Journal: International Journal of Molecular Sciences

Article Title: Acute Increases in Intracellular Zinc Lead to an Increased Lysosomal and Mitochondrial Autophagy and Subsequent Cell Demise in Malignant Melanoma

doi: 10.3390/ijms22020667

Figure Lengend Snippet: Mitochondrial membrane potential (Δψm) changes, ATP production and mitophagy in explant human melanoma cultures with lower free zinc stores (M5), average free zinc stores (M10) and higher free zinc stores (M9) exposed to 0.5 μM zinc pyrithione during 72 h. Cells were exposed to external zinc pyrithione and ( A ) loss of Δψm measured by decreased red fluorescence of JC-1 was determined in at least 1000 cells visualized by fluorescence microscopy. Results represent means ± SD of at least three independent experiments. # p < 0.05 significantly lower compared to the beginning of treatment with one-way ANOVA test and Dunnett’s post-test for multiple comparisons. ( B ) ATP production was measured in cell lysates by ATP bioluminescent assay kit ( C ). Mitophagy-specific fluorescence (Mitophagy Detection Kit) in cells exposed to zinc pyrithione alone or together with autophagy inhibitor chloroquine was determined fluorimetrically. Results represent means ± SD of at least three independent experiments. * p < 0.05 significantly higher compared to the beginning of treatment, # p < 0.05 significantly lower compared to the beginning of treatment with one-way ANOVA test and Dunnett’s post-test for multiple comparisons.

Article Snippet: Control and zinc pyrithione-treated cells of explant human melanoma grown in 96-well plates with black bottom were washed with PBS and incubated in 100 nM Mitophagy Dye Solution (Mitophagy Detection Kit (Dojindo Laboratories, Kumamoto, Japan)) for 35 min. After removal of the culture medium and washing with PBS, mitophagy-specific fluorescence was evaluated using a cell scoring module of MetaXpress ® Image Acquisition and Analysis Software.

Techniques: Membrane, Fluorescence, Microscopy, ATP Bioluminescent Assay

Journal: Scientific Reports

Article Title: Dietary iodine attenuates allergic rhinitis by inducing ferroptosis in activated B cells

doi: 10.1038/s41598-023-32552-1

Figure Lengend Snippet: KI promotes ferroptosis of activated B cells. Splenic B cells collected from C57BL/6 J mice were cultured with anti-CD40 antibody, IL-4, and serially diluted KI for three days. ( A ) Histogram of 7-ADD expression in B cells stimulated with DMSO or Fer-1. ( B , C ) Survival rate ( B ) and MFI of 7-AAD ( C ) in stimulated B cells with DMSO or Fer-1. Representative data from two independent experiments are shown as the mean ± SD ( n = 3 for each point). ( D ) MFI of ROS in the ROS + B220 + CD45 + B cells. Representative data from two independent experiments are shown as the mean ± SD ( n = 3 for each point). ( E ) Intracellular ferrous iron measurements in FerroFarRed + cells cultured with or without anti-CD40 and IL-4 for 3 days. ( F – I ) Quantitative polymerase chain reaction analysis of the relative expression levels of Steap3 ( F ), Slc11a2 ( G ), Fth1 , and Ftl1 ( H ) in anti-CD40-stimulated B cells. Data were pooled from two independent experiments and are shown as mean ± SEM ( n = 6 for each point). Welch’s t -test. n.s.: not significant, * p < 0.05, ** p < 0.01. IL, interleukin; KI, potassium iodide; MFI, mean fluorescence intensity; ROS, reactive oxygen species.

Article Snippet: To detect intracellular ROS and ferrous iron, ROS Assay Kit-High Sensitive DCFH-DA (DOJINDO) and FerroFarRed (Goryo Chemical, Inc.), were used, respectively, following the manufacturer’s protocol.

Techniques: Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Fluorescence

Journal: Biology Open

Article Title: Insulator speckles associated with long-distance chromatin contacts

doi: 10.1242/bio.019455

Figure Lengend Snippet: Long DNA FISH probes result in non-contiguous staining detected by structured illumination microscopy (SIM). (A) Schematic illustration of the Antennapedia complex (ANT-C) and the Bithorax complex (BX-C) on the right arm of chromosome 3 (3R) in Drosophila melanogaster . The DNA FISH probes are highlighted either in green, representing parts of ANT-C, or in red, representing parts of BX-C. For the exact genomic locations of each probe see Table S1 . (B) DNA FISH with long probes in wing imaginal discs imaged either with confocal laser scanning microscopy (CLSM) or with structured illumination microscopy (SIM). Increased resolution with SIM revealed the structured and incomplete appearance of the FISH probes in the case of kissing and non-kissing Hox genes (white arrows). Kissing was defined as distances between probe pair centres shorter than 350 nm . The scale bars represent 1 µm. (C) Orthogonal view of DNA FISH in wing imaginal discs displaying increased lateral (XY) and axial (XZ and YZ) resolution with SIM as compared to CLSM. Scale bars: 1 µm.

