Journal: Neoplasia (New York, N.Y.)

Article Title: A Preclinical Study Combining the DNA Repair Inhibitor Dbait with Radiotherapy for the Treatment of Melanoma 1

doi: 10.1016/j.neo.2014.08.008

Figure Lengend Snippet: Effect of Dbait on H2AX phosphorylation. (A) SK28 and 501mel melanoma cells were transfected with an inactive control oligonucleotide or Dbait ± NU7026 (DNA-PK inhibitor). Immunofluorescence of γ-H2AX (red) and chromatin (DAPI; blue) was visualized. Dbait treatment led to non-localized pan-nuclear H2AX phosphorylation evidencing Dbait activity. This activity was dependent on DNA-PK activation. Bar, 50 μm. (B) SK28 melanoma cells were transfected with an inactive control oligonucleotide or Dbait. Immunofluorescence of γ-H2AX (red) and chromatin (DAPI; blue) was visualized immediately after irradiation and/or Dbait treatment. Irradiation alone resulted in localized γ-H2AX foci representing radio-induced DNA DSBs; Dbait treatment with or without irradiation led to non-localized pan-nuclear H2AX phosphorylation evidencing Dbait activity. Bar, 30 μm. (C) SK28 cells were transduced with lentiviruses that express either control, non-targeting shRNA, or shRNA targeting DNA-PKcs. After Dbait transfection, cells were immunostained with mouse monoclonal anti–DNA-PKcs or anti–γ-H2AX. Dbait activity was not detected in cells transduced with shRNA targeting DNA-PKcs. Bar, 50 μm.

Article Snippet: Subconfluent SK28 cells were transduced with lentiviruses that expressed either the control, non-targeting shRNA (shCTL; sc-108080; Santa Cruz Biotechnology, (Dallas, Texas, USA)), or shRNA targeting DNA-PKcs (shDNA-PK; sc-35200-V; Santa Cruz Biotechnology) at a multiplicity of infection of 3 using polybrene (5 μg/ml).

Techniques: Phospho-proteomics, Transfection, Control, Immunofluorescence, Activity Assay, Activation Assay, Irradiation, Transduction, shRNA

Journal: Journal of Translational Medicine

Article Title: CCDC137 knockdown suppresses bladder cancer progression by downregulating SCD

doi: 10.1186/s12967-025-07033-w

Figure Lengend Snippet: Exploration of potential downstream mechanism and upstream regulators of CCDC137. A Heatmap shows DEGs identified by RNA sequencing in T24-shCtrl and T24-shCCDC137-1 cells. B Bar plot displays GO and KEGG functional enrichment results of downregulated DEGs from RNA sequencing. C GSVA-Hallmark pathway enrichment analysis displayed differences between CCDC137 positive (CCDC137 +) and CCDC137 negative (CCDC137 −) cells based on single-cell sequencing data. D mRNA and protein expression of stearoyl-CoA desaturase (SCD) were detected by qRT-PCR and Western blot. E DecoupleR was used to analyze differences in transcription factor activity between CCDC137 + and CCDC137 − epithelial cells in single-cell sequencing data. F TF-Target Finder was employed to identify potential upstream transcription factors of CCDC137. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, no statistical significance

Article Snippet: The shCCDC137 lentiviral particles and corresponding control lentiviral particles (shCtrl) were purchased from Genechem Co., Ltd (Shanghai, China).

Techniques: RNA Sequencing, Functional Assay, Sequencing, Expressing, Quantitative RT-PCR, Western Blot, Activity Assay

Journal: Journal of Translational Medicine

Article Title: CCDC137 knockdown suppresses bladder cancer progression by downregulating SCD

doi: 10.1186/s12967-025-07033-w

Figure Lengend Snippet: In vivo validation of CCDC137 regulating tumor growth. A Subcutaneous xenograft models were established in nude mice to validate the regulatory role of CCDC137 in tumor growth. B Tumor volume growth curves were plotted over 28 days after tumor cell inoculation. C Tumor weights of T24-shCtrl and T24-shCCDC137-1 groups were measured on day 28. D The Graphical Abstract described the key results in this study. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, no statistical significance

Article Snippet: The shCCDC137 lentiviral particles and corresponding control lentiviral particles (shCtrl) were purchased from Genechem Co., Ltd (Shanghai, China).

