EMBO/SourceData huggingface dataset:
Figure 2 (B) Bar diagram representation of the relative SUMO conjugation activity of Nse2, full-length Smc5-Nse2, ΔHinge/Smc5-Nse2, ΔHead/Smc5-Nse2 and Arm/Smc5-Nse2 truncation constructs (schematic representation above). Orange bars indicate the presence of ssDNA (virion ϕx174) and red bars absence of ssDNA. Reaction rates were performed at least in three different independent experiments (see Fig EV2). Data values are mean ± s.e.m.; and n=3 technical replicates. Significance was measured by a two-tailed unpaired t-test relative to wild-type. **P<0.01  (C) SYPRO-stained (left) and Western blot (right) time course SUMO conjugation reaction using and ΔHead/Smc5-Nse2 truncation construct in the presence of ssDNA. T7-tagged Smc5 and E1 were immunodetected by an anti-T7 antibody. Reactions were run at 30 °C and stopped at indicated times by adding SDS-loading buffer  (D) SYPRO-stained (left) and Western blot (right) time course SUMO conjugation reaction using and ΔHinge/Smc5-Nse2 truncation construct in the presence of ssDNA. T7-tagged Smc5 and E1 were immunodetected by an anti-T7 antibody. Reactions were run at 30 °C and stopped at indicated times by adding SDS-loading buffer  (E) Western blot of the time-course SUMO conjugation reaction in the presence of ssDNA (virion ϕx174) using either Nse2 (left) or Arm/Smc5-Nse2 complex (right). The reactions were run in the presence or absence of cNse4 external substrate. Reaction was run at 30°C and stopped at indicated minutes by adding SDS-loading buffer (N-S2, cNse4-SUMO2; N-2S2, cNse4-2SUMO2; N-3S2, cNse4-3SUMO2 and pS2, poly-SUMO2)
Figure 3 (D) Bar diagram representation of the SUMO conjugation rates of activity assays of Arm/Smc5-Nse2 KE mutants in the presence (orange bars) or absence (red bars) of ssDNA (virion ϕx174), relative to wild-type (set to 1). Reactions rates were performed at least in three different independent experiments. Data values are mean ± s.e.m. and n=3 technical replicates. Significance was measured by a two-tailed unpaired t-test relative to wild-type. *P<0.05, **P<0.01, ***P<0.001
Figure 4 (C) Degree of conformational changes in CM induced by 60b linear ssDNA. Blue bar, Arm/Smc5-Nse2 (wild-type); grey bar, Arm/Smc5-Nse2 (K333E/K344E); green bar, Arm/Smc5-Nse2 (K764E); pink bar, Arm/Smc5-Nse2 (K333E/K344E/K764E); red bar, Arm/Smc5-Nse2 (K743E/K745E). Reactions were performed at least in three different independent experiments. Data values are mean ± s.d. and n=3 technical replicates. Significance was measured by a two-tailed unpaired t-test relative to wild-type. ****P < 0.0001
Figure 5 (B) Growth test analysis of wild-type, nse2-CH and the indicated smc5-KE mutants; 10-fold serial dilutions of the liquid cultures were spotted in YPD and pictures taken after 48 hours  (C) Same as in (B) but using the nse2-CH, smc5-3KE, smc5-K743,745E, smc5-K743,745R, smc5-7KE and smc5-7KR mutants
Figure 1(b) RNAscope analysis of Lgr5 mRNA in DSS colitis demonstrating loss of Lgr5 transcript (N=3 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50µm(c) Hematoxylin and Eosin staining of the colonic epithelium following DSS induced damaged. Crypts are dramatically injured by d5. At d8 through d12, regenerating crypts are observed. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50µm(d-e) Compared to untreated controls, DSS treatment ablates Lgr5-GFP+ stem cells and results in functional loss of tdTomato+ lineage tracing as early as d5. Lgr5-GFP+ stem cells reappear by d12 (N=3-4 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50µm Data are represented as mean ± SD (e) analyzed using Student's t-test. *P≤0.05, ** P≤0.01.(f) Effects of DSS colitis injury on RNA expression levels of stem (Lgr5 and Krt19) and progenitor (Atoh1 and Notch1) cell markers. Lgr5 mRNA expression is significantly decreased after exposure to DSS (N=6-7 per condition). Data are represented as mean ± SEM analyzed using Student's t-test. *P≤0.05, ** P≤0.01.Mice body weight (h) are comparable between three treatments: DSS+DT (N=12), only DSS (N=8) or only DT (N=3). Data are represented as mean ± SEM analyzed using Student's t-test. *P≤0.05, ** P≤0.01.Mice survival (i) are comparable between three treatments: DSS+DT (N=12), only DSS (N=8) or only DT (N=3). Data are represented as mean ± SEM analyzed using Student's t-test. *P≤0.05, ** P≤0.01.(j) Hematoxylin and Eosin staining of the colonic epithelium shows no morphological difference between DSS alone and DSS+DT treated mice. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. scale bar (j) =500µm.(k) The extent of epithelial damage is similar in mice treated with DSS alone versus DSS+DT (to ablate Lgr5+ stem cells) (N=3 DSS; N=6 DSS+DT). Data are represented as mean ± SEM analyzed using Student's t-test. *P≤0.05, ** P≤0.01.(l) RNAscope for Lgr5 in Lgr5DTR-GFP mice showing loss of Lgr5 transcripts after DT treatment (N=3-4 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50µm
