Journal: Biochemical Journal

Article Title: Mitochondrion-associated protein LRPPRC suppresses the initiation of basal levels of autophagy via enhancing Bcl-2 stability

doi: 10.1042/BJ20130306

Figure Lengend Snippet: ( A ) Immunostaining analysis showing the co-localization of antibody-stained LRPPRC (green) with MitoTracker-labelled mitochondria (red) in HeLa cells. ( B ) Plot of intensities of LRPPRC (green) and mitochondrial signals (red) presented in ( A ). ( C ) Analysis of the impact of LRPPRC depletion on the mitochondrial potentials labelled with MitoTracker (red) in HeLa cells treated with mock or LRPPRC siRNA for 72 h. LRPPRC were labelled with anti-LRPPRC antibody (green). ( D ) Comparison of MitoTracker intensities between two neighbouring cells with (right) and without (left) LRPPRC suppressed. ( E ) Comparison of cytochrome c signals (red) between cells retaining normal levels of LRPPRC (*, green) and cells with LRPPRC completely silenced ( ∧ ). ( F ) Immunoblot (IB) of lysates from HeLa cells treated without siRNA (Mock) or with siRNA specifically targeting LRPPRC (LRPPRC). Molecular masses are indicated in kDa. Scale bars in ( A ) and ( C )–( E ), 10 μm.

Article Snippet: The IgG control antibodies from mouse (sc-2025) and rabbit (sc-2027), primary antibodies against β-actin (sc-47778), β-tubulin (sc-9104), cytochrome c (sc-7159), LRPPRC (mouse, sc-166178), ATG5 (sc-33210), LAMP1 (L1418), p27 (sc-528), Beclin 1 (sc-11427) and GFP (sc-8334), siRNA molecules specific to LRPPRC (sc-44734), p27 (sc-29429) and ATG5 (sc-41445), and random sequence control (sc-44234) were from Santa Cruz Biotechnology.

Techniques: Immunostaining, Staining, Comparison, Western Blot

Journal: Biochemical Journal

Article Title: Mitochondrion-associated protein LRPPRC suppresses the initiation of basal levels of autophagy via enhancing Bcl-2 stability

doi: 10.1042/BJ20130306

Figure Lengend Snippet: ( A ) Immunostaining of HeLa cells stably expressing GFP–LC3 treated with random siRNA (Mock) or LRPPRC-specific siRNA (LRPPRC) for 72 h in the absence (Ctrl) or presence of lysosomal inhibitor NH 4 Cl (20 mM overnight before harvest). ( B ) Enlarged views of GFP–LC3 punctate foci under similar treatments as shown in ( A ). ( C ) Quantification of GFP–LC3-labelled autophagosomes. The total area occupied by GFP–LC3 punctate foci is the mean±S.D. for ten randomly selected images in a field of 512 pixels×512 pixels. The significance of differences was determined by Student's t test. ( D ) Immunoblot (IB) of lysates from HeLa cells stably expressing GFP–LC3 similarly treated with random siRNA (Mock) or LRPPRC-specific siRNA (LRPPRC) in the absence (Ctrl) or presence of lysosomal inhibitor NH 4 Cl. Molecular masses are indicated in kDa.

Article Snippet: The IgG control antibodies from mouse (sc-2025) and rabbit (sc-2027), primary antibodies against β-actin (sc-47778), β-tubulin (sc-9104), cytochrome c (sc-7159), LRPPRC (mouse, sc-166178), ATG5 (sc-33210), LAMP1 (L1418), p27 (sc-528), Beclin 1 (sc-11427) and GFP (sc-8334), siRNA molecules specific to LRPPRC (sc-44734), p27 (sc-29429) and ATG5 (sc-41445), and random sequence control (sc-44234) were from Santa Cruz Biotechnology.

Techniques: Immunostaining, Stable Transfection, Expressing, Western Blot

Journal: Biochemical Journal

Article Title: Mitochondrion-associated protein LRPPRC suppresses the initiation of basal levels of autophagy via enhancing Bcl-2 stability

