organoid growth kit 1a  (ATCC)

Journal: Frontiers in Immunology

Article Title: Targeting Pin1 to overcome immunosuppressive tumor microenvironment in MSS colorectal cancer

doi: 10.3389/fimmu.2025.1677029

Figure Lengend Snippet: Pin1 promotes Tregs chemotactic migration through the CCL3-CCR5 axis. (A) Correlation analysis of Pin1 with CCL chemokines expression. (B, C) qRT-PCR and ELISA assessing CCL3 expression in SW480 and HT29 cells after Pin1 knockdown. (D) Schematic representation of PBMCs isolated from human peripheral blood; Treg cells sorted by flow cytometry, these populations co-cultured with siNC- or siPin1-CRC cells, respectively. (E, F) Level of Treg markers (CD25 + CD127 - ) on PBMCs in the co-culture system detected by flow cytometry. (G) Effect of supernatants from Pin1-knockdown cells treated with recombinant CCL3 protein on Treg chemotactic capacity. (H) Correlation between CCL3 and CCR5 expression in CRC tissues. (I) CCR5-neutralizing antibodies abolish differences in Treg chemotaxis between the siNC and siPin1 groups.

Article Snippet: The concentrations of CCL3 in culture media were evaluated using Human CCL3 (MIP-1a) ELISA Kit (liankebio, cat.no.

Techniques: Migration, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Knockdown, Isolation, Flow Cytometry, Cell Culture, Co-Culture Assay, Recombinant, Chemotaxis Assay

Journal: Frontiers in Immunology

Article Title: Targeting Pin1 to overcome immunosuppressive tumor microenvironment in MSS colorectal cancer

doi: 10.3389/fimmu.2025.1677029

Figure Lengend Snippet: Pin1 regulates CCL3 expression to promote Treg recruitment and CAFs activation through the NF-κB signaling pathway. (A) Validation of Pin1 interaction with p65 using CO-IP. (B) Co-localization of Pin1 with P65 confirmed using IF. (C) The main components of the NF-κB signaling pathway on knockdown of Pin1, as determined using WB. (D) Representative images of p65 IHC staining in subcutaneous tumors in mice (N.S. + IgG, Pin1 + IgG) (n=5 per group). (E) qRT-PCR assessed CCL3 expression in SW620 cells after Pin1 overexpression with/without NF-κB inhibitor treatment. (F) NF-κB inhibitor eliminated Pin1-overexpression-induced differences in Treg chemotaxis. (G) NF-κB inhibitor abrogated the activating effect of Pin1 overexpression on CAFs, as analyzed via IF.

Article Snippet: The concentrations of CCL3 in culture media were evaluated using Human CCL3 (MIP-1a) ELISA Kit (liankebio, cat.no.

Techniques: Expressing, Activation Assay, Biomarker Discovery, Co-Immunoprecipitation Assay, Knockdown, Immunohistochemistry, Quantitative RT-PCR, Over Expression, Chemotaxis Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: CXCL12/CXCR4 modulates macrophage efferocytosis to induce glomerular crescent formation and fibrosis via ELMO1/DOCK180/RAC1 signaling in ANCA-associated glomerulonephritis

doi: 10.1007/s00018-025-05750-5

Figure Lengend Snippet: Release of the chemokine CXCL12 by endothelial cells in AAGN promoted monocyte recruitment and infiltration. A Spatial transcriptome sequencing of renal tissues revealed that CCL2, CCL21 and CXCL12 were increased in AAGN group, of which CXCL12 was the highest. B The immunohistochemical staining of CXCL12 was increased in AAGN group (AAGN = 6, CTRL = 4, 80 ×, scale bar = 25 μm). C ELISA levels of CXCL12 were elevated in MPO + AAGN plasma ( n = 15) and urine ( n = 6). D ELISA levels of CXCL12 were not significantly increased in PR3 + AVV plasma ( n = 10) and PR3 + AAGN urine ( n = 9). E CXCL12 mRNA was increased in EA.hy926 after 3 days of incubation with AAGN serum medium, but not in HRMC, HPC and HK-2 ( n = 3). F Western blotting showed that after 3 days of incubation with AAGN serum medium, CXCL12 protein level was increased in EA.hy926, while not changed in HRMC, HPC and HK-2 ( n = 3). G CD14 immunofluorescence was increased in the crescents of AAGN group (AAGN = 6, CTRL = 4, PAS 80 ×, scale bar = 25 μm, IF 600 ×, scale bar = 100 μm). H Transwell assay confirmed that EA.hy926 incubated with AAGN serum in the lower chamber could recruit CD14. + monocytes from the upper chamber to migrate to the lower chamber ( n = 3, 400 ×, scale bar = 10 μm). * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: CXCL12 (CSB-EQ027490HU for human, CSB-E08729r for rat, Cusabio, Wuhan, China) and soluble CXCR4 (CSB- E12825 h for human, CSB-E12703r for rat, Cusabio) in serum and urine were measured with ELISA kits following the manufacturer’s instructions.

