Journal: Disease Models & Mechanisms

Article Title: A DUSP6 inhibitor suppresses inflammatory cardiac remodeling and improves heart function after myocardial infarction

doi: 10.1242/dmm.049662

Figure Lengend Snippet: BCI has no effect on CM proliferation, H 2 O 2 -induced CM death, CM hypertrophy and coronary vessel regeneration. (A) Released lactate dehydrogenase (LDH) was comparable in neonatal rat ventricular myocytes (NRVMs) treated with DMSO or BCI for 24 h in the presence or absence of H 2 O 2 ( n =3 per group). (B) Western blots and quantification of Bcl-2 (α-tubulin was used to normalize protein) and p-AKT (AKT was used to normalize protein) in NRVMs treated with DMSO or BCI for 24 h ( n =3 per group). (C) Western blots and quantification of H 2 O 2 -induced cleaved PARP (α-tubulin was used to normalize protein) and cleaved caspase 3 (total caspase 3 was used to normalize protein) in NRVMs treated with DMSO or BCI ( n =3 per group). (D) Immunofluorescent staining showing cTnT + and pH3 + CMs of sham-operated, vehicle-treated or BCI-treated LV tissues at 7 days after MI (scale bar: 100 μm). (E) Immunofluorescent staining showed that the numbers of either α-actinin + Ki67 + or α-actinin + pH3 + CMs were comparable in DMSO- and BCI-treated NRVMs (scale bars: 50 μm; n =3 per group). HPF, high-power field. (F) Immunofluorescent staining and statistics for CD31 + and α-SMA + vessels of sham-operated, vehicle-treated or BCI-treated LV tissues at 7 days after MI (scale bar: 100 μm; n =3 per group). (G) Wheat germ agglutinin staining and statistics for CM size in sham-operated, vehicle-treated or BCI-treated LV tissues at 28 days after MI (scale bar: 20 μm; n =5 per group). Mean±s.e.m.; ns, not significant.

Article Snippet: After blocking, the membranes were incubated with anti-Bcl-2 (1:1000; Cell Signaling Technology, 3498S), anti-AKT (1:1000; Cell Signaling Technology, 9272S), anti-p-AKT (1:1000; Cell Signaling Technology, 4060S), anti-cleaved PARP (1:1000; Cell Signaling Technology, 9548T), anti-caspase 3 (1:1000; Cell Signaling Technology, 9662S), anti-cleaved caspase 3 (1:1000; Cell Signaling Technology, 9664S), anti-NF-κB p65 (1:1000; Cell Signaling Technology, 8242S), anti-p-NF-κB p65 (1:1000; Cell Signaling Technology, 3033S), anti-ERK (1:1000; Cell Signaling Technology, 4695S), anti-p-ERK (1:1000; Cell Signaling Technology, 4370S), anti-p38 (1:1000; Cell Signaling Technology, 8690S), anti-p-p38 (1:1000; Cell Signaling Technology, 4511S), anti-JNK (1:1000; Abcam, Cambridge, UK, ab179461) or anti-p-JNK (1:1000; Abcam, ab76572).

Techniques: Western Blot, Staining

Journal: Gut Microbes

Article Title: Epithelial talin-1 protects mice from citrobacter rodentium -induced colitis by restricting bacterial crypt intrusion and enhancing t cell immunity

doi: 10.1080/19490976.2023.2192623

Figure Lengend Snippet: Loss of epithelial-specific talin-1 suppresses pathogen-induced epithelial apoptosis. (a) Representative images of colon tissues immunoperoxidase-stained for cleaved caspase-3. (b) the proportion of cleaved caspase-3-positive mucosa determined by measuring the total height of the mucosa and the height of the region with positive staining. Each dot represents measurements from a high-powered field; n = 12–15 fields from 3 different mice per group. All values reported with the median depicted as a thick line and the upper and lower quartiles as thin lines. (c) Expression of the gene encoding TNF-α analyzed by RT-qPCR. n = 4 uninfected mice and n = 6–7 infected mice per genotype. Each symbol is a different mouse. Values are reported as mean ± SEM. * P < 0.05 and ** P < 0.01 determined by (b) 1-way ANOVA and Tukey test and (c) 1-way ANOVA with Kruskal-Wallis test, followed by a Mann-Whitney U test. Scale bars represent 100 μm.

