Journal: Clinical Epigenetics

Article Title: The immunomodulatory anticancer agent, RRx-001, induces an interferon response through epigenetic induction of viral mimicry

doi: 10.1186/s13148-017-0312-z

Figure Lengend Snippet: RRx-001 induced IFN response through upregulation of type I and III IFN expression and JAK/STAT pathway. Cells were transiently (24 h) treated with 0.5 μM RRx-001 or 0.5 μM 5-AZA and subsequently maintained in drug-free medium for an additional 7 days. RRx-001 induced a significant increase in type I IFN (IFN-β) ( a ) and type III IFN (IL-29/IL-28B) ( b ) secretion into culture medium by HCT 116 cells as measured by ELISA. Transcript levels of IL29 / IL28A were also increased as determined by qPCR ( c ). ISGs ( IFI27 , IFi44 , IFI44L , and IFI6 ) were upregulated by 5-AZA and RRx-001 but blocked by the JAK/STAT inhibitor ruxolitinib (rux) at 2 μM concentration ( d ). Expression of ISGs ( IRF7 , ISG15 , DDX58 , and OASL ) was also upregulated in HCT 116 cells cultured in conditioned medium containing secreted IFNs induced by 5-AZA and RRx-001 as determined by qPCR ( e )

Article Snippet: Levels of type I IFN (IFN-β) and type III IFN (IL-29/IL-28B) were determined using a VeriKine Human IFN-beta ELISA Kit (PBL Assay Science, Piscataway Township, NJ, USA) and a Human IL-29/IL-28B (IFN-lambda 1/3) DuoSet ELISA Kit (R&D Systems, Minneapolis, MN, USA) according to manufactures’ instructions, respectively.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay, Cell Culture

Journal:

Article Title: Elevated Levels of Circulating Interleukin-18 in Human Immunodeficiency Virus-Infected Individuals: Role of Peripheral Blood Mononuclear Cells and Implications for AIDS Pathogenesis

doi: 10.1128/JVI.76.24.12448-12456.2002

Figure Lengend Snippet: Effect of TGF-β on IL-18 production from PBMC. PBMC (2 × 105) from HIV-seronegative donors were cultured in 200 μl of the culture medium in 96-well plate alone or in the presence of recombinant human (rh) TGF-β (20 ng per ml; R & D Systems), TGF-β-neutralizing, or control antibody (5 μg per ml each; R & D Systems). Culture supernatants were collected 24 h later and assayed for IL-18 content with the ELISA kit. The figure shows average ± standard error IL-18 contents from three replicate wells. Only the addition of the neutralizing antibody resulted in a significant (P < 0.05) increase in TGF-β production.

Article Snippet: PBMC (2 × 10 5 ) from HIV-seronegative donors were cultured in 200 μl of the culture medium in 96-well plate alone or in the presence of recombinant human (rh) TGF-β (20 ng per ml; R & D Systems), TGF-β-neutralizing, or control antibody (5 μg per ml each; R & D Systems).

Techniques: Cell Culture, Recombinant, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Elevated Levels of Circulating Interleukin-18 in Human Immunodeficiency Virus-Infected Individuals: Role of Peripheral Blood Mononuclear Cells and Implications for AIDS Pathogenesis

doi: 10.1128/JVI.76.24.12448-12456.2002

Figure Lengend Snippet: TGF-β contents of the plasma samples. The level of TGF-β was measured in the samples with a commercial ELISA kit. The figure depicts average ± standard error concentration of this cytokine in the samples after its activation (total TGF-β). N and P, HIV-seronegative healthy and HIV-infected donors, respectively. The star on the top of a bar indicates a significant (P < 0.05) difference between the two group means.

Article Snippet: PBMC (2 × 10 5 ) from HIV-seronegative donors were cultured in 200 μl of the culture medium in 96-well plate alone or in the presence of recombinant human (rh) TGF-β (20 ng per ml; R & D Systems), TGF-β-neutralizing, or control antibody (5 μg per ml each; R & D Systems).

