ::GFP lysates. Lysate samples = 3, where each sample was made from five third instar wandering larvae. unpaired t test with Welch correction showed P -value = 0.007. (B) Extracted ion chromatogram (XIC) of deacylated PI3P or GroPI3P (glycerophosphoinositol 3-phosphate) peak at R t = 7.37 min, separated from deacylated PI4P or GroPI4P (glycerophosphoinositol 4-phosphate) peak at R t = 9.12 min obtained from injecting WT larval lipid extract (details of sample preparation is discussed in methods). (C) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of da/+; dPIP4K 29 (green) and da> Mtm WT ::GFP; dPIP4K 29 (magenta). Biological samples = 3, where each sample was made from three third instar wandering larvae. unpaired t test with Welch correction showed P -value = 0.07. (D) Graph representing normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of da/+ (green) and da> Mtm WT ::GFP (magenta). Biological samples = 3, where each sample was made from three third instar wandering larvae. Unpaired t test with Welch correction showed P -value = 0.13.

    Article Snippet: diC16-PI3P (Echelon) was mixed with 20 μM phosphatidylserine (PS) (#P5660; Sigma-Aldrich) and dried in a centrifugal vacuum concentrator.

    Techniques: In Vitro, Phosphatase Assay, Sample Prep

    (A) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value as mean ± S.E.M. of WT (green) and dPIP4K 29 (magenta). Biological samples = 5, where each sample was made from five third instar wandering larvae. Unpaired t test with Welch correction showed P -value = 0.008. (B) Graph representing normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C/+ ; dPIP4K 29 (green) or Act5C> dPIP4K WT ::GFP; dPIP4K 29 (magenta). Biological samples = 5, where each sample was made from three third instar wandering larvae. unpaired t test with Welch correction showed P -value = 0.008. (C) Graph representing normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C/+ ; dPIP4K 29 (green) or Act5C> dPIP4K D271A; dPIP4K 29 (magenta). Biological samples = 6, where each sample was made from three third instar wandering larvae. Unpaired t test with Welch correction showed P -value = 0.818. (D) In vitro phosphatase assay on synthetic PI3P. Graph representing the response ratio of 17:0 20:4 PI (product)/17:0 20:4 PI3P (substrate) formed as mean ± S.E.M. on addition of either WT or dPIP4K 29 lysates for either a 5-min or a 15-min reaction. Lysate samples = 3, where each sample was made from five third instar wandering larvae. Multiple unpaired t test showed P -value = 0.26 for 5 min time point and P -value = 0.052. (E) qPCR measurements for mRNA levels of PI3K59F and PI3K68D from either WT (green) or dPIP4K 29 (magenta). unpaired t test showed P -value = 0.01 for PI3K59F and P -value = 0.58 for PI3K68D . qPCR measurements for mRNA levels of Mtm , CG3632, and CG3530 from either WT (green) or dPIP4K 29 (magenta). Unpaired t test showed P -value = 0.03 for Mtm , P -value = 0.23 for CG3632, and P -value = 0.0006 for CG3530 .

    Journal: Life Science Alliance

    Article Title: PI3P-dependent regulation of cell size and autophagy by phosphatidylinositol 5-phosphate 4-kinase

    doi: 10.26508/lsa.202301920

    Figure Lengend Snippet: (A) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value as mean ± S.E.M. of WT (green) and dPIP4K 29 (magenta). Biological samples = 5, where each sample was made from five third instar wandering larvae. Unpaired t test with Welch correction showed P -value = 0.008. (B) Graph representing normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C/+ ; dPIP4K 29 (green) or Act5C> dPIP4K WT ::GFP; dPIP4K 29 (magenta). Biological samples = 5, where each sample was made from three third instar wandering larvae. unpaired t test with Welch correction showed P -value = 0.008. (C) Graph representing normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C/+ ; dPIP4K 29 (green) or Act5C> dPIP4K D271A; dPIP4K 29 (magenta). Biological samples = 6, where each sample was made from three third instar wandering larvae. Unpaired t test with Welch correction showed P -value = 0.818. (D) In vitro phosphatase assay on synthetic PI3P. Graph representing the response ratio of 17:0 20:4 PI (product)/17:0 20:4 PI3P (substrate) formed as mean ± S.E.M. on addition of either WT or dPIP4K 29 lysates for either a 5-min or a 15-min reaction. Lysate samples = 3, where each sample was made from five third instar wandering larvae. Multiple unpaired t test showed P -value = 0.26 for 5 min time point and P -value = 0.052. (E) qPCR measurements for mRNA levels of PI3K59F and PI3K68D from either WT (green) or dPIP4K 29 (magenta). unpaired t test showed P -value = 0.01 for PI3K59F and P -value = 0.58 for PI3K68D . qPCR measurements for mRNA levels of Mtm , CG3632, and CG3530 from either WT (green) or dPIP4K 29 (magenta). Unpaired t test showed P -value = 0.03 for Mtm , P -value = 0.23 for CG3632, and P -value = 0.0006 for CG3530 .

