Journal: Molecular & Cellular Proteomics : MCP

Article Title: Insulin Induces Microtubule Stabilization and Regulates the Microtubule Plus-end Tracking Protein Network in Adipocytes *

doi: 10.1074/mcp.RA119.001450

Figure Lengend Snippet: CLASP2 and G2L1 colocalize in 3T3-L1 adipocytes. A–B, Live-cell imaging of adipocytes cultured in complete media coexpressing mRuby2-Tubulin (magenta) and GFP-CLASP2-HA (A, green) or mCherry-G2L1-myc (B, green). Live cells were imaged using TIRFM on a 2-s acquisition interval. Time series images to the right of the whole cell image are used to highlight +TIP dynamics within the indicated ROI. C, Immunofluorescence images of adipocytes cultured in complete media cooverexpressing GFP-CLASP2-HA (green) and mCherry-G2L1-myc (magenta). Cells were fixed and immunolabeled for tubulin (inverted white). Bottom row is magnified ROI. ROI, region of interest. Scale bar = 20 μm.

Article Snippet: To generate pLenti-iRFP670-Tubulin, tubulin was first subcloned out of the mRuby2-Tubulin-6 vector and into piRFP670-N1 (Addgene #45457, a gift from Vladislav Verkhusha). iRFP670-Tubulin was then subcloned out of the piRFP670-Tubulin vector and into the pLenti backbone to generate pLenti-iRFP670-Tubulin.

Techniques: Live Cell Imaging, Cell Culture, Immunofluorescence, Immunolabeling

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Insulin Induces Microtubule Stabilization and Regulates the Microtubule Plus-end Tracking Protein Network in Adipocytes *

doi: 10.1074/mcp.RA119.001450

Figure Lengend Snippet: The effect of insulin on CLASP2 +TIP dynamics. Live-cell imaging of adipocytes serum-starved for one hour and subsequently stimulated with 100 nm insulin. Live cells were imaged using TIRFM on a two-second acquisition interval. A, Single frame of live-cell imaging of adipocytes coexpressing GFP-CLASP2 (green) and mRuby2-Tubulin (magenta) at basal state (top row) or 10 mins (bottom row) following insulin stimulation. CLASP2 is displayed in inverted white to highlight +TIP density. B, Quantification of CLASP2-containing +TIP density per unit area (μm2) in adipocytes at basal state or 10 mins following insulin stimulation. Percent increase in CLASP2-containing +TIP density is indicated to the right. Statistical comparison made by paired parametric t test, n = 5 cells. C, Temporally color-coded projection of GFP-CLASP2 localization during a 30 s live-cell imaging interval. The length and extent of color overlap of time-projected CLASP2 localization indicates reduced displacement and hence lower velocity of CLASP2-containing +TIPs during the imaging interval in adipocytes after 10 mins of insulin stimulation. D, Quantification of CLASP2-containing +TIP velocity in adipocytes at basal state or 10 mins following insulin stimulation indicates reduced velocity of CLASP2-containing +TIPS after insulin treatment. Statistical comparison made by unpaired parametric t test, n = 123–125 CLASP2-containing +TIPS from five cells. Scale bars = 5 μm. E, Image of entire cells in the basal state (left panel) or 8 mins post insulin treatment (right panel) extracted from supplemental Video 2. Time series images under the whole cell image are used to highlight insulin-stimulated +TIP dynamics within the indicated ROI. F, Time series extracted from supplemental Video 2 of the indicated ROIs at either the basal state or at 4 mins post insulin treatment to present an example of changing CLASP2 microtubule plus-end dynamics with insulin stimulation. G, Live cell CLASP2-containing +TIP dynamics of the ROI extracted from supplemental Video 2 were captured in the basal state followed by stimulation with insulin. Each insulin-stimulated CLASP2-containing +TIP trail length time point was compared against the basal CLASP2-containing +TIP trail length to test for significant differences. t test; *p ≤ 0.05, **p ≤ 0.01. Red circles represent outlier data points. ROI, region of interest. BAS, basal. INS, insulin. Scale bar = 10 μm.

