Journal: Scientific Reports

Article Title: Selective targeting of KRAS oncogenic alleles by CRISPR/Cas9 inhibits proliferation of cancer cells

doi: 10.1038/s41598-018-30205-2

Figure Lengend Snippet: CRISPR/Cas9 targeting of KRAS c.35 G > T in SW620 and SW480 cells. ( a ) Indel frequencies in target KRAS region of SW620 and SW480 cells with sgKRAS-G12V, and in HEK293T cells with sgKRAS-WT. ( b ) Major indel patterns detected in the KRAS target region of SW620 cells with sgKRAS-G12V. Blue: sgRNA target sequence; bold blue: c.35 G > T single-nucleotide missense substitution; orange: PAM sequence; red: mismatched nucleotides. ( c ) Cell proliferation assays of indicated cell lines following transduction with lentiviral vector encoding Cas9, Cas9 and sgKRAS-G12V, or Cas9 and sgKRAS-WT. Red: sgRNA sample relevant to tested cell line. Bars represent the mean ± S.E.M. **** (P < 0.0001), ns: not significant.

Article Snippet: One day after seeding, the cells were transfected in GibcoTM Opti-MEMTM I Reduced Serum Medium (Life Technologies, USA) using 1 μg of target lentiviral vector (empty lentiCRISPR v2 or lentiCRISPR v2 containing sgKRAS-WT, sgKRAS-G12V, or sgKRAS-G12D), 0.25 μg of pMD2.G (Addgene, USA), 0.75 μg of psPAX2 (Addgene, USA) and 7 μl of LipofectamineTM 3000 (Life Technologies, USA).

Techniques: CRISPR, Sequencing, Transduction, Plasmid Preparation

Journal: Scientific Reports

Article Title: Selective targeting of KRAS oncogenic alleles by CRISPR/Cas9 inhibits proliferation of cancer cells

doi: 10.1038/s41598-018-30205-2

Figure Lengend Snippet: Indel ratios and transduced cell viability before and after puromycin selection. Indel frequencies in target KRAS region of AsPC-1 and SNU407 cells with sgKRAS-G12D were determined before ( a ) and after ( c ) antibiotic selection. Transduced cell viability was determined using the MTT assay before ( b ) and after ( d ) puromycin selection. For cell proliferation assays, AsPC-1 and SNU407 cells were transfected with lentiviral vectors encoding Cas9, Cas9 and sgKRAS-G12D, or Cas9 and sgKRAS-WT. Red: sgRNA sample relevant to tested cell line. Bars represent the mean ± S.E.M. **** (P < 0.0001), ns: not significant.

Article Snippet: One day after seeding, the cells were transfected in GibcoTM Opti-MEMTM I Reduced Serum Medium (Life Technologies, USA) using 1 μg of target lentiviral vector (empty lentiCRISPR v2 or lentiCRISPR v2 containing sgKRAS-WT, sgKRAS-G12V, or sgKRAS-G12D), 0.25 μg of pMD2.G (Addgene, USA), 0.75 μg of psPAX2 (Addgene, USA) and 7 μl of LipofectamineTM 3000 (Life Technologies, USA).

Techniques: Selection, MTT Assay, Transfection

Journal: Cell Death & Disease

Article Title: Novel protein complexes containing autophagy and UPS components regulate proteasome-dependent PARK2 recruitment onto mitochondria and PARK2-PARK6 activity during mitophagy

