human colon cancer cell line hct116  (ATCC)

Journal: Journal of Natural Products

Article Title: Paraisariamides: Cycloheptapeptide Toxins from Entomopathogenic Fungi ( Paraisaria spp.) That Inhibit Total Protein Synthesis

doi: 10.1021/acs.jnatprod.5c01111

Figure Lengend Snippet: Comparative biological activity of natural product Psds A–D ( 1 – 4 , 1 nM to 10 μM) from P. cascadensis relative to known elongation factor inhibitors, didemnin B (0.1 nM to 1 μM) and ternatin (0.1 nM to 1 μM). (A) Analysis of the secretory function of human U87-MG glioblastoma cells expressing the reporter Gaussia luciferase (GLuc). GLuc expression was assessed in the conditioned medium after 18 h in the presence of Psds 1 – 4 , didemnin B, ternatin, or vehicle (0.1% DMSO). (B) Viability of human HCT116 colon cancer cells and (C) wild-type human U87-MG glioblastoma cells exposed to pure compounds or 0.1% DMSO for 72 h. Cell viability was measured by quantification of ATP at the end point. Data points represent mean ± SE ( n = 3 wells per treatment), expressed as a % of vehicle-treated cells, from a comparison that was repeated at least three times.

Article Snippet: Human HeLa cervical cancer cells, human HCT116 colon cancer cells, and human U87-MG glioblastoma cells, were from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Activity Assay, Expressing, Luciferase, Comparison

Journal: Journal of Natural Products

Article Title: Paraisariamides: Cycloheptapeptide Toxins from Entomopathogenic Fungi ( Paraisaria spp.) That Inhibit Total Protein Synthesis

doi: 10.1021/acs.jnatprod.5c01111

Figure Lengend Snippet: Comparative biological activities of natural and synthetic Psds E-H (5–8) from P. insignis . Viability of human HCT116 colon cancer cells exposed to (A) natural Psds E–H ( 5 – 8 , 1 μM to 10 μM), ternatin (1 μM), and apratoxin A (300 nM); natural (NP) and synthetic (B) Psd A ( 1 ), (C) Psd E ( 5 ), (D) Psd F ( 6 ), (E) Psd G ( 7 ), and (F) Psd H ( 8 ) or 0.1% DMSO for 72 h. Cell viability was measured by quantification of ATP at the end point. Data points represent mean ± SE ( n = 3 wells per treatment), expressed as a % of vehicle-treated cells, from a comparison that was repeated at least three times.

Article Snippet: Human HeLa cervical cancer cells, human HCT116 colon cancer cells, and human U87-MG glioblastoma cells, were from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Comparison

Journal: Nature Communications

Article Title: Nucleoplasmic Lamin A/C controls replication fork restart upon stress by modulating local H3K9me3 and ADP-ribosylation levels

doi: 10.1038/s41467-025-66098-9

Figure Lengend Snippet: a Representative confocal microscopy image of HCT116 cells showing Lamin A/C in proximity to nascent DNA (EdU), detected by Lamin A/C:EdU PLA (magenta), Lamin A/C IF staining (yellow). While Lamin A/C is mainly detected at the nuclear periphery, its interaction with nascent DNA (EdU) is detected throughout the nucleus and in different axial perspectives (top left: XY view, bottom left: YZ view, top right: XZ view). Numerous comparable examples of this pattern were observed in two independent experiments. Scale bar, 5 µm. b Schematic representation of the Proximity Ligation assay used. EdU incorporation is followed by click chemistry with biotin-azide. Antibodies against the target protein and biotin are recognized by secondary antibodies carrying probes. When the target protein and EdU are in close proximity ( < 40 nm), probes are ligated and amplified giving rise to a fluorescent PLA signal c . Experimental design for the IF/PLA experiment in ( c ). The duration of the EdU pulse is adapted to allow comparable incorporation of EdU despite the genotoxic treatments. d Representative U2OS nuclei (DAPI) – untreated or treated for 1 h with 100 nM CPT or 20 nM ETP–and stained for DNA synthesis (EdU), Lamin A/C and its physical proximity to nascent DNA (Lamin A/C:EdU PLA). Scale bar, 10 μm. e Quantification of Lamin A/C PLA signals from c. Signal was quantified in at least 100 EdU+ nuclei, in 4 independent experiments. EdU- cells are used as negative control. Yellow circles indicate the median for each experiment, while the black bar indicates the mean of the median values +/- SD. Statistical analysis was applied on the median values, using one-way ANOVA test with Bonferroni’s post hoc correction.

Article Snippet: Human osteosarcoma U2OS cells and human colon cancer HCT116 cells were acquired from ATCC.

Techniques: Confocal Microscopy, Staining, Proximity Ligation Assay, Amplification, DNA Synthesis, Negative Control

Journal: Nature Communications

Article Title: Nucleoplasmic Lamin A/C controls replication fork restart upon stress by modulating local H3K9me3 and ADP-ribosylation levels

doi: 10.1038/s41467-025-66098-9

Figure Lengend Snippet: a , b Western Blot analysis of Lamin A levels upon siRNA-mediated or 5-Ph-IAA-mediated depletion in the indicated cell lines. H3 is used as loading control. c–e DNA fiber analysis of HCT116 and HCT116 mAID2-mClover-LMNA cells upon siRNA-mediated or 5-Ph-IAA-mediated depletion of Lamin A/C. c . Schematic CldU/IdU pulse-labeling protocol used to evaluate fork progression upon 20 nM ETP. 5-Ph-IAA was added 24 h before the assay. d Representative DNA fiber images for the experiment in ( c ). e IdU/CIdU ratio is plotted for a minimum of 100 forks from each of 3 (siRNA) and 4 (5-Ph-IAA) independent experiments. Yellow circles indicate the median for each experiment, while the black bar indicates the mean of the median values +/- SD. Statistical analysis was applied on the median values, using one-way ANOVA test with Bonferroni’s post hoc correction. f–h DNA fiber analysis of U2OS cells upon siRNA-mediated depletion of Lamin A/C or LAP2α. f Schematic CldU/IdU pulse-labeling protocol used to evaluate fork progression upon 20 nM ETP or 100 nM CPT. siRNA was transfected 48 h before the assay. g Representative DNA fiber images for the experiment in ( f ). h IdU/CIdU ratio is plotted for a minimum of 100 forks from each of three independent experiments. Yellow circles indicate the median for each experiment, while the black bar indicates the mean of the median values +/- SD. Statistical analysis was applied on the median values, using one-way ANOVA test with Bonferroni’s post hoc correction. i Representative metaphase spread images. Insets show magnified chromosomes, numbered in the overview images. Arrowheads point to chromosomal breaks. Scale bar: 10 µm, in inset 5 µm. j Schematic design of the metaphase spread experiment in ( i – k ). k Average number of chromosomal breaks in mock- or Lamin A/C-depleted (siRNA) U2OS cells, optionally treated with 100 nM CPT for 3 h followed by nocodazole treatment. Bar graph depicts mean +/- SD from 3 independent experiments (yellow dots). A minimum of 55 metaphases was analyzed per sample and experiment. Statistical analysis was applied on the median values, using one-way ANOVA test with Bonferroni’s post hoc correction.

Article Snippet: Human osteosarcoma U2OS cells and human colon cancer HCT116 cells were acquired from ATCC.

Techniques: Western Blot, Control, Labeling, Transfection

Journal: Nature Communications

Article Title: Nucleoplasmic Lamin A/C controls replication fork restart upon stress by modulating local H3K9me3 and ADP-ribosylation levels

doi: 10.1038/s41467-025-66098-9

Figure Lengend Snippet: a Representative PLA images illustrating G9a enrichment on nascent DNA upon mild RS (G9a-EdU PLA, red). RPE-1 cells were labeled with EdU for 20 min either before optional treatment with 1 mM HU (1 h) or at the end of 20 nM CPT (1 h) and 20 nM ETP (1 h) treatment. Scale bar: 10 µm. b Quantification of the total intensity of all G9a-EdU PLA spots per nucleus in ( a ). In ( b , d ), n > 800 S phase cells were analyzed in each condition; Kruskal- Wallis test followed by Dunn’s test were performed to test statistical significance for each of 2 independent PLA experiments. c Representative PLA images illustrating H3K9me3 deposition on nascent DNA upon mild RS (H3K9me3-EdU PLA, red). mAID2-LMNA HCT116 cells were labeled with EdU for 20 min at the end of optional treatment with 25 nM CPT (1 h) and 20 nM ETP (1 h). Scale bar: 10 µm. d Distribution of H3K9me3-EdU total PLA spot intensity per nucleus. Statistical analysis as in ( b ). e Schematic CldU/IdU pulse-labeling protocol used in f to evaluate fork progression upon treatment with 20 nM ETP, 100 nM CPT, G9ai (UNC0642, 1 μM) and/or PARGi (PDD0017272, 1 μM). G9ai and PARGi were added 2 h before the assay. f IdU/CIdU ratio is plotted for a minimum of 100 forks from each of 3 independent experiments. Similar results were observed in all independent experiments. Yellow circles indicate the median for each experiment, while the black bar indicates the mean of the median values +/- SD. Statistical analysis was applied on the median values, using one-way ANOVA test with Bonferroni’s post hoc correction. g Quantification of PAR:EdU PLA signals from U2OS cells, treated with 100 nM CPT, G9ai (UNC0642, 1 μM) and PARGi (PDD0017272, 1 μM). Experimental design as in Fig. . Signal was quantified in at least 100 EdU+ nuclei, in each of the 3 independent experiments. PARGi-treated and untreated samples were processed in parallel, but are displayed in different graphs due to different ranges of observed signal. Yellow circles indicate the median for each experiment, while the black bar indicates the mean of the median values +/- SD. Statistical analysis was applied on the individual experiments, using Kruskal-Wallis test with Dunn’s post hoc correction. h Schematic CldU/IdU pulse-labeling protocol used in i to evaluate fork progression upon treatment with 20 nM ETP and G9ai (UNC0642, 1 μM), and/or RECQ1 downregulation by siRNA. siRNA was transfected 48 h before the assay, while G9ai was added 2 h before. i IdU/CIdU ratio is plotted for a minimum of 100 forks from each of 3 independent experiments. Yellow circles indicate the median for each experiment, while the black bar indicates the mean of the median values +/- SD. Statistical analysis was applied on the median values, using one-way ANOVA test with Bonferroni’s post hoc correction. j Average frequency of reversed replication forks isolated from U2OS cells treated for 1 h with 100 nM CPT or 2 mM HU, combined with the indicated inhibitors. Yellow dots represent the observed percentage of reversed forks in each independent experiment ( n = 2; see Supplementary Fig. ). Total number of molecules analyzed per condition in brackets. A.U .: arbitrary units.

Article Snippet: Human osteosarcoma U2OS cells and human colon cancer HCT116 cells were acquired from ATCC.

Techniques: Labeling, Transfection, Isolation

Journal: Nature Communications

Article Title: Nucleoplasmic Lamin A/C controls replication fork restart upon stress by modulating local H3K9me3 and ADP-ribosylation levels

doi: 10.1038/s41467-025-66098-9

Figure Lengend Snippet: a Representative PLA images illustrating H3K9me3 deposition on nascent DNA upon mild RS (H3K9me3-EdU PLA, red) in mAID2-LMNA HCT116 cells. Cells were treated with 5-Ph-IAA for 24 h prior to the experiment, to induce Lamin A/C depletion. Cells were pulsed with EdU for 20 min at the end of the optional treatment with 25 nM CPT (1 h). Scale bar: 10 µm. b Quantification of H3K9me3-EdU total PLA spot intensity per nucleus. n > 800 S phase cells were analyzed in each condition; Kruskal−Wallis test followed by Dunn’s test were performed to test statistical significance for each of 2 independent PLA experiments. c Representative images of chromatin fibers acquired by ChromStretch stained for EdU (red), H3 (magenta) and H3K9me3 (green), from U2OS cells: untreated (top), treated with 25 nM CPT (1 h) (middle), and upon downregulation of LMNA prior to treatment with 25 nM CPT (1 h) (bottom). Cells were pulsed with EdU for 20 min at the end of the optional treatments with 25 nM CPT (1 h). Scale bar: 2 µm. d Moving average intensity profiles of EdU (red), H3K9me3 (green) and H3 (magenta) of the representative fibers shown in ( c ). e Quantification of H3K9me3 signal overlapping with EdU spots. n > 75 EdU tracks for each condition were analyzed in 3 independent experiments. f Quantification of H3 signal overlapping with EdU spots (normalized to the H3 signal outside EdU bubble). n > 75 EdU tracks for each condition were analyzed in 3 independent experiments. g Quantification of H3K9me3 signal overlapping with EdU spots upon optional treatment with 25 nM CPT (1 h) and optional downregulation of LMNA or KDM3A . n > 70 EdU tracks for each condition were analyzed in two independent experiments. Kruskal-Wallis test followed by Dunn’s test were performed to test statistical significance for PLA and chromatin fiber analysis. h Schematic CldU/IdU pulse-labeling protocol used in j to evaluate fork progression upon treatment with 100 nM CPT and/or KDM3A downregulation by siRNA. i Western Blot analysis of Lamin A and KDM3A levels upon siRNA-mediated depletion for the experiment in j. Ponceau S. is shown as loading control. j IdU/CIdU ratio is plotted for a minimum of 100 forks from each of 3 independent experiments. Yellow circles indicate the median for each experiment, while the black bar indicates the mean of the median values +/- SD. Statistical analysis was applied on the median values, using one-way ANOVA test with Bonferroni’s post hoc correction. A.U . arbitrary units.

Article Snippet: Human osteosarcoma U2OS cells and human colon cancer HCT116 cells were acquired from ATCC.

Techniques: Staining, Labeling, Western Blot, Control