Journal: Frontiers in Immunology

Article Title: Hyaluronic acid−CD44 signaling from decidual stromal cells orchestrates dNK1 differentiation and immune tolerance in early pregnancy

doi: 10.3389/fimmu.2026.1777567

Figure Lengend Snippet: HA-induced activation of the canonical Wnt pathway upregulates FOSL2 to expand the dNK1-like subpopulation. (A) Analysis of transcriptional activity and expression levels of transcription factors in three dNK subsets using scRNA-seq data from normal decidual tissue ( n = 11). (B) Transcriptional activity and expression levels of FOSL2 in decidual tissues from normal pregnancy ( n = 5) and RSA groups ( n = 3). (C) Changes in NK92MI cells of the five transcription factors ( STAT3, MAFB, HES1, FOSL2, ETV5 ) characterized by high transcriptional activity and expression in the dNK1 subset following HMW-HA treatment, as determined by RT−qPCR ( n = 6 per group). (D) FOSL2 expression in NK92MI cells following HMW-HA treatment, assessed by WB ( n = 3 per group). (E) Wnt1 and β-catenin protein expression in NK92MI cells treated with or without HMW-HA in the presence or absence of CD44 blockade ( n = 3 per group). (F) FOSL2 expression in HA−treated NK92MI cells after addition of CD44−blocking antibody or the Wnt pathway inhibitor IWP−2 to the culture system ( n = 3 per group). Data are expressed as mean ± SD; * P < 0.05; ** P < 0.01; ns, not significant. EP, early pregnancy; SA, spontaneous abortion; RSA, recurrent spontaneous abortion.

Article Snippet: The PVDF membranes were incubated overnight at 4 °C with primary antibodies against HAS2 (1:1000; 63kD; Santa Cruz, sc-514737), FOSL2 (1:1000; 45kD; Proteintech, M02615-2), Wnt1 (1:500; 45kD; Boster, PB0408), β-catenin (1:500; 92kD; CST, 8480T) and GAPDH (1:5000; 36kD; Proteintech, HRP-60004).

Techniques: Activation Assay, Activity Assay, Expressing, Quantitative RT-PCR, Blocking Assay

Journal: Scientific Reports

Article Title: Defective membrane repair machinery impairs survival of invasive cancer cells

doi: 10.1038/s41598-020-77902-5

Figure Lengend Snippet: Expression and subcellular localization of endogenous Anx in MDA-MB-231. ( a ) 10 µg of protein extracts from MDA-MB-231 and BeWo cells were analyzed by western-blot. The gel analysis plugin of ImageJ was used to quantify the density of bands. The histogram presents mean values (± SD) of the ratio Anx/GAPDH from ten independent experiments. Student t Test for independent samples. ** p < 0.01. ( b ) Revelation of AnxA5 in MDA-MB-231 and BeWo cells compared to 50 ng purified recombinant AnxA5 (Pure A5), presented as an example. The whole membrane, together with representative western-blot analysis for AnxA1, A2, A4 and A6, are presented in Supplementary Fig. . ( c ) MDA-MB-231 cells were immunostained for AnxA1, AnxA2, AnxA4, AnxA5 and AnxA6 (green), as indicated, and counterstained with DAPI (blue). The right-hand column presents magnified images extracted from the “Anx” column (indicated by inserts). Scale bar: 40 µm. ( d ) Quantification of fluorescence intensities after immunostaining of endogenous Anx in MDA-MB-231 cells. Fluorescence intensities were measured from 8-bit images by drawing a circular ROI inside the cytoplasm and measuring the mean pixel value. Mean pixel values from at least 20 cells are presented. Horizontal bars represent median values. The variance value is 323, 268, 224, 2372 and 2909 for AnxA1, A2, A4, A5 and A6, respectively. The coefficient of variation is 14%, 13%, 24%, 35% and 47% for AnxA1, A2, A4, A5 and A6, respectively. The expression level of AnxA5 or AnxA6 is heterogenous from one MDA-MB-231 cell to another. In some cells, AnxA5 and AnxA6 are expressed at a very low level.

Article Snippet: Semi-dry electrophoretic transfer (Bio-Rad, Hercules, CA, USA) onto PVDF membrane was performed for 1 h at 100 V. The cellular content in AnxA1 (37 kDa), AnxA2 (36 kDa), AnxA4 (32 kDa), AnxA5 (35 kDa), AnxA6 (68 kDa), and glyceraldehyde-3-phosphate deshydrogenase (GAPDH, loading control, 37 kDa) was detected with rabbit anti-AnxA1 polyclonal antibody (PA1006, BosterBio, Pleasanton, CA, USA), mouse anti-AnxA2 monoclonal antibody (3E8-B6, Sigma, Saint-Louis, MO, USA), mouse anti-AnxA4 monoclonal antibody (SAB4200121, Sigma), mouse anti-AnxA5 monoclonal antibody (AN5, Sigma), mouse anti-AnxA6 monoclonal antibody (sc-271859, Santa cruz Biotechnology), and rabbit anti-GAPDH polyclonal antibody (FL-335, Santa Cruz Biotechnology), respectively.

Techniques: Expressing, Western Blot, Purification, Recombinant, Membrane, Fluorescence, Immunostaining

Journal: Molecular Medicine

Article Title: Wnt5a-mediated autophagy contributes to the epithelial-mesenchymal transition of human bronchial epithelial cells during asthma

doi: 10.1186/s10020-024-00862-3

Figure Lengend Snippet: Schematic illustration of the mechanisms of Wnt5a-induced EMT in HBECs. This hypothesis illustrates a molecular pathway in HBECs leading to airway remodeling, typically associated with classical autophagy pathway and EMT. In short, Wnt5a induced by HDM or IL-4 leads to the overactivation of autophagy in HBECs through the Ca 2+ /CaMKII signaling pathway, subsequently promoting bronchial epithelial EMT and triggering airway remodeling

Article Snippet: The antibodies used in the present research were as follows: rabbit anti-α-Tubulin antibody (1:2000, Servicebio, Wuhan, China); rabbit anti-α-SMA antibody (1:1000, Cell Signaling Technology, USA); rabbit anti-E-cadherin antibody (1:2000, Cell Signaling Technology); rabbit anti-Beclin1 antibody (1:1500, Cell Signaling Technology); rabbit anti-Collagen I antibody (1:1000, Cell Signaling Technology); rabbit anti-LC3 antibody (1:1500, Cell Signaling Technology); rabbit anti-Wnt5a antibody (1:1000, BOSTER, Wuhan, China); rabbit anti-CaMKII antibody (1:1000, BOSTER); rabbit anti-p-CaMKII antibody (1:1000, Cell Signaling Technology).

Techniques:

Journal: Frontiers in Oncology

Article Title: Targeting endoplasmic reticulum stress-induced CLGN resensitizes hepatocellular carcinoma to apoptosis: paeonol synergistically enhances efficacy by dual inhibition of CLGN and NF-κB

doi: 10.3389/fonc.2025.1709962

Figure Lengend Snippet: ERS upregulates CLGN expression in HCC. (A) Volcano plot of differentially expressed genes from mRNA sequencing of Hep-G2 cells. Red and blue dots represent significantly up- and down-regulated genes, respectively (CLGN is labeled). (B) Heatmap of the top 25 up- and down-regulated genes from mRNA sequencing. (C) Expression levels of the top 25 upregulated genes in HCC and adjacent normal tissues from the TCGA database. (D–F) Kaplan-Meier survival analysis of HCC patients stratified by high and low expression of CLGN (D) , GPR1 (E) , and UNC5B (F) . (G) qRT–PCR analysis of candidate gene expression in Hep-G2 cells treated with or without TM (unpaired Student’s t-test). (H, I) Dose-dependent effects of the ERS inducer TM on CLGN and GRP78 expression in Hep-G2 cells, as determined by qRT–PCR (H) and Western blot (I) (one-way ANOVA with Dunnett’s post hoc test). (J) CLGN protein expression under UPR pathway inhibition. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Immunohistochemical staining was performed via a two-step method with a human monoclonal anti-rabbit CLGN antibody (1:100, BOSTER), a KI-67 antibody (1:400, CST), and an NF-κB antibody (1:400, CST).

Techniques: Expressing, Sequencing, Labeling, Quantitative RT-PCR, Gene Expression, Western Blot, Inhibition

Journal: Frontiers in Oncology

Article Title: Targeting endoplasmic reticulum stress-induced CLGN resensitizes hepatocellular carcinoma to apoptosis: paeonol synergistically enhances efficacy by dual inhibition of CLGN and NF-κB

