Journal: Cell Reports Medicine

Article Title: Vaccine-induced ICOS + CD38 + circulating Tfh are sensitive biosensors of age-related changes in inflammatory pathways

doi: 10.1016/j.xcrm.2021.100262

Figure Lengend Snippet:

Article Snippet: Anti-human Survivin (REA459) , Miltenyi , Cat # 130-106-741; RRID: AB_2653606.

Techniques: Recombinant, Staining, Gene Expression, Software

Journal: International Journal of Molecular Sciences

Article Title: Onconase Induces Apoptosis in Dabrafenib-Resistant Melanoma Cell Lines Through Dysregulation of ROS Homeostasis, Antioxidant Protein Expression, and Mitochondrial Dynamics

doi: 10.3390/ijms27041638

Figure Lengend Snippet: Effects of ONC on the expression of proteins involved in cell proliferation and cell death. A375P, A375R, FO-1P, and FO-1R melanoma cell populations were cultured for 24 h ( left ) or 48 h ( right ) in the presence or absence of 0.5 or 1 µM ONC. ( Top ): Representative immunoblots showing the expression levels of the phosphorylated forms of Signal Transducer and Activator of Transcription 3 (pSTAT3) and nuclear factor kappa-light-chain-enhancer of activated B cells (pP65-NF-κB), the phosphorylated and total forms of extracellular signal-regulated kinase (pERK and ERK), the phosphorylated forms of cyclin-dependent kinases 1, 2, and 4 (pCDK1, pCDK2 and pCDK4), cleaved poly(ADP-ribose) polymerase-1 (cleaved-PARP), cleaved caspase-9, survivin, PTEN-induced putative kinase 1 (PINK1), mitochondrial import receptor subunit TOM20 homolog (TOM20), Beclin1, Autophagy Related 3 (ATG3), and microtubule-associated protein 1 light chain 3 beta (LC3B). ( Bottom ): Histograms showing mean protein expression levels after 48 h ONC treatment ± S.D., as determined by densitometric analysis from three independent experiments. Histograms of different protein expression levels obtained after 24 h of ONC treatment are shown in the . All comparisons were performed relative to the corresponding control samples after normalization to β-actin expression; ANOVA test: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Membranes were then incubated overnight at 4 °C with gentle agitation in 5% bovine serum albumin (BSA) containing the following primary antibodies: FTH1 (A19544); p(Ser40)-NRF2 (AP1133); HO-1 (A19062); GCLM (A11444); SLC7A11 (A2413); GSTP1 (A19061); FSP1 (A22278); cleaved PARP (A19612); LC3B (AA19665); p(T161)CDK1 (AP0324); p(Thr160)CDK2 (AP1364); p(Thr172)CDK4 (AP1364); p(ser616)-DRP1 (AP1353); FIS1 (A19666); PINK1 (A24745); ATPB (A11214); pERK1/2 (AP0974); ERK1/2 (A16686); UQCRC1 (A26343); (Abclonal, Woburn, MA, USA); ATG3 (GTX128065); Beclin1 (GTX133555); SOD2 (GTX630559); Glutathione reductase (GTX114199); COX4 (GTX114330); c-Myc (GTX103436); (GeneTex, Irvine, CA, USA); Mitofusin-1 (#14739); Mitofusin-2 (#11925); DRP1 (#8570); OPA1 (#80471); TOM20 (#42406); GAPDH (#2118); p(Tyr605)-STAT3 (#9145); p(Ser536)P65-NF-kB (#3033); cleaved Caspase 9 (#9509) (Cell Signaling Technology, Danvers, MA, USA); PGC1α (PA5-38022) (Invitrogen, Life Technologies, Monza, Italy); γ-H2AX (29380-1); Survivin (66495-1-Ig); and β-actin (66031-1) (Proteintech, Manchester, UK).

