Journal: eLife

Article Title: UV irradiation alters TFAM binding specificity and compaction of DNA

doi: 10.7554/eLife.108862

Figure Lengend Snippet: ( A ) TFAM is one of the dominant proteins of the mitochondrial nucleoid and has been shown in vitro to be sufficient to coat and compact the genome. Compaction status of the mitochondrial nucleoid is associated with the activity of the nucleoid wherein loose and open genomes are accessible to polymerases and protein complexes required for transcription and replication, while compacted and closed-off genomes are inaccessible and inactive. ( B ) In its role as a transcription factor, TFAM serves to initiate both transcription and replication. TFAM binds upstream of one of the transcription start sites and recruits mitochondrial RNA polymerase (POLRMT). Transcription factor B2 mitochondrial (TFB2M) is then recruited to form the initiation complex. Synthesis of RNA by POLRMT then proceeds with transcription elongation factor mitochondrial (TEFM). Transcription of genome-length RNA transcripts can then occur. Production of a short RNA primer by POLRMT allows for the replication of the mitochondrial genome by the replicative polymerase γ, comprising the catalytic subunit (POLG) and its accessory subunit (POLG2). Created with BioRender.com .

Article Snippet: The GAPDH Endogenous Control (Taqman probe ID: Hs02786624_g1, VIC-MGB) and either TFAM (Taqman probe ID: Hs00273372_s1, FAM-MGB), POLG (Taqman probe ID: Hs00160298_m1, FAM-MGB), NRF-1 (Taqman probe ID: Hs00602161_m1, FAM-MGB), PGC1ɑ (Taqman probe ID: Hs00173304_m1, FAM-MGB), POLRMT (Taqman probe ID: Hs04187596_g1, FAM-MGB), or ND-1 (Taqman probe ID: Hs02596873_s1, FAM-MGB) probes were multiplexed in a 1:1 ratio for each sample.

Techniques: In Vitro, Activity Assay

Journal: eLife

Article Title: UV irradiation alters TFAM binding specificity and compaction of DNA

doi: 10.7554/eLife.108862

Figure Lengend Snippet: For panels A-F, x-axes represent recovery time, i.e., time following the UVC exposure. Doses of UVC used were 0, 10, and 30 J/m 2 . ( A ) Mitochondrial DNA damage levels following UVC exposure. The y-axis represents the level of damage (lesions/10 kb). Data analyzed via two-way ANOVA with a Dunnett’s post-hoc test for multiple comparisons (dose: p <0.0001, time: p =0.0001, interaction: p =0.0024). For panels B-E, y-axes represent fold change normalized to the control (0 J/m 2 ) at each time point. All data was analyzed via two-way ANOVA with Dunnett’s post-hoc test for multiple comparisons. ( B ) TFAM expression level assessed via qPCR following UVC exposure (dose: p =0.003, time: p <0.0001, interaction: p =0.01). ( C ) POLG expression level assessed via qPCR following UVC exposure (dose: p =<0.0001, time: p <0.0001, interaction: p =0.001). ( D ) POLRMT expression level assessed via qPCR following UVC exposure (Dose: p =0.002, time: p =0.008, interaction: p =0.05). ( E ) ND-1 expression level assessed via qPCR following UVC exposure (Dose: p <0.0001, time: p =0.01, interaction: p =0.002). ( F ) Mitochondrial membrane potential assessed via flow cytometry following exposure to 0, 10, 30, or 50 J/m 2 UVC at 6 and 24 hr after exposure. The x-axis represents the exposure group and time point, and the y-axis represents the change in Median Fluorescent Intensities (MFI) of tetramethylrhodamine, methyl ester (TMRM) normalized to the control for each time point. Cells were also exposed to FCCP, a well-known chemical that causes mitochondrial depolarization, as a positive control. Data was analyzed via a two-way ANOVA (treatment: p =0.0008, time: p <0.0001, interaction: p =0.14). ( G ) Cellular ATP levels following exposure to 0, 10, 30, or 50 J/m 2 UVC at 6 and 24 hr after exposure. The x-axis represents the exposure group and time point, and the y-axis represents the ATP content (μM). Data was analyzed via a two-way ANOVA (treatment: p =0.38, time: p <0.001, interaction: p =0.94).

Article Snippet: The GAPDH Endogenous Control (Taqman probe ID: Hs02786624_g1, VIC-MGB) and either TFAM (Taqman probe ID: Hs00273372_s1, FAM-MGB), POLG (Taqman probe ID: Hs00160298_m1, FAM-MGB), NRF-1 (Taqman probe ID: Hs00602161_m1, FAM-MGB), PGC1ɑ (Taqman probe ID: Hs00173304_m1, FAM-MGB), POLRMT (Taqman probe ID: Hs04187596_g1, FAM-MGB), or ND-1 (Taqman probe ID: Hs02596873_s1, FAM-MGB) probes were multiplexed in a 1:1 ratio for each sample.

Techniques: Control, Expressing, Membrane, Flow Cytometry, Positive Control

Journal: eLife

Article Title: UV irradiation alters TFAM binding specificity and compaction of DNA

doi: 10.7554/eLife.108862

Figure Lengend Snippet: ( A ) Schematic depicting the library generation. Each 60 nt sequence contains a 33 nt variable region from the human mitochondrial genome and a 27 nt primer. To ensure full coverage of the mitochondrial genome, the variable region of the sequence was generated using a sliding window with a width of 2 nt. ( B ) The custom DNA library was synthesized and double-stranded on a chip. Two of the chambers were subjected to ultraviolet-C (UVC) irradiation to induce UVC-associated lesions. Chambers were incubated with either 30 or 300 nM Transcription Factor A, Mitochondrial (TFAM) and a fluorophore-labeled antibody. The fluorescent signal associated with bound protein for each DNA spot was determined using a microarray scanner.

Article Snippet: The GAPDH Endogenous Control (Taqman probe ID: Hs02786624_g1, VIC-MGB) and either TFAM (Taqman probe ID: Hs00273372_s1, FAM-MGB), POLG (Taqman probe ID: Hs00160298_m1, FAM-MGB), NRF-1 (Taqman probe ID: Hs00602161_m1, FAM-MGB), PGC1ɑ (Taqman probe ID: Hs00173304_m1, FAM-MGB), POLRMT (Taqman probe ID: Hs04187596_g1, FAM-MGB), or ND-1 (Taqman probe ID: Hs02596873_s1, FAM-MGB) probes were multiplexed in a 1:1 ratio for each sample.

Techniques: Sequencing, Generated, Synthesized, Irradiation, Incubation, Labeling, Microarray

Journal: eLife

Article Title: UV irradiation alters TFAM binding specificity and compaction of DNA

doi: 10.7554/eLife.108862

Figure Lengend Snippet: X-axes represent the normalized median fluorescence intensity values and y-axes represent the count of sequences within each bin for 30 nM Transcription Factor A, Mitochondrial (TFAM) treatment without ultraviolet-C (UVC) irradiation ( A ), 300 nM TFAM treatment without UVC irradiation ( B ), 30 nM TFAM treatment with UVC irradiation ( C ), and 300 nM TFAM treatment with UVC irradiation ( D ). The red line is the Gaussian fit using the parameters in each plot and the equation below, where mu is the mean and sigma is the standard deviation.

Article Snippet: The GAPDH Endogenous Control (Taqman probe ID: Hs02786624_g1, VIC-MGB) and either TFAM (Taqman probe ID: Hs00273372_s1, FAM-MGB), POLG (Taqman probe ID: Hs00160298_m1, FAM-MGB), NRF-1 (Taqman probe ID: Hs00602161_m1, FAM-MGB), PGC1ɑ (Taqman probe ID: Hs00173304_m1, FAM-MGB), POLRMT (Taqman probe ID: Hs04187596_g1, FAM-MGB), or ND-1 (Taqman probe ID: Hs02596873_s1, FAM-MGB) probes were multiplexed in a 1:1 ratio for each sample.

Techniques: Fluorescence, Irradiation, Standard Deviation

Journal: eLife

Article Title: UV irradiation alters TFAM binding specificity and compaction of DNA

doi: 10.7554/eLife.108862

Figure Lengend Snippet: The median z-score is plotted to the coordinate of the middle nucleotide of the variable mitochondrial region of the sequence for the non-UVC-irradiated chamber containing 30 nM TFAM ( A ) and the chamber irradiated with 1500 J/m 2 UVC containing 30 nM TFAM ( B ). The gene map of the mitochondrial genome is shown in the center. Z-score variation is color-coded such that positive z-scores associated with high binding are in blue and progressively get lighter as the z-scores get higher. Negative z-scores associated with low binding are in red. Regions highlighted in yellow are the promoter sequences of the mitochondrial genome on the light strand (LSP1 and LSP2), while regions highlighted in pink are the promoter sequences on the heavy strand (HSP1 and HSP2). Plots of these regions can be found in .