Article Snippet: Tissues were then incubated overnight at 4°C using the following antibodies: prediluted rabbit polyclonal anti-Ki-67 (Biocare, PRM325AA), prediluted rabbit monoclonal anti-MPO (Biocare, PP023AA), rabbit polyclonal anti-CD3 (Abcam, ab5690; 1:150), or rabbit monoclonal anti-cleaved caspase-3 (Cell Signaling, 9664; 1:400).

Techniques: Staining, Expressing, Quantitative RT-PCR, Infection, MANN-WHITNEY

Journal: The American Journal of Pathology

Article Title: Lung remodeling regions in long-term Covid-19 feature basal epithelial cell reprogramming

doi: 10.1016/j.ajpath.2023.02.005

Figure Lengend Snippet: Cell proliferation and apoptosis in lung remodeling regions in Covid-19. A, Immunostaining for Ki-67 plus CD68 with DAPI counterstaining in lung sections from Covid-19 patients and non-disease controls (ND). Scale bar=100 μm; Original magnification, x4 (inset). B , Quantitation of staining for conditions in (A). C , Immunostaining for active caspase-3 with DAPI counterstaining in lung sections for conditions in (A). Scale bar=100 μm. D , Quantitation of staining for conditions in (C). E , Immunostaining for active-caspase-3 plus HT2-280 or CD68 with DAPI counterstaining for conditions in (A). Scale bar=50 μm; Original magnification, x3 (inset). F , Quantitation of staining for conditions in (E). G , Immunostaining for KRT5 plus CXCL17 with DAPI counterstaining for conditions in (A). h, Scale bar=50 μm; Original magnification, x3 (inset). H, Quantitation of staining for conditions in (G). Data are representative of 5 patients and 5 control subjects per staining condition. Values represent mean and s.e.m; * P <0.05 (n=5 patients or subjects per group).

Article Snippet: Immunostaining was performed using the following primary antibodies: rabbit anti-ACE-2 polyclonal Ab (pAb) (ab65863, Abcam), mouse anti-ACE-2 mAb (clone 171606, R&D Systems), rabbit anti-KRT5 pAb (ab53121, Abcam) and mAb (clone EP1601Y, ab52635, Abcam), rabbit anti-AQP3 pAb (ab125219, Abcam), mouse anti-CD68 mAb (clone Kp-1, Sigma-Aldrich), rabbit anti-CD163 mAb (clone D6U1J, Cell Signaling), rabbit anti-CD31 mAb (clone EPR17259, Abcam), rabbit anti-CollagenIV (CollIV) mAb (clone EPR20966, Abcam), mouse anti-SCGB1A1 mAb (clone E-11, Santa Cruz), mouse anti-acetylated Tubulin (clone 6-11B-1, Sigma-Aldrich), mouse anti-MUC5AC mAb (clone 45M1, ThermoFisher Scientific and Santa Cruz Biotechnology), mouse anti-HT2-280 pAb (TB-27AHT2-280, Terrace Biotech), rabbit anti-surfactant protein C (SFTPC) pAb (ab90716, Abcam), rabbit anti-podoplanin (PDPN) mAb (clone EPR7072, Abcam), rabbit anti-MUC5B pAb (ab87276, Abcam), rabbit anti-Ki-67 mAb (clone D2H10, Cell Signaling), rabbit active (cleaved) caspase-3 mAb (clone 5A1E, Cell Signaling), and mouse anti-CXCL17 mAb (clone 422204, R&D Systems).

Techniques: Immunostaining, Quantitation Assay, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: Estrogen downregulates CD73/adenosine axis hyperactivity via adaptive modulation PI3K/Akt signaling to prevent myocarditis and arrhythmias during chronic catecholamines stress

doi: 10.1186/s12964-023-01052-0

Figure Lengend Snippet: Antibody used in the study

Article Snippet: 8 , Cleaved Caspase-3 (Asp175) (D3E9) Rabbit mAb , Cell Signalling Technology , #9579.