    Article Snippet: diC16-PI3P (Echelon) was mixed with 20 μM phosphatidylserine (PS) (#P5660; Sigma-Aldrich) and dried in a centrifugal vacuum concentrator.

    Techniques: In Vitro, Phosphatase Assay

    (A) Schematic illustrating the methodology to assay PI3P by a dFab1-mediated radioactivity-based mass assay. dFab1 is purified from S2R+ cells by immunoprecipitation and used to convert PI3P from total lipid extracts obtained from larvae in the presence of ϒ 32 P-ATP to radiolabelled PI(3,5)P 2 product which is finally analysed using TLC. A portion of the total lipid extract is used for organic phosphate assay to normalise for sample size. (B) (i) Autoradiograph of TLC ran with lipid samples from in vitro PI3P mass assay using WT and dPIP4K 29 lipid samples. The first two lanes from the left are obtained from mass assay reactions using synthetic PI3P standard without or with addition of dFab1 enzyme, respectively. The origin spot and PI(3,5)P 2 spots are marked. (B) (ii) Graph represents normalised PI3P levels. Briefly, the spot marked as PI(3,5)P 2 on TLC in (B) is obtained by converting PI3P in the samples using immunoprecipitated dFab1 in the presence of ϒ 32 P-ATP are quantified using image analysis and then normalised to organic phosphate value (indicated in blue embedded text under TLC) to obtain normalised PI3P levels. Biological samples = 3, where each sample was made from five third instar wandering larvae. Unpaired t test with Welch correction showed P -value = 0.036. (C) Illustration depicting an indirect model where the increased PI3P levels in dPIP4K 29 (denoted by a red cross on dPIP4K) can be explained by either an activation of PI 3-kinase activity (green arrow) or an inhibition of PI3P 3-phosphatase activity (red stubbed arrow).

    Journal: Life Science Alliance

    Article Title: PI3P-dependent regulation of cell size and autophagy by phosphatidylinositol 5-phosphate 4-kinase

    doi: 10.26508/lsa.202301920

    Figure Lengend Snippet: (A) Schematic illustrating the methodology to assay PI3P by a dFab1-mediated radioactivity-based mass assay. dFab1 is purified from S2R+ cells by immunoprecipitation and used to convert PI3P from total lipid extracts obtained from larvae in the presence of ϒ 32 P-ATP to radiolabelled PI(3,5)P 2 product which is finally analysed using TLC. A portion of the total lipid extract is used for organic phosphate assay to normalise for sample size. (B) (i) Autoradiograph of TLC ran with lipid samples from in vitro PI3P mass assay using WT and dPIP4K 29 lipid samples. The first two lanes from the left are obtained from mass assay reactions using synthetic PI3P standard without or with addition of dFab1 enzyme, respectively. The origin spot and PI(3,5)P 2 spots are marked. (B) (ii) Graph represents normalised PI3P levels. Briefly, the spot marked as PI(3,5)P 2 on TLC in (B) is obtained by converting PI3P in the samples using immunoprecipitated dFab1 in the presence of ϒ 32 P-ATP are quantified using image analysis and then normalised to organic phosphate value (indicated in blue embedded text under TLC) to obtain normalised PI3P levels. Biological samples = 3, where each sample was made from five third instar wandering larvae. Unpaired t test with Welch correction showed P -value = 0.036. (C) Illustration depicting an indirect model where the increased PI3P levels in dPIP4K 29 (denoted by a red cross on dPIP4K) can be explained by either an activation of PI 3-kinase activity (green arrow) or an inhibition of PI3P 3-phosphatase activity (red stubbed arrow).