Article Snippet: The GAPDH Endogenous Control (Taqman probe ID: Hs02786624_g1, VIC-MGB) and either TFAM (Taqman probe ID: Hs00273372_s1, FAM-MGB), POLG (Taqman probe ID: Hs00160298_m1, FAM-MGB), NRF-1 (Taqman probe ID: Hs00602161_m1, FAM-MGB), PGC1ɑ (Taqman probe ID: Hs00173304_m1, FAM-MGB), POLRMT (Taqman probe ID: Hs04187596_g1, FAM-MGB), or ND-1 (Taqman probe ID: Hs02596873_s1, FAM-MGB) probes were multiplexed in a 1:1 ratio for each sample.

Techniques: Sequencing, Irradiation, Binding Assay

Journal: eLife

Article Title: UV irradiation alters TFAM binding specificity and compaction of DNA

doi: 10.7554/eLife.108862

Figure Lengend Snippet: The median z-score is plotted to the coordinate of the middle nucleotide of the variable mitochondrial region of the sequence for the non-UVC-irradiated chamber containing 300 nM TFAM ( A ) and the UVC-irradiated chamber containing 300 nM TFAM ( B ). The gene map of the mitochondrial genome is shown in the center. Z-score variation is color-coded such that positive z-scores associated with high binding are in blue and progressively get lighter as the z-scores get higher. Negative z-scores associated with low binding are in red. Regions highlighted in yellow are the promoter sequences of the mitochondrial genome on the light strand (LSP1 and LSP2), while regions highlighted in pink are the promoter sequences on the heavy strand (HSP1 and HSP2).

Article Snippet: The GAPDH Endogenous Control (Taqman probe ID: Hs02786624_g1, VIC-MGB) and either TFAM (Taqman probe ID: Hs00273372_s1, FAM-MGB), POLG (Taqman probe ID: Hs00160298_m1, FAM-MGB), NRF-1 (Taqman probe ID: Hs00602161_m1, FAM-MGB), PGC1ɑ (Taqman probe ID: Hs00173304_m1, FAM-MGB), POLRMT (Taqman probe ID: Hs04187596_g1, FAM-MGB), or ND-1 (Taqman probe ID: Hs02596873_s1, FAM-MGB) probes were multiplexed in a 1:1 ratio for each sample.

Techniques: Sequencing, Irradiation, Binding Assay

Journal: eLife

Article Title: UV irradiation alters TFAM binding specificity and compaction of DNA

doi: 10.7554/eLife.108862

Figure Lengend Snippet: The x-axis represents the median z-score for each probe in experiments performed at 30 nM TFAM and the y-axis represents the median z-score for each in experiments performed at 300 nM TFAM. Distributions to the right and above the graphs represent the distribution of z-scores for each concentration. The red line indicates the line of best fit. Data was analyzed via Pearson’s correlation.

Article Snippet: The GAPDH Endogenous Control (Taqman probe ID: Hs02786624_g1, VIC-MGB) and either TFAM (Taqman probe ID: Hs00273372_s1, FAM-MGB), POLG (Taqman probe ID: Hs00160298_m1, FAM-MGB), NRF-1 (Taqman probe ID: Hs00602161_m1, FAM-MGB), PGC1ɑ (Taqman probe ID: Hs00173304_m1, FAM-MGB), POLRMT (Taqman probe ID: Hs04187596_g1, FAM-MGB), or ND-1 (Taqman probe ID: Hs02596873_s1, FAM-MGB) probes were multiplexed in a 1:1 ratio for each sample.

Techniques: Concentration Assay

Journal: eLife

Article Title: UV irradiation alters TFAM binding specificity and compaction of DNA

doi: 10.7554/eLife.108862

Figure Lengend Snippet: The y-axis represents the z-score for each sequence, and the x-axis depicts the concentration of TFAM used in each experiment as well as the presence or absence of ultraviolet-C (UVC) exposure. The violin plots are color-coded to represent whether a GN 10 G motif was present (green, n=7397) or absent (blue, n=939) within each sequence. For all mitochondrial sequences on the array, sequences were classified as having a GN 10 G motif if the motif was present in the variable region and the data analyzed was the maximum z-score between the two orientations. Data was analyzed using a one-tailed Kolmogorov-Smirnov test where the null hypothesis was sequences containing GN 10 G motif have higher z-scores than those without.

Article Snippet: The GAPDH Endogenous Control (Taqman probe ID: Hs02786624_g1, VIC-MGB) and either TFAM (Taqman probe ID: Hs00273372_s1, FAM-MGB), POLG (Taqman probe ID: Hs00160298_m1, FAM-MGB), NRF-1 (Taqman probe ID: Hs00602161_m1, FAM-MGB), PGC1ɑ (Taqman probe ID: Hs00173304_m1, FAM-MGB), POLRMT (Taqman probe ID: Hs04187596_g1, FAM-MGB), or ND-1 (Taqman probe ID: Hs02596873_s1, FAM-MGB) probes were multiplexed in a 1:1 ratio for each sample.

Techniques: Sequencing, Concentration Assay, One-tailed Test

Journal: eLife

Article Title: UV irradiation alters TFAM binding specificity and compaction of DNA

doi: 10.7554/eLife.108862

Figure Lengend Snippet: ( A ) For all DNase Genomics Footprinting (DGF) sites reported by , that were identified in >90% of analyzed human samples, we selected all 33 bp mitochondrial DNA (mtDNA) probes in our on-chip DNA library that were contained entirely within DGFs. For DGFs shorter than 33 bp, we selected the DNA chip probes that contained the entire DGF. ( B ) The TFAM DNA-binding signal ( i.e . the fluorescence intensity measured on the DNA chip) was significantly higher at probes that overlap footprints (blue) vs. sites outside of footprints (gray); Mann-Whitney test p -value <2.2×10 –16 . ( C ) We also performed a randomization test where we shuffled the positions of the DGF sites 1000 times (keeping the number of DGFs and the distribution of their lengths constant). For each randomization, we repeated the analysis and found that in only 2 of 1000 random samples, the mean TFAM binding was at least as large as in the real DGF data ( p =0.002).

Article Snippet: The GAPDH Endogenous Control (Taqman probe ID: Hs02786624_g1, VIC-MGB) and either TFAM (Taqman probe ID: Hs00273372_s1, FAM-MGB), POLG (Taqman probe ID: Hs00160298_m1, FAM-MGB), NRF-1 (Taqman probe ID: Hs00602161_m1, FAM-MGB), PGC1ɑ (Taqman probe ID: Hs00173304_m1, FAM-MGB), POLRMT (Taqman probe ID: Hs04187596_g1, FAM-MGB), or ND-1 (Taqman probe ID: Hs02596873_s1, FAM-MGB) probes were multiplexed in a 1:1 ratio for each sample.

Techniques: Footprinting, Binding Assay, Fluorescence, MANN-WHITNEY

Journal: eLife

Article Title: UV irradiation alters TFAM binding specificity and compaction of DNA

doi: 10.7554/eLife.108862

Figure Lengend Snippet: For high and low TFAM binding regions reported by , determined using fiber-seq on linear mitochondrial DNA (mtDNA), we selected all 33 bp mtDNA probes in our on-chip DNA library that were contained entirely within the region. For regions shorter than 33 bp, we selected the DNA chip probes that contained the entire region. ( A ) For probes within the low binding regions, we observe lower TFAM DNA-binding signal ( i.e . the fluorescence intensity measured on the DNA chip) (red), and for probes within the high binding regions, we observe moderate to high levels of TFAM DNA-binding signal (blue) than all other probes that were not within the high and low TFAM regions reported by Isaac et al. Data was analyzed via Mann-Whitney test (all other probes vs. low regions p -value <3.0×10 –18 ; all other probes vs. high regions p -value <8.9×10 –05 ; low regions vs. high regions p -value <2.8×10 –13 ). We also performed a randomization test where we shuffled the positions of both the low ( B ) and high ( C ) TFAM binding regions 1000 times (keeping the number of regions and the distribution of their lengths constant). For the low binding regions, only 8 of the 1000 random samples had binding signal less than the mean for the probes within the low regions ( p =0.008). However, for the high binding regions, 81 of the 1000 random samples had binding signal higher than the mean for the probes within the high regions ( p =0.081).

Article Snippet: The GAPDH Endogenous Control (Taqman probe ID: Hs02786624_g1, VIC-MGB) and either TFAM (Taqman probe ID: Hs00273372_s1, FAM-MGB), POLG (Taqman probe ID: Hs00160298_m1, FAM-MGB), NRF-1 (Taqman probe ID: Hs00602161_m1, FAM-MGB), PGC1ɑ (Taqman probe ID: Hs00173304_m1, FAM-MGB), POLRMT (Taqman probe ID: Hs04187596_g1, FAM-MGB), or ND-1 (Taqman probe ID: Hs02596873_s1, FAM-MGB) probes were multiplexed in a 1:1 ratio for each sample.

Techniques: Binding Assay, Fluorescence, MANN-WHITNEY

Journal: eLife

Article Title: UV irradiation alters TFAM binding specificity and compaction of DNA

doi: 10.7554/eLife.108862

Figure Lengend Snippet: For panels A-D, the median z-score is plotted to the coordinate of the middle nucleotide of the variable mitochondrial region of the sequence for the non-UVC-irradiated chamber containing 30 nM TFAM (top panels, in blue) and the UVC-irradiated chamber containing 30 nM TFAM (bottom panels, in green). Z-score variation is color-coded such that positive z-scores associated with high binding are in blue and negative z-scores associated with low binding are in red. Panels represent the shaded regions in for LSP1 ( A ), HSP1 ( B ), LSP2 ( C ), and HSP2 ( D ). Regions highlighted in yellow are LSP TFAM binding sites, regions highlighted pink are HSP TFAM binding sites, and regions highlighted in green are transcription start sites.