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Activation Assay, Infection

Journal:

Article Title: Elevated Levels of Circulating Interleukin-18 in Human Immunodeficiency Virus-Infected Individuals: Role of Peripheral Blood Mononuclear Cells and Implications for AIDS Pathogenesis

doi: 10.1128/JVI.76.24.12448-12456.2002

Figure Lengend Snippet: Correlation between the levels of TGF-β in plasma and IL-18 in serum of HIV-infected individuals. The figure depicts a significant negative correlation between these two parameters as determined by the Pearson's method.

Article Snippet: PBMC (2 × 10 5 ) from HIV-seronegative donors were cultured in 200 μl of the culture medium in 96-well plate alone or in the presence of recombinant human (rh) TGF-β (20 ng per ml; R & D Systems), TGF-β-neutralizing, or control antibody (5 μg per ml each; R & D Systems).

Techniques: Infection

Journal: Cells

Article Title: Overexpression and Activation of αvβ3 Integrin Differentially Affects TGFβ2 Signaling in Human Trabecular Meshwork Cells

doi: 10.3390/cells10081923

Figure Lengend Snippet: Differentially expressed genes in WTβ3 and/or CAβ3 cells that are associated with glaucoma. Except where noted by *, Log 2 FC values were reported in DGE analysis using EdgeR. * Genes that were filtered out due to low abundance did not originally have a FC determined (ND). Log 2 FC was calculated from the raw count data. The values in parentheses were originally calculated using EdgeR.

Article Snippet: ELISA analysis was performed using an R&D Systems Human TGF-beta 2 Quantikine ELISA Kit (R & D Systems, Minneapolis, MN), and the procedure was performed according to the manufacturer’s instructions.

Techniques: Migration, Activity Assay, Binding Assay, Ubiquitin Proteomics

Journal: Cells

Article Title: Overexpression and Activation of αvβ3 Integrin Differentially Affects TGFβ2 Signaling in Human Trabecular Meshwork Cells

doi: 10.3390/cells10081923

Figure Lengend Snippet: Differentially expressed genes in WTβ3 and/or CAβ3 cells that are associated with IOP regulation. Log 2 FC values were reported in DGE analysis using EdgeR.

Article Snippet: ELISA analysis was performed using an R&D Systems Human TGF-beta 2 Quantikine ELISA Kit (R & D Systems, Minneapolis, MN), and the procedure was performed according to the manufacturer’s instructions.

Techniques:

Journal: Nature Microbiology

Article Title: Evolution of enhanced innate immune suppression by SARS-CoV-2 Omicron subvariants

doi: 10.1038/s41564-023-01588-4

Figure Lengend Snippet: a – g , Calu-3 infection with 2,000 E copies per cell of Delta (yellow, Ο), BA.1 (blue, Ο), BA.2 (blue, Δ), BA.4 (purple, O) and BA.5 (purple, Δ), n = 3: mean viral E copies at 2 h.p.i. across three independent experiments ( a ); viral replication over time measured by RT–qPCR for intracellular E copies per microgram RNA ( b ); infection levels measured by nucleocapsid expression (% N+ by flow cytometry) ( c ); expression of IFNB , CXCL10 , IFIT1 , IFIT2 , RSAD2 , MX1 , MX2 and DDX58 in infected cells over time ( d ); IFNβ ( e ) and CXCL10 ( f ) secretion from infected Calu-3 cells measured by ELISA at 48 h.p.i.; rescue of viral replication by JAK1-inhibitor ruxolitinib in Calu-3 cells at 48 h.p.i., where relative infection levels are shown across three independent experiments determined by E copies per microgram RNA normalized to the median infection level of the untreated control ( g ). h – k , Primary bronchial HAEs were infected with the indicated variants at 1,500 E copies per cell: viral replication measured by intracellular E copies at 72 h.p.i. ( h ) and viral release into apical washes over time ( i ), with three biological replicates shown; expression of IFNB , CXCL10 , IFIT1 , IFIT2 , DDX58 and RSAD2 in HAEs at 72 h.p.i., with six biological replicates shown ( j ); intracellular viral E copies in HAEs in the presence or absence of 5 μM ruxolitinib at 72 h.p.i., with three biological replicates shown ( k ). For a , one-way analysis of variance (ANOVA) with Bonferroni post-test was used. n.s., not significant at P > 0.05 for all comparisons. For b – h and j , one-way ANOVA and Dunnett’s post-test were used. For i , two-way ANOVA with a Bonferroni post-test was used. For k , one-tailed unpaired Student’s t -test was used. Replicate measurements from one of three independent experiments. Fold change over mock is shown. Mean ± s.e.m. or individual datapoints are shown. h.p.i., hours post infection.

