Journal: Translational Neurodegeneration

Article Title: Critical role of ROCK1 in AD pathogenesis via controlling lysosomal biogenesis and acidification

doi: 10.1186/s40035-024-00442-9

Figure Lengend Snippet: ROCK1 decreases lysosomal biogenesis and impairs lysosomal acid environment. a Schematic illustration of the experimental procedure. Image created with BioRender.com. b Heatmap of mRNA expression of ROCK1 and lysosome-related markers detected with qRT-PCR in HEK-293T cells transfected with control or ROCK1-overexpression plasmid for 48 h. n = 3. Red color, higher expression; blue color, lower expression. c Immunoblotting of lysosome-related markers in HEK-293T cells and quantitation of their protein levels. n = 3. d Transmission electron microscopy of lysosomes in HEK-293T cells. The number of lysosomes was analyzed. n = 10 cells per group. Scale bar, 2 μm. White arrows indicate lysosomes. e Lysotracker Red, Magic Red B and DQ BSA staining in HEK-293T cells. Fluorescence intensities were analyzed. n = 5. Scale bars, 25 μm. f Standard curve of LysoSensor to determine the lysosomal pH values of HEK-293T cells. g Lysosomal pH of HEK-293T cells was determined by LysoSensor. n = 5. h Schematic illustration of the experimental procedure. Image created with BioRender.com. i AAV-Vector and AAV-ROCK1 were successfully microinjected into the hippocampus of WT mice, respectively. GFP (green) was used to visualize viral diffusion. Scale bar, 200 μm. j qRT-PCR analysis of expression levels of indicated mRNAs. n = 3. k Transmission electron microscopy of lysosomes in the hippocampus. The number of lysosomes was analyzed. n = 9 slices from 3 mice. Scale bars, 1 μm. * P < 0.05, ** P < 0.01, *** P < 0.001 versus vector ( b - e , g ) or AAV-Vector group ( j , k ), Student’s t -test

Article Snippet: The AAV carrying either control shRNA with GFP (AAV-sh-Con) (4 µl, 5.8 × 10 12 viral genomes/µl, Sangon Biotech, Shanghai, China) or the ROCK1 shRNA with GFP (AAV-sh-ROCK1) (4 µl, 4.4 × 10 12 viral genomes/µl, Sangon Biotech) was microinjected into the hippocampus of mice using the following microinjection coordinates: anteroposterior − 2.06 mm, lateral ± 1.5 mm, and ventral + 2.0 mm (stereotaxic coordinates from bregma).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Control, Over Expression, Plasmid Preparation, Western Blot, Quantitation Assay, Transmission Assay, Electron Microscopy, Staining, Fluorescence, Diffusion-based Assay

Journal: Translational Neurodegeneration

Article Title: Critical role of ROCK1 in AD pathogenesis via controlling lysosomal biogenesis and acidification

doi: 10.1186/s40035-024-00442-9

Figure Lengend Snippet: Downregulation of ROCK1 promotes TFEB nuclear translocation and lysosomal biogenesis in the brains of APP/PS1 mice. a Timeline of experimental procedure and confocal images of brain sections microinjected with adeno-associated virus carrying control shRNA (AAV-sh-Con) or ROCK1 shRNA (AAV-sh-ROCK1). GFP (green) was used to visualize viral diffusion. Scale bar, 200 μm. b PLA and quantification of ROCK1 and TFEB in the hippocampal CA3 region of APP/PS1 mice microinjected with AAV-sh-Con/AAV-sh-ROCK1. n = 3. Scale bar, 50 μm. c Immunoblotting analysis and quantification of indicated proteins in the hippocampus of APP/PS1 mice microinjected with AAV-sh-Con/AAV-sh-ROCK1. n = 3. d Immunoblotting of cytoplasmic and nuclear TFEB. n = 3. e Transmission electron microscopy of lysosomes in the hippocampus. Number of lysosomes was analyzed. n = 9 slices from 3 mice. Scale bar, 2 μm. White arrows indicate lysosomes. f , g Immunostaining of Aβ (red) and CD68 (green) in the hippocampus of APP/PS1 mice microinjected with AAV-sh-Con/AAV-sh-ROCK1, and co-localization coefficient for Aβ and CD68. n = 3. Scale bar, 50 μm. h qRT-PCR analysis of expression of indicated mRNAs. n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001 versus APP/PS1-AAV-sh-Con group, Student’s t -test

Article Snippet: The AAV carrying either control shRNA with GFP (AAV-sh-Con) (4 µl, 5.8 × 10 12 viral genomes/µl, Sangon Biotech, Shanghai, China) or the ROCK1 shRNA with GFP (AAV-sh-ROCK1) (4 µl, 4.4 × 10 12 viral genomes/µl, Sangon Biotech) was microinjected into the hippocampus of mice using the following microinjection coordinates: anteroposterior − 2.06 mm, lateral ± 1.5 mm, and ventral + 2.0 mm (stereotaxic coordinates from bregma).