Techniques: Expressing, Cell Culture, Western Blot, Control

Journal: Genetics and molecular research : GMR

Article Title: Increased survivin expression and its association with clinical parameters of congenital choledochal cysts.

doi: 10.4238/gmr.15028032

Figure Lengend Snippet: Figure 1. Immunohistochemical staining results for the protein expression of survivin. The cytoplasm showed high survivin expression, which presented as granular form, with yellow or dark brown staining, while the nuclei were hardly stained (A. cytoplasm, 200X; B. nuclei, 400X).

Article Snippet: The primary mouse anti-human survivin monoclonal antibody was purchased from Wuhan Boster Biological Engineering Co., Ltd. (China).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Cell Death & Disease

Article Title: Dominant-negative ATF5 rapidly depletes survivin in tumor cells

doi: 10.1038/s41419-019-1872-y

Figure Lengend Snippet: a Transfection with GFP-FLAG-dn-ATF5 depletes survivin in PC3 prostate tumor cells. Cells were transfected as described in methods and assessed 3 days later for relative survivin levels (normalized to ACTIN) by western immunoblotting. Left panel shows representative blot, right panel shows quantification from 3 independent experiments. b Examples of survivin localization in T98G cells. Transfected cultures were immunostained for survivin and co-stained with Hoechst 33828 to visualize nuclear DNA. Blue/white arrowhead shows a cell without significant survivin expression. Red and blue arrowheads show cells with both cytoplasmic (red arrowheads) and nuclear (blue arrowheads) staining. Yellow arrowhead shows a cell with nuclear-only staining. Scale bar = 10 µm. c , d Transfection with GFP-FLAG-dn-ATF5 depletes nuclear-only localized survivin and increases the proportion of cells without detectable survivin expression in T89G and LN229 cultures at 24 h (C) or at 3–5 d (D). Transfected cells (GFP+) were blindly scored for survivin localization using criteria described in panel b . Data are from three independent experiments, each carried out in triplicate. In each experiment about 200–300 cells were evaluated. e Transfection with GFP-FLAG-dn-ATF5 does not increase the incidence of apoptosis in cultures of T89G and LN229 cultures at 24 h, a time when survivin is already depleted. Transfected cells (GFP+) in cultures described in panels c and d were scored for proportion with apoptotic nuclei. Data are from three independent experiments, each carried out in triplicate. In each experiment about 200–300 cells were evaluated. f Transfection with GFP-FLAG-dn-ATF5 promotes cell death at 3–5 days. As in e , but at 5 days for T98G and 3 days for LN229 cells.

Article Snippet: FLAG-tagged human survivin cDNA cloned into a pCMV6-entry vector was obtained from Origene, Rockville MD (#RC205935).

Techniques: Transfection, Western Blot, Staining, Expressing

Journal: Cell Death & Disease

Article Title: Dominant-negative ATF5 rapidly depletes survivin in tumor cells

doi: 10.1038/s41419-019-1872-y

Figure Lengend Snippet: a , b CP-dn-ATF5 depletes survivin in T98G, U87 and LN229 glioblastoma cell lines at 24 h after treatment. a Representative western blots. b Data are from three independent experiments. c CP-dn-ATF5 depletes survivin in T98G, U87 and LN229 glioblastoma cell lines at 48 and 72 h after treatment. Normalized survivin to ACTIN ratios are shown for each lane. d Treatment with CP-dn-ATF5 causes a rapid depletion of survivin protein in T98G cultures. Normalized survivin to ACTIN ratios are shown for each lane. e Cell death caused by CP-dn-ATF5 (100 µM) in T98G cultures occurs after survivin depletion. Relative numbers of cells per culture were determined at the indicated times. Three replicate cultures were evaluated at each time point

Article Snippet: FLAG-tagged human survivin cDNA cloned into a pCMV6-entry vector was obtained from Origene, Rockville MD (#RC205935).