doi: 10.1038/s41419-022-05339-x

Figure Lengend Snippet: A Representative western blot image showing endogenous PSMA7 and ATG5-12 protein levels in total cell lysate (Input) and ATG5-immunoprecipitated lysate (ATG5-IP) in HEK 293 T cells. Actin beta (ACTB) was used as loading control. B Graph representing the levels of immunoprecipitated PSMA7 protein with ATG5-12, normalized to ATG5-12 in HEK 293 T cells (mean ± SEM, n = 3). C Representative western blot image showing endogenous PSMA7 and ATG5-12 protein levels in total cell lysate (Input) and ATG5-immunoprecipitated lysate (ATG5-IP) in MEF cells. ACTB was used as loading control. D Graph representing the levels of immunoprecipitated PSMA7 protein with ATG5-12, normalized to ATG5-12 in MEF cells (mean ± SEM, n = 3). E Representative images of immunoprecipitation of FLAG and western blot analysis of PARK2 and PSMA7 in HEK 293 T cells co-transfected with MYC-PARK2 and/or FLAG-PSMA7. HEK 293 T cells were treated with STAURO (1 μM), CCCP (10 μM) or DMSO for 12 h after transfection and immunoprecipitation using Flag beads . F Quantification of PARK2 protein levels immunoprecipitated with PSMA7 from E , normalized to PSMA7 (mean ± SEM, n = 3). G and I , Representative western blot images showing the levels of PARK2 and PARK6 proteins respectively, immunoprecipitated with endogenous ATG5 in HA-PARK2 expressing HEK 293 T cells following DMSO or CCCP treatment (10 μM) for 12 h. H and J , Graphs showing the quantification of PARK2 (from G ) and PARK6 (from I ) protein levels, normalized to endogenous ATG5-12 (mean ± SEM, n = 3). K and L , SILAC-MS/MS-based interactome results as fold change graphs of ATG5 enrichment ( K ) and CCCP-induced PSMA7-ATG5 complex enrichment ( L ) as compared to control. M and N , Fold change graphs of PSMA7 enrichment ( M ) and CCCP-induced PARK2-PSMA7 complex enrichment ( N ). O and P , Confocal microscopy images of HEK 293 T cells co-transfected with GFP-PSMA7 (green) and pmCherry-ATG5 (red) constructs and treated with CCCP (10 μM) or STAURO for 12 h ( O ) and treated with CCCP (20 μM) or Oligomycin A/Antimycin A (O/A, 10 μM) for 2 h ( P ). R and S , overlap coefficiency graphs representing PSMA7 (green) and ATG5 (red) colocalization following 12 h (R) and 2 h ( S ) of treatments ( n = 60). T and U , Confocal microscopy images of HeLa cells that were co-transfected with pEGFP-PSMA7 (green) and pmCherry-PARK2 (red) constructs, and treated with staurosporine (1 μM, 12 h) or CCCP (10 μM, 12 h) ( T ) and CCCP or O/A for 2 h ( U ). V and Y , Overlap coefficiency graphs representing PSMA7 (green) and PARK2 (red) colocalization after 12 h ( V , n = 30) and 2 h ( Y , n = 91) of treatments. MERGE, overlay of green and red signals. ZOOM, zoomed images of particular area. Significance in R , S , V and Y was determined using one-way ANOVA.

Article Snippet: ATG5 KO lentiCRISPR V2 vector was created by cloning ATG5 targeting gRNA into lentiCRISPR V2 (Addgene, 52961) using BSMBI.

Techniques: Western Blot, Immunoprecipitation, Control, Transfection, Expressing, Multiplex sample analysis, Tandem Mass Spectroscopy, Confocal Microscopy, Construct

Journal: Cell Death & Disease

Article Title: Novel protein complexes containing autophagy and UPS components regulate proteasome-dependent PARK2 recruitment onto mitochondria and PARK2-PARK6 activity during mitophagy

doi: 10.1038/s41419-022-05339-x

Figure Lengend Snippet: A Representative western blot images showing PSMA7, PARK2, ATG5-12, TIM23 and Actin beta (ACTB) protein levels in cytoplasmic (Cytosol) and mitochondrial (Mito) fractions of YFP-PARK2 transfected HEK 293 T cells treated with DMSO or CCCP (10 µM, 12 h). B Quantification graph corresponding to mitochondrial protein levels of PARK2, PSMA7 and ATG5-12 in A . Mitochondrial protein levels were normalized to TIM23 (mean ± S.D., n = 3). C Representative western blot images showing PSMA7, PARK2, ATG5-12, VDAC1, and ACTB protein levels in cytoplasmic (Cytosol) and mitochondrial (Mito) fractions of HA-PARK2 overexpressing HeLa cells treated with DMSO or CCCP (20 µM, 2 h) or O/A (10 µM, 2 h). D Quantification graph corresponding to protein levels of PARK2, PSMA7 and ATG5-12 in C . Mitochondrial protein levels were normalized to VDAC1 (mean ± S.D., n = 3). E Confocal microscopy analysis of HeLa cells co-transfected with pEGFP-PSMA7 (green) and mito-dsRed (red) constructs, and treated with DMSO or CCCP for 12 h (upper panel) and DMSO, CCCP or O/A for 2 h (lower panel). MERGE, overlay of green and red signals. F and G Overlap coefficiency graphs representing PSMA7 (green) and mitochondria (red) colocalization during 12 h ( F , n = 68) and 2 h ( G , n = 62) treatments. H Confocal microscopy analysis of HeLa cells co-transfected with YFP-ATG5 (green) and mito-dsRed (red) constructs and treated with DMSO or CCCP for 12 h (upper panel) and CCCP or O/A for 2 h (lower panel). I and J Overlap coefficiency graphs representing ATG5 (green) and mitochondria (red) colocalization during 12 h ( I , n = 87) and 2 h ( J , n = 60) treatments. K Confocal microscopy analysis of HeLa cells that were co-transfected with YFP-PARK2 (green) and mito-dsRed (red) constructs and treated with DMSO or CCCP 12 h (upper panel) and DMSO, CCCP or O/A for 2 h (lower panel). L and M , Overlap coefficiency graphs representing PARK2 (green) and mitochondria (red) colocalization during 12 h ( L , n = 30) and 2 h (M , n = 30) treatments. MERGE, overlay of green and red signals. Ordinary one-way ANOVA test ( G, J and M ) or two-tail t-test ( F, I and L ) used for statistical analysis.

