Journal: Scientific Reports

Article Title: Discovery of a kleptoplastic ‘dinotom’ dinoflagellate and the unique nuclear dynamics of converting kleptoplastids to permanent plastids

doi: 10.1038/s41598-019-46852-y

Figure Lengend Snippet: Diatom nuclear dynamics in Durinskia capensis observed with CLSM under autotrophic conditions. SYBR-Green or DAPI stained nucleus = Green, Mitotracker Green stained mitochondria = Yellow, Chlorophyll a autofluorescence = Red. ( A ) D. capensis ethanol-fixed on the sampling day (day 0). There are two round nuclei, a host dinoflagellate nucleus (Hn), which contains condensed chromosomes, and a diatom nucleus (En). ( B ) By day 7, the round diatom nucleus has disappeared from the D. capensis cell; instead string-shaped DNA signals can be observed alongside the plastids (arrow). ( C ) The Mitotracker Green-stained cell on day 7 indicates that the string-shaped DNA signals are from diatom mitochondria. ( D , E ) On day 35, no DNA signals from the diatom organelles can be observed in D. capensis with SYBR-Green ( D ) nor with Mitotracker Green ( E ). Sclae bar = 10 µm.

Article Snippet: For staining the nuclei of Durinskia spp., 500X working solution of SYBR Green I (Lonza, Basel, for living cells) or 125 μg/ml of DAPI (Carl Roth, Karlsruhe, for ethanol-fixed day 0 samples of D. capensis ) were used for a final concentration of 3% (v/v) or 6% (v/v), respectively.

Techniques: SYBR Green Assay, Staining, Sampling

Journal: Oncology Letters

Article Title: Potential synergistic effects of sorafenib and CP-31398 for treating anaplastic thyroid cancer with p53 mutations

doi: 10.3892/ol.2020.11377

Figure Lengend Snippet: Effects of CP-31398 on the number of SW579 cells in the cell cycle. (A) Cell cycle distribution. (B) Statistical analysis of cell cycle phases. SW579 cells were incubated with 0, 0.3 or 3 µM CP-31398 for 72 h. The CP-31398-treated cells were loaded into a Via-1-Casette and analyzed for DAPI dyes, using a fluorescence image cytometer (NucleoCounter NC-3000). Data were expressed as the means ± standard deviation. Student's t-test was used. **P<0.01.

Article Snippet: The stained cells were loaded into an NC-Slide A8 (ChemoMetec) and evaluated using a Fixed Cell Cycle-DAPI/DNA fragmentation assay protocol in the NucleoCounter NC-3000 image cytometer (ChemoMetec) ( 19 ).

Techniques: Incubation, Fluorescence, Cytometry, Standard Deviation

Journal: Nature Communications

Article Title: Adaptive translational reprogramming of metabolism limits the response to targeted therapy in BRAF V600 melanoma

doi: 10.1038/s41467-022-28705-x

Figure Lengend Snippet: a Schematic summarizing screen workflow (see methods). b WM266.4 cells were transfected with the indicated siRNA and treated with DMSO or 300 nM Vem for 48 h. Cell number was calculated using high content image analysis of DAPI stained cells (top panel) and growth media was collected for determination of lactate levels. Lactate absorbance values were normalized to cell number to determine lactate production per cell (bottom panel). Data are presented as mean fold change relative to DMSO siOTP control, ±StDev; Data points are individual replicate wells from two technical replicate screening plates. c Functional annotation enrichment analysis was performed on 717 genes that enhanced the effects of Vem on lactate production (DMSO lactate per cell ratio <0.5-fold change and Vem lactate per cell ratio >0.5-fold change; see Supplementary Data ) using DAVID. Data is displayed as Log2 fold change versus −Log10 p -value. Annotations previously linked with BRAFi response and/or resistance are shown in blue, and the top 4 annotations linked with RNA binding and transport are shown in red. d Network analysis was performed on 622 viability screen hits and 717 hits that enhanced the effects of Vem on lactate production using String (see Supplementary Fig. ). Comparative network analysis was performed using Cytoscape, and hubs connecting the two networks are highlighted. e Heat map displaying viability and lactate screening data for the indicated genes. f Heatmap displaying percentage of melanoma patients with upregulation of the indicated mRNA transport and translation genes on progression following treatment with MAPK pathway inhibitors (data sourced from https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE65186 ; see Supplementary Data ). See also Supplementary Figs. – . Source data for b – f are provided as a Source Data file.

Article Snippet: After the assay, cells were fixed and stained with DAPI DNA dye and cells were imaged using a Cellomics Arrayscan automated microscope.

Techniques: Transfection, Staining, Control, Functional Assay, RNA Binding Assay

Journal: Nature Communications

Article Title: Adaptive translational reprogramming of metabolism limits the response to targeted therapy in BRAF V600 melanoma

doi: 10.1038/s41467-022-28705-x

Figure Lengend Snippet: WM266.4 and A375 cells were transfected with the indicated siRNA and treated with DMSO or 300 nM Vem for 48 h. a Oxygen consumption rate (OCR) was determined using Seahorse Extracellular Flux Analysis and representative profiles for WM266.4 (top panel) and A375 (bottom panel) cells are shown (Oligo = oligomycin; FCCP = Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone; Rot/Ant-A = rotenone + antimycin-A; representative of n = 4 biologically independent experiments). Effect of gene knockdown and Vem treatment on basal OCR ( b ), max OCR ( c ), spare respiratory capacity (SRC) ( d ), and ATP production ( e ) was determined following treatment with mitochondrial inhibitors as indicated in ( a ). Data are presented as mean values ± SEM ( n = 4 biologically independent experiments). Statistical significance was determined using a one-way ANOVA adjusted for multiple comparisons. f Mitochondrial number was determined using high content image analysis of Mitotracker stained melanoma cells treated as indicated. Data are presented as mean values ± SEM ( n = 3 biologically independent experiments). Statistical significance was determined using a one-way ANOVA adjusted for multiple comparisons. g Effect of gene knockdown and Vem treatment on expression of the indicated genes was determined using q-RT-PCR. Data are expressed as mean Log2 fold change ( n = 3 biologically independent experiments). h Whole-cell lysates were analysed by western blot analysis for the indicated proteins. Data are representative of n = 3 biologically independent experiments (SE = short exposure; LE = long exposure). i A375 cells were transfected with the indicated siRNA and treated with DMSO or 300 nM Vem, in the presence or absence of electron acceptors pyruvate (1 mM) or α-ketobutyrate (AKB; 1 mM). Cells were fixed and stained with DAPI 5 days post treatment and proliferation rate was calculated [(Log2(Day 5 count/Day 0 count)/4 days]. Data are presented as mean values ± SEM ( n = 4 biologically independent experiments). Statistical significance was determined using a two-way ANOVA adjusted for multiple comparisons. j Cell death was assessed in A375 cells treated as in ( i ) using a propidium iodide (PI) cell death assay. Data are normalized to % confluency and are presented as mean values ± SEM ( n = 4 biologically independent experiments). Statistical significance was determined using a two-way ANOVA adjusted for multiple comparisons. Source data are provided as a Source Data file.

Article Snippet: After the assay, cells were fixed and stained with DAPI DNA dye and cells were imaged using a Cellomics Arrayscan automated microscope.

Techniques: Transfection, Knockdown, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot