Journal: eLife

Article Title: Embryo-derive TNF promotes decidualization via fibroblast activation

doi: 10.7554/eLife.82970

Figure Lengend Snippet: ( A ) Immunofluorescence of SPARC and immunohistochemistry of TNC and S100A4 in mouse uteri on D4 (n=5), D4.5 (n=5), D5 (n=5), and D5.5 (n=5) of pregnancy. LE, luminal epithelium; St, stroma; * Embryo. Scale bar, 50 μm. ( B ) Western blot analysis of α-SMA, SPARC, TNC protein level under in vitro decidualization (EP) for 24 hr. *, p<0.05; **, p<0.01; ***, p<0.001. Figure 1—source data 1. Raw data of all western blots from . Figure 1—source data 2. Complete and uncropped membranes of all western blots from .

Article Snippet: Stromal cells were treated with TNC (5, 50 and 500 ng/ml, 3358-TC-050, R&D systems), S100A4 (50 and 500 ng/ml, HY-P71084, MedChemExpress), SPARC (1 and 10 μM, HY-P71086, MedChemExpress), AA (0.2, 2 and 20 μM, A3611, Sigma-Aldrich), PGI analogue ILOPROST (0.1, 1 and 10 μM, HY-A0096, MedChemExpress), SELEXIPAG (0.1, 1 and 10 μM, HY-14870, MedChemExpress), PPARδ agonist GW501516 (0.1, 1 and 10 μg/ml, 317318-70-0, Cayman Chemical, Ann Arbor, MI), COX-2 antagonist NS 398 (20 and 40 μM, S8433, Selleck, Shanghai, China), PPARδ antagonist GSK0660 (40 μM, 1014691-61-2, Selleck), and ACTIVIN A (1, 10 and 100 ng/ml, HY-P70311, MedChemExpress) in DMEM/F12 containing 2% cFBS, respectively.

Techniques: Immunofluorescence, Immunohistochemistry, Western Blot, In Vitro

Journal: eLife

Article Title: Embryo-derive TNF promotes decidualization via fibroblast activation

doi: 10.7554/eLife.82970

Figure Lengend Snippet: ( A ) Western blot analysis on the effects of TNC on decidualization markers (BMP2, WNT4, E2F8 and CYCLIN D3) after stromal cells were treatment with TNC for 72 hr. ( B ) QPCR analysis of Prl8a2 mRNA level after mouse stromal cells were treated with TNC for 72 hr. ( C ) Western blot analysisof the effects of S100A4 on decidualization markers after stromal cells were treated with S100A4 for 72 hr. ( D ) QPCR analysis of Prl8a2 mRNA level after mouse stromal cells were treated with S100A4 for 72 hr. ( E ) Western blot analysis on the effects after stromal cells were treated with SPARC for 72 hr. ( F ) QPCR analysis of Prl8a2 mRNA level after mouse stromal cells were treated SPARC for 72 hr. ( G ) Western blot analysis on ACTIVIN A protein levels in mouse uteri on D4, D4.5, PD4, and PD4.5, respectively. ( H ) Western blot analysis on the effects of ACTIVIN A on decidualization markers after stromal cells were treated with ACTIVIN A for 72 hr. ( I ) QPCR analysis of Prl8a2 mRNA level after mouse stromal cells were treated with ACTIVIN A for 72 hr. ( J ) Western blot analysis on the effects of ACTIVIN A on decidualization markers after stromal cells were treated with ACTIVIN A for 48 hr under in vitro decidualization. EP, 17β-estradiol+progesterone. All data were is presented as means ± SD. *, p<0.05; **, p<0.01; ***, p<0.001. CYC D3: CYCLIN D3; ACT-A: ACTIVIN A. Figure 2—source data 1. Raw data of all western blots from . Figure 2—source data 2. Complete and uncropped membranes of all western blots from .

Article Snippet: Stromal cells were treated with TNC (5, 50 and 500 ng/ml, 3358-TC-050, R&D systems), S100A4 (50 and 500 ng/ml, HY-P71084, MedChemExpress), SPARC (1 and 10 μM, HY-P71086, MedChemExpress), AA (0.2, 2 and 20 μM, A3611, Sigma-Aldrich), PGI analogue ILOPROST (0.1, 1 and 10 μM, HY-A0096, MedChemExpress), SELEXIPAG (0.1, 1 and 10 μM, HY-14870, MedChemExpress), PPARδ agonist GW501516 (0.1, 1 and 10 μg/ml, 317318-70-0, Cayman Chemical, Ann Arbor, MI), COX-2 antagonist NS 398 (20 and 40 μM, S8433, Selleck, Shanghai, China), PPARδ antagonist GSK0660 (40 μM, 1014691-61-2, Selleck), and ACTIVIN A (1, 10 and 100 ng/ml, HY-P70311, MedChemExpress) in DMEM/F12 containing 2% cFBS, respectively.

Techniques: Western Blot, In Vitro

Journal: eLife

Article Title: Embryo-derive TNF promotes decidualization via fibroblast activation

doi: 10.7554/eLife.82970

Figure Lengend Snippet: ( A ) AA concentration in uterine luminal fluid flushed on D3 (n=20 mice), D4 (n=20 mice), and D4.5 (n=20 mice) of pregnancy. ( B ) P-CPLA2α immunofluorescence in mouse uteri on D4 (n=6) and D4.5 (n=6). * Embryo. Scale bar = 50 μm. ( C ) P-CPLA2α immunofluorescence in mouse uteri at implantation sites and inter-implantation sites on D5 (n=6 mice). * Embryo. NI, inter-implantation site; IS, implantation site. Scale bar = 50 μm. ( D ) p-CPLA 2α immunofluorescence of in mouse uteri 12 and 24 h after delayed implantation was activated by estrogen treatment, respectively (n=4 mice). * Embryo. Scale bar = 0 μm. ( E ) Western blot analysis of CPLA 2α and P-CPLA 2α protein levels in mouse uteri on D4, D4.5 PD4 and PD4.5 (n=4 mice), respectively. ( F ) Western blot analysis of CPLA 2α and P-CPLA 2α protein levels in mouse uteri 12 and 24 hr after delayed implantation was activated by estrogen treatment (n=4 mice). ( G ) Immunostaining of TNC and S100A4 in mouse uteri 12, 24, 36, and 48 hr after delayed implantation was activated by estrogen treatment (n=4 mice). * Embryo. ( H ) Western blot analysis α-SMA, TNC, and SPARC protein levels in mouse uteri on D4 and PD4 (n=4 mice). *, p<0.05; **, p<0.01; ***, p<0.001. Figure 5—source data 1. Raw data of all western blots from . Figure 5—source data 2. Complete and uncropped membranes of all western blots from .

Article Snippet: Stromal cells were treated with TNC (5, 50 and 500 ng/ml, 3358-TC-050, R&D systems), S100A4 (50 and 500 ng/ml, HY-P71084, MedChemExpress), SPARC (1 and 10 μM, HY-P71086, MedChemExpress), AA (0.2, 2 and 20 μM, A3611, Sigma-Aldrich), PGI analogue ILOPROST (0.1, 1 and 10 μM, HY-A0096, MedChemExpress), SELEXIPAG (0.1, 1 and 10 μM, HY-14870, MedChemExpress), PPARδ agonist GW501516 (0.1, 1 and 10 μg/ml, 317318-70-0, Cayman Chemical, Ann Arbor, MI), COX-2 antagonist NS 398 (20 and 40 μM, S8433, Selleck, Shanghai, China), PPARδ antagonist GSK0660 (40 μM, 1014691-61-2, Selleck), and ACTIVIN A (1, 10 and 100 ng/ml, HY-P70311, MedChemExpress) in DMEM/F12 containing 2% cFBS, respectively.

Techniques: Concentration Assay, Immunofluorescence, Western Blot, Immunostaining

Journal: eLife

Article Title: Embryo-derive TNF promotes decidualization via fibroblast activation

doi: 10.7554/eLife.82970

Figure Lengend Snippet: ( A ) Immunostaining of TNC and S100A4 in mouse uteri after S100A9-soaked blue beads were transferred into uterine lumen of PD4 mice for 24 hr (n=6 mice). ( B ) Immunostaining of TNC and S100A4 in mouse uteri after HB-EGF-soaked blue beads were transferred into uterine lumen of PD4 mice for 24 hr (n=6 mice). ( C ) Immunostaining of TNC and S100A4 in mouse uteri after TNF-soaked blue beads were transferred into uterine lumen of PD4 mice for 24 hr (n=6 mice).* Bead; LE, luminal epithelium; M, muscular layer; St, stroma. Scale bar = 100 μm. ( D ) P-CPLA 2α immunofluorescence in mouse uteri after 0.1 and 1 μg/ml TNF-soaked blue beads were transferred into PD4 uterine lumen (n=3 mice). Scale bar = 50 μm. ( E ) Western blot analysis of CPLA 2α and P-CPLA 2α protein levels after cultured epithelial cells were treated with TNF for 3 hr. ( F ) Western blot analysis of CPLA 2α and P-CPLA 2α protein levels after cultured epithelial organoids were treated with TNF for 3 hr. ( G ) ELISA analysis of AA concentration in the cultured medium after cultured epithelial organoids were treated with TNF for 6 hr. *, p<0.05; **, p<0.01; ***, p<0.001. Figure 6—source data 1. Raw data of all western blots from . Figure 6—source data 2. Complete and uncropped membranes of all western blots from .

Article Snippet: Stromal cells were treated with TNC (5, 50 and 500 ng/ml, 3358-TC-050, R&D systems), S100A4 (50 and 500 ng/ml, HY-P71084, MedChemExpress), SPARC (1 and 10 μM, HY-P71086, MedChemExpress), AA (0.2, 2 and 20 μM, A3611, Sigma-Aldrich), PGI analogue ILOPROST (0.1, 1 and 10 μM, HY-A0096, MedChemExpress), SELEXIPAG (0.1, 1 and 10 μM, HY-14870, MedChemExpress), PPARδ agonist GW501516 (0.1, 1 and 10 μg/ml, 317318-70-0, Cayman Chemical, Ann Arbor, MI), COX-2 antagonist NS 398 (20 and 40 μM, S8433, Selleck, Shanghai, China), PPARδ antagonist GSK0660 (40 μM, 1014691-61-2, Selleck), and ACTIVIN A (1, 10 and 100 ng/ml, HY-P70311, MedChemExpress) in DMEM/F12 containing 2% cFBS, respectively.

Techniques: Immunostaining, Immunofluorescence, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: eLife

Article Title: Embryo-derive TNF promotes decidualization via fibroblast activation

doi: 10.7554/eLife.82970

Figure Lengend Snippet:

Article Snippet: Stromal cells were treated with TNC (5, 50 and 500 ng/ml, 3358-TC-050, R&D systems), S100A4 (50 and 500 ng/ml, HY-P71084, MedChemExpress), SPARC (1 and 10 μM, HY-P71086, MedChemExpress), AA (0.2, 2 and 20 μM, A3611, Sigma-Aldrich), PGI analogue ILOPROST (0.1, 1 and 10 μM, HY-A0096, MedChemExpress), SELEXIPAG (0.1, 1 and 10 μM, HY-14870, MedChemExpress), PPARδ agonist GW501516 (0.1, 1 and 10 μg/ml, 317318-70-0, Cayman Chemical, Ann Arbor, MI), COX-2 antagonist NS 398 (20 and 40 μM, S8433, Selleck, Shanghai, China), PPARδ antagonist GSK0660 (40 μM, 1014691-61-2, Selleck), and ACTIVIN A (1, 10 and 100 ng/ml, HY-P70311, MedChemExpress) in DMEM/F12 containing 2% cFBS, respectively.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Staining, Software

Journal: Journal of Nuclear Medicine

Article Title: Advancing Targeted Radionuclide Therapy Through the National Cancer Institute’s Small Business Innovation Research Pathway

doi: 10.2967/jnumed.118.214684

Figure Lengend Snippet: NCI SBIR–Funded TRT Contracts for 2015

Article Snippet: 2016 , Rockland Immunochemicals, Inc. , Phase I , HER2-positive cancers , 68 Ga and 177 Lu , HER2 and digoxigenin , Phase I: Develop bispecific recombinant single-domain antibodies with one arm targeting cancer biomarker HER2 and another arm against digoxigenin; investigate in vivo clearance, tumor accumulation, stability, and ability to capture digoxigenylated fluorescence dyes and radionuclides.

