Journal: Cell Communication and Signaling : CCS

Article Title: Netrin-1-UNC5B/neogenin axis enhances the stemness of colorectal cancer cells

doi: 10.1186/s12964-025-02489-1

Figure Lengend Snippet: Netrin-1-UNC5B/neogenin axis to enhance cancer stemness in MC38 cells. A The phosphorylation levels of NF-κB p65 and ERK1/2 after netrin-1 treatment. B The phosphorylation levels of NF-κB p65 and ERK1/2 in netrin-1 knockdown MC38 cells. C Western blot analysis of NF-κB p65 and ERK1/2 phosphorylation levels when knocked UNC5B and neogenin in MC38 cells with or without netrin-1 treatment. D The mRNA levels of stemness markers SOX2, Nanog, OCT-4 were detected by qRT-PCR. Cell populations of CD133 + ( E ) and ALDH + ( F ) were analyzed by FCM. G Transwell migration assay was conducted on MC38 cells with BAY 11–7085 and PD98059 treatment, scale bars: 100 μm. H Sphere formation assay of BAY 11–7085 and PD98059-treated MC38 cells, scale bars: 100 μm. I Colony formation ability of BAY 11–7085 and PD98059 treated MC38 cells. *** P < 0.001;** P < 0.01;* P < 0.05

Article Snippet: MC38 cells were incubated with the specific NF-κΒ p65 phosphorylation inhibitor BAY-11–7085 (10 μM) (MCE, HY-10257) or ERK1/2 phosphorylation antagonist PD98059 (20 μM) (MCE, HY-12028) in 10% serum medium for 24 h. Control cells were treated with the same volume of dimethyl sulfoxide (DMSO).

Techniques: Phospho-proteomics, Knockdown, Western Blot, Quantitative RT-PCR, Transwell Migration Assay, Tube Formation Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Netrin-1-UNC5B/neogenin axis enhances the stemness of colorectal cancer cells

doi: 10.1186/s12964-025-02489-1

Figure Lengend Snippet: Netrin-1 is a potential biomarker that correlates with stemness and poor prognosis in human colorectal cancer. A The Kaplan-Meier survival curve of OS and RFS based on UNC5B expression in CRC patients. B The Kaplan-Meier survival curve of OS and RFS based on neogenin expression in CRC patients. C The expression of netrin-1, UNC5B and neogenin in cancer tissue (abbr. T) and adjacent tissue (abbr. N) from CRC patients was detected by Western blot. D The expression of netrin-1, UNC5B and neogenin in colorectal cancer tissue (CRC) and adjacent tissue (Normal) from GSE24550 . E Immunofluorescence analysis of netrin-1 and CD133 in cancer tissues and adjacent tissues from patients with CRC. scale bars: 10 μm and 100 μm. F The mRNA expression of netrin-1 in human colorectal cancer cell lines from the Cancer Cell Line Encyclopedia database. G Western blot analysis of β-catenin, SOX2, Nanog, and OCT-4 protein expression in netrin-1-treated HCT116 cells. H The expression of β-catenin, SOX2, Nanog, and OCT-4 protein after netrin-1 knockdown in HCT116 cells. I Western blot analysis of NF-κB p65 and ERK1/2 phosphorylation levels after netrin-1 knockdown in HCT116 cells.*** P < 0.001; ns, no significance

Article Snippet: MC38 cells were incubated with the specific NF-κΒ p65 phosphorylation inhibitor BAY-11–7085 (10 μM) (MCE, HY-10257) or ERK1/2 phosphorylation antagonist PD98059 (20 μM) (MCE, HY-12028) in 10% serum medium for 24 h. Control cells were treated with the same volume of dimethyl sulfoxide (DMSO).