Techniques:

Journal: Cell Communication and Signaling : CCS

Article Title: Estrogen downregulates CD73/adenosine axis hyperactivity via adaptive modulation PI3K/Akt signaling to prevent myocarditis and arrhythmias during chronic catecholamines stress

doi: 10.1186/s12964-023-01052-0

Figure Lengend Snippet: In vivo results of estrogenic adaptive regulation of CD73/adenosine axis via PI3K/Akt pathways during CCS. A – D Representative immunoblots and graphical presentations of cleaved caspase 3, PI3K, p-PI3K and Akt, p-Akt, were assessed from Mice. S = Sham; S + I = Sham + ISO; O = OVX; O + I = OVX + ISO; O + E, OVX + E2; O + E + I = OVX + ISO + E2. E – H In vitro results of inhibition of PI3Kby LY294002. Representative immunoblots and graphical presentations of, PI3K, p-PI3K, Akt, p-Akt and CD73. I , J Representative immunofluorescence and plotted average values of CC3 expression. Data are presented as mean ± SEM; *** p < 0.001; ** p < 0.01; * p < 0.05

Article Snippet: 8 , Cleaved Caspase-3 (Asp175) (D3E9) Rabbit mAb , Cell Signalling Technology , #9579.

Techniques: In Vivo, Western Blot, In Vitro, Inhibition, Immunofluorescence, Expressing

Journal: bioRxiv

Article Title: Constitutive protein degradation induces acute cell death via proteolysis products

doi: 10.1101/2023.02.06.527237

Figure Lengend Snippet: a , Growth-rate (GR) drug sensitivity metrics of human cancer cell lines in response to the BET degrader MZ1 and the inactive epimer cis-MZ1, calculated based on dose-response curves from triplicate experiments. GRmax less than zero denotes cytotoxic range (efficacy). Log-transformed GR50 represents drug potency. NA denotes unavailable GR50 due to insensitivity to the treatments. b , GR metrics of the 22Rv1 cell line in response to MZ1, the inhibitor controls JQ1 and cis-MZ1, and the E3 ligand VH032. c , Immunoblot of the indicated proteins from 22Rv1 cells treated with BET siRNA, degrader or inhibitor in combination with the corresponding E3 ligand. C-term and N-term denote two separate BRD4 antibodies that recognize the amino acids 1312–1362 and 150–250 of BRD4, respectively. BET inhibitor -/+ E3 ligand: JQ1 and the paired JQ1 + VH032 are controls for MZ1 and ARV-771; JQ1 and JQ1 + thal.(thalidomide) for dBET1 and dBET6; HJB97 and HJB97 + lenal. (lenalidomide) for BETd-260 (also see for tabulated BETd molecules and the corresponding parental ligands). NT, non-targeting; n.s., non-specific bands. d , Kinetic analysis of siRNA-transfected 22Rv1 cells treated with the indicated compounds, along with Caspase-3/7 Green Detection Reagent (Invitrogen). Time zero denotes 4 days after siRNA transfection and the beginning of drug treatments. Data are presented as mean ± se; n = 3. e , siRNA-treated 22Rv1 cells were re-plated for DMSO or MZ1 treatment and collected for immunoblot by the indicated time point. f , Time course analysis of MZ1 treatments in siRNA-treated 22Rv1 cells. g , siRNA-treated 22Rv1 cells were re-plated for the treatment of DMSO or the indicated BETd compound. Unless stated otherwise in Reporting Summary, data represent two to four independent experiments. Source data are provided in Supplementary Information.

Article Snippet: Antibodies used for immunoblotting are as following: BRD4 N-term (ab128874, Abcam), BRD4 C-term (A700-004, Bethyl), BRD2 (ab139690, Abcam), BRD3 (A302-368A, Bethyl), Caspase-3 (9662, CST), Cleaved caspase-3 (9664, CST), Caspase-8 (ab32397, Abcam), Cleaved caspase-8 (9746, CST), GAPDH (2118, CST), cIAP1 (AF8181, R&D Systems), XIAP (610717, BD), VCP/p97 (ab109240, Abcam), Strep (SAB2702216, Sigma), PSMD4 (3336, CST), IgA (11449-1-AP, Proteintech), RNF114 (HPA021184, Sigma), CDK9 (2316, CST), Cyclin K (A301-939A, Bethyl), Wee1 (4936, CST), CRBN (HPA045910, Sigma).

