NOLC1 suppresses immunochemotherapy by inhibiting p53-mediated ferroptosis in gastric cancer
Figures

NOLC1 was upregulated in gastric cancer (GC) and a cisplatin (Cis)-resistant GC cell line.
(A, B) Immunohistochemistry (IHC) results of NOLC1 expression in gastric tumor (T) tissues and near-tumor (NT) tissues. (A) Representative images (n=3); scale bar = 50 μm. (B) Average optical density (AOD) of NOLC1 according to the IHC images (n=9). (C, D) Western blotting (WB) results of NOLC1 protein levels in gastric tumor (T) tissues and NT tissues. (C) Representative images. (D) Relative expression of NOLC1 (n=3). (E, F) Gene expression level of NOLC1 in TCGA GC tumor and matched TCGA normal stomach tissues. (E) Unpaired sample; (F) paired sample. (G–I) High NOLC1 expression predicts poor survival in GC patients. (G) Overall survival (OS); patients were divided into a NOLC1-high group (n=349) and a NOLC1-low group (n=526). (H) Time to first progression (FP); patients were divided into a NOLC1-high group (n=202) and a NOLC1-low group (n=438). (I) Post-progression survival (PPS); patients were divided into a NOLC1-high group (n=127) and a NOLC1-low group (n=371). (J) WB results of NOLC1, PARP, and cleaved PARP in MGC-803 and MGC-803-CR cell lines. (K) Immunofluorescence (IF) images of NOLC1 in MGC-803 and MGC-803-CR cell lines; scale bar = 15 μm. The data are presented as the means ± SDs. **p<0.01; ***p<0.001.
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Figure 1—source data 1
Original western blots for Figure 1C and J, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/103904/elife-103904-fig1-data1-v1.zip
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Figure 1—source data 2
Original files for western blot displayed in Figure 1C and J.
- https://cdn.elifesciences.org/articles/103904/elife-103904-fig1-data2-v1.zip

NOLC1 was highly expressed in gastric cancer (GC) tissues.
(A) Immunohistochemistry (IHC) results of NOLC1 expression in gastric tumor (T) tissues and near-tumor (NT) tissues; scale bar = 50 µm.

Cisplatin (Cis)-resistant gastric cancer (GC) cell line was successfully constructed.
(A) CCK-8 assay of MGC-803 and MGC-803-CR cells treated with different concentrations of Cis (n=3). (B, C) Colony formation assay of MGC-803 and MGC-803-CR cells treated with phosphate-buffered saline (PBS) or Cis (15 µM). (B) Representative images and (C) number of clones (n=3). (D, E) Annexin V-APC and 7-AAD staining of MGC-803 and MGC-803-CR cells treated with PBS or Cis (20 µM), as determined via fluorescence-activated cell sorting (FACS) (n=3). The data are presented as the means ± SDs. ns, nonsignificant; *p<0.05; **p<0.01; ***p<0.001.

NOLC1 was highly expressed in CR cells.
(A, B) Relative protein expression levels of (A) NOLC1 and (B) cleaved PARP (n=3). (C) Mean fluorescence intensity of NOLC1 (n=3). (D) Heatmap of mRNA-seq data from MGC-803 and MGC-803-CR cells. (E) KEGG analysis of the mRNA-seq data of MGC-803 and MGC-803-CR cells. The data are presented as the means ± SDs. ***p<0.001.