Article Snippet: To generate pLenti-iRFP670-Tubulin, tubulin was first subcloned out of the mRuby2-Tubulin-6 vector and into piRFP670-N1 (Addgene #45457, a gift from Vladislav Verkhusha). iRFP670-Tubulin was then subcloned out of the piRFP670-Tubulin vector and into the pLenti backbone to generate pLenti-iRFP670-Tubulin.

Techniques: Live Cell Imaging, Imaging

Journal: bioRxiv

Article Title: Coupling of translation quality control and mRNA targeting to stress granules

doi: 10.1101/2020.01.05.895342

Figure Lengend Snippet: A) U-2 OS cells stably expressing the stress granule marker mRuby2-G3BP1 were either untransfected (“None”) or transiently transfected with empty vector peGFP-N1 (“eGFP”), wild-type VCP fused to eGFP “VCP(WT)-eGFP”, or VCP alleles “VCP(A232E)-eGFP” and “VCP(R155H)-eGFP”. Cells were stressed for 45 min. with 0.5 mM arsenite then fixed, and smFISH performed to detect AHNAK mRNA (top) or NORAD lncRNA (bottom). Representative photomicrographs for are shown at left, with RNAs shown in white, stress granules in red and eGFP in green. Images were acquired on a Delta Vision microscope at 100x and maximum intensity projections of z-stacks shown. At right: avg percent AHNAK mRNA or NORAD lncRNA +/-s.e.m. that co-localize with stress granules. Three independent experiments were done and only cells expressing eGFP were counted for all transfected conditions. For AHNAK : None, n = 11 frames (31 cells); eGFP, n = 12 frames (25 cells); VCP(WT), n = 11 frames (17 cells); VCP(A323E), n = 12 frames (18 cells); VCP(R155H), n = 13 frames (22 cells). For NORAD : None, n = 8 frames (23 cells); eGFP, n = 10 frames (20 cells); VCP(WT), n = 10 frames (21 cells); VCP(A232E), n = 9 frames (12 cells); VCP(R155H), n = 9 frames (18 cells). Student’s t-test was done to assess significance between GFP and VCP(WT), VCP(A232E) or VCP(R155H) for AHNAK and NORAD with * indicating p < 0.05. Scale bars: 10 µm (whole cell) or 5 µm (magnified panels). B) Working model depicting three mechanisms by which RNAs may enter into stable associations with stress granules. Non-coding RNAs (green) can directly engage with a stress granule (large circle), while translating mRNAs exit translation via ribosome runoff (blue) or become stalled in translation (red) and must be removed by factors in the RQC complex including LTN1 (dark green circle), NEMF (light green circle), VCP (gray) and the proteasome (blue) in a process that may involve ubiquitination (“Ub”) of nascent peptides.

Article Snippet: U-2 OS cells stably expressing mRuby2-G3BP1 were plated on coverslips and untransfected or transiently transfected with peGFP-N1 (empty vector), VCP(WT)-EGFP, VCP(A232E)-EGFP or VCP(R155H)-EGFP (gifts from Nico Dantuma, Addgene #23971, #23973, and #23972) .

Techniques: Stable Transfection, Expressing, Marker, Transfection, Plasmid Preparation, Microscopy

Journal: Nature Genetics

Article Title: Hijacking of transcriptional condensates by endogenous retroviruses

