Journal: Frontiers in Molecular Biosciences

Article Title: Innate immunity mediator STING modulates nascent DNA metabolism at stalled forks in human cells

doi: 10.3389/fmolb.2022.1048726

Figure Lengend Snippet: STING affects the levels of RPA on parental and nascent DNA in HU-treated cells. (A) Examples of RPA32 to EdU PLA fluorescence in the control U2OS cells, treated as shown. Cells were labeled with EdU and harvested immediately or after a 5 h HU arrest, as shown by asterisks. Scale bar = 10 μm. (B) A schematic of the likely substrate for the RPA32/EdU PLA (red arc represents fluorescent signal) and representative distributions of RPA32/EdU PLA foci numbers in the U2OS cells with empty vector (e.v.) or the indicated STING transgenes. Cells were labeled as in (A) and EdU was Clicked to a mixture of biotin and Alexa488 azides at a molar ratio of 50:1 to enable simultaneous visualization of EdU-positive cells and PLA signals. PLA foci numbers are shown separately for EdU-negative (E−) and EdU-positive (E+) cells. Red lines are medians and their values are shown above the lines. p values were calculated in Wilcoxon tests. (C) Magnitudes of differences between RPA32/EdU PLA foci distributions in STING WT vs. control and STING S358A vs. control were calculated as Cliff’s delta values and plotted. Shown are Cliff’s delta values for each of four independent experiments performed and quantified as in (B) . Each experiment is identifiable by fill tone of the circle symbols. Red triangles are means. Blue arrowheads identify the values derived from the experiment shown in (B) . (D) Cliff’s delta values for two biological replicates of an experiment performed as in (B) and measuring RPA32/ssDNA PLA foci numbers. Likely substrates for the RPA32/ssDNA PLA are shown above the plot. (E) Distributions of RPA32 S33P/EdU PLA foci numbers in the U2OS cells with empty vector or the indicated STING transgenes. Cells were labeled with EdU and treated with HU as in (A) . All samples are HU-treated. PLA foci numbers are shown separately for EdU-negative (E−) and EdU-positive (E+) cells. Red lines are medians and their values are shown above the lines. p values were calculated in Wilcoxon tests. (F) Cliff’s delta values calculated for RPA32 S33P/EdU PLA foci distributions in STING WT vs. control and STING S358A vs. control pairs in each of eight independent experiments performed and quantified as in (E) and identifiable by fill tone or fill pattern of the circle symbols. Triangle symbols are means. Blue arrowheads identify the values from the experiment shown in (E) . (G) Cliff’s delta values calculated for differences of distributions of mean fluorescence intensities (MFI) per nucleus measured by IF in situ for RPA32 S33P in STING WT vs. control and STING S358A vs. control cells. MFI values were measured in EdU-positive, HU-arrested cells in three independent experiments. For all Cliff’s delta values, positive values mean that the values of the first distribution in the comparison (e.g., STING WT in the STING WT—e.v. comparison) are overall higher than those of the second distribution, and negative values mean that the reverse is true. Distribution medians are displayed as in (E) .

Article Snippet: Antibodies were as follows: Mouse α-biotin Cat. No. MB-9100 (Vector Laboratories); rat α-BrdU/CldU Cat. No. ab6326 (Abcam); mouse α-BrdU/IdU Cat. No.347580 (BD Biosciences); rabbit α-STING Cat. No.19851-1-AP (Proteintech), mouse α-NCL Cat. No. 396400 (Life Technologies), rabbit α-RPA32 Cat. No. A300-244A (Bethyl Labs), rabbit α-RPA32 S33P Cat. No. A300-246A (Bethyl Labs), rabbit CHK1 S345P Cat. No. 2348 (CST), mouse α-ssDNA MAB3034 (Millipore Sigma), rabbit α-cGAS Cat. No. 15102S (CST), mouse α-γH2AX clone JBW301, Cat. No. 05–636 (Millipore Sigma), rabbit α-DNA-PKcs Cat. No. 38168 (CST), mouse α-IFI16 Cat. No. sc-8023 (Santa Cruz Biotechnology), rabbit α-MRE11 Cat. No. NB100-142 (Novus biologicals), mouse α-LaminA/C Cat. No. sc-376248 (Santa Cruz Biotechnology), rabbit α-GAPDH Cat. No. 5174 (CST), rabbit α-Histone H3 Cat. No. 4499 (CST), rabbit α-FANCD2 Cat. No. A302-174A (Bethyl Labs), rabbit α-STING S366P Cat. No. 50907 (CST), rabbit α-ERp72 Cat. No. 5033 (CST), rabbit α-EMC1 Cat. No. A305-605A (Bethyl Labs), mouse α-WRN, Cat. No. W0393 (Millipore-Sigma).

