nod1 inhibitor  (MedChemExpress)

Journal: Journal of Inflammation Research

Article Title: Development and Verification of Diagnosis Model for Papillary Thyroid Cancer Based on Pyroptosis-Related Genes: A Bioinformatic and in vitro Investigation

doi: 10.2147/JIR.S478989

Figure Lengend Snippet: qRT-PCR Primer Sequences

Article Snippet: The NOD1 inhibitor Nodinitib-1 (ML130, Topscience) was dosed for 24 h and the cells were lysed in protein lysis buffer (Cell lysis buffer for Western and IP) (Beyotime, China) containing the protease inhibitor PMSF.

Techniques:

Journal: Journal of Inflammation Research

Article Title: Development and Verification of Diagnosis Model for Papillary Thyroid Cancer Based on Pyroptosis-Related Genes: A Bioinformatic and in vitro Investigation

doi: 10.2147/JIR.S478989

Figure Lengend Snippet: Validation of clinical specimens at our center. ( A ). Gene expression in our center: reverse transcription-polymerase chain reaction (RT-PCR) to detect the expression of NOD1, PRKACA, GSDMB, CASP6, and CASP9 genes in our samples (n = 87). ( B and C ). Immunofluorescence staining for CD68 and CD47 in the clinical tissue specimens. ( D and E ). Immunohistochemical staining for CD3 and CD8 in the clinical tissue specimens. ( F ). The PSF of high NOD1 expression and low NOD1 expression. ( G ). Immunofluorescence staining for PD-1 and NOD-1 in the clinical tissue specimens. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The NOD1 inhibitor Nodinitib-1 (ML130, Topscience) was dosed for 24 h and the cells were lysed in protein lysis buffer (Cell lysis buffer for Western and IP) (Beyotime, China) containing the protease inhibitor PMSF.

Techniques: Biomarker Discovery, Gene Expression, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunofluorescence, Staining, Immunohistochemical staining

Journal: Journal of Inflammation Research

Article Title: Development and Verification of Diagnosis Model for Papillary Thyroid Cancer Based on Pyroptosis-Related Genes: A Bioinformatic and in vitro Investigation

doi: 10.2147/JIR.S478989

Figure Lengend Snippet: A Total of 90 PTC Cases at Our Center: Correlation Between the NOD1 Expression and Clinicopathological Features

Article Snippet: The NOD1 inhibitor Nodinitib-1 (ML130, Topscience) was dosed for 24 h and the cells were lysed in protein lysis buffer (Cell lysis buffer for Western and IP) (Beyotime, China) containing the protease inhibitor PMSF.

Techniques: Expressing

Journal: Journal of Inflammation Research

Article Title: Development and Verification of Diagnosis Model for Papillary Thyroid Cancer Based on Pyroptosis-Related Genes: A Bioinformatic and in vitro Investigation

doi: 10.2147/JIR.S478989

Figure Lengend Snippet: NOD1 inhibitor ML130 showed significant in vitro anti-PTC activity. ( A and B ). q-PCR and Western blot results of high expression of TPC1 in PTC cells. ( C ). Effect of ML130 application on TPC-1 and BCPAP cell viability using CCK8 assay. ( D ). TPC-1 and BCPAP clone formation and scratch assay to detect the effect of ML130 on cell proliferation and migration ability. ( E ). The effect of ML130 on cell proliferation and migration ability. ( F ). Flow assay to detect the effect of ML130 on TPC-1 and BCPAP death. ( G ). Western blot results demonstrated that TPC-1 and BCPAP showed apoptosis and pyroptosis after ML130 application. Data are shown as mean ± SD for n = 3. * p < 0.05, *** p < 0.001.

Article Snippet: The NOD1 inhibitor Nodinitib-1 (ML130, Topscience) was dosed for 24 h and the cells were lysed in protein lysis buffer (Cell lysis buffer for Western and IP) (Beyotime, China) containing the protease inhibitor PMSF.

Techniques: In Vitro, Activity Assay, Western Blot, Expressing, CCK-8 Assay, Wound Healing Assay, Migration

Journal: Protein & Cell

Article Title: Microbiota enterotoxigenic Bacteroides fragilis -secreted BFT-1 promotes breast cancer cell stemness and chemoresistance through its functional receptor NOD1

doi: 10.1093/procel/pwae005

Figure Lengend Snippet: ETBF presence and NOD1 expression together in breast tumors predict a poor response to chemotherapy. (A) The sizes of 4T1 cell allograft tumors treated with or without ETBF were measured every four days in Balb/c mice and the tumor growth curve was shown. Mice were infected with ETBF (1 × 10 9 CFU) or water (CTRL) by intragastric gavage (IG) every two days for six times in total (All values were presented as mean ± SEM, *** P < 0.001, vs. CTRL). (B) The percentage of ALDH + cells was detected by ALDEFLUOR assay in tumor cells from 4T1 cell allograft tumors treated with or without ETBF. The bar graph was presented as mean ± SEM, ** P < 0.01. (C) The percentage of ALDH + cells was detected by ALDEFLUOR assay in SUM159 cells treated with or without ETBF. The bar graph was presented as mean ± SEM of three biological independent experiments, ** P < 0.01. (D) The mRNA was isolated from SUM159 cells treated with or without ETBF. The mRNA expression levels of stemness genes SOX2, OCT4 and NANOG were detected by qRT-PCR. The bar graph was presented as mean ± SEM, * P < 0.05, ** P < 0.01. (E) Pathway enrichment signatures were assessed by gene set enrichment analysis (GSEA) in T compared to PT from NR and CR to TNC. The rows corresponded to canonical pathways from the Reactome Pathway Database and the columns corresponded to tissue samples. The blue or red color of each cell referred to the − log 10 ( P -value). (F) The overlapped stemness-relative genes were analyzed in ALDH + cancer stem cells compared to ALDH - cancer cells from three breast cancer cell lines (SUM159, MCF7, and SUM149) and two breast cancer PDX (overlap gene list) models. Totally, eight overlapped genes were detected in all three cell lines and two PDX models. (G) A bubble chart showed the log P value and fold change (FC) at mRNA expression level of above overlapped stemness-relative genes in T from CR compared to T from NR as in (E). (H) The mRNA expression of NOD1 in both ALDH + and ALDH − cells sorted from MDA-MB-231, SUM159 and 4T1 cells was analyzed by qRT-PCR. The bar graph was presented as mean ± SEM of three biological independent experiments, ** P < 0.01, *** P < 0.001.(I) NOD1 protein level in both ALDH + and ALDH − cells sorted from MDA-MB-231, SUM159 and 4T1 cells was analyzed by Western blot. (J) The probability of overall survival (OS), relapse free survival (RFS), and distant metastasis-free survival (DMFS) of breast cancer patients received neoadjuvant chemotherapy was analyzed according to NOD1 mRNA expression and the statistical significance was analyzed by log-rank test. Patients were divided into a high-risk group and a low-risk group according to the median value of the NOD1 mRNA expression in tumor tissues. The numbers in the bottom part of the figure are “number at risk”. (K) NOD1 expression was analyzed by IHC staining in paired primary tumors (PR) and recurrent tumors (RE) from the same breast cancer patients. The representative IHC staining images of NOD1 were shown (left, Scale bar: 50 μm). The heatmap (right) showed the IHC staining intensity scores of NOD1 in PR and RE from 25 breast cancer patients. (L) NOD1 expression was analyzed by immunohistochemistry (IHC) staining in breast tumor tissues. The representative IHC staining images of NOD1 were shown (left, Scale bar: 50 μm). Violin plot (right) was shown for the comparison of IHC staining intensity scores of NOD1 between NR ( n = 27) and CR ( n = 41) to TNC. The bar graph was presented as mean ± SEM, * P < 0.05.(M) The correlation between NOD1 expression and ETBF presence in tumor tissues from NR or CR to TNC was assessed by IHC staining for NOD1 and FISH staining for ETBF 16S rRNA.

