Journal: Cell Death & Disease

Article Title: MiR-3622a-3p acts as a tumor suppressor in colorectal cancer by reducing stemness features and EMT through targeting spalt-like transcription factor 4

doi: 10.1038/s41419-020-02789-z

Figure Lengend Snippet: a Expression level of miR-3622a-3p was detected in CRC cell lines and NCM460 cell line by qRT-PCR. b Expression of miR-3622a-3p was increased in DLD-1 by miR-3622a-3p mimics transfection. c MiR-3622a-3p expression level was reduced in HCT116 by miR-3622a-3p inhibitor transfection. d , e The effect of miR-3622a-3p on proliferation of CRC cells was evaluated by CCK-8 cell proliferation assay. f , g Colony forming ability of CRC cells was negatively corelated with miR-3622a-3p expression level. h , i The results of EDU assay suggested overexpression of miR-3622a-3p suppressed proliferation of CRC cells while knockdown of miR-3622a-3p promoted CRC cell proliferation. All data are from three independent experiments and are presented as the means ± SD (* p < 0.05, ** p < 0.01).

Article Snippet: Human normal colon epithelial cell line NCM460 was obtained from American Type Culture Collection (ATCC, USA).

Techniques: Expressing, Quantitative RT-PCR, Transfection, CCK-8 Assay, Proliferation Assay, EdU Assay, Over Expression, Knockdown

Journal: Neural Regeneration Research

Article Title: Elucidation of the mechanism by which manganese-ferric Prussian blue nanozymes alleviate ischemic stroke damage in a mouse model

doi: 10.4103/nrr.nrr-d-24-00837

Figure Lengend Snippet: Figure 6 ｜Therapeutic mechanisms of MnPB nanozymes and SCFAs in MCAO model. (A) Illustration of the concentration-dependent cellular uptake of MnPB nanozymes, highlighting the biocompatibility and internalization efficiency in NCM460 cells. Scale bar: 50 μm. (B) Assessment of the toxic effects of MnPB nanozymes on NCM460 cells, demonstrating safety within a concentration range of 0–50 μg/mL (n = 5). (C) Assessment of the toxic effects of MnPB nanozymes on BV-2 cells, demonstrating safety within a concentration range of 0–50 μg/mL (n = 5) (D) Examination of the toxic impact of H2O2 on BV-2 cells, establishing a baseline for oxidative stress-induced injury (n = 5). (E) Evaluation of the protective effects of different concentrations of MnPB nanozymes and SCFAs on cell survival in an in vitro oxidative stress injury model induced by H2O2 (n = 5). (F–H) Determination of the cytotoxicity of different concentrations of sodium acetate (NaA), sodium propionate (NaP), and sodium butyrate (NaB) on BV-2 cells after a 12-hour treatment period using the CCK-8 kit (n = 5). (I) Assessment of the combined toxicity of NaA, NaP, NaB, and SCFAs mixtures on BV-2 cells after a 12-hour exposure, confirming the safety of the selected concentration ratios (n = 5). (J) Utilization of the DCFH-DA assay to measure the reactive oxygen species (ROS) scavenging effect of varying concentrations of SCFAs. (K) Quantitative analysis of ROS spectral intensity, providing insights into the antioxidant capacity of SCFAs (n = 3). (L) Flow cytometry results depicting the apoptotic effects in BV-2 cells under oxidative stress. (M) Statistical analysis of apoptosis rates, revealing the protective role of SCFAs against cell death (n = 3). (N) Analysis of LPS-induced inflammation in BV-2 cells and the subsequent impact of SCFAs supplementation on the expression of TLR4, IKKα, and p65, as detected by immunoblotting. (O) Presentation of protein expression level statistics, indicating the modulatory effects of SCFAs on the TLR4/NF-κB pathway (n = 3). (P) Detection of TLR4, IKKα, and pp65 expression in BV-2 cells following blockade of the TLR4 pathway, further elucidating the mechanism of action (n = 3). (Q) Quantitative analysis of protein expression levels, reinforcing the role of TLR4/NF-κB signaling in the observed effects (n = 3). (R) Immunoblotting detection of TLR4, IKKα, and pp65 expression in brain tissues, providing in vivo evidence of the pathway’s involvement. (S) Quantitative analysis of protein expression density relative to the control group, underscoring the significance of TLR4/NF-κB modulation in the therapeutic effects (n = 3). Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, vs. Control group; #P < 0.05, ##P < 0.01, ###P < 0.001, vs. MCAO group (one- way analysis of variance followed by Tukey’s post hoc test). CCK-8: Cell counting kit-8; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; IKKα: inhibitory kappa B kinase α; MCAO: middle cerebral artery occlusion; MnPB: manganese-iron Prussian blue; NF-κB: nuclear factor κB; RBITC: Rhodamine B isothiocyanate; SCFA: short-chain fatty acid; TLR4: Toll-like receptor 4.

Article Snippet: Cell culture and in vitro experimental design The human normal colonic epithelial cell line NCM460 (American Type Culture Collection, Manassas, VA, USA, CRL-1807, RRID:CVCL_2872) and BV-2 microglial cells (American Type Culture Collection, CRL-2467, RRID:CVCL_5744) were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Carlsbad, CA, USA, Cat# 11965092) containing 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA, Cat# A5669701) and 1% of an antibiotic mixture containing penicillin/streptomycin (Gibco, Cat# 15140122).

Techniques: Concentration Assay, In Vitro, CCK-8 Assay, DCFH-DA Assay, Flow Cytometry, Expressing, Western Blot, In Vivo, Control, Cell Counting

Journal: Autophagy

Article Title: Tumor-derived lactate promotes resistance to bevacizumab treatment by facilitating autophagy enhancer protein RUBCNL expression through histone H3 lysine 18 lactylation (H3K18la) in colorectal cancer.

doi: 10.1080/15548627.2023.2249762

Figure Lengend Snippet: Figure 2. Effects of histone lactylation inhibition on colorectal cancer cells proliferation and survival in hypoxia. Note: (A) Western blotting analysis of lactylation or H3K18la levels in normal colon epithelial cells and CRC cells. (B) Western blotting analysis of Pan- and site-specific histone lactylation in HCT116 and SW620 cells in normoxia compared with in hypoxia (1% oxygen, 24 h). (C) Lactylation and H3K18la levels were detected in HCT116 and SW620 cells cultured in different concentrations of 2-DG or oxamate for 24 h in hypoxia (1% oxygen) by western blot. (D) Lactylation and H3K18la levels were detected in HCT116 and SW620 cells cultured in hypoxia (1% oxygen) at the indicated time. (E and F) Proliferation of HCT116 and SW620 cells cultured in different

Article Snippet: Cell lines and cell cultures The CRC cells (SW480, SW620, RKO, Caco2, DLD1, Lovo, HT29 and HCT116), normal colon epithelial cells (NCM460 and HIEC-6) and HEK293T were purchased from American Type Culture Collection (CCL-228, CCL-227, CRL-2577, HTB-37, CCL-221, CCL-229, HTB-38, CCL-247, CRL-1831, CRL-3266, CRL-3216) and cultured in complete medium as recommended, supplemented with 100 μg/mL streptomycin, 100 IU/mL penicillin, and 10% fetal bovine serum (Gibco, 10099141) at 37°C in 5% CO2.

Techniques: Inhibition, Western Blot, Cell Culture