doi: 10.1042/BJ20130306

Figure Lengend Snippet: ( A ) Immunoblot analyses of LC3 isoforms in lysates from HeLa cells treated with random siRNA (Mock) or LRPPRC-specific siRNA (LRPPRC) in the absence (Ctrl) or presence of lysosomal inhibitor NH 4 Cl. Molecular masses are indicated in kDa. ( B ) Plots of relative intensities of LC3-I and LC3-II bands. The LC3-I and LC3-II intensities in samples treated with mock siRNA were set to 1. Results are means±S.D. of at least three repeats and the differences were compared using a paired Student's t test. * P ≤0.05. ( C ) Transmission electron microscopy imaging of HeLa cells treated with random siRNA (Mock) or LRPPRC-specific siRNA (LRPPRC) in the absence (Ctrl) or presence (BAF) of lysosomal inhibitor bafilomycin A1. *, autophagy vacuoles. ( D ) Plot of percentages of area occupied by autophagy vacuoles in the transmission electron microscopy images. Results are means±S.D. of at least three repeats and the differences were compared using Student's t test. * P ≤0.05. ( E ) Immunoblot (IB) analyses of p62 levels in lysates from HeLa cells treated with random siRNA (Mock) or LRPPRC-specific siRNA (LRPPRC) in the absence (Ctrl) or presence (BAF) of bafilomycin A1. ( F ) Plots of relative intensities of p62. The p62 intensities in samples treated with mock siRNA were set to 1. Results are means±S.D. of at least three repeats and the differences were compared using a paired Student's t test. * P ≤0.05. ( G ) Immunostaining analysis of p62 levels in HeLa cells treated with random siRNA (Mock) or LRPPRC-specific siRNA (LRPPRC) in the absence (Ctrl) or presence (BAF) of bafilomycin A1.

Article Snippet: The IgG control antibodies from mouse (sc-2025) and rabbit (sc-2027), primary antibodies against β-actin (sc-47778), β-tubulin (sc-9104), cytochrome c (sc-7159), LRPPRC (mouse, sc-166178), ATG5 (sc-33210), LAMP1 (L1418), p27 (sc-528), Beclin 1 (sc-11427) and GFP (sc-8334), siRNA molecules specific to LRPPRC (sc-44734), p27 (sc-29429) and ATG5 (sc-41445), and random sequence control (sc-44234) were from Santa Cruz Biotechnology.

Techniques: Western Blot, Transmission Assay, Electron Microscopy, Imaging, Immunostaining

Journal: Biochemical Journal

Article Title: Mitochondrion-associated protein LRPPRC suppresses the initiation of basal levels of autophagy via enhancing Bcl-2 stability

doi: 10.1042/BJ20130306

Figure Lengend Snippet: ( A ) Fluorescence imaging analysis of the co-localization of GFP–LC3 with the mitochondrial marker Tom20. HeLa cells stably expressing GFP–LC3 treated with random siRNA (Mock) or LRPPRC siRNA (LRPPRC) for 72 h and with (BAF) or without (Ctrl) bafilomycin A1 in the last 12 h. ( B ) Plots of ratio (percentages) of GFP–LC3-associated Tom20 to total Tom20 (upper panel) or Tom20-associated GFP–LC3 to total GFP–LC3 (lower panel) with representative images shown in ( A ). Results are means±S.D. of at least three repeats and the differences were compared using Student's t test. * P ≤0.05. ( C ) Immunostaining analysis of Tom20 levels in HeLa cells treated with random siRNA (Mock) or LRPPRC-specific siRNA (LRPPRC) in the absence (Ctrl) or presence (BAF) of bafilomycin A1. ( D ) Immunoblot (IB) analyses of Tom20 levels in lysates from HeLa cells treated with LRPPRC siRNA for 72 h and bafilomycin A1 (BAF) in the last 12 h. ( E ) Plots of relative intensities of Tom20 bands with representative images shown in ( D ). Results are means±S.D. of at least three repeats and the differences were compared using a paired Student's t test. * P ≤0.05. ( F ) Fluorescence imaging analysis showing the co-localization of Tom20 with lysosomal marker LAMP1. HeLa cells treated with random siRNA (Mock) or LRPPRC siRNA (LRPPRC) for 72 h and with (BAF) or without (Ctrl) bafilomycin A1 in the last 12 h. Scale bars, 10 μm. ( G ) Plots of ratio (percentages) of LAMP1-associated Tom20 to total Tom20 with representative images shown in ( F ). Results are means±S.D. of at least three repeats and the differences were compared using Student's t test. * P ≤0.05. ( H ) Fluorescence imaging analysis showing the co-localization of Tom20 with lysosomal marker LAMP2. HeLa cells treated with random siRNA (Mock) or LRPPRC siRNA (LRPPRC) for 72 h and with (BAF) or without (Ctrl) bafilomycin A1 in the last 12 h. ( I ) Plots of ratio (percentages) of LAMP2-associated Tom20 to total Tom20 with representative images shown in ( H ). Results are means±S.D. of at least three repeats and the differences were compared using Student's t test. * P ≤0.05.