Figure 2(b-c) Krt19+ cells lineage trace entire colonic crypts during both normal homeostasis and injury (N=3-4 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines outline the basement membrane. Scale bars (b) =45µm Data are represented as mean ± SEM *P≤0.05, **P≤0.01.DT ablation of Krt19+ cells (N≥3 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines outline the basement membrane. scale bar (e) =30µmDT ablation of Krt19+ cells significantly reduced the number of lineage labeled crypts (N≥3 per condition). Data are represented as mean ± SEM using Student's t-test *P≤0.05, **P≤0.01.(g) Mice body weight and survival remain unchanged between DSS and DSS+DT treated groups (N=6 control; N=7 DT). Data are represented as mean ± SEM using Student's t-test P≤0.05, **P≤0.01.Hematoxylin and Eosin staining of the colonic epithelium after DSS versus DSS+DT showing more damage is seen in mice treated with both DSS+DT treatment (N=6 control; N=7 DT). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines outline the basement membrane. scale bar (h) =1mm.quantification of damaged epithelial area (i), showing more damage is seen in mice treated with both DSS+DT treatment (N=6 control; N=7 DT). Data are represented as mean ± SEM using Student's t-test *P≤0.05, **P≤0.01.
Figure 3(b-c) Notch1+ progenitors are able to generate Lgr5+ stem cells and contribute to epithelial renewal in homeostasis; however, do not contribute to regeneration after colitis (N=4 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars=50μm. Data are represented as mean ± SD analyzed using two-way ANOVA with Sidak's multiple comparisons test. ****P≤0.0001.Images of whole mount showing Notch1 lineage labeled crypts. No partially labeled crypts were found after Lgr5+ stem cell ablation. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars=50μm.Images of tissue sections Notch1CreERT2; ROSA26tdTomato;Lgr5DTR-GFP mice (f) showing Notch1 lineage labeled crypts. No partially labeled crypts were found after Lgr5+ stem cell ablation. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars=50μm.
Figure 4(a-b) Atoh1+ progenitors show stem cell capacity during normal homeostasis and regeneration. At early stages of recovery, d10-12, Atoh1+ cells lineage trace the colonic crypt (N=4 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50μm Data are represented as mean ± SD analyzed using two-way ANOVA with Sidak's multiple comparisons test *P≤0.05, ***P≤0.001.(c-d) By d30 after DSS, Atoh1 derived stem cells continue to lineage trace complete colonic crypts (N=4 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars first two panels of c =50μm; scale bars (last panel of c =100μm. Data are represented as mean ± SD analyzed using two-way ANOVA with Sidak's multiple comparisons test *P≤0.05, ***P≤0.001.Whole mount (f) images of Atoh1 lineage labeled crypts. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50μmsection (g) images of Atoh1 lineage labeled crypts. Majority of crypts are labeled after Lgr5+ stem cell ablation and remain label with the appearance of Lgr5+ stem cells (inset, arrowheads). Atoh1 lineage labeled crypts remain by d30. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. scale bars =100μm.(h) Significantly increased labeling of crypt base was observed at d8 in DT treated mice (N=4 per condition). Data are represented as mean ± SD analyzed using Student's t-test with Welch's correction *P≤0.05, ***P≤0.001.(j-k) Scattered labeling of Atoh1+ cells in control conditions, while DSS injury lead to Atoh1 lineage labeled crypts lacking Lgr5+ stem cells. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50μm Data are represented as mean ± SD analyzed using Student's t-test with Welch's correction *P≤0.05, ***P≤0.001.
