Journal: Advanced Science

Article Title: Steroid Receptor Coactivator‐1 Drives Tumor‐Associated Macrophage Reprogramming by Mediating MMP12 Transcription in Pancreatic Cancer Perineural Invasion

doi: 10.1002/advs.202416575

Figure Lengend Snippet: TAMs reprogrammed by SRC‐1 secretion MMP12 induce PNI. A) BMDMs were induced into TAMs from WT and SRC‐1 ‐/‐ mice, and CM was collected for differential protein analysis by mass spectrometry (n = 3). B) Volcano plot of differentially expressed proteins between WT and SRC‐1 ‐/‐ groups. C) qPCR analysis revealed decreased MMP12 mRNA levels upon SRC‐1 downregulation.n = 3. Significance was calculated with Student's t ‐test. **** P< 0.0001. D, E) Western blotting detected the reduced MMP12 protein expression following SRC‐1 down‐regulation. n = 3. Significance was calculated with one‐way ANOVA with Tukey post‐test. * P< 0.05. F) Immunofluorescence staining to detect the expression of MMP12 in mouse tissues from the sciatic nerve injection models. G,H) Western blot confirmation demonstrated successful SRC‐1 knockdown and MMP12 over‐expression. n = 3. Significance was calculated with one‐way ANOVA with Tukey post‐test. * P< 0.05, **** P< 0.0001. I) CCK8 assays were utilized to indicate the enhanced proliferation of AsPC‐1 cells upon SRC‐1 down‐regulation and MMP12 over‐expression in TAMs. n = 3. Significance was calculated with one‐way ANOVA with Tukey post‐test. *** P< 0.001, **** P< 0.0001. J) Rescue experiments assessing changes in in vitro PNI following SRC‐1 down‐regulation and MMP12 over‐expression. The red arrow indicates the leading edge of pancreatic cancer cell invasion. K) The neural invasion index of the rescue experiments. n = 3. Significance was calculated with one‐way ANOVA with Tukey post‐test. * P< 0.05, ** P< 0.01. L–O) Wound healing and Trans‐well assays demonstrated the increased migration and invasion ability of AsPC‐1 cells upon SRC‐1 down‐regulation and MMP12 over‐expression in TAMs.n = 3. For wound healing assays: the scale bar is 500 µm. For the Transwell assay, the scale bar is 400 µm.n = 3. Significance was calculated with one‐way ANOVA with Tukey post‐test. * P <0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Wet transfer was performed at 350 mA for 1.5 h. The membrane was blocked with 5% non‐fat dry milk at room temperature(25 °C) for 1.5 h. Primary antibodies were diluted according to recommended ratios: SRC‐1 (CST, 2191, 1:1000), CD206 (Proteintech, 60 143, 1:5000), CD163 (WANLEI, WL03026, 1:1000), iNOS (Proteintech, 22 226, 1:1000), MMP12 (Proteintech, 22 989, 1:500), STAT1 (CST, 9172, 1:1000), p‐STAT1 (CST, 7649, 1:1000), and GAPDH (Proteintech, 10 494, 1:5000).

Techniques: Mass Spectrometry, Western Blot, Expressing, Immunofluorescence, Staining, Injection, Knockdown, Over Expression, In Vitro, Migration, Transwell Assay

Journal: Advanced Science

Article Title: Steroid Receptor Coactivator‐1 Drives Tumor‐Associated Macrophage Reprogramming by Mediating MMP12 Transcription in Pancreatic Cancer Perineural Invasion

doi: 10.1002/advs.202416575

Figure Lengend Snippet: SRC‐1 as a coactivator of STAT1, promotes MMP12 promoter activity. A, B) Western blot analysis of p‐STAT1 and STAT1 expression in TAMs under different treatments. n = 3. Significance was calculated with Student's t ‐test. * P< 0.05. C, D) Western blot analysis of p‐NF‐κB, NF‐κB, IRF4, and PPAR‐γ expression in TAMs under different treatments.n = 3. Significance was calculated with one‐way ANOVA with Tukey post‐test. * P< 0.05, ** P< 0.01, ns, no significant difference. E, F) Co‐immunoprecipitation experiments detecting the interaction between SRC‐1 and STAT1. n = 3. Significance was calculated with Student's t ‐test. * P< 0.05, ** P< 0.01, *** P< 0.001. G) Immunofluorescence validation of co‐localization of STAT1 (green) and SRC‐1 (red) in TAMs. H) Prediction by Jaspar of STAT1 binding sites on the MMP12 promoter region. I, J) ChIP assay confirming recruitment of STAT1 to the MMP12 promoter region. K, L) Luciferase assays demonstrating changes in MMP12 promoter luciferase activity upon knockdown or over‐expression of SRC‐1. n = 3. Significance was calculated with Student's t ‐test. * P < 0.05, ** P < 0.01.

