script direct rt qpcr probesmaster kit  (Jena Bioscience)

Journal: Nature Cell Biology

Article Title: Transcription-coupled DNA–protein crosslink repair by CSB and CRL4 CSA -mediated degradation

doi: 10.1038/s41556-024-01394-y

Figure Lengend Snippet: a , Top: schematic of experiment. Bottom: representative images of nascent transcription levels as determined by EU pulse labelling in MRC-5 cells treated with FA, 1 μM THZ1 or UV (8 J m –2 ). Scale bar, 50 μm. b , Quantification of transcription levels of RNA synthesis as shown in a . Relative fluorescence intensities (RFI) of EU were normalized to untreated levels and set to 100%. Black lines indicate the average integrated density ± s.e.m. n (left to right) = 635, 754, 708, 714, 669, 594, 519, 644, 617, 545, 722, 670 and 530 cells from 3 independent experiments. Unpaired two-tailed t -test. c , FRAP analysis of GFP–Pol II using MRC-5 GFP–RPB1 KI cells untreated or 1–2 h after a 30 min FA pulse. The RFI was measured over time, background-corrected and normalized to the pre-bleach fluorescence intensity. Graphs present mean values. n (top to bottom) = 49, 24, 23, 33 and 23 cells from n = 4 (FA) or n = 3 (UV) independent experiments. d , Relative immobile fractions of GFP–Pol II calculated from data indicated in the dashed box in c . Values represent the mean ± s.e.m. Unpaired two-tailed t -test. e , Left: representative images of GFP–DNMT1-expressing RPE1 cells treated with a 30 min 5-Aza-dC (50 μM) pulse and fixed after 120 min. Scale bars, 10 μm and 2 μm (magnification). Right: histogram of RFI for DNMT1, PCNA and EU at the indicated line 120 min after 5-Aza-dC treatment. f , Quantification of EU signals at DNMT1 foci and the surrounding nucleoplasm (global) as shown in e . RFI values were background-corrected and normalized to untreated samples, which was set at 1. Lines show the mean ± s.e.m. n (left to right) = 36, 43, 36, 36, 46, 43 and 43 cells from 3 independent experiments. Unpaired two-tailed t -test. g , Quantification of recovery of transcription after FA treatment shown in Extended Data Fig. . RFI values of EU were normalized to untreated levels and set to 100%. Black lines indicate the average integrated density ± s.e.m. n (left to right) = 626, 573, 877, 612, 601, 566, 592 and 549 cells from 3 independent experiments. Unpaired two-tailed t -test. h , GFP–Pol II FRAP as in c at the indicated time intervals after a 30 min FA (300 μM) pulse. Graphs represent the mean. n (top to bottom) = 45, 23, 24, 37 and 32 cells from 3 independent experiments. i , Relative immobile fractions of GFP–Pol II as in h . Values represent the mean ± s.e.m. Unpaired two-tailed t -test. Source numerical data are available in the source data.

Article Snippet: Transcription levels were measured by pulse labelling with 100 µM EU (Jena Bioscience) in regular culture medium, and cells were grown at 37 °C for 30 min before fixation with 3.6% FA (Sigma) in PBS for 10 min at room temperature.

Techniques: Fluorescence, Two Tailed Test, Expressing

Journal: Nature Cell Biology

Article Title: Transcription-coupled DNA–protein crosslink repair by CSB and CRL4 CSA -mediated degradation

