Journal: Cancers

Article Title: Voltage-Gated Sodium Channel Na V 1.7 Inhibitors with Potent Anticancer Activities in Medullary Thyroid Cancer Cells

doi: 10.3390/cancers15102806

Figure Lengend Snippet: Dose-dependent blockade of Na V 1.7 channels by SV188 . ( A ) Representative recordings showing stationary blockade of Na V 1.7 currents by increasing concentrations of SV188 in µM. Whole-cell patch-clamp recordings were made from Na V 1.7 channels transiently expressed in an HEK-293 cell. Sodium currents ( I Na ) were evoked by voltage steps to −10 mV from a holding potential (HP) of −120 mV applied every 10 s. Traces are the average of three consecutive recordings under the indicated experimental conditions. The black dotted line represents the zero-current level. ( B ) Time course of Na V 1.7 current blockade by SV188 . Data from the same cell shown in ( A ). Current recovery was incomplete in most cells; on average, 74 ± 3% of current was recovered after extensive wash out of SV188 . ( C ) Dose–response relationship of the effect of SV188 on Na V 1.7 channels. Fraction of the blocked current was calculated from peak current measurements from step voltages to −10 mV in the presence of several SV188 concentrations ( n = 3–16 cells). Data points (mean ± SEM) were fitted using a Hill equation (smooth line); the corresponding IC 50 and Hill slope ( n H ) parameters are shown in the graph. Pink points display the corresponding fraction of current blocked by 3 µM and 10 µM of SV188 when using an HP = −80 mV.

Article Snippet: Human embryonic kidney cells (HEK-293) were acquired from the American Type Cell Culture Collection (ATCC CRL-1573) and grown in DMEM/F12 mixture supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C in a CO 2 incubator.

Techniques: Patch Clamp

Journal: Antioxidants

Article Title: The Fate of Oxidative Strand Breaks in Mitochondrial DNA

doi: 10.3390/antiox12051087

Figure Lengend Snippet: Representative Southern blots of HEK 293 cells treated with ( A ) 0.5 mM H 2 O 2, or ( B ) 1 mM H 2 O 2 . The top label contains “M” molecular weight marker and the time point of the H 2 O 2 time course. Lanes 1–6 wild-type HEK 293 cells, lanes 7–12 POLGexo −/− line. Samples have been digested with Mlu I for the 18S nuclear loading control while leaving the mtDNA intact. The three main species of mtDNA can be seen: open circle (OC), full-length linear (FLL, 16.5 kb) and supercoiled (SC), as well as a smear of linear mtDNA fragments of smaller sizes. Before H 2 O 2 application (lanes 1 and 7), the main conformation of mtDNA is the supercoiled. After 30 min of H 2 O 2 treatment (lanes 2 and 8), the supercoiled species is lost, and the other open circle and linear forms increase. The supercoiled is recovered in wild-type cells starting at 2 h after 0.5 mM H 2 O 2 treatment and 6 h after 1 mM H 2 O 2 , but the recovery of the supercoiled is diminished in the POLGexo −/− cells. Note the higher background smear on the POLGexo −/− cell line indicating elevated levels of fragmented linear mtDNA. The quantification of band signal intensities from three different experiments is shown in panels ( C – J ). Blue bars—wild-type cells, gray bars—p.D274A POLG knock-in cell line. Band intensity of the three main mtDNA species (open circle, panels ( D , F ); full-length linear, panels ( G , I ); and supercoiled, panels ( C , E )) have been quantified, as well as the smear of smaller linear fragments (panels ( H , J )). Data for both cell lines ( n = 2 for 0.5 mM and n = 3 for 1.0 mM H 2 O 2 ) are shown as average ± SEM. Significances between time points were assessed by one-way ANOVA and between pairs of wild-type and POLGexo −/− samples by Student’s unpaired t -test. **, p < 0.01; *, p < 0.05. Note that after H 2 O 2 treatment of the POLGexo −/− line, a substantial amount of the signal is localized in the smear of fragmented mtDNA, as well as the recovery of supercoiled that is only present in wild-type cells.