Techniques: Fluorescence, Control, Labeling, Plasmid Preparation, Derivative Assay, In Situ, Comparison

Journal: Aging (Albany NY)

Article Title: Werner syndrome RECQ helicase participates in and directs maintenance of the protein complexes of constitutive heterochromatin in proliferating human cells

doi: 10.18632/aging.206132

Figure Lengend Snippet: WRN, HDAC2, and HP1α associations are reduced in replicatively senescing cells. ( A ) Normal human dermal fibroblasts were passaged to derive early and late passage cultures, which were cryopreserved and subsequently used in the same experiment. ( B ) SA β-gal levels in young (Y) and old (O) fibroblasts were quantified via fluorescent detection and microscopy. Cytoplasmic MFI was measured per digital image, normalized to the number of cells in each image determined by DAPI counterstaining, and plotted. ( C , D ) Quantitation of H3K9me3, WRN, HDAC2, and HP1α MFIs in young vs. old fibroblasts determined by IF in situ . ( E ) Quantitations of HDAC2/HP1α, WRN/HP1α, and WRN/HDAC2 PLA signals in young vs. old fibroblasts. Graphs in C, D, and E represent two independent experiments each. Crossbars in graphs are distribution means.

Article Snippet: Antibodies were as follows: rabbit α-biotin Cat. No. A150-109A (Bethyl); mouse α-BrdU/CldU Cat. No. NBP2-44055 (Novus); rabbit α-STING Cat. No. 19851-1-AP (Proteintech), mouse α-NCL Cat. No. 396400 (Life Technologies), mouse α-Lamin A/C Cat. No. sc-376248 (Santa Cruz Biotechnology), rabbit α-GAPDH Cat. No. 5174 (CST), rabbit α-Histone H3K9me3 Cat. No. 13969 (CST), rabbit α-Histone H3K9me3 Cat. No. A2217P (Diagenode), mouse α-WRN, Cat. No. W0393 (Millipore-Sigma), rabbit α-WRN, Cat. No. NB100-471 (Novus); mouse α-HP1α Cat. No. NBP2-52420 (Novus), rabbit α-HP1α Cat. No. 2616 (CST); rabbit α-V5 Cat. No. 3202 (CST); rabbit α-KAP1 Cat. No. 4124 (CST); mouse α-HDAC2 Cat. No. 5113 (CST); rabbit α-HDAC2 Cat. No. 57156 (CST); rabbit α-LBR Cat. No. A5468 (ABclonal); mouse α-Lamin B1 Cat. No. 66095-1 (Proteintech).

Techniques: Microscopy, Quantitation Assay, In Situ

Journal: Aging (Albany NY)

Article Title: Werner syndrome RECQ helicase participates in and directs maintenance of the protein complexes of constitutive heterochromatin in proliferating human cells

