Journal: Oncotarget

Article Title: Diagnostic and clinical relevance of the autophago-lysosomal network in human gliomas

doi: 10.18632/oncotarget.7910

Figure Lengend Snippet: ( A ) Schematic overview of the autophago-lysosomal pathway including modulators of the induction of autophagy as well as inhibitors of the autophagic flux used in Figure . ( B–D ) Immunohistochemistry with antibodies against (B) LC3B, (C) p62 and (D) LAMP2 in LNT-229 glioma cells treated with DMSO as control condition, the activators of autophagosome formation Torin1 (250 nM) or BafA1 (100 nM) as well as the combination of both Torin1 and BafA1 (treatment time 2 h in each condition). Negative controls of the immunocytochemical stainings are shown in the right upper corner of each picture. Scale bars: 50 μm. Corresponding immunoblots were performed from each treatment condition. ( E–G ) LNT-229 glioma cells were treated with Torin1 (2 h, 250 nM) and/or BafA1 (2 h, 100 nM), fixed and labeled with (E) anti-LC3B, (F) anti-p62 or (G) anti-LAMP2 antibodies, respectively. DRAQ5 was used to stain nuclei. Scale bars: 10 μm. Images were acquired with Opera High Content Screening System and antibody-labeled spots were automatically counted using Acapella software. Data represent mean of quadruplicates ± standard deviation, normalized to DMSO control.

Article Snippet: To analyze the cell culture derived lysates, the following antibodies were used: anti-human LC3B (mouse antibody, #2775, Cell Signaling), anti-human LC3B (rabbit antibody, PM036, MBL International), anti-human p62 (mouse antibody, M162-3, MBL International), anti-human PCNA (rabbit antibody, sc-7907, Santa Cruz), anti-human cleaved caspase 3 (rabbit antibody, #9665 Cell Signaling), anti-human CathepsinB (goat antibody, sc-6493, Santa Cruz), anti-human Actin (mouse antibody, A4700 Sigma Aldrich), anti-human LC3B (Figure – , rabbit antibody, L8918, Sigma Aldrich), anti-human p62 (Figure , mouse antibody, 610832, BD Transduction Laboratories), anti-human BAG3 (rabbit antibody, PAB0330, Biozol/Abnova), anti-human Beclin1 (mouse antibody, m612112, BD Pharmingen), anti-human LAMP2 (mouse antibody, ab25631, Abcam).

Techniques: Immunohistochemistry, Western Blot, Labeling, Staining, High Content Screening, Software, Standard Deviation

Journal: Oncotarget

Article Title: Diagnostic and clinical relevance of the autophago-lysosomal network in human gliomas

doi: 10.18632/oncotarget.7910

Figure Lengend Snippet: ( A ) Representative immunohistochemical staining against LC3B of normal appearing brain (NAB) tissue, pilocytic astrocytoma WHO grade I, diffuse astrocytoma WHO grade II, anaplastic astrocytoma WHO grade III and glioblastoma WHO grade IV. Scale bar: 50 μm. ( B ) Box blots showing LC3B scores (frequency × intensity) in 46 pilocytic astrocytomas WHO grade I (min: 0, max: 12, median: 1.5), 14 diffuse astrocytomas WHO grade II (min: 0, max: 6, median: 1), 33 anaplastic astrocytomas WHO grade III (min: 0, max: 8, median: 2) and 249 glioblastomas WHO grade IV (min: 0, max: 9, median: 1). ( C ) LC3B immunoblotting of normal grey matter, normal white matter and two astrocytomas of each WHO grade. ( D ) Relative LC3B mRNA levels of normal grey and white matter and two astrocytomas of each WHO grade normalized to normal white matter assessed by qPCR. ( E ) Representative immuno-histochemical staining against p62 of NAB, pilocytic astrocytoma WHO grade I, diffuse astrocytoma WHO grade II, anaplastic astrocytoma WHO grade III and glioblastoma WHO grade IV. Scale bar: 50 μm. ( F ) Box blots showing p62 scores (frequency × intensity) in 46 pilocytic astrocytomas WHO grade I (min: 0, max: 3, median: 0), 16 diffuse astrocytomas WHO grade II (min: 0, max: 3, median: 0), 34 anaplastic astrocytomas WHO grade III (min: 0, max: 3, median: 0) and 252 glioblastomas WHO grade IV (min: 0, max: 9, median: 1) ( G ) p62 immunoblotting of normal grey and white matter, and two astrocytomas of each WHO grade. ( H ) Relative p62 mRNA levels of normal and white matter and two astrocytomas of each WHO grade normalized to normal white matter assessed by qPCR.