Article Snippet: The GAPDH Endogenous Control (Taqman probe ID: Hs02786624_g1, VIC-MGB) and either TFAM (Taqman probe ID: Hs00273372_s1, FAM-MGB), POLG (Taqman probe ID: Hs00160298_m1, FAM-MGB), NRF-1 (Taqman probe ID: Hs00602161_m1, FAM-MGB), PGC1ɑ (Taqman probe ID: Hs00173304_m1, FAM-MGB), POLRMT (Taqman probe ID: Hs04187596_g1, FAM-MGB), or ND-1 (Taqman probe ID: Hs02596873_s1, FAM-MGB) probes were multiplexed in a 1:1 ratio for each sample.

Techniques: Sequencing, Irradiation, Binding Assay

Journal: eLife

Article Title: UV irradiation alters TFAM binding specificity and compaction of DNA

doi: 10.7554/eLife.108862

Figure Lengend Snippet: Panels A and B are kernel density estimates of the probes in the top 5% of z-scores ( A ) and the bottom 5% of z-scores ( B ) in the context of non-damaged DNA and UVC-irradiated DNA. X-axes represent the median z-score of the probe and y-axes represent the probability density. Note differences in y-axes between panels A and B. ( C ) The x-axis represents the median z-score for each probe in experiments performed at 30 nM TFAM and the y-axis represents the change in z-score for each probe between non-UVC and UVC-irradiated experiments. The distribution plot above of the graph indicates the distribution of median z-score values in the non-damaged probes performed at 30 nM TFAM. The distribution plot to the right of the graph indicates the distribution of the difference in z-scores calculated between non-UVC and UVC-irradiated experiments. The red line indicates the line of best fit. Data was analyzed via Pearson’s correlation.

Article Snippet: The GAPDH Endogenous Control (Taqman probe ID: Hs02786624_g1, VIC-MGB) and either TFAM (Taqman probe ID: Hs00273372_s1, FAM-MGB), POLG (Taqman probe ID: Hs00160298_m1, FAM-MGB), NRF-1 (Taqman probe ID: Hs00602161_m1, FAM-MGB), PGC1ɑ (Taqman probe ID: Hs00173304_m1, FAM-MGB), POLRMT (Taqman probe ID: Hs04187596_g1, FAM-MGB), or ND-1 (Taqman probe ID: Hs02596873_s1, FAM-MGB) probes were multiplexed in a 1:1 ratio for each sample.

Techniques: Irradiation

Journal: eLife

Article Title: UV irradiation alters TFAM binding specificity and compaction of DNA

doi: 10.7554/eLife.108862

Figure Lengend Snippet: For all graphs, the x-axes represent the Transcription Factor A, Mitochondrial (TFAM) concentration (nM) and the y-axes represent anisotropy. The three lines on each graph represent three replicates performed. Graphs in the top half (white) are the sequences tested without UV exposure. Graphs in the bottom half (gray) represent sequences that were first irradiated with ultraviolet-C (UVC). Sequences can be found in . K D values and n values can be found in .

Article Snippet: The GAPDH Endogenous Control (Taqman probe ID: Hs02786624_g1, VIC-MGB) and either TFAM (Taqman probe ID: Hs00273372_s1, FAM-MGB), POLG (Taqman probe ID: Hs00160298_m1, FAM-MGB), NRF-1 (Taqman probe ID: Hs00602161_m1, FAM-MGB), PGC1ɑ (Taqman probe ID: Hs00173304_m1, FAM-MGB), POLRMT (Taqman probe ID: Hs04187596_g1, FAM-MGB), or ND-1 (Taqman probe ID: Hs02596873_s1, FAM-MGB) probes were multiplexed in a 1:1 ratio for each sample.

Techniques: Concentration Assay, Irradiation

Journal: eLife

Article Title: UV irradiation alters TFAM binding specificity and compaction of DNA

doi: 10.7554/eLife.108862

Figure Lengend Snippet: ( A ) Top panel is a histogram plot for the volumes of TFAM protein only at 7.5 nM. The red line is aligned with the peak at 33 nm 3 in the TFAM only histogram to help show the shift in peaks for the TFAM with short DNA sequences in the two bottom panels. ( B ) Middle panel is the volume distribution of a 30 nM TFAM incubated with a final concentration of 10 nM of the low occupancy sequence ND4-473. ( C ) The bottom panel is the volume distribution of 30 nM TFAM in the presence of 10 nM high occupancy sequence ND1-353.

Article Snippet: The GAPDH Endogenous Control (Taqman probe ID: Hs02786624_g1, VIC-MGB) and either TFAM (Taqman probe ID: Hs00273372_s1, FAM-MGB), POLG (Taqman probe ID: Hs00160298_m1, FAM-MGB), NRF-1 (Taqman probe ID: Hs00602161_m1, FAM-MGB), PGC1ɑ (Taqman probe ID: Hs00173304_m1, FAM-MGB), POLRMT (Taqman probe ID: Hs04187596_g1, FAM-MGB), or ND-1 (Taqman probe ID: Hs02596873_s1, FAM-MGB) probes were multiplexed in a 1:1 ratio for each sample.

Techniques: Incubation, Concentration Assay, Sequencing

Journal: eLife

Article Title: UV irradiation alters TFAM binding specificity and compaction of DNA

doi: 10.7554/eLife.108862

Figure Lengend Snippet: ( A ) Atomic force microscopy (AFM) images of the pUC19 DNA only (control) and the 30 nM TFAM-DNA complexes in two different conditions: one with (+UV) or without (-UV) UVC irradiated DNA. Colored arrows on the AFM images represent the different TFAM-DNA complexes categorizations: dispersed (green), intermediate (yellow), punctate (red), and free DNA (blue). The white scale bar represents 1 μm. UV-irradiated plasmids were exposed to 100 J/m 2 UVC. ( B ) Histogram plot of the volumes distribution of plasmid DNA only (control) as well as the 15 nM and 30 nM TFAM-DNA complexes with (+UV) or without (-UV) UVC-damaged DNA. All axes in the histogram are scaled the same. The data for control pUC19 only was replicated for each TFAM concentration for clarity in comparisons. ( C ) Atomic force microscopy images of three different TFAM-DNA complex categorizations labeled as dispersed (small clusters with no protein tracts on DNA), intermediate (small clusters with protein tracts), and punctate (tightly associated punctate clusters). The white scale bar represents 200 nm. ( D ) A bar graph representing the percent total number of plasmids in the 15 nM TFAM concentration with (+UV) (N=66) or without (-UV) (N=136) UVC damaged DNA and the 30 nM TFAM concentration with (+UV) (N=65) or without (-UV) (N=91) UVC damaged DNA. Detailed counts of each classification can be found in . ( E ) Schematic of the TFAM-DNA binding and compaction mechanism.

Article Snippet: The GAPDH Endogenous Control (Taqman probe ID: Hs02786624_g1, VIC-MGB) and either TFAM (Taqman probe ID: Hs00273372_s1, FAM-MGB), POLG (Taqman probe ID: Hs00160298_m1, FAM-MGB), NRF-1 (Taqman probe ID: Hs00602161_m1, FAM-MGB), PGC1ɑ (Taqman probe ID: Hs00173304_m1, FAM-MGB), POLRMT (Taqman probe ID: Hs04187596_g1, FAM-MGB), or ND-1 (Taqman probe ID: Hs02596873_s1, FAM-MGB) probes were multiplexed in a 1:1 ratio for each sample.

Techniques: Microscopy, Control, Irradiation, Plasmid Preparation, Concentration Assay, Labeling, Binding Assay

Journal: eLife

Article Title: UV irradiation alters TFAM binding specificity and compaction of DNA

doi: 10.7554/eLife.108862

Figure Lengend Snippet: ( A ) 2D AFM images with TFAM-DNA complexes, with white arrows pointing at the tracts of TFAM along the DNA (white scale bars are all 100 nm). ( B ) 3D AFM images of two images in panel A (images labeled 1 and 2) showing the difference in areas along the DNA without protein and regions with DNA and protein (TFAM tracts). In the 3D AFM images, it is clear to see that the areas with DNA only (no protein) are much lower in height (nm), whereas the areas with protein along the DNA (TFAM tracts) are higher in height.

Article Snippet: The GAPDH Endogenous Control (Taqman probe ID: Hs02786624_g1, VIC-MGB) and either TFAM (Taqman probe ID: Hs00273372_s1, FAM-MGB), POLG (Taqman probe ID: Hs00160298_m1, FAM-MGB), NRF-1 (Taqman probe ID: Hs00602161_m1, FAM-MGB), PGC1ɑ (Taqman probe ID: Hs00173304_m1, FAM-MGB), POLRMT (Taqman probe ID: Hs04187596_g1, FAM-MGB), or ND-1 (Taqman probe ID: Hs02596873_s1, FAM-MGB) probes were multiplexed in a 1:1 ratio for each sample.

