htert expressing hdfs  (Addgene inc)

Journal: Scientific Reports

Article Title: Chronic replication stress invokes mitochondria dysfunction via impaired parkin activity

doi: 10.1038/s41598-024-58656-w

Figure Lengend Snippet: Constitutive replication stress and recovery causes fixed reduction in parkin expression in HDFs. ( A ) Schematics of exposure of HDFs with replication stress by APH treatment. ( B ) Representative image (left) and quantification (right) of immunoblots showing expression level of parkin. Ponceau indicates staining of a membrane before immunostaining used as loading control. Error bar: SEM of at least 3 independent experiments. ns: p > 0.05, *: p < 0.05, ***: p < 0.005, ****: p < 0.001 (One-way ANOVA). ( C ) Summary of RNA-seq experiments showing mRNA expression of genes of each category indicated in the HDF cells exposed to APH, normalized by non-treated controls. PRKN ( PARK2 ) is highlighted by orange rectangle. Error bars indicates SEM of 3 independent experiments. ns: p > 0.05, ***: p < 0.005, ****: p < 0.001 (One-way ANOVA). ( D ) Representative image (left) and quantification (right) of immunoblots showing expression level of parkin in the HEK293A cells treated with 50 µg/ml of cycloheximide (CHX) for indicated hours. APH indicates 300 nM APH for 96 h followed by recovery culture for 96 h. Error bars indicates SEM of 3 independent experiments.

Article Snippet: To introduce HA-parkin, hTERT expressing HDFs were transfected with pMXs-IP HA-Parkin (a gift from Dr. Noboru Mizushima, Addgene plasmid #38247) .

Techniques: Expressing, Western Blot, Staining, Membrane, Immunostaining, Control, RNA Sequencing

Journal: Scientific Reports

Article Title: Chronic replication stress invokes mitochondria dysfunction via impaired parkin activity

doi: 10.1038/s41598-024-58656-w

Figure Lengend Snippet: Constitutive replication stress and recovery causes elevated mtROS expression in HDFs. ( A ) Schematics of exposure of HDFs with acute replication stress (APH) or 100 nM of Rotenone, an inhibitor of mitochondrial electron transport chain complex I. ( B ) Sequential progression of mtROS indicated by mitoSOX signal normalized by mitochondrial signal indicated by MitoTracker green. Error bar: SEM of at least 3 independent experiments. ns: p > 0.05, *: p < 0.05, **; p < 0.01, (One-way ANOVA). ( C ) Representative image of microscopic observation of the HDFs labeled with MitoTracker and MitoSOX. Scale bar: 20 µm. ( D ) Schematics of exposure of HDFs with constitutive replication stress (APH). ( E ) Sequential progression of mtROS indicated by mitoSOX signal normalized by mitochondrial signal indicated by MitoTracker green. Error bar: SEM of at least 3 independent experiments. ns: p > 0.05, *: p < 0.05, **; p < 0.01, (One-way ANOVA). ( F ) Representative image of microscopic observation of the HDFs labeled with MitoTracker and MitoSOX. Scale bar: 20 µm. ( G ) Representative image (left) and quantification (right) of immunoblots showing expression level of HA-parkin. Ponceau indicates staining of a membrane before immunostaining used as loading control. Error bar: SEM of at least 3 independent experiments. ns: p > 0.05, student’s t-test.

Article Snippet: To introduce HA-parkin, hTERT expressing HDFs were transfected with pMXs-IP HA-Parkin (a gift from Dr. Noboru Mizushima, Addgene plasmid #38247) .

Techniques: Expressing, Labeling, Western Blot, Staining, Membrane, Immunostaining, Control

Journal: Scientific Reports

Article Title: Chronic replication stress invokes mitochondria dysfunction via impaired parkin activity

doi: 10.1038/s41598-024-58656-w

Figure Lengend Snippet: Constitutive replication stress and recovery causes fixed reduction in parkin expression in HDFs. ( A ) Representative image (left) of immunoblots showing expression level of parkin and NDUFA9 in the HDF cells. HDFs were treated with indicated dose of APH for 96 h, followed by recovery culture without APH for 96 h and harvest for western blot. Ponceau indicates staining of a membrane before immunostaining used as loading control. Right bar graph shows mitophagy index indicated by % degradation of NDUFA9 in the cells with indicated dose of CCCP for 24 h. Error bar: SEM of at least 3 independent experiments. ns: p > 0.05. *: p < 0.05, Student’s t-test. ( B ) Representative image (left) and quantification (right) of immunoblots showing DRP1 expression or phosphorylation level of DRP1. HDFs were treated with indicated dose of APH for 96 h, followed by recovery culture without APH for 96 h and harvest for western blot. Ponceau indicates staining of a membrane before immunostaining used as loading control. Error bar: SEM of at least 3 independent experiments. *: p > 0.05, Student’s t-test. ( C ) Representative image (left) of immunoblots showing expression level of parkin and NDUFA9 in HEK293A cells. HEK293A cells were treated with indicated dose of APH for 96 h, followed by recovery culture without APH for 96 h and harvest for western blot. Error bar: SEM of at least 3 independent experiments. ns: p > 0.05. Student’s t-test. ( D ) Representative images (top) and quantifications (bottom) of LC3 (autophagosome) and TOM20 (mitochondria) localizations in the HEK293A cells treated with 20 µM CCCP for 6 h. The # of LC3 dots and co-localization of LC3 with TOM20 were analyzed. Error bar: SEM of at least 3 independent experiments. ns: no significance, Student’s t-test. Scale bar: 20 µm. ( E ) Representative images (left) and quantifications (right) of 8-oxoguanine and MitoTracker Red in the HEK293A cells. Both 8-oxoguanine signals on MitoTracker Red and DAPI (nuclei) were analyzed. ns: p > 0.05, ***: p < 0.005, Student’s t-test. Scale bar: 20 µm.

Article Snippet: To introduce HA-parkin, hTERT expressing HDFs were transfected with pMXs-IP HA-Parkin (a gift from Dr. Noboru Mizushima, Addgene plasmid #38247) .

Techniques: Expressing, Western Blot, Staining, Membrane, Immunostaining, Control, Phospho-proteomics

Journal: Scientific Reports

Article Title: Chronic replication stress invokes mitochondria dysfunction via impaired parkin activity

doi: 10.1038/s41598-024-58656-w

Figure Lengend Snippet: Constitutive replication stress and recovery causes morphological alteration in HDFs. ( A ) Representative images of morphological changes of mitochondria labeled with mitoTracker green. A arrow in the magnified image indicates a representation of donut mitochondria. Scale bar: 20 µm. ( B–D ) Summarized bar graphs of mean branch length ( B ), mean branches per network ( C ) or number of donut mitochondria relative to NT condition ( D ) in HDFs. Error bars indicates SEM of at least 60 cells for each condition were analyzed. ns: p > 0.05, *: p < 0.05 (one-way ANOVA). ( E–G ) Summarized bar graphs of mean branch length ( E ), mean branches per network ( F ) or number of donut mitochondria relative to NT condition ( G ) in HEK293A cells. Error bars indicates SEM of at least 60 cells for each condition were analyzed. ns: p > 0.05, ***: p < 0.005 (one-way ANOVA).

Article Snippet: To introduce HA-parkin, hTERT expressing HDFs were transfected with pMXs-IP HA-Parkin (a gift from Dr. Noboru Mizushima, Addgene plasmid #38247) .

Techniques: Labeling