Article Snippet: Wet transfer was performed at 350 mA for 1.5 h. The membrane was blocked with 5% non‐fat dry milk at room temperature(25 °C) for 1.5 h. Primary antibodies were diluted according to recommended ratios: SRC‐1 (CST, 2191, 1:1000), CD206 (Proteintech, 60 143, 1:5000), CD163 (WANLEI, WL03026, 1:1000), iNOS (Proteintech, 22 226, 1:1000), MMP12 (Proteintech, 22 989, 1:500), STAT1 (CST, 9172, 1:1000), p‐STAT1 (CST, 7649, 1:1000), and GAPDH (Proteintech, 10 494, 1:5000).

Techniques: Activity Assay, Western Blot, Expressing, Immunoprecipitation, Immunofluorescence, Biomarker Discovery, Binding Assay, Luciferase, Knockdown, Over Expression

Journal: Advanced Science

Article Title: Steroid Receptor Coactivator‐1 Drives Tumor‐Associated Macrophage Reprogramming by Mediating MMP12 Transcription in Pancreatic Cancer Perineural Invasion

doi: 10.1002/advs.202416575

Figure Lengend Snippet: Inhibition of MMP12 can effectively alleviate PNI both in vivo and in vitro. A, B) Paralysis severity in the hind limbs of mice from various groups following sciatic nerve co‐injection of tumor and macrophage mixtures. Groups: (1) WT BMDM, (2) SRC‐1‐KO BMDM, (3) WT BMDM + vehicle, and (4) WT BMDM + MMP408 (5 mg/kg). n = 19 C) Tumor growth and invasion in the sciatic nerve of mice were recorded. D) Body weight changes in mice over a 21‐day period following sciatic nerve co‐injection of tumor and macrophage mixtures were recorded. E) Sciatic functional index in mice over 21 days following co‐injection of tumor and macrophage mixtures was recorded. WT‐BMDM+KRAS/p53 /m/+ group: n = 4, the other groups:n = 5. Significance was calculated with one‐way ANOVA with Tukey post‐test at the final time point. ** P< 0.01. F) Sciatic functional scores in mice over 21 days following co‐injection of tumor and macrophage mixtures were recorded. WT‐BMDM+KRAS/p53 /m/+ group:n = 4, the other groups:n = 5. Significance was calculated with one‐way ANOVA with Tukey post‐test at the final time point. **** P< 0.0001. G) Tumor weight in mice harvested at 21 days post‐injection was recorded. WT‐BMDM+KRAS/p53 /m/+ group:n = 4, the other groups:n = 5. Significance was calculated with one‐way ANOVA with Tukey post‐test. * P< 0.05, ** P< 0.01, ns, no significant difference. H) The length of invasion in the mouse groups was recorded. WT‐BMDM+KRAS/p53 /m/+ group:n = 4, the other groups:n = 5. Significance was calculated with one‐way ANOVA with Tukey post‐test. ** P< 0.01. I) Quantitative analysis of CD206 + SRC‐1 + dual‐positive cells was performed in murine tissue using dual‐immunofluorescence staining.WT‐BMDM+KRAS/p53 /m/+ group:n = 4, the other groups:n = 5. Significance was calculated with Student's t ‐test. * P< 0.05. J) Neural invasion index was recorded in the DRG‐tumor cells co‐culture assay. WT‐BMDM+KRAS/p53 /m/+ group:n = 4, the other groups:n = 5. Significance was calculated with one‐way ANOVA with Tukey post‐test. ** P< 0.01, *** P< 0.001. K) Multiplex immunofluorescence co‐staining demonstrating expression of SRC‐1 (red), CD206 (yellow), and S100β (green) in murine tumor tissues. L) A DRG and tumor cell (red) co‐culture model was utilized to quantitatively assess tumor cell PNI capacity ex vivo.

Article Snippet: Wet transfer was performed at 350 mA for 1.5 h. The membrane was blocked with 5% non‐fat dry milk at room temperature(25 °C) for 1.5 h. Primary antibodies were diluted according to recommended ratios: SRC‐1 (CST, 2191, 1:1000), CD206 (Proteintech, 60 143, 1:5000), CD163 (WANLEI, WL03026, 1:1000), iNOS (Proteintech, 22 226, 1:1000), MMP12 (Proteintech, 22 989, 1:500), STAT1 (CST, 9172, 1:1000), p‐STAT1 (CST, 7649, 1:1000), and GAPDH (Proteintech, 10 494, 1:5000).

