Journal: Cancer & Metabolism

Article Title: Epigenetic repression of de novo cysteine synthetases induces intra-cellular accumulation of cysteine in hepatocarcinoma by up-regulating the cystine uptake transporter xCT

doi: 10.1186/s40170-024-00352-4

Figure Lengend Snippet: Epigenetic modifications of the 5’-flanking region of the cysteine synthetic genes in the hepatic tumors. A and B , The mRNA levels of the enzymes involved in cysteine synthesis ( A ) and DNA methyltransferases ( B ) in the liver and BNL 1ME A.7 R.1-formed tumors in mice. The expression levels were normalized to those of 18s . The values in the liver were set at 1.0. Each value represents the mean with S.D. ( n = 6). ** P < 0.01; significant difference between the two groups ( t 10 = − 15.397, P < 0.001 for Cbs ; t 10 = − 7.424, P < 0.001 for Cth ; t 10 = − 3.258, P = 0.009 for Dnmt1 ; t 10 = 5.041, P = 0.001 for Dnmt3a ; t 10 = − 3.438, P = 0.006 for Dnmt3b ; unpaired t -test, two sided). C and D , Methylation status of the 5’-flanking region in mice Cbs (C) and Cth (D) genes in the BNL 1ME A.7 R.1-formed tumors. Left panels show representative electropherograms of direct-bisulfite sequencing. Triangles indicate the methylation sites. Right panels show the quantification of methylation levels. Each value represents the mean with S.D. ( n = 4). ** P < 0.01, * P < 0.05; significant difference between the two groups (unpaired t -test, two sided)

Article Snippet: Small hairpin RNA (shRNA) expressing vectors against the mouse Slc7a11 , Dnmt1 , Dnmt3a , or Dnmt3b gene were purchased from VectorBuilder (Chicago, IL).

Techniques: Expressing, Methylation, Methylation Sequencing

Journal: Cancer & Metabolism

Article Title: Epigenetic repression of de novo cysteine synthetases induces intra-cellular accumulation of cysteine in hepatocarcinoma by up-regulating the cystine uptake transporter xCT

doi: 10.1186/s40170-024-00352-4

Figure Lengend Snippet: DNA methyltransferase suppresses the expression of cysteine synthetic enzymes in BNL 1ME A.7 R.1 cells. A and B , Induction of CBS and CTH expression by pharmacological inhibition of DNA methyltransferase activity. BNL 1ME A.7 R.1 cells were treated with 500 nM decitabine for 24 h. The mRNA and protein levels were normalized to those of 18s and β-ACTIN, respectively. The values in vehicle-treated cells were set at 1.0. Each value represents the mean with S.D. ( n = 4). * P < 0.05; significant difference between the two groups ( t 6 = 2.719, P = 0.035 for Cth mRNA; t 6 = 3.498, P = 0.013 for CBS protein; t 6 = 3.270, P = 0.017 for CTH protein; unpaired t -test, two sided). C and D , Induction of CBS and CTH expressions by down-regulation of DNA methyltransferase. BNL 1ME A.7 R.1 cells were transduced with lentivirus expressing shRNA against Dnmt1 , Dnmt3a , or Dnmt3b . The mRNA and protein levels in mock-transduced and Dnmt -knockdown (KD) cells were normalized to those of 18s and β-ACTIN, respectively. The values in mock-transduced cells were set at 1.0. Each value represents the mean with S.D. ( n = 3). ** P < 0.01, * P < 0.05; significant difference between the indicated groups ( F 3,8 = 160.673, P < 0.001 for Cbs mRNA; F 3,8 = 13.969, P = 0.002 for Cth mRNA; F 3,8 = 13.491, P = 0.002 for CBS protein; F 3,8 = 24.930, P < 0.001 for CTH protein; ANOVA with Tukey–Kramer’s post hoc test)

Article Snippet: Small hairpin RNA (shRNA) expressing vectors against the mouse Slc7a11 , Dnmt1 , Dnmt3a , or Dnmt3b gene were purchased from VectorBuilder (Chicago, IL).