Article Snippet: IFNβ, IFNλ1/IFNλ3 and CXCL10 were measured using Human IFN-β Quantikine ELISA Kit, Human IL-29/IL-28B (IFNλ1/IFNλ3) DuoSet ELISA or Human CXCL10/IP-10 DuoSet ELISA reagents (Bio-Techne R&D Systems) according to the manufacturer’s instructions.

Techniques: Infection, Quantitative RT-PCR, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, One-tailed Test

Journal: Cancer discovery

Article Title: Active CREB1 promotes a malignant TGFβ2 autocrine loop in glioblastoma.

doi: 10.1158/2159-8290.CD-14-0275

Figure Lengend Snippet: Figure 2. TGFβ induces TGFβ2 expression in GBM and non-GBM cell lines. A, qRT-PCR of TGFB 1 , TGFB 2 , and TGFB 3 in LN229 cells treated with TGFβ1 for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. B, qRT-PCR of TGFB 2 in LN229 cells treated with TGFβ1, TGFβ2, and TGFβ3 for 3 hours. GAPDH mRNA levels were used as an internal normalization control. *, P < 0.05, using the Student t test; data, mean ± SD. C, secreted TGFβ2 protein levels determined by ELISA in culture supernatant from LN229 cells treated with TGFβ for 72 hours. ***, P < 0.005, using the Student t test; data, mean ± SD. D, immunoblot analysis and qRT-PCR of TGF b 2 in LN229 cells treated with TGFβ1 and/or the TβRI inhibitor (TβRI inh.) LY-2109761 for 3 hours. GAPDH mRNA levels were used as an internal normalization control. *, P < 0.05, using the Student t test; data, mean ± SD. E, qRT-PCR of TGFB 2 in GBM and non-GBM cell lines treated with TGFβ for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD.

Article Snippet: For the quantitative determination of TGFβ2 protein levels secreted to the media, we used the Human TGFβ2 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s specifi cations.

Techniques: Expressing, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Cancer discovery

Article Title: Active CREB1 promotes a malignant TGFβ2 autocrine loop in glioblastoma.

doi: 10.1158/2159-8290.CD-14-0275

Figure Lengend Snippet: Figure 3. CREB1 regulates the autocrine induction of TGFβ2 by TGFβ. A, nucleotide sequence of the proximal region of the TGFB 2 promoter. The SBEs and CREB1 site (CRE) are indicated relative to the transcription start site. ClustalW sequence alignment for 3 animal species [ Homo sapiens ( H.s .), Pan troglodytes ( P.t. ), and Mus musculus ( M.m .)] shows the conservation of the binding sites. B, qRT-PCR of TGFB 2 and CREB1 in LN229 cells expressing an shRNA targeting CREB1 treated with TGFβ for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. C, qRT-PCR of TGFB 2 and CREB1 in LN229 cells expressing an siRNA targeting CREB1 treated with TGFβ for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. D, immunoblot analysis and qRT-PCR of TGFβ2 in LN229 cells expressing ICER treated with TGFβ for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. The molecular weights are shown.

Article Snippet: For the quantitative determination of TGFβ2 protein levels secreted to the media, we used the Human TGFβ2 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s specifi cations.