Article Snippet: To analyze the cell culture derived lysates, the following antibodies were used: anti-human LC3B (mouse antibody, #2775, Cell Signaling), anti-human LC3B (rabbit antibody, PM036, MBL International), anti-human p62 (mouse antibody, M162-3, MBL International), anti-human PCNA (rabbit antibody, sc-7907, Santa Cruz), anti-human cleaved caspase 3 (rabbit antibody, #9665 Cell Signaling), anti-human CathepsinB (goat antibody, sc-6493, Santa Cruz), anti-human Actin (mouse antibody, A4700 Sigma Aldrich), anti-human LC3B (Figure – , rabbit antibody, L8918, Sigma Aldrich), anti-human p62 (Figure , mouse antibody, 610832, BD Transduction Laboratories), anti-human BAG3 (rabbit antibody, PAB0330, Biozol/Abnova), anti-human Beclin1 (mouse antibody, m612112, BD Pharmingen), anti-human LAMP2 (mouse antibody, ab25631, Abcam).

Techniques: Immunohistochemical staining, Staining, Western Blot

Journal: Oncotarget

Article Title: Diagnostic and clinical relevance of the autophago-lysosomal network in human gliomas

doi: 10.18632/oncotarget.7910

Figure Lengend Snippet: Overview about ( A ) LC3B, ( B ) p62, ( C ) LAMP2 and ( D ) CTSB immunohistochemistry in glioblastoma (N: necrosis, T: tumor center). (E–H) Double immunofluorescent staining against LC3B and ( E ) GFAP, ( F ) Iba1, ( G ) Glut1 as well as ( H ) cCasp3 in glioblastoma. ( I ) Overview of cCasp3 immunohistochemistry in glioblastoma. (A*, B*, C*, D* and I* are higher magnifications of A, B, C, D and I respectively; all scale bars: 50 μm). ( J ) Schematic overview of the border zone of necrotic foci with different nutrition levels in glioblastoma (arrows: apoptotic cell, *cells expressing autophagy-associated and lysosomal markers, N: necrosis).

Article Snippet: To analyze the cell culture derived lysates, the following antibodies were used: anti-human LC3B (mouse antibody, #2775, Cell Signaling), anti-human LC3B (rabbit antibody, PM036, MBL International), anti-human p62 (mouse antibody, M162-3, MBL International), anti-human PCNA (rabbit antibody, sc-7907, Santa Cruz), anti-human cleaved caspase 3 (rabbit antibody, #9665 Cell Signaling), anti-human CathepsinB (goat antibody, sc-6493, Santa Cruz), anti-human Actin (mouse antibody, A4700 Sigma Aldrich), anti-human LC3B (Figure – , rabbit antibody, L8918, Sigma Aldrich), anti-human p62 (Figure , mouse antibody, 610832, BD Transduction Laboratories), anti-human BAG3 (rabbit antibody, PAB0330, Biozol/Abnova), anti-human Beclin1 (mouse antibody, m612112, BD Pharmingen), anti-human LAMP2 (mouse antibody, ab25631, Abcam).

Techniques: Immunohistochemistry, Staining, Expressing

Journal: Oncotarget

Article Title: Diagnostic and clinical relevance of the autophago-lysosomal network in human gliomas

doi: 10.18632/oncotarget.7910

Figure Lengend Snippet: ( A–B ) LC3B immunohistochemistry of LNT-229 cells cultured in serum-free medium (A) with 25 mM glucose and (B) without glucose (scale bar: 50 μm). ( C ) Percentage of LC3B positive cells in immunocytochemistry of cell culture conditions with 0 (min: 2, max: 51, median: 20) and 25 mM glucose (min: 0, max: 13, median: 3) from 28 differently treated LNT-229 cytopellets. ( D ) LC3B punctae per 100 cells in immunocytochemistry of cell culture conditions with 0 (min: 2, max: 240, median: 100.5) and 25 mM glucose (min: 0, max: 17, median: 4.5) from 28 differently treated LNT-229 cytopellets. Different treatment options: see . (E, F) Correlation of Glut1 (1 = weak, 2 = moderate, 3 = strong) expression and ( E ) LC3B-positive cells or ( F ) LC3B-positive punctae per 100 cells in 28 differently treated LNT-229 cell culture experiments. ( G ) Immunoblotting of LNT-229 cells cultured in serum-free medium at different oxygen, glucose or amino acid (arginin, lysine, L-glutamine) levels.

Article Snippet: To analyze the cell culture derived lysates, the following antibodies were used: anti-human LC3B (mouse antibody, #2775, Cell Signaling), anti-human LC3B (rabbit antibody, PM036, MBL International), anti-human p62 (mouse antibody, M162-3, MBL International), anti-human PCNA (rabbit antibody, sc-7907, Santa Cruz), anti-human cleaved caspase 3 (rabbit antibody, #9665 Cell Signaling), anti-human CathepsinB (goat antibody, sc-6493, Santa Cruz), anti-human Actin (mouse antibody, A4700 Sigma Aldrich), anti-human LC3B (Figure – , rabbit antibody, L8918, Sigma Aldrich), anti-human p62 (Figure , mouse antibody, 610832, BD Transduction Laboratories), anti-human BAG3 (rabbit antibody, PAB0330, Biozol/Abnova), anti-human Beclin1 (mouse antibody, m612112, BD Pharmingen), anti-human LAMP2 (mouse antibody, ab25631, Abcam).