doi: 10.1038/s41556-024-01394-y

Figure Lengend Snippet: a : Representative images of transcription levels in MRC-5 cells treated for 30 min with the indicated concentrations of formaldehyde (FA), Quantification is shown in Fig. . Scale bar = 50 μm. b : Close up of cells exposed to mock- or formaldehyde treatment with 300 μM FA. Scale bar = 10 μm. c : MRC-5 cells treated with Actinomycin D (ActD, 50 ng/ml) or Flavopiridol (Flavo, 1 μM) for 1 hr. Scale bar = 20 μm and 10 μm (magnification). d : MRC-5 cells, treated with the inhibitors as in ( c ), were subsequently treated with 1 mM FA for 30 min. Relative fluorescence intensities (RFI) of EU was normalized to levels without FA and set to 100%. Black lines indicate average integrated density ±S.E.M. n = 849, 1025, 745, 792, 686, 600 cells (left to right) from 3 independent experiments. Unpaired two-tailed t-test. e : Representative pictures of transcription levels in WT and RPB1 K1268R cells after a 1 mM FA pulse (30 min). Scale bar = 50 μm. f : Quantification of transcription as shown in ( b ). RFI of EU was normalized to untreated levels and set to 100%. Black lines indicate average integrated density ±S.E.M. n = 1311, 1169, 1224, 1439 cells (left to right) from 3 independent experiments. Unpaired two-tailed t-test. g : Immunoblot of chromatin fractions from HeLa WT and RPB1-K1268R mutant stained for pSer2 Pol II and SSRP as a loading control. This experiment was performed twice with similar results. h : Representative pictures of transcription levels in MRC-5 cells treated with either 1 mM FA or 1 μM Flavopiridol during a 30 min EU pulse for the indicated times. Scale bar = 50 μm. i : Quantification of transcription levels as shown in ( e ). RFI of EU was normalized to untreated levels and set to 100%. Black lines indicate average integrated density ±S.E.M. n = 807, 964, 852, 859, 852, 757, 829 cells (left to right) from 3 independent experiments. Unpaired two-tailed t-test. j : Representative pictures of transcription levels in MRC-5 cells, treated with 1 μM Flavopiridol for 2 hr prior to treatment with 1 mM FA for 30 min when indicated. Scale bar = 50 μm. k : Quantification of transcription levels as shown in ( j ). RFI of EU were normalized Flavopiridol-treated levels and set at 1. Black lines indicate average integrated density ±S.E.M. n = 967, 902, 937, 873, 856, 824 cells (left to right) from 3 independent experiments. Unpaired two-tailed t-test. l : Quantification of nascent RNA transcription after the Flavopiridol wash-out. After a 2 hr, 1 μM Flavopiridol pretreatment, MRC-5 cells were treated 1 mM FA for 30 min. After FA wash-out, RNA was collected at the indicated timepoints and the expression was assayed by RT-qPCR. Values represent the mean ±S.E.M. from 3 independent experiments. m : Representative images of mock-treated GFP-DNMT1 expressing RPE1 cells, which were fixed after 120 min. Right: Histogram of fluorescence signal for DNMT, PCNA and EU at the indicated line. This experiment was performed 3 times independently with similar results. Scale bar = 10 μm and 2 μm in the magnification. Source numerical data and unprocessed blots are available in source data.

Article Snippet: Transcription levels were measured by pulse labelling with 100 µM EU (Jena Bioscience) in regular culture medium, and cells were grown at 37 °C for 30 min before fixation with 3.6% FA (Sigma) in PBS for 10 min at room temperature.

Techniques: Fluorescence, Two Tailed Test, Western Blot, Mutagenesis, Staining, Control, Expressing, Quantitative RT-PCR

Journal: Nature Cell Biology

Article Title: Transcription-coupled DNA–protein crosslink repair by CSB and CRL4 CSA -mediated degradation

doi: 10.1038/s41556-024-01394-y

Figure Lengend Snippet: a : Representative images of recovery of transcription for the indicated recovery times after a 30 min pulse of 1mM FA as determined by a 30 min pulse labelling with EU. This is quantified in Fig. . Scale bar = 50 μm. b : Representative images of replication levels in RPE1 cells treated with 1 µM CDK4/6 inhibitor Palbociclib for 24 hr prior to pulse-labeling with EdU for 3 hr. Scale bar = 50 μm. c : Quantification of the number of EdU positive cells shown in ( b ). Black bars indicate average from 2 independent experiments. d : Representative images of transcription levels in RPE1 cells treated with 1 µM CDK4/6 inhibitor Palbociclib for 24 hr prior to EU pulse-labelling for 30 min. Scale bar = 50 μm. e : Quantification of the transcription levels as shown in ( d ). Relative fluorescence intensities (RFI) of EU were normalized to untreated levels and set to 100%. Black bars indicate average integrated density ±S.E.M. n = 314 or 231 cells from 2 independent experiments. Unpaired two-tailed t-test. f : Representative images of transcription levels in cycling and non-cycling cells. Cells were arrested with 1 µM CDK4/6 inhibitor Palbociclib for 24 hr prior to exposure to 1 mM FA for 30 min. Transcription levels were assayed by EU incorporation and visualized with click-chemistry. Scale bar = 50 μm. g : Quantification of the recovery of transcription in cycling cells or non-cycling RPE1 cells as shown in ( f ). RFI of EU at the indicated time points after 1 mM FA pulse treatments was normalized to untreated levels and set to 100%. Black lines indicate average integrated density ±S.E.M. n = 985, 928, 967, 974, 900, 691, 691, 752 cells (left to right) from 3 independent experiments. Unpaired two-tailed t-test. Source numerical data are available in source data.

Article Snippet: Transcription levels were measured by pulse labelling with 100 µM EU (Jena Bioscience) in regular culture medium, and cells were grown at 37 °C for 30 min before fixation with 3.6% FA (Sigma) in PBS for 10 min at room temperature.