Techniques: Labeling

Journal: eLife

Article Title: UV irradiation alters TFAM binding specificity and compaction of DNA

doi: 10.7554/eLife.108862

Figure Lengend Snippet: The top panels show examples of 15 nM TFAM in the presence of undamaged (-UV) pUC19 DNA and the bottom panels show examples of 15 nM TFAM in the presence of damaged (+UV) pUC19 DNA. Colored arrows on the AFM images represent the different TFAM-DNA complexes categorizations dispersed (green), intermediate (yellow), punctate (red), and protein-free DNA (blue). Each AFM image is 2×2 μm and 512×512 size pixels.

Article Snippet: The GAPDH Endogenous Control (Taqman probe ID: Hs02786624_g1, VIC-MGB) and either TFAM (Taqman probe ID: Hs00273372_s1, FAM-MGB), POLG (Taqman probe ID: Hs00160298_m1, FAM-MGB), NRF-1 (Taqman probe ID: Hs00602161_m1, FAM-MGB), PGC1ɑ (Taqman probe ID: Hs00173304_m1, FAM-MGB), POLRMT (Taqman probe ID: Hs04187596_g1, FAM-MGB), or ND-1 (Taqman probe ID: Hs02596873_s1, FAM-MGB) probes were multiplexed in a 1:1 ratio for each sample.

Techniques:

Journal: eLife

Article Title: UV irradiation alters TFAM binding specificity and compaction of DNA

doi: 10.7554/eLife.108862

Figure Lengend Snippet: ( A ) Exposure paradigm for TFAM overexpression experiments. Cells were exposed to doxycycline for 48 hr prior to UVC exposure to ensure upregulation of TFAM at the time of exposure. Protein was quantified in control cells only to ensure TFAM upregulation. ( B ) Representative western blot of TFAM-tetON cell lysates following 48, 72, and 96 hr of doxycycline treatment to confirm TFAM upregulation. TFAM-tetON cells contain an HA tag that when expressed, results in a second band. ( C ) Pixel quantifications of n=3 western blots shown in panel ( B ) at each time point. TFAM protein levels were normalized to β-actin and then normalized to the non-doxycycline-treated controls. The x-axis represents the doxycycline treatment and y-axis represents the fold change relative to the non-doxycycline-treated cells. Data was analyzed via two-way ANOVA (doxycycline treatment p =0.0006, time p =0.25, interaction p =0.25). ( D ) TFAM mRNA quantification following doxycycline treatment. The x-axis represents the doxycycline treatment, and the y-axis represents the fold change relative to the non-doxycycline-treated cells. Data was analyzed via two-way ANOVA (doxycycline treatment p =0.0003, time p =0.52, interaction p =0.52). ( E ) Lesion frequency following UVC exposure in TFAM-tetON cells immediately after the exposure (0 hr recovery time). The x-axis represents the doxycycline treatment across a range of UVC doses and y-axis represents the lesion frequency (lesions per 10 kb). Data was analyzed via two-way ANOVA (UVC dose: p <0.0001, doxycycline treatment p =0.15, interaction: p =0.77). ( F ) Lesion frequency following UVC exposure in TFAM-tetON cells 24 hr after the exposure (24 hr recovery time). The x-axis represents the doxycycline treatment across a range of UVC doses and y-axis represents the lesion frequency (lesions per 10 kb). Data was analyzed via two-way ANOVA (UVC dose: p <0.0001, doxycycline treatment p =0.71, interaction: p =0.65). ( G ) Lesion frequency following UVC exposure in TFAM-tetON cells 48 hr after the exposure (48 hr recovery time). The x-axis represents the doxycycline treatment across a range of UVC doses and y-axis represents the lesion frequency (lesions per 10 kb). Data was analyzed via two-way ANOVA (UVC dose: p <0.0001, doxycycline treatment p =0.01, interaction: p =0.61). ( H ) Representative atomic force microscopy (AFM) images of in vitro nucleoids generated using purified TFAM and PCR amplified human mtDNA at 0, 100, 250, and 1000 nM TFAM. All scale bars represent 500 nm. ( I ) Lesion frequency following UVC exposure in in vitro nucleoids. The x-axis represents the UVC dose and y-axis represents the lesion frequency (lesions per 10 kb). Data was analyzed via two-way ANOVA (UVC dose: p <0.0001, TFAM concentration p =0.20, interaction: p =0.86). ( J ) Lesion frequency following UVC exposure in cells with and without a TFAM knockdown. The x-axis represents the recovery time, i.e., time following exposure to UVC, and the y-axis represents the level of mtDNA damage (lesions per 10 kb). Data was analyzed via a three-way ANOVA (UVC dose: p <0.0001, recovery time: p =0.002, siRNA treatment: p <0.0001, recovery time*UVC dose: p <0.0001, recovery time*siRNA treatment: p =0.001, UVC dose*siRNA treatment: p =0.08, recovery time*UVC dose*siRNA treatment: p =0.66). ( K ) Ratio of mtDNA copy number to nuclear DNA copy number following UVC exposure in cells with and without a TFAM knockdown. The x-axis represents the recovery time, i.e., time following exposure to UVC, and the y-axis represents the level of mtDNA damage (lesions per 10 kb). Data was analyzed via a three-way ANOVA (UVC dose: p =0.01, recovery time: p <0.01, siRNA treatment: p <0.0001, recovery time*UVC dose: p <0.001, recovery time*siRNA treatment: p <0.0001, UVC dose*siRNA treatment: p =0.04, recovery time*UVC dose*siRNA treatment: p =0.01). Figure 5—source data 1. Original files for western blot analysis displayed in . Figure 5—source data 2. PDF file containing original western blot analysis displayed in , indicating relevant bands, treatment groups, and time points.

Article Snippet: The GAPDH Endogenous Control (Taqman probe ID: Hs02786624_g1, VIC-MGB) and either TFAM (Taqman probe ID: Hs00273372_s1, FAM-MGB), POLG (Taqman probe ID: Hs00160298_m1, FAM-MGB), NRF-1 (Taqman probe ID: Hs00602161_m1, FAM-MGB), PGC1ɑ (Taqman probe ID: Hs00173304_m1, FAM-MGB), POLRMT (Taqman probe ID: Hs04187596_g1, FAM-MGB), or ND-1 (Taqman probe ID: Hs02596873_s1, FAM-MGB) probes were multiplexed in a 1:1 ratio for each sample.

Techniques: Over Expression, Control, Western Blot, Microscopy, In Vitro, Generated, Purification, Amplification, Concentration Assay, Knockdown

Journal: eLife

Article Title: UV irradiation alters TFAM binding specificity and compaction of DNA

doi: 10.7554/eLife.108862

Figure Lengend Snippet: The in vitro data provided in this study indicate that TFAM more readily compacts DNA harboring UV-induced lesions. While there is currently no in vivo evidence to suggest damaged mtDNA is more compacted, future work should determine whether this phenomenon is occurring in cells. In vivo, this feature may serve to ‘tag’ mitochondrial genomes as damaged, which could lend itself to repression of the replication of damaged genomes, flagging damaged genomes for targeted degradation, or both. Both active removal and repression of replication would allow for removal of damaged genomes during mtDNA turnover processes and provide a mechanism for preventing mtDNA mutagenesis. Created with BioRender.com .

Article Snippet: The GAPDH Endogenous Control (Taqman probe ID: Hs02786624_g1, VIC-MGB) and either TFAM (Taqman probe ID: Hs00273372_s1, FAM-MGB), POLG (Taqman probe ID: Hs00160298_m1, FAM-MGB), NRF-1 (Taqman probe ID: Hs00602161_m1, FAM-MGB), PGC1ɑ (Taqman probe ID: Hs00173304_m1, FAM-MGB), POLRMT (Taqman probe ID: Hs04187596_g1, FAM-MGB), or ND-1 (Taqman probe ID: Hs02596873_s1, FAM-MGB) probes were multiplexed in a 1:1 ratio for each sample.

Techniques: In Vitro, In Vivo, Mutagenesis

Journal: bioRxiv

Article Title: AURKA promotes malignant properties of neuroblastoma and is downregulated by the PP2A pathway

doi: 10.1101/2025.07.29.665837

Figure Lengend Snippet: Shown is mRNA expression of AURKA (A), CDKN2A (B), PPP2R4 (C) and of an AURKA signature (D) consisting of expression of AURKA and 18 genes important for regulating AURKA during progression of NB in ganglia of TH-MYCN mice compared to ganglia from wild-type mice. The means and standard deviations of four samples depending on age are depicted. The difference in the regression slope between transgenic and wild-type samples was calculated. The multiple testing-corrected p -value for the linear regression analysis is shown. E) Enhanced expression of AURKA after transfection and stable selection of SH-EP-MYCN-ER cells. Representative Western blot of AURKA in transfected SH-EP-MYCN-ER cells. AURKA expression was quantified by densitometry relative to β-Actin. F) Enhanced anchorage-independent growth of SH-EP-MYCN-ER cells overexpressing AURKA upon induced nuclear translocation of MYCN-ER. Stably selected SH-EP-MYCN-ER cells were seeded into soft agar in 24-well plates and grown for 2 weeks. 4-hydroxytamoxifen (4-OHT) was added to culture media to translocate MYCN-ER into the nucleus. Colonies of more than 30 cells were counted. Means of more than 20 wells are shown. ***, p < 0.001 and **** p < 0.0001 by one-way ANOVA.