Article Snippet: Immortalized human embryonic kidney (HEK 293) cells were commercially obtained from ATCC, catalogue Nr. CRL-1573.

Techniques: Molecular Weight, Marker, Knock-In

Journal: Antioxidants

Article Title: The Fate of Oxidative Strand Breaks in Mitochondrial DNA

doi: 10.3390/antiox12051087

Figure Lengend Snippet: Sequence motifs at the ends of mtDNA fragments detected by deep sequencing in wild-type HEK 293 cells. Nucleotides within a 40 bp distance relative to detected ends were counted. Position-specific nucleotide frequencies were normalized to overall nucleotide frequencies. Upper panels, Illumina sequencing results from natively blunt linker-ligatable mtDNA ends. Lower panels, PacBio sequencing of linear mtDNA. mtDNA samples before treatment, 30 min after H 2 O 2 pulse, and 24 h after H 2 O 2 were investigated. Ends associated with GC-stretches are present only 30 min after the H 2 O 2 but absent before treatment and after 24 h of recovery (cf. Figure 4a in Reference ).

Article Snippet: Immortalized human embryonic kidney (HEK 293) cells were commercially obtained from ATCC, catalogue Nr. CRL-1573.

Techniques: Sequencing

Journal: Antioxidants

Article Title: The Fate of Oxidative Strand Breaks in Mitochondrial DNA

doi: 10.3390/antiox12051087

Figure Lengend Snippet: BrdU southern blot and quantification of BrdU incorporation into HEK 293 cell lines. ( A , B ): Experiments without hydrogen peroxide addition. ( C , D ): Experiments in the presence of 1 mM hydrogen peroxide. Southern blots ( A , C ) have been labeled with a BrdU antibody to visualize BrdU-incorporated DNA. “M” is for the molecular weight marker, and time points are indicated. All samples have been digested with Mlu I for the 18S nuclear loading control while keeping mtDNA intact. The molecular weight marker and the 18S have been visualized with a DIG-labeled 18S probe. Blots have been assembled from portions of the same membrane. The three main species of mtDNA can be seen: open circle (OC), full-length linear (FLL), and supercoiled (SC) (see for mtDNA probe confirmation). The large dark cloud smear in the 24 h time points is from nuclear DNA BrdU incorporation due to an incomplete block by aphidicolin. Quantification ( B , D ) of the relative intensity of BrdU incorporated into mtDNA as the sum of all three major mtDNA species. Time after BrdU application is on the x -axis in hours, and the y -axis is the relative intensity of BrdU incorporated into mtDNA. Band intensity is normalized to the 18S loading control and the 24-h control sample. Blue bars—wild-type HEK 293 cells, gray bars—POLGexo −/− cells. Error bars, ±SEM ( B ): n = 4 for wild-type and n = 1 for POLGexo −/− . BrdU begins to be incorporated into mtDNA at 2 h in the non-H 2 O 2 condition, which is slightly reduced in the POLGexo −/− cells. ( D ): n = 5 for wild-type and n = 2 for POLGexo −/− . BrdU is apparently incorporated at a lower rate in the presence of H 2 O 2 , only reaching comparable amounts to the non-H 2 O 2 samples after 24 h (however, the difference between the treatment conditions did not reach statistical significance). The observed rate of BrdU incorporation in the presence of H 2 O 2 indicates that increased mtDNA replication is very unlikely to be responsible for the fast recovery of intact mtDNA in wild-type cells after H 2 O 2 damage.

Article Snippet: Immortalized human embryonic kidney (HEK 293) cells were commercially obtained from ATCC, catalogue Nr. CRL-1573.

Techniques: Southern Blot, BrdU Incorporation Assay, Labeling, Molecular Weight, Marker, Blocking Assay