Techniques: Labeling, Fluorescence, Two Tailed Test

Journal: Nature Cell Biology

Article Title: Transcription-coupled DNA–protein crosslink repair by CSB and CRL4 CSA -mediated degradation

doi: 10.1038/s41556-024-01394-y

Figure Lengend Snippet: a : DPC repair % in expressed bins was plotted against their expression level. Spearman correlation between DPC repair and transcription levels was 0.61997. b : A graphic representation of the RT-qPCR primers used in the DPC removal assay along the CRIM1 gene. c : Relative DPC recovery in untreated (NT) or after the indicated times after a 30 min 1 mM FA pulse using K-SDS precipitation followed by qPCR of genomic DNA in the CRIM1 gene and CRIM1 intergenic region as well a silent genomic region on chromosome Y. Values were normalized to 0 hr conditions for each primer set at 1 and represent mean with S.E.M. from 4 independent experiments. d : Relative DPC recovery at 4 hr after a 30 min 1 mM FA pulse using K-SDS precipitation followed by qPCR of genomic DNA in the CRIM1 gene and CRIM1 intergenic region. Values were normalized to 0 hr conditions (not shown) for each primer set at 1 and represent mean with S.E.M. from 4 independent experiments. Source numerical data are available in source data.

Article Snippet: Transcription levels were measured by pulse labelling with 100 µM EU (Jena Bioscience) in regular culture medium, and cells were grown at 37 °C for 30 min before fixation with 3.6% FA (Sigma) in PBS for 10 min at room temperature.

Techniques: Expressing, Quantitative RT-PCR

Journal: Nature Cell Biology

Article Title: Transcription-coupled DNA–protein crosslink repair by CSB and CRL4 CSA -mediated degradation

doi: 10.1038/s41556-024-01394-y

Figure Lengend Snippet: a , Top: scheme of experiment. Bottom: quantification of recovery of transcription after a 30 min pulse of 1 mM FA. RFI of EU were normalized to untreated levels and set to 100%. Black lines indicate the average integrated density ± s.e.m. n (left to right) = 779, 683, 759, 758, 801, 647, 676, 786, 692, 524, 697, 615, 696, 625, 609, 673, 368, 277, 364 and 334 cells from 3 independent experiments. Unpaired two-tailed t -test at the 6 h time point. b , Relative immobile fractions of GFP–Pol II in MRC-5 GFP–RPB1 KI WT, XPA KO or CSB KO cells at the indicated time intervals after a 30 min pulse with 300 μM FA. Values represent the mean ± s.e.m. n (left to right) = 23, 23, 26, 24, 22, 22, 25, 22 and 24 cells from 3 independent experiments. Unpaired two-tailed t -test. c , Relative immobile fractions of CSB FRAP after a 30 min pulse with 300 μM FA followed over time in KO cells. Values represent the mean ± s.e.m. n (left to right) = 122, 129, 82, 82, 96, 61, 52, 67, 47, 56, 68, 44, 53, 57 and 48 cells from 3 (WT and XPA KO) or 4 ( CSA KO) independent experiments. Unpaired two-tailed t -test. d , TCR-UDS in non-replicating RPE1 XPC KO by 1 μM palbociclib treatment for 24 h before treatment with the indicated doses of FA or with 8 J m –2 UV. Cells were pre-treated with 2 μM THZ1 where indicated. Values represent the mean ± s.e.m. n (left to right) = 648, 997, 502, 754 and 610 cells from 3 independent experiments. Unpaired two-tailed t -test. Source numerical data are available in the source data.

Article Snippet: Transcription levels were measured by pulse labelling with 100 µM EU (Jena Bioscience) in regular culture medium, and cells were grown at 37 °C for 30 min before fixation with 3.6% FA (Sigma) in PBS for 10 min at room temperature.

Techniques: Two Tailed Test

Journal: Nature Cell Biology

Article Title: Transcription-coupled DNA–protein crosslink repair by CSB and CRL4 CSA -mediated degradation

doi: 10.1038/s41556-024-01394-y

Figure Lengend Snippet: a : Representative images of transcription levels in MRC-5 KO cells treated for 30 min with 1 mM FA as determined by EU pulse-labelling for 30 min. Quantification is shown in Fig. . Scale bar = 50 μm. b : GFP-Pol II FRAP in MRC-5 GFP-RPB1 KI WT of XPA and CSB KO cells at the indicated time-intervals after a pulse treatment with 300 μM FA for 30 min. Line represent mean of n = 23, 24, 25 cells (WT), n = 23, 22, 22 cells (CSB), n = 26, 22, 24 cells (XPA) (up to down) from 3 independent experiments. Relative immobile fraction as shown in Fig. . c : CSB-mScarlet-I FRAP with in HCT116 CSA KO cells treated with 300 μM FA for 30 min. Graph is an average of n = 129, 96, 67, 68, 57, 87 cells (up to down) from 4 independent experiments. Relative immobile fraction as shown in Fig. . d : CSB-mScarlet-I FRAP with in HCT116 XPA KO cells treated with 300 μM FA for 30 min. Graph is an average of n = 82, 61, 47, 44, 48, 98 cells (up to down) from 4 independent experiments. Relative immobile fraction as shown in Fig. . Source numerical data are available in source data.

Article Snippet: Transcription levels were measured by pulse labelling with 100 µM EU (Jena Bioscience) in regular culture medium, and cells were grown at 37 °C for 30 min before fixation with 3.6% FA (Sigma) in PBS for 10 min at room temperature.