Techniques: In Vivo, Biomarker Discovery

Journal: bioRxiv

Article Title: DECR1 is an androgen-repressed survival factor that regulates PUFA oxidation to protect prostate tumor cells from ferroptosis

doi: 10.1101/865626

Figure Lengend Snippet: (A) Schematic of DECR1 function in fatty acid (FA) β-oxidation. In order to translocate FAs into the mitochondria, CPT1 converts long-chain acyl-CoA species to their corresponding long-chain acylcarnitine species. This is followed by a dehydrogenation step mediated by acyl CoA dehydrogenase (ACAD) to generate trans-2-enoyl-CoA, the only intermediate that can be processed by downstream enzymes in the β-oxidation process. Many FAs have unsaturated bonds either on an odd-numbered carbon or in the cis-configuration, resulting in the generation of enoyl-CoA intermediates that cannot be directly processed via the downstream β-oxidation enzymes. These FAs require the activity of 3 auxiliary enzymes, ECI1, ECH1 and DECR1 in order to form trans-2-enoyl-CoA before undergoing β-oxidation. DECR1 catalyzes the conversion of either 2-trans,4-cis-dienoyl or 2-trans,4-trans-dienoyl-CoA to 3-trans-enoyl-CoA. A complete cycle of β-oxidation results in the release of the first two carbon units as acetyl-CoA, and a fatty-acyl-CoA minus two carbons. The acetyl-CoA enters the TCA cycle to produce energy (ATP). The shortened fatty-acyl-CoA is processed again starting with the ACADs to form trans-2-enoyl-CoA either directly or with the aid of the auxiliary enzymes depending on the presence of double bonds. This process continues until all carbons in the fatty acid chain are turned into acetyl-CoA. (B) DECR1 protein expression after 72 hours or 96 hours siRNA transfection. Densitometry quantification of relative DECR1 protein expression was normalized to the HSP90 internal control. (C) Linoleic acid level in LNCaP cells quantified in following 96 hours DECR1 knockdown using GC QQQ targeted metabolomics. (D) Relative quantities of the C10:2 acylcarnitine species in LNCaP cell conditioned medium (left) or cell lysates (right) (n=3). (E) Quantification of ATP levels in LNCaP cell lysates. LNCaP cells were transfected with DECR1 siRNAs for 48 hours and then starved in no-glucose medium and treated with the lipolysis inhibitor DEUP (100µM) in the presence (BSA-LA) or absence (BSA) of the PUFA linoleic acid for 48 hours before measuring ATP levels. ( F) Oxygen consumption rate (OCR) was assessed in LNCaP cells supplemented with the PUFA linoleic acid (LA) or (G) the saturated fatty acid palmitic acid (PA). Each data point represents an OCR measurement. ATP production, maximal mitochondrial respiration and mitochondrial spare capacity were assessed. (H) Extracellular acidification rate (ECAR) was assessed in LNCaP cells. Each data point represents an ECAR measurement. For experiments (F-H) LNCaP cells were transfected with DECR1 siRNAs for 72 hours, then starved in substrate limited medium for 24 hours; the assay was run in FAO assay medium. (I & J) Metabolites were quantified in LNCaP cells following 96 hours DECR1 knockdown using GC QQQ targeted metabolomics. Data in bar graphs are represented as the mean ± s.e.m (n=3). Statistical analysis was performed using two-tailed Student’s t -test: * p <0.05, ** p <0.01 and **** p <0.0001.