Techniques: Sequencing, Immunohistochemical staining, Staining, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Incubation, Western Blot, Immunofluorescence, Transwell Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: CXCL12/CXCR4 modulates macrophage efferocytosis to induce glomerular crescent formation and fibrosis via ELMO1/DOCK180/RAC1 signaling in ANCA-associated glomerulonephritis

doi: 10.1007/s00018-025-05750-5

Figure Lengend Snippet: Glomerular endothelial cell apoptosis in AAGN resulted in CXCL12 release. A Immunofluorescence levels of CD31 and ERG were decreased in AAGN crescents, whereas TUNEL was increased (AAGN = 6, CTRL = 4, PAS 80 ×, scale bar = 50 μm, IF 600 ×, scale bar = 20 μm). B The mRNA and protein levels of BCL-2/BAX were decreased in endothelial cells incubated with AAGN serum ( n = 3). C The mRNA and protein levels of cleaved-Caspase-3 were decreased in endothelial cells incubated with AAGN serum ( n = 3). D The mRNA and protein levels of CD31 and ERG were decreased in endothelial cells incubated with AAGN serum ( n = 3). E CCK-8 assay showed that with the prolonged incubation time of AAGN serum, the proliferation ability of endothelial cells decreased, and the most obvious was observed at 72–84h ( n = 3). F Flow cytometry verified that the proportion of apoptotic endothelial cells was significantly increased when incubated with AAGN serum for 3 days ( n = 3). * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: CXCL12 (CSB-EQ027490HU for human, CSB-E08729r for rat, Cusabio, Wuhan, China) and soluble CXCR4 (CSB- E12825 h for human, CSB-E12703r for rat, Cusabio) in serum and urine were measured with ELISA kits following the manufacturer’s instructions.

Techniques: Immunofluorescence, TUNEL Assay, Incubation, CCK-8 Assay, Flow Cytometry

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: CXCL12/CXCR4 modulates macrophage efferocytosis to induce glomerular crescent formation and fibrosis via ELMO1/DOCK180/RAC1 signaling in ANCA-associated glomerulonephritis

doi: 10.1007/s00018-025-05750-5

Figure Lengend Snippet: Inhibition of CXCL12/CXCR4 alleviates AAGN progression. A Suitable concentrations for LIT927 (CXCL12 neutral ligand antagonist) and AMD3100 (CXCR4 inhibitor) were determined as 30 μM and 100 μM, respectively, using CCK-8 assays ( n = 3). B When mixed individually with AAGN serum to stimulate macrophages (Mφs), both drugs reduced MERTK, CD163, TGF-β1 and ELMO1/DOCK180/RAC1 axis components at transcriptional and protein levels ( n = 3). C Schematic of the EAV rat model establishment. Drug administration, either concurrently with modeling or from the third week post-modeling, did not significantly affect rat body weight D but alleviated hematuria E and proteinuria F , with AMD3100 showing greater efficacy. Starting treatment at the third week was as effective as concurrent administration. G LIT927 administration at the onset of modeling reduced cellular and fibrous crescent formation. When started from the third week, it alleviated fibrocellular and fibrous crescents without affecting cellular crescents. AMD3100 reduced various types of crescent formation when given at modeling onset but primarily alleviated fibrocellular and fibrous crescents when started three weeks later (80 ×, scale bar = 10 μm, n = 4). * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001

Article Snippet: CXCL12 (CSB-EQ027490HU for human, CSB-E08729r for rat, Cusabio, Wuhan, China) and soluble CXCR4 (CSB- E12825 h for human, CSB-E12703r for rat, Cusabio) in serum and urine were measured with ELISA kits following the manufacturer’s instructions.