Figure 5(b-c) Sporadic labeling of Atoh1+ cells after 4-OHT induction. Following irradiation, entire colonic organoids exhibit full lineage labeling of Atoh1+ cells, characteristic of multipotent stem cells (N=3 per condition). Data information: Scale bars =100μm Data are represented as mean ± SD analyzed using Student's t-test (c)(d-e) Flow cytometry for tdTomato+ cells in crypt epithelium from DT treated Atoh1CreERT2;Lgr5DTR-GFP;ROSA26tdTomato mice.(f) tdTomato+ colonic organoids from single Atoh1-tdTomato+/Lgr5-negative cells (N=1; n=3 technical replicates per condition). Data information: Scale bars =100μm(g) Post-passage colonic organoids from RFP+ cells of control or DT treated mice continue growing. tdTomato-negative cells of control or DT treated mice also form colonic organoids after passaging. Data information scale bars (g) =50μm.(h) tdTomato+ colonic organoids established from mice in which Lgr5+ stem cells have been ablated show higher growth efficiency compared to their colonic organoid counterparts (N=1; n=3 technical replicates per condition). Data are represented as mean ± SD analyzed using two-way ANOVA with Sidak's multiple comparisons test (h). *P≤0.05, **P≤0.01, ***P≤0.001.
Figure 6(b) Atoh1+ lineage labelling post-DSS and DSS+DT. Data information: Scale bars (b)=200μm (c) DSS+DT treated-mice exhibit significant weight loss and lower survival compared to DSS alone (N=4-7 control; N=3-4 DSS). Data are represented as mean ± SEM analyzed using Student's t-test. *P≤0.05.(d-e) Hematoxylin and Eosin staining of colonic epithelium after DSS versus DSS+DT (d). Extensive epithelial damage was observed (d, arrow) and quantified of in DSS+DT treated mice (N=7 control; N=4 DSS). Data information scale bars (d)=400μ Data are represented as mean ± SEM analyzed using Student's t-test. *P≤0.05.(g-h) DT treatment effectively ablates Atoh1+ cells shown by reduced number of tdTomato+ cells per organoid. Data information scale bars =100μm. Data are represented as mean ± SEM analyzed using Student's t-test. *P≤0.05.(i-j) Concurrent irradiation injury and Atoh1+ cell ablation results in significantly reduced organoid survival (N=3; n=3 technical replicates per condition). Data information: ; scale bars =100μm. Data are represented as mean ± SEM analyzed using Student's t-test. *P≤0.05.
Figure 1B: Double-IHC of coronal brain sections (40 μm) of a littermate (LM) and a NexCre cTKO mouse (age: 5 months) stained for Ctip2 (green, expressed in layer V/VI) and Calbindin (red, expressed in layer II/III and to some extent in layer V). Note that no gross abnormalities in cortical lamination were observed. Layers of DAPI stained cell nuclei are denoted on the right (I-VI), scale bar: 100 µm.C: Giemsa-stained, glycolmethacrylate-embedded coronal brain sections (bregma -1.9) displaying the hippocampus and adjacent callosal fiber tracts of a wildtype (WT, top), littermate control (LM, middle) and a NexCre cTKO (cTKO, bottom; age: 5-6 months). Note the agenesis of the corpus callosum (no CC) in NexCre cTKO mice, which should always be present at this bregma and in sections in which the medial habenula (mHb) is present (CC in WT and LM). While WTs and LMs show a compact layer of CA1 cells, NexCre cTKOs display a bilaminar cytoarchitecture (boxed regions marking higher magnification to the right) with scattered ectopic cells in the stratum radiatum (white arrowheads). In addition, the CA3 appears less compact (red arrowheads). Layer identity visualized via Calbindin staining (right panel, scale bar: 50 µm).