Article Snippet: LNCaP cells were transduced with the universal negative control shRNA lentiviral particles (shControl), DECR1 shRNA lentiviral particles (shDECR1) or hDECR1 (GFP-Puro) designed by GenTarget Inc. (San Diego, CA, USA) according to the manufacturer’s protocol.

Techniques: Activity Assay, Expressing, Transfection, Control, Knockdown, Two Tailed Test

Journal: bioRxiv

Article Title: DECR1 is an androgen-repressed survival factor that regulates PUFA oxidation to protect prostate tumor cells from ferroptosis

doi: 10.1101/865626

Figure Lengend Snippet: (A) Cell viability after DECR1 knockdown in non-malignant PNT1 prostate cells; hormone-responsive PCa cell lines (LNCaP and VCaP); castrate-resistant V16D and 22RV1 cell lines and enzalutamide-resistant MR94F cells cultured in full serum media. (B) Cell viability of stable DECR1-overexpressed LNCaP cells cultured in full serum media. Cell viability and cell death were measured using trypan blue exclusion following 96 hours DECR1 knockdown. Percentages are represented relative to the control siRNA; n = 3 independent experiments per cell line. (C) Clonogenic cell survival of LNCaP cells were assessed using colony formation assay. Stable DECR1-overexpressed cells or (D) stable DECR1 knockdown was achieved using two different short hairpin (sh) vectors and DECR1 expression was confirmed using western blot. Cells were cultured for 2 weeks, washed with PBS, fixed with paraformaldehyde and stained with 1% crystal violet for 30 minutes. Colonies with more than 50 cells were counted manually; data shown is representative of n = 2 independent experiments. (E) LNCaP and 22RV1 cell growth in 3D spheres. Spheroids were prepared using the hang drop assay following 48 hours DECR1 knockdown. Spheroid volumes were determined after five days of culturing the cells in 20 µl drops; at least 25 spheres per cell line were assessed using the ReViSP software, n = 3 independent experiments per cell line. (F) LNCaP, 22RV1 and MR49F cell migration and (G) 22RV1 cell invasion were assessed using transwell migration/invasion assay. Cells were transfected with DECR1 siRNA or control siRNA for 48 hours. Equal number of cells were transferred to the upper inserts in serum free medium; lower chambers were filled with medium containing 5% serum as a chemoattractant. Plates were incubated for a further 48 hours. Migrated/invaded cells on the lower face of the inserts were washed with PBS, fixed with paraformaldehyde, stained with 1% crystal violet for 30 minutes, and counted manually; data shown is representative of n = 3 independent experiments. (H) Violin plots of mKi67 and DECR1 mRNA expression in LNCaP tumors (n = 5 mice, shControl; n = 4 mice, shDECR1). (I) Representative KI67 IHC staining of LNCaP tumors. Scale bar, 100µm. Data in bar graphs are represented as the mean ± s.e.m. Statistical analysis was performed using one-way ANOVA, followed by Dunnett’s multiple comparisons test: * p <0.05, ** p <0.01, *** p <0.001 and **** p <0.0001.

Article Snippet: LNCaP cells were transduced with the universal negative control shRNA lentiviral particles (shControl), DECR1 shRNA lentiviral particles (shDECR1) or hDECR1 (GFP-Puro) designed by GenTarget Inc. (San Diego, CA, USA) according to the manufacturer’s protocol.

Techniques: Knockdown, Cell Culture, Control, Colony Assay, Expressing, Western Blot, Staining, Software, Migration, Invasion Assay, Transfection, Incubation, Immunohistochemistry

Journal: Cell Proliferation

Article Title: Linc RNA ‐p21 suppresses development of human prostate cancer through inhibition of PKM 2

doi: 10.1111/cpr.12395

Figure Lengend Snippet: Depletion of pyruvate kinase M2 (PKM2) reduces the tumorigenic capacity of cells with lowly expressed lincRNA‐p21. (A) LincRNA‐p21‐silenced LNCaP cells expressing shPKM2 or shCtrl were implanted into nude mice (n = 8) and tumours developed. The time point of 0 day represented 3 weeks after LNCaP cells injection. (B) LincRNA‐p21‐silenced DU145 cells expressing shPKM2 or shCtrl were implanted into nude mice (n = 8) and tumours developed. Immunoblotting was preformed to examine PKM2 expression (left). Tumour initiation were recorded (middle) and survival was analysed (right)

Article Snippet: ShlincRNA‐p21, shPKM2 and control shRNA (shCtrl) lentivirus particles were obtained from GenePharma.