Techniques: Translocation Assay, Virus, Control, shRNA, Diffusion-based Assay, Western Blot, Transmission Assay, Electron Microscopy, Immunostaining, Quantitative RT-PCR, Expressing

Journal: Cell Death & Disease

Article Title: Acquisition of meiotic DNA repair regulators maintain genome stability in glioblastoma

doi: 10.1038/cddis.2015.75

Figure Lengend Snippet: GBM cells express components of the meiotic HR machinery. ( a and b ) Oncomine analysis of the Sun database demonstrates elevated ( a ) HOP2 ( P =<0.0001) and ( b ) MND1 ( P =0.0005) mRNA expression in GBM compared with normal brain ( n =23 normal brain, n =81 GBM; analyzed with unpaired t -test). ( c and d ) Oncomine analysis of the Phillips database indicates that elevated ( c ) HOP2 (P=0.0201) and ( d ) MND1 (P=0.0023) mRNA expression correlates with increased glioma tumor grade ( n =23 astrocytomas, n =76 GBM; analyzed with unpaired t -test). ( e and f ) Analysis of the Phillips data set available through Oncomine indicates a significant correlation between high ( e ) HOP2 expression ( n =35 HOP2 low, n =36 HOP2 high; P =0.002 with log-rank analysis), and ( f ) high MND1 expression ( n =35 MND1 low, n =36 MND1 high; P <0.0001 with log-rank analysis) and poor survival. ( g ) DMC1 expression was analyzed by immunoblot analysis in three non-neoplastic brain (NM32, NM33 and NM53) and four GBM cell lines (U87, LN229, T98 and D54)

Article Snippet: For intracranial implantation studies, GFP-Luciferase-shRNA (control or shDMC1) expressing U87 cells were implanted into the right frontal lobes of immunocompromised NSG (NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ) mice (Charles River Laboratories, Wilmington, MA, USA).

Techniques: Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: Acquisition of meiotic DNA repair regulators maintain genome stability in glioblastoma

doi: 10.1038/cddis.2015.75

Figure Lengend Snippet: DMC1 depletion inhibits proliferation of GBM cells with minimal effects on non-neoplastic brain cells. ( a and b ) U87 ( a ) and LN229 ( b ) cells were transduced with lentivirus expressing either control shRNA (shControl-black) or DMC1-directed shRNA sh1068 (red) and sh826 (blue) and knockdown efficiency was measured by immunoblot analysis. RAD51 protein expression was evaluated in response to DMC1 depletion by immunoblot analysis. Proliferation changes in response to DMC1 depletion was measured in transduced U87 ( c ), LN229 ( d ), by pulsing cells for 4 h with radiolabeled thymidine at the indicated times post-transduction. ( e and f ) NM32 ( e ) and NM53 ( f ) cells were transduced with lentivirus expressing either control shRNA (shControl-black) or DMC1-directed shRNA sh1068 (red) and sh826 (blue) and knockdown efficiency was measured by immunoblot analysis. RAD51 protein expression was evaluated in response to DMC1 depletion by immunoblot analysis. Proliferation changes in response to DMC1 depletion was measured in transduced NM32 ( g ) and NM53 ( h ), by pulsing cells for 4 h with radiolabeled thymidine at the indicated time points. n =3, errors bars represent S.D.; **** P <0.0001 with ANOVA

Article Snippet: For intracranial implantation studies, GFP-Luciferase-shRNA (control or shDMC1) expressing U87 cells were implanted into the right frontal lobes of immunocompromised NSG (NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ) mice (Charles River Laboratories, Wilmington, MA, USA).

Techniques: Transduction, Expressing, Control, shRNA, Knockdown, Western Blot

Journal: Cell Death & Disease

Article Title: Acquisition of meiotic DNA repair regulators maintain genome stability in glioblastoma

doi: 10.1038/cddis.2015.75

Figure Lengend Snippet: DMC1 depletion reduces clonogenic survival cells and alters the cell cycle profile of GBM cells. ( a – d ) DMC1-depleted U87 ( a and b ) and LN229 ( c and d ) cells were plated at low density and stained with crystal violet for evaluation of clonogenic survival. Representative images ( a and c ) are shown and the data quantified ( b and d ) by counting the number of colonies formed. ( e – h ) DMC1-depleted U87 ( e and g ) and LN229 ( f and h ) cells were fixed, stained with PI, and cell cycle profiles evaluated using flow cytometric analysis. Percentage of S-phase ( e and f ) and aneuploid cells ( g and h ) are shown. n =3, error bars represent S.D.; NS, no significance; * P <0.05; ** P <0.01; **** P <0.0001 with ANOVA

Article Snippet: For intracranial implantation studies, GFP-Luciferase-shRNA (control or shDMC1) expressing U87 cells were implanted into the right frontal lobes of immunocompromised NSG (NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ) mice (Charles River Laboratories, Wilmington, MA, USA).