Techniques: In Vitro, In Vivo, Marker, Fluorescence, Drug discovery, Imaging

Journal: Journal of Nuclear Medicine

Article Title: Advancing Targeted Radionuclide Therapy Through the National Cancer Institute’s Small Business Innovation Research Pathway

doi: 10.2967/jnumed.118.214684

Figure Lengend Snippet: NCI SBIR–Funded TRT Contracts from 2016 to 2017

Article Snippet: 2016 , Rockland Immunochemicals, Inc. , Phase I , HER2-positive cancers , 68 Ga and 177 Lu , HER2 and digoxigenin , Phase I: Develop bispecific recombinant single-domain antibodies with one arm targeting cancer biomarker HER2 and another arm against digoxigenin; investigate in vivo clearance, tumor accumulation, stability, and ability to capture digoxigenylated fluorescence dyes and radionuclides.

Techniques: Radioactivity, Drug discovery, Recombinant, Biomarker Discovery, In Vivo, Fluorescence, Binding Assay, Labeling, In Vitro, Produced, Software

Journal: Cancer Research

Article Title: Slit2 Inhibits Breast Cancer Metastasis by Activating M1-Like Phagocytic and Antifibrotic Macrophages

doi: 10.1158/0008-5472.can-20-3909

Figure Lengend Snippet: Figure 1. rSlit-N treatment inhibits breast cancer growth and metastases. A, Mammary glands were isolated from MMTV-PyMT at different stages of tumor progression and tissue sections were stained with hematoxylin and eosin (H&E) or immunostained with Slit2 antibody using the IHC technique. B, Eight-weeks-old MMTV-PyMT mice bearing spontaneous mammary tumors were treated with rSlit2-N or PBS for 4 weeks. C, Tumor volume was measured every week up to 12 weeks of age. D, Representative pictures of the lungs harvested from B. E, The number of metastatic nodules in the lungs. F–M, MVT1 cells were orthotopically implanted into the 6-week-old FVB wild-type mice. F, Schematics of mice treatment with rSlit2-N or PBS. N ¼ 6 mice in each group. G, Tumor volume was measured every week. H, Representative pictures of the lungs harvested from F. I, The number of metastatic nodules in the lungs. J, Schematics of mice treatment with rSlit2-N or PBS, showing Slit2-treated tumors were allowed to grow for additional 2 weeks. N ¼ 5 mice in each group. K, At the end, tumors were harvested and weight was measured. L, Representative pictures of the lungs harvested from J. M, The number of metastatic nodules in the lungs. N, Human breast cancer cell line MDA-MB-231 overexpressing Slit2 (231-Slit2) or vector control (231-Vec) implanted orthotopically into the NSG mice. O, After 2 weeks of tumor injection, tumor volume was measured every week up to the age of 12 weeks. P, The images of lungs harvested from N. Q, The harvested lungs were analyzed for the number of metastatic nodules using a dissection microscope. R–U, NSG females were injected with MDA-MB-231 cells and treated with Adeno-Slit2 or Adeno-Null. R, Schematics of mice treatment with Adeno-Slit2 or Adeno-Null. S, Starting from 8 weeks of age, tumor volume was measured every week. T, Representative pictures of the lungs harvested from R. U, The number of metastatic nodules in the lungs. , P < 0.05; , P < 0.01; , P < 0.001; NS, P value not significant using Student t test.

Article Snippet: To analyze Slit2 expression in human samples, we purchased two tissue microarrays (TMA) having normal, invasive, and lymph node metastatic primary tumors from Imgenex (IMH-364 and BR1008).