doi: 10.1038/s41588-022-01132-w

Figure Lengend Snippet: a , Models of the TRIM28/HP1α pathway and enhancers. b , Heatmap of ChIP–seq read densities within a 2-kb window around full-length IAP ERVs and enhancers in mESC. The genomic elements were length normalized. Enhancers include the constituent enhancers of SEs and typical enhancers. Rpm, reads per million. c , Scheme of the dTAG system to degrade TRIM28 in mESCs. d , Western blot validation of the FKBP degron tag and its ability to degrade TRIM28. e , FC in read density of TT-SLAM-seq and RNA-seq data after the indicated duration of dTAG-13 treatment, normalized to the level in the DMSO control. Data are presented as mean values ± s.d. from three biological replicates. P values are from unpaired two-sided t -tests. ** P < 0.01. f , Genome browser tracks of ChIP–seq data (H3K27Ac, OCT4, SOX2, NANOG) in control mESCs and TT-SLAM-seq data upon 0 h, 2 h, 6 h and 24 h dTAG-13 treatment at the Klf4 locus. Chr, chromosome. g , FC of gene transcription (TT-SLAM-seq data) upon dTAG-13 treatment. The number of significantly deregulated genes (DESeq2) and example pluripotency genes are highlighted. h , Gene set enrichment analysis: genes are ranked according to their FC in transcription (TT-SLAM-seq) after 24 h of dTAG-13 treatment. SE genes are marked with black ticks. P denotes a nominal P value. i , Log 2 FC in TT-SLAM-seq read density at SEs and typical enhancers upon dTAG-13 treatment normalized to untreated control mESCs. P values are from two-sided Wilcoxon–Mann–Whitney tests. **** P = 5 × 10 −8 , *** P = 5 × 10 −4 . j , Representative images of individual z-slices (same z) of RNA-FISH and IF signal, and an image of the merged channels. The nuclear periphery determined by DAPI staining is highlighted as a white contour (scale bars, 2.5 μm). Also shown are averaged signals of either RNA-FISH or RNAPII IF centered on the FISH foci or randomly selected nuclear positions (scale bars, 0.5 μm). r denotes a Spearman’s correlation coefficient. k , Live-cell PALM imaging of Dendra2-RNAPII and nascent RNA transcripts of Sox2 -MS2 in mESCs after 24 h dTAG-13 treatment. Left, size of the nearest RNAPII cluster around Sox2 ; middle left, distance between the Sox2 locus and the nearest RNAPII cluster; middle right, average RNAPII cluster size globally; right, number of RNAPII clusters per cell. Data are presented as mean values ± s.d. P values are from Wilcoxon–Mann–Whitney tests.

Article Snippet: The repair template included a mRuby2 fluorescent protein sequence, P2A linker and the FKBP tag sequence (Supplementary Fig. ) . mRuby2 sequence was amplified from the mRuby2-N1 plasmid (Addgene, catalog no. 54614), and the P2A-FKBP sequence was amplified from the PITCh dTAG donor vector (Addgene, catalog no. 91792).

Techniques: ChIP-sequencing, Western Blot, Biomarker Discovery, RNA Sequencing, Control, MANN-WHITNEY, Staining, Imaging

Journal: Nature Genetics

Article Title: Hijacking of transcriptional condensates by endogenous retroviruses

doi: 10.1038/s41588-022-01132-w

Figure Lengend Snippet: a . Acute reduction of transcription at the miR290-295 super-enhancer locus upon TRIM28-degradation. Displayed are genome browser tracks of ChIP-seq data (H3K27Ac, OCT4, SOX2, NANOG) in control mESCs, and TT-SLAM-seq data upon 0 h, 2 h, 6 h and 24 h dTAG-13 treatment at the miR290-295 locus. Rpm: reads per million. Co-ordinates are mm10 genome assembly co-ordinates. b . Acute reduction of transcription at the Mycn super-enhancer locus upon TRIM28-degradation. Displayed are genome browser tracks of ChIP-seq data (H3K27Ac, OCT4, SOX2, NANOG) in control mESCs, and TT-SLAM-seq data upon 0 h, 2 h, 6 h and 24 h dTAG-13 treatment at the Mycn locus. Rpm: reads per million. Co-ordinates are mm10 genome assembly co-ordinates. c . qRT-PCR validation of the TT-SLAM-seq data at the miR290-295 and Klf4 loci. Displayed are transcript levels after the indicated duration of dTAG-13 treatment. Values are displayed as mean ± SD from three independent experiments and are normalized to the level at 0 h. P values are from two-tailed t -tests. ****: P < 10 −4 , ***: P < 10 −3 , **: P < 10 −2 , *: P < 0.05. d . Visualization of nascent transcripts at super-enhancers, enhancers and de-repressed LTR retrotransposons. Displayed are TT-SLAM-seq read densities from both strands within 4 kb around the indicated sites. The genomic features (the middle part of the plot) were length normalized. Meta-analyses of the mean signals are displayed above the heatmaps.

Article Snippet: The repair template included a mRuby2 fluorescent protein sequence, P2A linker and the FKBP tag sequence (Supplementary Fig. ) . mRuby2 sequence was amplified from the mRuby2-N1 plasmid (Addgene, catalog no. 54614), and the P2A-FKBP sequence was amplified from the PITCh dTAG donor vector (Addgene, catalog no. 91792).