Article Snippet: The commercial NOD1 inhibitor Nodinitib-1 (HY-18639, MedChemExpress) was used at 5–10 μmol/L for 48–72 h in vitro and 20 mg/kg in vivo .

Techniques: Expressing, Infection, Isolation, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Comparison, Staining

Journal: Protein & Cell

Article Title: Microbiota enterotoxigenic Bacteroides fragilis -secreted BFT-1 promotes breast cancer cell stemness and chemoresistance through its functional receptor NOD1

doi: 10.1093/procel/pwae005

Figure Lengend Snippet: NOD1 is a functional receptor for BFT-1 and mediates ETBF-enhanced cancer cell stemness. (A) The percentage of ALDH + cells was detected by ALDEFLUOR assay in the control (shCTRL) or NOD1-knockdown (shNOD1-1, shNOD1-2) SUM159 cells treated with or without ETBF. The bar graph was presented as mean ± SEM of three biological independent experiments, ** P < 0.01. (B) The mRNA was isolated from shCTRL or NOD1-knockdown (shNOD1-1, shNOD1-2) SUM159 cells treated with or without ETBF. The mRNA expression levels of stemness genes SOX2, OCT4, and NANOG were detected by qRT-PCR. The bar graph was presented as mean ± SEM, * P < 0.05, ** P < 0.01, ns: no significance. (C) The percentage of ALDH + cells was detected by ALDEFLUOR assay in the control (shCTRL) or NOD1-knockdown (shNOD1-1, shNOD1-2) SUM159 cells treated with or without BFT-1. The bar graph was presented as mean ± SEM of three biological independent experiments, * P < 0.05, ** P < 0.01, **** P < 0.0001, ns: no significance.(D) The mRNA was isolated from shCTRL or NOD1-knockdown (shNOD1-1, shNOD1-2) SUM159 cells treated with or without BFT-1. The mRNA expression levels of stemness genes SOX2, OCT4, and NANOG were detected by qRT-PCR. The bar graph was presented as mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001, ns: no significance. (E and F) The direct interaction between BFT-1 and NOD1 was assessed by GST pull-down assay. (G) The confocal microscopic images were shown for NOD1-overexpressing SUM159 cells. NOD1-overexpressing SUM159 cells were treated with HIS-peptide or HIS-tagged BFT-1 for two h and stained with anti-HIS antibody. An isotype-matched IgG was used as a control. Cell nuclei were counterstained with DAPI. Scale bar, 20 μm. (H) The empty vector (EM) or NOD1-overexpressing (NOD1) 4T1 cells were treated with or without graded concentrations (0–20 nmol/L) of HIS-tagged BFT-1 for 30 min, and the cells were stained with anti-HIS antibody and the HIS-tagged BFT-1 binding properties were analyzed by flow cytometry. Dead cells were excluded prior to analysis with DAPI staining. All values were presented as mean ± SEM, *** P < 0.001.(I) The shCTRL or NOD1-knockdown (shNOD1-1, shNOD1-2) MDA-MB-231 cells were treated with or without graded concentrations (0–20 nmol/L) of HIS-tagged BFT-1 for 30 min, and the cells were stained with anti-HIS antibody and the HIS-tagged BFT-1 binding properties were analyzed by flow cytometry. Dead cells were excluded prior to analysis with DAPI staining. All values were presented as mean ± SEM, * P < 0.05, ** P < 0.01.(J) The protein level of NOD1 in MDA-MB-231 cells treated with BFT-1 at indicated concentrations was analyzed by Western blot. (K) The protein level of NOD1 in MDA-MB-231cells treated with 10 nmol/L BFT-1 for 2 or 4 h was analyzed by Western blot.(L) MDA-MB-231 cells were pretreated with or without 10 nmol/L BFT-1 for 4 h. The culture medium was then replaced with medium containing CHX, and the cells were harvested at the indicated times after CHX treatment. The protein level of was analyzed by Western blot (left) and the NOD1 density was quantified by densitometry using image J (right). Cycloheximide (CHX): 10 μmol/L. All values were presented as mean ± SEM, ** P < 0.01, ns: no significance.