Article Snippet: FISH probes were directly labelled by nick translation using the FISH Tag DNA Green Kit (Alexa Fluor 488 dye, Thermo Fisher Scientific) and the FISH Tag DNA Red Kit (Alexa Fluor 594 dye, Thermo Fisher Scientific).

Techniques: Staining, Microscopy, Confocal Laser Scanning Microscopy

Journal: Biology Open

Article Title: Insulator speckles associated with long-distance chromatin contacts

doi: 10.1242/bio.019455

Figure Lengend Snippet: Probe pair ANT-C9kb/BX-C10kb is in closest proximity to each other. (A) Specificity of short probes verified by DNA FISH on polytene chromosomes. Each of the probes showed a distinct single band as magnified in the inset. (B) Hybridization of each of the short probes of one Hox gene cluster together with the long probe of the other Hox gene cluster revealed small but significant differences in distance. BX-C9kb and BX-C10kb as well as ANT-C8kb and ANT-C9kb showed the shortest distances to their corresponding long probe. Significant differences (two-tailed Mann–Whitney-U test) in distance are indicated (*). (C) Hybridization of one of the ANT-C probes ANT-C8kb or ANT-C9kb with one of the BX-C probes BX-C9kb or BX-C10kb. Examples of nuclei representing the mean distances between the short probes. Mean distances are highlighted in the merge view. Scale bars: 1 µm. Imaged with SIM. (D) Box-plot mean values indicated that the probe pair ANT-C9kb/BX-C10kb showed a significantly (two-tailed Mann-Whitney-U test) closer distance than the others, indicated (*). (E) Distance between ANT-C.9kb and BX-C.10kb in wing imaginal disc nuclei. Hox gene kissing occurred in only 1.9% of the nuclei. n =total number of analysed wing imaginal disc nuclei in B, D and E. For P values of the pairwise comparison in B and D see Table S2 .

Article Snippet: FISH probes were directly labelled by nick translation using the FISH Tag DNA Green Kit (Alexa Fluor 488 dye, Thermo Fisher Scientific) and the FISH Tag DNA Red Kit (Alexa Fluor 594 dye, Thermo Fisher Scientific).

Techniques: Hybridization, Two Tailed Test, MANN-WHITNEY, Comparison

Journal: Biology Open

Article Title: Insulator speckles associated with long-distance chromatin contacts

doi: 10.1242/bio.019455

Figure Lengend Snippet: Hox gene kissing is a rare event in interphase nuclei of Drosophila melanogaster. (A) Schematic illustration of the location of the positive and negative control probes on chromosome 3R. The colour of the probe indicates the colour used for FISH detection. Beat-Vc and wake have been described as non-kissing sites , and are separated by approximately 10 Mb. The distance of the probe pair ANT-C9kb and 26 MB is twice as long (20 Mb). (B) Specificity of probes verified by DNA FISH on polytene chromosomes. Each of the probes showed a single band (yellow arrow in overview and band in magnified inset). (C) SIM examples of nuclei representing the mean distance between the probes pairs. Mean distances are indicated in the merge view and determined in (D). The scale bars represent 1 µm. (D) Small, but significant differences in the distances between the analysed probe pairs. All three pairs showed an average distance of 1.2 to 1.3 µm. * P <0.05 (two-tailed Mann–Whitney-U test), n =total number of analysed nuclei in three different wing imaginal discs. (E) No significant differences in percentage of Hox gene kissing for the positive probe pair ANT-C9kb/BX-C10kb and the negative control pairs beat-Vc/wake and ANT-C9kb/26MB. In all cases a range of 1.5% to 2.5% was found.

Article Snippet: FISH probes were directly labelled by nick translation using the FISH Tag DNA Green Kit (Alexa Fluor 488 dye, Thermo Fisher Scientific) and the FISH Tag DNA Red Kit (Alexa Fluor 594 dye, Thermo Fisher Scientific).

Techniques: Negative Control, Two Tailed Test, MANN-WHITNEY