Article Snippet: Membranes were probed with the primary antibodies rabbit anti-human AURKA (#4718, 1:1000, Cell Signaling Danvers, MA), rabbit anti-human-PPP2R4 (PTPA) (#HPA005695, 1:5000, Sigma-Aldrich), mouse anti-mePPP2CA (sc-81603, 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-Actin (A5441, 1:5000, Sigma-Aldrich) and mouse anti-Tubulin (clone DM1A1, 1:5000, Sigma-Aldrich).

Techniques: Expressing, Transgenic Assay, Transfection, Selection, Western Blot, Translocation Assay, Stable Transfection

Journal: bioRxiv

Article Title: AURKA promotes malignant properties of neuroblastoma and is downregulated by the PP2A pathway

doi: 10.1101/2025.07.29.665837

Figure Lengend Snippet: Kaplan-Meier analysis of overall survival of 498 clinically annotated NB patients (SECQ-GSE62564) depending on transcript levels of AURKA , PPP2R4 and PPP2CA (A) or of the combination of AURKA and PPP2R4 or AURKA and PPP2CA (B) are shown. Statistical analysis of Kaplan-Meier curves was performed using the log-rank test with Bonferroni correction. The cut-off was determined by the scanning method.

Article Snippet: Membranes were probed with the primary antibodies rabbit anti-human AURKA (#4718, 1:1000, Cell Signaling Danvers, MA), rabbit anti-human-PPP2R4 (PTPA) (#HPA005695, 1:5000, Sigma-Aldrich), mouse anti-mePPP2CA (sc-81603, 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-Actin (A5441, 1:5000, Sigma-Aldrich) and mouse anti-Tubulin (clone DM1A1, 1:5000, Sigma-Aldrich).

Techniques:

Journal: bioRxiv

Article Title: AURKA promotes malignant properties of neuroblastoma and is downregulated by the PP2A pathway

doi: 10.1101/2025.07.29.665837

Figure Lengend Snippet: A) Bayesian analysis of mRNA expression in ganglia and NB of TH-MYCN mice suggests a role of PPP2R4 in dysregulation of AURKA in neuroblastomagenesis. Microarray mRNA data previously described in were used. 85 genes implicated in the AURKA network were subjected to Bayesian analysis. The direction (arrows) and probability (edge thickness) of influence of the top 20 genes according to the differences of mean expression in NB vs. ganglia are shown. Genes are color-coded according to function in relation to AURKA. B) Loss of PPP2R4 depletes mePPP2CA protein and increases AURKA protein in KELLY cells. KELLY cells were transiently transfected with a CRISPR/Cas9 construct targeting PPP2R4 , sorted and seeded as single cells. Single-cell clones were expanded and classified as PPP2R4 wild-type or homozygous knockout. Shown are Western blots of PPP2R4, methylated PPP2CA (mePPP2CA), AURKA and Tubulin as loading control. C) Schematic cartoon depicting the relationship between AURKA, PPP2R4, MYCN and INK4A/ARF in promoting malignant properties of NB.

Article Snippet: Membranes were probed with the primary antibodies rabbit anti-human AURKA (#4718, 1:1000, Cell Signaling Danvers, MA), rabbit anti-human-PPP2R4 (PTPA) (#HPA005695, 1:5000, Sigma-Aldrich), mouse anti-mePPP2CA (sc-81603, 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-Actin (A5441, 1:5000, Sigma-Aldrich) and mouse anti-Tubulin (clone DM1A1, 1:5000, Sigma-Aldrich).

Techniques: Expressing, Microarray, Transfection, CRISPR, Construct, Clone Assay, Knock-Out, Western Blot, Methylation, Control

Journal: EBioMedicine

Article Title: NFIB Mediates BRN2 Driven Melanoma Cell Migration and Invasion Through Regulation of EZH2 and MITF

doi: 10.1016/j.ebiom.2017.01.013

Figure Lengend Snippet: NFIB expression correlates with BRN2 in melanocytic and melanoma cells. (A–D) QPCR analysis on A2058 melanoma cells following lentiviral transduction to produce stable over-expression of MITF or BRN2, investigating NFIA , NFIB , NFIC , and NFIX expression. Data represented as fold change relative to the empty vector control and normalized to B2M gene. (E–F) Western Blot analysis on neonatal foreskin-derived QF1236 and QF1566 primary human melanoblast cells induced to differentiate into pigmented melanocytes over a 5-day period. Antibodies were used against BRN2, NFIB, MITF, and GAPDH. (G) Whole cell lysates from six human melanoma cell lines immunoblotted for NFIB, BRN2, and MITF. (H) ChIP-ChIP analysis data in 501 Mel human melanoma cells investigating BRN2 binding to chromatin regions, reveals BRN2 binds to a 2Kb intronic region located upstream of the NFIB promoter. *: P < 0.05, **: P < 0.01, ***: P < 0.001. Data representative of three independent experiments. Band expression intensity of Western Blots was normalized to the first lane (GAPDH used as a loading control) using ImageJ software and indicated below each blot. Data from (A–D) is represented as the mean ± SEM and analysed using a one-way ANOVA with Dunnett's multiple comparisons test. See also Fig. S3.

Article Snippet: Immunoblots were probed using rabbit monoclonal BRN2 antibody D2C1L (Cell Signaling #12137), rabbit monoclonal NFIB antibody (Sigma Aldrich; HPA003956), rabbit monoclonal MITF D567V (Cell Signaling #12590), mouse monoclonal EZH2 (Active Motif #39639), and Rabbit monoclonal H3K27 tri-methyl C36B11 (Cell Signaling #9733), with anti-GAPDH 6C5 (Santa Cruz #sc-32233) used as a loading control.

Techniques: Expressing, Transduction, Over Expression, Plasmid Preparation, Control, Western Blot, Derivative Assay, ChIP-chip, Binding Assay, Software

Journal: EBioMedicine

Article Title: NFIB Mediates BRN2 Driven Melanoma Cell Migration and Invasion Through Regulation of EZH2 and MITF

doi: 10.1016/j.ebiom.2017.01.013

Figure Lengend Snippet: BRN2 positively regulates NFIB and EZH2 expression. (A) Cell lysates from A2058, MM96L, and HT144 human melanoma cells treated with three different siRNA directed against BRN2 were analyzed by Western Blot with antibodies against BRN2, NFIB, EZH2, and GAPDH (B) Cell lysates from A2058, MM96L, and HT144 human melanoma cells treated with lentivirus to create stable overexpression of BRN2 were analyzed by Western Blot with antibodies against BRN2, NFIB, EZH2, and GAPDH (C) Cell lysates from doxocycline-off inducible BRN2 expressing A2058, MM96L, and HT144 cells treated with and without dox for 48 h were analyzed by Western Blot with antibodies against BRN2, NFIB, EZH2, and GAPDH. (D) NFIB immunofluorescence (red) on A2058 parental cells grown on coverslips and treated with siRNA directed against BRN2. DAPI used to stain cell nuclei. (E) NFIB immunofluorescence (red) on A2058 BRN2 over-expressing cells. DAPI used to stain cell nuclei. (F) Cell lysates from A2058 and MM96L melanoma cells stably over-expressing BRN2 were analyzed by Western Blot for H3K27 tri methylation status (EZH2 global methylation marker). Band expression intensity of Western Blots was normalized to the first lane (GAPDH used as a loading control) using ImageJ software and indicated below each blot. All data representative of three independent experiments.

Article Snippet: Immunoblots were probed using rabbit monoclonal BRN2 antibody D2C1L (Cell Signaling #12137), rabbit monoclonal NFIB antibody (Sigma Aldrich; HPA003956), rabbit monoclonal MITF D567V (Cell Signaling #12590), mouse monoclonal EZH2 (Active Motif #39639), and Rabbit monoclonal H3K27 tri-methyl C36B11 (Cell Signaling #9733), with anti-GAPDH 6C5 (Santa Cruz #sc-32233) used as a loading control.