Techniques: Inhibition, In Vivo, In Vitro, Injection, Functional Assay, Immunofluorescence, Staining, Co-culture Assay, Multiplex Assay, Expressing, Co-Culture Assay, Ex Vivo

Journal: Advanced Science

Article Title: Steroid Receptor Coactivator‐1 Drives Tumor‐Associated Macrophage Reprogramming by Mediating MMP12 Transcription in Pancreatic Cancer Perineural Invasion

doi: 10.1002/advs.202416575

Figure Lengend Snippet: Schematic illustrating working model for the mechanisms by which SRC‐1 drives TAMs reprogramming by mediating MMP12 transcription in pancreatic cancer PNI. SRC‐1 binds to STAT1 to enhance the transcriptional activity of the MMP12 promoter, thereby increasing MMP12 secretion from TAMs.

Article Snippet: Wet transfer was performed at 350 mA for 1.5 h. The membrane was blocked with 5% non‐fat dry milk at room temperature(25 °C) for 1.5 h. Primary antibodies were diluted according to recommended ratios: SRC‐1 (CST, 2191, 1:1000), CD206 (Proteintech, 60 143, 1:5000), CD163 (WANLEI, WL03026, 1:1000), iNOS (Proteintech, 22 226, 1:1000), MMP12 (Proteintech, 22 989, 1:500), STAT1 (CST, 9172, 1:1000), p‐STAT1 (CST, 7649, 1:1000), and GAPDH (Proteintech, 10 494, 1:5000).

Techniques: Activity Assay

Journal: MedComm

Article Title: Novel aspect of neprilysin in kidney fibrosis via ACSL4‐mediated ferroptosis of tubular epithelial cells

doi: 10.1002/mco2.330

Figure Lengend Snippet: Genetic knockdown of neprilysin improved the fibrotic and ferroptotic phenotype in TGFβ1‐induced TCMK‐1 cells. (A and B) The knockdown efficiency of NEP siRNA (siNEP) in TCMK‐1 cells evaluated by RT‐PCR and Western blot analysis. (C) Representative Western blot images and quantitative analysis of NEP expression in TCMK‐1 cells. (D) RT‐PCR was performed to detect the mRNA level of Fn, α‐SMA, and Col Iα1. (E) RT‐PCR was performed to detect the mRNA level of ACSL4, GPX4, and Ptgs2 (COX2). (F) Western blot analysis was performed to detect protein expression of Fn, α‐SMA, and Col Iα1. (G) Western blot analysis was performed to detect protein expression of ACSL4, GPX4, and COX2. (H) The accumulation of lipid peroxidation in TCMK‐1 cells was analyzed by BODIPY C11 staining (×200, scale bar = 50 μm). NS p > 0.05, versus control. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, versus siNC. ### p < 0.001, ## p < 0.01, # p < 0.05, versus siNC+TGF‐β1. siNC, negative control.

Article Snippet: Primary antibodies against NEP (1:200, 18008‐1‐AP; Proteintech, USA), α‐SMA (1:100, ET1607‐43; Huabio, China), and ACSL4 (1:200, 81196‐1‐RR; Proteintech) were used in the study.

Techniques: Knockdown, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Staining, Control, Negative Control

Journal: MedComm

Article Title: Novel aspect of neprilysin in kidney fibrosis via ACSL4‐mediated ferroptosis of tubular epithelial cells

doi: 10.1002/mco2.330

Figure Lengend Snippet: Neprilysin aggravated fibrosis by ACSL4‐mediated ferroptosis in TGF‐β1‐induced TCMK‐1 cells. (A and B) The efficiency of NEP overexpression in TCMK‐1 cells evaluated by RT‐PCR and Western blot analysis. (C and D) The overexpression efficiency of ACSL4 in TCMK‐1 cells evaluated by RT‐PCR and Western blot analysis. (E and F) The knockdown efficiency of ACSL4 siRNA in TCMK‐1 cells evaluated by RT‐PCR and Western blot analysis. (G) RT‐PCR was performed to detect the mRNA expression of ACSL4, GPX4, and Ptgs2 (COX2). (H) Western blot analysis was performed to detect protein expression of ACSL4, GPX4, and COX2. (J) RT‐PCR was performed to detect the mRNA expression of Fn, α‐SMA, and Col Iα1. (I) Western blot analysis was performed to detect protein expression of Fn, α‐SMA, and Col Iα1. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, versus oeNC or siNC or oeNC+siNC. #### p < 0.0001, ### p < 0.001, ## p < 0.01, # p < 0.05, versus oeNC+siNC+TGF‐β1. $$$$ p < 0.0001, $$ p < 0.01, $ p < 0.05, versus oeNEP+siNC+TGF‐β1. siNC, silencing negative control; siNEP, NEP siRNA; siACSL4, ACSL4 siRNA; oeNC, overexpression negative control; oeNEP, overexpression of NEP; oeACSL4, overexpression of ACSL4.