Article Snippet: The commercial NOD1 inhibitor Nodinitib-1 (HY-18639, MedChemExpress) was used at 5–10 μmol/L for 48–72 h in vitro and 20 mg/kg in vivo .

Techniques: Functional Assay, Control, Knockdown, Isolation, Expressing, Quantitative RT-PCR, Pull Down Assay, Staining, Plasmid Preparation, Binding Assay, Flow Cytometry, Western Blot

Journal: Protein & Cell

Article Title: Microbiota enterotoxigenic Bacteroides fragilis -secreted BFT-1 promotes breast cancer cell stemness and chemoresistance through its functional receptor NOD1

doi: 10.1093/procel/pwae005

Figure Lengend Snippet: NOD1 positively regulates breast cancer cell stemness and chemoresistance. (A) The cell proliferation was detected by MTT assay in MDA-MB-231 cells treated with or without 10 nmol/L BFT-1. All values between both groups were presented as mean ± SEM, *** P < 0.001. (B) The percentage of ALDH + cells was detected by ALDEFLUOR assay in MDA-MB-231 cells treated with or without 10 nmol/L BFT-1. The bar graph was presented as mean ± SEM of three biological independent experiments, * P < 0.05. (C) The self-renewal ability was determined by primary mammosphere formation assay (sphere number and sphere diameter) in MDA-MB-231 cells treated with or without 10 nmol/L BFT-1. The bar graph was presented as mean ± SEM of three biological independent experiments, **** P < 0.0001. (D) The self-renewal ability was determined by secondary mammosphere formation (sphere formation efficiency) assay in MDA-MB-231 cells treated with or without 10 nmol/L BFT-1. The bar graph was presented as mean ± SEM of three biological independent experiments, *** P < 0.001. (E) The cell proliferation was detected by MTT assay in EM or NOD1-overexpressing MDA-MB-231 cells. All values between both groups were presented as mean ± SEM, *** P < 0.001. (F) The percentage of ALDH + cells was detected by ALDEFLUOR assay in EM or NOD1-overexpressing MDA-MB-231 cells. The bar graph was presented as mean ± SEM of three biological independent experiments, ** P < 0.01. (G) The self-renewal ability was determined by primary mammosphere formation assay (sphere number and sphere diameter) in EM or NOD1-overexpressing MDA-MB-231 cells. The bar graph was presented as mean ± SEM of three biological independent experiments, *** P < 0.001, **** P < 0.0001. (H) The self-renewal ability was determined by secondary mammosphere formation (sphere formation efficiency) assay in EM or NOD1-overexpressing MDA-MB-231 cells. The bar graph was presented as mean ± SEM of three biological independent experiments, *** P < 0.001. (I) The cell proliferation was detected by MTT assay in shCTRL or NOD1-knockdown (shNOD1-1, shNOD1-2) MDA-MB-231 cells. All values between both groups were presented as mean ± SEM, *** P < 0.001. (J) The percentage of ALDH + cells was detected by ALDEFLUOR assay in shCTRL or NOD1-knockdown (shNOD1-1, shNOD1-2) MDA-MB-231 cells. The bar graph was presented