Techniques: Isolation, Staining, Plasmid Preparation, Control, Injection, Dissection, Microscopy

Journal: Cancer Research

Article Title: Slit2 Inhibits Breast Cancer Metastasis by Activating M1-Like Phagocytic and Antifibrotic Macrophages

doi: 10.1158/0008-5472.can-20-3909

Figure Lengend Snippet: Figure 6. Slit2 expression levels predict breast cancer patient survival. A, Human breast tissue microarray (pilot TMA) was immunostained with Slit2 antibody using IHC. Representative images showing Slit2 expression levels in normal (n ¼ 10), infiltrative ductal carcinoma (IDC; n ¼ 50), and lymph node-positive metastatic (LNþ metastatic) (n ¼ 40) breast tissues. B, The graph depicts the percentage of human breast samples expressing different levels of Slit2. C, The expression level of Slit2 in human breast tissues using UALCAN database. BL1, basal-like 1; BL2, basal-like 2; IM, immunomodulatory; LAR, luminal androgen receptor; M, mesenchymal; MSL, mesenchymal stem-like; UNS, unspecified. D, Analysis of Slit2 gene copy number in The Cancer Genome Atlas dataset using Oncomine. DC, ductal carcinoma (n-5); IDC & ILC, invasive ductal and invasive lobular carcinoma (n-5); IDC & LC, invasive ductal and lobular carcinoma (n-14); IDC, invasive ductal carcinoma (n-639); N, normal breast (n-111). E, Using experimental TMA, the association of Slit2 protein expression levels with patient survival was analyzed. The Kaplan–Meier graph demonstrating the association of Slit2 expression level with patient survival. F, Analysis of breast cancer patient overall survival based on expression levels of Slit2 mRNA in the Kaplan–Meier plotter database (n ¼ 1115). G, Analysis of breast cancer lymph node-positive (LNþ) metastases patient survival based on expression levels of Slit2 in the Kaplan–Meier plotter database (n ¼ 936).

Article Snippet: To analyze Slit2 expression in human samples, we purchased two tissue microarrays (TMA) having normal, invasive, and lymph node metastatic primary tumors from Imgenex (IMH-364 and BR1008).

Techniques: Expressing, Microarray

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Inhibition of Tumor Growth and Immunomodulatory Effects of Flavonoids and Scutebarbatines of Scutellaria barbata D. Don in Lewis-Bearing C57BL/6 Mice

doi: 10.1155/2015/630760

Figure Lengend Snippet: Effect of flavonoids and scutebarbatines in SB on the levels of immune factors in Lewis tumor-bearing mice. All data were expressed as means ± SD ( n = 9).

Article Snippet: Antibodies to these immunoregulatory factors including IL-10, IL-17, FOXP3, IFN- γ , IL-2, ROR γ t, TGF- β 1, IL-6, and β -actin were obtained from Wuhan BOSTER Biological Engineering Co., Ltd. (Wuhan, China).

Techniques:

Journal: eLife

Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

doi: 10.7554/eLife.64977

Figure Lengend Snippet: ( A ) HER2 (diamonds), TNBC (circles), and luminal (squares and triangles) breast cancer lines were seeded into 96-well plates and treated with increasing doses of MAL3-101 for 72 hr. Viability is expressed as the average of three or more independent experiments, ± SEM. ( B ) MAL3-101-sensitive (MCF7 and MDA MB 231, denoted in blue) and resistant (MDA MB 453 and MDA MB 361, denoted in black) cells were treated with 12 µM MAL3-101 for the indicated times, and lysates were prepared and immunoblotted for cleaved caspase-3, caspase-7, and caspase-8. β-actin serves as a loading control. ( C ) The corresponding fold-increase of the indicated apoptotic markers relative to the DMSO control are plotted, ± SEM (n≥3 for cleaved caspase-3, n=3 for cleaved caspase-7, and n≥4 for cleaved caspase-8). Black asterisks correspond to statistical significance between MDA MB 231 cells (closed circle) and MDA MB 453 and MDA MB 361 (open circle and triangle, respectively), and the red asterisk represents statistical significance between MCF7 (closed triangle) and MDA MB 453 and MDA MB 361 (open circle and triangle) cells; * denotes p<0.05, ** denotes p<0.005. Figure 1—source data 1. Source data for cell viability assay and apoptotic marker accumulation in .