doi: 10.18632/aging.206132

Figure Lengend Snippet: WRN, HDAC2, and HP1α associations are reduced in RAF oncogene-induced senescence. ( A ) 4HT-inducible expression of RAF oncogene in WI38hTERT leads to cessation of proliferation and onset of senescence marked by the formation of SAHF after three days. ( B ) Quantitations of H3K9me3 (left panel, three independent experiments), and WRN, HP1α, and HDAC2 (center and right panels, two independent experiments each) nuclear levels in WI38hTERT after 4 days in 4HT compared to the contemporaneous control without 4HT, determined by IF in situ . ( C ) Quantitations of nuclear area (in pixels) of WI38hTERT treated with 4HT as in ( B ). The graph represents three independent experiments. ( D ) Total levels of the WRN, HP1α, and HDAC2 proteins in WI38hTERT under the same conditions, determined by Western blotting. Quantitations below the image average three independent experiments. The values were normalized to NCL as loading control and shown relative to no-4HT conditions. ( E ) An example of HDAC2/WRN PLA data in WI38hTERT with and without 4HT. Scale, 10 µm. ( F ) Quantitations of HDAC2/HP1α, WRN/HP1α, and HDAC2/WRN PLA in WI38hTERT after 4 days in 4HT compared to the contemporaneous no-4HT control. The left and center panels represent two independent experiments each, and the right panel represents two biological replicas. Crossbars in graphs are distribution means.

Article Snippet: Antibodies were as follows: rabbit α-biotin Cat. No. A150-109A (Bethyl); mouse α-BrdU/CldU Cat. No. NBP2-44055 (Novus); rabbit α-STING Cat. No. 19851-1-AP (Proteintech), mouse α-NCL Cat. No. 396400 (Life Technologies), mouse α-Lamin A/C Cat. No. sc-376248 (Santa Cruz Biotechnology), rabbit α-GAPDH Cat. No. 5174 (CST), rabbit α-Histone H3K9me3 Cat. No. 13969 (CST), rabbit α-Histone H3K9me3 Cat. No. A2217P (Diagenode), mouse α-WRN, Cat. No. W0393 (Millipore-Sigma), rabbit α-WRN, Cat. No. NB100-471 (Novus); mouse α-HP1α Cat. No. NBP2-52420 (Novus), rabbit α-HP1α Cat. No. 2616 (CST); rabbit α-V5 Cat. No. 3202 (CST); rabbit α-KAP1 Cat. No. 4124 (CST); mouse α-HDAC2 Cat. No. 5113 (CST); rabbit α-HDAC2 Cat. No. 57156 (CST); rabbit α-LBR Cat. No. A5468 (ABclonal); mouse α-Lamin B1 Cat. No. 66095-1 (Proteintech).

Techniques: Expressing, Control, In Situ, Western Blot

Journal: Aging (Albany NY)

Article Title: Werner syndrome RECQ helicase participates in and directs maintenance of the protein complexes of constitutive heterochromatin in proliferating human cells

doi: 10.18632/aging.206132

Figure Lengend Snippet: H3K9me3 heterochromatic mark is reduced on satellite tandem repeats in WRN-depleted cells. ( A ) Quantitation of IF in situ for H3K9me3 levels in WI38hTERT expressing shRNA against WRN or non-specific control (NS). Crossbars are distribution means. The graph represents three independent experiments. ( B ) ChIP qPCR analyses of H3K9me3 levels on the indicated genomic loci, performed in the same cells as in ( A ). The graph summarizes results of five independent experiments with technical triplicates. ( C ) ChIP qPCR analyses of histone H3 levels on the indicated genomic loci, performed in the same cells as in ( A ). The graph summarizes results of three independent experiments with technical triplicates. In ( B - C ), red triangles are means, and boxplots mark first and fourth quartiles and the distributions’ medians. ( D ) Quantitations of the indicated PLA in situ analyses in WI38hTERT cells expressing shRNA against WRN or control shRNA. Graphs represent two (HP1α/KAP1) and four (HP1α/LBR) independent experiments each. ( E ) Quantitations of the indicated IF in situ analyses performed on the same cells as in ( D ). Graphs represent two (KAP1), and three (HP1α) independent experiments each.

