Journal: Disease Markers

Article Title: PFKFB4 Overexpression Facilitates Proliferation by Promoting the G1/S Transition and Is Associated with a Poor Prognosis in Triple-Negative Breast Cancer

doi: 10.1155/2021/8824589

Figure Lengend Snippet: PFKFB4 overexpression facilitates the proliferation of breast cancer cells by promoting the G1/S phase transition. (a) A WB assay revealed that 231 cells had elevated levels of HIF-1 α and PFKFB4 following hypoxia treatment. Stable PFKFB4 overexpression (b) promoted the growth of TNBC cancer cells (c) and these cells exhibited more resistance to cisplatin (d) than negative control (231 NC) and empty vector (Vector) cells. (e, f, ∗ p < 0.05) Overexpression of PFKFB4 remarkably promoted the formation of cell clones compared with control expression. (g, h) Flow cytometric analysis found that PFKFB4 overexpression increased the percentage of S-phase cells. Each bar represents the mean of three independent experiments ( ∗ p < 0.05). (i) A WB assay showed increases in CDK6 and pRb Ser795 expression, which hinted that PFKFB4 overexpression might promote TNBC cell proliferation by facilitating the G1/S phase transition.

Article Snippet: The following antibodies were used: anti-HIF-1 α (#61959) from BD Transduction Laboratories (USA); anti-PFKFB4 (ab137785) from Abcam (USA); and anti-phosphor-Rb (Ser795) (#9301) and anti-CDK6 (#3136) from Cell Signaling Technology, Inc. (USA).

Techniques: Over Expression, Sublimation, Negative Control, Plasmid Preparation, Clone Assay, Control, Expressing

Journal: Oncology Research

Article Title: Long non-coding RNA H19 promotes proliferation in hepatocellular carcinoma cells via H19/miR-107/CDK6 axis

doi: 10.32604/or.2023.030395

Figure Lengend Snippet: H19 suppression downregulates the expression of CDK6 in HCC cells. (A) qRT-PCR analysis of key cell cycle genes in HCC cells upon shRNA-mediated depletion of H19 induced by the presence of doxycycline (Dox). (B) Western blot of CDK6 in HCC cells (HepG2 and JHH-4) with or without Dox treatment. GAPDH was used as a loading control. (C) Western blot of phosphorylated Rb (p-Rb) and total Rb (t-Rb) in HCC cells with or without Dox treatment. Data are presented as the mean ± SD and expressed relative to those of-Dox (set as 1.0) for qRT-PCR experiments. N = 3 replicates per group from at least two independent experiments. Student’s t -test; * p < 0.05 and ** p < 0.01.

Article Snippet: Membranes were then probed overnight with the following primary antibodies against CDK6 (1:1,000; Santa Cruz; Cat #sc-7961), Cyclin A2 (CST, Danvers, MA, United States; Cat #4656T), PLK1 (CST; Cat #4513T), GAPDH (1:2,500; Invitrogen; Cat #437000), total Rb (1:500; Santa Cruz; Cat #sc-102), and phosphorylated Rb (1:1,000; CST; Cat #8516T for Ser807/811, #9307T for Ser780, and #9301T for Ser795).

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

Journal: Oncology Research

Article Title: Long non-coding RNA H19 promotes proliferation in hepatocellular carcinoma cells via H19/miR-107/CDK6 axis

doi: 10.32604/or.2023.030395

Figure Lengend Snippet: Rb phosphorylation and expression of E2F target genes upon shRNA-mediated knockdown of H19 (sh-H19) induced by doxycycline (Dox). (A) Western blot of phosphorylated Rb at the Ser780 and Ser795 residues upon H19 knockdown. (B) Western blot analysis of Cyclin A2 and PLK1 upon H19 knockdown. Data are presented as the mean ± SD. N = 3 replicates per group. Student’s t -test; * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Membranes were then probed overnight with the following primary antibodies against CDK6 (1:1,000; Santa Cruz; Cat #sc-7961), Cyclin A2 (CST, Danvers, MA, United States; Cat #4656T), PLK1 (CST; Cat #4513T), GAPDH (1:2,500; Invitrogen; Cat #437000), total Rb (1:500; Santa Cruz; Cat #sc-102), and phosphorylated Rb (1:1,000; CST; Cat #8516T for Ser807/811, #9307T for Ser780, and #9301T for Ser795).

Techniques: Phospho-proteomics, Expressing, shRNA, Knockdown, Western Blot

Journal: Nature Communications

Article Title: STAT3 serine phosphorylation is required for TLR4 metabolic reprogramming and IL-1β expression

doi: 10.1038/s41467-020-17669-5

Figure Lengend Snippet: a Schematic representation of the putative TRAF6-binding domains and phosphorylation sites of STAT3. ND: N-terminal domain: SH2: Src homology domain TAD: transactivation domain. b BMDMs (1 × 10 7 cells) were seeded in 10 cm culture dishes and grown for 24 h prior to stimulation with LPS (1 μg/ml) for indicated times. TRAF6 was immunoprecipitated from cellular lysates and probed with anti-TRAF6, or STAT3 antibody ( n = 3) c HEK293T cells (1 × 10 6 ) were transfected with indicated plasmid vectors for 24 h. Cells were lysed and probed with recombinant GST-TRAF6 to immunoprecipitated interacting proteins, separated by SDS-PAGE and probed with anti-FLAG antibody ( n = 3).

Article Snippet: To assess the role of kinases in STAT3 phosphorylation, BMDMs were pretreated with either 1 μM BX-795 (Caymen Chemicals, Cat# 14932) or 1 μM TG10073 (Selleck Chemicals, Cat#S2870) for 60 min prior to LPS challenge and assayed as described above.

Techniques: Binding Assay, Phospho-proteomics, Immunoprecipitation, Transfection, Plasmid Preparation, Recombinant, SDS Page

Journal: Nature Communications

Article Title: STAT3 serine phosphorylation is required for TLR4 metabolic reprogramming and IL-1β expression

doi: 10.1038/s41467-020-17669-5

Figure Lengend Snippet: a Immortalised BMDMs (iBMDMs) (1 × 10 6 ) were treated with IFNα (1000 U) for indicated times, b BMDMs (1 × 10 6 cells/well) were treated with LPS for indicated times, and c BMDMs were also treated with TLR agonists, Pam 3 Cys (TLR2, 100 ng/ml), Poly I:C (TLR3, 500 ng/ml), LPS (TLR4, 100 ng/ml), Loxoribine (TLR7, 10 mM), CpG-DNA (TLR9, 2.5 μM) and IFNα (IFNAR1, 1000 U) for 60 min. STAT3 phosphorylation status was detected by immunoblot of cellular lysates with anti-STAT3, anti-STAT3 pS727 and anti-STAT3 pY705 antibodies as indicated ( n = 3). d MyD88 −/− /TRIF −/− iBMDM or e IFNAR1 −/− BMDMs were treated with TLR ligands ( n = 2) as described and immunoblot analysis with anti-STAT3 pS727 and anti-STAT3 antibodies of cellular lysates show ablated TLR-induced STAT3 Ser727 phosphorylation compared with IFNAR1-deficient BMDMs (Fig. ), but not IFNα ( n = 2). (F) iBMDMs were treated with PBS or LPS (100 ng/mL) for 1 h and whole cell lysate, S100 or clean mitochondrial fractions isolated and separated by SDS-PAGE. Abundance of STAT3 and pS727 STAT3 in each fraction was observed as was fraction purity by probing with antibodies against Erk2 (S100), VDAC (mitochondria) or Lamin A/C (nucleus). Data shown are the mean ± S.E.M of a n = 1, b n = 5, c, e n = 3, d, f n = 2 independent experiments.

Article Snippet: To assess the role of kinases in STAT3 phosphorylation, BMDMs were pretreated with either 1 μM BX-795 (Caymen Chemicals, Cat# 14932) or 1 μM TG10073 (Selleck Chemicals, Cat#S2870) for 60 min prior to LPS challenge and assayed as described above.

Techniques: Phospho-proteomics, Western Blot, Isolation, SDS Page

Journal: Nature Communications

Article Title: STAT3 serine phosphorylation is required for TLR4 metabolic reprogramming and IL-1β expression

doi: 10.1038/s41467-020-17669-5

Figure Lengend Snippet: a iBMDMs were seeded at 4 × 10 4 cells per well in triplicate 24 h prior to pretreatment with kinase inhibitors (Y-axis; 500 nM) for 30 min. MitoSOX was added for 10 min prior to challenge with LPS (100 ng/ml; 0, 1, 2 and 4 h). LPS-induced production of superoxide by mitochondria was analysed by measuring oxidised MitoSOX fluorescence at 580 nm. See also Supplementary Fig. . Cell lysates from LPS stimulated BMDMs (0–120 min) were immunoprecipitated with b anti-TRAF6 and c anti-STAT3 antibodies. TBK-1 interaction with TRAF6 and STAT3 was identified by immunoblot with anti-TBK-1 and anti-TBK-1 pS172 antibodies respectively. b , c Data represents three independent experiments with similar results. d TBK-1-deficient and WT macrophages were treated with LPS or IFNα for indicated times and STAT3 phosphorylation assayed in cellular lysates by immunoblot with indicated antibodies. TBK-1 expression was ascertained by immunoblot with anti-TBK-1 antibody. e BMDMs were pretreated with the TBK-1/IKKε (1 μM) inhibitor BX-795 for 60 min prior to LPS stimulation for indicated times. STAT3 and TBK-1 phosphorylation was observed by immunoblot with indicated antibodies. DMSO was added to lanes 13–14 as vehicle control. Immunoblot results are representative of three independent experiments and data presented ( d , e ) as mean ± S.E.M.

Article Snippet: To assess the role of kinases in STAT3 phosphorylation, BMDMs were pretreated with either 1 μM BX-795 (Caymen Chemicals, Cat# 14932) or 1 μM TG10073 (Selleck Chemicals, Cat#S2870) for 60 min prior to LPS challenge and assayed as described above.

Techniques: Fluorescence, Immunoprecipitation, Western Blot, Phospho-proteomics, Expressing, Control

Journal: Nature Communications

Article Title: STAT3 serine phosphorylation is required for TLR4 metabolic reprogramming and IL-1β expression

doi: 10.1038/s41467-020-17669-5

Figure Lengend Snippet: a Peritoneal macrophages (1 × 10 5 /well) obtained from WT vs STAT3 SA mice ( n = 3 per genotype). Real-time changes in the extracellular acidification rate (ECAR) of WT and STAT3 SA macrophages treated with LPS ( b , c ) WT and STAT3 SA peritoneal macrophages were stimulated with LPS or vehicle as indicated for 24 h. b *** p < 0.0001, and c ** p = 0.0034, *** p < 0.0001. a , b The ECAR and oxygen consumption rate (OCR) were analysed as indicators of oxidative phosphorylation and glycolysis, using a Seahorse XFp analyser. d Peritoneal macrophages were assayed for real-time changes in the OCR by sequential treatment with sequential treatment of cells with electron train chain inhibitors (oligomycin: adenosine triphosphate (ATP) synthase inhibitor; FCCP: H+ ionophore cyanide p-triflurmethooxyphenyl-hydrozone; and Complex I inhibitor rotenone). e Comparison of maximal respiratory capacity (MRC) between WT and STAT3 SA peritoneal macrophages stimulated with LPS or vehicle for 24 h. Results presented ( a – e ) as the mean ± S.E.M of peritoneal macrophages obtained from three individual mice per genotype, *** p = 0.0004, One-way ANOVA with Tukey’s multiple comparisons test ( f – g ) Peritoneal macrophages obtained from individual mice were seeded at 1 × 10 5 cells/well and stimulated with LPS (100 ng/ml) for 24 h prior to analysis of metabolites succinate and lactate produced in WT and STAT3 SA cultured supernatants. Data presented as mean values of duplicate technical replicates ± SEM of five individual mice per genotype, f * p = 0.0292, *** p < 0.0001 g *** p = 0.0004, One-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: To assess the role of kinases in STAT3 phosphorylation, BMDMs were pretreated with either 1 μM BX-795 (Caymen Chemicals, Cat# 14932) or 1 μM TG10073 (Selleck Chemicals, Cat#S2870) for 60 min prior to LPS challenge and assayed as described above.