Article Snippet: For the remaining proteins aliquots from the same lysates were instead heated to 37°C for 30 min prior to SDS-PAGE using 4–20% Tris-Glycine gradient gels (XP04202Box, Thermo Fisher Scientific), and after transfer on nitrocellulose membranes (#84–874, Prometheus Laboratories Inc, California), the blots were incubated with anti-cleaved Caspase-8 (18C8, #9496; at 1:1000), anti-BiP (C50B12, #3177S; at 1:1000), anti-Hsp70 (smc-113, StressMarq Biosciences, Victoria, British Columbia; at 1:1000), anti-Hsc/Hsp70 (#4872S; at 1:1000), anti-HER2 (29D8, #2165; at 1:2000), anti-eIF2α (#9722S; at 1:2000), anti-phospho-eIF2α (#9721L; at 1:500), anti-PERK (D11A8, #5683S; at 1:2000), anti-PKR (D7F7, #12297S; at 1:2000), anti-Ire1α (14C10, #3294S; at 1:1000), anti-HRI (MBS2538114, MyBioSource, San Diego, CA; at 1:500) and anti-GCN2 and anti-GCN2 pT899 antibody (ab-134053 and ab-75836, Abcam, Cambtidge, UK; at 1:2000).

Techniques: Viability Assay, Marker

Journal: eLife

Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

doi: 10.7554/eLife.64977

Figure Lengend Snippet: Breast cancer cells exhibit a range of sensitivities to MAL3-101, a specific Hsp70 inhibitor. The indicated breast cancer lines were seeded into 96-well plates and treated with increasing doses of the indicated compounds for 72 hr. Viability was measured using the CellTiter-Glo assay. IC 50 values were generate using a sigmoidal nonlinear regression with SigmaPlot 11.0. ND stands for an undetermined value. MAL3-101 sensitivities of Hsp70 inhibitor resistant cells are in bold.

Article Snippet: For the remaining proteins aliquots from the same lysates were instead heated to 37°C for 30 min prior to SDS-PAGE using 4–20% Tris-Glycine gradient gels (XP04202Box, Thermo Fisher Scientific), and after transfer on nitrocellulose membranes (#84–874, Prometheus Laboratories Inc, California), the blots were incubated with anti-cleaved Caspase-8 (18C8, #9496; at 1:1000), anti-BiP (C50B12, #3177S; at 1:1000), anti-Hsp70 (smc-113, StressMarq Biosciences, Victoria, British Columbia; at 1:1000), anti-Hsc/Hsp70 (#4872S; at 1:1000), anti-HER2 (29D8, #2165; at 1:2000), anti-eIF2α (#9722S; at 1:2000), anti-phospho-eIF2α (#9721L; at 1:500), anti-PERK (D11A8, #5683S; at 1:2000), anti-PKR (D7F7, #12297S; at 1:2000), anti-Ire1α (14C10, #3294S; at 1:1000), anti-HRI (MBS2538114, MyBioSource, San Diego, CA; at 1:500) and anti-GCN2 and anti-GCN2 pT899 antibody (ab-134053 and ab-75836, Abcam, Cambtidge, UK; at 1:2000).

Techniques:

Journal: eLife

Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

doi: 10.7554/eLife.64977

Figure Lengend Snippet: The cell numbers and autophagy or proteasome inhibitor concentrations used for the cell viability assay in combination with increasing doses of MAL3-101 are shown. The concentrations of bortezomib, CQ, and bafilomycin to induce no greater than 30% of cell death in each line after 72 hr treatment are shown. ND stands for undetermined value.

Article Snippet: For the remaining proteins aliquots from the same lysates were instead heated to 37°C for 30 min prior to SDS-PAGE using 4–20% Tris-Glycine gradient gels (XP04202Box, Thermo Fisher Scientific), and after transfer on nitrocellulose membranes (#84–874, Prometheus Laboratories Inc, California), the blots were incubated with anti-cleaved Caspase-8 (18C8, #9496; at 1:1000), anti-BiP (C50B12, #3177S; at 1:1000), anti-Hsp70 (smc-113, StressMarq Biosciences, Victoria, British Columbia; at 1:1000), anti-Hsc/Hsp70 (#4872S; at 1:1000), anti-HER2 (29D8, #2165; at 1:2000), anti-eIF2α (#9722S; at 1:2000), anti-phospho-eIF2α (#9721L; at 1:500), anti-PERK (D11A8, #5683S; at 1:2000), anti-PKR (D7F7, #12297S; at 1:2000), anti-Ire1α (14C10, #3294S; at 1:1000), anti-HRI (MBS2538114, MyBioSource, San Diego, CA; at 1:500) and anti-GCN2 and anti-GCN2 pT899 antibody (ab-134053 and ab-75836, Abcam, Cambtidge, UK; at 1:2000).