Techniques: ChIP-sequencing, Control, Quantitative RT-PCR, Biomarker Discovery, Two Tailed Test

Journal: Nature Genetics

Article Title: Hijacking of transcriptional condensates by endogenous retroviruses

doi: 10.1038/s41588-022-01132-w

Figure Lengend Snippet: a . Analyses of cells used in Fig. . (left) RNAPII IF intensity at the miR290-295 FISH foci (n DMSO = 61, n dTAG-13 = 50). (middle) RNAPII mean fluorescence intensity at random nuclear positions (n DMSO = 61, n dTAG-13 = 50). (right) Distance between the FISH focus and the nearest RNAPII puncta (n DMSO = 67, n dTAG-13 = 53). Data presented as mean values ± SD from one staining experiment. P values are from two-sided Mann-Whitney tests. NS: not significant. b . Images of RNA-FISH and IF signal. Nuclear periphery determined by DAPI staining is highlighted as a white contour. Also shown are averaged signal of either RNA FISH or RNAPII IF centered on the miR290-295 FISH foci or randomly selected nuclear positions. Data were collected as an independent replicate of experiments displayed in Fig. . Scale bars: 2.5 μm. c . Analysis of cells used in panel ‘ b ’. (left) RNAPII IF intensity at the miR290-295 FISH foci (n DMSO = 43, n dTAG-13 = 25). (center) RNAPII mean fluorescence intensity (n DMSO = 30, n dTAG-13 = 40). (right) Number of RNAPII puncta on a representative set of cells (n DMSO = 22, n dTAG-13 = 22). Data are presented as mean values ± SD from one staining experiment. P values are from two-sided Mann-Whitney tests. NS: not significant. d . Images of individual z-slices (same z) of the Fgf4 RNA-FISH and IF signal. Nuclear periphery determined by DAPI staining is highlighted as a white contour. Also shown are averaged signals of either RNA FISH or RNAPII IF centered on the FISH foci or randomly selected positions. Scale bars: 2.5 μm. e . Analyses of cells used in panel ‘ d .’ (left) RNAPII IF intensity at the Fgf4 FISH foci (n DMSO = 53, n dTAG-13 = 37). (right) RNAPII mean fluorescence intensity at random nuclear positions (n DMSO = 53, n dTAG-13 = 29). Data presented as mean values ± SD from one staining experiment. P values are from two-sided Mann-Whitney tests. NS: not significant. f . (left) Scheme of FKBP knock-in strategy in the R1 mESCs used in the PALM experiments. (right) TRIM28 Western blot in the R1 mESCs. Western blot was done once.

Article Snippet: The repair template included a mRuby2 fluorescent protein sequence, P2A linker and the FKBP tag sequence (Supplementary Fig. ) . mRuby2 sequence was amplified from the mRuby2-N1 plasmid (Addgene, catalog no. 54614), and the P2A-FKBP sequence was amplified from the PITCh dTAG donor vector (Addgene, catalog no. 91792).

Techniques: Fluorescence, Staining, MANN-WHITNEY, Knock-In, Western Blot

Journal: Nature Genetics

Article Title: Hijacking of transcriptional condensates by endogenous retroviruses