Techniques: Western Blot

Journal: Cell Death & Disease

Article Title: Dominant-negative ATF5 rapidly depletes survivin in tumor cells

doi: 10.1038/s41419-019-1872-y

Figure Lengend Snippet: a – d CP-dn-ATF5 depletes survivin mRNA in multiple cancer cell lines at 48 h of treatment. Data are from three independent experiments. e CP-dn-ATF5 (100 µM) causes rapid depletion of survivin mRNA in T98G cells. Values are from one experiment carried out in triplicate

Article Snippet: FLAG-tagged human survivin cDNA cloned into a pCMV6-entry vector was obtained from Origene, Rockville MD (#RC205935).

Techniques:

Journal: Cell Death & Disease

Article Title: Dominant-negative ATF5 rapidly depletes survivin in tumor cells

doi: 10.1038/s41419-019-1872-y

Figure Lengend Snippet: a CP-dn-ATF5 decreases the expression of exogenous FLAG-survivin. Indicated cell lines were infected with lentivirus expressing FLAG-survivin and 48 h later were treated with 100 µM CP-dn-ATF5 for 72 h and then assessed for relative (to ACTIN) FLAG-survivin levels by western immunoblotting. Normalized survivin to ACTIN ratios are shown for each lane. b CP-dn-ATF5 accelerates the turnover of survivin protein in T98G cells. Replicate cultures were treated for 24 h with or without (Control) 100 µM CP-dn-ATF5 and then exposed to 50 µM cycloheximide for the indicated times in the continued presence or absence of CP-dn-ATF5 and then assessed for relative survivin levels by western immunoblotting. c CP-dn-ATF5 accelerates the turnover of survivin protein in T98G cells. Cultures were treated as in b and relative survivin expression determined vs ACTIN and normalized to the zero time value in each independent experiment. Numbers of independent experiments for various points are as follows: Control, 1 h, n = 5; 2 h, n = 6; 3 h, n = 5; 4 h, n = 6; 12 h, n = 1; 24 h, = 5. CP-dn-ATF5: 1 h, n = 5; 2 h, n = 6; 3 h, n = 5; 4 h, n = 5; 12 h, n = 1, 24 h, n = 6. Mean data points were fitted to an exponential curve

Article Snippet: FLAG-tagged human survivin cDNA cloned into a pCMV6-entry vector was obtained from Origene, Rockville MD (#RC205935).

Techniques: Expressing, Infection, Western Blot

Journal: Cell Death & Disease

Article Title: Dominant-negative ATF5 rapidly depletes survivin in tumor cells

doi: 10.1038/s41419-019-1872-y

Figure Lengend Snippet: a Proteasomal inhibitor epoxomicin suppresses loss of survivin expression promoted by CP-dn-ATF5. T98G cells stably expressing FLAG-survivin were treated for 100 µM CP-dn-ATF5 for 24 h in presence or absence (Control, CTR) of 10 nM epoxomicin (EPOX) and assessed by western immunoblotting for levels of FLAG-survivin and ACTIN. Left panel shows representative Western immunoblot. Right panel shows quantification of relative FLAG-survivin levels under each condition. Data are from three independent experiments. b Knockdown of USP9X reduces levels of survivin protein in T98G cells. Cultures were transfected with control (CTR) or USP9X siRNA for 72 h and assessed 3 days later for USP9X, survivin and ACTIN protein levels by western immunoblotting (left panel). Right panel shows relative survivin protein levels for five independent experiments. c USP9X knockdown does not affect survivin mRNA levels in T98G cells. Cultures were treated as in b and assessed for relative survivin mRNA levels. Data are from 3 independent experiments

Article Snippet: FLAG-tagged human survivin cDNA cloned into a pCMV6-entry vector was obtained from Origene, Rockville MD (#RC205935).

