Journal: Neural Regeneration Research

Article Title: Srgap2 suppression ameliorates retinal ganglion cell degeneration in mice

doi: 10.4103/1673-5374.369122

Figure Lengend Snippet: Downstream signaling pathways after inhibition of Srgap2 . (A) Detection of SRGAP2 (green, Alexa Fluor 488) expression in PC12 cells by immunofluorescence staining. n = 3 independent cell culture preparations. Scale bars: 50 μm. (B) SRGAP2 protein level was decreased after siRNA transfection and Rac1 and Cdc42 were increased after inhibiting Srgap2. (C) Quantification of B. n = 3 independent cell culture preparations. (D) mTOR signaling pathway was activated after the downregulation of Srgap2. (E) Quantification of D. n = 3 independent cell culture preparations. (F, G) ONC induced activation of the mTOR signaling pathway 7 days after ONC. Srgap2 +/– mice showed increased mTOR activation compared with WT. n = 3 independent animals in each group. Data (normalized by WT-ctrl group) are presented as mean ± SEM. ** P < 0.01, *** P < 0.001, **** P < 0.0001 (two-tailed Student’s t -test). Cdc42: Cell division cycle 42; Ctrl: control; DAPI: 4′,6-diamidino-2-phenylindole; mTOR: mammalian target of rapamycin; ONC: optic nerve crush; Rac1: Ras-related C3 botulinum toxin substrate 1; siRNA: small interfering RNA; Srgap2: Slit-Robo GTPase activating protein 2; WT: wild type.

Article Snippet: PC12, one of the most commonly used cell lines in neuroscience research (You et al., 2022), was purchased from CHI Scientific (Jiangyin, China; RRID: CVCL_0481).

Techniques: Protein-Protein interactions, Inhibition, Expressing, Immunofluorescence, Staining, Cell Culture, Transfection, Activation Assay, Two Tailed Test, Control, Small Interfering RNA

Journal: Basic and Clinical Neuroscience

Article Title: Effectiveness of Nicotinamide Phosphoribosyltransferase/Pre-B Cell Colony-enhancing Factor/Visfatin in preventing High Glucose-induced Neurotoxicity in an In-vitro Model of Diabetic Neuropathy

doi: 10.32598/bcn.2021.2870.2

Figure Lengend Snippet: A) The effects of different concentrations of glucose on pheochromocytoma PC12 cell viability, B) The effects of visfatin on high-glucose damaged PC12 cells Data are shown as Mean±SEM; * P<0.05, ** P<0.01 vs control, # P<0.001, ## P<0.0001 vs HG treatment.

Article Snippet: PC12 cells were purchased from the Buali (Avicenna) Research Institute.

Techniques: Control

Journal: Basic and Clinical Neuroscience

Article Title: Effectiveness of Nicotinamide Phosphoribosyltransferase/Pre-B Cell Colony-enhancing Factor/Visfatin in preventing High Glucose-induced Neurotoxicity in an In-vitro Model of Diabetic Neuropathy

doi: 10.32598/bcn.2021.2870.2

Figure Lengend Snippet: A) The effect of visfatin on HG-induced intracellular ROS level in PC12 cells, B) The effects of visfatin on HG-induced apoptosis of PC12 cell, C) The apoptosis evaluation of PC12 cells under different treatments Note: Data are shown as Mean±SEM; * P<0.05 vs control, # P<0.01, ## P<0.0001vs HG treatment.

Article Snippet: PC12 cells were purchased from the Buali (Avicenna) Research Institute.

Techniques: Control

Journal: International Journal of Molecular Sciences

Article Title: Network Pharmacology-Guided Discovery of Traditional Chinese Medicine Extracts for Alzheimer’s Disease: Targeting Neuroinflammation and Gut–Brain Axis Dysfunction