Techniques: Expressing, Inhibition, Activity Assay, Transduction, shRNA, Knockdown

Journal: Oxidative Medicine and Cellular Longevity

Article Title: RIP3 Contributes to Cardiac Hypertrophy by Influencing MLKL-Mediated Calcium Influx

doi: 10.1155/2022/5490553

Figure Lengend Snippet: The protein level of RIP3 is elevated in hypertrophic hearts. (a) Representative Western blots and quantitation of RIP3 expression in heart tissue from patients with dilated cardiomyopathy (DCM) and normal controls. (b) (Up) Western blots of RIP3, ANP, β -MHC, and GAPDH and (down) relative quantitation of RIP3 expression in heart tissue from rats after sham or AB operation. (c) (Up) Western blots of RIP3, ANP, β -MHC, and GAPDH and (down) relative quantitation of RIP3 in NRCMs stimulated with Ang-II (1 μ M) or PBS. (d) (up) Western blots of RIP3, ANP, β -MHC, and GAPDH and (down) relative quantitation of RIP3 in NRCMs stimulated with PE (50 μ M) or PBS. kDa: kilo-Dalton; ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: Empty rAAV vector and rAAV vector expressing RIP3 (oe-RIP3) or RIP3-specific small hairpin RNA (shRIP3) were ordered from GeneChem company (Shanghai, China).

Techniques: Western Blot, Quantitation Assay, Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: RIP3 Contributes to Cardiac Hypertrophy by Influencing MLKL-Mediated Calcium Influx

doi: 10.1155/2022/5490553

Figure Lengend Snippet: RIP3 contributes to cardiac hypertrophy in vitro. (a) Representative Western blot analysis and relative quantitation of RIP3 in NRCMs stably transfected with scramble (negative control), sh-RIP3, vector (empty), and oe-RIP3 plasmid. (b) Cell morphology of NRCMs was determined by actin staining. NRCMs in (a) (lanes 1-4) were stimulated by Ang-II (1 μ M), PE (50 μ M), and PBS for 24 hrs. (c) ANP and β -MHC mRNA levels were determined by real-time qPCR. NRCMs treated as in (b) were used. (d) Cell morphology of NRCMs was determined by actin staining. NRCMs in (a) (lanes 6-7) were used and stimulated by Ang-II (1 μ M), PE (50 μ M), and PBS for 24 hrs. (e) ANP and β -MHC mRNA levels were determined by real-time qPCR. NRCMs treated as in (d) were used. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: Empty rAAV vector and rAAV vector expressing RIP3 (oe-RIP3) or RIP3-specific small hairpin RNA (shRIP3) were ordered from GeneChem company (Shanghai, China).

Techniques: In Vitro, Western Blot, Quantitation Assay, Stable Transfection, Transfection, Negative Control, Plasmid Preparation, Staining

Journal: Oxidative Medicine and Cellular Longevity

Article Title: RIP3 Contributes to Cardiac Hypertrophy by Influencing MLKL-Mediated Calcium Influx

doi: 10.1155/2022/5490553

Figure Lengend Snippet: RIP3 contributes to cardiac hypertrophy in vivo. (a–c) Heart weight/body weight ratio (HW/BW), lung weight/body weight ratio (LW/BW), and HW/tibia length (HW/TL, mg/mm) of the indicated rats. Rats were infected with RIP3-containing or VEC (empty vector) rAVVs and then underwent sham or AB surgery. 4 weeks later, these parameters of rats were determined. (d) The gross appearance, size, and hematoxylin-eosin (H&E) staining of the whole heart from cardiomyocyte-specific oe-RIP3 and VEC rats. The rats as in (a) were used, which received sham or AB surgery, respectively. (e–g) Heart weight/body weight ratio (HW/BW), lung weight/body weight ratio (LW/BW), and HW/tibia length (HW/TL, mg/mm) of the indicated rats. The control rats and rats infected with scramble or shRIP3-containing rAVVs were subjected to sham or AB surgery, and 4 weeks later, the indicated parameters were determined. (h) The gross appearance, size, and H&E staining of the whole heart from rats in (e–g). -: control; Scr: scramble. n = 6 per experimental group, ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: Empty rAAV vector and rAAV vector expressing RIP3 (oe-RIP3) or RIP3-specific small hairpin RNA (shRIP3) were ordered from GeneChem company (Shanghai, China).