Techniques: Expressing, Western Blot, Over Expression, Immunofluorescence, Staining, Stable Transfection, Methylation, Marker, Control, Software

Journal: EBioMedicine

Article Title: NFIB Mediates BRN2 Driven Melanoma Cell Migration and Invasion Through Regulation of EZH2 and MITF

doi: 10.1016/j.ebiom.2017.01.013

Figure Lengend Snippet: NFIB manipulation increases migration and EZH2 expression and decreases MITF expression. (A) Cell lysates from A2058, MM96L, and HT144 human melanoma cells treated with three different siRNA directed against NFIB were analyzed by Western Blot with antibodies against BRN2, NFIB, EZH2, MITF, and GAPDH. (B) Cell lysates from A2058, MM96L, and HT144 human melanoma cells treated with lentivirus to create stable overexpression of NFIB were analyzed by Western Blot with antibodies against BRN2, NFIB, EZH2, MITF and GAPDH. (C) A2058 stables for MITF, BRN2, NFIB, and empty control cells transfected with two luciferase reporter constructs; A wild type construct containing a region of the EZH2 promoter containing the NFIB putative binding site (WT) and a mutant construct with the NFI binding site mutated out (Mut). Data represented as relative luciferase fold activity following normalisation against the empty-Mut. (D) Cell lysates from stable NFIB overexpressing A2058 and MM96L melanoma cells were analyzed by Western Blot for H3K27 tri methylation status (EZH2 global methylation marker). (E) Quantification of wound healing assay performed on A2058 human melanoma cells treated with siRNA against BRN2, MITF or a scrambled control 24 h prior to wound initiation. (F) Quantification of wound healing assay performed in A2058 stable BRN2, MITF and empty control cells. (G) Quantification of wound healing assay performed in A2058 human melanoma cells treated with siRNA against BRN2, MITF or a scrambled control 24 h prior to commencement of the experiment. (H) Quantification of wound healing assay performed in A2058 stable NFIB and empty control cells. Data represented as the mean ± SEM. *: P < 0.05, ***: P < 0.001, ****: P < 0.0001. A two-way ANOVA with a Tukey's post-hoc test was performed in (C) and (E–H). All data representative of three independent experiments. See also Figs. S1, S2, S3 and S5.

Article Snippet: Immunoblots were probed using rabbit monoclonal BRN2 antibody D2C1L (Cell Signaling #12137), rabbit monoclonal NFIB antibody (Sigma Aldrich; HPA003956), rabbit monoclonal MITF D567V (Cell Signaling #12590), mouse monoclonal EZH2 (Active Motif #39639), and Rabbit monoclonal H3K27 tri-methyl C36B11 (Cell Signaling #9733), with anti-GAPDH 6C5 (Santa Cruz #sc-32233) used as a loading control.

Techniques: Migration, Expressing, Western Blot, Over Expression, Control, Transfection, Luciferase, Construct, Binding Assay, Mutagenesis, Activity Assay, Methylation, Marker, Wound Healing Assay

Journal: EBioMedicine

Article Title: NFIB Mediates BRN2 Driven Melanoma Cell Migration and Invasion Through Regulation of EZH2 and MITF

doi: 10.1016/j.ebiom.2017.01.013

Figure Lengend Snippet: NFIB drives migration downstream of BRN2 through interactions with EZH2. (A–B) Lightphase images and quantification of wound healing assays performed in A2058 stable BRN2 cells treated with siRNA against BRN2, NFIB or a scrambled control 24 h prior to wound initiation (0 h), with images taken at 0, 24, and 48 h. (C) Cell lysates from A2058 BRN2 stable melanoma cells treated with siRNA against BRN2, NFIB or a scrambled control were analyzed by Western Blot with antibodies against BRN2, NFIB, EZH2, and GAPDH. (D-E) Lightphase images and quantification of wound healing assays performed in A2058 stable NFIB cells treated with EZH2 inhibitor GSK343 at 1 μM or a vehicle control (DMSO) 24 h prior to wound inititation (0 h), with images taken at 0, 24, and 48 h. (F) Cell lysates from A2058 NFIB stable melanoma cells treated with GSK343 at 0.1 μM, 1 μM or a vehicle control (DMSO) were analyzed by Western Blot with antibodies against BRN2, NFIB, EZH2, MITF and GAPDH. (G-H) Lightphase images and quantification of wound healing assays performed in A2058 stable NFIB melanoma cells treated with MITF or empty control lentivirus 24 h prior to commencement of the experiment, with images taken at 0, 24, and 48 h. Data represented as the mean ± SEM and analysed with two-way ANOVA with a Tukey's post hoc test. **: P < 0.01, ***: P < 0.001, ****: P < 0.0001. All data representative of three independent experiments. Band expression intensity of Western Blots was normalized to the first lane (GAPDH used as a loading control) using ImageJ software and indicated below each blot. See also Figs. S2 and S6.

Article Snippet: Immunoblots were probed using rabbit monoclonal BRN2 antibody D2C1L (Cell Signaling #12137), rabbit monoclonal NFIB antibody (Sigma Aldrich; HPA003956), rabbit monoclonal MITF D567V (Cell Signaling #12590), mouse monoclonal EZH2 (Active Motif #39639), and Rabbit monoclonal H3K27 tri-methyl C36B11 (Cell Signaling #9733), with anti-GAPDH 6C5 (Santa Cruz #sc-32233) used as a loading control.

Techniques: Migration, Control, Western Blot, Expressing, Software

Journal: EBioMedicine

Article Title: NFIB Mediates BRN2 Driven Melanoma Cell Migration and Invasion Through Regulation of EZH2 and MITF

doi: 10.1016/j.ebiom.2017.01.013

Figure Lengend Snippet: Overexpression of BRN2 decreases melanoma cell tumourigenicity but increases invasion. (A–B) 2 × 10 5 A2058 human melanoma cells with stable over-expression of empty control or NFIB were injected subcutaneously into the hind flanks of five 5-week old male immunocompromised BALB/c Foxn1 nu mice. Three-dimensional measurement was performed two times per week, with tumour volume expressed as mm 3 . (C) Analysis of 471 melanoma samples in the TCGA dataset comparing NFIB expression and its correlation with a previously reported invasive gene signature . Grey bars represent average expression of NFIB in each individual tumour, while the black line is a rank based on the average expression of the invasive gene signature used for initial sorting of the samples from low invasive to high invasive phenotypes. The purple line represents a moving average of NFIB expression per 20 tumours. Linear regression analysis reveals a Spearman P -value = 3.553e − 15 indicating a positive correlation between NFIB and invasiveness. (D) MM96L stable BRN2, MITF, NFIB, or empty human melanoma cells grown on agarose to generate 3D non-adherent melanoma spheroids. Spheroids were embedded in a collagen-media mixture and left to grow over a 72 h time-frame, with light phase photographs taken every 24 h. (E) Spheroid invasion was calculated from (D) by determining the change in the area of the invading cells disseminating away from the spheroid at 24 h time intervals relative to the 0 h timepoint. (F) The change in spheroid size was determined in (D) by measuring the change in area occupied by the spheroid alone (not the invasive populations) at 0 h vs. 72 h. *: P < 0.001, **: P < 0.001 ****: P < 0.0001. A two-way ANOVA with Tukey's post hoc test was performed on E–F. Changes in area occupied by invading cells and spheroid growth were calculated using ImageJ software. Data from (D–F) is representative of three independent experiments and is represented as the mean ± SEM. See also Fig. S3.

Article Snippet: Immunoblots were probed using rabbit monoclonal BRN2 antibody D2C1L (Cell Signaling #12137), rabbit monoclonal NFIB antibody (Sigma Aldrich; HPA003956), rabbit monoclonal MITF D567V (Cell Signaling #12590), mouse monoclonal EZH2 (Active Motif #39639), and Rabbit monoclonal H3K27 tri-methyl C36B11 (Cell Signaling #9733), with anti-GAPDH 6C5 (Santa Cruz #sc-32233) used as a loading control.

Techniques: Over Expression, Control, Injection, Expressing, Software

Journal: EBioMedicine

Article Title: NFIB Mediates BRN2 Driven Melanoma Cell Migration and Invasion Through Regulation of EZH2 and MITF

doi: 10.1016/j.ebiom.2017.01.013

Figure Lengend Snippet: NFIB shows colocalisation with BRN2 in melanoma tumours and shows increased expression in aggressive/metastatic melanoma models. (A) Immunofluorescence microscopy on A2058 xenograft tumours surgically excised, formalin-fixed, and embedded in paraffin. Tumours were sectioned at 5 μm thickness and antigen-retrieved before labeling with BRN2 (red) and NFIB (green) antibody. DAPI was used to stain nuclei. (B-C) Immunofluorescence microscopy as described above on patient derived subcutaneous primary melanoma tumours and Lymph node metastatic melanoma tumours. (D) Microarray analysis of melanoma clinical samples representing 31 primary melanomas and 52 melanoma metastases from a previously published data set . Relative RNA expression was plotted and linear regression analysis was performed investigating the relationship between BRN2 and NFIB expression in metastatic samples. (E) Regression analysis on the above dataset looking at a correlation between MITF and NFIB expression in metastatic tumours. (F) Analysis of relative NFIB expression (log2 transformed) in 102 primary and 368 metastatic tumours from the TCGA dataset. Data represented as a violin plot and analyzed using the Mann-Whitney rank test. (G) Microarray analysis of subcutaneous tumours or lung metastases from immunodeficient mice injected subcutaneously or intravenously with a poorly-metastatic A375 melanoma cell line or with highly-metastatic derivative cell lines from a previously published dataset . Relative NFIB RNA expression was investigated in three specific groups; Poorly metastatic (PM), Subcutaneous tumours (SC), and the resultant lung metastases from the aforementioned subcutaneous tumours (LM). (H) Microarray analysis of relative NFIB expression (log2 transformed) in primary cutaneous melanomas derived from iMet (metastasis-capable) and iHRAS (non-metastatic) models from a previously published dataset . *: P < 0.05, ***: P < 0.001. A one-way ANOVA with a Tukey's post-hoc test was performed on (H). Data is represented as the mean ± SEM. Scale bars in white represent 200 μm. See also Figs. S3 and S6.