Techniques: Staining

Journal: Cell Death & Disease

Article Title: Acquisition of meiotic DNA repair regulators maintain genome stability in glioblastoma

doi: 10.1038/cddis.2015.75

Figure Lengend Snippet: Depletion of DMC1 induces replication stress. ( a and b ) Protein expression of phosphorylated CHK1 (S345) and total CHK1 in U87 ( a ) and LN229 ( b ) DMC1-depleted cells was evaluated by immunoblot analysis. ( c and d ) Formation of nuclear RPA foci was monitored using immunostaining analysis for RPA (green; nuclei counterstained with DAPI, blue) in U87 ( c ) and LN229 ( d ) DMC1-depleted cells. ( e and f ) Quantification of RPA foci in U87 ( e ) and LN229 ( f ) was obtained through ScanR analysis. RPA foci signal was normalized to DAPI per cell. n =3, error bars represent S.D.; **** P <0.0001. Scale bar=10 μ m

Article Snippet: For intracranial implantation studies, GFP-Luciferase-shRNA (control or shDMC1) expressing U87 cells were implanted into the right frontal lobes of immunocompromised NSG (NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ) mice (Charles River Laboratories, Wilmington, MA, USA).

Techniques: Expressing, Western Blot, Immunostaining

Journal: Cell Death & Disease

Article Title: Acquisition of meiotic DNA repair regulators maintain genome stability in glioblastoma

doi: 10.1038/cddis.2015.75

Figure Lengend Snippet: DMC1 depletion radiosensitizes GBM cells to IR. ( a and b ) DMC1-depleted U87 ( a ) and LN229 ( b ) cells were plated at low density and evaluated for clonogenic survival in combination with increasing doses of radiation. Colonies were counted for each group and plotted on a log-transformed graph. ( c – h ) Combinatorial effects of DMC1 depletion and radiation on apoptosis and viability were evaluated by co-staining with Annexin V-FITC and DAPI, respectively. U87 ( c ) and LN229 ( f ) DMC1-depleted cells were exposed to 4 Gy IR and analyzed by flow cytometry 24 h post-radiation. U87 Annexin V ( d ) and DAPI ( e ) positive cell graphs. LN229 Annexin V ( g ) and DAPI ( h ) positive cell graphs. n =3, error bars represent S.D.; NS, no significance; * P <0.05; ** P <0.01; **** P <0.0001 with ANOVA

Article Snippet: For intracranial implantation studies, GFP-Luciferase-shRNA (control or shDMC1) expressing U87 cells were implanted into the right frontal lobes of immunocompromised NSG (NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ) mice (Charles River Laboratories, Wilmington, MA, USA).

Techniques: Transformation Assay, Staining, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Acquisition of meiotic DNA repair regulators maintain genome stability in glioblastoma

doi: 10.1038/cddis.2015.75

Figure Lengend Snippet: DMC1 depletion attenuates activation of the DDR. ( a and b ) Activation of the DDR was assessed by immunoblot analysis of phosphorylated CHK2 (T68), total CHK2, DMC1, p21 CIP1/WAF1 and γ -H2AX in U87 ( a ) and LN229 ( b ) DMC1-depleted cells exposed to 4 Gy radiation and harvested at the indicated time points. ( c – f ) DNA damage of baseline and radiation-treated DMC1-depleted cells was evaluated at the indicated time points following 4 Gy radiation with the alkaline comet assay in U87 ( c and d ) and LN229 ( e and f ) cells. Representative images ( c and e ) are shown. Quantification of comet tails for U87 ( d ) and LN229 ( f ) was done using the Casplab software and is represented as OTM. n =3, error bars represent S.D.; ** P <0.01; **** P <0.0001 with two-way ANOVA

Article Snippet: For intracranial implantation studies, GFP-Luciferase-shRNA (control or shDMC1) expressing U87 cells were implanted into the right frontal lobes of immunocompromised NSG (NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ) mice (Charles River Laboratories, Wilmington, MA, USA).

Techniques: Activation Assay, Western Blot, Alkaline Single Cell Gel Electrophoresis, Software

Journal: Cell Death & Disease

Article Title: Acquisition of meiotic DNA repair regulators maintain genome stability in glioblastoma

doi: 10.1038/cddis.2015.75

Figure Lengend Snippet: Depletion of DMC1 attenuates tumor growth in vivo . ( a and b ) GFP-Luciferase expressing U87 cells transduced with control shRNA or DMC1-directed shRNA were implanted intracranially in NSG mice, five mice per group ( a ) Mice were imaged in vivo using the IVIS Lumina at the indicated days post-implantation. ( b ) Relative luminescence values for all three groups were plotted and analyzed over time. Errors bars represent S.E.M.; * P <0.05; ** P <0.01 with ANOVA. ( c ) Mice were killed at first sign of neurological deficit and Kaplan–Meier survival curves plotted. **** P <0.0001 with log-rank test. ( d ) Table with medial survival in days for each experimental arm

Article Snippet: For intracranial implantation studies, GFP-Luciferase-shRNA (control or shDMC1) expressing U87 cells were implanted into the right frontal lobes of immunocompromised NSG (NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ) mice (Charles River Laboratories, Wilmington, MA, USA).

Techniques: In Vivo, Luciferase, Expressing, Transduction, Control, shRNA