doi: 10.3389/fonc.2025.1709962

Figure Lengend Snippet: High CLGN expression correlates with aggressive clinicopathological features and poor prognosis in HCC. (A) CLGN mRNA expression in unpaired HCC and normal liver tissues from the TCGA-LIHC cohort (unpaired Student’s t-test). (B–F) Analysis of CLGN mRNA expression levels in the TCGA cohort stratified by (B) tumor status, (C) age, (D) sex, (E) serum AFP level, and (F) histological grade (unpaired Student’s t-test or one-way ANOVA). (G) Sankey diagram illustrating the flow and association between TNM stage, histological grade, CLGN expression level, and tumor status. (H) IHC images of CLGN staining in HCC tissues, classified into four grades (0-3) based on staining intensity. (I) Statistical analysis of CLGN IHC scores in HCC tissues compared with adjacent non-tumor tissues (paired Student’s t-test). (J–L) Analysis of CLGN IHC scores stratified by (J) hepatitis status, (K) liver cirrhosis status, and (L) tumor size (unpaired Student’s t-test). (M, N) Correlation between CLGN protein expression and the ERS markers (M) GRP78 and (N) ATF6. Patients were grouped based on the median IHC score of each ERS marker (unpaired Student’s t-test). (O) Kaplan-Meier analysis of overall survival based on CLGN IHC staining in our institutional cohort (n=35, Log-rank test). (P, Q) Kaplan-Meier survival analysis of the TCGA-LIHC cohort based on CLGN mRNA expression levels, showing (P) disease-specific survival and (Q) overall survival (Log-rank test). (R) Western blot analysis of CLGN protein expression in 8 paired fresh-frozen HCC (T) and adjacent non-tumor (N) tissues. GAPDH was used as a loading control. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Immunohistochemical staining was performed via a two-step method with a human monoclonal anti-rabbit CLGN antibody (1:100, BOSTER), a KI-67 antibody (1:400, CST), and an NF-κB antibody (1:400, CST).

Techniques: Expressing, Staining, Marker, Immunohistochemistry, Western Blot, Control

Journal: Frontiers in Oncology

Article Title: Targeting endoplasmic reticulum stress-induced CLGN resensitizes hepatocellular carcinoma to apoptosis: paeonol synergistically enhances efficacy by dual inhibition of CLGN and NF-κB

doi: 10.3389/fonc.2025.1709962

Figure Lengend Snippet: CLGN promotes HCC cell proliferation in vitro . (A, B) Proliferation of Hep-G2 cells with stable CLGN knockdown was assessed by (A) colony formation assay and (B) CCK-8 assay. (C, D) Proliferation of Huh-7 cells with stable CLGN knockdown was assessed by (C) colony formation assay and (D) CCK-8 assay. (E, F) Proliferation of Hep-3B cells with stable CLGN overexpression was assessed by (E) colony formation assay and (F) CCK-8 assay. (G) Proliferation of CLGN-knockdown Hep-G2 and Huh-7 cells was assessed by EdU assay. Scale bar, 50 μm. (H) Proliferation of CLGN-overexpressing Hep-3B cells was assessed by EdU assay. Scale bar, 50 μm. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-test or one-way ANOVA).

Article Snippet: Immunohistochemical staining was performed via a two-step method with a human monoclonal anti-rabbit CLGN antibody (1:100, BOSTER), a KI-67 antibody (1:400, CST), and an NF-κB antibody (1:400, CST).

Techniques: In Vitro, Knockdown, Colony Assay, CCK-8 Assay, Over Expression, EdU Assay

Journal: Frontiers in Oncology

Article Title: Targeting endoplasmic reticulum stress-induced CLGN resensitizes hepatocellular carcinoma to apoptosis: paeonol synergistically enhances efficacy by dual inhibition of CLGN and NF-κB

doi: 10.3389/fonc.2025.1709962

Figure Lengend Snippet: CLGN promotes invasion, migration, and suppresses apoptosis in HCC cells in vitro . (A, B) Effects of CLGN knockdown in Hep-G2 cells on (A) wound healing migration and (B) Transwell invasion. (C, D) Effects of CLGN knockdown in Huh-7 cells on (C) wound healing migration and (D) Transwell invasion. (E, F) Effects of CLGN overexpression in Hep-3B cells on (E) wound healing migration and (F) Transwell invasion. (G) Apoptosis analysis by flow cytometry in CLGN-knockdown Hep-G2 and Huh-7 cells. (H) Apoptosis analysis by flow cytometry in CLGN-overexpressing Hep-3B cells. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-test or one-way ANOVA).

Article Snippet: Immunohistochemical staining was performed via a two-step method with a human monoclonal anti-rabbit CLGN antibody (1:100, BOSTER), a KI-67 antibody (1:400, CST), and an NF-κB antibody (1:400, CST).