Techniques: Transformation Assay, Western Blot, Transfection

Journal: bioRxiv

Article Title: Constitutive protein degradation induces acute cell death via proteolysis products

doi: 10.1101/2023.02.06.527237

Figure Lengend Snippet: a , Immunoblot to confirm BRD4 depletion. 22Rv1 cells transfected with the indicated siRNA were collected after three days, or re-plated and cultured for additional two days before harvest. b , As described in , time course analysis of percent cell confluence in response to the indicated treatments. Data are presented as mean ± se; n = 3. c , As described in , representative images of siRNA-treated 22Rv1 cells in response to MZ1 treatment at hour 20. Magenta masks denote caspase-3/7 cleavage. d , siRNA-transfected 22Rv1 cells were re-plated for the indicated treatments, and cell lysates were analyzed by immunoblot using the indicated antibodies. Unless stated otherwise in Reporting Summary, data represent two to three independent experiments. Source data are provided in Supplementary Information.

Article Snippet: Antibodies used for immunoblotting are as following: BRD4 N-term (ab128874, Abcam), BRD4 C-term (A700-004, Bethyl), BRD2 (ab139690, Abcam), BRD3 (A302-368A, Bethyl), Caspase-3 (9662, CST), Cleaved caspase-3 (9664, CST), Caspase-8 (ab32397, Abcam), Cleaved caspase-8 (9746, CST), GAPDH (2118, CST), cIAP1 (AF8181, R&D Systems), XIAP (610717, BD), VCP/p97 (ab109240, Abcam), Strep (SAB2702216, Sigma), PSMD4 (3336, CST), IgA (11449-1-AP, Proteintech), RNF114 (HPA021184, Sigma), CDK9 (2316, CST), Cyclin K (A301-939A, Bethyl), Wee1 (4936, CST), CRBN (HPA045910, Sigma).

Techniques: Western Blot, Transfection, Cell Culture

Journal: bioRxiv

Article Title: B3GALT6 Promotes Dormant Breast Cancer Cell Survival and Recurrence by Enabling Heparan Sulfate-Mediated FGF Signaling

doi: 10.1101/2023.01.31.526529

Figure Lengend Snippet: A. Inference of CRISPR edits (ICE) analysis for sg B3galt6 _3 and B. sg B3galt6 _1 displaying the proportion of indels in the population. Wild-type sequence is represented by the orange + sign at 0. Dotted line indicates the Cas9 cut site. C. Immunofluorescence and D. associated quantification for Ki67 (red) or E, F. cleaved caspase 3 (cc3, red) in Her2-dependent-Cas9 sg Rosa and sg B3galt6 _3 (green) PTs, D4, and D7 RLs. Scale bar=100μm. Quantification in the sg Rosa (grey) and sg B3galt6 _3 (dark blue) groups is represented as mean ± SD. * p <0.05.

Article Snippet: Immunofluorescence samples were blocked with 5% goat serum in 1x PBS with mouse-on-mouse block (Vector Laboratories, Cat. # BMK-2202), washed 3 times in 1x PBS and incubated overnight at 4°C with primary antibodies or matched isotype controls diluted in 5% goat serum in 1x PBS with mouse-on-mouse diluent (Vector Laboratories, Cat. # BMK-2202) as follows: Primary antibodies - Rat anti-Ki67 (Thermo Fisher Scientific, Cat. # 14-5698; 1:100), Rabbit anti-Her2 (Cell Signaling, Cat. # 2165; 1:100), Rabbit anti-cleaved Caspase-3 (Cell Signaling, Cat. # 9664; 1:250), Mouse anti-Chondroitin sulfate (Sigma, Cat. # SAB4200696), Mouse anti-Heparan sulfate (Amsbio, Cat. # 370225), Rabbit anti-GFP (Cell Signaling, Cat. # 2956; 1:200), and Mouse anti-GFP (Living Colors, Cat. # 632381; 1:250).