as mean ± SEM of three biological independent experiments, ** P < 0.01. (K) The self-renewal ability was determined by primary mammosphere formation assay (sphere number and diameter) in shCTRL or NOD1-knockdown (shNOD1-1, shNOD1-2) MDA-MB-231 cells. The bar graph was presented as mean ± SEM of three biological independent experiments, *** P < 0.001, **** P < 0.0001. (L) The self-renewal ability was determined by secondary mammosphere formation assay (sphere formation efficiency) in shCTRL or NOD1-knockdown (shNOD1-1, shNOD1-2) MDA-MB-231 cells. The bar graph was presented as mean ± SEM of three biological independent experiments, *** P < 0.001. (M and N) 1 × 10 6 EM or NOD1-overexpressing MDA-MB-231 cells were injected at the fourth mammary fat pads of nude mice ( n = 5 for each group) and the tumor size was measured once a week. At the end of the experiments, the mice were sacrificed, and the tumor images (M) and the tumor growth curve (N) were shown. All values were presented as mean ± SEM, *** P < 0.001, vs. the EM. (O) The percentage of ALDH + cells was detected by ALDEFLUOR assay in EM or NOD1-overexpressing MDA-MB-231 xenograft tumors. The bar graph was presented as mean ± SEM, *** P < 0.001. (P) The stem cell frequency in EM or NOD1-overexpressing MDA-MB-231 xenograft tumors was calculated by the limited dilution assay. The stem cell frequency and P value were calculated based on the positive tumor sites per group. (Q and R) 1 × 10 6 shCTRL or NOD1-knockdown (shNOD1-1, shNOD1-2) MDA-MB-231 cells were injected at the fourth mammary fat pads of nude mice ( n = 5 for each group) and the tumor size was measured once a week. At the end of the experiments, the mice were sacrificed, and the tumor images (Q) and the tumor growth curve (R) were shown. All values were presented as mean ± SEM, *** P < 0.001. (S) The percentage of ALDH + cells was detected by ALDEFLUOR assay in tumor cells from shCTRL or NOD1-knockdown (shNOD1-1, shNOD1-2) MDA-MB-231 cell xenograft tumors. The bar graph was presented as mean ± SEM, *** P < 0.001. (T) The stem cell frequency in tumor cells from shCTRL or NOD1-knockdown (shNOD1-1, shNOD1-2) MDA-MB-231 cell xenograft tumors was calculated by the limited dilution assay. The stem cell frequency and P value were calculated based on the positive tumor sites per group. (U) Half maximal inhibitory concentration (IC 50 ) values of Docetaxel (DTX) were evaluated by MTT assay in MDA-MB-231 cells treated with or without 10 nmol/L BFT-1. All values were presented as mean ± SEM. (V) Half maximal inhibitory concentration (IC 50 ) values of Docetaxel (DTX) were evaluated by MTT assay in EM or NOD1-overexpressing MDA-MB-231 cells. All values were presented as mean ± SEM. (W) Half maximal inhibitory concentration (IC 50 ) values of Docetaxel (DTX) were evaluated by MTT assay in shCTRL or NOD1-knockdown (shNOD1-1, shNOD1-2) MDA-MB-231 cells. All values were presented as mean ± SEM.

Article Snippet: The commercial NOD1 inhibitor Nodinitib-1 (HY-18639, MedChemExpress) was used at 5–10 μmol/L for 48–72 h in vitro and 20 mg/kg in vivo .

Techniques: MTT Assay, Tube Formation Assay, Knockdown, Injection, Dilution Assay, Concentration Assay

Journal: Protein & Cell

Article Title: Microbiota enterotoxigenic Bacteroides fragilis -secreted BFT-1 promotes breast cancer cell stemness and chemoresistance through its functional receptor NOD1

doi: 10.1093/procel/pwae005

Figure Lengend Snippet: NOD1 promotes NUMB lysosomal degradation and subsequently activates the NOTCH1-HEY1 pathway. (A) Pathway enrichment signatures were assessed by gene set enrichment analysis (GSEA) in EM and NOD1-overexpressing breast cancer cells. The normalized enrichment score (NES) and P value were shown in the pictures. (B) The mRNA was isolated from EM or NOD1-overexpressing and shCTRL or NOD1-knockdown (shNOD1-1, shNOD1-2) SUM159 cells. The mRNA expression levels of HEY1 were detected by qRT-PCR. The bar graph was presented as mean ± SEM of three biological independent experiments, ** P < 0.01, *** P < 0.001. (C) The correlation between NOD1 expression and HEY1 expression was analyzed in breast tumor tissues by IHC scores using Pearson’s correlation analysis. Representative IHC images of NOD1 and HEY1 in tumor tissues from the same patient (left, scale bar, 100 μm) and violin plot of IHC score of NOD1 or HEY1 (right) were shown ( n = 80). (D) The protein level of NICD1 and NUMB in EM or NOD1-overexpressing SUM159 or 4T1 cells was analyzed by Western blot. (E) The protein level of NICD1 and NUMB in shCTRL or NOD1-knockdown (shNOD1-1, shNOD1-2) SUM159 or 4T1 cells was analyzed by Western blot. (F) The protein level of NICD1 and NUMB in EM or NOD1-overexpressing SUM159 cells treated with or without 10 nmol/L BFT-1 was analyzed by Western blot. (G) NUMB in two daughter cells was detected by immunofluorescence (IF) staining with NUMB antibody in ALDH + cells sorted from EM or NOD1-overexpressing SUM159 cells. Bar chart showed the frequency of NUMB − /NUMB + (S/D), NUMB + /NUMB + (D/D), or NUMB − /NUMB − (S/S) cell pairs. The bar graph was presented as mean ± SEM of three biological independent experiments ( n = 50 ALDH + cells/replicate), * P < 0.05, ns: no significance. (H) The degradation rate of NUMB was analyzed by Western blot in EM or NOD1-overexpressing SUM159 cells. The protein level of was analyzed by Western blot (left) and the NUMB density was quantified by densitometry using image J (right). Cycloheximide (CHX): 10 μmol/L. All values were presented as mean ± SEM, **** P < 0.0001. (I) NUMB protein level was analyzed by Western blot in EM or NOD1-overexpressing SUM159 cells treated with or without the lysosomal inhibitor chloroquine (CQ, 1 μmol/L) or the proteasome inhibitor MG132 (1 μmol/L) for 9 h. (J) The localization of NUMB (green) and the lysosomal tracker (red) was analyzed by IF staining in EM or NOD1-overexpressing HEK293T cells treated with CQ for 9 h. Scale bar, 20 μm.

Article Snippet: The commercial NOD1 inhibitor Nodinitib-1 (HY-18639, MedChemExpress) was used at 5–10 μmol/L for 48–72 h in vitro and 20 mg/kg in vivo .

Techniques: Isolation, Knockdown, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining

Journal: Protein & Cell

Article Title: Microbiota enterotoxigenic Bacteroides fragilis -secreted BFT-1 promotes breast cancer cell stemness and chemoresistance through its functional receptor NOD1

doi: 10.1093/procel/pwae005

Figure Lengend Snippet: NOD1 interacts with cyclin G-associated kinase (GAK) to promote NUMB lysosomal degradation by phosphorylating NUMB. (A) The direct interaction between NOD1 and GAK was assessed by Co-Immunoprecipitation assay in SUM159 cells. (B) The direct interaction between NOD1 and GAK was assessed by GST pull-down assay. (C) The direct interaction between NOD1 and GAK was assessed by Co-immunoprecipitation assay in NOD1-overexpressing SUM159 cells treated with ETBF or BFT-1. (D) NUMB protein level was analyzed by Western blot in NOD1-overexpressing SUM159 cells with or without GAK-knockdown (shGAK-2, shGAK-5). (E) The localization of NUMB (green) and the lysosomal tracker (red) was analyzed by IF staining in EM or NOD1-overexpressing HEK293T cells with or without GAK-knockdown (shGAK-2, shGAK-5) in the presence of CQ for 9 h. Scale bar, 20 μm. (F) NUMB phosphorylation on Thr residues was analyzed by Western blot in EM or NOD1-overexpressing SUM159 cells. Immunoprecipitation of NUMB in EM or NOD1-overexpressing SUM159 cells was performed and followed by immunoblot analysis for p-Thr. (G) The kinase-substrate interaction between GAK and NUMB protein was analyzed by in vitro phosphorylation assay.

Article Snippet: The commercial NOD1 inhibitor Nodinitib-1 (HY-18639, MedChemExpress) was used at 5–10 μmol/L for 48–72 h in vitro and 20 mg/kg in vivo .

Techniques: Co-Immunoprecipitation Assay, Pull Down Assay, Western Blot, Knockdown, Staining, Immunoprecipitation, In Vitro, Phosphorylation Assay

Journal: Protein & Cell

Article Title: Microbiota enterotoxigenic Bacteroides fragilis -secreted BFT-1 promotes breast cancer cell stemness and chemoresistance through its functional receptor NOD1

doi: 10.1093/procel/pwae005

Figure Lengend Snippet: NOD1 inhibition and ETBF clearance together increase breast cancer chemosensitivity. (A) Schematic design for treatment regimen. A 25-day “Trinity” therapeutic strategy was designed for breast cancer cell xenograft tumors. 1000 tumor cells from PDX Lsl17 were injected at the fourth mammary fat pads of Nude mice and were treated with Metronidazole (MNZ, 25 mg/kg) by intragastric gavage (IG) every two days for four times in total and continuously treated with MNZ (0.5 g/L) via drinking water (DW) throughout the experiment. Docetaxel (DTX, 15 mg/kg), Nodinitib-1 (20 mg/kg), or a combination of both drugs were given by intraperitoneal injection (IP) every other day starting at day 11 ( n = 5 for each group). (B) At the end of experiments, the mice were sacrificed and the tumors were taken out. Representative images of tumors were shown. (C) The tumor weights of PDX Lsl17 tumors at the end of experiments were analyzed and graphed. The bar graph was presented as mean ± SEM, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns no significance. (D) The tumor sizes were measured every two days ( n = 5) and the tumor growth curve was shown. All values were presented as mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns no significance. (E) The percentage of ALDH + cells was analyzed by ALDEFLUOR assay in cells from PDX Lsl17 tumors subjected to “Trinity” therapeutic strategy. The bar graph was presented as mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001, ns no significance. (F) The stem cell frequency was calculated by the limited dilution assay. Tumor cells isolated from PDX Lsl17 tumors subjected to “Trinity” therapeutic strategy were engrafted into mammary fat pads of nude mice at limiting dilution. The stem cell frequency and P value were calculated based on the positive tumor sites per group. (G) Schematic design for treatment regimen. 3 × 10 4 4T1 cells were injected at the fourth mammary fat pads of Balb/c mice and were treated with ATBs by DW, and infected with ETBF (1 × 10 9 CFU) by IG for six times. Mice were treated with MNZ (25 mg/kg) for four times by IG and continuously treated with MNZ (0.5 g/L) via DW. Nodinitib-1 (20 mg/kg) alone or combined with DTX (20 mg/kg) were given by IP every five days starting at day 16 ( n = 6 for each group). (H) The tumor weights of 4T1 allograft tumors at the end of experiments were analyzed. The bar graph was presented as mean ± SEM, *** P < 0.001, **** P < 0.0001, ns no significance. (I) The tumor sizes of 4T1 allograft tumors were measured every three days ( n = 5) and the tumor growth curve was shown. All values were presented as mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns: no significance.

Article Snippet: The commercial NOD1 inhibitor Nodinitib-1 (HY-18639, MedChemExpress) was used at 5–10 μmol/L for 48–72 h in vitro and 20 mg/kg in vivo .

Techniques: Inhibition, Injection, Dilution Assay, Isolation, Infection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Fusobacterium nucleatum -triggered neutrophil extracellular traps facilitate colorectal carcinoma progression

doi: 10.1186/s13046-023-02817-8

Figure Lengend Snippet: Fn stimulates NETs generation by activating TLR4-ROS signaling and NOD1/2 receptor. A Flow cytometry analysis for ROS levels in neutrophils treated with Fn or PMA for 8 h. The mean fluorescence intensity (MFI) of three independent experiments is quantified and shown below. B Representative IF images for DNA / NE / MPO in neutrophils treated with Fn or Fn + DPI for 8 h. C Western blot analysis of CitH3 and PAD4 in neutrophils treated with Fn or Fn + DPI for 8 h. Protein levels were quantified by using densitometry and normalized to β-actin and are shown as fold changes compared to the control (Right). Each bar displays the means ± SD of three independent experiments. D ELISA analysis was performed to detect MPO-DNA levels in the supernatant of neutrophils treated with Fn or Fn + DPI for 8 h. E Representative IF images for DNA / NE / MPO in neutrophils treated with Fn or Fn + TAK-242 for 8 h. F Western blot analysis of CitH3 and PAD4 in neutrophils treated with Fn or Fn + TAK-242 for 8 h. Protein levels were quantified by using densitometry and normalized to β-actin and are shown as fold changes compared to the control (Right). Each bar displays the means ± SD of three independent experiments. G ELISA analysis was performed to detect MPO-DNA levels in the supernatant of neutrophils treated with Fn or Fn + TAK-242 for 8 h. H Flow cytometry analysis was used to detect ROS levels in neutrophils treated with Fn or Fn + TAK-242 for 8 h. The MFI of three independent experiments is quantified and shown (Right). I Representative IF images for DNA / NE / MPO in neutrophils treated with Fn , Fn + ML130 or Fn + GSK717 for 8 h. J Western blot analysis of CitH3 and PAD4 in neutrophils treated with Fn , Fn + ML130 or Fn + GSK717 for 8 h. Protein levels were quantified by using densitometry and normalized to β-actin and are shown as fold changes compared to the control (Right). Each bar displays the average + SD of three independent experiments. K ELISA analysis was performed to detect MPO-DNA levels in the supernatant of neutrophils treated with Fn , Fn + ML130 or Fn + GSK717 for 8 h. L Flow cytometry analysis was used to detect ROS levels in neutrophils treated with Fn , Fn + ML130 or Fn + GSK717 for 8 h. The MFI of three independent experiments is quantified and shown (Right). None, no treatment. White scale bars: 50 μm. ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The inhibitors used for this study were as follows: TLR4 inhibitor TAK-242 (10 μmol/L, S7455, Selleck, Houston, Texas, USA), ROS inhibitor DPI (10 μmol/L, S8639, Selleck, Houston, Texas, USA), NOD1 inhibitor ML130 (20 ug/mL, HY-18639, MedChemExpress, New Jersey, USA), NOD2 inhibitor GSK717 (20 ug/mL, HY136555, MedChemExpress, New Jersey, USA).

Techniques: Flow Cytometry, Fluorescence, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Fusobacterium nucleatum -triggered neutrophil extracellular traps facilitate colorectal carcinoma progression

doi: 10.1186/s13046-023-02817-8

Figure Lengend Snippet: A working model illustrating that activation of TLR4-ROS and NOD1/2 signalings by Fn -mediated NETs formation, which subsequently facilitated the growth and metastasis of CRC cells. Additionally, monitoring circulating NETs, especially combined with CEA may have potential to be a biomarker for predicting CRC metastasis

Article Snippet: The inhibitors used for this study were as follows: TLR4 inhibitor TAK-242 (10 μmol/L, S7455, Selleck, Houston, Texas, USA), ROS inhibitor DPI (10 μmol/L, S8639, Selleck, Houston, Texas, USA), NOD1 inhibitor ML130 (20 ug/mL, HY-18639, MedChemExpress, New Jersey, USA), NOD2 inhibitor GSK717 (20 ug/mL, HY136555, MedChemExpress, New Jersey, USA).

Techniques: Activation Assay, Biomarker Assay