Techniques: Sequencing, Binding Assay, Quantitative RT-PCR, Expressing, shRNA, Control, Western Blot

Journal: Cancer discovery

Article Title: Active CREB1 promotes a malignant TGFβ2 autocrine loop in glioblastoma.

doi: 10.1158/2159-8290.CD-14-0275

Figure Lengend Snippet: Figure 5. PI3K and RSK regulate the TGFβ- mediated induction of TGFβ2 through CREB1. A, immunoblot analysis and qRT-PCR of TGFB2 in LN229 cells treated with TGFβ for 3 hours and the PI3K inhibitor (inh) LY-294002 for 24 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. B, immunoblot analysis and qRT-PCR of TGFB2 in LN229 cells treated with increasing amounts of the RSK inhibitor BI-D1870 for 24 hours and TGFβ for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. C, secreted TGFβ2 protein levels determined by ELISA in LN229 cells treated with TGFβ for 48 hours and the PI3K inhibitor for 72 hours. *, P < 0.05, using the Student t test; data, mean ± SD. D, secreted TGFβ2 protein levels determined by ELISA in LN229 cells treated with the RSK inhibitor BI-D1870 for 72 hours and TGFβ for 48 hours. *, P < 0.05, using the Student t test; data, mean ± SD.

Article Snippet: For the quantitative determination of TGFβ2 protein levels secreted to the media, we used the Human TGFβ2 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s specifi cations.

Techniques: Western Blot, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay

Journal: Cancer discovery

Article Title: Active CREB1 promotes a malignant TGFβ2 autocrine loop in glioblastoma.

doi: 10.1158/2159-8290.CD-14-0275

Figure Lengend Snippet: Figure 6. TGFβ2 correlates with CREB1 expression in GBM patient tumors. A and B, graphs showing the correlation between CREB1 and TGFB 1 (B) or TGFB 2 (A) mRNA levels in patient GBM tumor samples. Data obtained from the REMBRANDT database. A Spearman test was used, and the correlation coeffi cient (ρ) and the two-tailed P value are shown. C, graph showing the correlation between p-CREB1 and TGFβ2 protein levels in tissue microarrays (TMA) from patient GBM samples. Not all spots were evaluable in all stainings. A Spearman test was used, and the correlation coeffi cient (ρ) and the two- tailed signifi cance are shown. Representative images from the TMAs are shown; scale bar, 50 μm. D, Kaplan–Meier curves showing the OS of patients with TGFB2 mRNA levels upregulated ≥3-fold and CREB1 mRNA levels upregulated ≥2-fold. Statistical signifi cance was assessed by the log-rank test. Data obtained from the REMBRANDT database.

Article Snippet: For the quantitative determination of TGFβ2 protein levels secreted to the media, we used the Human TGFβ2 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s specifi cations.

Techniques: Expressing, Two Tailed Test

Journal: Cancer discovery

Article Title: Active CREB1 promotes a malignant TGFβ2 autocrine loop in glioblastoma.

doi: 10.1158/2159-8290.CD-14-0275

Figure Lengend Snippet: Figure 7. CREB1 regulates TGFβ2 expression in PDX models. A, scheme showing the experimental procedure. B, IHC of p-CREB1 and TGFβ2 from mouse tumors 60 days after inoculation with neurospheres expressing shRNAs targeting CREB1 and control shRNAs; scale bar, 50 μm (C). Kaplan–Meier survival curves from mice in B. D, the TGFβ2 malignant autocrine loop. In GBM, TGFβ collaborates with the PI3K and RSK pathways through a CREB1– SMAD3 transcriptional complex to induce TGFβ2 expression. This leads to the generation of an autocrine loop and accumulation of TGFβ2 in the tumor, hyperactivation of TGFβ, and tumor progression.

Article Snippet: For the quantitative determination of TGFβ2 protein levels secreted to the media, we used the Human TGFβ2 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s specifi cations.