Article Snippet: Immunoblots were probed using rabbit monoclonal BRN2 antibody D2C1L (Cell Signaling #12137), rabbit monoclonal NFIB antibody (Sigma Aldrich; HPA003956), rabbit monoclonal MITF D567V (Cell Signaling #12590), mouse monoclonal EZH2 (Active Motif #39639), and Rabbit monoclonal H3K27 tri-methyl C36B11 (Cell Signaling #9733), with anti-GAPDH 6C5 (Santa Cruz #sc-32233) used as a loading control.

Techniques: Expressing, Immunofluorescence, Microscopy, Labeling, Staining, Derivative Assay, Microarray, RNA Expression, Transformation Assay, MANN-WHITNEY, Injection

Journal: EBioMedicine

Article Title: NFIB Mediates BRN2 Driven Melanoma Cell Migration and Invasion Through Regulation of EZH2 and MITF

doi: 10.1016/j.ebiom.2017.01.013

Figure Lengend Snippet: BRN2 and NFIB colocalise with EZH2 populations in vivo. (A) Immunofluorescence microscopy on A2058 xenograft tumours surgically excised, formalin-fixed, and embedded in paraffin. Tumours were sectioned at 5 μm thickness and antigen-retrieved before labeling with BRN2 (red) and EZH2 (green) antibody. DAPI was used to stain nuclei. (B–C) Immunofluorescence microscopy as described above on patient derived subcutaneous primary melanoma tumours and Lymph node metastatic melanoma tumours, labeled with BRN2 (red) and EZH2 (green). (D) Immunofluorescence microscopy on A2058 xenograft tumours as described above, labeled with NFIB (red) and EZH2 (green) antibody. (E–F) Immunofluorescence microscopy as described above on patient derived subcutaneous primary melanoma tumours and Lymph node metastatic melanoma tumours, labeled with NFIB (red) and EZH2 (green).

Article Snippet: Immunoblots were probed using rabbit monoclonal BRN2 antibody D2C1L (Cell Signaling #12137), rabbit monoclonal NFIB antibody (Sigma Aldrich; HPA003956), rabbit monoclonal MITF D567V (Cell Signaling #12590), mouse monoclonal EZH2 (Active Motif #39639), and Rabbit monoclonal H3K27 tri-methyl C36B11 (Cell Signaling #9733), with anti-GAPDH 6C5 (Santa Cruz #sc-32233) used as a loading control.

Techniques: In Vivo, Immunofluorescence, Microscopy, Labeling, Staining, Derivative Assay

Journal: Molecular Cancer

Article Title: DNA methylation-associated dysregulation of transfer RNA expression in human cancer

doi: 10.1186/s12943-022-01532-w

Figure Lengend Snippet: tDNA methylation is associated with different overall survival in TCGA cohorts. A Dot plots summarizing the logrank tests ( left ) and univariate Cox regression models ( right ) used to compare the overall survival of patients according to methylation status of the 71 tDNAs. Cases that are significantly associated with different prognosis are represented in large-sized bullets. Yellow and blue represent favorable and unfavorable prognosis according to univariate Cox regression analyses ( right ), respectively. HR, hazard ratio. B Kaplan-Meier curves show that tRNA-Arg-TCT-4-1 hypomethylation is associated with a shorter overall survival in KIRP ( top ) and UCEC ( below ) TCGA cohorts. HR, hazard ratio; CI, confidence interval. p -values correspond to logrank tests. C Bisulfite genomic sequencing confirms tRNA-Arg-TCT-4-1 hypomethylation in HEC1 cell line. The tDNA sequence is indicated with a blue bracket. The orange rectangles correspond to the A and B boxes of the tDNA. The TSS is marked with a black arrow. CpG dinucleotides are represented as short vertical lines, and their methylation status is denoted with black (methylated) or white (unmethylated) squares. The CpG represented in the HM450 microarray is marked with a red asterisk. D qRT-PCR exposes higher tRNA-Arg-TCT-4-1 levels in HEC1 compared to DND41 and SW48 cells. Data represent the mean ± SD of biological triplicates analyzed by an unpaired two-tailed Student’s t-test. *** p < 0.001. E Cell cycle analysis reveals an accumulation of tRNA-Arg-TCT-4-1 knockout HEC1 cells in G0/G1 phase. Data shown represent the mean ± SD of biological triplicates analyzed by unpaired two-tailed Student’s t-test. * p < 0.05. F SRB assay shows a reduced growth of HEC1 knockout cells. Data at each time points are the mean ± SD of four biological replicates. Statistical differences were determined using an unpaired two-tailed Student’s t-test at the 144 h final time point. * p < 0.05. G Transwell assay shows a reduced migration of tRNA-Arg-TCT-4-1-silenced HEC1 cells. Data represent the mean ± SD of biological triplicates analyzed by an unpaired two-tailed Student’s t-test. *** p < 0.001. Representative images of the Transwell insert membranes are shown.

Article Snippet: To identify DNA methylation defects in tDNA, we retrieved the Infinium HumanMethylation450 (HM450) methylation microarray probes’ identity that mapped into the loci corresponding to the 416 high confidence tDNA that are found in the human nuclear genome [ ].

Techniques: Methylation, Genomic Sequencing, Sequencing, Microarray, Quantitative RT-PCR, Two Tailed Test, Cell Cycle Assay, Knock-Out, Sulforhodamine B Assay, Transwell Assay, Migration

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Dendritic cell fate is determined by BCL11A

doi: 10.1073/pnas.1319228111

Figure Lengend Snippet: BCL11A regulates ID2, ID3, MTG16, and E2-2/TCF4 in human B and pDC cell lines. (A) BCL11A isoforms (XS or XL) retrovirally overexpressed in Raji and BJAB Burkitt’s lymphoma B-cell lines altered transcription (average threefold) of the HLH proteins ID2 and ID3 compared with mock-transduced control cells. Multiple (n > 4) independent experiments tracked by multiple probe elements spotted per LymphoChip cDNA microarray. (B) Inducible shRNA silencing of BCL11A down-regulates transcription of ID3 and E2-2. (Left) CAL-1 pDC cell line stably transduced with inducible shRNA targeted to exon 2 of Bcl11a under the control of a doxycycline (DOX)-inducible H1 promoter containing TETR binding sites (59). RT-PCR with optimal cycling conditions in the CAL-1 pDC cell line revealed strong knockdown of BCL11A transcripts beginning 6 h postinduction and continuing for >24 h. ID3 and E2-2 transcripts were correspondingly reduced throughout the 24-h experiment, whereas ID2 was consistently, although transiently, induced; the irrelevant target gene CD37 was unaffected. GAPDH amplification shows equivalence of mRNA amplification and loading. (Right) At 24 h, shRNA-mediated BCL11A knockdown resulted in ID3 transcriptional inhibition >80%, and E2-2 of 60%, when normalized to the GAPDH housekeeping control gene. Mean ± SD depicted; n ≥ 3 experiments. (C) BCL11A is recruited to 5′ regulatory regions of ID3, E2-2, and itself. ChIP from CAL-1 human pDC cells using anti-BCL11A or control IgG antibodies was analyzed by endpoint PCR. Primers annealing 10 kb upstream of the ID3 TSS failed to amplify and thus served as a negative control. (D) Overexpression of BCL11A (XL) up-regulates ID3 promoter-driven reporter transcription in CAL1 pDCs and Raji B cells. (Upper) Schematic of the ID3 proximal promoter indicating the species-conserved EICE-like BCL11A DNA binding motif (Fig. S4); the location of BLIMP-1 and E-box motifs; and the HpaII methylation site. An 820-bp fragment spanning this region (indicated by arrows) that was consistently PCR amplified in ChIP experiments (indicated by wedges) was inserted upstream of luciferase in the pGL2 luciferase vector. (Lower) Dual luciferase activity was determined following transient transfection with increasing DNA levels (in nanograms) of the reporter into CAL1 human pDC cells or Raji B cells. Mean relative luciferase activity ± SEM obtained depicted; n = 2 independent experiments read in triplicate. (E) Knockdown of BCL11A results in MTG16 knockdown 24 h post-DOX induction in CAL-1 pDC cells.

Article Snippet: Total RNA was extracted from Bcl11a −/− fetal liver cells (or Bcl11a cKO spleen cells) using TRIzol reagent (Invitrogen) and oligo-dT cDNA was prepared using SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen).

Techniques: Control, Microarray, shRNA, Stable Transfection, Transduction, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Knockdown, Amplification, Inhibition, Negative Control, Over Expression, Methylation, Luciferase, Plasmid Preparation, Activity Assay, Transfection

Journal: Mutation Research. Reviews in Mutation Research

Article Title: Advances in ligase chain reaction and ligation-based amplifications for genotyping assays: Detection and applications

doi: 10.1016/j.mrrev.2017.05.001

Figure Lengend Snippet: Comparative Evaluation For Various Versions Of Ligase Chain Reaction (LCR) And Ligation-Based Amplifications .