Techniques: In Vivo, Infection, Plasmid Preparation, Staining

Journal: Oxidative Medicine and Cellular Longevity

Article Title: RIP3 Contributes to Cardiac Hypertrophy by Influencing MLKL-Mediated Calcium Influx

doi: 10.1155/2022/5490553

Figure Lengend Snippet: RIP3 is implicated in the MLKL-mediated calcium influx. (a) Co-IP of RIP3 and MLKL in NRCMs stimulated with PE. (b) Western blot showed the cell membrane fraction assay of WT and RIP3 −/− H9c2 cells, RIP3 −/− , RIP3 knockout. WT and RIP3 −/− H9c2 cells were stimulated with Ang-II (1 μ M), PE (50 μ M), and PBS for 24 hrs. (c) Immunofluorescence of MLKL in WT and RIP3 −/− H9c2 cells stimulated with Ang-II or PE. The same cells in (b) were used. (d) Intracellular calcium concentration of H9c2 cells was determined by Fluo4 staining. The same cells in (c) were stimulated with Ang-II (1 μ M) or PE (50 μ M) for 24 hrs. (e) The surface area of the indicated NRCM cell lines. Cells were stimulated with Ang-II (1 μ M) or PE (50 μ M) for 24 hrs. VEC: infected with vector plenti; VEC/shMLKL: infected with vector/shMLKL plenti; oe-RIP3: infected with RIP3 plenti; oe-RIP3/shMLKL: infected with RIP3/shMLKL plenti. kDa: kilo-Dalton; ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: Empty rAAV vector and rAAV vector expressing RIP3 (oe-RIP3) or RIP3-specific small hairpin RNA (shRIP3) were ordered from GeneChem company (Shanghai, China).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Knock-Out, Immunofluorescence, Concentration Assay, Staining, Infection, Plasmid Preparation

Journal: Oxidative Medicine and Cellular Longevity

Article Title: RIP3 Contributes to Cardiac Hypertrophy by Influencing MLKL-Mediated Calcium Influx

doi: 10.1155/2022/5490553

Figure Lengend Snippet: Blockage of calcium influx reverses RIP3-mediated exacerbation of cardiac remodeling. (a–c) Heart hypertrophy parameters including HW/BW, LW/BW, and HW/TL of rats. Rats (VEC and oe-RIP3) were treated with PBS, LaCl 3 (5 mg/kg body weight, BW), and 2-APB (5 mg/kg BW) by intraperitoneal for 4 weeks. (d) The gross appearance and H&E staining of the whole heart from the rats in (a–c). (e–g) Cell morphology of NRCMs was determined by actin staining. NRCMs were stably infected with empty (VEC) or RIP3 plasmid, which were prestimulated by LaCl 3 (2.5 μ M) and 2-APB (10 μ M) for 24 hrs and then treated with Ang-II (1 μ M), PE (50 μ M), and PBS for 24 hrs. n = 6 per experimental group, ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: Empty rAAV vector and rAAV vector expressing RIP3 (oe-RIP3) or RIP3-specific small hairpin RNA (shRIP3) were ordered from GeneChem company (Shanghai, China).

Techniques: Staining, Stable Transfection, Infection, Plasmid Preparation

Journal: Cell Biology International

Article Title: Homocysteine facilitates endoplasmic reticulum stress and apoptosis of hepatocytes by suppressing ERO1α expression via cooperation between DNMT1 and G9a

doi: 10.1002/cbin.11805

Figure Lengend Snippet: Primer sequences for qRT‐PCR

Article Snippet: The vectors expressing small interfering RNA (siRNA) for ERO1α , DNMT1, and G9a were purchased from Genechem Co. Ltd. (Genechem Co. Ltd.).

Techniques: Sequencing

Journal: Cell Biology International

Article Title: Homocysteine facilitates endoplasmic reticulum stress and apoptosis of hepatocytes by suppressing ERO1α expression via cooperation between DNMT1 and G9a

doi: 10.1002/cbin.11805

Figure Lengend Snippet: ERO1α primer sequences for nMS‐PCR

Article Snippet: The vectors expressing small interfering RNA (siRNA) for ERO1α , DNMT1, and G9a were purchased from Genechem Co. Ltd. (Genechem Co. Ltd.).

Techniques: Sequencing, Methylation

Journal: Cell Biology International

Article Title: Homocysteine facilitates endoplasmic reticulum stress and apoptosis of hepatocytes by suppressing ERO1α expression via cooperation between DNMT1 and G9a