Techniques: Expressing, Injection, Western Blot

Journal: Cell Proliferation

Article Title: Linc RNA ‐p21 suppresses development of human prostate cancer through inhibition of PKM 2

doi: 10.1111/cpr.12395

Figure Lengend Snippet: LincRNA‐p21 down‐regulates pyruvate kinase M2 (PKM2) to suppress aerobic glycolysis. (A) Relative glucose consumption, pyruvate concentration and lactate production were quantified in lincRNA‐p21 knockdown or control DU145 or LNCaP cells. (B) Relative glucose consumption, pyruvate concentration and lactate production were quantified in lincRNA‐p21‐silenced DU145 and LNCaP cells expressing shPKM2 or shCtrl. (C) DU145 cells with lowly expressed lincRNA‐p21 were treated with or without rapamycin, and then glucose consumption, pyruvate concentration and lactate production were examined. *P< .05; **P< .01; ***P< .001

Article Snippet: ShlincRNA‐p21, shPKM2 and control shRNA (shCtrl) lentivirus particles were obtained from GenePharma.

Techniques: Concentration Assay, Knockdown, Control, Expressing

Journal: Cell Proliferation

Article Title: Linc RNA ‐p21 suppresses development of human prostate cancer through inhibition of PKM 2

doi: 10.1111/cpr.12395

Figure Lengend Snippet: Pyruvate kinase M2 (PKM2) is critical for lincRNA‐p21 suppression of cell proliferation and colony formation of prostate cancer cells. (A) LincRNA‐p21‐silenced DU145 cells expressing shPKM2 or shCtrl were subjected to cell viability analysis at indicated time. (B and C) Cells described in (A) were subjected to colony formation assay. Representative images of cell colonies (B) and relative colony number were calculated (C). (D) LincRNA‐p21‐silenced LNCaP cells expressing shPKM2 or shCtrl were subjected to cell viability analysis at indicated time. (E and F) Cells described in (D) were subjected to colony formation assay. Representative images of cell colonies (E) and relative colony number were calculated (F). (G and H) DU145 cells with lowly expressed lincRNA‐p21 were treated with or without rapamycin (G) or 3‐Brpa (H) at different dosages for 48 hours, and cell viability was examined using CCK8 kit. *P< .05; **P< .01; ***P< .001

Article Snippet: ShlincRNA‐p21, shPKM2 and control shRNA (shCtrl) lentivirus particles were obtained from GenePharma.

Techniques: Expressing, Colony Assay

Journal: Molecular Metabolism

Article Title: Inhibition of mitochondrial fission and iNOS in the dorsal vagal complex protects from overeating and weight gain

