Journal: Brain

Article Title: Pathophysiology of protein aggregation and extended phenotyping in filaminopathy

doi: 10.1093/brain/aws200

Figure Lengend Snippet: Primary antibodies used in double immunofluorescence studies

Article Snippet: Hsp60 , D307 , Rabbit (pAb) , Cell Signalling , 1/50.

Techniques: Immunofluorescence

Journal: Cells

Article Title: Loss of Peter Pan (PPAN) Affects Mitochondrial Homeostasis and Autophagic Flux

doi: 10.3390/cells8080894

Figure Lengend Snippet: PPAN is decreased by CCCP and PPAN knockdown further sensitizes cells to mitochondrial loss. ( A ) Endogenous PPAN localizes to nucleoli and mitochondria in HEK293A GFP-LC3 cells. The confocal image is representative of 3 experiments. GFP-LC3 expressing cells (GFP fluorescence not shown) were stained with PPAN antibodies and DAPI (see merge). Scale bar, 10 µm. ( B – D ) CCCP-mediated activation of mitophagy significantly reduces COX IV and PPAN levels as monitored by Western blotting in HEK293A GFP-LC3 cells. PPAN knockdown in HEK293A GFP-LC3 cells as indicated above panels reveals decrease of COX IV upon CCCP treatment. In addition, PPAN is decreased following CCCP treatment (10 µM) for 24 h. Western blots of whole cell lysates probed with COX IV, PPAN and GAPDH antibodies. Lysates of the same experiment were loaded twice (see GAPDH placed below respective blots). ( C ) Quantification of COX IV normalized to GAPDH as shown in (B). Note that 4 out of 5 experiments were treated without DMSO (n = 4) and one experiment with DMSO. The si control in absence of CCCP was set to 1. ( D ) Quantification of PPAN normalized to GAPDH as shown in ( B ). The si control was set to 1. ( E – H ) PPAN knockdown in HCT116 cells as indicated above panels reveals decrease of COX IV, which is accompanied by increased LC3-II accumulation upon CCCP treatment. In addition, PPAN is decreased following CCCP treatment (20 µM) for 17 h. Controls were treated with DMSO as indicated. ( E ) Whole cell lysates were probed with COX IV, PPAN, GAPDH and total LC3 antibodies. Whole cell lysates of the same experiment were loaded twice. ( F ) Quantification of COX IV normalized to GAPDH as shown in ( E ) by densitometry. The DMSO treated si control cells were set to 1. ( G ) Quantification of PPAN normalized to GAPDH as shown in ( E ). The DMSO treated si control cells were set to 1. ( H ) Quantification of LC3-II normalized to GAPDH as shown in ( E ). The DMSO treated si control cells were set to 1. Error bars represent S.D., *, p < 0.05; **, p < 0.01; ***, p < 0.001; n = number of independent experiments.

Article Snippet: Cell Signaling: COX IV (3E11), HSP60 (D307), GAPDH (1410C), LC3A/B (#4108), PARP (#9542), Ubiquitin (P4D1); nanoTools: LC3 (#0321-100); Proteintech: PPAN (#11006-1-AP); BD: TIM23 (#611222); Santa Cruz: LAMP2 (H4B3), TOM20 (FL-145); Sigma: GST (G1160, clone2); Abcam: p62 (EPR4844).

Techniques: Knockdown, Expressing, Fluorescence, Staining, Activation Assay, Western Blot, Control

Journal: Cells

Article Title: Loss of Peter Pan (PPAN) Affects Mitochondrial Homeostasis and Autophagic Flux

doi: 10.3390/cells8080894

Figure Lengend Snippet: PPAN knockdown decreases mitochondrial mass and mtDNA to nuclear DNA ratio in U2OS cells. ( A – C ) COX IV and PPAN protein levels in wildtype U2OS cells upon 24 h O/A treatment as indicated. Controls were treated with EtOH (ethanol) as indicated. ( A ) Whole cell lysates were subjected to Western blotting. Membranes were probed with PPAN, COX IV and GAPDH antibodies. ( B ) Quantification of COX IV normalized to GAPDH as shown in ( A ). The EtOH treated cells were set to 1. ( C ) Quantification of PPAN normalized to GAPDH as shown in ( A ) by densitometry; ns, non-significant differences. ( D – F ) PPAN knockdown in U2OS cells decreases COX IV protein levels. ( D ) Whole cell lysates were subjected to Western blotting and membranes were probed with COX IV, PPAN and GAPDH antibodies. Lysates of the same experiment were loaded twice (see GAPDH placed below respective blots). ( E ) Quantification of COX IV normalized to GAPDH as shown in ( D ). ( F ) Quantification of PPAN normalized to GAPDH as shown in ( D ). The si control was set to 1. ( G ) qPCR analysis of U2OS cells transfected with control and PPAN siRNAs as indicated. Transfection of both PPAN siRNAs results in significant down-regulation of PPAN mRNA levels. PPAN was normalized to GAPDH (=relative expression, R). ( H ) Measurement of mtDNA/nDNA ratio by qPCR analysis of U2OS cells transfected with control and PPAN siRNAs as indicated. ( I ) Measurement of mtDNA/nDNA ratio of U2OS cells treated with EtOH or O/A for 24 h as indicated. The si control was set to 1. Error bars represent S.D.; p values were calculated by t-test. Statistically significant differences are indicated by asterisks, *, p < 0.05, ***, p < 0.001, ****, p < 0.0001; n = number of independent experiments.

Article Snippet: Cell Signaling: COX IV (3E11), HSP60 (D307), GAPDH (1410C), LC3A/B (#4108), PARP (#9542), Ubiquitin (P4D1); nanoTools: LC3 (#0321-100); Proteintech: PPAN (#11006-1-AP); BD: TIM23 (#611222); Santa Cruz: LAMP2 (H4B3), TOM20 (FL-145); Sigma: GST (G1160, clone2); Abcam: p62 (EPR4844).

Techniques: Knockdown, Western Blot, Control, Transfection, Expressing