Techniques: Viability Assay

Journal: eLife

Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

doi: 10.7554/eLife.64977

Figure Lengend Snippet: Breast cancer cells exhibit a range of sensitivities to MAL3-101 in the presence of either autophagy or proteasome inhibitors. Cells were seeded into 96-well plates and treated with increasing doses of MAL3-101 in the presence or absence of subcritical doses of bortezomib (proteasome inhibitor), or CQ or bafilomycin (autophagy inhibitors) for 72 hr. Viability was measured using the CellTiter-Glo assay. IC 50 values were generate using a sigmoidal nonlinear regression with SigmaPlot 11.0. ND stands for undetermined value. MAL3-101 resistant cells are highlighted in yellow.

Article Snippet: For the remaining proteins aliquots from the same lysates were instead heated to 37°C for 30 min prior to SDS-PAGE using 4–20% Tris-Glycine gradient gels (XP04202Box, Thermo Fisher Scientific), and after transfer on nitrocellulose membranes (#84–874, Prometheus Laboratories Inc, California), the blots were incubated with anti-cleaved Caspase-8 (18C8, #9496; at 1:1000), anti-BiP (C50B12, #3177S; at 1:1000), anti-Hsp70 (smc-113, StressMarq Biosciences, Victoria, British Columbia; at 1:1000), anti-Hsc/Hsp70 (#4872S; at 1:1000), anti-HER2 (29D8, #2165; at 1:2000), anti-eIF2α (#9722S; at 1:2000), anti-phospho-eIF2α (#9721L; at 1:500), anti-PERK (D11A8, #5683S; at 1:2000), anti-PKR (D7F7, #12297S; at 1:2000), anti-Ire1α (14C10, #3294S; at 1:1000), anti-HRI (MBS2538114, MyBioSource, San Diego, CA; at 1:500) and anti-GCN2 and anti-GCN2 pT899 antibody (ab-134053 and ab-75836, Abcam, Cambtidge, UK; at 1:2000).

Techniques:

Journal: BioMed research international

Article Title: Effects of Hypoxic Environment on Periodontal Tissue through the ROS/TXNIP/NLRP3 Inflammasome Pathway.

doi: 10.1155/2022/7690960

Figure Lengend Snippet: Figure 2: Effects of systemic hypoxia stimulation on the expression of TXNIP/NLRP3 signaling pathway-related factors in periodontal tissues of rats: (a) immunofluorescence staining (IF) showed that TXNIP, NLRP3, ASC, and caspase-1 in periodontal tissues. Ab: alveolar; PL: periodontal ligament; R: root. Scale bars = 25 μm. (b) RT-PCR analysis of TXNIP, NLRP3, ASC, caspase-1, and IL-1β in periodontal tissues (n = 5). (c) Western blotting analysis of TXNIP, NLRP3, ASC, caspase-1, and IL-1β in periodontal tissues (n = 5 for each group). The above data are presented as the mean ± SEM, ∗p < 0:05.

Article Snippet: For IF, the sections were incubated overnight at 4°Cwith primary antibodies (1 : 250) diluted in PBS: TXNIP (Rabbit IgG, Boster, China), NLRP3 (Rabbit IgG, Boster, China), ASC (Rabbit IgG, Immunoway, USA), and caspase-1 (Rabbit IgG, Immunoway, China) and then incubated with Alexa Fluor-488 and Alexa Fluor-555 conjugate secondary antibodies at room temperature for 1h.

Techniques: Expressing, Staining, Reverse Transcription Polymerase Chain Reaction, Western Blot