Journal: Nucleic Acids Research

Article Title: DNA-PK cs promotes chromatin decondensation to facilitate initiation of the DNA damage response

doi: 10.1093/nar/gkz694

Figure Lengend Snippet: Generation and initial characterization of the DNA-PK cs kinase-dead (KD) HCT116 cell line. ( A ) Basic schematic of the method used to generate the HCT116 DNA-PK cs kinase dead (KD/−) cell line. ( B ) Expression level of DNA-PK cs in HCT116 DNA-PK cs +/+, +/−, −/−, and KD/− cells as assessed by western blotting. ( C ) Measurement of DNA-PK cs in vitro kinase activity. Nuclear extracts from the HCT116 DNA-PK cs +/+, +/−, −/−, and KD/− cells were examined for their ability to phosphorylate a biotin-tagged H2AX peptide. H2AX phosphorylation was observed in the −/− cell line and this was subtracted from the other samples’ readouts. The 100% kinase activity was normalized using the +/+ cell lysate results. The data are presented as the mean ± SD from three individual experiments. ( D ) Measurement of DNA-PK cs in vivo kinase activity. The HCT116 DNA-PK cs +/+, +/−, −/−, and KD/− cell lines were mock-treated or γ-irradiated with a dose of 10 Gy and allowed to recover for 30 min. Cell extracts were prepared and western blot analysis was performed to assess autophosphorylation of DNA-PK cs at serine 2056. Immunoblotting of Ku70 and Ku80 were used as loading controls. ( E ) The interaction between DNA-PK cs and the Ku70/80 heterodimer is not affected by inactivating the kinase activity of DNA-PK cs . DNA-PK cs was immunoprecipitated from the HCT116 +/+, +/−, −/−, and KD/− cell lines 5 min after being irradiated with 10 Gy of γ-rays. The immunoprecipitates were analyzed by western blotting with anti-DNA-PK cs , Ku80, and Ku70 antibodies. Tubulin was used as a loading control for the input of each immunoprecipitation assay.

Article Snippet: Briefly, the HCT116 DNA-PK cs +/−, −/−, and KD/− cells were mock-treated or irradiated with 10 Gy and allowed to recover for 10 min. Next, the cells were harvested after trypsinization and processed with the Thermo Fisher Subcellular Protein Fractionation Kit according to the manufacturer's instructions.

Techniques: Expressing, Western Blot, In Vitro, Activity Assay, Phospho-proteomics, In Vivo, Irradiation, Immunoprecipitation, Control

Journal: Nucleic Acids Research

Article Title: DNA-PK cs promotes chromatin decondensation to facilitate initiation of the DNA damage response

doi: 10.1093/nar/gkz694

Figure Lengend Snippet: DNA-PK cs kinase activity is important for cell survival and DSB repair following DSB induction. ( A ) Colony formation assays were performed to compare radiation sensitivities of the HCT116 DNA-PK cs +/+, +/−, −/−, and KD/− cell lines. Cells lines were left cycling and irradiated at the indicated doses and plated for analysis of survival and colony-forming ability. The data is presented as mean ± SD from three independent experiments. ( B ) DSB repair proficiency of HCT116 DNA-PK cs +/+, +/−, −/−, and KD/− was evaluated using neutral Comet assay. Cells were irradiated with 10 Gy of IR, allowed to recover for 30 min, harvested, and then Comet assays were performed. Tail moment values for >400 mock treated and irradiated cells were calculated and plotted via a distribution dot plot. ( C ) Dynamic 53BP foci distribution in HCT116 DNA-PK cs +/+, +/−, −/−, and KD/− cells after IR. The cells were irradiated with 1 Gy of γ-rays and 53BP1 foci formation and resolution was assessed 0.5, 1, 2, 4 and 8 h later. Data were normalized to the foci number enumerated at 30 min post IR. Remaining foci number per time point were calculated and plotted. Error bars denote SEM of three independent experiments. ( D ) NHEJ-mediated and ( E ) HR-mediated DSB repair were evaluated in HCT116 DNA-PK cs +/+, +/−, −/−, and KD/− cells using GFP-based reporter assays. The data were presented as Mean ± SEM with P -values from at least three biological repeats.

Article Snippet: Briefly, the HCT116 DNA-PK cs +/−, −/−, and KD/− cells were mock-treated or irradiated with 10 Gy and allowed to recover for 10 min. Next, the cells were harvested after trypsinization and processed with the Thermo Fisher Subcellular Protein Fractionation Kit according to the manufacturer's instructions.