Techniques: CRISPR, Sequencing, Immunofluorescence

Journal: Toxics

Article Title: L-Ascorbic Acid 2-Phosphate Attenuates Methylmercury-Induced Apoptosis by Inhibiting Reactive Oxygen Species Accumulation and DNA Damage in Human SH-SY5Y Cells

doi: 10.3390/toxics11020144

Figure Lengend Snippet: A cell damage model of MeHg was created by the treatment of SH-SY5Y cells with 0–4 μM MeHg at indicated time intervals. ( A ) MTT assay showing cell viability of SH-SY5Y cells treated with 0–4 μM MeHg for 6–48 h ( B ) The live cell workstation used to observe cell morphology of SH-SY5Y cells treated with 0–2 μM MeHg for 24 h. ( C ) Flow cytometry detection of apoptosis in 0–2 μM MeHg treated SH-SY5Y cells for 24 h. ( D ) GraphPad Prism 8.0.2 software was used to analyze the total apoptosis rate of SH-SY5Y cells treated with 0–2 μM MeHg for 24 h. Statistical significance was determined using ANOVA; p < 0.05. ( E ) Western blot analysis of the protein expression levels of PARP1, AIF, PAR, Cyt C, and cleaved Caspase-3 in SH-SY5Y cells treated with 0–2 μM MeHg for 24 h. ( F ) The Tanon-2500 fully automated digital gel image analysis system was used to analyze the net optical density of the Western blot strips. All values are represented as means ± SD ( n = 3). Superscript denotes a statistically significant difference between groups ( p < 0.05). Abbreviations: AIF, apoptosis-inducing factor; ANOVA, analysis of variance; Cyto C, cytochrome C; MeHg, methylmercury; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PAR, poly (adp-ribose); PARP1, poly (adp-ribose) polymerase 1; SH-SY5Y, neuroblastoma cells.

Article Snippet: The membranes were incubated in 5% non-fat milk powder (Anchor, Auckland City, New Zealand) at room temperature for 1 h. The membranes were then incubated with the following primary antibodies: PARP1 rabbit mAb (1:1000; CST), AIF rabbit mAb (1:1000, CST), Cleave Caspase-3 rabbit mAb (1:1000, CST), γH2AX rabbit mAb (1:1000, CST), Cyt C rabbit mAb (1:1000; CST), KU70 rabbit mAb (1:1000; CST), BRCA1 rabbit mAb (1:1000; CST), RAD51 rabbit mAb (1:1000; CST), GAPDH mouse mAb (1:5000; ProteinTech Group Inc.), HRP-conjugated AffiniPure Goat Anti-Rabbit IgG (H + L) (1:10,000; ProteinTech Group Inc., Wuhan East Lake New Technology Development Zone, China), and HRP-conjugated AffiniPure Goat Anti-Mouse IgG (H + L) (1:10,000; Protein-Tech Group Inc., Wuhan East Lake New Technology Development Zone, China).

Techniques: MTT Assay, Flow Cytometry, Software, Western Blot, Expressing

Journal: Toxics

Article Title: L-Ascorbic Acid 2-Phosphate Attenuates Methylmercury-Induced Apoptosis by Inhibiting Reactive Oxygen Species Accumulation and DNA Damage in Human SH-SY5Y Cells

doi: 10.3390/toxics11020144

Figure Lengend Snippet: MeHg induces DNA damage and participates in both NHEJ repair and induced apoptosis in SH-SY5Y cells ( A ) The alkaline comet assay detects cell trailing in 1 μM MeHg-treated SH-SY5Y cells from 0–24 h. The images were photographed with a Leica fluorescence microscope. ( B ) CASP software analyses of comet image and the Olive tail moment can be used to determine the extent of DNA damage. ( C ) Western blot analysis detected the protein expression levels of γH2AX, BRCA1, RAD51, KU70, PARP1, AIF, PAR, Cyt C, and cleaved Caspase-3 in SH-SY5Y cells treated with 1 μM MeHg from 0–24 h. ( D ) The Tanon-2500 fully automated digital gel image analysis system was used to analyze the net optical density of the Western blot strips. The mean was calculated using GraphPad Prism 8.0.2 software. The data shown represent the mean from one of three independent experiments. Statistical significance was determined using variance (ANOVA). All values are represented as means ± SD ( n = 3). Superscript denotes a statistically significant difference between groups ( p < 0.05). Abbreviations: BRCA1 and RAD51, homologous repair-associated repair proteins; HR, homologous recombination; KU70, non-homologous end joining-associated repair protein; NHEJ, nonhomologous end joining; γH2AX, phosphorylated histone H2AX.