doi: 10.3390/ijms26178545

Figure Lengend Snippet: Cell viability assessment of PC12/Caco-2 co-cultures treated with TCM extracts and active compounds. ( A ) Cell viability of PC12/Caco-2 co-cultures exposed to Aβ and LPS damage, followed by treatment with 20 μg/mL concentrations of VT, PM, AVF, and EF extracts. ( B ) Cell viability of PC12/Caco-2 co-cultures exposed to Aβ and LPS damage, followed by treatment with 2 μM concentrations of bioactive compounds. PC12 cells were initially stimulated with NGF to enhance neuronal differentiation (1.5–2-fold increase in proliferation). Co-cultured cells were then pre-treated with 10 μM Aβ or LPS for 1 h to induce cytotoxicity. Subsequently, PC12 cells (basal layer) were treated with herbal extracts (10, 20, 50 μg/mL) or bioactive compounds (2, 5 μM) in low NGF medium, while Caco-2 cells (upper layer) received the same concentrations of LPS treatment. Donepezil hydrochloride served as positive control. After 24 h treatment, cell viability was assessed using the MTT assay. Data are presented as mean values. Different superscript letters (a, b, c) indicate significant differences between groups within each parameter (Tukey’s test, p < 0.05). Abbreviations: Aβ, β-amyloid; AVF, Apocyni Veneti Folium ; EF, Eucommiae folium ; LPS, lipopolysaccharide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NGF, nerve growth factor; PM, Plantago major ; TCM, traditional Chinese medicine; VT, Vitex trifolia .

Article Snippet: An in vitro gut–brain axis model was established using a co-culture system of PC12 neuronal cells and Caco-2 intestinal epithelial cells (KCLB 21721, Korean Cell Line Bank, Seoul, Republic of Korea), as illustrated in .

Techniques: Cell Culture, Positive Control, MTT Assay

Journal: International Journal of Molecular Sciences

Article Title: Network Pharmacology-Guided Discovery of Traditional Chinese Medicine Extracts for Alzheimer’s Disease: Targeting Neuroinflammation and Gut–Brain Axis Dysfunction

doi: 10.3390/ijms26178545

Figure Lengend Snippet: Relative mRNA expression of PC12 cells/Caco-2 cells co-culture. ( A ) Inflammatory cytokine mRNA expression ( TNF-α , IL-1β , IL-6 ) in PC12 and Caco-2 cells following treatment with 20 μg/mL concentrations of VT, PM, AVF, and EF extracts. ( B ) Neurodegeneration-related mRNA expression ( Tau and BDNF ) in PC12 cells following treatment with 20 μg/mL concentrations of VT, PM, AVF, and EF extracts. ( C ) Inflammatory cytokine mRNA expression ( TNF-α , IL-1β , IL-6 ) in PC12 and Caco-2 cells following treatment with 2 μM concentrations of active ingredients. ( D ) Neurodegeneration-related mRNA expression ( Tau and BDNF ) in PC12 cells following treatment with 2 μM concentrations of active ingredients. PC12 and Caco-2 cells were maintained in co-culture. PC12 cells were exposed to 10 μM Aβ for 1 h to induce neurodegeneration, while Caco-2 cells were exposed to 10 μM LPS for 1 h to induce intestinal inflammation. Following damage induction, cells were treated for 24 h with vehicle control (cell water), donepezil hydrochloride (positive control), TCM extracts (VT, PM, AVF, EF), or bioactive compounds. mRNA was then extracted from both cell types for gene expression analysis. Data are presented as mean ± standard error. Different letters (a, b, c, d) above bars indicate significant differences between treatment groups (Tukey’s test, p < 0.05). Identical letters indicate no significant difference. Abbreviations: Aβ, β-amyloid; AVF, Apocyni Veneti Folium ; BDNF, brain-derived neurotrophic factor; EF, Eucommiae folium ; IL-1β, interleukin-1β; IL-6, interleukin-6; LPS, lipopolysaccharide; PM, Plantago major ; TCM, traditional Chinese medicine; TNF-α, tumor necrosis factor-α; VT, Vitex trifolia .

Article Snippet: An in vitro gut–brain axis model was established using a co-culture system of PC12 neuronal cells and Caco-2 intestinal epithelial cells (KCLB 21721, Korean Cell Line Bank, Seoul, Republic of Korea), as illustrated in .