Techniques: Expressing, Stable Transfection, Western Blot, Transfection

Journal: Cell Death & Disease

Article Title: Dominant-negative ATF5 rapidly depletes survivin in tumor cells

doi: 10.1038/s41419-019-1872-y

Figure Lengend Snippet: a CP-dn-ATF5 reduces expression of over-expressed FLAG-survivin, but to levels far in excess of endogenous survivin. T98G cultures were infected with lentivirus expressing either pLVX-EF1α-IRES-mCherry (pLV in Figure) or pLVX-EF1α-FLAG-survivin-IRES-mCherry (pLV-survivin in Figure) as indicated and 24 h later treated with or without 100 µM CP-dn-ATF5 for 3 d and assessed for exogenous and endogenous survivin by western immunoblotting with anti-survivin. Long exposure shows levels of endogenous survivin. b Survivin over-expression does not rescue the effects of CP-dn-ATF5 on number or appearance of T98G cells. Cultures were infected with lentiviruses described in panel a as indicated and 24 h later were treated with 100 µM CP-dn-ATF5 for 3 days. Panels show immunofluoresence for mcherry (red) or phase contrast images. Scale bars = 50 µm. c Survivin over-expression does not rescue T98G cells from apoptotic death promoted by CP-dn-ATF5. Cultures were infected with lentivirus described in panel a and 24 h later were treated with 100 µM CP-dn-ATF5 for 3 days. Cultures were harvested and analyzed for proportion of apoptotic cells by flow cytometry. Left panel shows the representative cytometry data and right panel shows quantitative results from three independent experiments, each in triplicate. d Survivin over-expression does not rescue cell number in T98G cultures treated with CP-dn-ATF5. Cultures were infected with above described lentiviruses and 24 h later were treated with or without 100 µM CP-dn-ATF5 as indicated for 3 days. Cultures were harvested and analyzed for total cell numbers. Data are from three independent experiments, each in triplicate. e Survivin over-expression does not rescue promotion of apoptosis by FLAG-GFP-dn-ATF5 in multiple cancer cell lines. Indicated cell lines were co-transfected as shown with a ratio of pLVX:pLE constructs of 3:1 and transfected (GFP+) cells were assessed for proportion with apoptotic nuclei 3 days later. Data are from three independent experiments

Article Snippet: FLAG-tagged human survivin cDNA cloned into a pCMV6-entry vector was obtained from Origene, Rockville MD (#RC205935).

Techniques: Expressing, Infection, Western Blot, Over Expression, Flow Cytometry, Cytometry, Transfection, Construct

Journal: Cell Death & Disease

Article Title: Dominant-negative ATF5 rapidly depletes survivin in tumor cells

doi: 10.1038/s41419-019-1872-y

Figure Lengend Snippet: Proposed role of survivin regulation in the mechanism by which dn-ATF5 kills tumor cells

Article Snippet: FLAG-tagged human survivin cDNA cloned into a pCMV6-entry vector was obtained from Origene, Rockville MD (#RC205935).

Techniques:

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Transcriptomics based identification of S100A3 as the key anti-hepatitis B virus factor of 16F16.

doi: 10.1016/j.biopha.2023.114904

Figure Lengend Snippet: Fig. 8. qPCR validation of DEGs. (A) qPCR analysis of upregulated genes selected from transcriptomics analysis. qPCR analysis results were consistent with the transcriptomics analysis. (B) Validation of downregulated genes through qPCR analysis, results were consistent with the transcriptomics analysis which shows ac curacy of transcriptomics data. (C) Western blot validation of EREG, PDIA2 and PDIA6.

Article Snippet: The membranes were incubated with the primary S100A3 antibody (sc-514339, 1:1000), PDIA2 antibody (BST19364904, BOSTER Biological Technology), PDIA6 antibody (A03813–2, BOSTER Biological Technology), EREG antibody (ZP3681BP81, BOSTER Biological Technology) and GAPDH (GB12002, 1/1000) overnight at 4◦C.

Techniques: Biomarker Discovery, Western Blot