Article Snippet: ATG5 KO lentiCRISPR V2 vector was created by cloning ATG5 targeting gRNA into lentiCRISPR V2 (Addgene, 52961) using BSMBI.

Techniques: Western Blot, Transfection, Confocal Microscopy, Construct

Journal: Cell Death & Disease

Article Title: Novel protein complexes containing autophagy and UPS components regulate proteasome-dependent PARK2 recruitment onto mitochondria and PARK2-PARK6 activity during mitophagy

doi: 10.1038/s41419-022-05339-x

Figure Lengend Snippet: A and C , Western blot images representing the levels of PARK2, PARK6, VDAC1, PSMA7, PSMB5 and ATG5-12 following gel filtration chromatography performed using mitochondrial lysates collected from YFP-PARK2 expressing HEK 293 T cells treated with either DMSO ( A ) , or CCCP ( C) for 12 h. M, mitochondrial extracts before fractionation. L-PARK6, uncleaved/unprocessed form of PARK6 (63 kDa). S-PARK6, cleaved/processed form of PARK6 (55 kDa). B and D , Quantifications of PARK2 relative intensity in A and C respectively ( n = 3). E and F , Graphs showing SILAC-MS/MS enrichments of ATG5 ( E ) and PARK2-ATG5 complex ( F ) in mitochondrial fraction ( n = 3).

Article Snippet: ATG5 KO lentiCRISPR V2 vector was created by cloning ATG5 targeting gRNA into lentiCRISPR V2 (Addgene, 52961) using BSMBI.

Techniques: Western Blot, Filtration, Chromatography, Expressing, Fractionation, Multiplex sample analysis, Tandem Mass Spectroscopy

Journal: Cell Death & Disease

Article Title: Novel protein complexes containing autophagy and UPS components regulate proteasome-dependent PARK2 recruitment onto mitochondria and PARK2-PARK6 activity during mitophagy

doi: 10.1038/s41419-022-05339-x

Figure Lengend Snippet: A, Representative western blot images of PARK2, VDAC1, ATG5-12 and ACTB in mitochondrial fractions of YFP-PARK2 expressing HEK 293 T cells transfected with control vector or ATG5 specific shRNA vector (shATG5), and treated with DMSO or CCCP (10 µM) for 12 h. B Graph representing the quantification of mitochondria localized PARK2 from A (mean ± SEM, n = 3). C Representative confocal images of YFP-PARK2 (green) and mito-dsRed (red) expressing HeLa cells co-transfected with control vector (CNT) or shATG5 vector, and treated with DMSO or CCCP (10 µM) for 12 h. MERGE, overlay of green and red signals. D Graph showing colocalization coefficiency of PARK2 overlap with mitochondria from C (mean ± S.D., n≥30, one-way ANOVA). E Representative western blot images of MFN2, PARK2, ATG5-12 and ACTB in MYC-PARK2 over expressing wild type (WT) and ATG5 knockout (KO) HeLa cells treated with DMSO or CCCP (10 µM) for 12 h. ACTB was used as loading control. F , Quantification of MFN2 levels from E (mean ± SEM, n = 3). G , Representative western blot images of phosphoSer65-ubiquitin (pSer65-UB) and total ubiquitin (UB) levels in MYC-PARK2 expressing wild type (WT) and ATG5 knockout (KO) HeLa cells treated with DMSO, CCCP (20 μM, 2 h) or MG132 (30 μM, 2.5 h) and CCCP (20 μM, 2.5 h). Band intensities were marked below ( n = 3). H Confocal images of GFP-optineurin (OPTN, green) and mito-dsRed (red) expressing HeLa cells co-transfected with control vector or shATG5 vector, and treated with DMSO or CCCP (10 μM) for 12 h. MERGE, overlay of green and red signals. ZOOM, zoomed images of particular area. I Quantification graph showing the overlap coefficiency values of optineurin dots (green) and mitochondria (red) from H (mean ± S.D., n ≥ 30, one-way ANOVA).

Article Snippet: ATG5 KO lentiCRISPR V2 vector was created by cloning ATG5 targeting gRNA into lentiCRISPR V2 (Addgene, 52961) using BSMBI.

Techniques: Western Blot, Expressing, Transfection, Control, Plasmid Preparation, shRNA, Knock-Out, Ubiquitin Proteomics