NOLC1 promoted cisplatin (Cis) resistance in gastric cancer (GC).
(A) CCK-8 assay of GC cells transduced with shNC or shNOLC1 lentivirus and treated with different concentrations of Cis (n=6). (B, C) Colony formation assay of GC cells transduced with shNC or shNOLC1 lentivirus and treated with phosphate-buffered saline (PBS) or Cis (15 μM). (B) Representative images of the colony formation assay. (C) Quantitative analysis of colony formations assay (n=3). (D, E) Annexin V-APC and 7-AAD staining of GC cells transduced with shNC or shNOLC1 lentivirus and treated with the indicated concentrations of Cis, as analyzed via fluorescence-activated cell sorting (FACS); (D) MGC-803 cells and (E) MKN-45 cells. (F, G) Immunofluorescence staining assays of γ-H2AX in GC cells transduced with shNC or shNOLC1 lentivirus and treated with the indicated concentrations of Cis; (F) MGC-803 cells; scale bar = 10 μm; (G) MKN-45 cells; scale bar = 5 μm. (H) Comet assay of MGC-803 cells transduced with shNC or shNOLC1 lentivirus and treated with PBS or Cis (15 μM). (I) Western blotting (WB) results of cleaved PARP, PARP, cleaved caspase-3, and caspase-3 in GC cells transduced with shNC or shNOLC1 lentivirus and treated with indicated concentrations of Cis. The data are presented as the means ± SDs. ns, nonsignificant; *p<0.05; **p<0.01; ***p<0.001.
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Figure 2—source data 1
Original western blots for Figure 2I, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/103904/elife-103904-fig2-data1-v1.pdf
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Figure 2—source data 2
Original files for western blot displayed in Figure 2I.
- https://cdn.elifesciences.org/articles/103904/elife-103904-fig2-data2-v1.zip

Knockdown or overexpression of NOLC1 in gastric cancer (GC) cell lines.
(A, B) Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay of NOLC1 mRNA levels in (A) MGC-803 cells and (B) MKN-45 cells (n=3). (C, D) Immunoblotting assay of NOLC1 in GC cells transduced with shNC or shNOLC1 lentivirus. (E) Immunoblotting assay of NOLC1 in MGC-803 cells transduced with vector or NOLC1 plasmid. The data are presented as the means ± SDs. ns, nonsignificant; ***p<0.001.
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Figure 2—figure supplement 1—source data 1
Original western blots for Figure 2—figure supplement 1C-E, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/103904/elife-103904-fig2-figsupp1-data1-v1.pdf
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Figure 2—figure supplement 1—source data 2
Original files for western blot displayed in Figure 2—figure supplement 1C-E.
- https://cdn.elifesciences.org/articles/103904/elife-103904-fig2-figsupp1-data2-v1.zip

NOLC1 promoted cisplatin (Cis) resistance in gastric cancer (GC) cells.
(A) Viability of MGC-803 cells transduced with vector or NOLC1 plasmid and treated with the indicated concentrations of Cis (n=3). (B, C) Relative cleaved caspase-3 expression level in GC cells transduced with shNC or shNOLC1 lentivirus and treated with indicated concentrations of Cis (n=3); (B) MGC-803 cells and (C) MKN-45 cells. The data are presented as the means ± SDs. ns, nonsignificant; *p<0.05; **p<0.01; ***p<0.001.

NOLC1 promoted cisplatin (Cis) resistance in vivo.
(A) Schematic representation showing the treatment of the subcutaneous xenograft tumor models. (B) Photographs of the corresponding tumors after indicated treatments. (C) Relative tumor volume after different treatments. (D, E) Tumor growth curves after indicated treatments. (F) Hematoxylin and eosin (H&E) staining and immunohistochemical (IHC) analysis of cleaved caspase-3 and Ki-67; scale bar = 50 μm. (G) Quantification analysis of the necrotic area and cleaved caspase-3 and Ki-67 expression in tumor tissue from mice given the indicated treatments (n=3). The data are presented as the means ± SDs. ns, nonsignificant; *p<0.05; **p<0.01; ***p<0.001.