Techniques: Expressing, Paraffin-embedded Immunohistochemistry, Control

Journal: Nature Communications

Article Title: A programmable synthetic lineage-control network that differentiates human IPSCs into glucose-sensitive insulin-secreting beta-like cells

doi: 10.1038/ncomms11247

Figure Lengend Snippet: ( a ) Schematic of the synthetic lineage-control network. The constitutively expressed, vanillic acid-sensitive olfactory G protein-coupled receptor MOR9-1 (pCI-MOR9-1; P hCMV -MOR9-1-pA) senses extracellular vanillic acid levels and triggers a synthetic signalling cascade, inducing P CRE -driven expression of the transcription factor VanA 1 (pSP1, P CRE -VanA 1 -pA). At medium vanillic acid concentrations (purple arrows), VanA 1 binds and activates the bidirectional vanillic acid-responsive promoter P 3VanO2 (pSP12, pA-Ngn3 cm ←P 3VanO2 →mFT-miR30Pdx1 g-shRNA -pA), which drives the induction of codon-modified Neurogenin 3 ( Ngn3 cm ) as well as the coexpression of both the blue-to-red medium fluorescent timer (mFT) for precise visualization of the unit's expression dynamics and miR30pdx1 g-shRNA (a small hairpin RNA programming the exclusive destruction of genomic pancreatic and duodenal homeobox 1 ( Pdx1 g ) transcripts). Consequently, Ngn3 cm levels switch from low to high (OFF-to-ON), and Pdx1 g levels toggle from high to low (ON-to-OFF). In addition, Ngn3 cm triggers the transcription of Ngn3 g from its genomic promoter, which initiates a positive-feedback loop. At high vanillic acid levels (orange arrows), VanA 1 is inactivated, and both Ngn3 cm and miR30pdx1 g-shRNA are shut down. At the same time, the MOR9-1-driven signalling cascade induces the modified high-tightness and lower-sensitivity P CREm promoter that drives the co-cistronic expression of the codon-modified variants of Pdx1 ( Pdx1 cm ) and V-maf musculoaponeurotic fibrosarcoma oncogene homologue A ( MafA cm ; pSP17, P CREm -Pdx1 cm -2A-MafA cm -pA). Consequently, Pdx1 cm and MafA cm become fully induced. As Pdx1 cm expression ramps up, it initiates a positive-feedback loop by inducing the genomic counterparts Pdx1 g and MafA g . Importantly, Pdx1 cm levels are not affected by miR30Pdx1 g-shRNA because the latter is specific for genomic Pdx1 g transcripts and because the positive feedback loop-mediated amplification of Pdx1 g expression becomes active only after the shutdown of miR30Pdx1 g-shRNA . Overall, the synthetic lineage-control network provides vanillic acid-programmable, transient, mutually exclusive expression switches for Ngn3 (OFF-ON-OFF) and Pdx1 (ON-OFF-ON) as well as the concomitant induction of MafA (OFF-ON) expression, which can be followed in real time . ( b ) Schematic illustrating the individual differentiation steps from human IPSCs towards beta-like cells. The colours match the cell phenotypes reached during the individual differentiation stages programmed by the lineage-control network shown in a .

Article Snippet: Human islets and beta-like cells differentiated using the lineage-control network (day 11) were dissociated into single cells using 0.5 ml of StemPro Accutase Cell Dissociation Reagent (Invitrogen) and prepared for electron-microscopic analysis using a standard procedure .