Techniques:

Journal: Nature Cell Biology

Article Title: Transcription-coupled DNA–protein crosslink repair by CSB and CRL4 CSA -mediated degradation

doi: 10.1038/s41556-024-01394-y

Figure Lengend Snippet: a , Quantification of recovery of transcription in RPE1 WT and CSB KO cells transfected with short interfering RNA (siRNA) targeting SPRTN (siSPRTN) or a control siRNA (siCtrl) after a 30 min pulse of 1 mM FA. After FA washout, cells were left to recover for the indicated times, including a 30 min pulse labelling with EU before fixation. RFI of EU were normalized to untreated levels and set to 100%. Black lines indicate the average integrated density ± s.e.m. from three independent experiments. n (left to right) = 777, 634, 742, 757, 509, 481, 491, 458, 494, 480, 448, 616, 333, 371, 356 and 349 cells. Unpaired two-tailed t -test. b , Relative immobile fraction of CSB FRAP in non-replicating RPE1 CSB–mScarlet-I cells transfected with the indicated siRNAs and treated with 1 μM palbociclib 24 h before FRAP. Cells were treated with 300 μM FA for 30 min and followed for the indicated times. Values represent the mean ± s.e.m. from three independent experiments. n (left to right) = 49, 51, 55, 46, 37, 41, 34, 39, 45, 55, 56, 55, 46, 60 and 34 cells. Unpaired two-tailed t -test. c , Relative survival of non-replicating RPE1 cells as determined by Alamar Blue staining. RPE1 cells, arrested in G1 by treatment with 1 μM palbociclib, were treated with the indicated doses of FA for 1 h and allowed to recover for 4 days. Metabolic activity was assayed as a measure of cell viability. Values represent the mean ± s.e.m. from four independent experiments and were normalized to untreated cells. Unpaired two-tailed t -test at 5 mM FA. d , GFP–Pol II FRAP in WT and CSB KO neurons. GFP–RPB1 KI iPS cells were differentiated into post-mitotic neurons through neurogenin-2 induction. FRAP in two different clones (A and B) was performed after treatment with 300 μM FA for 90 min. Graph represents values from eight cells. Source numerical data are available in the source data.

Article Snippet: Transcription levels were measured by pulse labelling with 100 µM EU (Jena Bioscience) in regular culture medium, and cells were grown at 37 °C for 30 min before fixation with 3.6% FA (Sigma) in PBS for 10 min at room temperature.

Techniques: Transfection, Small Interfering RNA, Control, Two Tailed Test, Staining, Activity Assay, Clone Assay

Journal: Nature Cell Biology

Article Title: Transcription-coupled DNA–protein crosslink repair by CSB and CRL4 CSA -mediated degradation

doi: 10.1038/s41556-024-01394-y

Figure Lengend Snippet: a : Immunoblot of RPE1 cells, transfected with siRNA against SPRTN, stained with antibodies against SPRTN and Tubulin. This experiment was performed twice with similar results. b : Representative pictures of transcription levels in RPE1 WT and CSB KO cells transfected with siRNAs against SPRTN. Quantification is shown in Fig. . Scale bar, 50 = μm. c : Sequencing of UVSSA and SPRTN mutations in RPE1 cells. d : Immunoblot for the indicated proteins in various RPE1 KO cell lines. This experiment was performed twice with similar results. e : Clonogenic survival assay in RPE1 cells after varying doses of UV and FA. Graph represents the average ±S.E.M. from 3 independent experiments. Unpaired two-tailed t-test at 3 J m -2 UV and 2 mM FA. f : Representative pictures of transcription levels in WT, CSA KO, CSB KO, UVSSA KO and SPRTN −/+ RPE1 cells. Scale bar = 50 μm. g : Quantification of the transcription levels as shown in ( f ). Relative fluorescence intensities (RFI) of EU were normalized to mock-treated levels and set to 100%. Black lines indicate average integrated density ±S.E.M. n = 424, 407, 408, 654, 352, 308, 345, 345, 420, 385, 493, 413, 508, 482, 576, 626, 302, 283, 294, 304 cells (left to right) from 2 independent experiments. h : CSB-mScarlet-I FRAP with in cells non-replicating cells which were treated with 1 μM Palbociclib (CDKi) for 24 hr prior to FRAP after 1 mM FA for 30 min. Graph is an average of n = 35, 37, 40, 40, 40 cells (up to down) from 4 independent experiments. i : Relative immobile fractions of mScarlet-I-CSB FRAP as in ( h ). Values represent mean ± S.E.M. Unpaired two-tailed t-test. j : CSB-mScarlet-I FRAP with in cells transfected with control (Ctrl) siRNA. Cells were treated with 1 μM Palbociclib (CDKi) for 24 hr prior to FRAP after 1 mM FA for 30 min. Relative immobile fraction as shown in Fig. . Graph is an average of n = 49, 46, 34, 55, 46 cells (up to down) from 3 independent experiments. k : CSB-mScarlet-I FRAP with in cells transfected with siCSA RNA. Cells were treated with 1 μM Palbociclib (CDKi) for 24 hr prior to FRAP after 1 mM FA for 30 min. Relative immobile fraction as shown in Fig. . Graph is an average of n = 51, 37, 39, 56, 60 cells (up to down) from 4 independent experiments. l : CSB-mScarlet-I FRAP with in cells transfected with siSPRTN RNA. Cells were treated with 1 μM Palbociclib (CDKi) for 24 hr prior to FRAP after 1 mM FA for 30 min. Relative immobile fraction as shown in Fig. . Graph is an average of n = 55, 41, 45, 55, 34 cells (up to down) from 3 independent experiments. m : Characterization of the CSB mutation in iPS GFP-RPB1 cells. This experiment was performed twice with similar results. Source numerical data and unprocessed blots are available in source data.