Techniques: Activity Assay, Irradiation, Neutral Comet Assay

Journal: Nucleic Acids Research

Article Title: DNA-PK cs promotes chromatin decondensation to facilitate initiation of the DNA damage response

doi: 10.1093/nar/gkz694

Figure Lengend Snippet: DNA-PK cs initiates phosphorylation of H2AX and KAP1 after IR. ( A ) IR-induced phosphorylation of KAP1 and H2AX is attenuated in the KD/− cell line at early time points. HCT116 DNA-PK cs +/−, −/−, and KD/− cell lines were mock-treated or irradiated with a dose of 10 Gy and allowed to recover for 1, 3, or 5 min. Whole cell lysates were obtained and immunoblotting was performed to assess the phosphorylation status of DNA-PK cs at serine 2056, ATM at serine 1981, KAP1 at serine 824, and H2AX at serine 139. Tubulin was used as a loading control. ( B ) H2AX and KAP1 are phosphorylated in ATM-deficient cells (AT5) after IR, which is dependent on DNA-PK cs . AT5 cells were mock-treated or incubated for 2 h prior to irradiation with 10 μM NU7441 to inhibit DNA-PK cs and then AT5 cells and ATM-expressing AT5 cells (AT5+ATM) were mock-treated or irradiated with a dose of 10 Gy and allowed to recover for 3, 10, or 30 min. Whole cell lysates were obtained and immunoblotting was performed to assess the phosphorylation status of DNA-PK cs at serine 2056, KAP1 at serine 824, and H2AX at serine 139. Tubulin was used as a loading control. ( C – E ) Time courses of IR-induced KAP1 and H2AX phosphorylation in the HCT116 DNA-PK cs +/−, −/−, and KD/− cell lines. The cells were mock-treated or irradiated with a dose of 10 Gy and allowed to recover for 1, 3, 5, 10, 15, 30, or 60 min. (C) Whole cell lysates were obtained and immunoblotting was performed to assess the phosphorylation status of DNA-PK cs at serine 2056, KAP1 at serine 824, and H2AX at serine 139. Tubulin was used as a loading control. All immunoblots for each protein shown in (C) come from the same exposure of a single film. The relative phosphorylation level of H2AX and KAP1 at each time point are shown in (D) and (E), respectively. Following quantification of the protein levels, the relative level of H2AX and KAP1 phosphorylation were calculated by taking γH2AX/H2AX and KAP1 S824/KAP1 and then normalizing each time point to value of γH2AX/H2AX and KAP1 S824/KAP1 at 1 h after IR in the (+/−) control cell line. The data are presented as the mean ± SEM from three independent experiments. The P -values were generated by comparing +/− with −/− or KD/− with Student t -test. *, P < 0.05; **, P < 0.01. All data (A–C) are a representative image of three independent experiments.

Article Snippet: Briefly, the HCT116 DNA-PK cs +/−, −/−, and KD/− cells were mock-treated or irradiated with 10 Gy and allowed to recover for 10 min. Next, the cells were harvested after trypsinization and processed with the Thermo Fisher Subcellular Protein Fractionation Kit according to the manufacturer's instructions.

Techniques: Phospho-proteomics, Irradiation, Western Blot, Control, Incubation, Expressing, Generated

Journal: Nucleic Acids Research

Article Title: DNA-PK cs promotes chromatin decondensation to facilitate initiation of the DNA damage response

doi: 10.1093/nar/gkz694

Figure Lengend Snippet: Chromatin decondensation in response to DNA damage at early time points requires DNA-PK cs kinase activity. ( A ) IR-induced chromatin relaxation is attenuated in the HCT116 DNA-PK cs KD/− and −/− cells. The HCT116 DNA-PK cs +/−, −/−, and KD/− cell lines were mock-treated or irradiated with a dose of 10 Gy and allowed to recover for 10 min. Chromatin decondensation was then determined by examining micronuclease (MNase) accessibility. Nuclei were processed and the DNA was visualized by resolving it via agarose gel electrophoresis. Panels on the right show quantified signal as the percent of total for each lane across a distance from the well to the end of gel. ( B ) ATM kinase activity is not required for the initial chromatin relaxation after IR-induced DNA damage. The HCT116 DNA-PK cs +/− and KD/− cell lines were mock-treated or incubated for 2 h prior to irradiation with 10 μM KU60019 to inhibit ATM and then the cells were mock-treated or irradiated with a dose of 10 Gy and allowed to recover for 10 min. Samples were processed and quantified as described in (A). ( C ) Inhibition of DNA-PK cs suppresses IR-induced chromatin decondensation in ATM-deficient AT5 cells. The AT5 cells were mock-treated or incubated for 2 h prior to irradiation with 10 μM NU7441 to inhibit DNA-PK cs and then the cells were mock-treated or irradiated with a dose of 10 Gy and allowed to recover for 10 min. Samples were processed and quantified as described in (A). All data (A–C) are a representative image of three independent experiments.

Article Snippet: Briefly, the HCT116 DNA-PK cs +/−, −/−, and KD/− cells were mock-treated or irradiated with 10 Gy and allowed to recover for 10 min. Next, the cells were harvested after trypsinization and processed with the Thermo Fisher Subcellular Protein Fractionation Kit according to the manufacturer's instructions.

Techniques: Activity Assay, Irradiation, Agarose Gel Electrophoresis, Incubation, Inhibition

Journal: Nucleic Acids Research

Article Title: DNA-PK cs promotes chromatin decondensation to facilitate initiation of the DNA damage response

doi: 10.1093/nar/gkz694

Figure Lengend Snippet: DNA-PK cs catalytic activity facilitates the initial recruitment of the DDR machinery to DSBs. ( A ) Recruitment of HR and NHEJ factors to the chromatin after IR is attenuated in the HCT116 DNA-PK cs KD/− cell line. HCT116 DNA-PK cs +/−, −/−, and KD/− cells were mock-treated or irradiated with a dose of 10 Gy and allowed to recover for 10 min. Subsequently, soluble nuclear and the chromatin-enriched fractions were isolated for immunoblotting to assess the recruitment of the proteins listed in the figure to the chromatin after irradiation. ( B ) IR-induced focus formation of MDC1 is attenuated in the HCT116 DNA-PK cs KD/− cell line. The HCT116 DNA-PK cs +/−, −/−, and KD/− cell lines were mock-treated or irradiated with a dose of 1 Gy and MDC1 foci formation was assessed 5 and 10 min later. MDC1 focus formation was examined in at least 50 cells and the number of MDC1 IR-induced foci per nucleus is shown. ****, P value < 0.0001. ( C – F ) Recruitment of GFP-tagged (C) NBS1, (D) EXO1, (E) XLF, and (F) XRCC4 to laser-generated DSBs is attenuated in the HCT116 DNA-PK cs KD/− cell line compared to the +/− cells. Relative fluorescent intensity of GFP-tagged NBS1, EXO1, XLF, and XRCC4 are presented as the mean ± SEM. *, P value < 0.05.

Article Snippet: Briefly, the HCT116 DNA-PK cs +/−, −/−, and KD/− cells were mock-treated or irradiated with 10 Gy and allowed to recover for 10 min. Next, the cells were harvested after trypsinization and processed with the Thermo Fisher Subcellular Protein Fractionation Kit according to the manufacturer's instructions.

Techniques: Activity Assay, Irradiation, Isolation, Western Blot, Generated

Journal: bioRxiv

Article Title: Integration of Patient-Derived Organoids and Organ-on-Chip Systems: Investigating Colorectal Cancer Invasion within the Mechanical and GABAergic Tumor Microenvironment

doi: 10.1101/2023.09.14.557797

Figure Lengend Snippet: (A.) Schematic representing the initial events of the metastatic cascade that can be measured using the CRC-OOC. Tumor cells in the top channel (1) can be visualized and analyzed separately from tumor cells that have invaded and adhered in the endothelial compartment (2). Additionally, tumor cells that are found in the endothelial effluent (3; circulating tumor cells (“CTC-like” cells)) can also be collected and analyzed. (B.) Circulating tumor cells were collected from the endothelial effluent of stretched and not stretched HCT116 CRC-Chips and RNAseq was performed. GO Pathway analysis was performed on a subset of genes with either a 2-fold difference between stretched and not stretched CTCs or an FDR-adjusted p-value <0.1. N=2 biological replicates with 3 pooled chips in each replicate. (C.) Gene expression of neurotransmitter-related genes were measured by a neurotransmitter-specific PCR array. HCT116 tumor cells of stretched and not stretched chips were harvested on day 6 from the top epithelial channel and isolated via FACs. Data is displayed as the gene expression fold change of stretch versus not stretch conditions Expression was normalized to the average of 5 housekeeping genes. Genes that had a 1.5-fold increase or decrease in the stretched condition are displayed. N=3 biological replicates with 3 chips pooled per biological replicate. (D.) Schematic of the production of GABA from glutamate by the enzyme GAD1. In this figure, experiments related to GABA are indicated in purple and experiments related to GAD1 are indicated in light blue. (E.) Representative confocal immunofluorescent images of the epithelial (top; 1) or endothelial (bottom; 2) channel of the CRC-Chips stained for GABA (purple) on day 6. Invaded HCT116 H2B-GFP stain positive for GABA, while HCT116 H2B-GFP tumor cells that are in the top channel stain weakly for GABA. DAPI stains the nuclei of Caco2 C2BBe1 cells in the top channel and endothelial cells in the bottom channel. Scale bars represent 200 μm in the top channel image and 100 μm in the bottom channel images. Top channel images are maximum projections that span a 35 μm Z-height with a 5 μm step size. Bottom channel images are maximum projections that span a 10 μm Z-height with a 5 μm step size. (F.) RNAseq analysis was performed on CRC organoids and normalized GAD1 expression is shown. N=5 independent donors with 2-3 replicates each. Individual data points are shown and mean ± SEM is displayed. Analysis between US and UP data was performed using an unpaired t-test; ***p<0.001 ( G.) CRC organoids were isolated from stretched chips and qPCR analysis of GAD1 gene expression was performed. N=5 independent doners with 3 replicates each. Individual data points are shown and mean ± SEM is displayed. Analysis between US and UP data was performed using an unpaired t-test p<0.05. ( H.) GAD1 mRNA expression from TCGA in KRAS, NRAS, or BRAF mutant primary colon cancer tumors. N=196 patients with KRAS, NRAS, or BRAF mutant tumors; N=201 patients with KRAS, NRAS, or BRAF wildtype tumors. Individual data points are shown and median with interquartile range is represented. Data was analyzed with an unpaired t-test; ****p<0.0001. ( I.) Kaplan-Meier curve with univariate analysis of the survival of patients with KRAS, NRAS, or BRAF mutated CRC tumors based on high versus low expression of GAD1 (defined as above or below the median GAD1 mRNA expression z-score of 0.3). Data was extracted from the TCGA. N=254 patients. Data was analyzed using a log-rank (Mantel-Cox test). ( J.) Effluent from the epithelial channel of the patient-derived organoids was collected on day 0 (D0) and day 6 (D6). GABA intensity was analyzed from extracted metabolites N=6 chips per timepoint per patient; n=4 on D0 and 2 on D6 for UK. Data was analyzed using a two-way ANOVA; ***p<0.001; ****p<0.0001. ( K .) US-H2B-GFP (top) and UP-H2B-GFP (bottom) stretched tumor-chips were stained for GABA (purple). Scale bars represent 200 μm. L. Representative 10x immunofluorescence images of the 5 tumors stained for EpCAM (green), CK20 (red), and GABA (purple). Scale bars represent 500 μm and 200 μm for UK. All schematics were made in or are from BioRender.

Article Snippet: The primary antibodies used for the HCT116 H2B-GFP ABAT knock down validation were mouse anti-ABAT (1:500; Santa Cruz Technology, Inc., #sc-393769) and mouse anti-Actin (1:10000; Millipore Sigma, #A5441).

Techniques: Gene Expression, Isolation, Expressing, Staining, Mutagenesis, Derivative Assay, Immunofluorescence

Journal: bioRxiv

Article Title: Integration of Patient-Derived Organoids and Organ-on-Chip Systems: Investigating Colorectal Cancer Invasion within the Mechanical and GABAergic Tumor Microenvironment

doi: 10.1101/2023.09.14.557797

Figure Lengend Snippet: (A.) Invasion of HCT116 tumor-chips in the presence or absence of exogenous GABA (flowed through the epithelial channel) was measured on day 6 (D6) of the experiment and normalized to day 0 (D0) invasion. N=6 chips. Individual data are shown, with mean ± SEM represented and analyzed using a one-way ANOVA; ****p<0.0001. (B.) Intracellular [ 13 C 4 ]GABA or unlabeled GABA was measured via mass spectrometry-based metabolomics in the HCT116 tumor-chips after the addition of exogenous GABA for six days. N=3 chips. (C.) Schematic of GABA catabolism by ABAT, subsequent entry into the TCA cycle, and inhibition of ABAT activity by vigabatrin. In this figure, experiments related to GABA are indicated as purple, and experiments related to ABAT are indicated as teal. (D.) Western blot analysis of ABAT in shRNA control or ABAT shRNA HCT116 tumor cells. Cropped western blot (left) and quantification (right) confirm knockdown of ABAT. (E.) Growth rate of ABAT-knockdown or control HCT116 tumor cells when grown in traditional cell culture methods. N=3. Individual data are shown and mean ± SEM are represented. Data was analyzed using a t-test; **p<0.01. (F.) Numbers of ABAT-knockdown or control HCT116 tumor cells in the top channel on-chip as measured via fluorescence microscopy and quantified on day 0 (D0) and day 6 (D6). N=5-6 chips. Individual data are shown and mean ± SEM are represented. Data was analyzed using a two-way ANOVA; *p<0.05;***p<0.001. (G.) Invasion of ABAT knockdown (KD) or control shRNA HCT116 tumor-chips in the presence or absence of stretching was measured on day 6 (D6) of the experiment and normalized to day 0 (D0) invasion. N=5-6 chips. Individual data points are shown and mean ± SEM are represented. Data was analyzed using a one-way ANOVA; *p<0.05. (H.) Numbers of HCT116 tumor cells in the top channel on-chip in the presence or absence of stretching, with or without vigabatrin was measured via fluorescence microscopy and quantified on day 0 (D0) and day 6 (Day 6). Individual data are shown and mean ± SEM are represented. N=4 chips. Data was analyzed using a two-way ANOVA; **p<0.01. (I.) Invasion of HCT116 tumor-chips in the presence or absence of stretching, with or without vigabatrin was measured on day 6 (D6) of the experiment and normalized to day 0 (D0) invasion. N=4 chips. Individual data points are shown and mean ± SEM are represented. Data was analyzed using a one-way ANOVA; ***p<0.001. (J.) Numbers of US-H2B-GFP (red) or UP-H2B-GFP (blue) tumor cells in the top channel on-chip in the presence or absence of stretching, with or without vigabatrin was measured via fluorescence microscopy and quantified on day 0 (D0) and day 6 (Day 6). N=4-5 chips. Individual data are shown and mean ± SEM are represented. Data was analyzed using a two-way ANOVA; ns=p>0.05. (K.) Invasion of US-H2B-GFP or UP-H2B-GFP organoid-tumor chips in the presence or absence of stretching, with or without vigabatrin was measured on day 6 (D6) of the experiment and normalized to day 0 (D0) invasion. N=4-5 chips. Individual data are shown and mean ± SEM are represented. Data was analyzed using a one-way ANOVA; **p<0.01. All schematics were made in or are from BioRender.

Article Snippet: The primary antibodies used for the HCT116 H2B-GFP ABAT knock down validation were mouse anti-ABAT (1:500; Santa Cruz Technology, Inc., #sc-393769) and mouse anti-Actin (1:10000; Millipore Sigma, #A5441).

Techniques: Mass Spectrometry, Inhibition, Activity Assay, Western Blot, shRNA, Control, Knockdown, Cell Culture, Fluorescence, Microscopy

Journal: Nature Immunology

Article Title: Enhancing human NK cell antitumor function by knocking out SMAD4 to counteract TGFβ and activin A suppression

doi: 10.1038/s41590-025-02103-z

Figure Lengend Snippet: a , Mean ± s.e.m. percentage of activated caspase-3-positive HCT116 cells after coculture with control NK and SMAD4 KO NK cells treated with IL-2 ± TGFβ1 at the indicated effector:target (E:T) ratios analyzed by flow cytometry. Spontaneous active caspase-3 levels were subtracted. Data are from four independent experiments. b , c , Amount of CCL5 ( b ) and IFNγ ( c ) in cell-free culture supernatants of control NK and SMAD4 KO NK cells after coculture with the HCT116 cell line by ELISA. Each dot shows data from an independent experiment ( n = 4). d – f , HCT116-GFP + -Luc + spheroids were cocultured with PKH26-labeled control NK or SMAD4 KO NK cells previously exposed or not to TGFβ1. Images were taken at 6 h and 24 h. d , Representative image of one spheroid in each coculture at 24 h. e , Mean ± s.e.m. GFP intensity along time in each coculture. Data are from two independent experiments including five technical replicates each. Only the significance by two-way ANOVA followed by Tukey’s multiple-comparisons test between TGFβ1-treated control and SMAD4 KO NK cells at 30 h is indicated. f , Mean ± s.e.m. luciferase activity of remaining HCT116 spheroids after 24 h of coculture with NK cells. Each dot represents the average cytotoxic activity of NK cells in four independent experiments. g , h , Tumor growth kinetics of HCC1954 xenografts in NSG mice treated with: (i) trastuzumab (Tz)/pertuzumab (Pt) ( n = 4); (ii) control NK cells (2 × 10 5 , n = 5); (iii) SMAD4 KO NK cells (2 × 10 5 , n = 5); (iv) control NK cells (1 × 10 5 ) and trastuzumab/pertuzumab ( n = 5); or (v) SMAD4 KO NK cells (1 × 10 5 ) and trastuzumab/pertuzumab ( n = 5). g , Treatment schedule. h , Tumor volume fold change in each treatment group. Only differences between control and SMAD4 KO NK cells at last measurement are indicated. i , Tumor growth kinetics of HCT116 xenografts in NSG mice treated with either control or SMAD4 KO NK cells (2 × 10 5 ). Tumor volume fold change in each treatment group ( n = 5 in NT group, n = 6 in control and SMAD4 KO NK cell groups). Statistical significance by two-way ANOVA followed by Tukey’s multiple-comparisons test at last measurement for a , e , h and i ; and one-way ANOVA followed by Tukey’s multiple-comparisons test for b , c , f and g . RLUs, relative light units.

Article Snippet: Production of CCL5, TNF and IFNγ was measured after 2 h coculture with HCT116 cells and analyzed by ELISA using DY278, DY210 (R&D Systems) and 88-7316-88 (Invitrogen), following the manufacturer’s instructions.

Techniques: Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Labeling, Luciferase, Activity Assay

Journal: Nature Immunology

Article Title: Enhancing human NK cell antitumor function by knocking out SMAD4 to counteract TGFβ and activin A suppression

doi: 10.1038/s41590-025-02103-z

Figure Lengend Snippet: a ) Mean ± SEM TNF in culture supernatants of control- and SMAD4 KO NK cells after 2 h coculture with HCT116 cells as analysed by ELISA. Statistical significance calculated by one-way ANOVA followed by Fisher’s test. b ) HCT116-Luc + -GFP+ spheroids were cocultured with control- or SMAD4 KO NK cells previously treated with IL-2 ± TGF-β. Mean ± SEM Luciferase activity of remaining HCT116 cells after 24 h coculture. Data from 3 independent experiments using NK cells from different donors. Each dot represents data from the 5 technical replicates included in each condition. Statistical significance calculated by one-way ANOVA followed by Turkey’s multiple comparisons test. c ) HCC1954 xenografts in NSG mice were treated with either: i) trastuzumab (Tz)/pertuzumab (Pt)(n = 4); ii) control NK cells (2 × 10 5 ,n = 5); iii) SMAD4 KO NK cells (2 × 10 5 ,n = 5); iv) control NK cells (1 × 10 5 ) and trastuzumab/pertuzumab (n = 5) or v) SMAD4 KO NK cells (1 × 10 5 ) and trastuzumab/pertuzumab (n = 5). Tumor volume in each treatment group. Statistical significance between control and SMAD4 KO NK cells treatments at the last measurment by two-way ANOVA followed by Turkey’s multiple comparisons test are indicated. d ) HCT116 xenografts in NSG mice were treated with either control or SMAD4 KO NK cells (2 × 10 5 ). Tumor volume in each treatment group (n = 5 in NT and n = 6 in NK cell treated groups). e ) HCT116-GFP + -Luc+ and HCC1954 GFP + -Luc+ spheroids were cultured with rIFNɣ, rIFN-β or rTNF. Mean ± SEM Luciferase activity of HCT116 nd HCC1954 spheroids after 24 h coculture. Statistical significance by one-way ANOVA followed by Turkey’s multiple comparisons test.

Article Snippet: Production of CCL5, TNF and IFNγ was measured after 2 h coculture with HCT116 cells and analyzed by ELISA using DY278, DY210 (R&D Systems) and 88-7316-88 (Invitrogen), following the manufacturer’s instructions.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Luciferase, Activity Assay, Cell Culture

Journal: Nature Immunology

Article Title: Enhancing human NK cell antitumor function by knocking out SMAD4 to counteract TGFβ and activin A suppression

doi: 10.1038/s41590-025-02103-z

Figure Lengend Snippet: a , Mean transcript levels of adhesion molecules by RNA-seq analysis. b , Mean ± s.e.m. percentage or intensity of surface expression levels of CD11a ( n = 4), CD18 ( n = 3), CD49d ( n = 5), CD103 ( n = 6) and CD29 ( n = 5) by flow cytometry in control and SMAD4 KO NK cells treated or not with TGFβ1. Each dot indicates the results from an independent experiment with NK cells from different individuals. c – e , HCT116 spheroids were cocultured with control NK or SMAD4 KO NK cells previously exposed or not to TGFβ1. After 1 h coculture, spheroids and attached NK cells were fixed and processed for light-sheet imaging. HCT116 cells were labeled with an anti-EpCAM-FITC antibody and NK cells with an anti-CD45-Vio R667. c , Image of a representative HCT116 spheroid (green surface) cocultured with SMAD4 KO NK cells (red dots). d , Number of NK cells counted in spheroids in the indicated conditions. e , Quantification of the distance between the spheroid surface and each infiltrating NK cell. Each dot represents the measurement of one infiltrating NK cell in one experiment. f , Mean ± s.e.m. fluorescence intensity of surface CXCR3 ( n = 9), CCR5 ( n = 6) and CXCR4 ( n = 4) in control and SMAD4 KO NK cells treated or not with TGFβ1 by flow cytometry. Each dot shows data from independent experiments. g , Transcript expression levels of chemokine receptors in control NK and SMAD4 KO NK cells exposed or not to TGFβ1, according to RNA-seq data from three independent donors. The dashed lines in a and g separate genes downregulated from those upregulated by TGFβ in control NK cells. h , Mean ± s.e.m. percentage of transmigrating control or SMAD4 KO NK cells treated with TGFβ1 to CCL5, CXCL9, SDF-1/CXCL12 or the indicated chemokine combinations. Data are from four independent experiments with NK cells from different donors. In all assays, statistical significance was calculated by one-way ANOVA followed by Fisher’s test.

Article Snippet: Production of CCL5, TNF and IFNγ was measured after 2 h coculture with HCT116 cells and analyzed by ELISA using DY278, DY210 (R&D Systems) and 88-7316-88 (Invitrogen), following the manufacturer’s instructions.

Techniques: RNA Sequencing, Expressing, Flow Cytometry, Control, Imaging, Labeling, Fluorescence

Journal: Nature Immunology

Article Title: Enhancing human NK cell antitumor function by knocking out SMAD4 to counteract TGFβ and activin A suppression

doi: 10.1038/s41590-025-02103-z

Figure Lengend Snippet: a) Surface expression of LFA-1 (CD11a and CD18), VLA-4 (CD49d and CD29) and CD103 in control and SMAD4 KO NK cells treated with IL-2 ± TGF-β as analysed by flow cytometry. Representative histograms of each marker staining in NK cells from a representative individual. b ) HCT116 spheroids were cocultured with control- or SMAD4 KO NK cells previously treated with IL2 ± TGF-β. After 1 h coculture, spheroids and attached NK cells were fixed and processed for lightsheet imaging. HCT116 cells were labeled with an anti-Epcam-FITC antibody and NK cells with an anti-CD45-VioR667 antibody. Image of a representative HCT116 spheroid for the indicated conditions. Inset numbers correspond to the number of NK cells counted in each spheroid.

Article Snippet: Production of CCL5, TNF and IFNγ was measured after 2 h coculture with HCT116 cells and analyzed by ELISA using DY278, DY210 (R&D Systems) and 88-7316-88 (Invitrogen), following the manufacturer’s instructions.

Techniques: Expressing, Control, Flow Cytometry, Marker, Staining, Imaging, Labeling

Journal: Nature Immunology

Article Title: Enhancing human NK cell antitumor function by knocking out SMAD4 to counteract TGFβ and activin A suppression

doi: 10.1038/s41590-025-02103-z

Figure Lengend Snippet: a – d , SMAD4 KO and control NK cells were expanded in the presence of TGFβ1 for 7 days. Control NK cells were treated with the TGFBR-I inhibitor SB-431542 (SB inhib). a , b , SMAD4, pSMAD2 and β-actin levels in total cell extracts by western blot in SMAD4 KO and control NK cells. a , Representative western blots including NK cells from two different donors. b , Quantification of mean ± s.e.m. levels of SMAD4/β-actin and pSMAD2/β-actin ratios in NK cells from three different donors. Statistical significance was calculated by a two-tailed, unpaired Student’s t -test. c , Representative histograms showing the expression of GzmB, NKG2D and CD103 in the indicated NK cells by flow cytometry. d , e , SMAD4 KO and control NK cells treated with SB-431542 were cocultured with HCT116-GFP + -Luc + spheroids. Luciferase counts were analyzed at 24 h of coculture. d , Mean ± s.e.m. luciferase counts from one representative experiment. Dots show data from five technical replicates. e , Mean ± s.e.m. luciferase counts from three independent experiments using NK cells from different individuals. Each dot indicates the mean of five technical replicates for each condition/experiment. Statistical significance was calculated by one-way ANOVA followed by Tukey’s multiple-comparisons test for d and e .

Article Snippet: Production of CCL5, TNF and IFNγ was measured after 2 h coculture with HCT116 cells and analyzed by ELISA using DY278, DY210 (R&D Systems) and 88-7316-88 (Invitrogen), following the manufacturer’s instructions.

Techniques: Control, Inhibition, Western Blot, Two Tailed Test, Expressing, Flow Cytometry, Luciferase

Journal: Nature Immunology

Article Title: Enhancing human NK cell antitumor function by knocking out SMAD4 to counteract TGFβ and activin A suppression

doi: 10.1038/s41590-025-02103-z

Figure Lengend Snippet: a , Relative transcript expression of activin receptor genes in the indicated NK cells. Average expression in NK cells from three independent individuals as analyzed by RNA-seq. b – d , SMAD4 KO and control NK cells were incubated with TGFβ1 or activin A for 7 days. b , Expression of GzmB at day 7 by flow cytometry. c , Mean ± s.e.m. granzyme B levels in SMAD4 KO and control NK cells. Data are from experiments with NK cells from eight different individuals. d , SMAD4 KO and control NK cells previously incubated with TGFβ1 or activin A (Activ) were cocultured with HCT116 cells for 2 h. Mean ± s.e.m. percentage of active caspase-3 + (aCasp3) HCT116 cells in the indicated conditions. Data are from five independent experiments with NK cells from different individuals. Statistical significance was calculated by one-way ANOVA followed by Fisher’s test.

Article Snippet: Production of CCL5, TNF and IFNγ was measured after 2 h coculture with HCT116 cells and analyzed by ELISA using DY278, DY210 (R&D Systems) and 88-7316-88 (Invitrogen), following the manufacturer’s instructions.

Techniques: Expressing, RNA Sequencing, Control, Incubation, Flow Cytometry

Journal: Nature Immunology

Article Title: Enhancing human NK cell antitumor function by knocking out SMAD4 to counteract TGFβ and activin A suppression

doi: 10.1038/s41590-025-02103-z

Figure Lengend Snippet: a – c , Control and SMAD4 KO NK cells transduced with anti-CD19-CAR lentivirus were cultured with IL-2 ± TGFβ1 for 6 days. a , Mean ± s.e.m. percentage of control or SMAD4 KO NK cells transduced with the anti-CD19-CAR by flow cytometry. Data are from three independent experiments. b , c , CAR19-transuced control or SMAD4 KO NK cells treated with IL-2 ± TGFβ1 were cocultured with Nalm6-GFP + -Luc + for 3 h at a 4:1 E:T ratio. b , Mean ± s.e.m. luciferase counts at the end of the coculture in the indicated conditions. Dots show data from technical replicates ( n = 4) in a representative experiment. c , Mean ± s.e.m. percentage of Nalm6 cell killing in the indicated conditions. Dots show data of independent experiments with CD19-CAR NK cells from different donors ( n = 4). d – f , Control and SMAD4 KO GTA002 cells were cultured in IL-2 ± TGFβ1 for 5 days. Data are from four independent experiments. GTA002 cells from different cord-blood units are presented as independent dots. d , SMAD4 MFI in control and SMAD4 KO GTA002 cells at day 5. e , Mean ± s.e.m. percentage of NKG2D + control or SMAD4 KO GTA002 cells in the indicated conditions. f , Mean luciferase activity remaining after 24 h coculture of HCT116-GFP + -Luc + spheroids with control or SMAD4 KO GTA002 cells in the indicated conditions. Sph, spheroid. g – i , Control or SMAD4 KO ADAPT-NK cells were cultured for 5 days with IL-2 ± TGFβ1. Degranulation and IFNγ production were analyzed by coculturing control or SMAD4 KO ADAPT-NK cells with K562 cells at a 1:1 E:T ratio for 4 h. Dots represent data from independent experiments using ADAPT-NK cells from different donors. g , SMAD4 MFI in control and SMAD4 KO ADAPT-NK cells at day 5. h , i , Mean ± s.e.m. percentage of CD107 + ( h ) and IFNγ + ( i ) control and SMAD4 KO ADAPT-NK cells in the indicated conditions. Basal degranulation and IFNγ production in the absence of target were subtracted. Statistical significance was calculated by a two-tailed, paired Student’s t -test in d and g , and by one-way ANOVA followed by Fisher’s test in b , c , e , f , h and i .

Article Snippet: Production of CCL5, TNF and IFNγ was measured after 2 h coculture with HCT116 cells and analyzed by ELISA using DY278, DY210 (R&D Systems) and 88-7316-88 (Invitrogen), following the manufacturer’s instructions.

Techniques: Control, Transduction, Cell Culture, Flow Cytometry, Luciferase, Activity Assay, Two Tailed Test

Journal: Journal of the American Chemical Society

Article Title: Hijacking Methyl Reader Proteins for Nuclear-Specific Protein Degradation

doi: 10.1021/jacs.2c00874

Figure Lengend Snippet: A) Structure of the L3MBTL3 antagonist, UNC1215. B) Structure of biotin-UNC1215. C) UNC1215 does not disrupt the L3MBTL3-Cul4DCAF5 interaction. HEK293T (293T) cells were transiently transfected with L3MBTL3-HA and myc-DCAF5, lysed after 48 h and incubated in the presence or absence of 20 μM UNC1215 followed by biotin probe (200 nM) pulldown (n = 2). D) L3MBTL3 interacts with DCAF5 and DDB1 in HEK293T cells and E) HCT116 cells. HEK293T or HCT116 cells were co-transfected with L3MBTL3-HA and myc-DCAF5, lysed after 48 h and incubated in the presence or absence of 5 μM UNC1215 followed by immunoprecipitation of L3MBTL3-HA (n = 2).

Article Snippet: Cell lines HEK293T cells, THP1 and HCT116 cells were purchased from ATCC.

Techniques: Transfection, Incubation, Immunoprecipitation

Journal: Journal of the American Chemical Society

Article Title: Hijacking Methyl Reader Proteins for Nuclear-Specific Protein Degradation

doi: 10.1021/jacs.2c00874

Figure Lengend Snippet: A) Schematic of BRD and L3MBTL3 proteins in complex with the JQ1-UNC1215 PROTAC. Figure was created with BioRender.com Structure of KL-7. B) KL-7 degrades BRD2 in a dose-dependent manner in HCT116 cells. HCT116 cells were treated with increasing concentrations of KL-7 for 12 h. Quantitation is on the right and the quantified data represent mean ± SEM (n = 3, ****P<0.0001). C) BRD2 forms a ternary complex with L3MBTL3 in a KL-7 dependent manner. HEK293T cells were co-transfected with L3MBTL3-HA and GFP-BRD2-V5 for 48 h and lysates were incubated with 5 μM KL-7 for 2 h, followed by V5 immunoprecipitation (n = 2). D) Excess JQ1 ligand blocks KL-7 induced BRD2 degradation. HCT116 cells were pre-treated 100 μM JQ1 for 1.5 h, and then co-treated with 5 μM KL-7 for 12 h. Quantitation is below and the quantified data represent mean ± SEM (n = 2, **P<0.005). E) KL-7 induced BRD2 degradation is L3MBTL3-dependent. L3MBTL3 wildtype (WT) and Knockout (KO) 293T cells were treated with 5 μM KL-7 for 8 h. Quantitation is below and the quantified data represent mean ± SEM (n = 2, ****P<0.0001). F) KL-7 induced BRD2 degradation is neddylation and proteasome-dependent. HCT116 cells were pre-treated with 1 μM MLN4924 and 0.5 μM epoxomicin for 1.5 h, and then co-treated with 5 μM KL-7 for 12 h. Quantitation is on the right and the quantified data represent mean ± SEM (n = 2, **P<0.005).

Article Snippet: Cell lines HEK293T cells, THP1 and HCT116 cells were purchased from ATCC.

Techniques: Quantitation Assay, Transfection, Incubation, Immunoprecipitation, Knock-Out