Techniques: Control, Expressing, shRNA, Modification, Amplification

Journal: Nature Communications

Article Title: A programmable synthetic lineage-control network that differentiates human IPSCs into glucose-sensitive insulin-secreting beta-like cells

doi: 10.1038/ncomms11247

Figure Lengend Snippet: Quantitative RT–PCR-based expression profiling of the following genes in glucose-sensitive insulin-secreting beta-like cells differentiated by the lineage-control network: ( a ) the key pancreatic beta-cell-specific transcription factors Glis3, MafA, MafB, Mnx1, NeuroD, Pax4, Pdx1 and Nkx6.1 , ( b ) the glucose- and insulin-processing factors Gck, Glut2, G6pc2, Pcsk1, Pcsk2, Slc30a8, Snap25, Stx1A, Stxbp1, Syt4 , ( c ) the channels essential for the secretion of insulin such as Abcc8, Cacna1D, Kcnk1/3 and Kcnj11 and ( d ) the human islet peptide hormones Chgb, Ghrelin, Glucagon, Iapp, Insulin and Somatostatin , and ( e ) immature as well as mature human pancreatic beta-cell markers Acox2, Ck19, Dpp4, FoxA1, Fzd2, Gcgr, Irx2, Mmp2, Onecut2, Sftpd and Ucn3 . The transcript levels were profiled at day 11 relative to hIPSCs and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data are the means±s.d. of triplicate experiments ( n =9). Abcc8, ATP-binding cassette transporter sub-family C member 8; Acox2, Acyl-CoA oxidase 2; Cacna1D, voltage-dependent, L-type alpha 1D subunit; Chgb, chromogranin B; Ck19, cytokeratin-19; Dpp4, dipeptidyl-peptidase 4; FoxA1, forkhead box protein A1; Fzd2, frizzled 2; Gcgr, glucagon receptor; Gck, glucokinase; Glis3, glis family zinc finger 3; Glut2, glucose transporter 2; G6pc2, glucose-6-phosphatase 2; Iapp, islet amyloid polypeptide; Irx2, iroquois homeobox 2; Kcnk1/3, potassium channel, subfamily K, member 1/3; Kcnj11, potassium inwardly-rectifying channel, subfamily J, member 11; MafA/B, V-maf musculoaponeurotic fibrosarcoma oncogene homologue A/B; Mmp2, matrix metalloproteinase 2; Mnx1, motor neuron and pancreas homeobox 1; NeuroD1, neurogenic differentiation factor 1; Nkx6.1, NK6 homeobox 1; Onecut2, onecut homeobox 2; Pax4, paired box gene 4; Pcsk1/2, proprotein convertase 1/2; Sftpd, surfactant protein D; Pdx1, pancreatic and duodenal homeobox 1; Slc30a8, solute carrier family 30, member 8; Snap25, synaptosomal-associated protein; Stx1A (Syntaxin-1A), Stxbp1, syntaxin binding protein 1; Syt4 synaptotagmin-4; Ucn3, urocortin 3.

Article Snippet: Human islets and beta-like cells differentiated using the lineage-control network (day 11) were dissociated into single cells using 0.5 ml of StemPro Accutase Cell Dissociation Reagent (Invitrogen) and prepared for electron-microscopic analysis using a standard procedure .

Techniques: Quantitative RT-PCR, Expressing, Control, Binding Assay

Journal: Nature Communications

Article Title: A programmable synthetic lineage-control network that differentiates human IPSCs into glucose-sensitive insulin-secreting beta-like cells

doi: 10.1038/ncomms11247

Figure Lengend Snippet: ( a ) Quantitative analysis of lineage-controlled beta-like cells co-stained for VanA 1 and either insulin (C-peptide), glucagon or somatostatin. The cells staining positive for VanA 1 are containing the lineage-control network. Data are the means±s.d. ( n =3). ( b , c ) Human pancreatic islets and beta-like cells produced by programming hIPSC-derived pancreatic progenitor cells using the synthetic lineage-control network or the growth-factor/chemical-based differentiation technique were exposed to low (2.8 mM), medium (10 mM), high (20 mM) glucose as well as high glucose and potassium chloride (30 mM) before intracellular ( b ) and secreted ( c ) insulin (C-peptide) levels were profiled using ELISA. Data are the means±s.d. of duplicate experiments ( n =6). Statistics by Student's t -test; * P <0.05, ** P <0.005, **** P <0.0001; ns, not significant. ( d ) Beta-like cells produced by programming hIPSC-derived pancreatic progenitor cells using the synthetic lineage-control network or the growth-factor/chemical-based differentiation technique and cultivated for 4 weeks were exposed to low (2.8 mM), medium (10 mM), high (20 mM) glucose as well as high glucose and potassium chloride (30 mM) before secreted insulin (C-peptide) levels were profiled using ELISA. Data are the means±s.d. of duplicate experiments ( n =6). Statistics by Student's t -test; ** P <0.005; ns, not significant. ( e ) Transmission-electron micrographs of human pancreatic islets and lineage-controlled beta-like cells. Scale bars, 1 μm (human pancreatic islets) and 2 μm (beta-like cells differentiated by the synthetic lineage-control network).