Article Snippet: The IgG control antibodies from mouse (sc-2025) and rabbit (sc-2027), primary antibodies against β-actin (sc-47778), β-tubulin (sc-9104), cytochrome c (sc-7159), LRPPRC (mouse, sc-166178), ATG5 (sc-33210), LAMP1 (L1418), p27 (sc-528), Beclin 1 (sc-11427) and GFP (sc-8334), siRNA molecules specific to LRPPRC (sc-44734), p27 (sc-29429) and ATG5 (sc-41445), and random sequence control (sc-44234) were from Santa Cruz Biotechnology.

Techniques: Fluorescence, Imaging, Marker, Stable Transfection, Expressing, Immunostaining, Western Blot

Journal: Biochemical Journal

Article Title: Mitochondrion-associated protein LRPPRC suppresses the initiation of basal levels of autophagy via enhancing Bcl-2 stability

doi: 10.1042/BJ20130306

Figure Lengend Snippet: ( A ) Immunoblot analyses showing the impact of LRPPRC on the levels of p27 and ATG5. HeLa cells were treated with siRNA specific to LRPPRC or p27 either individually or in combination for 72 h in the absence (Ctrl) or presence (BAF) of bafilomycin A1 (10 nM overnight overnight before harvest). ( B ) Immunoblot analyses showing the impact of p27 and ATG5 on LRPPRC-suppressed autophagy initiation. HeLa cells were treated with siRNA specific to p27, ATG5 and/or LRPPRC individually or in combination for 72 h in the absence (Ctrl) or presence (BAF) of bafilomycin A1 (10 nM overnight overnight before harvest). Molecular masses are indicated in kDa.

Article Snippet: The IgG control antibodies from mouse (sc-2025) and rabbit (sc-2027), primary antibodies against β-actin (sc-47778), β-tubulin (sc-9104), cytochrome c (sc-7159), LRPPRC (mouse, sc-166178), ATG5 (sc-33210), LAMP1 (L1418), p27 (sc-528), Beclin 1 (sc-11427) and GFP (sc-8334), siRNA molecules specific to LRPPRC (sc-44734), p27 (sc-29429) and ATG5 (sc-41445), and random sequence control (sc-44234) were from Santa Cruz Biotechnology.

Techniques: Western Blot

Journal: Biochemical Journal

Article Title: Mitochondrion-associated protein LRPPRC suppresses the initiation of basal levels of autophagy via enhancing Bcl-2 stability

doi: 10.1042/BJ20130306

Figure Lengend Snippet: ( A ) Immunoblot (IB) analyses of lysates prepared from HeLa or HEK-293T cells treated with mock or LRPPRC siRNA for 72 h showing the impact of LRPPRC depletion on levels of proteins controlling autophagy initiation through the PI3K/Akt/mTOR pathway. Molecular masses are indicated in kDa. ( B ) Plots of relative intensities of Beclin 1, PI3KCIII and Bcl-2 bands as shown in ( A ). The intensities in samples treated with mock siRNA were set to 1. Results are means±S.D. of at least three repeats and the differences were compared using a paired Student's t test. * P ≤0.05. ( C ) Immunoblot (IB) analyses of lysates prepared from COS7 cells overexpressing LRPPRC showing the impact of LRPPRC overexpression on levels of proteins controlling autophagy initiation through the PI3K/Akt/mTOR pathway. Expression levels of LRPPRC were confirmed by immunoblot with antibodies against LRPPRC (top panel) or GFP (second panel). Molecular masses are indicated in kDa. ( D ) Plots of relative intensities of Beclin 1, PI3KCIII and Bcl-2 bands in COS7 cells overexpressing LRPPRC as shown in ( C ). The intensities in cells overexpressing GFP were set to 1. Results are means±S.D. of at least three repeats and the differences were compared using a paired Student's t test. * P ≤0.05.

Article Snippet: The IgG control antibodies from mouse (sc-2025) and rabbit (sc-2027), primary antibodies against β-actin (sc-47778), β-tubulin (sc-9104), cytochrome c (sc-7159), LRPPRC (mouse, sc-166178), ATG5 (sc-33210), LAMP1 (L1418), p27 (sc-528), Beclin 1 (sc-11427) and GFP (sc-8334), siRNA molecules specific to LRPPRC (sc-44734), p27 (sc-29429) and ATG5 (sc-41445), and random sequence control (sc-44234) were from Santa Cruz Biotechnology.

Techniques: Western Blot, Over Expression, Expressing

Journal: Biochemical Journal

Article Title: Mitochondrion-associated protein LRPPRC suppresses the initiation of basal levels of autophagy via enhancing Bcl-2 stability

doi: 10.1042/BJ20130306

Figure Lengend Snippet: ( A ) Representative result of co-immunoprecipitation analyses of the LRPPRC–Beclin 1 interaction. The same amount of HeLa cell lysates was used to perform immunoprecipitation with the same amount of anti-LRPPRC antibody or mouse IgG control. ( B ) Representative result of co-immunoprecipitation analyses of the LRPPRC–Bcl-2 interaction. The same amount of HeLa cell lysates was used to perform immunoprecipitation with the same amount of anti-LRPPRC antibody or mouse IgG control. ( C ) Representative result of co-immunoprecipitation analyses of the interaction of Bcl-2 with LRPPRC and Beclin 1. The same amount of HeLa cell lysates was used to perform immunoprecipitation with the same amount of anti-Bcl-2 antibody or mouse IgG control. ( D ) Representative result of co-immunoprecipitation analyses of interaction of LRPPRC with PI3KCIII. The same amount of HeLa cell lysates were used to perform immunoprecipitation with the same amount of anti-LRPPRC antibody or mouse IgG control. ( E and F ) Co-immunoprecipitation analyses of the impact of LRPPRC depletion on Beclin 1–Bcl-2 and Beclin 1–PI3KCIII interactions. Lysates containing equal amounts of total proteins prepared from HeLa ( E ) or HEK-293T cells ( F ) treated with mock or LRPPRC siRNA were immunoprecipitated with anti-Beclin 1 or control IgG antibody and the precipitates were immunoblotted with antibodies against Beclin 1, Bcl-2 and PI3KCIII. ( A )–( F ) IB, immunoblot; IP, immunoprecipitation. Molecular masses are indicated in kDa. ( G ) Impact of LRPPRC depletion on the interaction of Beclin 1 with Bcl-2 or PI3KCIII.

Article Snippet: The IgG control antibodies from mouse (sc-2025) and rabbit (sc-2027), primary antibodies against β-actin (sc-47778), β-tubulin (sc-9104), cytochrome c (sc-7159), LRPPRC (mouse, sc-166178), ATG5 (sc-33210), LAMP1 (L1418), p27 (sc-528), Beclin 1 (sc-11427) and GFP (sc-8334), siRNA molecules specific to LRPPRC (sc-44734), p27 (sc-29429) and ATG5 (sc-41445), and random sequence control (sc-44234) were from Santa Cruz Biotechnology.

Techniques: Immunoprecipitation, Control, Western Blot

Journal: Current Opinion in Pharmacology

Article Title: Drugging PI3K in cancer: refining targets and therapeutic strategies

doi: 10.1016/j.coph.2015.05.016

Figure Lengend Snippet: The PI3K pathway with respective PI3K inhibitors. When PI3K is activated, phosphatidylinositol 3,4,5-trisphosphate (PIP3) is generated from phosphatidylinositol 3,4-bisphosphate (PIP2), and recruits AKT to the cell membrane [ , ]. This leads to a conformational change and phosphorylation of AKT and its subsequent activation. AKT then translocates to the cytoplasm and nucleus, where phosphorylation of various downstream substrates involved in the regulation of multiple cellular functions, including proliferation, survival and growth occurs. The PI3K pathway is one of the most frequently activated signalling pathways in human cancers, affecting 30–50% of tumours, making it a rational target for novel anticancer drug development. The red arrows indicate the respective mechanisms of action of different PI3K inhibitors, which include the dual PI3K/mTOR inhibitors, pan-Class I PI3K inhibitors and isoform-selective PI3K inhibitors. Individual examples of different PI3K inhibitors in clinical testing are shown in the figure. The table in the figure lists the regulatory and catalytic subunits of the respective PI3K classes.

Article Snippet: A major step forward in recent years has been the progression of over 30 small molecule PI3K inhibitors into clinical trials and the first regulatory approval of one such agent, idelalisib (Zydelig, CAL-101; Gilead Sciences) (see for representative chemical structures) [ , , ].

Techniques: Generated, Activation Assay

Journal: Current Opinion in Pharmacology

Article Title: Drugging PI3K in cancer: refining targets and therapeutic strategies

doi: 10.1016/j.coph.2015.05.016

Figure Lengend Snippet: Chemical structures of PI3K inhibitors highlighted in this article.

Article Snippet: A major step forward in recent years has been the progression of over 30 small molecule PI3K inhibitors into clinical trials and the first regulatory approval of one such agent, idelalisib (Zydelig, CAL-101; Gilead Sciences) (see for representative chemical structures) [ , , ].

Techniques: