Journal: BMC Medicine

Article Title: Upregulation of NOD1 and NOD2 contribute to cancer progression through the positive regulation of tumorigenicity and metastasis in human squamous cervical cancer

doi: 10.1186/s12916-022-02248-w

Figure Lengend Snippet: NOD1 and NOD2 are upregulated in human CSCC tissues and associated with poor survival. A Partial heat map showing differentially expressed NLR genes including NOD1 and NOD2 in the CSCC ( n = 4) and normal cervix ( n = 6) tissues. B NOD1 and NOD2 mRNA copy numbers in unpaired CSCC tissues (NOD1, n = 59; NOD2, n = 24) and normal cervix (NOD1, n = 33; NOD2, n = 31). C NOD1 and NOD2 mRNA expression in the CSCC (NOD1, n = 75; NOD2 n = 75) and normal cervix samples (non-tumoral adjacent tissue, NOD1, n = 188; NOD2 n = 188) extracted from TCGA database. D Representative immunofluorescence images showing co-staining of AE1/AE3 and NOD1/NOD2 in paired CSCC tumors and normal cervix tissues (data were from two independent experiments with eight samples). E Representative IHC images showing in situ expression of NOD1and NOD2 in paired human CSCC tissues of different pathological stages (early and late stages and LVSI) and adjacent non-tumor tissues (scale bar = 100 μm and magnification—× 10 or × 20). F–H IHC scores of NOD1 and NOD2 in F paired tumor and adjacent non-tumor tissues (NOD1, n = 75; NOD2, n = 70), G tumors with and without LVSI (NOD1: LVSI = 58, non-LVSI = 45; NOD2: LVSI = 48, non-LVSI = 49), and H tumors with and without LM (NOD1: LM = 48, non-LM = 48; NOD2: LM = 55, non-LM = 39). I Kaplan-Meier curves showing overall survival of CSCC patients demarcated on the basis of in situ NOD1 expression ( http://www.proteinatlas.org ). J NOD1 and NOD2 mRNA levels and representative immunofluorescence images showing respective protein levels in Siha and CasKi cell lines. K Immunofluorescence images showing respective NOD1 and NOD2 protein levels in primary CSC cells. For cell lines, the experiments were performed in two wells with three replicates; for primary cells, the experiments were performed by two independent experiments with four samples; the picture is a representative one. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The slices were then incubated overnight with rabbit anti-human vimentin (without diluted, Origene, Beijing, China), mouse anti-human pan-cytokeratin AE1/AE3 (without diluted, Origene, Beijing, China), mouse anti-human Ki67 (diluted 1:200, ZSGB-BIO, Beijing, China), mouse anti-human P16 (diluted 1:200, ZSGB-BIO, Beijing, China), mouse anti-human NOD1 (B-4; dilution: 1:100; sc-398696, Santa Cruz, USA), and mouse anti-human NOD2 (2D9; dilution: 1:100; sc-56168, Santa Cruz, USA) antibodies and the isotype control at 4 °C.

Techniques: Expressing, Immunofluorescence, Staining, In Situ

Journal: BMC Medicine

Article Title: Upregulation of NOD1 and NOD2 contribute to cancer progression through the positive regulation of tumorigenicity and metastasis in human squamous cervical cancer

doi: 10.1186/s12916-022-02248-w

Figure Lengend Snippet: NOD1 and NOD2 enhanced the proliferation of CSCC cells. A Number of colonies formed by Siha, CasKi, and C33a cells overexpressing NOD1 or NOD2 (each group was performed triplicate wells, reproducible in three independent experiments). B Time-dependent increase in the number of Siha/LV-NOD1 and Siha/LV-NOD2 cells (results were highly reproducible triplicate: representative data of triplicate). C Number of colonies formed by cell lines and primary CSCC cells treated with Trip-DAP or MDP (10 ng/mL). D Proliferation of cell lines treated with various concentrations of NOD1 or NOD2 ligands. The assay of C and D was repeated thrice for the cell lines and twice for the primary cells using three independent samples. E Cell cycle distribution of control and NOD1/NOD2 overexpressing Siha, CasKi, and C33a cells. The percentage of cells in each phase is shown on the right. F Representative images of EDU-stained Siha/LV-NOD1, Siha/LV-NOD2, and Siha/LV-Ctrl cells (scale bar—30 μm, triplicate independent experiments). G Representative images of tumors in BALB/C nude mice subcutaneously injected with Siha/LV-NOD1, Siha/LV-NOD2 (right), and respective control cells (left). The tumor volume and weight of the indicated groups are shown on the right. All data are presented as mean ± SD ( n = 10 for each group). *** P < 0.001; ** P < 0.01; * P < 0.05

Article Snippet: The slices were then incubated overnight with rabbit anti-human vimentin (without diluted, Origene, Beijing, China), mouse anti-human pan-cytokeratin AE1/AE3 (without diluted, Origene, Beijing, China), mouse anti-human Ki67 (diluted 1:200, ZSGB-BIO, Beijing, China), mouse anti-human P16 (diluted 1:200, ZSGB-BIO, Beijing, China), mouse anti-human NOD1 (B-4; dilution: 1:100; sc-398696, Santa Cruz, USA), and mouse anti-human NOD2 (2D9; dilution: 1:100; sc-56168, Santa Cruz, USA) antibodies and the isotype control at 4 °C.

Techniques: Control, Staining, Injection

Journal: BMC Medicine

Article Title: Upregulation of NOD1 and NOD2 contribute to cancer progression through the positive regulation of tumorigenicity and metastasis in human squamous cervical cancer

doi: 10.1186/s12916-022-02248-w

Figure Lengend Snippet: NOD1 and NOD2 promoted migration and invasion of CSCC cells. A Representative images showing wound closure of control and NOD1/2-overexpressing Siha and CasKi cells at 24 h after scratching. The percentage of wound healing is shown on the right. Data represent triplicates of one representative experiment. B, C The representative images indicating migration (panel B ) (Siha, CasKi and C33a) and invasion (panel C ) (Siha and CasKi) ability of NOD1/NOD2-overexpressing cell lines by transwell assay (reproducible in three independent experiments in triplicate wells). D Representative migration and invasion images of the primary CSCC cells by transwell members (The cells were pretreated with 10 ng/mL Tri-DAP or MDP at 24 h) (three independent experiments with three primary CSCC cells, triplicate wells; magnification × 100). E Number of lung metastatic nodules (The yellow arrows indicate representatively metastatic nodes in fresh lung; the red arrows indicate the nodes in fixed lung) in mice injected with Siha/LV-Ctrl, Siha/LV-NOD1, and Siha/LV-NOD2 cells (mouse harboring Siha/LV-Ctrl: n = 6, mouse harboring Siha/LV-NOD1: n = 6, mouse harboring Siha/LV-NOD2: n = 6). F Kaplan-Meier curves showing OS of the above tumor-bearing groups ( n = 10 for each group). All data are presented as mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: The slices were then incubated overnight with rabbit anti-human vimentin (without diluted, Origene, Beijing, China), mouse anti-human pan-cytokeratin AE1/AE3 (without diluted, Origene, Beijing, China), mouse anti-human Ki67 (diluted 1:200, ZSGB-BIO, Beijing, China), mouse anti-human P16 (diluted 1:200, ZSGB-BIO, Beijing, China), mouse anti-human NOD1 (B-4; dilution: 1:100; sc-398696, Santa Cruz, USA), and mouse anti-human NOD2 (2D9; dilution: 1:100; sc-56168, Santa Cruz, USA) antibodies and the isotype control at 4 °C.

Techniques: Migration, Control, Transwell Assay, Injection

Journal: BMC Medicine

Article Title: Upregulation of NOD1 and NOD2 contribute to cancer progression through the positive regulation of tumorigenicity and metastasis in human squamous cervical cancer

doi: 10.1186/s12916-022-02248-w

Figure Lengend Snippet: NOD1/NOD2 promote CSCC tumorigenesis by activating the ERK and NF-κB signaling pathways. A Proliferation rates of Siha/LV-NOD1 and Siha/LV-NOD2 treated with 0.56 μM (upper) and 25 μM (lower) ML-130. B Proliferation of Siha/LV-NOD1 and Siha/LV-NOD2 cells transfected with the NOD1 and NOD2 siRNAs. C Representative images of transwell assay showing the migration and invasion of Siha/LV-NOD1 and Siha/LV-NOD2 cells with NOD1 or NOD2 knockdown. D Protein arrays showing the molecules and signaling pathways involved in the upregulation of NOD1 and NOD2 (data was merged with Siha and CasKi cells)—viral protein interactions (left), bacterial response (right), MAPK, and NF-κB signaling pathways (left) and ERK cascade (right). E GO analysis showing enriched proteins in Siha and CasKi cells transfected with LV-NOD1 or LV-NOD2 compared to LV-Ctrl. F Immunoblots showing expression of ERK/p-ERK and NF-κB/p-NF-κB in LV-NOD1 or LV-NOD2 CSCC cell lines (Siha and CasKi). G Representative cytokine array panels for Siha/LV-NOD1 and Siha/LV-NOD2 cells (upper panel) and cytokine levels validated by ELISA (lower panel). Data of CSCC cell lines represent triplicate independent experiment of representative one. The data of C and G were performed with triplicate wells. All data are presented as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: The slices were then incubated overnight with rabbit anti-human vimentin (without diluted, Origene, Beijing, China), mouse anti-human pan-cytokeratin AE1/AE3 (without diluted, Origene, Beijing, China), mouse anti-human Ki67 (diluted 1:200, ZSGB-BIO, Beijing, China), mouse anti-human P16 (diluted 1:200, ZSGB-BIO, Beijing, China), mouse anti-human NOD1 (B-4; dilution: 1:100; sc-398696, Santa Cruz, USA), and mouse anti-human NOD2 (2D9; dilution: 1:100; sc-56168, Santa Cruz, USA) antibodies and the isotype control at 4 °C.

Techniques: Protein-Protein interactions, Transfection, Transwell Assay, Migration, Knockdown, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: BMC Medicine

Article Title: Upregulation of NOD1 and NOD2 contribute to cancer progression through the positive regulation of tumorigenicity and metastasis in human squamous cervical cancer

doi: 10.1186/s12916-022-02248-w

Figure Lengend Snippet: Reparixin inhibits NOD1/2-induced tumorigenesis of CSCC cells by inhibiting the secretion of IL-8. A IL-8 levels secreted by Siha/LV-NOD1 and Siha/LV-NOD2 cells with NOD1 and NOD2 knockdown (left panels), or ML-130 pretreatment (right panels). B IL-8 levels secreted by Siha/LV-NOD1 and Siha/LV-NOD2 treated with EVP4593 (NF-κB inhibitor). C Proliferation rates of Siha/LV-NOD1 and Siha/LV-NOD2 cells treated with Reparixin (R), EVP4593 (E), SCH772984 (S), and their combination. D, E Representative images of transwell assays showing migration and invasion of Siha/LV-NOD1 and Siha/LV-NOD2 cells treated with Reparixin or EVP4593. The percentages are shown on the right. ELISA and transwell assays were performed in triplicates and CCK8 assay in five replicates. F, G Immunoblot showing expression of ERK/p-ERK and NF-κB/p-NF-κB in the Siha/LV-NOD1 and Siha/LV-NOD2 CSCC cells (data are from three independent experiment) transfected with siRNAs. All data are presented as mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: The slices were then incubated overnight with rabbit anti-human vimentin (without diluted, Origene, Beijing, China), mouse anti-human pan-cytokeratin AE1/AE3 (without diluted, Origene, Beijing, China), mouse anti-human Ki67 (diluted 1:200, ZSGB-BIO, Beijing, China), mouse anti-human P16 (diluted 1:200, ZSGB-BIO, Beijing, China), mouse anti-human NOD1 (B-4; dilution: 1:100; sc-398696, Santa Cruz, USA), and mouse anti-human NOD2 (2D9; dilution: 1:100; sc-56168, Santa Cruz, USA) antibodies and the isotype control at 4 °C.

Techniques: Knockdown, Migration, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Western Blot, Expressing, Transfection

Journal: BMC Medicine

Article Title: Upregulation of NOD1 and NOD2 contribute to cancer progression through the positive regulation of tumorigenicity and metastasis in human squamous cervical cancer

doi: 10.1186/s12916-022-02248-w

Figure Lengend Snippet: Reparixin inhibited growth of NOD1 high and NOD2 high CSCC tumors in vivo . A Volume of Siha/LV-NOD1 and Siha/LV-NOD2 tumors in the untreated ( n = 5), Reparixin ( n = 10), and EVP4593 ( n = 10) groups. B Tumor weight in the Reparixin ( n = 10), EVP4593 ( n = 10), and PBS ( n = 5) groups. C OS Kaplan-Meier curves of mice in the above groups. D Representative fluorescence images showing metastasis of GFP+ Siha/LV-NOD1 or Siha/LV-NOD2 cells in the indicated groups (mouse harboring GFP+ Siha/LV-NOD1: Group treated by PBS: n = 9; group treated by Reparixin: n = 8; group treated by EVP4593: n = 6. mouse harboring GFP+ Siha/LV-NOD2: Group treated by PBS: n = 9; group treated by Reparixin: n = 8; group treated by EVP4593: n = 6). Quantification of GFP intensity is shown on the right. E Survival curves of the above tumor-bearing mice treated with PBS, Reparixin, or EVP4593. The results are presented as mean ± SD. *** P < 0.001; ** P < 0.01; * P < 0.05

Article Snippet: The slices were then incubated overnight with rabbit anti-human vimentin (without diluted, Origene, Beijing, China), mouse anti-human pan-cytokeratin AE1/AE3 (without diluted, Origene, Beijing, China), mouse anti-human Ki67 (diluted 1:200, ZSGB-BIO, Beijing, China), mouse anti-human P16 (diluted 1:200, ZSGB-BIO, Beijing, China), mouse anti-human NOD1 (B-4; dilution: 1:100; sc-398696, Santa Cruz, USA), and mouse anti-human NOD2 (2D9; dilution: 1:100; sc-56168, Santa Cruz, USA) antibodies and the isotype control at 4 °C.

Techniques: In Vivo, Fluorescence

Journal: Journal of Cellular and Molecular Medicine

Article Title: NOD2 deficiency confers a pro‐tumorigenic macrophage phenotype to promote lung adenocarcinoma progression

doi: 10.1111/jcmm.16790

Figure Lengend Snippet: NOD2 was the key immune gene in the TME and acted as a tumour suppressor in LUAD. (A) Gene expression correlations of 13 PDEIRGs. Lines between two genes indicate the correlation value calculated by Pearson coefficient of correlation test. (B and C) PPI networks of 13 PDEIRGs from STRING database (B) and ordered by the number of connected nodes (C). (D) NOD2 expressions in different immunity clusters of the entire TCGA LUAD patients ( n = 422). (E) NOD2 expressions in the high‐risk group and the low‐risk group of the entire TCGA LUAD patients ( n = 422). (F) NOD2 expressions of the entire TCGA LUAD patients with different tumour sizes (T stage, n = 422). (G) NOD2 expressions of the entire TCGA LUAD patients with and without metastasis (M stage, n = 422). (H) NOD2 expressions of the entire TCGA LUAD patients with different TNM stages ( n = 422)

Article Snippet: The sections were incubated overnight at 4°C with rabbit anti‐human NOD2 polyclonal antibody (1:800, Proteintech, Wuhan, China) and rabbit anti‐human CD68 polyclonal antibody (1:800, Bioss, Beijing, China).

Techniques: Gene Expression

Journal: Journal of Cellular and Molecular Medicine

Article Title: NOD2 deficiency confers a pro‐tumorigenic macrophage phenotype to promote lung adenocarcinoma progression

doi: 10.1111/jcmm.16790

Figure Lengend Snippet: NOD2 was mainly expressed in the macrophages. (A) The fractions of 22 kinds of immune cells of patients with high NOD2 expressions (red) and low NOD2 expressions (green) in the TCGA LUAD cohort ( n = 422). (B–E) Gene expression correlations between NOD2 and monocytes, macrophages M0, macrophages M1, macrophages M2, respectively, in the TCGA LUAD cohort ( n = 422). (F and G) Representative pictures of the expressions of CD68 and NOD2 in LUAD patients tested by immunohistochemistry ( n = 3)

Article Snippet: The sections were incubated overnight at 4°C with rabbit anti‐human NOD2 polyclonal antibody (1:800, Proteintech, Wuhan, China) and rabbit anti‐human CD68 polyclonal antibody (1:800, Bioss, Beijing, China).

Techniques: Gene Expression, Immunohistochemistry

Journal: Journal of Cellular and Molecular Medicine

Article Title: NOD2 deficiency confers a pro‐tumorigenic macrophage phenotype to promote lung adenocarcinoma progression

doi: 10.1111/jcmm.16790

Figure Lengend Snippet: Decreased NOD2 expressions in TAMs were induced by LUAD cells. (A) Total NOD2 expression values of mo‐Mac in nLung, tLung and mBrain samples of GEO cohort. (B) Average NOD2 expression values of mo‐Mac in nLung, tLung and mBrain samples of GEO cohort (mean + SEM). (C) The schemes of the in vitro cellular co‐cultured experiment. (D) The level of NOD2 expressions in the co‐culture system was tested by Western blot. Representative pictures are shown. (E and F): The average densitometric values of NOD2 expressions are shown by histograms normalized to the levels of β‐tubulin (mean + SD). Abbreviations: mBrain, metastatic brain tissue samples; nLung, distant normal lung samples; SD, standard deviation; SEM, standard error of mean; tLung, primary tumour samples; TPM, transcripts per million

Article Snippet: The sections were incubated overnight at 4°C with rabbit anti‐human NOD2 polyclonal antibody (1:800, Proteintech, Wuhan, China) and rabbit anti‐human CD68 polyclonal antibody (1:800, Bioss, Beijing, China).

Techniques: Expressing, In Vitro, Cell Culture, Co-Culture Assay, Western Blot, Standard Deviation

Journal: Journal of Cellular and Molecular Medicine

Article Title: NOD2 deficiency confers a pro‐tumorigenic macrophage phenotype to promote lung adenocarcinoma progression

doi: 10.1111/jcmm.16790

Figure Lengend Snippet: Tumour cells spurred the conversion of the protective M1 phenotype to pro‐tumorigenic M2 subtype by down‐regulating NOD2 expression in TAMs. (A) Average fractions of M1 and M2 macrophages in different TNM stage patients of TCGA cohort. (B) Gene expressions of TNF‐α, IL‐1β, CD206 and CD163 in the high NOD2 expression group and the low NOD2 group in the GEO malignant samples. X‐axis represents the high NOD2 expression group and the low NOD2 group. Y‐axis represents normalized TPM values of targeted genes (mean + SEM). (C–F) The correlations of gene expressions between NOD2 and marker genes of M1 or M2 macrophages in the GEO LUAD cohort. (G) The schemes of the in vitro RNA interfered THP‐1 co‐cultured with LPS or IL‐4. (H–K) Gene expressions of TNF‐α, IL‐1β, CD206 and arginase 1 in RNA interfered THP‐1 co‐cultured with LPS or IL‐4 were tested by Q‐PCR (mean + SD)

Article Snippet: The sections were incubated overnight at 4°C with rabbit anti‐human NOD2 polyclonal antibody (1:800, Proteintech, Wuhan, China) and rabbit anti‐human CD68 polyclonal antibody (1:800, Bioss, Beijing, China).

Techniques: Expressing, Marker, In Vitro, Cell Culture