Article Snippet: Transcription levels were measured by pulse labelling with 100 µM EU (Jena Bioscience) in regular culture medium, and cells were grown at 37 °C for 30 min before fixation with 3.6% FA (Sigma) in PBS for 10 min at room temperature.

Techniques: Western Blot, Transfection, Staining, Sequencing, Clonogenic Cell Survival Assay, Two Tailed Test, Fluorescence, Control, Mutagenesis

Journal: Nature Cell Biology

Article Title: Transcription-coupled DNA–protein crosslink repair by CSB and CRL4 CSA -mediated degradation

doi: 10.1038/s41556-024-01394-y

Figure Lengend Snippet: a , Top: scheme of experiment. Bottom: relative GFP–RPB1 protein levels measured by flow cytometry in the presence of 100 µM cycloheximide after a pulse of 1 mM FA for 30 min. RFI of GFP were normalized to mock-treated levels and set to 1. Bars represent the mean fluorescence ± s.e.m. from three independent experiments. Unpaired two-tailed t -test. b , Chromatin-bound elongating Pol II as determined by immunoblotting with the indicated antibodies. MRC-5 cells were treated with 1 mM FA for 30 min and collected at the indicated times. SSRP1 was used as the loading control. This experiment was performed three times with similar results. c , Quantification of pSer2-modified RPB1 levels as shown in b . Values indicate the average integrated density ± s.e.m. from three independent experiments. Unpaired two-tailed t -test. d , Relative survival of WT, CSB KO or K1268R mutated RPB1 HeLa cells treated with the indicated doses of FA for 1 h. Values represent the mean ± s.e.m. from three independent experiments. Unpaired two-tailed t -test at 1.5 mM FA. e , Quantification of recovery of transcription in WT, CSB KO or K1268R mutated RPB1 HeLa cells after a 30 min pulse of 1 mM FA. After FA washout, cells were left to recover for the indicated times, including a 30 min pulse labelling with EU. RFI of EU were normalized to mock-treated levels and set to 100%. Black lines indicate the average integrated density ± s.e.m. n (left to right) = 1,311, 1,169, 1,162, 1,224, 1,439, 1,360, 1,236, 1,386, 973, 1,286, 1,310 and 1,107 cells from 3 independent experiments. Source numerical data ( a , c – e ) and unprocessed blots ( b ) are available in the source data.

Article Snippet: Transcription levels were measured by pulse labelling with 100 µM EU (Jena Bioscience) in regular culture medium, and cells were grown at 37 °C for 30 min before fixation with 3.6% FA (Sigma) in PBS for 10 min at room temperature.

Techniques: Flow Cytometry, Fluorescence, Two Tailed Test, Western Blot, Control, Modification

Journal: Nature Cell Biology

Article Title: Transcription-coupled DNA–protein crosslink repair by CSB and CRL4 CSA -mediated degradation

doi: 10.1038/s41556-024-01394-y

Figure Lengend Snippet: a : Flow cytometry of GFP-RPB1 after a pulse of 1 mM FA for 30 min in the presence of proteasome inhibitor MG132. Cells were harvested at the indicated times and the fluorescent levels were determined by flow cytometry. Values were normalized to untreated levels and represent the average ±S.E.M. from 3 independent experiments. Unpaired two-tailed t-test. b : Representative pictures of transcription levels in WT, CSB KO and RPB1 K1268R cells, which are quantified in Fig. . Scale bar = 50 μm. Source numerical data are available in source data.

Article Snippet: Transcription levels were measured by pulse labelling with 100 µM EU (Jena Bioscience) in regular culture medium, and cells were grown at 37 °C for 30 min before fixation with 3.6% FA (Sigma) in PBS for 10 min at room temperature.