Figure 2C: Representative 3D reconstructions of CA1 pyramidal neurons from deep (left, dark blue) and superficial (right, light blue) radial layers.D: Sholl analysis reveals no genotype effect on basal dendritic segments of deep CA1 pyramidal neurons (dCA1; LM: n=21, N=7; cTKO: n=21/N=6). E: Compared to littermate controls, dCA1 neurons of NexCre cTKO animals show no significant reduction in total dendritic length in basal dendrites (unpaired Student's t-test, nsp = 0.4979). F: The number of primary basal dendrites is significantly reduced in NexCre cTKO animals compared to LM in dCA1 (Mann-Whitney test, **p = 0.0032). G:
Figure 3A, B: Spine density of deep CA1 pyramidal cells is significantly reduced in basal dendritic segments (A; unpaired Student's t-test, **p = 0.0067), while it is unchanged in midapical dendritic segments (B; unpaired Student's t-test, nsp = 0.4949). C: Representative images of basal and midapical dendritic segments from both radial layers. Brightness and contrast were adjusted to ensure a uniform appearance. Scale bar: 5 µm. D
Figure 4A: mEPSC and mIPSC sample traces of recordings from deep CA1 pyramidal cells. B - E: Bar graphs of mEPSC and mIPSC frequencies and amplitudes in deep CA1 pyramidal cells. mEPSC frequency was not different in deep CA1 pyramidal neurons of adult (4 months) NexCre cTKO versus littermate controls (B, p = 0.6415). mEPSC amplitude was increased in deep CA1 pyramidal cells of NexCre cTKO compared to LM control cells (C, *p = 0.0363). mIPSC frequency was not different in deep CA1 pyramidal neurons of adult (4 months) NexCre cTKO versus littermate controls (D, p = 0.0564) and mIPSC amplitude was increased in deep CA1 pyramidal cells of NexCre cTKO compared to LM (E, *p = 0.0356). F: mEPSC and mIPSC sample traces of recordings from superficial CA1 pyramidal cells. G
Figure 5A: Diurnal behavior in the home cage (HC). NexCre cTKOs show overshooting activity in the dark phase. Line graphs illustrate the average locomotion per hour. [Sidak's multiple comparisons test, geno F(1,32) = 9.503, p = 0.0042; timepoint F(6.102,195.3) = 10.51, p < 0.0001; timepoint × geno F(47,1504) = 5.874, p < 0.0001].I: Sociability in the three chambers test (3CT). LM controls spend more time with mouse stranger 1 (S1) whereas NexCre cTKOs spend comparable time exploring the novel object (NO) or S1. Bottom: schematic representation of test. [Paired Student's t-test comparison between chambers LM ***p = 0.0002; Wilcoxon test between chambers NexCre cTKO ns p = 0.5412].
Figure 6C: T-Maze alternation test shows that spontaneous alternation is absent in NexCre cTKOs. [geno F(1,16) = 18.29, p = 0.0006]. Chance level is indicated by a dotted line.N: Morris Water Maze (MWM), escape latency. During place navigation training, NexCre cTKO animals showed no evidence of learning. [geno F(1,18) = 30.53, p < 0.0001; day F(4,72) = 13.35, p < 0.0001 ; day × geno F(4,72) = 11.77 p < 0.0001].
Figure 1A The expression of LINC00839 in CRC tissues and normal mucosa tissues in the UALCAN database. B, C, D The expression of LINC00839 in unpaired CRC and normal samples in the LnCAR database (GSE37364, GSE71187, and GSE33113). Data information: In A, B, C, D middle lines represent the median, and the upper and lower lines represent the upper and lower quartiles. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.E, F The expression levels of LINC00839 in tumour tissues and NATs from the CRC cohort (n = 45). nmCRC denotes CRC patients without metastases, and mCRC denotes CRC patients with metastases. Data information: In E, F, the data are presented as the mean ± SD and were analysed by Student's t test, n = 3 technical replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.G Representative ISH staining of LINC00839 in paraffin-embedded samples of CRC tumour tissues and NATs. (a) Expression of LINC00839 in tumour tissues and NATs. Scale bars, 100 μm. (b) Magnification of normal mucosal area and (c) magnification of the tumour area. Scale bars, 50 μm. (d) Weakly positive staining of LINC00839 in normal mucosa. Scale bars, 50 μm. (e) Strong positive staining of LINC00839 in tumours and (f) magnification of the local area. Scale bars, 100 μm.H The expression score of the tumours and NATs in the ISH cohort (n = 222). Data information: In H the data are shown as the mean ± SD and were analysed by Student's t test. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.I Differential expression of LINC00839 in nonmetastatic CRC patients (nmCRC) and metastatic CRC patients (mCRC). Data information: In I, the data are shown as the mean ± SD and were analysed by Student's t test. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.J Expression of LINC00839 in CRC patients with early-stage (stage I and II) and advanced-stage (stage III and IV) disease. Data information: In J, middle lines represent the median, and the upper and lower lines represent the upper and lower quartiles. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.Kaplan-Meier analysis of overall survival rate of the ISH CRC cohort.L Kaplan-Meier analysis of overall disease-free survival rate of the ISH CRC cohort. M Kaplan-Meier analysis of the disease-free survival rate of CRC patients in the LnCAR database (GSE33113).
