Journal: Sensors (Basel, Switzerland)

Article Title: Cyber-Physical System for Smart Traffic Light Control

doi: 10.3390/s23115028

Figure Lengend Snippet: Comparison of various intelligent traffic light systems.

Article Snippet: Abbas et al. [ ] , No , No , SIRDA intersection simulator, MATLAB , No , Yes , No.

Techniques: Comparison

Journal: PLoS Genetics

Article Title: The H3.3K27M oncohistone affects replication stress outcome and provokes genomic instability in pediatric glioma

doi: 10.1371/journal.pgen.1009868

Figure Lengend Snippet: (A) Experimental outline for determining the number of replication stress-induced DNA ultrafine bridges (UFBs) in RPE1 cells. Doxycycline induced asynchronized cells were treated with Aphidicolin (APH) for 24 hrs and subsequently blocked in mitosis with Nocodazole to enrich for mitotic cells. After release, cells were fixed in anaphase enabling visualisation of DNA UFBs. Note that for U251 and BT-174 UFB experiments, no Doxycycline pre-treatment was performed. (B) Confocal images showing representative examples of Bloom (BLM) coated DNA UFBs (indicated by arrowheads) in RPE1 cells (upper panels), U251 cells (middle panels) and BT-174 cells (lower panels). CREST immunolabeling reveals kinetochores, counterstaining was performed with DAPI. Scale bars represent 10 μm. (C) Quantification of Bloom coated DNA UFBs upon Aphidicolin (APH) and Nocodazole treatment of RPE1 cells. Data are represented as means ±SD (n = 3 experiments with +/- 30 mitoses per condition), **P = 0.0013, 0.0012 and 0.0055, (one-way ANOVA, Tukey correction for multiple comparisons). (D) Western blot showing FLAG and H3K27me3 expression in U251 human glioblastoma cells transfected with either H3.3WT or H3.3K27M expression constructs. H4, histone H4, loading control. (E) Quantification of Bloom coated DNA UFBs upon Aphidicolin (APH) and Nocodazole treatment of U251 cells. Data are represented as means ±SD (n = 2 experiments with > 20 mitoses per condition), *P = 0.0377 (paired t-test). (F) Confocal images showing representative stainings of neural markers in BT-174 H3.3K27M pediatric high-grade glioma cells. Counterstaining was performed with DAPI. Scale bars represent 20 μm. (G) Western blot showing H3.3K27M expression in BT-174 parental K27M cells or bulk BT-174 CRISPR-KO cells (Bulk KO #1, #4, #5, and #6). H4, histone H4, loading control. (H) Quantification of Bloom coated DNA UFBs upon Aphidicolin (APH) and Nocodazole treatment of parental BT-174 K27M cells or bulk BT-174 CRISPR-KO cells. Data are represented as means ±SD (n = 3–5 experiments with > 20 mitoses per condition), *P = 0.0357 and **P = 0.0051 (unpaired t-test). (I) Western blots showing ( i ) H3.3K27M and H3.3 expression, and ( ii ) H3K27me3 expression in BT-174 parental K27M cells or single-cell derived BT-174 CRISPR-KO clones (sc-Clone #12, #15, and #25). H4, histone H4, loading control. (J) Quantification of Bloom coated DNA UFBs upon Aphidicolin (APH) and Nocodazole treatment of parental BT-174 K27M cells or single-cell derived BT-174 CRISPR-KO clones. Data are represented as means ±SD (n = 3 experiments with > 30 mitoses per condition), *P = 0.0381 and P = 0.0497 (unpaired t-test).

Article Snippet: Two hrs prior to imaging, cells were refreshed with media containing 150 or 500 ng/ml Doxycycline and 20 nM fluorescent DNA binding dye (SirDNA, Spirochrome TebuBio) [ ].

Techniques: Immunolabeling, Western Blot, Expressing, Transfection, Construct, CRISPR, Derivative Assay, Clone Assay

Journal: PLoS Genetics

Article Title: The H3.3K27M oncohistone affects replication stress outcome and provokes genomic instability in pediatric glioma

doi: 10.1371/journal.pgen.1009868

Figure Lengend Snippet: (A) Experimental outline for measuring 53BP1 nuclear body formation by time lapse microscopy in replication stress conditions (Low dose Aphidicolin (0.2 μM) treatment for 24 hrs following 48 hrs of Doxycycline induction). (B) Immunofluorescent images depicting typical mirrored 53BP1 nuclear bodies in two daughter cells commonly seen after rupture of an unresolved DNA UFB. Scale bars represents 10 μm. ( C) Quantification of 53BP1 nuclear bodies (NB) following 24 hrs Aphidicolin treatment by time lapse microscopy. Chart representing the percentage of cells with 53BP1 nuclear body formation after mitosis. Data are represented as means ±SD (n = 3 experiments with > 20 mitoses per condition), *P = 0.0463 and 0.0430 (one-way ANOVA, Tukey correction for multiple comparisons). (D) Western blot showing EZH2, GAPDH and H3K27me3 expression in H3.3 WT or H3.3 K27M RPE1 cells treated with a scrambled shRNA (SCR), or an shRNA against EZH2 (shEZH2). H4, histone H4, loading control. (E) Quantification of 53BP1 nuclear bodies (NB) following 24 hrs Aphidicolin treatment of H3.3 WT or H3.3 K27M RPE1 cells treated with a scrambled shRNA (SCR), or an shRNA against EZH2 (shEZH2). Chart representing the average number of 53BP1 nuclear bodies per nucleus. Data are represented as means ±SD (n = 3 experiments with > 70 nuclei per condition), **P = 0.0055 (one-way ANOVA, Dunnett correction for multiple comparisons). (F) Representative images of two untreated pediatric HGG biopsy samples stained for proliferation marker PCNA (red arrows) and 53BP1 (yellow arrows) to reveal the nuclear body load. Dashed squares indicate the zoomed area. Scale bar represents 10 μm (5 μm on zooms). (G) Quantification of the proliferation rate ( i . e ., percentage of PCNA positive cells) and 53BP1 nuclear body (NB) load (percentage 53BP1 positive cells) per HGG tumor biopsy. Each circle represents the number for one tumor (HGG Histone WT , n = 5; HGG Histone H3.3 Mutant , n = 6). Data are represented as means per histone group ±SD (with > 200 cells analyzed per tumor). **P = 0.0087 (Mann Whitney non-parametric t-test).

Article Snippet: Two hrs prior to imaging, cells were refreshed with media containing 150 or 500 ng/ml Doxycycline and 20 nM fluorescent DNA binding dye (SirDNA, Spirochrome TebuBio) [ ].

Techniques: Time-lapse Microscopy, Western Blot, Expressing, shRNA, Staining, Marker, Mutagenesis, MANN-WHITNEY