doi: 10.1002/cbin.11805

Figure Lengend Snippet: Hcy induces ER stress and apoptosis by downregulation of ERO1α in hepatocyte. (a and b) Western blot and qRT‐PCR were used to detect the mRNA and protein expression of ERO1α in the liver tissue of cbs +/+ and cbs +/ − mice ( n = 6) and hepatocytes treated with 100 μM Hcy ( n = 3). (c) The expression of ERO1α was examined by qRT‐PCR and western blot in hepatocytes transfected with siRNAs targeting ERO1α (si‐ ERO1α ‐1/2/3) ( n = 3). (d) ERO1α expression in hepatocytes transfected with ERO1α ‐overexpressed plasmids (p ERO1α ) or negative control vector (EGFP‐N1) was detected by qRT‐PCR and western blot ( n = 3). (e) The protein expression of GRP78, ATF6, PERK, and XBP‐1 were measured by western blot in hepatocytes transfected with p ERO1α or si‐ ERO1α in the presence of Hcy ( n = 3). (f) Flow cytometry was used to detect the apoptosis rate of hepatocytes transfected with p ERO1α or si‐ ERO1α in the presence of Hcy ( n = 3). Data represents mean ± SD , * p < 0.05, ** p < 0 .01. ER, endoplasmic reticulum; ERO1α , endoplasmic reticulum oxidoreductase 1α; Hcy, homocysteine; qRT‐PCR, quantitative real‐time polymerase chain reaction; SD , standard deviation

Article Snippet: The vectors expressing small interfering RNA (siRNA) for ERO1α , DNMT1, and G9a were purchased from Genechem Co. Ltd. (Genechem Co. Ltd.).

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Transfection, Negative Control, Plasmid Preparation, Flow Cytometry, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Cell Biology International

Article Title: Homocysteine facilitates endoplasmic reticulum stress and apoptosis of hepatocytes by suppressing ERO1α expression via cooperation between DNMT1 and G9a

doi: 10.1002/cbin.11805

Figure Lengend Snippet: Hcy inhibits ERO1α expression through DNA methylation. (a) Bioinformatics was used to analyze the methylation sites of ERO1α promoter from −2000 to −1 region on the MethPrimer website ( http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi ), and the predicted CpG islands were indicated by blue color. (b) Several fragments (−1 to −600, −1 to −900, −1 to −1100, −1 to −1700) of the ERO1α promoter were cloned into a pGL3‐basic reporter vector and cotransfected with a Renilla reporter plasmid in HEK293T cells, and the ERO1α promoter activity was analyzed by dual‐luciferase reporter assay ( n = 3). (c) Methylation levels of ERO1α promoter in liver tissues from cbs +/ − and cbs +/+ mice were detected by nMS‐PCR after bisulfite modification of the DNA ( n = 6). M: methylation; U: unmethylation. (d) MassARRAY methylation analysis was used to measure the methylation level of ERO1α promoter in hepatocytes cells treated with 100 μM Hcy ( n = 3). (e) The mRNA and protein expression of ERO1α were determined by qRT‐PCR and western blot in hepatocytes treated with DNMT1, DNMT3a, and DNMT3b specific inhibitors (DC‐05, Theaflavin‐3 (TF‐3), and Nanaomycin A (NA), respectively ( n = 3). (f) DNMT1 protein expression was analyzed by western blot in liver tissues of cbs +/ − and cbs +/+ mice ( n = 6). (G) Protein expression of DNMT1 was analyzed by western blot in hepatocytes treated with Hcy ( n = 3). (h) Protein expression of DNMT1 was analyzed in hepatocytes after transfection with negative control vector (EGFP‐N1) or DNMT1‐overexpressed plasmid (pDNMT1) by western blot ( n = 3). (i) Western blot was used to detect DNMT1 protein expression in hepatocytes transfected with DNMT1 siRNAs (si‐DNMT1‐1/2/3) ( n = 3). (j) ERO1α methylation level was measured with MassARRAY methylation analysis after hepatocytes transfected with pDNMT1 or si‐DNMT1 in the presence of Hcy ( n = 3). (k) Protein expression of ERO1α was analyzed by western blot in Hcy‐treated hepatocytes after transfection with pDNMT1 or si‐DNMT1 ( n = 3). Data represents mean ± SD , * p < 0.05, ** p < 0 .01. DNMT1, DNA methyltransferase 1; ER, endoplasmic reticulum; ERO1α , endoplasmic reticulum oxidoreductase 1α; Hcy, homocysteine; nMS‐PCR, nested methylation‐specific polymerase chain reaction; qRT‐PCR, quantitative real‐time polymerase chain reaction; SD , standard deviation

Article Snippet: The vectors expressing small interfering RNA (siRNA) for ERO1α , DNMT1, and G9a were purchased from Genechem Co. Ltd. (Genechem Co. Ltd.).