NOLC1 silence rendered gastric cancer (GC) susceptible to ferroptosis.
(A) Transmission electron microscopy (TEM) images of MGC-803 cells transduced with shNC or shNOLC1 lentivirus and treated with phosphate-buffered saline (PBS) or cisplatin (Cis) (15 µM); scale bar = 400 nm. (B) Lactate dehydrogenase (LDH) release analysis of MGC-803 cells transduced with shNC or shNOLC1 lentivirus and treated with indicated concentrations of Cis (n=5). (C) Representative JC-1 fluorescence images of MGC-803 cells transduced with shNC or shNOLC1 lentivirus and treated with indicated concentrations of Cis; scale bar = 20 µm. (D) Fluorescence intensity of 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) measured by fluorescence-activated cell sorting (FACS) in GC cells transduced with shNC or shNOLC1 lentivirus and treated with indicated concentrations of Cis. (E) Fluorescence intensity of MitoPeDPP measured by FACS in GC cells transduced with shNC or shNOLC1 lentivirus and treated with Cis (15 µM) for different times. (F) Representative MitoPeDPP fluorescence images of MGC-803 cells transduced with shNC or shNOLC1 lentivirus and treated with indicated concentrations of Cis; scale bar = 10 µm. (G) Relative H2O2 content of GC cells transduced with shNC or shNOLC1 lentivirus and treated with indicated concentrations of Cis (n=3). (H) Western blotting (WB) result of ACSL4, FSP1, and GPX4 protein levels in GC cells transduced with shNC lentivirus or shNOLC1 lentivirus and treated with different concentrations of Cis. The data are presented as the means ± SDs. ns, nonsignificant; *p<0.05; **p<0.01; ***p<0.001.
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Figure 4—source data 1
Original western blots for Figure 4H, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/103904/elife-103904-fig4-data1-v1.pdf
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Figure 4—source data 2
Original files for western blot displayed in Figure 4H.
- https://cdn.elifesciences.org/articles/103904/elife-103904-fig4-data2-v1.zip

NOLC1 silence rendered gastric cancer (GC) cells susceptible to ferroptosis.
(A) Transmission electron microscopy (TEM) image of MGC-803 cells transduced with shNC or shNOLC1 lentivirus and treated with phosphate-buffered saline (PBS) or cisplatin (Cis) (15 µM); scale bar = 400 nm. (B) Viability of MGC-803 cells transduced with shNC or shNOLC1 lentivirus and treated with the indicated concentrations of erastin (n=3). (C) Viability of MGC-803 cells transduced with shNC or shNOLC1 lentivirus and treated with the Cis (0 or 10 µM) and indicated concentrations of Fer-1 (n=5). (D) Lactate dehydrogenase (LDH) release analysis of MKN-45 cells transduced with shNC or shNOLC1 lentivirus and treated with different concentrations of Cis (n=5). (E) LDH release analysis of MGC-803 cells transduced with shNC or shNOLC1 lentivirus and treated with different concentrations of Fer-1 (n=3). (F) JC-1 fluorescence staining analysis of GC cells transduced with shNC lentivirus or shNOLC1 lentivirus and treated with indicated concentrations of Cis analyzed via fluorescence-activated cell sorting (FACS). (G) The ratio of red to green fluorescence of JC-1 in GC cells (n=3). (H) Representative 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence images of MGC-803 cells transduced with shNC or shNOLC1 lentivirus and treated with indicated concentrations of Cis; scale bar = 75 µm. (I) Relative mean fluorescence intensity (MFI) of DCFH-DA in GC cells transduced with shNC or shNOLC1 lentivirus and treated with indicated concentrations of Cis (n=3). (J) Relative MFI of MitoPeDPP in GC cells transduced with shNC or shNOLC1 lentivirus and treated with Cis (15 µM) for different times (n=3). The data are presented as the means ± SDs. ns, nonsignificant; *p<0.05; **p<0.01; ***p<0.001.

NOLC1 silence rendered gastric cancer (GC) cells susceptible to ferroptosis.
(A) Immunohistochemical (IHC) staining analysis of GPX4 in MGC-803 tumor tissues after the indicated treatments; scale bar = 50 µm. (B) Relative protein expression of GPX4 in GC cells transduced with shNC or shNOLC1 lentivirus and treated with different concentrations of cisplatin (Cis) (n=3). The data are presented as the means ± SDs. *p<0.05; **p<0.01; ***p<0.001.

NOLC1 decreased p53 transcriptional activity.
(A) Western blotting (WB) analysis of p53, BCL-2, and BAX in MGC-803 cells transduced with shNC or shNOLC1 lentivirus and treated with indicated concentrations of cisplatin (Cis). (B, C) Luciferase assay of p53 transcription activity (B) in MGC-803 cells transduced with shNC or shNOLC1 lentivirus and treated with phosphate-buffered saline (PBS) or Cis (15 µM) and (C) in HEK-293T cells transfected with 0.00, 0.25, or 0.50 µg NOLC1 plasmid and treated with PBS or Cis (15 µM). (D‒F) Docking analysis of NOLC1 and p53; (D) The backbones of the proteins were shown in tubes and colored red (NOLC1) and cyan (p53); (E) The NOLC1 and p53 proteins were shown on the surface; (F) The detailed binding mode of NOLC1 with p53. The yellow dash represented the hydrogen bond. (G) Coimmunoprecipitation (Co-IP) assay of NOLC1-FLAG with p53-HA. (H) Representative NOLC1-Flag and p53-HA fluorescence images of HEK-293T transfected with indicated amount of NOLC1-Flag and p53-HA plasmids and treated with PBS or Cis (15 µM); scale bar = 10 µm. (I) WB analysis of p53 protein levels in the cytoplasm or nucleus of MGC-803 cells transduced with shNC or shNOLC1 lentivirus and treated with PBS or Cis (15 µM). (J) Schematic map of the p53 functional domain. (K) Co-IP assay of NOLC1 with different p53 functional domains. (L) Co-IP assay of p53 DNA-binding domain (DBD)-HA with NOLC1-Flag. (M) Schematic map of the chromatin immunoprecipitation (ChIP-seq) analysis. (N) GEO analysis of the unique p53 peak identified via ChIP-seq. The data are presented as the means ± SDs. ns, nonsignificant; **p<0.01; ***p<0.001.
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Figure 5—source data 1
Original western blots for Figure 5A, G, I, K, and L, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/103904/elife-103904-fig5-data1-v1.zip
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Figure 5—source data 2
Original files for western blot displayed in Figure 5A, G, I, K, and L.
- https://cdn.elifesciences.org/articles/103904/elife-103904-fig5-data2-v1.zip

NOLC1 inhibited p53 transcriptional activity.
(A, B) Relative protein expression levels of (A) p53 and (B) BAX in MGC-803 cells transduced with shNC or shNOLC1 lentivirus and treated with different concentrations of cisplatin (Cis) (n=3). (C) Immunohistochemical (IHC) staining of p53 in MGC-803 tumor tissues after different treatments; scale bar = 50 µm. (D) Luciferase assay of WT p53 reporter transcription activity in MKN-45 cells transduced with shNC or shNOLC1 lentivirus and treated with phosphate-buffered saline (PBS) or Cis (15 µM) (n=3). (E) Luciferase assay of negative control (NC) reporter transcription activity in HEK-293T cells transduced with shNC or shNOLC1 lentivirus and treated with PBS or Cis (15 µM) (n=3). (F) Luciferase assay of mut p53 reporter transcription activity in HEK-293T cells transduced with shNC or shNOLC1 lentivirus and treated with PBS or Cis (15 µM) (n=3). (G, H) Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis of CDKN1A, BAX, FAS, and PTEN mRNA levels (G) in MGC-803 cells transduced with shNC or shNOLC1 lentivirus and treated with Cis (0 or 15 µM) (n=3) and (H) in MGC-803 tumor tissues after the indicated treatment (n=3). The data are presented as the means ± SDs. ns, nonsignificant; *p<0.05; **p<0.01; ***p<0.001.

NOLC1 combined with p53 and inhibited p53 nuclear accumulation.
(A) Representative NOLC1 and p53 fluorescence images of HEK-293T and MGC-803 cells transfected with NOLC1-Flag and p53-HA plasmids; scale bar = 5 µm or 10 µm, respectively. (B) The nuclear/cytoplasm ratio of p53 in HEK-293T cells transfected with different amounts of NOLC1 plasmid. (C) Representative immunofluorescence images of p53 and NOLC1-stained HEK-293T cells transduced with shNC or shNOLC1 lentivirus and treated with phosphate-buffered saline (PBS) or cisplatin (Cis) (15 µM); scale bar = 10 µm. The data are presented as the means ± SDs. *p<0.05; ***p<0.001.

NOLC1 inhibited p53 ubiquitination and degradation.
(A) Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis of TP53 mRNA in MGC-803 cells transduced with shNC or shNOLC1 lentivirus and treated with phosphate-buffered saline (PBS) or cisplatin (Cis) (15 µM) (n=3). (B) RT-qPCR analysis of MDM2 mRNA in MGC-803 cells transduced with shNC or shNOLC1 lentivirus and treated with PBS or Cis (15 µM) (n=3). (C, D) Western blotting (WB) assay of MDM2 in MGC-803 cells transduced with shNC or shNOLC1 lentivirus and treated with PBS or Cis (15 µM); (C) Representative images and (D) relative MDM2 protein levels (n=3). (E, F) Immunohistochemical (IHC) staining of MDM2 in MGC-803 tumors from BALB/c-nu mice; (E) Representative images; scale bar = 50 µm. (F) MDM2-positive area in MGC-803 tumors (n=3). (G) p53 turnover rate analysis in HEK-293T cells transfected with vector or NOLC1 plasmids and treated with CHX (40 µM) for the indicated times (n=3). (H) Ubiquitination assay in MGC-803 and HEK-293T cells transfected with the indicated plasmids. The data are presented as the means ± SDs. ns, nonsignificant; *p<0.05; **p<0.01; ***p<0.001.
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Figure 6—source data 1
Original western blots for Figure 6C, G, and H, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/103904/elife-103904-fig6-data1-v1.pdf
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Figure 6—source data 2
Original files for western blot displayed in Figure 6C, G, and H.
- https://cdn.elifesciences.org/articles/103904/elife-103904-fig6-data2-v1.zip

NOLC1 promoted cisplatin (Cis) resistance by blocking p53 function in gastric cancer (GC).
(A, B) Annexin V-APC and 7-AAD staining of GC cells transduced with shNC or shNOLC1 lentivirus and transfected with si-NC or si-p53 siRNA and treated with phosphate-buffered saline (PBS) or Cis (15 µM), as analyzed via fluorescence-activated cell sorting (FACS). (C) Viability of MGC-803 cells transduced with shNC or shNOLC1 lentivirus and transfected with si-NC or si-p53 siRNA and treated with different concentrations of Cis (n=3). (D) Immunoblotting analysis of GPX4 protein levels in MGC-803 cells transduced with shNC or shNOLC1 lentivirus and transfected with si-NC or si-p53 siRNA and treated with 0 or 10 µM Cis. The data are presented as the means ± SDs. ns, nonsignificant; *p<0.05; **p<0.01.
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Figure 6—figure supplement 1—source data 1
Original western blots for Figure 6—figure supplement 1D, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/103904/elife-103904-fig6-figsupp1-data1-v1.pdf
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Figure 6—figure supplement 1—source data 2
Original files for western blot displayed in Figure 6—figure supplement 1D.
- https://cdn.elifesciences.org/articles/103904/elife-103904-fig6-figsupp1-data2-v1.zip

NOLC1 knockdown increased the efficacy of cisplatin (Cis) combined with anti-programmed cell death-1 (anti-PD-1) treatment.
(A) Schematic representation showing the treatment of the subcutaneous tumors. (B) Corresponding tumor photographs after different treatments (n=5). (C) Tumor growth curves after different treatments (n=5). (D) Tumor weight after the indicated treatment (n=5). (E) Representative images of Liperfluo staining and immunohistochemical (IHC) analysis of cleaved caspase-3, Ki-67, GPX4, and p53 in tumor tissues; IHC analysis scale bar = 25 μm, Liperfluo staining scale bar = 50 μm. The data are presented as the means ± SDs. ns, nonsignificant; *p<0.05; **p<0.01.

NOLC1 knockdown increased the release of damage-associated molecular patterns (DAMPs) induced by ferroptosis.
(A, B) HMGB1 immunofluorescence staining of MGC-803 cells transduced with shNC or shNOLC1 lentivirus and treated with different concentrations of cisplatin (Cis). (A) Representative images; scale bar = 10 µm. (B) Mean fluorescence intensity of HMGB1 (n=3). (C, D) CRT immunofluorescence staining of MGC-803 cells transduced with shNC or shNOLC1 lentivirus and treated with different concentrations of Cis. (C) Representative images; scale bar = 10 µm. (D) Mean fluorescence intensity of CRT (n=3). The data are presented as the means ± SDs. ns, nonsignificant; *p<0.05; **p<0.01; ***p<0.001.

NOLC1 knockdown increased the combination treatment efficacy of anti-programmed cell death-1 (anti-PD-1) plus cisplatin (Cis).
(A) Growth curves of MFC tumors transduced with shNC or shNOLC1 lentivirus and subjected to different treatments. (B) Hematoxylin and eosin (H&E) staining of MFC tumors transduced with shNC or shNOLC1 lentivirus after the indicated treatments. (C) Quantification of the necrotic area in the tumor tissue of the mice given the different treatment (n=3). (D–G) Quantification of protein expression in tumor tissue from mice given the indicated treatment (n=3); (D) Cleaved caspase-3, (E) Ki-67, (F) GPX4, and (G) p53 nuclear positive ratio. (H) The serum levels of HMGB1 (n=5). The data are presented as the means ± SDs. ns, nonsignificant; *p<0.05; **p<0.01; ***p<0.001.

Knockdown of NOLC1 reprogrammed the tumor microenvironment after cisplatin (Cis) and anti-programmed cell death-1 (anti-PD-1) combination therapy.
(A) Fluorescence-activated cell sorting (FACS) analysis of peripheral blood lymphocytes (n=3). (B) Representative immunofluorescence staining of CD-3 and CD-8 in MFC tumors after indicated treatments; scale bar = 50 µm. (C) The serum levels of inflammatory factors (interleukin 6 [IL-6], interferon-γ [IFN-γ], and tumor necrosis factor-α [TNF-α]) (n=5). (D) Schematic illustration of the mechanism by which NOLC1 inhibits p53-mediated ferroptosis to increase the efficacy of anti-PD-1 plus Cis in gastric cancer (GC). The data are presented as the means ± SDs. ns, nonsignificant; *p<0.05; **p<0.01.

Gating strategy of flow cytometry for peripheral blood lymphocyte analysis.

Biosafety of anti-programmed cell death-1 (anti-PD-1) and cisplatin (Cis) combination therapy.
(A) Biochemical analysis of the serum of mice subjected to various treatments (n=5). (B) Hematoxylin and eosin (H&E) staining of mouse heart, liver, spleen, lung, and kidney samples from different treatment groups. Scale bar = 100 μm. The data are presented as the means ± SDs. ns, nonsignificant.
Tables
The docking results of the two target proteins – NOLC1 and p53.
Protein 1 | Protein 2 | Binding energy (kcal/mol) | Contact sites (protein 1) | Contact sites (protein 2) | Combination type |
---|---|---|---|---|---|
NOLC1 | TP53 | –236.13 | SER-60, SER-12, LYS-59, TYR-15, ASP-52, ARG-23, PRO-11, PHE-56 | GLN-104, ARG-283, ASN-131, SER-269, TYR-126, HIS-115, SER-116, LEU-289, TRP-146 | Hydrogen bond |
Hydrophobic interaction |
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/103904/elife-103904-mdarchecklist1-v1.docx
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Source data 1
ChIP results in HKE293T cells tranduced with p53-HA plasmid.
- https://cdn.elifesciences.org/articles/103904/elife-103904-data1-v1.xlsx
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Source data 2
ChIP results in HKE293T cells tranduced with p53-HA and NOLC1-Flag plasmid.
- https://cdn.elifesciences.org/articles/103904/elife-103904-data2-v1.xlsx