doi: 10.1016/j.molmet.2020.101123

Figure Lengend Snippet: Knockdown of iNOS in the DVC protects from developing HFD-dependent insulin resistance and decreases body weight and food intake. ( A ) Experimental design and feeding study protocol. ( B ) Western blot analysis of the iNOS knockdown in the DVC. iNOS levels of n = 8 for shControl and n = 8 shiNOS are shown in the bar graph. A representative Western blot image is shown at the bottom. ( C ) Representative confocal image of iNOS labelling in animals expressing ShRNA for iNOS or the shControl in the NTS of the DVC. Bar = 20 μm ( D ) NO levels in the DVC of RC-fed rats compared with HFD-fed rats expressing the control virus and HFD-fed rats expressing shiNOS. Data are shown as mean ± SEM, with each single point highlighted of n = 9 RC rats and n = 6 HFD-fed rats expressing either shControl or shiNOS. ( E ) Acute feeding study: total food intake at 4 h, comparing animals treated with insulin or a vehicle in the DVC. Data are shown as mean ± SEM, with each single point highlighted of n = 10 rats for control vehicle, n = 7 for control insulin, n = 11 for shiNOS vehicle, n = 7 for shiNOS insulin. ( F ) Chronic cumulative food intake, from day 1 (see schematic in A). ( G ) Chronic data showing body weight increase from day 1. ( H ) White adipose tissue: epididymal, retroperitoneal, and visceral fat collected on the day of sacrifice. Data are shown as mean ± SEM, with each single point highlighted. Data from F to H are representative of n = 10 for shControl and n = 8 for shiNOS. ( I ) Western blot analysis of p-PERK levels in the DVC of animals expressing either ShiNOS or ShControl of n = 8 rats per group. A representative western blot image is shown at the bottom. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: On day 0, a lentiviral system was used to deliver ShRNA to knockdown of iNOS (shiNOS) or a control scramble ShRNA (shControl; Santa Cruz Biotechnology, sc-29417-V and sc-108080, respectively), or on day 1, an adenoviral system was used to deliver either a constitutively active form of Drp1 (Drp1-S637A), a catalytically inactive form of Drp1 (Drp1-K38A), or a control of GFP expressed under CMV [ ] or GFAP promoters.

Techniques: Knockdown, Western Blot, Expressing, shRNA, Control, Virus

Journal: Nucleic acids research

Article Title: Overlapping and distinct functions of SPT6, PNUTS, and PCF11 in regulating transcription termination.

doi: 10.1093/nar/gkaf179

Figure Lengend Snippet: Figure 1. SPT6 And IWS1 chromatin binding and transcriptional roles. ( A ) A schematic representation of SPT6 N-terminal (1–283 amino acids), core (284–1287 amino acids), and C-terminal (1288–1726 amino acids) regions. The illustration denotes N to C terminal protein domains. ( B ) Overexpressed Flag-tagged SPT6 from HEK293T total protein extracts was immunoprecipitated using an anti-Flag antibody and analyzed by western blotting using indicated antibodies. ( C ) SPT6 and IWS1 mean ChIP-seq signal intensities plotted at RefSeq-annotated genes. The solid line represents the mean. The dark area represents the standard error and light area represents the 95% confidence interval. ( D ) Heatmap representing the Pearson correlation coefficients calculated for IWS1, SPT6, and RNAPII merged ChIP-seq signals, on RefSeq-annotated genes ( ±1 kb). ( E ) SPT6 / RNAPII and IWS1 / RNAPII mean ChIP-seq signal ratios calculated on RefSeq-annotated genes in HeLa cells. The solid line represents the mean. The dark area represents the standard error and light area represents the 95% confidence interval. ( F ) Total protein extracts from HeLa cells were separated using a 5%–60% glycerol gradient. A total of 12 fractions were recovered after ultracentrifugation and analyzed by western blotting. ( G ) Western blot showing SPT6 and IWS1 depletions upon siRNA transfection in RNA-seq e xperiments. L o w er panel sho ws protein quantification relative to GAPDH and to the siCT condition ( n = 3). ( H ) SPT6 and IWS1 target genes in HeLa cells. Positive targets correspond to genes having a |FC| > | ±1.5| and a padj < 0.05. ( I ) Density plot showing the gene count of SPT6 and IWS1 readthrough targets, highlighting the distribution of their log 2 fold-change (FC) values. ( J ) SEC22B gene locus featuring RNAPII, SPT6, and IWS1 ChIP-seq profiles, as well as RNA-seq profiles upon depletion of SPT6 and IWS1 (negative strand). The arrow highlights readthrough transcription ( K ) Western blot showing SPT6 and IWS1 depletion upon siRNA transfection (see the “Materials and methods” section for details). ( L ) SEC22B readthrough and mRNA levels were assayed by RT-qPCR in three independent experiments. Values were normalized to the siCT condition arbitrarily set to 1. ( M ) SEC22B readthrough le v els w ere assa y ed b y nuclear run-on e xperiments. T he input represents total nuclear RNAs. The siSPT6 (-BrdU) condition is used to control the specificity of the anti-BrdU immunoprecipitation. Values were normalized to the siCT condition arbitrarily set to 1, and to the KDSR and 18S housekeeping genes.