doi: 10.1038/s41588-022-01132-w

Figure Lengend Snippet: a , Representative images of individual z-slices (same z) of RNA-FISH and RNAPII IF signal, and an image of the merged channels. The nuclear periphery determined by DAPI staining is highlighted as a white contour. The zoom column displays the region of the images highlighted in a yellow box (enlarged for greater detail). Merge of the nuclear z-projections is displayed, and overlapping pixels between the RNA-FISH and IF channels are highlighted in white. Displayed M OC and Pearson’s correlation coefficient ( r ) values are an average obtained from 24 analyzed nuclei. Scale bars, 2.5 μm. b, Same as a , except with MED1 IF. c , Distance of IAP RNA-FISH foci to the nearest RNAPII or MED1 IF puncta. Each dot represents one IAP RNA-FISH focus. d , Meta representations of RNAPII ChIP with reference exogenous genome (ChIP-RX) (left) and MED23 ChIP–seq (right) read densities at IAP, MMERVK and MMETn ERVs in control (DMSO-) and dTAG-13 (24 h)-treated mESCs. The mean read densities are displayed ±2 kb around the indicated elements. The genomic elements were length normalized. e , Genome browser tracks at the Cthrc1 locus. Note the independent transcription initiation events at Cthrc1 and MMETn, ruling out that the MMETn acts as an alternative Cthrc1 promoter. Rpm, reads per million. f , Representative images of individual z-slices (same z) of RNA-FISH and IF signal, and an image of the merged channels. The nuclear periphery determined by DAPI staining is highlighted as a white contour (scale bars, 2.5 μm). Also shown are averaged signals of either RNA-FISH or IF centered on the Cthrc1 FISH foci or randomly selected nuclear positions (scale bars, 0.5 μm). r denotes a Spearman’s correlation coefficient. g, Same as f , except with NFYA IF. h , qRT–PCR data for IAP RNA, Cthrc1 mRNA and the Pri-miR-290-295 transcript in control and ERV-triple knockout (TKO) cells. Data are presented as mean values ± s.d. from six biological replicates. P values are from two-tailed t -tests. **** P < 1 × 10 −4 . i , Principal component (PC) plot of Hi-C interactions at an ERV-rich locus on chromosome 12. j , Pile-up analysis of contacts between IAPs, MMERVKs, MMETns and transcribed genes in wild-type and TRIM28-degraded mESCs.

Article Snippet: The repair template included a mRuby2 fluorescent protein sequence, P2A linker and the FKBP tag sequence (Supplementary Fig. ) . mRuby2 sequence was amplified from the mRuby2-N1 plasmid (Addgene, catalog no. 54614), and the P2A-FKBP sequence was amplified from the PITCh dTAG donor vector (Addgene, catalog no. 91792).

Techniques: Staining, ChIP-sequencing, Control, Quantitative RT-PCR, Triple Knockout, Two Tailed Test, Hi-C

Journal: Nature Genetics

Article Title: Hijacking of transcriptional condensates by endogenous retroviruses

doi: 10.1038/s41588-022-01132-w

Figure Lengend Snippet: a, b . Co-localization between the IAP RNA and (a) RNAPII puncta and (b) MED23 puncta in TRIM28-degraded mESCs. Displayed are separate images of the RNA-FISH and IF signal, and an image of the merged channels. The nuclear periphery determined by DAPI staining is highlighted as a white contour. The zoom column displays the region of the images highlighted in a yellow box zoomed in for greater detail. After 24 h dTAG-13 treatment, small nuclear puncta appear, and after 48 h of dTAG-13 treatment, large nuclear foci are visible. Scale bars: 2.5 μm. c . Scheme of the 1-6 hexanediol (1-6 HD) treatment experiments. d . Representative images of RNAPII immunofluorescence in control and 1-6 HD-treated cells. 1-6 HD partially dissolved the punctate localization of RNAPII. Scale bars: 5 μm. e . Transcription of the nascent Cthcr1 RNA is reduced by 30 min 1% 1-6 hexanediol-treatment in TRIM28-degraded cells. The bar plots show qRT-PCR data as fold change normalized to the DMSO control across 6 and 3 biological replicates for 24 h and 48 h timepoints, respectively. Note that the IAP RNA does not contain introns; thus, the IAP RNA qRT-PCR detects the steady state pool of IAP RNAs. Each dot represents a data point, and bar indicates the mean. P values are from two-tailed t tests. NS: not significant.

Article Snippet: The repair template included a mRuby2 fluorescent protein sequence, P2A linker and the FKBP tag sequence (Supplementary Fig. ) . mRuby2 sequence was amplified from the mRuby2-N1 plasmid (Addgene, catalog no. 54614), and the P2A-FKBP sequence was amplified from the PITCh dTAG donor vector (Addgene, catalog no. 91792).