Techniques: Expressing, DNA Methylation Assay, Methylation, Clone Assay, Plasmid Preparation, Activity Assay, Luciferase, Reporter Assay, Modification, Quantitative RT-PCR, Western Blot, Transfection, Negative Control, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Cell Biology International

Article Title: Homocysteine facilitates endoplasmic reticulum stress and apoptosis of hepatocytes by suppressing ERO1α expression via cooperation between DNMT1 and G9a

doi: 10.1002/cbin.11805

Figure Lengend Snippet: G9a mediates H3K9me2 at ERO1α promoter in hepatocytes with Hcy treatment. (a and b) The H3K9me2 levels in liver tissues of cbs +/ − mice ( n = 6) and hepatocytes treated with Hcy ( n = 3) were analyzed by western blot. (c) The enrichment of H3K9me2 on ERO1α promoter was assayed by ChIP assay in hepatocytes after Hcy treatment ( n = 6). (d and e) G9a protein expression was detected by western blot in liver tissue of cbs +/ − mice ( n = 6) and hepatocytes treated with Hcy ( n = 3). (f) G9a protein expression was measured by western blot in hepatocytes transfected with G9a‐overexpressed plasmid (pG9a) ( n = 3). (g) Protein expression of G9a was examined by western blot in hepatocytes after transfection with three G9a siRNAs (si‐G9a‐1/2/3) ( n = 3). (h) ChIP assay for analysis of the enrichment of H3K9me2 on ERO1α promoter in hepatocytes after transfection with pG9a or si‐G9a in the presence of Hcy ( n = 3). (i) Western blot was used to measure the protein expression of ERO1α in Hcy‐treated hepatocytes after transfection with pG9a and si‐G9a ( n = 3). Data represents mean ± SD , * p < 0 .05, ** p < 0 .01. ChIP, chromatin immunoprecipitation; ERO1α , endoplasmic reticulum oxidoreductase 1α; Hcy, homocysteine; siRNA, small interfering RNA

Article Snippet: The vectors expressing small interfering RNA (siRNA) for ERO1α , DNMT1, and G9a were purchased from Genechem Co. Ltd. (Genechem Co. Ltd.).

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Chromatin Immunoprecipitation, Small Interfering RNA

Journal: Cell Biology International

Article Title: Homocysteine facilitates endoplasmic reticulum stress and apoptosis of hepatocytes by suppressing ERO1α expression via cooperation between DNMT1 and G9a

doi: 10.1002/cbin.11805

Figure Lengend Snippet: DNMT1 cooperates with G9a to regulate ERO1α expression in hepatocytes treated with Hcy. (a) DNA methylation level of ERO1α promoter was measured by nMS‐PCR in hepatocytes after transfection with siRNA of DNMT1 and G9a in the presence of Hcy ( n = 3). (b) Enrichment of H3K9me2 on ERO1α promoter was detected by ChIP assay in hepatocytes after transfection with si‐DNMT1 and si‐G9a in the presence of Hcy ( n = 3). (c) Protein expression of ERO1α was measured by western blot in hepatocytes after transfection with si‐DNMT1 and si‐G9a in the presence of Hcy ( n = 3). (d) DNA methylation of ERO1α was detected by nMS‐PCR in hepatocytes after treatment with DC‐05 and Bix (Bix01294, G9a specific inhibitor) in the presence of Hcy ( n = 3). (e) ChIP assay was used to detect the enrichment of H3K9me2 on ERO1α promoter in hepatocytes after treatment with DC‐05 and Bix in the presence of Hcy ( n = 3). (f) Detection of ERO1α protein expression in hepatocytes treated with DC‐05 and Bix in the presence of Hcy ( n = 3). Data represents mean ± SD , * p <0 .05, ** p < 0.01. DNMT1, DNA methyltransferase 1; ChIP, chromatin immunoprecipitation; ERO1α , endoplasmic reticulum oxidoreductase 1α; Hcy, homocysteine; nMS‐PCR, nested methylation‐specific polymerase chain reaction

Article Snippet: The vectors expressing small interfering RNA (siRNA) for ERO1α , DNMT1, and G9a were purchased from Genechem Co. Ltd. (Genechem Co. Ltd.).