Article Snippet: Human islets and beta-like cells differentiated using the lineage-control network (day 11) were dissociated into single cells using 0.5 ml of StemPro Accutase Cell Dissociation Reagent (Invitrogen) and prepared for electron-microscopic analysis using a standard procedure .

Techniques: Staining, Control, Produced, Derivative Assay, Enzyme-linked Immunosorbent Assay, Transmission Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Recurrent Hypoglycemia Impaired Vascular Function in Advanced T2DM Rats by Inducing Pyroptosis

doi: 10.1155/2022/7812407

Figure Lengend Snippet: NF- κ B, NLRP3, and cGAS–STING pathway activity of the aortas in response to acute and recurrent hypoglycemia in aged T2DM rats. (a) p-p65, NLRP3, ASC, (b) cleaved caspase-1, cGAS, and (e) STING expression were assayed by western blotting. The expression and location of NLRP3 were determined by (c) immunohistochemistry and (d) immunofluorescence; ∗ p < 0.05 DM vs. control; # p < 0.05 H-DM, RH-DM vs. DM; & p < 0.05 H-DM vs. RH-DM.

Article Snippet: Proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and incubated with primary antibodies: eNOS (1 : 1000, ab300071, Abcam), iNOS (1 : 1000, ab283655, Abcam), NOX2 (1 : 2000, 19013-1-AP, ProteinTech), NOX4 (1 : 2000, 14347-1-AP, ProteinTech), p-p65 (1 : 1000, 3033, Cell Signaling Technology), p65 (1 : 1000, 8242, Cell Signaling Technology), NLRP3 (1 : 1000, NBP2-12446, NOVUS), ASC (1 : 1000, sc-514414, Santa Cruz), Caspase-1 (1 : 1000, bs-10743R, Bioss), cGAS (1 : 1000, NBP3-16666, NOVUS), STING (1 : 1000, CST50494, Cell Signaling Technology), GSDMD (1 : 1000, NBP2-33422, NOVUS), Bax (1 : 1000, 50599-2-Ig, ProteinTech), Bcl-2 (1 : 1000, 26593-1-AP, ProteinTech), and β -actin (1 : 5000, 20536-1-AP, ProteinTech).

Techniques: Activity Assay, Expressing, Western Blot, Immunohistochemistry, Immunofluorescence

Journal: Advanced Science

Article Title: Human Platelet Lysates‐Based Hydrogels: A Novel Personalized 3D Platform for Spheroid Invasion Assessment

doi: 10.1002/advs.201902398

Figure Lengend Snippet: Protein and growth factor release from PLMA hydrogels. A) Total protein quantification and B,C) ELISA quantification of TGF‐β1 and VEGF‐A release from PLMA hydrogels at 10, 15, and 20% (w/v). Data are presented as mean ± SD ( n ≥ 3).

Article Snippet: ELISA assays were performed to quantify the release of vascular endothelial growth factor (VEGF Human ELISA Kit, Invitrogen, ThermoFisher Scientific, USA), transforming growth factor β1 (TGF‐β1 Human ELISA Kit, Invitrogen, ThermoFisher Scientific, USA), and epidermal growth factor (Human EGF Quantikine ELISA Kit, R&D systems, Minneapolis, USA).

Techniques: Enzyme-linked Immunosorbent Assay