Journal: PLoS ONE

Article Title: Resistance to Inhibitors of Cholinesterase (Ric)-8A and Gα i Contribute to Cytokinesis Abscission by Controlling Vacuolar Protein-Sorting (Vps)34 Activity

doi: 10.1371/journal.pone.0086680

Figure Lengend Snippet: ( A ) Vps34, GFP and actin immunoblots of cell lysates immunoprecipitate from HeLa cells transiently transfected with vectors expressing YFP, Gα i3 wt-YFP, or Gα i3 QL-YFP. Experiments were repeated twice with similar results. ( B ) 3D reconstruction of confocal images of HeLa cells expressing GFP-Ric-8A immunostained for endogenous Vps34. Scale bar is 5 µm. The right panels show an electronic magnification of midbody area (white square). ( C–D ) Co-immunoprecipitation of endogenous Vps34 and endogenous Ric-8A in HeLa cells. The experiment was repeated 3 times with similar results. ( E ) Asynchronous HeLa cells lysates treated with PTX (200 ng/mL, 3 h) or with its vehicle were immunoprecipitated using anti-Vps34 antibody and immunoblotted for Ric-8A or Vps34. ( F ) Quantification of PtdIns(3)P production after purification of endogenous Vps34 kinase in asynchronous or G2/M enriched HeLa cells treated with PTX (200 ng/mL, 3 h) or siRNA control versus siRNA Ric-8A. Quantification was performed from 4 independent experiments (*, p<0.05).

Article Snippet: IP was performed with anti-Vps34 antibody (Echelon).

Techniques: Western Blot, Transfection, Expressing, Immunoprecipitation, Purification

Journal: PLoS ONE

Article Title: Resistance to Inhibitors of Cholinesterase (Ric)-8A and Gα i Contribute to Cytokinesis Abscission by Controlling Vacuolar Protein-Sorting (Vps)34 Activity

doi: 10.1371/journal.pone.0086680

Figure Lengend Snippet: ( A ) Vps34, GFP and actin immunoblots of cell lysates immunoprecipitate from HeLa cells transiently transfected with vectors expressing YFP, Gα i3 wt-YFP, or Gα i3 QL-YFP. Experiments were repeated twice with similar results. ( B ) 3D reconstruction of confocal images of HeLa cells expressing GFP-Ric-8A immunostained for endogenous Vps34. Scale bar is 5 µm. The right panels show an electronic magnification of midbody area (white square). ( C–D ) Co-immunoprecipitation of endogenous Vps34 and endogenous Ric-8A in HeLa cells. The experiment was repeated 3 times with similar results. ( E ) Asynchronous HeLa cells lysates treated with PTX (200 ng/mL, 3 h) or with its vehicle were immunoprecipitated using anti-Vps34 antibody and immunoblotted for Ric-8A or Vps34. ( F ) Quantification of PtdIns(3)P production after purification of endogenous Vps34 kinase in asynchronous or G2/M enriched HeLa cells treated with PTX (200 ng/mL, 3 h) or siRNA control versus siRNA Ric-8A. Quantification was performed from 4 independent experiments (*, p<0.05).

Article Snippet: For immunoprecipitation, supernatants (50 µg) were incubated with 1 µg anti-GFP antibody (Cell Signaling), 0.5 µg anti-p190 antibody (Invitrogen), 0.5 µg anti-Vps34 (Echelon) antibody, 0.5 µg anti-Vps15 antibody (Bethyl) or 0.5 µg anti-Ric-8A antibody (Abcam) overnight at 4°C and then incubated with protein G-Sepharose (Sigma) beads for 1 h at 4°C.

Techniques: Western Blot, Transfection, Expressing, Immunoprecipitation, Purification

Journal: Experimental & Molecular Medicine

Article Title: NRBF2-mediated autophagy contributes to metabolite replenishment and radioresistance in glioblastoma

doi: 10.1038/s12276-022-00873-2

Figure Lengend Snippet: a Autophagy-related protein levels after treatment confirmed by western blot. b Schematic images of the autophagy initiation step involved in NRBF2. c Coimmunoprecipitation of NRBF2 and the VPS34 complex confirmed by western blot in GBM cell lines. d Autophagic flux analysis using the mCherry-EGFP reporter. A172 and U-87 MG cells transfected with mCherry-GFP-LC3B were treated with IR (3 Gy), siNRBF2 or both. The mean numbers of autophagosomes and autolysosomes are represented by yellow and red dots, respectively, in the merged images. The images were quantified using ImageJ ( n = 5). Scale bars, 10 µm. e IHC of LC3B in coronal sections from U-87 WT or NRBF2 knockout orthotopic xenograft mouse models treated with radiation (2 Gy × 5). Scale bars, 10 μm. ** p < 0.01, *** p < 0.001, **** p < 0.0001 using an unpaired t test.

Article Snippet: Antibodies specific for VPS34 were purchased from Echelon Biosciences (Echelon Biosciences, Salt Lake City, UT, USA), and antibodies specific for ATG14L were purchased from MBL (MBL, Tokyo, Japan).