Article Snippet: Construction of shRNA plasmids and production of virus-like particles Control short hairpin RNA (shRNA) sequence (shCT) or shRNA sequence targeting PNUTS (see references in Supplementary Table S1 ) were cloned into the pLK O .1- TRC cloning vector (gift from David Root, Addgene plasmid #10878; http:// n2t.net/ addgene:10878 ; RRID: Addgene_10 878) according to the manufacturer’s instructions.

Techniques: Binding Assay, Immunoprecipitation, Western Blot, ChIP-sequencing, Transfection, RNA Sequencing, Quantitative RT-PCR, Control

Journal: Nucleic acids research

Article Title: Overlapping and distinct functions of SPT6, PNUTS, and PCF11 in regulating transcription termination.

doi: 10.1093/nar/gkaf179

Figure Lengend Snippet: Figure 3. SPT6 interacts with IWS1, PNUTS, and PCF11. ( A ) Upper panel: Diagram depicting PNUTS domains: the TND (1–147 amino acids), the PP1-binding domain (348–418 amino acids) and the RNA-binding domain (674–750 amino acids). L o w er panel: Diagram depicting PCF11 domains: the CTD-interacting domain (CID) (1 4–1 42 amino acids), and the RNA-binding zinc fingers (between 1343 and 1478 amino acids). The illustration denotes N-to-C terminal protein domains. ( B ) Nuclear protein extracts from WT or IWS1-knockout (IWS1- / -) J-Lat A1 cells were used to immunoprecipitate endogenous SPT6. Co-immunoprecipitation of IWS1, PCF11 and PNUTS was assessed ( n = 3). ( C ) Overexpressed Flag-tagged WT PNUTS (WT) or Flag-tagged PNUTS–W401A (W401A), a mutant for PP1 interaction, were immunoprecipitated using an anti-Flag antibody from HEK293T total protein e xtracts. SPT6 co-immunoprecipitation w as e xamined f or both WT and mutant PNUTS ( n = 3). ( D ) HeLa cells infected with lentiviruses containing either a control shRNA (shCT) or an shRNA targeting PNUTS (shPNUTS). Cells nuclear fractions were used to immunoprecipitate endogenous SPT6 ( n = 2). “C yto ” stands for Cytoplasmic fraction, and “Nuc” stands for Nuclear fraction used as input.

Article Snippet: Construction of shRNA plasmids and production of virus-like particles Control short hairpin RNA (shRNA) sequence (shCT) or shRNA sequence targeting PNUTS (see references in Supplementary Table S1 ) were cloned into the pLK O .1- TRC cloning vector (gift from David Root, Addgene plasmid #10878; http:// n2t.net/ addgene:10878 ; RRID: Addgene_10 878) according to the manufacturer’s instructions.