Techniques: Flow Cytometry, Two Tailed Test

Journal: Nature Cell Biology

Article Title: Transcription-coupled DNA–protein crosslink repair by CSB and CRL4 CSA -mediated degradation

doi: 10.1038/s41556-024-01394-y

Figure Lengend Snippet: a , Relative immobile fractions of non-replicating mScarlet-I–CSB FRAP in cells arrested following treatment with 1 µM palbociclib for 24 h. Cells were mock treated or treated with 50 µM proteasome inhibitor (MG132), 10 µM VCP inhibitor (VCPi; NMS873) or 20 µM neddylation inhibitor (NAEi; MLN4924) before treatment with 1 mM FA for 30 min. Values represent the mean ± s.e.m. n (left to right) = 25, 24, 34, 26, 24, 30, 40, 31, 27, 30, 39, 34, 27, 30, 39, 34, 27, 30, 40 and 33 cells from 3 (NAEi) or 4 (VCPi) independent experiments. Unpaired two-tailed t -test. b , Top: scheme of experiment. Bottom: IP of pSer2-modified elongating Pol II followed by immunoblotting with the indicated antibodies either directly after a 30 min FA pulse (1 mM) or after recovery for the indicated time. This experiment was performed twice with similar results. Untr., untreated. c , Relative immobile fractions of non-replicating mScarlet-I–CSB FRAP in siRNA-transfected cells, which were arrested in G1 with 1 μM palbociclib for 24 h before treatment with 1 mM FA for 30 min. Graphs represent the mean ± s.e.m. n (left to right) = 36, 28, 38, 24, 35, 28, 36, 28, 29 and 28 cells from 3 independent experiments. Unpaired two-tailed t -test. d , Quantification of recovery of transcription in RPE1 cells transfected with the indicated siRNA. Cells were treated with a 30 min FA (1 mM) pulse and left to recover for the indicated times, including a 30 min pulse labelling with EU. RFI of EU were normalized to untreated levels and set to 100%. Black lines indicate the average integrated density ± s.e.m. n (left to right) = 688, 652, 331, 633, 611, 466, 726, 391, 265, 721, 418 and 313 cells from 2 (siCSB) or 3 (siCtrl and siDDB1) independent experiments. Unpaired two-tailed t -test. e , Relative clonogenic survival assay in WT (MRC-5 sv40), CS-A (CS3BE sv40), CS-B (CS1AN sv40) and UVSS-A (TA-24 sv40) cells with the indicated doses of FA for a 1 h pulse. Graphs were normalized to the untreated colony number, which was set at 100%. Graphs represent the mean ± s.e.m. from five independent experiments. Unpaired two-tailed t -test. f , TC-DPC repair model. TC-DPC repair is initiated when DPC-stalled Pol II is recognized by CSB, which recruits CRL4 CSA E3 ligase. UVSSA stabilizes CSB through USP7. Ubiquitin (Ub)-DPC ubiquitylation by CRL4 CSA drives VCP-dependent and proteasome-dependent DPC degradation followed by transcription restart. Functional TC-DPC repair may explain the phenotypic differences in the TC-NER syndromes CS and UV-sensitive syndrome. Source numerical data ( a , c – e ) and unprocessed blots ( b ) are available in the source data. Scheme in f was created using BioRender ( https://biorender.com ).

Article Snippet: Transcription levels were measured by pulse labelling with 100 µM EU (Jena Bioscience) in regular culture medium, and cells were grown at 37 °C for 30 min before fixation with 3.6% FA (Sigma) in PBS for 10 min at room temperature.

Techniques: Two Tailed Test, Modification, Western Blot, Transfection, Clonogenic Cell Survival Assay, Functional Assay

Journal: Nature Cell Biology

Article Title: Transcription-coupled DNA–protein crosslink repair by CSB and CRL4 CSA -mediated degradation

