Journal: Molecular Cancer

Article Title: Epigenetic regulation of CpG promoter methylation in invasive prostate cancer cells

doi: 10.1186/1476-4598-9-267

Figure Lengend Snippet: Validation of methylated targets in LNcaP and DU145 cells . A) DNA was extracted using the DNeasy kit and total of 1 μg from parental (total) LNCaP and DU145 cells was bisulfite modified using the EpiTect Bisulfite kit from Qiagen. MS-PCR was performed using Platinum Taq Polymerase (Invitrogen) and 200 ng of either genomic of bisulfite treated DNA was used. The samples were visualized using a 1% agarose gel and ethidium bromide. Both Sox1 and Bmx are methylated in the LNCaP and DU145 cell lines. B) Total RNA was isolated using TRIzol and qRT-PCR analysis was performed using a StepOne Real-time PCR machine with TaqMan Gene Expression Assay reagents and probes. Isolation of DNA and cDNA from non-invasive and invasive cells was carried out as previously described in materials and methods. Relative fold induction of mRNA was compared between non-invasive and invasive cells using the Delta-Delta CT method of quantitation where the parental lines were set at 1.0 as the control, and 18S rRNA was used as a loading control. Increased levels of both Sox1 and Bmx are seen in invasive LNCaP and DU145 cells compared to the non-invasive and parental lines. Normal human prostate RNA was used as a control. A Two-way ANOVA with a Bonferroni post-test was performed to compare groups and * represents a p-value of < 0.05 comparing parental to non-invasive cells and ** comparing parental to invasive cells. C) Staining of invasive or non-invasive cells was performed directly on the Matrigel membrane. Cells were incubated with either anti-pBMX antibody or SOX1 overnight and goat anti-rabbit Alexa-488 was added for 1 hour. Membranes were mounted on glass slides with Vectashield containing DAPI and visualized with a Zeiss-510 L5 confocal microscope. Images were analyzed using the Zeiss LSM5 Image Browser (20×) and further prepared in Adobe Photoshop CS. Increased levels of pBMX and SOX1 are seen in invasive cells compared to the non-invasive cells on top of the membrane.

Article Snippet: Quantitative real time polymerase chain reaction (qRT-PCR) analysis was performed using a StepOne Real-time PCR machine (Applied Biosystems, Foster City, CA) with TaqMan Gene Expression Assay reagents and probes (Applied Biosystems).

Techniques: Methylation, Modification, Agarose Gel Electrophoresis, Isolation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Expressing, Quantitation Assay, Staining, Incubation, Microscopy

Journal: The FASEB Journal

Article Title: Protease-activated receptor 2 activates airway apical membrane chloride permeability and increases ciliary beating

doi: 10.1096/fj.201700114RRR

Figure Lengend Snippet: Expression of PAR-2 in airway epithelial cells. A) PAR-2 expression in primary airway epithelial cell lines and primary cells from 3 patients was assayed by reverse transcription-PCR. Oral squamous epithelium cDNA was used as a control for PAR-4. b.p., base pair; Pt, patient; rt, reverse transcription. B) PAR-2 expression was confirmed by Western blot in primary sinonasal ALI culture samples (6 separate patients, unrelated to patients in A. C) Immunofluorescence using a PAR-2 antibody in submerged BEAS-2B cells revealed plasma membrane–localized staining. GLUT1 was a control for membrane localization. D, E) Immunofluorescence of PAR-2 in primary dissociated sinonasal ciliated cells showing colocalization with NKCC1 (D) and Na+K+ ATPase (E). A distinct gap was noted (arrows) between the base of the cilia (labeled with β-tubulin IV), corresponding to the apical cell body membrane and the start of basolateral NKCC1/Na+K+ ATPase immunofluorescence. PAR-2 rabbit pAb was used with mouse mAb anti-NKCC1 (top). PAR-2 mouse mAb was used with rabbit mAb anti-Na+K+ATPase (bottom). Scale bars, 10 µm. F) Representative scatter plots of PAR-2 intensity and either NKCC or Na+K+ ATPase. For all dissociated cells imaged (n = 15 with NKCC1 and n = 17 with Na+K+ ATPase), Pearson’s correlation coefficient (Pearson’s R) and Mander’s overlap coefficient (Mander’s R) for basolateral marker and PAR-2 were both ≥0.95.

Article Snippet: Human PAR-2 and PAR-4 samples were amplified by PCR using an iCycler (Bio-Rad, Hercules, CA, USA), RedTaq DNA Polymerase (MilliporeSigma), and the following primers: 5′-GCTGGTCACCATCCCTTTGT-3′ and 5′-TCTGCTTTACAGTGCGGACA-3′ (hPAR-2) and 5′-CCTGGTTTATCTCTACCGGCG-3′ and 5′-CTCTCGTCGTAGACGCTGGG-3′ (hPAR-4) from Integrated DNA Technologies (Coralville, IA, USA). cDNA was prepared from cultured cells using Trizol (Thermo Fisher Scientific) with rDNAse I (Ambion, Austin, TX, USA) and an iScript cDNA Synthesis Kit (Bio-Rad).

Techniques: Expressing, Reverse Transcription, Control, Western Blot, Immunofluorescence, Clinical Proteomics, Membrane, Staining, Labeling, Marker

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: Cell reports

Article Title: Btla signaling in conventional and regulatory lymphocytes coordinately tempers humoral immunity in the intestinal mucosa

doi: 10.1016/j.celrep.2022.110553

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: QIAmo Fast DNA Stool kit , Qiagen , 51604.

Techniques: Control, Plasmid Preparation, Recombinant, Cell Isolation, cDNA Synthesis, SYBR Green Assay, Staining, Enzyme-linked Immunosorbent Assay, Cloning, Library Quantification, Software

Journal: Cell host & microbe

Article Title: Altered immunity of laboratory mice in the natural environment is associated with fungal colonization

doi: 10.1016/j.chom.2020.02.015

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: RNeasy Plus Mini Kit , QIAGEN , Cat#74136.

Techniques: Activation Assay, Recombinant, Red Blood Cell Lysis, Staining, RNA Sequencing, Sequencing, RNA Expression, Gene Expression, Flow Cytometry, Software

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