Techniques: Inhibition, CCK-8 Assay

Journal: Journal of cellular and molecular medicine

Article Title: The Impact of METTL3 on MDM2 Promotes Podocytes Injury During Diabetic Kidney Disease.

doi: 10.1111/jcmm.70627

Figure Lengend Snippet: FIGURE 5 | Inhibiting podocytes MDM2 expression alleviates renal pathological damage, and reduces podocytes dedifferentiation levels. (A) Representative images showing PAS and Masson staining (scale bar = 20 μm) in control, STZ, and shMDM2-injected STZ groups. (B) IF staining of Ki-67 (red), Cyclin B1 (green), Podocin (yellow), and MDM2 (pink), counterstained with DAPI (blue) in control, STZ and STZ injected with shMDM2 groups. (C) Protein levels of p21, Cyclin B1 and Podocin in control, STZ and STZ injected with shMDM2 groups, analysed by western blot. Data rep- resent mean ± SD of three independent experiments.

Article Snippet: The following antibodies were used at these dilutions for western blot analysis: METTL3 (Abcam ab195352, 1:1000), METTL14 (Sigma, HPA038002, 1:1000), FTO (Proteintech, 27226- 1- AP, 1:1000), synaptopodin (synap) (Sigma- Aldrich, SAB3500585, 1:500), podocin (Abcam, ab181143, 1:500), caspase 3 (Abcam, ab184787, 1:500), B- cell lymphoma/leukaemia- 2 (Bcl2) (Abcam, ab182858, 1:1000), MDM2 (Santa Cruz Biotechnology, sc- 965, 1:500), IGF2BP2 (Proteintech, 11,601- 1- AP, 1:1000), proliferating cell nuclear antigen (PCNA) (Abcam, ab92552, 1:1000), cyclin B1 (Abcam, ab181593, 1:500), cyclin- dependent kinase inhibitor 1A (P21) (Proteintech, 28,248- 1- AP for mouse, 1:1000), Notch intracellular domain (NICD) (Abcam, ab52627, 1:500), and hairy and enhancer of split 1 (Hes1) (Abcam, ab71559, 1:500).

Techniques: Expressing, Staining, Control, Injection, Western Blot

Journal: Journal of cellular and molecular medicine

Article Title: The Impact of METTL3 on MDM2 Promotes Podocytes Injury During Diabetic Kidney Disease.

doi: 10.1111/jcmm.70627

Figure Lengend Snippet: FIGURE 6 | MDM2 regulates the Notch1 signalling pathway in podocytes during AGE-induced abnormal cell cycle regulation. (A, B) Western blot analysis and semi-quantitative assessment of NICD and Hes1 protein levels in BSA-treated, AGE-treated, and siMDM2-transfected AGE-treated groups. (C, D) Western blot analysis and semi-quantitative assessment of NICD and Hes1 protein levels in control, STZ-treated, and shMDM2-injected STZ-treated groups. (E) Evaluation of NICD and Hes1 protein levels in control, STZ-treated and shMDM2-injected STZ-treated groups by IHC as- say (scale bar = 50 μm). (F, G) Western blot analysis and semi-quantitative assessment of Hes1, Cyclin B1 and p-H3 protein levels in AGE-treated, AGE + siMDM2-treated, and Jagged1-treated AGE + siMDM2 groups. Data represent mean ± SD of three independent experiments. **p < 0.01 versus AGE group (B), or STZ group (D), or AGE + siMDM2 group (G) by one-way ANOVA.

Article Snippet: The following antibodies were used at these dilutions for western blot analysis: METTL3 (Abcam ab195352, 1:1000), METTL14 (Sigma, HPA038002, 1:1000), FTO (Proteintech, 27226- 1- AP, 1:1000), synaptopodin (synap) (Sigma- Aldrich, SAB3500585, 1:500), podocin (Abcam, ab181143, 1:500), caspase 3 (Abcam, ab184787, 1:500), B- cell lymphoma/leukaemia- 2 (Bcl2) (Abcam, ab182858, 1:1000), MDM2 (Santa Cruz Biotechnology, sc- 965, 1:500), IGF2BP2 (Proteintech, 11,601- 1- AP, 1:1000), proliferating cell nuclear antigen (PCNA) (Abcam, ab92552, 1:1000), cyclin B1 (Abcam, ab181593, 1:500), cyclin- dependent kinase inhibitor 1A (P21) (Proteintech, 28,248- 1- AP for mouse, 1:1000), Notch intracellular domain (NICD) (Abcam, ab52627, 1:500), and hairy and enhancer of split 1 (Hes1) (Abcam, ab71559, 1:500).

Techniques: Western Blot, Transfection, Control, Injection