Figure 2C Colony formation assay was performed with LINC00839-overexpressing cells and vector control cells. , the data are presented as the mean ± SD and were analysed by Student's t test, n = 3 biological replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.000D Wound-healing assay was performed with LINC00839-overexpressing cells and vector control cells. Scale bars, 100 μm. , the data are presented as the mean ± SD and were analysed by Student's t test, n = 3 biological replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.000E Matrigel invasion assay was performed with LINC00839-overexpressing cells. Scale bars, 100 μm. , the data are presented as the mean ± SD and were analysed by Student's t test, n = 3 biological replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.000F The CDX model showing the effect of LINC00839 on tumour growth. Representative image of tumours harvested from the mouse model (upper panel), the quantitative analysis of tumour weight and tumour volume (medium panel), and representative images and quantification of IHC staining of Ki-67 in tumours (bottom panel) (n = 6 for each cohort, and the results are presented as the mean ± SD, Student's t test (medium left and low right panel) and ANOVA (medium right). Scale bars, 100 μm (n = 6 biological replicates . Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.000. G The CRC orthotopic mouse model revealed the effect of LINC00839 on tumour development. Representative images of primary tumours (yellow arrowheads) and liver metastasis (blue arrowheads) in the model (upper panel), analysis of tumour volume and the number of mice with liver metastasis (medium panel), and H&E staining of tumours and metastases (n = 6 for each cohort). Scale bars, 100 μm (n = 6 biological replicates . Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.000
Figure 3A An RNA pull-down assay was performed to identify proteins that bind to LINC00839 in HCT116 cells.B RNA pull-down assay and WB showed that biotinylated LINC00839 can bind to Ruvb1 in HCT116 cells. Dot blot of RNA-protein interactions indicating equal amounts of RNA used in the assay.C RIP experiments were performed to confirm the binding of LINC00839 to Ruvb1. IgG was used as a negative control antibody. Primers specific for U6 and GAPDH were used as negative control primers. Data information: In C, the data are presented as the mean ± SD and were analysed by unpaired t test, n = 3 technical replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.D Different fragments of LINC00839 (solid line) and its antisense sequence (dotted line) were used for the RNA pull-down assay and the subsequent WB.E Full length LINC00839, antisense sequence of LINC00839, and LINC00839 sequence lacking nucleotides 1033-1290 were used for the RNA pull-down assay and the subsequent WB.F RIP experiments were performed by incubating Tip60, INO80, SRCAP, and PIH1D1 antibodies with HCT116 cell lysates. Data information: In F, the data are presented as the mean ± SD and were analysed by unpaired t test, n = 3 technical replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.G The effect of LINC00839 on the expression of Tip60 and Ruvb1 was determined by WB.H Interaction of endogenous Ruvb1 and Tip60 in CRC cells was confirmed by Co-IP.I The interaction between Ruvb1 and Tip60 in cells overexpressing wild-type LINC00839 and LINC-Δ5 was confirmed by Co-IP.J The interaction between Ruvb1 and Tip60 in LINC00839-KD cells and cells expressing LINC-Δ5 was confirmed by Co-IP.K Acetylase activity assays were performed with LINC00839-overexpressing and LINC00839-knockdown cells. Data information: In the data are presented as the mean ± SD and were analysed by unpaired t test, n = 3 technical replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.L Acetylase activity was measured in SW480 and LoVo cells subjected to different treatments. Data information: In L, the data are presented as the mean ± SD and were analysed by unpaired t test, n = 3 technical replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.