Techniques: Biomarker Discovery, Expressing, Western Blot, Immunofluorescence, Knockdown, Phospho-proteomics

Journal: The EMBO Journal

Article Title: A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

doi: 10.15252/embj.2020104712

Figure Lengend Snippet: Representative confocal micrographs of co‐cultures of wild‐type (WT) and LUZP1 knockout (LUZP1 KO) Eph4 cells in the apical plane. Scale bar, 10 μm. Bar plots with dot density plots showing that ROCK1 mean intensities within circumferential rings (CRs) are similar between WT and LUZP1 KO cells (40.87 ± 9.95 arbitrary units [a.u.] [WT] vs. 39.48 ± 6.04 a.u. [LUZP1 KO]). n = 3. P = 0.54 (Mann–Whitney U test). Bars and error bars represent the mean ± standard deviation (SD). In vitro myosin light chain (MLC) phosphorylation assay using 25 ng GST‐MLC, 4 ng GST‐ROCK1 catalytic domain, 1 mM ATP, and 0–5 μg GST‐LUZP1. Quantification of the ppMLC/MLC ratio relative to the control showed that LUZP1 did not change the ratio (1.00 [1 st lane, control] vs. 1.13 ± 0.24 [2 nd lane] vs. 1.01 ± 0.44 [3 rd lane] vs. 1.08 ± 0.73 [4 th lane]). n = 4. P = 0.49 (Kruskal–Wallis test). Bars and error bars represent the mean ± SD. IB, immunoblotting. Representative confocal micrographs of co‐cultures of Venus‐LUZP1‐expressing LUZP1 KO (REV) and LUZP1 KO Eph4 cells treated with 100 nM calyculin A for 30 min. Scale bar, 10 μm. Bar plots with dot density plots showing that calyculin A reversed the difference in ppMLC levels within CRs between REV and LUZP1 KO cells (control, 21.14 ± 16.80 a.u. [WT] vs. 3.10 ± 1.72 a.u. [LUZP1 KO]; calyculin A, 25.24 ± 10.54 a.u. [WT] vs. 20.65 ± 5.62 a.u. [LUZP1 KO]; washout, 22.09 ± 7.90 a.u. [WT] vs. 7.92 ± 4.01 a.u. [LUZP1 KO]). ** P < 0.01 (Mann–Whitney U test). Bars and error bars represent the mean ± SD. n = 3. Representative immunoblot of WT, LUZP1 KO, and Venus‐LUZP1‐expressing LUZP1 knockout (REV) Eph4 cells treated with 100 nM calyculin A for 30 min. Quantification of the ppMLC/MLC ratio relative to WT control, confirming the reversal of the difference in ppMLC levels within CRs between WT and LUZP1 KO cells by calyculin A (WT, 1.00 [control] vs. 1.40 ± 0.06 [calyculin A] vs. 1.14 ± 0.33 [washout]; KO, 0.09 ± 0.04 [control] vs. 1.49 ± 0.06 [calyculin A] vs. 0.81 ± 0.99 [washout]; REV, 2.06 ± 1.78 [control] vs. 1.82 ± 1.50 [calyculin A] vs. 1.80 ± 1.14 [washout]). n = 3. Bars and error bars represent the mean ± SD. Source data are available online for this figure.

Article Snippet: GST‐ROCK1‐catalytic domain , Carna biosciences , Cat#01‐109.

Techniques: Knock-Out, MANN-WHITNEY, Standard Deviation, In Vitro, Phosphorylation Assay, Western Blot, Expressing