Techniques: Immunohistochemistry, Cell Culture, Immunocytochemistry, Expressing, Western Blot

Journal: Oncotarget

Article Title: Diagnostic and clinical relevance of the autophago-lysosomal network in human gliomas

doi: 10.18632/oncotarget.7910

Figure Lengend Snippet: ( A ) LC3B, ( B ) p62 and ( C ) cCasp3 immunocytochemistry of primary glioblastoma spheres (scale bars: 100 μm). ( D ) Correlation cCasp3-positive cells (in %) and p62 Score (frequency × intensity) in 28 differently treated LNT-229 cell culture experiments. ( E ) Correlation analysis of cCasp3-positive cells (in %) and absolute numbers of LC3B-positive punctae per cell in 28 differently treated LNT-229 cell culture experiments. ( F ) Correlation analysis of LC3B punctae per cell and p62 score (frequency × intensity) in 28 differently treated LNT-229 cell culture experiments. ( G ) Immunoblotting of LNT-229 cells treated with Torin2 (250 nM, 8 h), BafA1 (100 nM, 8 h), Epoxomicin (50 nM, 18 h) or TRAIL (250 ng/ml, 18 h). DMSO served as a control.

Article Snippet: To analyze the cell culture derived lysates, the following antibodies were used: anti-human LC3B (mouse antibody, #2775, Cell Signaling), anti-human LC3B (rabbit antibody, PM036, MBL International), anti-human p62 (mouse antibody, M162-3, MBL International), anti-human PCNA (rabbit antibody, sc-7907, Santa Cruz), anti-human cleaved caspase 3 (rabbit antibody, #9665 Cell Signaling), anti-human CathepsinB (goat antibody, sc-6493, Santa Cruz), anti-human Actin (mouse antibody, A4700 Sigma Aldrich), anti-human LC3B (Figure – , rabbit antibody, L8918, Sigma Aldrich), anti-human p62 (Figure , mouse antibody, 610832, BD Transduction Laboratories), anti-human BAG3 (rabbit antibody, PAB0330, Biozol/Abnova), anti-human Beclin1 (mouse antibody, m612112, BD Pharmingen), anti-human LAMP2 (mouse antibody, ab25631, Abcam).

Techniques: Immunocytochemistry, Cell Culture, Western Blot

Journal: bioRxiv

Article Title: Rapamycin induced autophagy enhances lipid breakdown and ameliorates lipotoxicity in Atlantic salmon cells

doi: 10.1101/2024.12.11.627915

Figure Lengend Snippet: A. Schematic overview of experiments performed in this study. B. LD count and size measurements in SHK-1 cells following 24, 48 and 72 hr treatments under basal, lipid overload, lipid overload with RAPA, and lipid overload with RAPA+MRT. C. Quantification of LD numbers at 48 hr (30 cells counted from 3 independent experiments). D. Quantification of LD size at 48 hr (30 cells counted from 3 independent experiments). E. Representative confocal images showing the colocalisation of LC3 (green puncta) with LDs stained with BOPIPY (red). Baf-A was added to all treatments to block the autophagic. Scale bar = 10μm, n=3. Data shown as mean +/− SD, ***p < 0.001.

Article Snippet: A standard immunoblotting protocol was used for rabbit polyclonal antibodies for LC3 (1:3000, PM036), SQSTM1/p62 (1:500, Ab 264313), ATGL (1:500, Proteintech, 55190-1-AP), and MGL (1:500, Proteintech, 20494-1-AP).

Techniques: Staining, Blocking Assay

Journal: bioRxiv

Article Title: Rapamycin induced autophagy enhances lipid breakdown and ameliorates lipotoxicity in Atlantic salmon cells

doi: 10.1101/2024.12.11.627915

Figure Lengend Snippet: A. Representative immunoblot and B. Immunoblot quantification showing LC3 abundance in SHK-1 cells after 72 hr treatment with no lipid overload, lipid overload, lipid overload with RAPA, and lipid overload with RAPA and MRT treatments, with and without Baf-A (n=3). C . Representative immunoblot and D. Immunoblot quantification showing SQSTM1/p62 abundance in SHK-1 cells after 72 hr treatment with no lipid overload, lipid overload, lipid overload with RAPA, and lipid overload with RAPA and MRT treatments. E. Total protein blot stained with simply blue safe stain for protein abundance quantification. Data shown as mean +/− SD, * p < 0.05; ** p < 0.01.

Article Snippet: A standard immunoblotting protocol was used for rabbit polyclonal antibodies for LC3 (1:3000, PM036), SQSTM1/p62 (1:500, Ab 264313), ATGL (1:500, Proteintech, 55190-1-AP), and MGL (1:500, Proteintech, 20494-1-AP).

Techniques: Western Blot, Staining, Quantitative Proteomics