Techniques: Migration, In Vitro, Knockdown, Over Expression, Flow Cytometry

Journal: Frontiers in Oncology

Article Title: Targeting endoplasmic reticulum stress-induced CLGN resensitizes hepatocellular carcinoma to apoptosis: paeonol synergistically enhances efficacy by dual inhibition of CLGN and NF-κB

doi: 10.3389/fonc.2025.1709962

Figure Lengend Snippet: CLGN knockdown enhances the anti-tumor efficacy of Pae by modulating ERS. (A, B) Hep-G2 control and CLGN-knockdown cells were treated with TM and/or Pae, followed by analysis of (A) apoptosis via flow cytometry and (B) clonogenic survival. (C) Representative images of resected tumors from the xenograft mouse model under different treatment conditions. (D) Tumor weights from each treatment group at the endpoint. (E) IHC analysis of Ki67, CLGN, and NF-κB expression in xenograft tumor tissues. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 (B, D: one-way ANOVA with Tukey’s post hoc test; A: two-way ANOVA).

Article Snippet: Immunohistochemical staining was performed via a two-step method with a human monoclonal anti-rabbit CLGN antibody (1:100, BOSTER), a KI-67 antibody (1:400, CST), and an NF-κB antibody (1:400, CST).

Techniques: Knockdown, Control, Flow Cytometry, Expressing

Journal: Frontiers in Oncology

Article Title: Targeting endoplasmic reticulum stress-induced CLGN resensitizes hepatocellular carcinoma to apoptosis: paeonol synergistically enhances efficacy by dual inhibition of CLGN and NF-κB

doi: 10.3389/fonc.2025.1709962

Figure Lengend Snippet: CLGN suppresses apoptosis through activation of the NF-κB pathway. (A) Volcano plot of DEGs from RNA sequencing of control versus CLGN-knockdown Hep-G2 cells. (B) Chord plot illustrating the results of combined GO/KEGG and logFC enrichment analysis for the identified DEGs. (C) Bar graph of the most significantly enriched KEGG pathways. (D) Western blot analysis of key NF-κB pathway proteins in Hep-G2 with CLGN knockdown and Hep-3B cells with CLGN overexpression. (E) Western blot analysis of CLGN, NF-κB, and Bcl-2 expression in control and CLGN-knockdown Hep-G2 cells treated with TM or TM+Pae. (F) Western blot analysis of CLGN, NF-κB, and Bcl-2 expression in vector-control and CLGN-overexpressing Hep-3B cells treated with the NF-κB inhibitor PDTC or Pae. GAPDH was used as a loading control for all Western blot analyses.

Article Snippet: Immunohistochemical staining was performed via a two-step method with a human monoclonal anti-rabbit CLGN antibody (1:100, BOSTER), a KI-67 antibody (1:400, CST), and an NF-κB antibody (1:400, CST).

Techniques: Activation Assay, RNA Sequencing, Control, Knockdown, Western Blot, Over Expression, Expressing, Plasmid Preparation

Journal: Scientific Reports

Article Title: A novel immune-related gene signature for diagnosis and potential immunotherapy of microsatellite stable endometrial carcinoma

doi: 10.1038/s41598-024-53338-z

Figure Lengend Snippet: The immunochemistry stain results of four novel screened IRGs.

Article Snippet: The slides were incubated with rabbit anti-AGTR1 primary antibody (Boster, BA0582) in a dilution of 1:200, rabbit anti-HTR3C antibody (Solarbio, K006945P) in a dilution of 1:50, rabbit anti-SERPIND1 antibody (Immunoway, YT2109) in a dilution of 1:200, and mouse anti-CD3E antibody (ZSGB-BIO, TA506064) in a dilution of 1:150 overnight at 4 °C separately.

Techniques: Staining

Journal: Scientific Reports

Article Title: A novel immune-related gene signature for diagnosis and potential immunotherapy of microsatellite stable endometrial carcinoma

doi: 10.1038/s41598-024-53338-z

Figure Lengend Snippet: Expression verification of the IRGS. ( A – B ) Gene expression analysis of each gene in IRGS between Microsatellite stable (MSS), Microsatellite instable (MSI) and non-endometrial carcinoma status. ( C ) Immunohistochemistry results of each gene in IRGS (AGTR1, HTR3C, CD3E, SERPIND1) between endometrial carcinoma and normal endometrium tissues respectively.

Article Snippet: The slides were incubated with rabbit anti-AGTR1 primary antibody (Boster, BA0582) in a dilution of 1:200, rabbit anti-HTR3C antibody (Solarbio, K006945P) in a dilution of 1:50, rabbit anti-SERPIND1 antibody (Immunoway, YT2109) in a dilution of 1:200, and mouse anti-CD3E antibody (ZSGB-BIO, TA506064) in a dilution of 1:150 overnight at 4 °C separately.

Techniques: Expressing, Gene Expression, Immunohistochemistry