Journal: The EMBO Journal

Article Title: A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

doi: 10.15252/embj.2020104712

Figure Lengend Snippet: A schematic drawing of myosin phosphatase. Myosin phosphatase consists of PP1c β/δ, myosin phosphatase targeting subunit 1 (MYPT1), and a small 20‐kDa regulatory subunit (M20). PP1c β/δ represents a catalytic subunit responsible for dephosphorylating myosin light chain (MLC), whereas MYPT1 targets myosin phosphatase to MLC by binding both PP1c β/δ and MLC. Representative confocal micrographs of co‐cultures of wild‐type (WT) and LUZP1 knockout (LUZP1 KO) Eph4 cells in the apical plane. Scale bar, 10 μm. Bar plots with dot density plots showing that PP1c mean intensities within CRs are similar between WT and LUZP1 KO cells (28.68 ± 9.60 arbitrary units [a.u.] [WT] vs. 25.04 ± 9.47 a.u. [LUZP1 KO]). P = 0.09 [Mann–Whitney U test]. n = 3. Bars and error bars represent the mean ± standard deviation (SD). Co‐immunoprecipitation of HA‐PP1c β/δ and GFP‐LUZP1. LUZP1 binds to PP1c β/δ. IB, immunoblotting. In vitro MLC phosphorylation assay using 1 μg GST‐PP1c β/δ in addition to 25 ng GST‐MLC, 4 ng GST‐ROCK1 catalytic domain, 1 mM ATP, and 0–5 μg GST‐LUZP1. Quantification of the di‐phosphorylated MLC (ppMLC)/MLC ratio relative to the control showed that LUZP1 upregulated ppMLC/MLC levels in a dose‐dependent manner (1.00 [1 st lane, control] vs. 1.27 ± 0.33 [2 nd lane] vs. 1.76 ± 0.68 [3 rd lane] vs. 2.53 ± 1.65 [4 th lane] vs. 2.93 ± 2.45 [5 th lane]). n = 3 or 6. ** P < 0.01 (Kruskal–Wallis test followed by Steel test [compared with 1 st lane]). Bars and error bars represent the mean ± SD. In vitro Merlin phosphorylation assay using 1 μg GST‐PP1c β/δ, 100 ng GST‐Merlin, 2 pg p21‐activated kinase 1 (PAK1), and 5 μg GST‐LUZP1. Quantification of the phosphorylated Merlin (pMerlin)/Merlin ratio relative to the control showed that LUZP1 upregulated pMerlin/Merlin levels (0.23 ± 0.15 [1 st lane] vs. 1.00 [2 nd lane, control] vs. 0.32 ± 0.17 [3 rd lane] vs. 0.97 ± 0.42 [4 th lane] vs. 1.25 ± 0.39 [5 th lane]). n = 4 or 9. * P < 0.05, ** P < 0.01 (Kruskal–Wallis test followed by Steel test [compared with 3 rd lane]). Bars and error bars represent the mean ± SD. A schematic drawing of the relationships among ppMLC, LUZP1, and myosin phosphatase at tight junction (TJ)‐associated CRs to promote apical constriction. Source data are available online for this figure.

Article Snippet: GST‐ROCK1‐catalytic domain , Carna biosciences , Cat#01‐109.

Techniques: Binding Assay, Knock-Out, MANN-WHITNEY, Standard Deviation, Immunoprecipitation, Western Blot, In Vitro, Phosphorylation Assay

Journal: The EMBO Journal

Article Title: A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

doi: 10.15252/embj.2020104712

Figure Lengend Snippet: Box plots with dot density plots showing the ratio of the apical area/basal area in co‐cultures of Venus‐LUZP1‐expressing LUZP1 knockout (REV) and LUZP1 knockout (LUZP1 KO) Eph4 cells; 2 μM nocodazole treatment for 30 min partially reversed apical constriction of REV cells (REV, 0.65 ± 0.16 [control] vs. 0.90 ± 0.18 [nocodazole] vs. 0.64 ± 0.16 [washout]; KO, 1.30 ± 0.17 [control] vs. 1.07 ± 0.13 [nocodazole] vs. 1.32 ± 0.19 [washout]). ** P < 0.01 (Kruskal–Wallis test followed by Steel–Dwass test). The solid lines represent the medians, and the boxes represent the interquartile ranges. The error bars extending from the box represent the data within 1.5 times of the interquartile range. Representative confocal micrographs of co‐cultures of LUZP1‐expressing wild‐type (WT) and LUZP1 KO Eph4 cell treated with 2 μM nocodazole for 30 min. Nocodazole treatment partially reversed the difference in di‐phosphorylated MLC (ppMLC) levels within circumferential rings (CRs) between WT and LUZP1 KO cells. Scale bar, 10 μm. Bar plots with dot density plots showing that ppMLC levels within CRs were significantly downregulated in WT Eph4 cells after nocodazole treatment. Importantly, ppMLC levels in LUZP1 KO Eph4 cells were unchanged after nocodazole treatment (WT, 21.43 ± 6.96 arbitrary units [a.u.] [control] vs. 17.67 ± 5.40 a.u. [nocodazole] vs. 20.84 ± 7.19 a.u. [washout]; KO, 8.74 ± 1.71 a.u. [control] vs. 8.67 ± 1.89 a.u. [nocodazole] vs. 7.96 ± 2.35 a.u. [washout]). n = 3. ** P < 0.01 (Kruskal–Wallis test followed by Steel–Dwass test). Bars and error bars represent the mean ± standard deviation (SD). In vitro MLC phosphorylation assay using 1 μg MTs in addition to 25 ng GST‐MLC, 4 ng GST‐ROCK1 catalytic domain, 1 mM ATP, 1 μg GST‐protein phosphatase 1c β/δ (PP1c β/δ), and 0–5 μg GST‐LUZP1. Quantification of the relative ppMLC/MLC ratio to the control showed that MTs promote LUZP1‐mediated inhibition of PP1c β/δ (1.00 [1 st ‐lane, control] vs. 1.42 ± 0.59 [2 nd ‐lane] vs. 1.72 ± 0.76 [3 rd ‐lane] vs. 1.99 ± 0.56 [4 th ‐lane] vs. 1.14 ± 0.37 [5 th ‐lane] vs. 2.87 ± 1.51 [6 th ‐lane] vs. 2.74 ± 1.19 [7 th ‐lane] vs. 2.50 ± 0.88 [8 th ‐lane]). n = 6. * P < 0.05 (Kruskal–Wallis test followed by Steel test [compared with 1 st lane]). Bars and error bars represent the mean ± SD. A schematic drawing of the relationships among MTs, ppMLC, LUZP1, and myosin phosphatase at TJ‐associated CRs to promote apical constriction. Source data are available online for this figure.