Techniques: Expressing, DNA Methylation Assay, Transfection, Western Blot, Chromatin Immunoprecipitation, Methylation, Polymerase Chain Reaction

Journal: Cells

Article Title: Aquaporin-3 and Aquaporin-5 Facilitate Migration and Cell–Cell Adhesion in Pancreatic Cancer by Modulating Cell Biomechanical Properties

doi: 10.3390/cells11081308

Figure Lengend Snippet: AQP3 and AQP5 are the most abundant AQPs in pancreatic BxPC-3 cells. ( A ) mRNA expression levels of the AQP paralogs naturally expressed in BxPC-3 cells. Values normalized to HPRT-1. ( B – G ) Validation of the loss-of-function model. ( B , C ) mRNA expression levels of BxPC-3 cells silenced for AQP3 ( B ) and AQP5 ( C ). Values normalized to HPRT-1. ( D , F ) Time course of cell volume changes caused by osmotic and solute challenges with mannitol ( D ) and glycerol ( F ) in control, siAQP3, siAQP5, and siAQP3/5 cells. ( E , G ) Water (P f ) ( E ) and glycerol (P gly ) permeabilities ( G ). ( H ) Representative traces of intracellular ROS accumulation, given by H 2 -DCFDA fluorescence increase after addition of 100 μM H 2 O 2 . ( I ) First-order kinetic rate constant of H 2 O 2 influx through endogenous AQP3 and AQP5 in BxPC-3 cells after a 100 μM H 2 O 2 challenge. Data represent a mean of n = 3 independent experiments ± standard error of the mean (SEM); n = 10 for permeability assays. ** p < 0.01; *** p < 0.001; both silenced vs. control cells; siAQP3, AQP3-silenced cells; siAQP5, AQP5-silenced cells; and, siAQP3/5, AQP3-, and AQP5-silenced cells.

Article Snippet: For AQPs knock-down, small interfering RNA (siRNA)-containing expression vectors targeting human AQP3 and AQP5 (ID: s1521 and ID: s1527, respectively, Ambion, Thermo Fisher Scientific) combined with Lipofectamine RNAiMAX Reagent (Invitrogen) were used according to the manufacturer protocol.

Techniques: Expressing, Biomarker Discovery, Control, Fluorescence, Permeability

Journal: Cells

Article Title: Aquaporin-3 and Aquaporin-5 Facilitate Migration and Cell–Cell Adhesion in Pancreatic Cancer by Modulating Cell Biomechanical Properties

doi: 10.3390/cells11081308

Figure Lengend Snippet: Effect of transient silencing of AQP3 and AQP5 in BxPC-3 migration. ( A ) Representative images of wound closure progression in control, AQP3-, AQP5-, and AQP3/5-silenced cells at 0, 12, and 24 h. ( B ) Wound extension progression in control, AQP3-, AQP5-, and AQP3/5-silenced cells at 0, 12, and 24 h past wound opening. ( C ) Cell migration rate of control, AQP3-, AQP5-, and AQP3/5-silenced cells. Results are expressed as mean ± SEM of three independent experiments. § p < 0.05; §§§ p < 0.001; both times after wound opening vs. initial measurement (0 h); *** p < 0.001, silenced vs. control cells.

Article Snippet: For AQPs knock-down, small interfering RNA (siRNA)-containing expression vectors targeting human AQP3 and AQP5 (ID: s1521 and ID: s1527, respectively, Ambion, Thermo Fisher Scientific) combined with Lipofectamine RNAiMAX Reagent (Invitrogen) were used according to the manufacturer protocol.

Techniques: Migration, Control

Journal: Cells

Article Title: Aquaporin-3 and Aquaporin-5 Facilitate Migration and Cell–Cell Adhesion in Pancreatic Cancer by Modulating Cell Biomechanical Properties

doi: 10.3390/cells11081308

Figure Lengend Snippet: AQP5 is crucial for cell viability. ( A ) Cell viability analysis of control, siAQP3, siAQP5, and siAQP3/5 BxPC-3 cells stained with YO-PRO-1 and PI. Cells in the lower right quadrant are in early apoptosis, and those in the upper right quadrant are in mid and late apoptosis. ( B ) Percentage of viable, apoptotic, and necrotic cells. Results are expressed as mean ± SEM of three independent experiments. *** p < 0.001, silenced vs. control cells.

Article Snippet: For AQPs knock-down, small interfering RNA (siRNA)-containing expression vectors targeting human AQP3 and AQP5 (ID: s1521 and ID: s1527, respectively, Ambion, Thermo Fisher Scientific) combined with Lipofectamine RNAiMAX Reagent (Invitrogen) were used according to the manufacturer protocol.

Techniques: Control, Staining