Article Snippet: 10- SNPlex Genotyping System , - High multiplexing potential with high throughput capability as it can detect several hundred SNPs in a hundred or more samples at the same time. (80) - Highly sensitive - Highly specific - High throughput - Commonly used and commercially available , - Requires special and expensive equipment (80) - Requires 2 days for running the full assay , - Less than 1 ng , - Expensive , - 2 days for the whole assay (80) , - Yes - 48 SNPs at a time , - 3730xl DNA Analyzer (Applied Biosystems) (80) , - Very common , - Capillary electrophoresis - Genetic Analyzer 3730xl DNA Analyzer (Applied Biosystems) (80) , - 521 SNPs in Human (79) - SNPs in grapevine (Vitis vinifera L.) (80) - 29 insertion/deletion SNPs in Chinese Han samples (81) , - Yes , - Yes SNPlexTM Genotyping System 48-plex.

Techniques: Ligation, Multiplexing, Produced, Amplification, High Throughput Screening Assay, Comparison, Spectrophotometry, MicroChIP Assay, Electrophoresis, Autoradiography, Fluorescence, Virus, Mutagenesis, Preserving, Microarray, Labeling, Magnetic Beads, Generated, Multiplex Assay, Real-time Polymerase Chain Reaction, Reverse Transcription, Recombinant, RNA Detection, Detection Assay, Multiplex Ligation-dependent Probe Amplification, Sequencing, Hybridization, Transgenic Assay, Methylation, Luminex, Flow Cytometry, Bacteria, Mass Spectrometry, Colorimetric Assay, Clinical Proteomics, SPR Assay, Imaging

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Molecular events for CRBP1 gene in cervical epithelium samples. A: In order to know the gain of copy number of the CRBP1 gene, DNA of healthy cervix and CC samples, were subjected to real time PCR with specific Taqman probes. White bar (healthy cervix samples) represents the mean of the normal cervices (n = 26) without extra copies of CRBP1 gene. Black bars show CC samples with gain of copy number (2-20X); while gray dotted line bars are showing CC samples that do not change in the copies number. Values above the cut-off line (as 1), being assigned as increased gene copy number compared with normal cervical epithelium. CRBP1 Hs01437985_cn probe, and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Hs00894322_cn probe were used as reference; the relative genomic copy number was calculated using the comparative Ct methods [26]. In X-axis represents cervical samples, Y-axis relative copies fold change of CRBP1 gene. B: CRBP1 expression was observed as positive immunostaining result on tissue microarray as mentioned in Methods section The DNAs used for gain of copy number (panel A) were also used for the methylation assay. Methylation result represents the methylation of the CRBP1 promoter. In this case, each healthy or CC sample, correspond to each column for CRBP1 expression and methylation status. Interestingly, in most of the cases, there was an association between the lack of expression of the CRBP1 gene and its methylation status.

Article Snippet: CRBP1 Hs01437985_cn probe, and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Hs00894322_cn probe were used as reference; the relative genomic copy number was calculated using the comparative Ct methods [ 26 ].

Techniques: Real-time Polymerase Chain Reaction, Expressing, Immunostaining, Microarray, Methylation

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: CRBP1 immunodetection in the uterine cervix samples. A: (1) Cytoplasmic CRBP1 expression is present in cells of the basal layer of normal cervical epithelium (healthy tissue); (2) the immunodetection in the transformed cells of a cervical cancer (CC03) tissue harboring gain of CRBP1 gene. (3) CC samples without gain CRBP1 gene showing negative immunostaining (CC16 sample). A kidney tissue section (4) was used as positive control, while a heart tissue section for negative control (5). B: cervical progression spectrum. The tissue section shows a brownish reaction (positive reaction) in the basal cell layer of the “normal” region, in the high-grade lesion, and also in the invasive region. All tissue sections were hematoxylin counterstained, 200X original amplification.

Article Snippet: CRBP1 Hs01437985_cn probe, and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Hs00894322_cn probe were used as reference; the relative genomic copy number was calculated using the comparative Ct methods [ 26 ].

Techniques: Immunodetection, Expressing, Transformation Assay, Immunostaining, Positive Control, Negative Control, Amplification

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Immunolocalization of CRBP1 by immunofluorescence in cervical cells. Nuclei were Dapi stained in blue color (A-C). The immunodetection of CRBP1 was observed in green color (D-F). Cytoplasmic immunodetection of CRBP1 in the merge imaging (G-I). 100X original amplification.

Article Snippet: CRBP1 Hs01437985_cn probe, and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Hs00894322_cn probe were used as reference; the relative genomic copy number was calculated using the comparative Ct methods [ 26 ].

Techniques: Immunofluorescence, Staining, Immunodetection, Imaging, Amplification

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Methylation promoter of CRBP1 gene in cervical cancer samples. Example of CRBP1 gene promoter methylation analysis. Lanes: Healthy cervix sample, CC03 and CC06 samples with un-methylated status; lanes CC 10 and CC 16 with methylated status; HeLa cells as un-methylated control (109 bp), or MCF-7 cells as methylated control (99 bp). MW: molecular weight marker of 100 bp.

Article Snippet: CRBP1 Hs01437985_cn probe, and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Hs00894322_cn probe were used as reference; the relative genomic copy number was calculated using the comparative Ct methods [ 26 ].

Techniques: Methylation, Control, Molecular Weight, Marker

Journal: International Journal of Molecular Sciences

Article Title: Effects and Mechanism of Particulate Matter on Tendon Healing Based on Integrated Analysis of DNA Methylation and RNA Sequencing Data in a Rat Model

doi: 10.3390/ijms23158170

Figure Lengend Snippet: ( A ) Kyoto Encyclopedia of Genes and Genome (KEGG) pathways associated with the significantly upregulated and downregulated differentially expressed genes (DEGs) between particulate matter (PM)-exposed and control (non-PM) rats ( p < 0.05). ( B ) Canonical pathways with significant Z-scores (>2) identified using ingenuity pathway analysis. Heatmaps showing the DEGs in the ( C ) cAMP response element-binding protein (CREB) and ( D ) cAMP signaling pathways. ( E ) Schematic diagram showing the methods and criteria for gene analysis. ( F ) Venn diagram showing the combined DNA methylation and transcriptome data. The red circle indicates 67 genes exhibiting hypomethylated/upregulated expression. ( G ) The top five KEGG pathways associated with the hypomethylated/upregulated genes.

Article Snippet: A DNA methylation microarray was performed by Genomictree (Daejeon, Korea) using the tendon tissues (n = 3 per group) and a custom-designed Agilent-based microarray platform with 2 × 400 K probes per slide (popular_2X400K_chip; Agilent Design ID: 086791; Agilent Technologies, Santa Clara, CA, USA).

Techniques: Control, Binding Assay, Protein-Protein interactions, DNA Methylation Assay, Expressing

Journal: Journal of Inflammation Research

Article Title: Comprehensive Bioinformatics Analyses and Experimental Validation of the Cell Cycle Related Protein SAPCD2 as a New Biomarker and Potential Therapeutic Target in Pancreatic Cancer

doi: 10.2147/JIR.S501850

Figure Lengend Snippet: Genomic alterations, expression, and epigenetic regulation of SAPCD2 in gastrointestinal tumors. ( A ) The landscape of the copy number amplification and mutation frequency of SAPCD2 gene in various tumor types. ( B ) Potential mutations sites across the SAPCD2 protein in various cancers. Green dots, missense mutations; Black dots, truncating mutations; Brown dots, in-frame mutations. The hotspot mutation (E234K) is observed in the conserved Suppressor_APC domain. ( C ) The CNV percentage in each GI tumor. ( D ) The correlation between CNV and RNA-seq derived SAPCD2 gene expression in digestive tumors. ( E ) The differences of the patients’ survival between the CNV group and the wild type group in digestive tumors. ( F ) The diagram of the genomic structure of human SAPCD2 gene. The green box indicates the CpG island locus; the Orange boxes indicate the 6 exons’ locus; the red boxes indicate the positions of the 8 probes for evaluating the DNA methylation status; the grey arrow indicates the transcription direction of SAPCD2 gene whose cDNA is 8431 base pairs long. The width of each box indicates its relative length, and numbers within the square brackets indicates the start and end sites respectively. The 8 probes in this study are: cg15733507, cg21241219, cg21066537, cg15785720, cg03180426, cg01448891, cg14607755, cg14156314. ( G ) The summarized view of the relationship between the RNA-seq derived SAPCD2 gene expression and DNA methylation at 8 probes sites in TCGA-PAAD cohort. Each probe (Infinium Human Methylation 450 microarray data) was indicated on the left side, and the numbers on the right side indicated the correlation coefficient or P value. *p<0.05, **p<0.01, ***p<0.001. ( H ) The detailed view of the relationship between of SAPCD2 gene expression and DNA methylation at 8 probes sites in TCGA-PAAD cohort. The methylation level differed in SAPCD2 gene high and low expression group at 3 probe sites: cg21241219*, cg03180426 ***, cg14156314 ***.

Article Snippet: Each probe (Infinium Human Methylation 450 microarray data) was indicated on the left side, and the numbers on the right side indicated the correlation coefficient or P value.

Techniques: Expressing, Amplification, Mutagenesis, RNA Sequencing, Derivative Assay, Gene Expression, DNA Methylation Assay, Methylation, Microarray