Techniques: Western Blot, Transfection, Knock-Out

Journal: American Journal of Physiology - Renal Physiology

Article Title: The expression level of class III phosphatidylinositol-3 kinase controls the degree of compensatory nephron hypertrophy

doi: 10.1152/ajprenal.00381.2019

Figure Lengend Snippet: Generation of a Pik3c3 hypomorphic mouse model. Schematic illustration of the wild-type allele, targeting vector, and hypomorphic allele of the mouse Pik3c3 gene, which enabled us to generate homozygous Pik3c3 hypomorphic (Pik3c3Hypo/Hypo) mice and heterozygous Pik3c3-hypomorphic (Pik3c3Hypo/WT) mice to compare with their wild-type (Pik3c3WT/WT) littermates (A). Both immunoblot (B) and immunohistochemistry staining (C) indicated that Pik3c3Hypo/Hypo mice express a markedly lower level of Pik3c3 than Pik3c3Hypo/WT littermates, which already express a lower level of Pik3c3 than Pik3c3WT/WT littermates. E, embroynic day. Shown are representative blots and images from n = 6–8 mice/genotype group with similar results.

Article Snippet: Antibodies against Pik3c3 (Z-R016) were purchased from Echelon Biosciences (Salt Lake City, UT).

Techniques: Plasmid Preparation, Western Blot, Immunohistochemistry, Staining

Journal: American Journal of Physiology - Renal Physiology

Article Title: The expression level of class III phosphatidylinositol-3 kinase controls the degree of compensatory nephron hypertrophy

doi: 10.1152/ajprenal.00381.2019

Figure Lengend Snippet: The reduction of Pik3c3 expression also occurs in other organs of the Pik3c3 hypomorphic mouse model. Immunoblot analysis indicated that homozygous Pik3c3 hypomorphic (Pik3c3Hypo/Hypo) mice (Homo) express a markedly lower level of Pik3c3 than heterozygous Pik3c3 hypomorphic (Pik3c3Hypo/WT) littermates (Het), which already express a lower level of Pik3c3 than wild-type (Pik3c3WT/WT) littermates (WT). Of note, although an equal amount (10 or 20 μg) of total protein from different tissue homogenates was loaded, both β-actin and α-tubulin (two commonly used loading controls) were expressed at considerably different levels in different tissues, reminiscent to a previous report (28). However, expression levels of either β-actin or α-tubulin in the same type of tissues examined (including spleen, brain, lung, heart, liver, pancreas, testis, and adipose tissue) were comparable among WT, Het, and Homo Pik3c3 hypomorphic mice, validating the comparison of Pik3c3 expression levels as well as the equal loading of total protein. Shown are representative blots from n = 6 mice/genotype group with similar results.

Article Snippet: Antibodies against Pik3c3 (Z-R016) were purchased from Echelon Biosciences (Salt Lake City, UT).

Techniques: Expressing, Western Blot

Journal: American Journal of Physiology - Renal Physiology

Article Title: The expression level of class III phosphatidylinositol-3 kinase controls the degree of compensatory nephron hypertrophy

doi: 10.1152/ajprenal.00381.2019

Figure Lengend Snippet: The reduction of Pik3c3 expression in Pik3c3 hypomorphic mice did not cause any apparent phenotype. Pik3c3Hypo/Hypo mice had a mean body weight (A), left kidney weight (B), right kidney weight (C), and left and right kidney-to-body weight ratio (D) that were not statistically different from those of Pik3c3Hypo/WT and Pik3c3WT/WT littermates. Values are means ± SE; n = 7 mice for each group. Furthermore, hematoxylin and eosin staining revealed no difference in renal histology among Pik3c3Hypo/Hypo, Pik3c3Hypo/WT, and Pik3c3WT/WT mice (E). Shown are representative images from n = 6 for each group with similar results.

Article Snippet: Antibodies against Pik3c3 (Z-R016) were purchased from Echelon Biosciences (Salt Lake City, UT).

Techniques: Expressing, Staining

Journal: American Journal of Physiology - Renal Physiology

Article Title: The expression level of class III phosphatidylinositol-3 kinase controls the degree of compensatory nephron hypertrophy

doi: 10.1152/ajprenal.00381.2019

Figure Lengend Snippet: Body weight, left kidney weight, left kidney-to-body weight ratio, BUN, and serum creatinine levels in Pik3c3 WT/WT , Pik3c3 Hypo/WT , and Pik3c3 Hypo/Hypo mice 7 days after sham or uninephrectomy surgery

Article Snippet: Antibodies against Pik3c3 (Z-R016) were purchased from Echelon Biosciences (Salt Lake City, UT).