Techniques: Binding Assay, RNA Binding Assay, Zinc-Fingers, Knock-Out, Immunoprecipitation, Mutagenesis, Infection, Control, shRNA

Journal: Nucleic acids research

Article Title: Overlapping and distinct functions of SPT6, PNUTS, and PCF11 in regulating transcription termination.

doi: 10.1093/nar/gkaf179

Figure Lengend Snippet: Figure 5. Differential readthrough transcripts upon depletion of SPT6, PNUTS, and PCF11. ( A ) Mean RNA-seq signal intensity shown at SPT6 readthrough targets (TES + 5 kb) in the different conditions. ( B ) Heatmap displaying differentially expressed readthrough transcripts in HeLa cells f ollo wing SPT6 and / or PNUTS depletion. Readthrough transcripts with a log 2 FC > 1 in the siSPT6 condition compared to the control condition were considered positive. The readthrough regions were identified as described in Supplementary Fig. S1 F. The heatmap plots the average log2FC scores (siRNA / siCT) of these regions. Three distinct clusters were defined to characterize the varying responses to SPT6 and / or PNUTS depletion. The analysis was conducted using three independent biological replicates. ( C ) Box-plots showing the median log 2 FC of readthrough transcripts relative to the control condition for the three clusters shown in panel (B). ( D –F ) Same as in panels (A)–(C) but for SPT6 and / or PCF11 depletion. ( G ) CTNND1 gene locus featuring RNA-seq data (positive strand) following the use of the indicated siRNAs. The lower panel (PAS) displays the locations of human PASs as defined by Zhang et al. [ 82 ]. The arrow highlights readthrough transcription. The scale is shown at the top right corner of the figure.

Article Snippet: Construction of shRNA plasmids and production of virus-like particles Control short hairpin RNA (shRNA) sequence (shCT) or shRNA sequence targeting PNUTS (see references in Supplementary Table S1 ) were cloned into the pLK O .1- TRC cloning vector (gift from David Root, Addgene plasmid #10878; http:// n2t.net/ addgene:10878 ; RRID: Addgene_10 878) according to the manufacturer’s instructions.

Techniques: RNA Sequencing, Control

Journal: Nucleic acids research

Article Title: Overlapping and distinct functions of SPT6, PNUTS, and PCF11 in regulating transcription termination.

doi: 10.1093/nar/gkaf179

Figure Lengend Snippet: Figure 6. Differential PROMPTs regulation upon depletion of SPT6, PNUTS, and PCF11. ( A ) Mean RNA-seq signal intensity shown at SPT6 PROMPT targets (TSS, -5 kb) in the different conditions. ( B ) Heatmap displaying differentially expressed PROMPTs in HeLa cells following SPT6 and / or PNUTS depletion. PROMPTs with a log 2 FC > 1 in the siSPT6 condition compared to the control condition were considered positive. PROMPT regions were identified as described in Supplementary Fig. S6 A. The heatmap plots the average log 2 FC scores (siRNA / siCT) of these regions. Three distinct clusters were defined to characterize the varying responses to SPT6 and / or PNUTS depletion. The analysis was conducted using three independent biological replicates. ( C ) B o x-plots sho wing the median log 2 FC of PR OMPTs relativ e to the control condition f or the three clusters sho wn in panel (B). ( D ) PXDN PR OMPT le v els w ere assa y ed b y R T-qPCR in three independent e xperiments. Values w ere normaliz ed to the siCT condition arbitrarily set to 1. ( E –G ) Same as in panels (A)–(C) but for SPT6 and / or PCF11 depletion. ( H ) Same as in panel (D) but for the GGCT gene. ( I ) PVT1 gene locus featuring RNA-seq data (positive strand is in positive values and negative strand in negative values) following the use of the indicated siRNAs. The lower panel (PAS) displays the locations of human PASs as defined by Zhang et al. [ 82 ]. The arrow highlights PROMPTs. The scale is shown at the top right corner of the figure.

Article Snippet: Construction of shRNA plasmids and production of virus-like particles Control short hairpin RNA (shRNA) sequence (shCT) or shRNA sequence targeting PNUTS (see references in Supplementary Table S1 ) were cloned into the pLK O .1- TRC cloning vector (gift from David Root, Addgene plasmid #10878; http:// n2t.net/ addgene:10878 ; RRID: Addgene_10 878) according to the manufacturer’s instructions.

Techniques: RNA Sequencing, Control