Article Snippet: The membranes were incubated in 5% non-fat milk powder (Anchor, Auckland City, New Zealand) at room temperature for 1 h. The membranes were then incubated with the following primary antibodies: PARP1 rabbit mAb (1:1000; CST), AIF rabbit mAb (1:1000, CST), Cleave Caspase-3 rabbit mAb (1:1000, CST), γH2AX rabbit mAb (1:1000, CST), Cyt C rabbit mAb (1:1000; CST), KU70 rabbit mAb (1:1000; CST), BRCA1 rabbit mAb (1:1000; CST), RAD51 rabbit mAb (1:1000; CST), GAPDH mouse mAb (1:5000; ProteinTech Group Inc.), HRP-conjugated AffiniPure Goat Anti-Rabbit IgG (H + L) (1:10,000; ProteinTech Group Inc., Wuhan East Lake New Technology Development Zone, China), and HRP-conjugated AffiniPure Goat Anti-Mouse IgG (H + L) (1:10,000; Protein-Tech Group Inc., Wuhan East Lake New Technology Development Zone, China).

Techniques: Alkaline Single Cell Gel Electrophoresis, Fluorescence, Microscopy, Software, Western Blot, Expressing, Homologous Recombination, Non-Homologous End Joining

Journal: Toxics

Article Title: L-Ascorbic Acid 2-Phosphate Attenuates Methylmercury-Induced Apoptosis by Inhibiting Reactive Oxygen Species Accumulation and DNA Damage in Human SH-SY5Y Cells

doi: 10.3390/toxics11020144

Figure Lengend Snippet: AA2P regulation of the PARP1/AIF and Caspase-3 pathways during MeHg-induced apoptosis of SH-SY5Y cells. ( A ) Flow cytometry detection of apoptosis in SH-SY5Y cells co-treated with 300 μM AA2P and 1 μM MeHg for 12 h. ( B ) GraphPad Prism 8 software analysis of the apoptosis rate of SH-SY5Y cells co-treated with 300 μM AA2P and 1 μM MeHg for 12 h. Statistical significance was determined using ANOVA, p < 0.05. ( C ) Flow cytometry detection of apoptosis in SH-SY5Y cells co-treated with 300 μM AA2P and 1 μM MeHg for 24 h. ( D ) GraphPad Prism 8.0.2 software analysis of the apoptosis rate of SH-SY5Y cells co-treated with 300 μM AA2P and 1μM MeHg for 24 h. Statistical significance was determined using ANOVA, p < 0.05. ( E ) Western blot analysis of PARP1, PAR, AIF, Cyt C, and cleaved Caspase-3 in SH-SY5Y cells co-treated with 300 μM AA2P and 1 μM MeHg for 24 h. ( F ) The Tanon-2500 fully automated digital gel image analysis system was used to analyze the net optical density of the Western blot strips. All values are represented as means ± SD ( n = 3).

Article Snippet: The membranes were incubated in 5% non-fat milk powder (Anchor, Auckland City, New Zealand) at room temperature for 1 h. The membranes were then incubated with the following primary antibodies: PARP1 rabbit mAb (1:1000; CST), AIF rabbit mAb (1:1000, CST), Cleave Caspase-3 rabbit mAb (1:1000, CST), γH2AX rabbit mAb (1:1000, CST), Cyt C rabbit mAb (1:1000; CST), KU70 rabbit mAb (1:1000; CST), BRCA1 rabbit mAb (1:1000; CST), RAD51 rabbit mAb (1:1000; CST), GAPDH mouse mAb (1:5000; ProteinTech Group Inc.), HRP-conjugated AffiniPure Goat Anti-Rabbit IgG (H + L) (1:10,000; ProteinTech Group Inc., Wuhan East Lake New Technology Development Zone, China), and HRP-conjugated AffiniPure Goat Anti-Mouse IgG (H + L) (1:10,000; Protein-Tech Group Inc., Wuhan East Lake New Technology Development Zone, China).

Techniques: Flow Cytometry, Software, Western Blot