Techniques:

Journal: American Journal of Physiology - Renal Physiology

Article Title: The expression level of class III phosphatidylinositol-3 kinase controls the degree of compensatory nephron hypertrophy

doi: 10.1152/ajprenal.00381.2019

Figure Lengend Snippet: The enlargement of proximal tubules and glomeruli induced by unilateral nephrectomy (UNX) was inhibited in Pik3c3 hypomorphic (Hypo) mice. UNX-induced renal glomerular hypertrophy and proximal tubular hypertrophy were assessed by double immunofluorescence staining with synaptopodin to highlight glomeruli in red and Lotus tetragonolobus lectin (LTL) to label renal proximal tubules in green (A). The area of proximal tubules (B) and area of glomeruli (C) were measured as detailed in methods (n = 7 mice/group). Values are means ± SE. P values for the compared groups are indicated. N.S., not statistically significant; WT, wild type.

Article Snippet: Antibodies against Pik3c3 (Z-R016) were purchased from Echelon Biosciences (Salt Lake City, UT).

Techniques: Double Immunofluorescence Staining

Journal: American Journal of Physiology - Renal Physiology

Article Title: The expression level of class III phosphatidylinositol-3 kinase controls the degree of compensatory nephron hypertrophy

doi: 10.1152/ajprenal.00381.2019

Figure Lengend Snippet: The reduction of Pik3c3 expression in Pik3c3 hypomorphic mice inhibits the mammalian target of rapamycin complex 1 signaling pathway. Immunoblot analysis of renal cortical homogenates revealed a graded reduction of unilateral nephrectomy (UNX)-induced ribosomal protein S6 (rpS6) phosphorylation in Pik3c3Hypo/WT and Pik3c3Hypo/Hypo mice compared with Pik3c3WT/WT mice (A). Each lane represents one sample from the left kidney of a right uninephrectomized or sham-operated individual mouse. Immunohistochemistry (IHC) staining confirmed the graded inhibition of UNX-induced rpS6 phosphorylation in Pik3c3Hypo/WT and Pik3c3Hypo/Hypo mice compared with Pik3c3WT/WT mice (B). Shown are representative blots and images from n = 7 mice/group with similar results.

Article Snippet: Antibodies against Pik3c3 (Z-R016) were purchased from Echelon Biosciences (Salt Lake City, UT).

Techniques: Expressing, Western Blot, Immunohistochemistry, Inhibition

Journal: American Journal of Physiology - Renal Physiology

Article Title: The expression level of class III phosphatidylinositol-3 kinase controls the degree of compensatory nephron hypertrophy

doi: 10.1152/ajprenal.00381.2019

Figure Lengend Snippet: A schematic summary illustrating the main concept of the present work demonstrating that Pik3c3 plays a pivotal role in mediating nephron deficit-induced compensatory nephron hypertrophy (CNH). Specifically, using a mouse model of uninephrectomy (UNX) to induce a 50% nephron reduction, the present study provides the first definitive evidence showing that Pikc3c plays an indispensable role in regulating the degree of CNH by generating Pik3c3 hypomorphic mouse model. Our hypertrophy data indicate that the degree of UNX-induced CNH is tightly regulated by the expression level of Pik3c3. Together with our previous demonstration that phosphorylated ribosomal protein S6 (rpS6) is a downstream effector of the mammalian target of rapamycin complex 1 (mTORC1)-S6 kinase 1 (S6K1) signaling pathway mediating CNH (53), our signaling data examining the level of rpS6 phosphorylation in the present study indicate that Pik3c3 functions upstream of the mTORC1-S6K1-rpS6 pathway mediating increased protein synthesis to drive the development of CNH.

Article Snippet: Antibodies against Pik3c3 (Z-R016) were purchased from Echelon Biosciences (Salt Lake City, UT).

Techniques: Expressing

Journal: Scientific Reports

Article Title: Renal developmental genes are differentially regulated after unilateral ureteral obstruction in neonatal and adult mice

doi: 10.1038/s41598-020-76328-3

Figure Lengend Snippet: Phosphatidylinositol 3 kinase class III (Pik3c3) expression is upregulated in intact opposite kidneys. Neonatal and adult mice were subjected to UUO or sham operation. Whole kidneys were processed for western blot analysis at day 1, 5, 12, and 19 after surgery. Pik3c3 expression, which controls compensatory hypertrophy, was strongly upregulated in adult intact opposite kidneys ( a and b ) and less in neonatal IO-kidneys ( a and c ). The shown western blot images are cropped, for uncropped western blots see Supplementary Fig. – online. For additional blots on Pik3c3 expression in UUO and sham-operated kidneys see Supplementary Figure . For details on significance between groups see Supplementary Table online. n = 3; * p < 0.05. Significance markings reflect the differences between groups, compared to each other on the same points in time. The images for ( a ) were rearranged for uniform presentation. The brightness of the western blot images was changed after the analysis for uniform presentation.

Article Snippet: Gdnf antibody (Abcam plc, Cambridge, UK, ab 17732; 1:1000), BMP4 antibody (clone V9, Sigma, Germany, V-6630; 1:200), Pik3c3 antibody (Echelon Biosciences, Z-R016, 1:100) were used for western blot analysis.

Techniques: Expressing, Western Blot