Techniques: Expressing, Co-Culture Assay, Control, Positive Control, Gene Expression, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Network Pharmacology-Guided Discovery of Traditional Chinese Medicine Extracts for Alzheimer’s Disease: Targeting Neuroinflammation and Gut–Brain Axis Dysfunction

doi: 10.3390/ijms26178545

Figure Lengend Snippet: Insulin signaling pathway in PC12 cells by Western blot. ( A ) Representative Western blot images showing protein expression levels of key insulin signaling pathway components (Akt, GSK-3β, AMPK) and their phosphorylated forms in PC12 cells from co-culture experiments. ( B ) Quantitative analysis of total and phosphorylated protein levels of Akt, GSK-3β, and AMPK in PC12 cells following treatment with TCM extracts. Experimental protocol: PC12 and Caco-2 cells were maintained in co-culture with PC12 cells in the basal layer and Caco-2 cells in the upper layer. PC12 cells were exposed to 10 μM Aβ for 1 h to induce neuronal damage, while Caco-2 cells were simultaneously exposed to 10 μM LPS for 1 h to simulate intestinal inflammation. Following damage induction, cells were treated for 24 h with vehicle control, donepezil hydrochloride (positive control), or 20 μg/mL concentrations of VT, PM, AVF, and EF extracts. PC12 cells were then harvested for Western blot analysis of insulin signaling pathway proteins, including total and phosphorylated forms of Akt, AMPK, and GSK-3β, with β-actin serving as loading control. Data are presented as mean ± standard error. Different letters (a, b, c) above bars indicate significant differences between treatment groups (Tukey’s test, p < 0.05). Identical letters indicate no significant difference. Abbreviations: Akt, AKT serine/threonine kinase; AMPK, AMP-activated protein kinase; AVF, Apocyni Veneti Folium ; EF, Eucommiae folium ; GSK-3β, glycogen synthase kinase-3β; LPS, lipopolysaccharide; PM, Plantago major ; TCM, traditional Chinese medicine; VT, Vitex trifolia ; Aβ, β-amyloid.

Article Snippet: An in vitro gut–brain axis model was established using a co-culture system of PC12 neuronal cells and Caco-2 intestinal epithelial cells (KCLB 21721, Korean Cell Line Bank, Seoul, Republic of Korea), as illustrated in .

Techniques: Western Blot, Expressing, Co-Culture Assay, Control, Positive Control

Journal: International Journal of Molecular Sciences

Article Title: Network Pharmacology-Guided Discovery of Traditional Chinese Medicine Extracts for Alzheimer’s Disease: Targeting Neuroinflammation and Gut–Brain Axis Dysfunction

doi: 10.3390/ijms26178545

Figure Lengend Snippet: Morphological and mitochondrial analysis of PC12/Caco-2 co-cultures. ( A ) Hematoxylin and eosin (H&E) staining of Caco-2 cells following treatment with 20 μg/mL TCM extracts in co-culture experiments (100× magnification). ( B ) Toluidine blue staining of Caco-2 cells showing mast cell infiltration (indicated by red circles) following treatment with 20 μg/mL TCM extracts in co-culture experiments (100× magnification). ( C ) JC-1 fluorescent staining of PC12/Caco-2 co-cultures demonstrating mitochondrial membrane potential and cellular protection following treatment with TCM extracts (20 μg/mL) or active ingredients (2 μM) (200× magnification). Experimental protocol: PC12 and Caco-2 cells were maintained in co-culture with PC12 cells in the basal layer and Caco-2 cells in the upper layer. PC12 cells were exposed to 10 μM Aβ for 1 h, while Caco-2 cells were exposed to 10 μM LPS for 1 h to induce cellular damage. Following damage induction, cells were treated for 24 h with vehicle control, donepezil hydrochloride (positive control), VT, PM, AVF, or EF extracts (20 μg/mL), or active ingredients (2 μM). JC-1 fluorescent staining was used to examine the degree of apoptosis in Caco-2 cells and PC12 cells. The mitochondrial membrane potential was expressed as red fluorescence. The more fluorescent red (Caco-2 cells) and fluorescent green (PC12-cells), the higher the intensity of cell protection and the inhibition of cell apoptosis. Data are presented as mean ± standard error. Different letters (a, b, c) above bars indicate significant differences between treatment groups (Tukey’s test, p < 0.05). Identical letters indicate no significant difference. Abbreviations: AVF, Apocyni Veneti Folium; EF, Eucommiae folium; H&E, hematoxylin and eosin; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; LPS, lipopolysaccharide; PM, Plantago major ; VT, Vitex trifolia .

Article Snippet: An in vitro gut–brain axis model was established using a co-culture system of PC12 neuronal cells and Caco-2 intestinal epithelial cells (KCLB 21721, Korean Cell Line Bank, Seoul, Republic of Korea), as illustrated in .

Techniques: Staining, Co-Culture Assay, Membrane, Control, Positive Control, Fluorescence, Inhibition

Journal: Signal Transduction and Targeted Therapy

Article Title: Olverembatinib, a multikinase inhibitor that modulates lipid metabolism, in advanced succinate dehydrogenase-deficient gastrointestinal stromal tumors: a phase 1b study and translational research

doi: 10.1038/s41392-025-02456-9

Figure Lengend Snippet: SDHB knockout enhanced lipid uptake in GIST cells, which could be inhibited by olverembatinib treatment, and reoverexpression of SDHB increased the resistance of these cells to olverembatinib and abolished the suppression of lipid uptake by olverembatinib. a SDHB knockout enhanced lipid accumulation in GIST-T1 cells. Representative images showing lipid droplets stained with BODIPY-labeled GIST-T1 cells and GIST-T1-SDHB-KO cells. Lipid droplet counts per cell were then measured via ImageJ according to the fluorescence image ( n = 6). b Compared with GIST-T1 cells, GIST-T1-SDHB-KO cells were more sensitive to olverembatinib treatment in lipid-depleted medium. GIST-T1 cells and GIST-T1-SDHB-KO cells were treated with 3 nM olverembatinib in RPMI 1640 with 10% or 3% FBS, and then, cell viability was measured with a CellTiter 96 ® Aqueous Non-Radioactive Cell Proliferation Assay. c SDH-deficient GIST primary cells cultured in lipid-depleted medium were more sensitive to olverembatinib treatment than those cultured in lipid-rich medium. SDH-deficient GIST primary cells from Patient #1 were treated with 1.25 and 2.5 μM olverembatinib in RPMI 1640 with 10%, 3% and 1% FBS, and then, cell viability was measured with a CellTiter 96 ® Aqueous Non-Radioactive Cell Proliferation Assay. d Olverembatinib inhibited lipid droplet accumulation and lipid uptake in SDH-deficient GIST primary cells. Representative images showing lipid droplets stained with BODIPY in primary SDH-deficient GIST (patient #3) cells. Green, BODIPY (493 nm/503 nm) for lipid droplet staining; blue, Hoechst 33342 (350 nm/461 nm) for nuclear staining; red, MitoTracker Red FM (581 nm/644 nm) for mitochondrial staining. e Effects of olverembatinib and other TKIs on lipid uptake in SDH-deficient GIST primary cells from Patient #2. After treatment with DMSO and TKIs at the indicated concentrations for 24 h in basic medium without FBS, the intracellular BODIPY™ FL C16 level in the cells was detected by a multifunction microplate reader at 490 nm/535 nm ( n = 4). f Olverembatinib, but not other TKIs, downregulated CD36 in primary SDH-deficient GIST cells. After the cells were treated with the indicated concentrations of olverembatinib, imatinib, regorafenib, and sunitinib for 24 h, the cell lysates were collected for western blot analysis. g Viability response to olverembatinib after SDHB re-expression. Primary SDH-deficient GIST cells from Patients #2 and #3, transfected as in ( f ), were treated with 100 nM olverembatinib 48 h post-transfection. Cell viability was measured to assess olverembatinib sensitivity before and after SDHB restoration. h Western blot analysis confirmed SDHB overexpression post-transfection. Reoverexpression of SDHB was carried out by transient transfection of pcDNA3.1-SDHB into primary SDH-deficient GIST cells (patients #2 and #3). Western blots showing the overexpression of SDHB after transfection. NC, cells transfected with the pcDNA3.1 vector plasmid. SDHB-OE, cells transfected with the SDHB-overexpressing pcDNA3.1 plasmid. i Lipid uptake response to olverembatinib after SDHB re-expression. Primary SDH-deficient GIST cells from Patients #2 and #3, transfected as in ( f ), were treated with 100 nM olverembatinib 48 h post-transfection. Lipid uptake capacity was evaluated before and after SDHB restoration. ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The PC12 SDHB knockdown (KD) cell line (PC12#5F7) was obtained from GENEWIZ, Inc. (Azenta Life Sciences; Suzhou, China) via a sgRNA-mediated clustered regularly interspersed short palindromic repeat (CRISPR)/Cas9 system.

Techniques: Knock-Out, Staining, Labeling, Fluorescence, Proliferation Assay, Cell Culture, Western Blot, Expressing, Transfection, Over Expression, Plasmid Preparation