Article Snippet: Primary antibodies against NEP (1:200, 18008‐1‐AP; Proteintech, USA), α‐SMA (1:100, ET1607‐43; Huabio, China), and ACSL4 (1:200, 81196‐1‐RR; Proteintech) were used in the study.

Techniques: Over Expression, Reverse Transcription Polymerase Chain Reaction, Western Blot, Knockdown, Expressing, Negative Control

Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: CD10 , REAL318 , 50 , 130-118-368 , FITC , Miltenyi Biotec.

Techniques: Imaging

Journal: Scientific Reports

Article Title: ADAMDEC1 promotes the malignant progression of cholangiocarcinoma by regulating NF-κB signaling pathway

doi: 10.1038/s41598-025-85241-6

Figure Lengend Snippet: The expression correlation between ADAMDEC1 and MMP12 in CCA. A According to the String and TIMER2.0 databases, the protein-protein interaction network indicates that the ADAMDEC1 gene has a direct interaction with MMP12, with findings consistent across both databases. B CO-IP experiment tested the co-expression of ADAMDEC1 and MMP12 in cell lines. C IF assay detected the co-localization of MMP12 and ADAMDEC1 protein expression in each group (×400). D IHC assay assessed MMP12 protein expression in five pairs of human CCA and paratumor tissues (×400). IHC assay measured the MMP12 protein expression level in a nude mouse xenograft model of CCA between two groups (×200).

Article Snippet: The primary antibodies used in this study included GAPDH (Proteintech, 60004-1-Ig, 36 kDa), ADAMDEC1 (17899-1-AP, 44 kDa), TNFR2 (19272-1-AP, 70-75 kDa), p65 (10745-1-AP, 65 kDa), p-p65 (ab76302, 65 kDa), inhibitor of kappa B alpha (IκBα) (ab32518, 35 kDa), phosphorylated (p)-IκBα (ab133462, 35 kDa), MMP12 (ab52897, 54 kDa), and NF-κB activator 1 (HY-134476, MCE).

Techniques: Expressing, Co-Immunoprecipitation Assay

Journal: Scientific Reports

Article Title: ADAMDEC1 promotes the malignant progression of cholangiocarcinoma by regulating NF-κB signaling pathway

doi: 10.1038/s41598-025-85241-6

Figure Lengend Snippet: The interference efficiency of MMP12 and the biological behavior of CCA cells in vitro. A Interference sequences were transfected into QBC939 and RBE cell lines. qRT-PCR and Western blot assays were conducted to detect the mRNA and protein expression levels of MMP12 in the interference target groups (siMMP12-1, -2, -3). B The CCK-8 assay was used to analyze the proliferation properties of the cell lines. Cell proliferation was assessed at five time points to obtain relative growth data (** P < 0.01). C Flow cytometry analysis was performed to assess apoptosis properties. The proportion of apoptotic cells in the siMMP12 group was compared with the siCtrl group in QBC939 and RBE cell lines, as shown in the histogram(*** P < 0.001). D The wound healing assay was used to analyze migration ability. The migration ability was observed in the siMMP12 group at 24 h and 48 h in the QBC939 and RBE cell lines(* P < 0.05, ** P < 0.01). E Transwell chamber technology (+ ECM) was employed to determine invasive properties. A total of five different view fields were used to count the number of invading cells. The transwell assay demonstrated that invasive ability was affected in both the siMMP12 and siCtrl groups. The count was repeated thrice (** P < 0.01, *** P < 0.001).

Article Snippet: The primary antibodies used in this study included GAPDH (Proteintech, 60004-1-Ig, 36 kDa), ADAMDEC1 (17899-1-AP, 44 kDa), TNFR2 (19272-1-AP, 70-75 kDa), p65 (10745-1-AP, 65 kDa), p-p65 (ab76302, 65 kDa), inhibitor of kappa B alpha (IκBα) (ab32518, 35 kDa), phosphorylated (p)-IκBα (ab133462, 35 kDa), MMP12 (ab52897, 54 kDa), and NF-κB activator 1 (HY-134476, MCE).

Techniques: In Vitro, Transfection, Quantitative RT-PCR, Western Blot, Expressing, CCK-8 Assay, Flow Cytometry, Wound Healing Assay, Migration, Transwell Assay