    Article Snippet: diC16-PI3P (Echelon) was mixed with 20 μM phosphatidylserine (PS) (#P5660; Sigma-Aldrich) and dried in a centrifugal vacuum concentrator.

    Techniques: Radioactivity, Mass Assay, Purification, Immunoprecipitation, Autoradiography, In Vitro, Activation Assay, Activity Assay, Inhibition

    (A) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+; dPIP4K 29 (n = 9), AB1>PI3K59F RNAi; dPIP4K 29 (n = 9). Sample size is represented on individual bars. Unpaired t test with Welch correction showed P -value < 0.0001. (B) Graph representing normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C /+; dPIP4K 29 (green) or Act5C > PI3K59F RNAi; dPIP4K 29 (magenta). Biological samples = 5, where each sample was made from five third instar wandering larvae. Unpaired t test with Welch correction showed P -value = 0.011. (C) (i) Representative confocal z-projections depicting a subpopulation of early endosomal compartment using 2XFYVE-mCherry in the salivary glands from wandering third instar larvae of AB1> 2XFYVE::mCherry (Control) and AB1> 2XFYVE::mCherry; dPIP4K 29 . Scale bar indicated at 20 μm. (ii) Graph representing 2XFYVE punctae measurement in the salivary glands from wandering third instar larvae of AB1> 2XFYVE::mCherry (N = 8, n = 40) and AB1>2XFYVE::mCherry; dPIP4K 29 (N = 8, n = 40). Unpaired t test with Welch correction showed P -value = 0.4057. (D) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+; dPIP4K 29 (n = 8), AB1>dPIP4K 2XFYVE ; dPIP4K 29 (n = 8). Sample size is represented on individual bars. Unpaired t test with Welch correction showed P -value = 0.171. (E) qPCR measurements for mRNA levels of UVRAG from either da/+ (green) or da> UVRAG RNAi (magenta). Unpaired t test showed P -value = 0.0046. (n = 6, where each n is derived from five third instar wandering larvae). (F) Graph representing average cell size measurement as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+; dPIP4K 29 (n = 12) and AB1> UVRAG RNAi; dPIP4K 29 (n = 12). unpaired t test with Welch correction showed P -value = 0.4306. (G) (i) Representative confocal z-projections depicting 2XFYVE punctae using 2XFYVE::mCherry in the salivary glands from the genotypes a. AB1> 2XFYVE :: mCherry (Control) b. AB1> 2XFYVE::mCherry; UVRAG RNAi. (ii) Graph representing measurement of 2XFYVE punctae numbers per unit cell area of the salivary glands from wandering third instar larvae of AB1> 2XFYVE::mCherry (N = 3, n = 11), AB1> 2XFYVE::mCherry; UVRAG RNAi (N = 3,n = 11).

    Journal: Life Science Alliance

    Article Title: PI3P-dependent regulation of cell size and autophagy by phosphatidylinositol 5-phosphate 4-kinase

