Journal: Cell Death Discovery

Article Title: NTRK1 knockdown induces mouse cognitive impairment and hippocampal neuronal damage through mitophagy suppression via inactivating the AMPK/ULK1/FUNDC1 pathway

doi: 10.1038/s41420-023-01685-7

Figure Lengend Snippet: A , B DCFH-DA staining results suggestedthatNTRK1 knockdown enhanced ROS accumulation in mouse neurons. n = 3. C The ATP content analysis indicated that NTRK1 knockdown resulted in reduced ATP content in mouse neurons. n = 3. D , E The results of JC-1 staining indicated thatNTRK1 knockdown decreased the mitochondrial membrane potential in mouse neurons. n = 3. F , G The mRFP-eGFP-LC3 assay revealed that NTRK1 knockdown suppressed the autophagic flux in mouse neurons. n = 3. ** P < 0.01.

Article Snippet: Mitochondria were isolated from mouse hippocampus tissues and mouse hippocampal neurons using the Mitochondrial Isolation Kit (C3601, Beyotime, Shanghai, China) in accordance with the directions.

Techniques: Staining, Knockdown, Membrane

Journal: Molecules

Article Title: Chlorahololide D, a Lindenane-Type Sesquiterpenoid Dimer from Chloranthus holostegius Suppressing Breast Cancer Progression

doi: 10.3390/molecules28207070

Figure Lengend Snippet: Chlorahololide D triggered apoptosis in MCF-7 cells. Various concentrations of chlorahololide D (7.5, 15, and 30 μM) were administrated to MCF-7 cells and incubated for 48 h. Cells were stained with Annexin V and propidium iodide (PI), and detected by flow cytometry subsequently. ( A ) Flow cytometric analysis of MCF-7 cells with the treatment of chlorahololide D. ( B ) Histogram of the proportions of apoptotic cells at 48 h after being treated with chlorahololide D. The results are expressed as mean ± SD. ** p < 0.01 and *** p < 0.001 vs. control group.

Article Snippet: Annexin V-FITC apoptosis detection kit, cell cycle and apoptosis kit, and BCA protein assay kit were supplied by Beyotime Biotechnology Co., Ltd. (Shanghai, China).

Techniques: Incubation, Staining, Flow Cytometry, Control

Journal: Molecules

Article Title: Chlorahololide D, a Lindenane-Type Sesquiterpenoid Dimer from Chloranthus holostegius Suppressing Breast Cancer Progression

doi: 10.3390/molecules28207070

Figure Lengend Snippet: Chlorahololide D regulated apoptosis-related proteins. MCF-7 cells were pre-treated with chlorahololide D for 36 h, and Western blotting analysis was carried out. ( A ) The expression of Bcl-2 and Bax. ( B ) Histogram of the protein relative expression levels. β -actin protein was used as an internal reference. The results are presented as mean ± SD. ** p < 0.01 and *** p < 0.001 vs. control group.

Article Snippet: Annexin V-FITC apoptosis detection kit, cell cycle and apoptosis kit, and BCA protein assay kit were supplied by Beyotime Biotechnology Co., Ltd. (Shanghai, China).

Techniques: Western Blot, Expressing, Control

Journal: Cancer Research

Article Title: ACSL4-Mediated Membrane Phospholipid Remodeling Induces Integrin β1 Activation to Facilitate Triple-Negative Breast Cancer Metastasis