Article Snippet: Antibodies were as follows: rabbit α-biotin Cat. No. A150-109A (Bethyl); mouse α-BrdU/CldU Cat. No. NBP2-44055 (Novus); rabbit α-STING Cat. No. 19851-1-AP (Proteintech), mouse α-NCL Cat. No. 396400 (Life Technologies), mouse α-Lamin A/C Cat. No. sc-376248 (Santa Cruz Biotechnology), rabbit α-GAPDH Cat. No. 5174 (CST), rabbit α-Histone H3K9me3 Cat. No. 13969 (CST), rabbit α-Histone H3K9me3 Cat. No. A2217P (Diagenode), mouse α-WRN, Cat. No. W0393 (Millipore-Sigma), rabbit α-WRN, Cat. No. NB100-471 (Novus); mouse α-HP1α Cat. No. NBP2-52420 (Novus), rabbit α-HP1α Cat. No. 2616 (CST); rabbit α-V5 Cat. No. 3202 (CST); rabbit α-KAP1 Cat. No. 4124 (CST); mouse α-HDAC2 Cat. No. 5113 (CST); rabbit α-HDAC2 Cat. No. 57156 (CST); rabbit α-LBR Cat. No. A5468 (ABclonal); mouse α-Lamin B1 Cat. No. 66095-1 (Proteintech).

Techniques: Quantitation Assay, In Situ, Expressing, shRNA, Control, ChIP-qPCR

Journal: Aging (Albany NY)

Article Title: Werner syndrome RECQ helicase participates in and directs maintenance of the protein complexes of constitutive heterochromatin in proliferating human cells

doi: 10.18632/aging.206132

Figure Lengend Snippet: Reduction of Lamin B1, LBR-associated interactions in WRN-depleted cells. ( A ) An example of LBR and WRN IF in situ in WI38hTERT cells expressing the indicated shRNAs. Scale, 10 µm. ( B ) Quantitations of the indicated IF in situ analyses in the same WI38hTERT cells as in ( A ). Left panel, mean nuclear levels of LBR (per cell) background-corrected by the means of the entire fluorescent signal outside the nuclei (per image). Right panel, ratios of mean nuclear to mean cytoplasmic LBR IF signals per cell. Note the log scale. Red triangles are distribution means. The graphs represent four (LBR, left panel), two (LBR, right panel) and over five (WRN) independent experiments each. ( C ) A Western blot of WI38hTERT expressing the indicated shRNAs and probed with antibodies against NCL (internal control), LBR, and HP1α. Quantitations below the image average two independent experiments. The values were normalized to NCL and shown relative to shNS control. ( D , E ) Quantitations of the indicated PLA or IF in situ analyses in the same WI38hTERT cells as above. The panels represent two (LBR/Lamin B1), three (Lamin B1/H3K9me3), and three (Lamin B1) independent experiments each. Nuclear MFI values were used instead of PLA foci numbers in cases where the latter numbers were too high to robustly count individual foci. ( F ) ChIP qPCR analyses of LBR levels on the indicated genomic loci performed in the same cells as elsewhere in the figure. The graphs summarize results of two independent experiments.

Article Snippet: Antibodies were as follows: rabbit α-biotin Cat. No. A150-109A (Bethyl); mouse α-BrdU/CldU Cat. No. NBP2-44055 (Novus); rabbit α-STING Cat. No. 19851-1-AP (Proteintech), mouse α-NCL Cat. No. 396400 (Life Technologies), mouse α-Lamin A/C Cat. No. sc-376248 (Santa Cruz Biotechnology), rabbit α-GAPDH Cat. No. 5174 (CST), rabbit α-Histone H3K9me3 Cat. No. 13969 (CST), rabbit α-Histone H3K9me3 Cat. No. A2217P (Diagenode), mouse α-WRN, Cat. No. W0393 (Millipore-Sigma), rabbit α-WRN, Cat. No. NB100-471 (Novus); mouse α-HP1α Cat. No. NBP2-52420 (Novus), rabbit α-HP1α Cat. No. 2616 (CST); rabbit α-V5 Cat. No. 3202 (CST); rabbit α-KAP1 Cat. No. 4124 (CST); mouse α-HDAC2 Cat. No. 5113 (CST); rabbit α-HDAC2 Cat. No. 57156 (CST); rabbit α-LBR Cat. No. A5468 (ABclonal); mouse α-Lamin B1 Cat. No. 66095-1 (Proteintech).

Techniques: In Situ, Expressing, Western Blot, Control, ChIP-qPCR