doi: 10.1038/s41556-024-01394-y

Figure Lengend Snippet: a : CSB-mScarlet-I FRAP with in non-replicating cells treated 1 μM Palbociclib (CDKi) for 24 hr prior to treatment with 50 µM proteasome inhibitor (MG132) and subsequently with 1 mM FA for 30 min. Relative immobile fraction as shown in Fig. . Graph is an average of n = 24, 30, 30, 30, 30 cells (up to down) from 3 independent experiments. b : CSB-mScarlet-I FRAP with in non-replicating cells treated 1 μM Palbociclib (CDKi) for 24 hr prior to treatment with 10 µM VCP inhibitor (VCPi: NMS873) and subsequently with 1 mM FA for 30min. Relative immobile fraction as shown in Fig. . Graph is an average of n = 34, 40, 39, 39, 40 cells (up to down) from 3 independent experiments. c : CSB-mScarlet-I FRAP with in non-replicating cells treated 1 μM Palbociclib (CDKi) for 24 hr prior to treatment with 20 µM neddylation inhibitor (NAEi: MLN4924) and subsequently with 1 mM FA for 30 min. Relative immobile fraction as shown in Fig. . Graph is an average of n = 26, 31, 34, 34, 33 cells (up to down) from 3 independent experiments. d : Relative immobile fractions of mScarlet-I-CSB FRAP in HCT116 cells mock-treated, or treated with 50 µM MG132 and 10 µM VCP inhibitor (VCPi: NMS873) prior to treatment with 300 μM FA for 30 min. Values represent mean ± S.E.M., whereby 122, 50, 46, 82, 45, 48, 52, 48, 49, 56, 45, 52, 53, 51, 54 cells respectively (left to right) from 3 independent experiments were analyzed. Unpaired two-tailed t-test. e : CSB-mScarlet-I FRAP in HCT116 cells treated with 50 µM proteasome inhibitor MG132 prior to treatment with 300 μM FA for 30 min. Relative immobile fraction as shown in ( d ). Graph is an average of n = 46, 46, 48, 49, 52, 54 cells (up to down) from 3 independent experiments. f : CSB-mScarlet-I FRAP in HCT116 cells treated with 10 µM VCP inhibitor NMS873 (VCPi) prior to treatment with 300 μM FA for 30 min. Relative immobile fraction as shown in ( d ). Graph is an average of n = 38, 50, 45, 48, 45, 51 cells (up to down) from 3 independent experiments. g : CSB-mScarlet-I FRAP in non-replicating RPE1 cells treated 1 μM Palbociclib (CDKi) for 24 hr prior to treatment with 2 μM SUMO inhibitor (SUMOi: ML792) and subsequently with 1 mM FA for 30 min. Graph is an average of n = 29, 28, 27, 28, 28, 27 cells (up to down) from 3 independent experiments. h : Relative immobile fractions of mScarlet-I-CSB FRAP in non-cycling RPE1 cells mock treated, or treated with 2 μM SUMO inhibitor (SUMOi: ML792) prior to treatment with 1 mM FA for 30 min quantified from ( h ). Values represent mean ± S.E.M. Unpaired two-tailed t-test. i : Immunostaining with the indicated antibodies of chromatin fraction after formaldehyde treatment. Cells were pre-treated for 2 hr pretreatment with 2 μM sumoylation inhibitor (SUMOi) before treatment with 1 mM FA for 30 min. Cells were harvested immediately after FA treatment for chromatin fractionation. This experiment was performed twice with similar results. j : Relative immobile fractions of mScarlet-I-CSB FRAP in WT and CSA KO HCT116 cells and WT cells treated with 20 µM neddylation inhibitor (NAEi: MLN4924) or 2 μM SUMO inhibitor (SUMOi: ML792) prior to treatment with 300 μM FA for 30 min. Values represent mean ± S.E.M., whereby 122, 129, 61, 25, 82, 96, 57, 37, 52, 67, 49, 26, 56, 68, 74, 30, 53, 57, 65, 27 cells respectively (left to right) from a 3 (NAEi and Sumoi) or 4 (CSA KO) independent experiments were analyzed. Unpaired two-tailed t-test. k : CSB-mScarlet-I FRAP in HCT116 cells treated with 20 µM neddylation inhibitor (NAEi: MLN4924) prior to treatment with 300 μM FA for 30 min. Relative immobile fraction as shown in ( j ). Graph is an average of n = 66, 56, 61, 57, 49, 74, 65 cells (up to down) from 3 independent experiments. l : CSB-mScarlet-I FRAP in HCT116 cells treated with 2 μM SUMO inhibitor (SUMOi: ML792) prior to treatment with 300 μM FA for 30 min. Relative immobile fraction as shown in (j) . Graph is an average of n = 25, 37, 26, 30, 27 cells (up to down) from 3 independent experiments. m : Immunoblot of cells transfected with siRNA against DDB1, stained with antibodies against DDB1 and Tubulin. This experiment was performed twice with similar results. n : CSB-mScarlet-I FRAP in cells transfected with siRNAs against DDB1. Cells were treated with 1 μM Palbociclib (CDKi) for 24 hr prior to FRAP after 1 mM FA for 30 min. Relative immobile fraction as shown in Fig. . Graph is an average of n = 28, 24, 28, 28, 28 cells (up to down) from 3 independent experiments. o : Representative pictures of the recovery of transcription in siRNA transfected RPE1 cells. Quantification is shown in Fig. . Scale bar = 50 μm. Source numerical data and unprocessed blots are available in source data.

Article Snippet: Transcription levels were measured by pulse labelling with 100 µM EU (Jena Bioscience) in regular culture medium, and cells were grown at 37 °C for 30 min before fixation with 3.6% FA (Sigma) in PBS for 10 min at room temperature.