Techniques: Staining, Immunofluorescence, Control, Quantitative RT-PCR, Two Tailed Test

Journal: Nature Genetics

Article Title: Hijacking of transcriptional condensates by endogenous retroviruses

doi: 10.1038/s41588-022-01132-w

Figure Lengend Snippet: a , Genotype of the iPSC line and scheme of the experimental setup. The iPSC line contains degradation-sensitive Trim28-FKBP alleles and doxycycline-inducible Oct4 , Sox2 , Klf4 and c-Myc (OSKM) transgenes. b , Western blot validation of the FKBP degron tag and OSKM ectopic expression in iPSCs. c , Representative images of IAP RNA-FISH staining. The number and percentage refer to cells with detectable IAP foci, pooled from two biological replicates. Scale bars, 10 μm; inset scale bars, 2 μm. d . Quantification of cells with detectable IAP foci (IAP + cells) at the indicated treatment regimes. e , IAP RNA expression is reduced in TRIM28-degraded iPSCs that ectopically express OSKM factors. The line plot shows qRT–PCR data of IAP RNA levels normalized to 0 h of dTAG-13 treatment. Data are from three independent biological replicates (three wells on a tissue culture plate) and are presented as mean values ± s.d. The experiment was repeated three times, and data from one representative experiment are shown. P value is from two-tailed t -tests. *** P < 1 × 10 −4 . f , Colocalization between the nascent RNA of miR290-295 and RNAPII puncta in TRIM28-degraded iPSCs that ectopically express OSKM factors. Separate images of individual z-slices (same z) of the RNA-FISH and IF signal are shown along with an image of the merged channels. The nuclear periphery determined by DAPI staining is highlighted as a white contour (scale bars, 2.5 μm). Also shown are averaged signals of either RNA-FISH or RNAPII IF centered on the miR290-295 RNA FISH foci or randomly selected nuclear positions. r denotes a Spearman’s correlation coefficient (scale bars, 0.5 μm). g , Elevated levels of miR290-295 SE transcript and Pri-miR290-295 nascent transcript in TRIM28-degraded iPSCs that ectopically express OSKM factors. qRT–PCR data was normalized to the 0 h of dTAG-13 treatment. Data are from three independent biological replicates (three wells on a tissue culture plate) and are presented as mean values ± s.d. The experiment was repeated three times, and data from one representative experiment are shown. P values are from two-tailed t -tests. * P = 0.027, *** P = 1 × 10 −4 .

Article Snippet: The repair template included a mRuby2 fluorescent protein sequence, P2A linker and the FKBP tag sequence (Supplementary Fig. ) . mRuby2 sequence was amplified from the mRuby2-N1 plasmid (Addgene, catalog no. 54614), and the P2A-FKBP sequence was amplified from the PITCh dTAG donor vector (Addgene, catalog no. 91792).

Techniques: Western Blot, Biomarker Discovery, Expressing, Staining, RNA Expression, Quantitative RT-PCR, Two Tailed Test

Journal: Nature Genetics

Article Title: Hijacking of transcriptional condensates by endogenous retroviruses

doi: 10.1038/s41588-022-01132-w

Figure Lengend Snippet: a . Western blot validation of the FKBP degron tag and its ability to degrade TRIM28 in iPSCs. Washout of the dTAG-13 ligand (24 h) indicates reversibility of degradation. Western blot experiments were performed twice and one representative image is shown. Actin is shown as the loading control. b . Western blot validation of the OSKM ectopic expression in the iPSC line. Western blot experiments were performed three times and one representative image is shown. HSP90 is shown as the loading control. c . dTAG-13 treatment leads to reduced RNAPII immunofluorescence signal at miR290-295 FISH foci which is rescued by OSKM ectopic expression, while overall RNAPII levels do not change. (top) Quantification of RNAPII mean fluorescence intensity (n = 117 for DMSO, n = 138 for dTAG-13, n = 110 for Dox+dTAG-13) in the cells used in Fig. . (bottom) Quantification of RNAPII IF intensities at the miR290-295 FISH foci (n = 128 for DMSO, n = 39 for dTAG-13, n = 89 for Dox+dTAG-13) detected in the cells used in Fig. . Data are presented as mean values ± SD from one staining experiment. P value is from a two-sided Mann-Whitney test. d . Mass spectrometry-detected protein abundance for three individual replicate samples after 0 h, 24 h, 48 h, 72 h and 96 h dTAG-13 treatment of mESCs. RNAPII subunits are highlighted in green. Mediator complex subunits are highlighted in red. e–h . qRT-PCR data normalized to the 0 h of dTAG-13 treatment. Data are from three independent biological replicates (that is, three wells on a tissue culture plate) and are presented as mean values ± SD. The experiment was repeated three times, and data from one representative experiment are shown. P values are from two-tailed t -tests. *: P < 0.05, ***: P < 10 −3 , ****: P < 10 −4 .