doi: 10.1158/0008-5472.CAN-23-2491

Figure Lengend Snippet: The enhanced ACSL4 correlates with poor TNBC prognosis and contributes to metastasis. A and B, Representative images ( A ) and quantification ( B ) of ACSL4 IHC staining in TNBC tumor samples ( n = 30) and paired adjacent normal tissues ( n = 30). C–F, ACSL4 IHC staining in a TMA (TMA-1) containing primary tumor samples from patients with breast cancer. Representative images ( C ) and quantification ( D ) of ACSL4 IHC staining in breast cancer tissues from patients with different subtypes. E, IHC scores of ACSL4 in breast cancer tissues from patients with different N stages. F, Kaplan–Meier analysis of the overall survival of patients with breast cancer in the TMA-1 cohort. G and H , Representative images ( G ) and quantification ( H ) of ACSL4 IHC staining in primary breast tumor tissues from patients with (M+) or without (M−) distant metastasis. I, Relative mRNA levels of ACSL4 in the primary TNBC tumor tissues from patients with (M+) or without (M−) metastasis. The data are shown as the normalized fold change of TNBC M+ compared with TNBC M−. J and K, Representative IHC staining images ( J ) and quantification ( K ) of TMA-2 cohort ACSL4 expression in the primary and metastatic breast tumors. Primary and metastatic tumor tissues from NCG mice injected with MDA-MB-231 cells are shown in top panel; unpaired primary and metastases from the TMA-2 cohort containing primary and metastatic breast cancer tissues from patients are shown in the bottom panel. L, IHC staining images of ACSL4 expression in the paired primary and metastases tumors from two patients with TNBC. M, Representative images of TUNEL staining and quantification of TUNEL-positive circulating MDA-MB-231 cells (CTC) to evaluate anoikis resistance of CTCs. CTCs (GFP-positive) were isolated from mouse blood and selected by flow cytometry. N–O, MDA-MD-231 cells expressing two independent, nonoverlapping shRNA against ACSL4 (shACSL4#1, shACSL4#2) or nontargeting shRNA (shNC) were injected into the tail vein of NCG mice. Lungs and livers were harvested at day 30 after injection. N, Representative images of lung and liver and Ki-67 IHC staining in metastases ( n = 6/group). O, Quantification of metastatic nodules of lung surfaces (left), metastatic foci of liver sections (middle), and Ki-67 levels in lung or liver metastatic areas (right). For Ki-67 quantification, 9 random microscopic fields from three mice of each group were captured. All data represent mean ± SD. Fisher exact test ( B ), Student t test ( E, H, I, M, and O ), one-way ANOVA ( D and K ), and log-rank test ( F ) were utilized. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: GFP-positive tumor cells were selected by flow cytometry and stained using the One-step TUNEL In Situ Apoptosis Kit (#E-CK-A325; Elabscience) according to the manufacturer's instructions.

Techniques: Immunohistochemistry, Expressing, Injection, TUNEL Assay, Staining, Isolation, Flow Cytometry, shRNA

Journal: Liver Transplantation

Article Title: The Impact of Dabigatran Treatment on Sinusoidal Protection Against Hepatic Ischemia/Reperfusion Injury in Mice

doi: 10.1002/lt.25929

Figure Lengend Snippet: Characterization of thrombin generation after hepatic IRI and cytotoxicity of thrombin for hepatic SECs. (A) In the clinical study of patents who underwent hepatectomy (n = 14), plasma TAT levels were plotted at 2 time points: before and 1 day after hepatectomy. TAT levels were significantly elevated after operation. (B) In in vivo hepatic IRI models of mice, plasma TAT levels measured by ELISA was significantly increased after IRI compared with the naïve group (n = 6 in each group). (C‐a) In hepatic SECs cultured in the nonischemic condition, LDH cytotoxicity levels in the supernatant of cell cultures were elevated in a dose‐dependent manner with the addition of thrombin. The addition of 100 U/mL thrombin significantly increased LDH cytotoxicity compared with both groups of naïve and 10 U/mL thrombin (n = 4 in each group). (C‐b) In H‐R models of SECs in vitro, the addition of 100 U/mL thrombin also significantly increased LDH cytotoxicity compared with vehicle (n = 5 in each group). H‐R was established using an anaeropack jar system.

Article Snippet: Cell cytotoxicity was assessed by measuring lactate dehydrogenase (LDH) levels in the supernatant using a Cytotoxicity LDH Assay Kit‐WST (Dojindo, Japan) following the manufacturer’s instructions.

Techniques: Clinical Proteomics, In Vivo, Enzyme-linked Immunosorbent Assay, Cell Culture, In Vitro

Journal: Liver Transplantation

Article Title: The Impact of Dabigatran Treatment on Sinusoidal Protection Against Hepatic Ischemia/Reperfusion Injury in Mice

doi: 10.1002/lt.25929

Figure Lengend Snippet: Direct effect of dabigatran treatment on pure cultured hepatic SECs in an H‐R model in vitro. (A) In H‐R models of pure cultured hepatic SECs, dabigatran treatment significantly decreased LDH cytotoxicity levels in the supernatant compared with vehicle (n = 5 in each group). (B‐a) Dabigatran treatment significantly increased TM expression in cell lysate measured by ELISA compared with vehicle (n = 5 in each group). (B‐b) By contrast, in TM expression in the supernatant of cell cultures measured by ELISA, there was no significant difference between the vehicle and dabigatran groups (n = 5 in each group). (C‐a) Based on Western blot analysis, dabigatran treatment did not significantly affect HMGB‐1 expression in cell lysate compared with the vehicle group (n = 5 in each group). Quantification of HMGB‐1 band intensities normalized to β‐actin. (C‐b) By contrast, HMGB‐1 levels in the supernatant of cell cultures measured by ELISA were markedly reduced by dabigatran treatment compared with the vehicle (n = 5 in each group). In H‐R models of this study, SECs were cultured in a serum‐starved medium containing 100 U/mL thrombin and exposed to H‐R using an anaeropack jar system.

Article Snippet: Cell cytotoxicity was assessed by measuring lactate dehydrogenase (LDH) levels in the supernatant using a Cytotoxicity LDH Assay Kit‐WST (Dojindo, Japan) following the manufacturer’s instructions.

Techniques: Cell Culture, In Vitro, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Liver Transplantation

Article Title: The Impact of Dabigatran Treatment on Sinusoidal Protection Against Hepatic Ischemia/Reperfusion Injury in Mice

doi: 10.1002/lt.25929

Figure Lengend Snippet: Direct effect of dabigatran treatment on pure cultured hepatocytes in an H‐R model in vitro. In H‐R models of pure cultured hepatocytes, there was no significant difference between the H‐R + vehicle group and the H‐R + dabigatran group (A) in LDH cytotoxicity levels in the supernatant of cell cultures, in TM levels in cell lysate measured by ELISA (B‐a), in TM levels in the supernatant of cell cultures measured by ELISA (B‐b), in HMGB‐1 expression in cell lysate evaluated by Western blot analysis (C‐a), or in HMGB‐1 levels in the supernatant of cell cultures measured by ELISA (C‐b) (n = 5 in each group). In Western blot analysis, quantification of HMGB‐1 band intensities normalized to β‐actin. In H‐R models of this study, cells were cultured in a serum‐starved medium containing 100 U/mL thrombin and exposed to H‐R using an anaeropack jar system.

Article Snippet: Cell cytotoxicity was assessed by measuring lactate dehydrogenase (LDH) levels in the supernatant using a Cytotoxicity LDH Assay Kit‐WST (Dojindo, Japan) following the manufacturer’s instructions.

Techniques: Cell Culture, In Vitro, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot

Journal: Liver Transplantation

Article Title: The Impact of Dabigatran Treatment on Sinusoidal Protection Against Hepatic Ischemia/Reperfusion Injury in Mice

doi: 10.1002/lt.25929

Figure Lengend Snippet: Effects of dabigatran for hepatocytes in a paracrine communication model between hepatic SECs and hepatocytes. (A) A scheme of our paracrine communication model between hepatic SECs and hepatocytes. SECs were pretreated with or without dabigatran followed by incubation in a serum‐starved medium in the H‐R condition; thereafter the supernatant of the SECs was harvested. Hepatocytes were pretreated with the supernatant of SECs followed by incubation in a serum‐starved medium in the H‐R condition. (B) LDH cytotoxicity levels in the supernatant of hepatocyte cultures were significantly lower in the group of H‐R + the supernatant of SECs pretreated with dabigatran than in the group of H‐R + the supernatant of SECs pretreated with vehicle (n = 5 in each group). TUNEL staining detected cellular death of hepatocytes after H‐R (C‐a) in the group of H‐R + the supernatant of SECs pretreated with vehicle and (C‐b) in the group of H‐R + the supernatant of SECs pretreated with dabigatran (original magnification ×200). (C‐c) The supernatant of SECs pretreated with dabigatran markedly reduced the number of TUNEL‐positive hepatocytes compared with the supernatant of SECs pretreated with the vehicle (n = 5 in each group).

Article Snippet: Cell cytotoxicity was assessed by measuring lactate dehydrogenase (LDH) levels in the supernatant using a Cytotoxicity LDH Assay Kit‐WST (Dojindo, Japan) following the manufacturer’s instructions.

Techniques: Incubation, TUNEL Assay, Staining

Journal: BMC oral health

Article Title: Keratinocyte necroptosis promotes the progression of radiation-induced oral mucositis.

doi: 10.1186/s12903-025-06278-7

Figure Lengend Snippet: Fig. 4 Pretreatment with RIPK3 inhibitor or MLKL inhibitor reduces radiation-induced cell death and inflammation. (A) Lactate dehydrogenase (LDH) release was measured in HaCaT cells cultured for 2.5 days post-irradiation with a dose of 12 Gy, in the presence or absence of the RIPK3 inhibitor GSK’872 or the MLKL inhibitor GW806742X. (B) Flow cytometry analysis of HaCaT cells stained with propidium iodide (PI) and annexin V, and (C) quantification of double-positive cells (PI and annexin V staining). (D) Representative fluorescent images of Hoechst 33342 (blue) and PI (red) double staining. Scale bar: 100 μm. (E, F) The mRNA expression levels of inflammatory cytokines by qRT-PCR, including IL-1β, IL-6, and TNF-α. Results are shown as ± SD means of three replicates from three independent experiments. Statistical significance was determined using one-way ANOVA and LSD post hoc tests. *P < 0.05, **P < 0.01 and ***P < 0.001

Article Snippet: Cytotoxicity was evaluated using the Lactate Dehydrogenase (LDH) Assay Kit (Beyotime).

Techniques: Cell Culture, Irradiation, Flow Cytometry, Staining, Double Staining, Expressing, Quantitative RT-PCR