NOLC1 was up-regulated in GC and a 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 density (AOD) of NOLC1 according to the IHC images.

C, D Western blotting (WB) results of NOLC1 protein levels in gastric tumor (T) tissues and near-tumor (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) Postprogression 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 IF images of NOLC1 in MGC-803 and MGC-803-CR cell lines; scale bar = 15 μm.

**p < 0.01; ***p < 0.001.

NOLC1 promoted Cis resistance in 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 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 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 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. ns, nonsignificant; *p < 0.05; **p < 0.01; ***p < 0.001.

NOLC1 promoted 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 H&E staining and 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). ns, nonsignificant; *p < 0.05; **p < 0.01; ***p < 0.001.

NOLC1 deletion rendered GC susceptible to ferroptosis.

A TEM image of MGC-803 cells transduced with shNC or shNOLC1 lentivirus and treated with PBS or Cis (15 µM); scale bar = 400 nm.

B 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 DCFHA-DA measured by 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 time.

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 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. ns, nonsignificant; *p < 0.05; **p < 0.01; ***p < 0.001.

NOLC1 decreased p53 transcriptional activity.

A WB analysis of p53 and BAX in MGC-803 cells transduced with shNC or shNOLC1 lentivirus and treated with indicated concentrations of Cis.

B, C Luciferase assay of p53 transcription activity (B) in MGC-803 cells transduced with shNC or shNOLC1 lentivirus and treated with 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 Co-IP assay of NOLC1 with p53.

H 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.

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 Schematic map of the ChIP-seq analysis.

M GEO analysis of the unique p53 peak identified via ChIP-seq. ns, nonsignificant;

**p < 0.01; ***p < 0.001.

Docking results of the two target proteins

NOLC1 inhibited p53 ubiquitination and degradation.

A RT‒qPCR analysis of p53 mRNA in MGC-803 cells transduced with shNC or shNOLC1 lentivirus and treated with PBS or Cis (15 µM).

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).

C, D 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.

E, F 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.

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.

H Ubiquitination assay in HEK-293T cells transfected with the indicated plasmids. ns, nonsignificant; *p < 0.05; **p < 0.01; ***p < 0.001.

NOLC1 knockdown increased the efficacy of Cis combined with anti-PD-1 treatment.

A Schematic representation showing the treatment of the subcutaneous tumors.

B Corresponding tumor photographs after different treatments.

C Tumor growth curves after different treatments.

D Tumor weight after the indicated treatment.

E Representative images of Liperfluo staining and 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. ns, nonsignificant; *p < 0.05; **p < 0.01.

Knockdown of NOLC1 reprogrammed the tumor microenvironment after Cis and anti-PD-1 combination therapy.

A FACS analysis of peripheral blood lymphocytes.

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 (IL-6, IFN-γ, and TNF-α) (n = 5).

D Schematic illustration of the mechanism of NOLC1 inhibits p53 mediate ferroptosis to increase the efficacy of anti-PD-1 plus Cis in GC. ns, nonsignificant; *p < 0.05; **p < 0.01.