Article Snippet: GST‐ROCK1‐catalytic domain , Carna biosciences , Cat#01‐109.

Techniques: Expressing, Knock-Out, Standard Deviation, In Vitro, Phosphorylation Assay, Inhibition

Journal: The EMBO Journal

Article Title: A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

doi: 10.15252/embj.2020104712

Figure Lengend Snippet:

Article Snippet: GST‐ROCK1‐catalytic domain , Carna biosciences , Cat#01‐109.

Techniques: Recombinant, Plasmid Preparation, Sequencing, Transfection, Protease Inhibitor, Purification, Western Blot, Blocking Assay, Software, Imaging, Modification

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: Cytoplasmic HMGB1 promotes the activation of JAK2-STAT3 signaling and PD-L1 expression in breast cancer.

doi: 10.1186/s10020-025-01235-0

Figure Lengend Snippet: Fig. 2 HMGB1 regulates the expression of PD-L1 through the STAT3 signaling. (A, B) Immunoblots representing the expression of p-STAT3, STAT3, p-ERK1/2, ERK1/2, p-p65, p65, p-AKT, and AKT in MDA-MB-231 cells transfected with empty vector or Myc-HMGB1 plasmid (A) or siCtrl or siHMGB1 (B). (C) MDA-MB-231 cells were transfected with Myc-HMGB1 for 24 h and treated with 20 µM Stattic for 16 h. (D) MDA-MB-231 cells were transfected with Myc- HMGB1 for 48 h and treated with 0.31 µM SCH772984 for 42 h. (E) MDA-MB-231 cells were transfected with Myc-HMGB1 for 48 h and treated with 0.62 µM BMS-345541 for 42 h. (F) MDA-MB-231 cells were transfected with Myc-HMGB1 for 24 h and treated with 5 µM MK-2206 for 16 h. (G) Immunoblots repre senting the expression of p-STAT3, STAT3, PD-L1 in MEFHMGB1 KO cells transfected with empty vector or Myc-HMGB1. n = 3 samples per group for all immu noblot data. (H) GSEA enrichment plot for the STAT3 signaling target gene set (STAT3_02) comparing the top 10% and bottom 10% of HMGB1 expression in the TCGA Firehose Legacy dataset. Data are presented as the mean ± SEM. The two-tailed unpaired Student t-test was used for statistical analysis. ns, not significant. *p < 0.05 and *** p < 0.001. E/V, Empty Vector; H, Myc-HMGB1; GSEA, Gene Set Enrichment Analysis; NES, normalized enrichment score

Article Snippet: Stattic, a STAT3 inhibitor (S7024; Selleckchem, Houston, TX, USA); Inflachromene (ICM), an HMGB1 nucleo-cytoplasmic translocation inhibitor (AOB6225; Gloucester, MA, USA); MK-2206, an AKT inhibitor (S1078; Selleckchem); BMS-345541, a NF-κB p65 phosphorylation inhibitor (S8044; Selleckchem); SCH772984, an ERK1/2 inhibitor (S7101; Selleckchem); dimethyl sulfoxide (D2650; Sigma-Aldrich, St. Louis, MO, USA) were used.

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Two Tailed Test