    doi: 10.26508/lsa.202301920

    Figure Lengend Snippet: (A) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+; dPIP4K 29 (n = 9), AB1>PI3K59F RNAi; dPIP4K 29 (n = 9). Sample size is represented on individual bars. Unpaired t test with Welch correction showed P -value < 0.0001. (B) Graph representing normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C /+; dPIP4K 29 (green) or Act5C > PI3K59F RNAi; dPIP4K 29 (magenta). Biological samples = 5, where each sample was made from five third instar wandering larvae. Unpaired t test with Welch correction showed P -value = 0.011. (C) (i) Representative confocal z-projections depicting a subpopulation of early endosomal compartment using 2XFYVE-mCherry in the salivary glands from wandering third instar larvae of AB1> 2XFYVE::mCherry (Control) and AB1> 2XFYVE::mCherry; dPIP4K 29 . Scale bar indicated at 20 μm. (ii) Graph representing 2XFYVE punctae measurement in the salivary glands from wandering third instar larvae of AB1> 2XFYVE::mCherry (N = 8, n = 40) and AB1>2XFYVE::mCherry; dPIP4K 29 (N = 8, n = 40). Unpaired t test with Welch correction showed P -value = 0.4057. (D) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+; dPIP4K 29 (n = 8), AB1>dPIP4K 2XFYVE ; dPIP4K 29 (n = 8). Sample size is represented on individual bars. Unpaired t test with Welch correction showed P -value = 0.171. (E) qPCR measurements for mRNA levels of UVRAG from either da/+ (green) or da> UVRAG RNAi (magenta). Unpaired t test showed P -value = 0.0046. (n = 6, where each n is derived from five third instar wandering larvae). (F) Graph representing average cell size measurement as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+; dPIP4K 29 (n = 12) and AB1> UVRAG RNAi; dPIP4K 29 (n = 12). unpaired t test with Welch correction showed P -value = 0.4306. (G) (i) Representative confocal z-projections depicting 2XFYVE punctae using 2XFYVE::mCherry in the salivary glands from the genotypes a. AB1> 2XFYVE :: mCherry (Control) b. AB1> 2XFYVE::mCherry; UVRAG RNAi. (ii) Graph representing measurement of 2XFYVE punctae numbers per unit cell area of the salivary glands from wandering third instar larvae of AB1> 2XFYVE::mCherry (N = 3, n = 11), AB1> 2XFYVE::mCherry; UVRAG RNAi (N = 3,n = 11).

    Article Snippet: diC16-PI3P (Echelon) was mixed with 20 μM phosphatidylserine (PS) (#P5660; Sigma-Aldrich) and dried in a centrifugal vacuum concentrator.

    Techniques: Derivative Assay

    (A) qPCR measurements for mRNA levels of Mtm , CG3632, and CG3530 from either control ( da /+, in green) or da > Mtm RNAi, in magenta. Multiple t test with post hoc Holm-Sidak’s test showed P -value < 0.0001 between da/+ and da> Mtm RNAi for Mtm and P -value = 0.35 between da/+ and da> Mtm RNAi for CG3632 , and P -value = 0.04 between da/+ and da> Mtm RNAi for CG3530 . (B) (i) Representative confocal images of salivary glands from the genotypes a. AB1/+ , b. AB1>Mtm RNAi. Cell body is marked magenta by BODIPY-conjugated lipid dye, and the nucleus is marked by DAPI shown in green. Scale bar indicated at 50 μm. (ii) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+ (n = 7), AB1> Mtm RNAi (n = 7). Sample size is represented on individual bars. Unpaired t test with Welch correction showed P -value = 0.0005. (C) Graph representing normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value as mean ± S.E.M. of da/+ (green) and da> Mtm RNAi (magenta). Biological samples = 4, where each sample was made from five third instar wandering larvae. Unpaired t test with Welch correction showed P -value = 0.07. (D) (i) Representative confocal z-projections depicting autophagosomal levels using Atg8a-mCherry in the salivary glands from the genotypes a. AB1>Atg8a::mCherry (control), b. AB1>Atg8a::mCherry; Mtm RNAi. Scale bar indicated at 20 μm. (ii) Graph representing Atg8a punctae measurement in the salivary glands from wandering third instar larvae of AB1>Atg8a::mCherry (N = 7, n = 40), AB1>Atg8a::mCherry; Mtm RNAi (N = 7, n = 40). Unpaired t test with Welch correction showed P -value < 0.0001. (E) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+ (n = 11), AB1>Mtm RNAi (n = 8), AB1>Mtm RNAi, Atg8a RNAi (n = 12). Sample size is represented on individual bars. One-way ANOVA with post hoc Tukey’s test showed P -value < 0.0001 between AB1/+ and AB1>Mtm RNAi and P -value = 0.0002 between AB1/+; dPIP4K 29 and AB1>Mtm RNAi, Atg8a RNAi.

    Journal: Life Science Alliance

    Article Title: PI3P-dependent regulation of cell size and autophagy by phosphatidylinositol 5-phosphate 4-kinase

    doi: 10.26508/lsa.202301920

    Figure Lengend Snippet: (A) qPCR measurements for mRNA levels of Mtm , CG3632, and CG3530 from either control ( da /+, in green) or da > Mtm RNAi, in magenta. Multiple t test with post hoc Holm-Sidak’s test showed P -value < 0.0001 between da/+ and da> Mtm RNAi for Mtm and P -value = 0.35 between da/+ and da> Mtm RNAi for CG3632 , and P -value = 0.04 between da/+ and da> Mtm RNAi for CG3530 . (B) (i) Representative confocal images of salivary glands from the genotypes a. AB1/+ , b. AB1>Mtm RNAi. Cell body is marked magenta by BODIPY-conjugated lipid dye, and the nucleus is marked by DAPI shown in green. Scale bar indicated at 50 μm. (ii) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+ (n = 7), AB1> Mtm RNAi (n = 7). Sample size is represented on individual bars. Unpaired t test with Welch correction showed P -value = 0.0005. (C) Graph representing normalised PI3P levels which is the peak area of GroPI3P/peak area of GroPI4P normalised to organic phosphate value as mean ± S.E.M. of da/+ (green) and da> Mtm RNAi (magenta). Biological samples = 4, where each sample was made from five third instar wandering larvae. Unpaired t test with Welch correction showed P -value = 0.07. (D) (i) Representative confocal z-projections depicting autophagosomal levels using Atg8a-mCherry in the salivary glands from the genotypes a. AB1>Atg8a::mCherry (control), b. AB1>Atg8a::mCherry; Mtm RNAi. Scale bar indicated at 20 μm. (ii) Graph representing Atg8a punctae measurement in the salivary glands from wandering third instar larvae of AB1>Atg8a::mCherry (N = 7, n = 40), AB1>Atg8a::mCherry; Mtm RNAi (N = 7, n = 40). Unpaired t test with Welch correction showed P -value < 0.0001. (E) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+ (n = 11), AB1>Mtm RNAi (n = 8), AB1>Mtm RNAi, Atg8a RNAi (n = 12). Sample size is represented on individual bars. One-way ANOVA with post hoc Tukey’s test showed P -value < 0.0001 between AB1/+ and AB1>Mtm RNAi and P -value = 0.0002 between AB1/+; dPIP4K 29 and AB1>Mtm RNAi, Atg8a RNAi.

    Article Snippet: diC16-PI3P (Echelon) was mixed with 20 μM phosphatidylserine (PS) (#P5660; Sigma-Aldrich) and dried in a centrifugal vacuum concentrator.

    Techniques:

    Schematic showing the regulation of PI3P at an autophagic membrane downstream of Atg1. In this study, dPIP4K which metabolizes PI3P has been shown to genetically interact with Vps34 and Mtm, two established PI3P regulators. Atg8a-marked autophagosomes form, mature to autolysosomes, and function in maintaining normal cell size in WT cells. In dPIP4K 29 cells, PI3P levels increase, leading to an increase in Atg8a punctae by increased autophagy initiation, which in turn leads to decreased cell size phenotype.

    Journal: Life Science Alliance

    Article Title: PI3P-dependent regulation of cell size and autophagy by phosphatidylinositol 5-phosphate 4-kinase

    doi: 10.26508/lsa.202301920

    Figure Lengend Snippet: Schematic showing the regulation of PI3P at an autophagic membrane downstream of Atg1. In this study, dPIP4K which metabolizes PI3P has been shown to genetically interact with Vps34 and Mtm, two established PI3P regulators. Atg8a-marked autophagosomes form, mature to autolysosomes, and function in maintaining normal cell size in WT cells. In dPIP4K 29 cells, PI3P levels increase, leading to an increase in Atg8a punctae by increased autophagy initiation, which in turn leads to decreased cell size phenotype.

    Article Snippet: diC16-PI3P (Echelon) was mixed with 20 μM phosphatidylserine (PS) (#P5660; Sigma-Aldrich) and dried in a centrifugal vacuum concentrator.

    Techniques:

    Multiple reaction monitoring (MRM) values for commercial lipids used.

    Journal: Life Science Alliance

    Article Title: PI3P-dependent regulation of cell size and autophagy by phosphatidylinositol 5-phosphate 4-kinase

    doi: 10.26508/lsa.202301920

    Figure Lengend Snippet: Multiple reaction monitoring (MRM) values for commercial lipids used.

    Article Snippet: diC16-PI3P (Echelon) was mixed with 20 μM phosphatidylserine (PS) (#P5660; Sigma-Aldrich) and dried in a centrifugal vacuum concentrator.

    Techniques: