Journal: Redox Biology

Article Title: Hypoxia-induced cysteine metabolism reprogramming is crucial for the tumorigenesis of colorectal cancer

doi: 10.1016/j.redox.2024.103286

Figure Lengend Snippet: Cystine/Cysteine transporters are upregulated in CRC cells to promote tumor growth . (A) Schematic diagram of exogenous 13 C 2 -cystine or 13 C 3 -cysteine uptake through various transporters. (B) Colorectal cancer cells import both cystine and cysteine. HCT116 or DLD1 cells was treated by either 13 C 2 -cystine (100 μM) or 13 C 3 -cysteine (100 μM). Intracellular cysteine was labeled as 13 C 1 -cysteine (M1) by 13 C 2 -cystine treatment, or as 13 C 3 -cysteine (M3) by 13 C 3 -cysteine treatment. Both unlabeled and 13 C-labeled cysteine were presented (n = 6). (C) The transporter genes of both cystine and cysteine were significantly upregulated in CRC compared with paired non-tumor colon tissues in TCGA database (n = 50). SLC7A11, the light chain of cystine/glutamate antiporter system; SLC3A2, the heavy chain of cystine/glutamate antiporter system; SLC1A4, alanine/serine/cysteine/threonine transporter 1 (ASCT1); SLC1A5, alanine/serine/cysteine/threonine transporter 2 (ASCT2). mRNA levels represent FPKM value downloaded from TCGA database. (D and E) Knockdown of SLC1A4, SLC1A5, SLC7A11 or SLC3A2 reduced intracellular cysteine abundance in CRC cells. SLC1A4, SLC1A5, SLC7A11 or SLC3A2 was knocked down by siRNAs mixture in HCT116 or DLD1 cells. The knockdown efficiency was validated by qRT-PCR (n = 3) (D), and intracellular cysteine abundance was measured by UHPLC-QTRAP/MS (n = 6 for DLD1 SLC1A4 knockdown cells; n = 3 for others) (E). (F–H) Knockdown of SLC7A11 or SLC1A5 reduced the proliferation of CRC cells. SLC1A5 or SLC7A11 was knocked down by shRNA in HCT116 or DLD1 cells. The knockdown efficiency of SLC1A5 (F) and SLC7A11 (G) was validated by qRT-PCR (n = 3) and Western blots. The proliferation rates were measured by CCK-8 (n = 3) (H). (I–L) Knockdown of SLC1A5 on top of SLC7A11 knockdown further reduced intracellular cysteine content and significantly impaired CRC tumor growth. SLC1A5 was knockdown by siRNAs in DLD1 cells or DLD1 shRNA-SLC7A11 knockdown cells, and the cells were assayed for abundance of intracellular cysteine by UHPLC-QTRAP/MS (n = 3) (I); cell proliferation by CCK-8 (n = 3) (J); xenograft tumor (n = 10) growth rate (K) and weight (L). Two-way ANOVA was used for statistical analyses of H, J, K. Student t-test was used for others. Data of K is presented as mean ± SEM, and data of others are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.001; n.s., not significant.

Article Snippet: HCT116 or DLD1 cells were transduced with the lentivirus expressing mCherry-EGFP-LC3B fusion protein (GeneCopoeia, Guangzhou, China) at 15 MOI, and stable mCherry-EGFP-LC3B cells were selected with puromycin (Invitrogen) (0.8 μg/ml for HCT116 and 2.0 μg/ml for DLD1).

Techniques: Labeling, Knockdown, Quantitative RT-PCR, shRNA, Western Blot, CCK-8 Assay

Journal: Redox Biology

Article Title: Hypoxia-induced cysteine metabolism reprogramming is crucial for the tumorigenesis of colorectal cancer

doi: 10.1016/j.redox.2024.103286

Figure Lengend Snippet: Hypoxia induces the transcription of cystine/cysteine transporters through ATF4 . (A) SLC1A4, SLC1A5, SLC7A11 and SLC3A2 were upregulated concurrently in CRC tumors. The RNA-seq data of SLC1A4, SLC1A5, SLC7A11 and SLC3A2 in CRC and paired non-tumor tissues from TCGA (n = 50) and GSE223120 (n = 20) were analyzed. The fold change (tumor/non-tumor) >1.1 was considered as upregulation. The percentage of tumors which upregulated 4 genes, 3 genes, 2 genes, or 1 gene were presented. (B and C) Hypoxia induced the expression of SLC1A4, SLC1A5, SLC7A11 and SLC3A2. HCT116 and DLD1 cells were incubated in hypoxia chamber (1.0 % O 2 , 5.0 % CO 2 , 94 % N 2 37 °C) or normoxia chamber overnight respectively. qRT-PCR was performed to examine the expression levels of SLC1A4, SLC1A5, SLC7A11 and SLC3A2 (n = 3) (B). Two independent sets of HCT116 cells were transfected with indicated pGL3-promoter constructs and internal control Renilla luciferase construct. 2 days post-transfection, two sets of cells were incubated under hypoxia or normoxia conditions respectively overnight. Luciferase activities were then assayed by dual-luciferase reporter assay system (n = 3) (C). (D) The flowchart to screen transcription factors which are responsible for co-expression of SLC1A4, SLC1A5, SLC7A11 and SLC3A2. ChEA3, ChIP-X Enrichment Analysis Version 3; GTRD, Gene Transcription Regulation Database; Hypoxia, the transcription factors which were reported as hypoxia inducible genes; STRING, functional protein association networks. (E and F) Hypoxia-related ATF4 regulates the expression of SLC1A4, SLC1A5, SLC7A11 and SLC3A2. ATF4 was knocked down by shRNAs in HCT116 cells. Cells were incubated under hypoxia condition overnight and the expression levels of indicated genes were examined by qRT-PCR (n = 3) (E). pCMV-ATF4 or empty vector was transfected into HCT116 cells. 48 h post-transfection, the expression levels of indicated genes were examined by qRT-PCR (n = 3) (F). (G) ROS induced the expression of SLC1A4, SLC1A5, SLC7A11 and SLC3A2. Cells were treated with H 2 O 2 , and the expression levels of indicated genes were examined by qRT-PCR (n = 3). (H) ER stress regulates the expression of SLC1A4, SLC1A5, SLC7A11 and SLC3A2 via ATF4. HCT116 cells were treated with Tg (thapsigargin), and the expression levels of indicated genes were examined by Western blots. (I) Fold enrichment of DNA fragments surrounding putative ATF4 binding sites of indicated gene promoters by ChIP-qPCR with or without H 2 O 2 (250 μM) treatment (n = 3). (J) Knockdown of ATF4 significantly inhibited the expression of cystine and cysteine transporter genes induced by hypoxia (overnight), H 2 O 2 (250 μM, 6 h), or Tg (250 nM, 6 h). (K and L) Hypoxia induced ATF4 expression through PERK. ATF4 protein levels in PERK knockdown cells (K) or ISRIB-treated cells ( p -eIF2α inhibition) (L) were detected. Student t-test was used for statistical analyses. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.001; n.s., not significant.

Article Snippet: HCT116 or DLD1 cells were transduced with the lentivirus expressing mCherry-EGFP-LC3B fusion protein (GeneCopoeia, Guangzhou, China) at 15 MOI, and stable mCherry-EGFP-LC3B cells were selected with puromycin (Invitrogen) (0.8 μg/ml for HCT116 and 2.0 μg/ml for DLD1).

Techniques: RNA Sequencing, Expressing, Incubation, Quantitative RT-PCR, Transfection, Construct, Control, Luciferase, Reporter Assay, Functional Assay, Plasmid Preparation, Western Blot, Binding Assay, ChIP-qPCR, Knockdown, Inhibition

Journal: Redox Biology

Article Title: Hypoxia-induced cysteine metabolism reprogramming is crucial for the tumorigenesis of colorectal cancer

doi: 10.1016/j.redox.2024.103286

Figure Lengend Snippet: The flux from cysteine to GSH increases in CRC . (A) Volcano plots of differential expressed metabolites of HCT116 cells cultured with or without cystine/cysteine. Metabolites in cysteine-GSH pathway are highlighted as red. (B) KEGG pathway analysis of DEMs from HCT116 cells with or without cystine/cysteine. Y axis represents -log 10 (p value) of pathway analysis. (C) GSH rescued the growth of CRC cells in cystine/cysteine depletion condition. 200 μM of cystine, cysteine, or NAC (N-acetyl cysteine), 400 μM GSH (reduced glutathione) or taurine, nucleotides (1 × EmbyoMax Nucleosides), NEAA (1 × non-essential amino acids), 2 mM glutamate or α-KG (dimethyl α-ketoglutarate), 1 mM sodium pyruvate, 200 μM cystathionine or homocysteine were individually supplemented into cystine/cysteine deficient medium. 48 h later, cell numbers were counted with hemocytometer by Trypan-Blue exclusive assay (n = 3). (D–F) The flux from cysteine to GSH is elevated in CRC cells. Cells were supplied with both 100 μM 13 C 2 -cystine (GSH labeled as M1) and 100 μM 13 C 3 -cysteine (GSH labeled as M3) for indicated time points, fraction of 13 C-labeled GSH was measured (n = 6) (D). Schematic diagram of 13 C 3 -cysteine flux and related enzymes is shown in (E). Cells were cultured in the presence of 100 μM 13 C 3 -cysteine for 2 h, and percentages of labeled metabolites in total pool were plotted (n = 6) (F). (G) The flux from cysteine to GSH is elevated by hypoxia induced ER stress. DLD1 cells were treated with Tg (250 nM), H 2 O 2 (250 μM), or hypoxia overnight, and then incubated with 100 μM 13 C 3 -cysteine for 30 min. Percentages of labeled GSH in total pool were plotted (n = 8). (H) Spontaneous colon tumors were generated with CAKP mice. After infusion with 13 C 3 -cysteine for 4 h, the serum, colorectal tumors and adjacent non-tumor colon tissues were collected, and percentages of labeled metabolites were plotted (n = 8). Student t-test was used for statistical analyses. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.001; n.s., not significant. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: HCT116 or DLD1 cells were transduced with the lentivirus expressing mCherry-EGFP-LC3B fusion protein (GeneCopoeia, Guangzhou, China) at 15 MOI, and stable mCherry-EGFP-LC3B cells were selected with puromycin (Invitrogen) (0.8 μg/ml for HCT116 and 2.0 μg/ml for DLD1).

Techniques: Cell Culture, Labeling, Incubation, Generated

Journal: Redox Biology

Article Title: Hypoxia-induced cysteine metabolism reprogramming is crucial for the tumorigenesis of colorectal cancer

doi: 10.1016/j.redox.2024.103286

Figure Lengend Snippet: Overexpression of GSS plays important role in CRC growth . (A and B) Glutathione synthetase (GSS) was significantly upregulated in CRC samples compared with normal colon tissues. The mRNA levels of indicated genes of paired CRC and normal tissues in TCGA (n = 50, A) and GSE223120 (n = 20, B) datasets were plotted. (C–H) Knockdown of GSS reduced the growth of colorectal cancer cells. HCT116 or DLD1 cells were transfected with siRNAs against GSS or control siRNA. 48 h post-transfection, the cells were assayed for knockdown efficiency by qRT-PCR (n = 3) (C) or Western blots (D); relative abundance of γ-glutamylcysteine (E) or GSH (F) by LC-MS (n = 6); cell proliferation rate by CCK-8 (n = 3) (G); subcutaneous xenograft tumor growth (n = 10) (H). Two-way ANOVA was used for statistical analyses of G, H. Student t-test was used for others. Data of H is presented as mean ± SEM, and data of others are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.001; n.s., not significant.

Article Snippet: HCT116 or DLD1 cells were transduced with the lentivirus expressing mCherry-EGFP-LC3B fusion protein (GeneCopoeia, Guangzhou, China) at 15 MOI, and stable mCherry-EGFP-LC3B cells were selected with puromycin (Invitrogen) (0.8 μg/ml for HCT116 and 2.0 μg/ml for DLD1).

Techniques: Over Expression, Knockdown, Transfection, Control, Quantitative RT-PCR, Western Blot, Liquid Chromatography with Mass Spectroscopy, CCK-8 Assay

Journal: Redox Biology

Article Title: Hypoxia-induced cysteine metabolism reprogramming is crucial for the tumorigenesis of colorectal cancer

doi: 10.1016/j.redox.2024.103286

Figure Lengend Snippet: Scavenging exogenous cystine/cysteine represses CRC growth . (A) Relative survival of colorectal cancer cells and normal colon epithelial cells under cystine/cysteine depletion condition (n = 3). (B and C) Cyst(e)inase efficiently depletes cystine and cysteine. HCT116 cells were treated with 1 mg/ml cyst(e)inase for 24 h. Intracellular (cells) and exogenous (medium) cystine or cysteine were measured by UHPLC-QTRAP/MS (n = 6) (B). C57BL/6 mice (n = 6) were intraperitoneally injected with cyst(e)inase (50 mg/kg). 24 h later, mice serum was collected to measure cystine or cysteine levels by UHPLC-QTRAP/MS (C). (D) The fold changes of cysteine-related metabolites abundance after HCT116 cells were treated with cystine/cysteine depletion condition or cyst(e)inase for 24 h. (E–G) Cyst(e)inase inhibited the CRC tumor growth. Nude mice were subcutaneous injected with either HCT116 cells (E) or small pieces of patient-derived xenograft tumors (F). Once tumors reached 100–150 mm 3 , mice were divided into two groups to intraperitoneally inject with PBS vehicle or cyst(e)inase (50 mg/kg) thrice a week. CAKP mice were administrated with tamoxifen. 40 days post injection, mice were divided into two groups to intraperitoneally inject with PBS vehicle or cyst(e)inase (50 mg/kg) thrice a week. Kaplan-Meier analyses was used to show the survival of mice with or without cyst(e)inase treatment (G). Student t-test was used for statistical analyses. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.001; n.s., not significant.

Article Snippet: HCT116 or DLD1 cells were transduced with the lentivirus expressing mCherry-EGFP-LC3B fusion protein (GeneCopoeia, Guangzhou, China) at 15 MOI, and stable mCherry-EGFP-LC3B cells were selected with puromycin (Invitrogen) (0.8 μg/ml for HCT116 and 2.0 μg/ml for DLD1).

Techniques: Injection, Derivative Assay

Journal: Redox Biology

Article Title: Hypoxia-induced cysteine metabolism reprogramming is crucial for the tumorigenesis of colorectal cancer

doi: 10.1016/j.redox.2024.103286

Figure Lengend Snippet: Depletion of cystine/cysteine induces autophagy of CRC cells through mTOR-ULK pathway . (A) Depletion of cystine/cysteine affects cellular autophagy, apoptosis or ferroptosis. Cells were cultured under cystine/cysteine depletion condition (-cyst(e)ine) or treated with cyst(e)inase (+cyst(e)inase) for 48 h. Cell lysates were collected and cell death markers were detected by Western blot assays. (B) Depletion of cystine/cysteine, but not glutamine or methionine, significantly induced autophagy in CRC cells but not in normal epithelial cells. (C–E) Cystine/cysteine depletion significantly induces autophagy in CRC cells. mCherry-GFP-LC3-expressing HCT116 and DLD1 cells were cultured under cystine/cysteine depletion condition or treated with cyst(e)inase for 24 h. Representative images of fluorescent LC3 puncta were show in (C). Mean number of GFP and mcherry dots per cell (n = 3) (D); and mean number of autophagosomes (yellow dots in merged images) and autolysosomes (red dots in merged images) per cell were counted (n = 3) (E). (F and G) Cystine/cysteine depletion induces autophagy of CRC cells through mTOR-ULK1 pathway. The dose-dependence (F) and time-dependence (G) of autophagy (the expression of LC3A and LC3B), mTOR activity (p-p70S6K) and ULK activity ( p -ULK) upon cystine/cysteine depletion were assayed by Western blots. Student t-test was used for statistical analyses. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.001; n.s., not significant. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: HCT116 or DLD1 cells were transduced with the lentivirus expressing mCherry-EGFP-LC3B fusion protein (GeneCopoeia, Guangzhou, China) at 15 MOI, and stable mCherry-EGFP-LC3B cells were selected with puromycin (Invitrogen) (0.8 μg/ml for HCT116 and 2.0 μg/ml for DLD1).

Techniques: Cell Culture, Western Blot, Expressing, Activity Assay

Journal: Nature Communications

Article Title: ORC1 binds to cis -transcribed RNAs for efficient activation of replication origins

doi: 10.1038/s41467-023-40105-3

Figure Lengend Snippet: a Schematic representation of human ORC1 protein domains , , . b Cross-correlation between endogenous ORC1, and unlabeled (control) or EU-labeled RNA (short or long pulse), comparing STORM experimental (EXP) and randomized (RND) analysis in the chromatin fraction of U2OS cells synchronized in G1. Data were presented as mean values ( n > 50 cells). Indicated p values (ns denoting p value >0.05) derive from unpaired two-sample t -test. c Schematic of RIP-seq and iCLIP experimental approaches, where endogenous or Flag-tagged ORC1 is immunoprecipitated from native or UV-crosslinked nuclear extracts, followed by recovery of full-length or digested bound RNAs. Below, the number of genes identified by both methods, with different iCLIP stringencies, and hypergeometric test p values of the experimental overlap (RIP-iCLIP) on top of the bars; red for selected high confidence (HC) ORC1-RNAs. d Genomic distributions of ORC1 iCLIP crosslinks, and (below) their density around TSSs (−/+ 10 kb) of ORC1-bound genes. e Gene length and expression level of high confidence (HC) ORC1-RNAs (iCLIP-RIP overlap) and ORC1-RNAs (iCLIP-RIP union), relative to sample size-matched control genes with different fold changes (FC) in ORC1 RIP-seq. n = number of genes in each category (Supplementary Data ; from iCLIP data [>5 crosslink sites and <0.05 FDR] and RIP-seq data [log2 fold change >1 and p value <0.05]). Box plots show the median distribution between Q1 and Q3. *** denotes p value <0.001, derived from unpaired two-tailed Student’s t -test. f Gene biotypes of ORC1-RNAs. g Percentage of ORC1-RNA and high confidence ORC1-RNA (HC) genes with mutual interactions according to Hi-C analysis, compared to controls shown in Fig. 1e. Bars represent mean values. **** denotes p value <0.0001, derived from a two-proportion z -test. h , i Density plots of h ORC1 ChIP-seq and i SNS-seq normalized reads across six quantiles (Q) of ORC1-RNA genes, defined by ORC1 iCLIP, centered around their TSSs (−/+ 5 kb). j Browser snapshot of representative high confidence ORC1-RNA genes, showing data of ORC1 RNA-binding (ORC1 RIP-seq or iCLIP crosslinks), and replication origins (SNS-seq) at their TSSs, in HCT116 cells. Green arrows indicate positions of GAA repeats.

Article Snippet: Samples were de-crosslinked overnight at 65 °C, followed by phenol:chloroform DNA extraction and ethanol precipitation. qPCR of precipitated DNA was done as cDNA samples (see RNA processing section), with self-designed primers at genomic DNA replication origins or control regions (Supplementary Data ), having SNS-seq data in wild-type HCT116 cells as a reference and purchased from Metabion.

Techniques: Labeling, Immunoprecipitation, Expressing, Derivative Assay, Two Tailed Test, Hi-C, ChIP-sequencing, RNA Binding Assay

Journal: Nature Communications

Article Title: ORC1 binds to cis -transcribed RNAs for efficient activation of replication origins

doi: 10.1038/s41467-023-40105-3

Figure Lengend Snippet: a RNA-FISH representative images, and signal quantification (below), of RNAs containing GAA repeats, in ASO-transfected HCT116 cells. Dots represent mean values ( n = 4 biologically independent samples) ±SEM. * denotes p value < 0.05, derived from unpaired two-tailed Student’s t -test. b DNA fiber quantification of inter-origin distances and fork rates of ASO-transfected HCT116 cells. Red lines indicate the median. ns denotes p value > 0.05, ** denotes p value < 0.01, **** denotes p value <0.0001, derived from unpaired two-tailed Mann–Whitney t -test. c SNS-seq peak count frequency and distribution relative to TSS positions (±3 kb) in ASO control and anti-GAA treated HCT116 cells. d GSEA showing the reduction of SNS-seq signal (peaks enriched in the control condition) in anti-GAA downregulated genes. Statistical significance (adjusted p value 0.038) of the enrichment score (ES) derives from a permutation test. e CDC45 and PCNA chromatin immunofluorescences per cell (HCT116) upon ASO knockdown, after soluble protein washout. Data were presented as mean values ( n > 100 cells) ±SEM. *** denotes p value < 0.001, derived from unpaired two-tailed Mann–Whitney t -test. f ORC1 western blot and protein quantification, in chromatin extracts of ASO-transfected HCT116 cells. Data were presented as mean values ( n = 5 biologically independent experiments) ±SEM. ns denotes p value > 0.05, * denotes p value < 0.05, derived from paired two-tailed Student’s t -test. g Browser snapshot at DDX5-CEP95 locus showing ORC1 RIP-seq enrichment, ORC1 iCLIP peaks, and SNS-seq reads in HCT116 cells, with the position of qPCR primers (#) indicated, and origins highlighted in blue. h Enrichment of nascent strands determined by SNS-qPCR at genomic positions indicated in Fig. 2g, in siRNA-treated HCT116 cells. Data were presented as mean values ( n = 5 biologically independent experiments) ±SEM. ns denotes p value > 0.05, * denotes p value <0.05, ** denotes p value < 0.01, derived from paired two-tailed Student’s t -test.

Article Snippet: Samples were de-crosslinked overnight at 65 °C, followed by phenol:chloroform DNA extraction and ethanol precipitation. qPCR of precipitated DNA was done as cDNA samples (see RNA processing section), with self-designed primers at genomic DNA replication origins or control regions (Supplementary Data ), having SNS-seq data in wild-type HCT116 cells as a reference and purchased from Metabion.

Techniques: Transfection, Derivative Assay, Two Tailed Test, MANN-WHITNEY, Western Blot

Journal: Nature Communications

Article Title: ORC1 binds to cis -transcribed RNAs for efficient activation of replication origins

doi: 10.1038/s41467-023-40105-3

Figure Lengend Snippet: a 3D model of human ORC1 showing domains in colors, and residues R441, R444, and R465 (involved in RNA-binding) in red. Below, the vertebrate consensus of ORC1 RNA-binding region, circles indicating mutated residues in MUT-ORC1. b RNA staining of EMSA assays, with GST-tagged purified WT and MUT-ORC1 (amino acids 413–511) (2.5 µM) incubated with fragmented cellular RNA (2.5 µM). Below, the silver staining of proteins used in the assay. c Cross-correlation between ORC1 and EU-labeled RNA (long pulse) in G1-synchronized U2OS cells, untransfected or transfected with Halo-tagged WT and MUT-ORC1, comparing STORM experimental (EXP) and randomized (RND) samples. Data were presented as mean values ( n > 50 cells) ± SEM. ns denotes p value >0.05, ** denotes p value <0.01, derived from unpaired two-sample t -test. d DNA fiber quantification of inter-origin distances and fork rates in HCT116 cells transfected with the indicated siRNAs, ±plasmids expressing Flag-tagged WT or MUT-ORC1. Black lines indicate the median. ns denotes p value >0.05, * denotes p value <0.05, ** denotes p value <0.01, **** denotes p value <0.0001, derived from unpaired two-tailed Mann–Whitney t -test. e Browser snapshot at ORC1-RNA PABPC1 , NFAT5 , and DDX5-CEP95 loci, showing SNS-seq normalized signal of HCT116 cells stably expressing WT or MUT-ORC1. f GSEA showing enrichment of ORC1-RNAs in merged iCLIP-defined quantiles (Q), toward ranked genes according to their WT vs MUT (log2 fold change) SNS-seq coverage at TSSs. Statistical significance (adjusted p value 1.13e-18 or 0.003) of the enrichment scores (ES) were calculated by permutation tests. g CDC45 and PCNA chromatin immunofluorescences per cell, in HCT116 cells stably expressing WT or MUT-ORC1, after soluble protein washout. Data were presented as mean values ( n > 100 cells) ± SEM. *** denotes p value <0.001, derived from unpaired two-tailed Mann–Whitney t -test. h Coverage plot of CDC45 ChIP-seq data at TSSs, in WT or MUT-ORC1 HCT116 stably expressing cells, and two-tailed t -test statistical results between their coverage at TSSs of ORC1 iCLIP-defined gene quantiles (Q).

Article Snippet: Samples were de-crosslinked overnight at 65 °C, followed by phenol:chloroform DNA extraction and ethanol precipitation. qPCR of precipitated DNA was done as cDNA samples (see RNA processing section), with self-designed primers at genomic DNA replication origins or control regions (Supplementary Data ), having SNS-seq data in wild-type HCT116 cells as a reference and purchased from Metabion.

Techniques: RNA Binding Assay, Staining, Purification, Incubation, Silver Staining, Labeling, Transfection, Derivative Assay, Expressing, Two Tailed Test, MANN-WHITNEY, Stable Transfection, ChIP-sequencing

Journal: Nature Communications

Article Title: ORC1 binds to cis -transcribed RNAs for efficient activation of replication origins

doi: 10.1038/s41467-023-40105-3

Figure Lengend Snippet: a p53 and ORC1-3xFlag protein quantification from western blots with total extracts of HCT116 cells, transfected with WT or MUT-ORC1, and treated with cycloheximide (CHX) or MG-132. Dots represent mean values ( n = 3 biologically independent experiments) ± SEM. ns denotes p value >0.05, * denotes p value <0.05, ** denotes p value <0.01, derived from paired two-tailed Student’s t -test. b Western blot on chromatin extracts of HCT116 cells, transfected with Flag-tagged WT-ORC1 and MUT-ORC1, unsynchronized (Uns) or synchronized in G1/S and released at different times (T as in Supplementary Fig. ). Below, normalized protein quantification. Dots represent mean values ( n = 4 biologically independent experiments) ± SEM. ns denotes p value >0.05, * denotes p value <0.05, derived from paired two-tailed Student’s t -test. c Western blot and quantification of endogenous ORC1 on chromatin in different stages of the cell cycle (T as in Supplementary Fig. ), upon depletion of GAA-RNAs (ASO anti-GAA) or control conditions (ASO CTRL). Bars represent mean values ( n = 3 biologically independent experiments) ± SEM. ns denotes p value >0.05, * denotes p value <0.05, ** denotes p value <0.01, derived from paired two-tailed Student’s t -test. d Western blot showing the effect of RNase A treatment on WT and MUT-ORC1 chromatin association, along the cell cycle of synchronized cells (T as in Supplementary Fig. ). Quantification of independent biological replicates ( n = 4) is shown in Supplementary Fig. . e Representation of the IDR in WT and MUT-ORC1, showing RNA-binding regions (orange), and the discrete positions of RNA-binding mutations (black) and phosphorylated residues (red) detected by mass spectrometry, in control or GAA-knockdown conditions. Source data are provided as a Source Data file.

Article Snippet: Samples were de-crosslinked overnight at 65 °C, followed by phenol:chloroform DNA extraction and ethanol precipitation. qPCR of precipitated DNA was done as cDNA samples (see RNA processing section), with self-designed primers at genomic DNA replication origins or control regions (Supplementary Data ), having SNS-seq data in wild-type HCT116 cells as a reference and purchased from Metabion.

Techniques: Western Blot, Transfection, Derivative Assay, Two Tailed Test, RNA Binding Assay, Mass Spectrometry