Article Snippet: The repair template included a mRuby2 fluorescent protein sequence, P2A linker and the FKBP tag sequence (Supplementary Fig. ) . mRuby2 sequence was amplified from the mRuby2-N1 plasmid (Addgene, catalog no. 54614), and the P2A-FKBP sequence was amplified from the PITCh dTAG donor vector (Addgene, catalog no. 91792).

Techniques: Western Blot, Biomarker Discovery, Control, Expressing, Immunofluorescence, Fluorescence, Staining, MANN-WHITNEY, Mass Spectrometry, Quantitative Proteomics, Quantitative RT-PCR, Two Tailed Test

Journal: Nature Genetics

Article Title: Hijacking of transcriptional condensates by endogenous retroviruses

doi: 10.1038/s41588-022-01132-w

Figure Lengend Snippet: a . Scheme of knockdown experiments with simultaneous TRIM28 degradation. b . qRT-PCR analysis from three independent biological replicates. Data presented as mean values ± SD. Experiment was performed twice, and the data shown are from one representative experiment. P values are from two-tailed t tests. ****: P < 10 −4 , ***: P < 10 −3 , **: P < 10 −2 , *: P < 0.05. c . Scheme of IAP knockdown experiments. d . qRT-PCR data displayed as fold change normalized to the 0 h control from three independent biological replicates. Data are presented as mean values ± SD. Each dot represents the mean of the three biological replicates of an individual experiment. P values are from two-tailed t -tests. ****: P < 10 −4 , ***: P < 10 −3 , **: P < 10 −2 , *: P < 0.05. e . Scheme of the experiment in which shRNAs are induced for 24 h and then treated either with DMSO (yellow), dTAG-13 (orange) or with dTAG-13 and Dox (maroon) for additional 24 h. f . qRT-PCR data as fold change normalized to the Dox (24 h) treatment control from three independent biological replicates. Data are presented as mean values ± SD. Experiment was performed twice, and data from one representative experiment is shown. P values are from two-tailed t -tests. ****: P < 10 −4 , ***: P < 10 −3 , **: P < 10 −2 , *: P < 0.05. g . RNA levels detected with total RNA-seq. Values from three biological replicates are normalized to levels detected at 0 h. Red arrowheads highlight the ERV taxa against whose sequences the shRNAs were designed. h . Representative images of IAP RNA FISH in cells described in panels ( e–f) . Scale bar: 2.5 μm. i . Analyses of cells used in Fig. . (left) RNAPII IF intensities at the miR290-295 FISH foci (n DMSO = 100, n dTAG-13 = 52, n Dox+dTAG-13 = 60). (right) RNAPII mean fluorescence intensity at random nuclear positions (n DMSO = 97, n dTAG-13 = 88, n Dox+dTAG-13 = 60). Data presented as mean values ± SD from one staining experiment. P values are from two-sided Mann-Whitney tests. NS: not significant. j . Images of individual z-slices (same z) of the RNA-FISH and IF signal. Nuclear periphery determined by DAPI staining is highlighted as a white contour. Also shown are averaged signals of either RNA FISH or RNAPII IF centered on the miR290-295 FISH foci or randomly selected positions. Scale bars: 2.5 μm. The experiment is an independent biological replicate of the experiments shown in Fig. .

Article Snippet: The repair template included a mRuby2 fluorescent protein sequence, P2A linker and the FKBP tag sequence (Supplementary Fig. ) . mRuby2 sequence was amplified from the mRuby2-N1 plasmid (Addgene, catalog no. 54614), and the P2A-FKBP sequence was amplified from the PITCh dTAG donor vector (Addgene, catalog no. 91792).

Techniques: Knockdown, Quantitative RT-PCR, Two Tailed Test, Control, RNA Sequencing, Fluorescence, Staining, MANN-WHITNEY