Techniques: Two Tailed Test, Immunostaining, Fractionation, Western Blot, Transfection, Staining

Journal: bioRxiv

Article Title: Dynamics of RNA localization to nuclear speckles are connected to splicing efficiency

doi: 10.1101/2024.02.29.581881

Figure Lengend Snippet: (A) Scheme of ARTR-seq. Specific speckle scaffold protein is immunostained by primary and secondary antibodies sequentially. pAG-RTase is then allowed to bind to the antibody to initiate reverse transcription in situ. The generated biotinylated cDNAs are collected and prepared for sequencing. (B) Representative image showing colocalization of pAG-RTase labeled with Alexa Fluor 647 (AF647) (red), secondary antibody labeled with Alexa Fluor 568 (AF568, yellow) against anti-SON primary antibody, and generated biotinylated-cDNA detected by Alexa Fluor 488 (AF488) labeled antibody against biotin (green). Scale bar: 3 µm. (C) Venn diagram of overlapped speckle-enriched genes identified through targeting SON and SRRM2 in HeLa cells using Method 1 or Method 2. Speckle-enriched transcripts are defined by I NSE >2 and adjusted p-value < 0.05. (D) Genome tracks showing ARTR-seq reads generated from targeting SON or SRRM2 proteins, and from control samples without primary antibody, mapped to gene locus encoding lncRNA MALAT1 . (E) RNA FISH images showing speckle-enriched LAMA5 transcript in comparison with speckle non-enriched P4HB transcript. RNA FISH probes are labeled with AF647 (red). Nucleus was stained with DAPI. Scale bar: 10 μm. (F) Correlation between speckle partition coefficient (R NS/NU ) measured by RNA FISH imaging and I NSE values determined by ARTR-seq using Method 1.

Article Snippet: Samples were then incubated with pAG-RTase for an additional 30 min. A reverse transcription reaction mixture was prepared by mixing 2 μM adapter-RT primer, 0.05 mM biotin-16-dUTP (Jena Bioscience), 0.05 mM biotin-16-dCTP (Jena Bioscience), 0.05 mM dTTP (Thermo Fisher Scientific), 0.05 mM dCTP (Thermo Fisher Scientific), 0.1 mM dATP (Thermo Fisher Scientific), 0.1 mM dGTP (Thermo Fisher Scientific), 1 U/μL RNaseOUT (Thermo Fisher Scientific) in 50 μL buffer of DPBS supplemented with 3 mM MgCl 2 .

Techniques: Reverse Transcription, In Situ, Generated, Sequencing, Labeling, Control, Comparison, Staining, Imaging

Journal: bioRxiv

Article Title: Dynamics of RNA localization to nuclear speckles are connected to splicing efficiency

doi: 10.1101/2024.02.29.581881

Figure Lengend Snippet: (A) Scatter plot showing randomly selected genes from Group A, B and C genes, and corresponding I NSE(exon) under NT and Plad B treatment conditions in HeLa cells. (B) Genome tracks showing polyA RNA-seq (pink) and nuclear RNA-seq (blue) under NT and Plad B treatment conditions for selected genes: THOC6 in Group A gene, TUBB4B in Group B gene, and NCL in Group C gene. Selected efficiently spliced or inefficiently spliced introns for RT-PCR assay are highlighted in cyan and red boxes respectively. Genome tracks of other selected genes for RT-PCR assays are shown in Figure S8. (C) Schematic description of the RT-PCR assay. After reverse transcription of extracted total RNA, primers located on two adjacent exons of selected introns were used for amplification and the PCR products were analyzed by electrophoresis. (D) Representative immunofluorescence images showing nuclear speckles upon SON / SRRM2 double knockdown (KD) and treated with control siRNA (siC). Nuclear speckles were stained with AF488 labeled antibody against SRRM2 antibody (blue); and nuclei were stained with DAPI (grey). Scale bar: 10 μm. (E) Histogram of SRRM2 immunofluorescence intensity distribution of cells with double knockdown (KD) or treatment with control siRNA (siC). (F) Violin plot showing the number of speckles per cell for KD and siC treatment. Total number of cells included in each data set is indicated by “N” in (E) and (F). P-values calculated with unpaired t-tests are reported above each violin plot. (G) Representative electrophoresis analysis of RT-PCR products from THOC6 , TUBB4B and NCL upon KD and siC treatment. Gels of other selected genes for RT-PCR assays are shown in Figure S8. Gels were imaged with Chemidoc Imaging System. (H) Apparent unspliced fractions of selected introns were calculated by ratios of the intensity of the unspliced band and the sum of the unspliced band and spliced band. The intensity of bands was quantified using Fiji. Error bars report standard deviation from two biological replicates.

Article Snippet: Samples were then incubated with pAG-RTase for an additional 30 min. A reverse transcription reaction mixture was prepared by mixing 2 μM adapter-RT primer, 0.05 mM biotin-16-dUTP (Jena Bioscience), 0.05 mM biotin-16-dCTP (Jena Bioscience), 0.05 mM dTTP (Thermo Fisher Scientific), 0.05 mM dCTP (Thermo Fisher Scientific), 0.1 mM dATP (Thermo Fisher Scientific), 0.1 mM dGTP (Thermo Fisher Scientific), 1 U/μL RNaseOUT (Thermo Fisher Scientific) in 50 μL buffer of DPBS supplemented with 3 mM MgCl 2 .

Techniques: RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Amplification, Electrophoresis, Immunofluorescence, Knockdown, Control, Staining, Labeling, Imaging, Standard Deviation