Techniques: Phospho-proteomics, Comparison, Produced, Cell Culture

Journal: Nature Communications

Article Title: STAT3 serine phosphorylation is required for TLR4 metabolic reprogramming and IL-1β expression

doi: 10.1038/s41467-020-17669-5

Figure Lengend Snippet: a Peritoneal macrophages obtained from three mice per genotype were seeded at 5 × 10 5 cells/well in and stimulated with LPS (100 ng/ml) for 24 h prior to analysis IL-1β mRNA expression. Data are presented as mean values ± SEM, *** p < 0.0001, n.s.—not significant, one-way ANOVA, Tukey’s multiple comparisons test. Peritoneal macrophages generated from individual WT and STAT3 mice (3 mice per genotype) were seeded at 1.5 × 10 5 in triplicate. Macrophages were stimulated for 4, 12, and 24 h with 100 ng/ml of LPS and b cell lysates assayed for IL-1β and presented as IL-1β/mg of total protein ** p = 0.0011, *** p < 0.0001, and cultured supernatants assayed for c TNF (4 h, *** p = 0.0007; 12 h ** p = 0.0015; 24 h *** p = 0.0001), d IL-6 (*** p < 0.0001), and e IL-10 (* p = 0.0168, *** p < 0.0001) by ELISA. Data presented are the mean of triplicate values from three individual mice ( n = 3) per genotype per time point, Analyses 2 way ANOVA, Sidak’s multiple comparison tests. WT and STAT3 SA mice ( n = 9 mice per genotype) were intraperitoneally treated with 10 mg/kg of LPS for 90 min and serum analysed for f IL-1β (* p = 0.0444), g IL-6 (* p = 0.0313), h MCP-1 (** p = 0.0046), i TNF ( p = 0.3994) and j IL-10 ( p = 0.8204) protein expression by ELISA or cytometric bead array. Data are presented as mean values ± SEM, Students unpaired t test, two-tailed.

Article Snippet: To assess the role of kinases in STAT3 phosphorylation, BMDMs were pretreated with either 1 μM BX-795 (Caymen Chemicals, Cat# 14932) or 1 μM TG10073 (Selleck Chemicals, Cat#S2870) for 60 min prior to LPS challenge and assayed as described above.

Techniques: Expressing, Generated, Cell Culture, Enzyme-linked Immunosorbent Assay, Comparison, Two Tailed Test

Journal: Free Radical Biology and Medicine

Article Title: CELL CYCLE ARREST BY MONOCHLORAMINE THROUGH THE OXIDATION OF RETINOBLASTOMA PROTEIN

doi: 10.1016/j.freeadbiomed.2003.10.013

Figure Lengend Snippet: Fig. 2. Phosphorylation status of pRB after NH2Cl treatment. Jurkat cells were treated with 70 AM NH2Cl for 10 min at 37jC. Then the medium was replaced with fresh medium, and incubation proceeded for the indicated times. The phosphorylation status of pRB was studied by the Western blotting method. (A) Immunostaining with anti-pRB antibody. Hypo-phosphorylated pRB migrates slightly faster than hyperphos- phorylated pRB on SDS–PAGE. (B) Immunostaining with phospho- specific antibodies that react only when pRB is phosphorylated at the specific serine residue. Optical densities were measured with Scion image software and are expressed as percentages of ‘‘time 0’’ samples.

Article Snippet: Antibodies specific to the phosphorylated pRB (Ser780, Ser795 and Ser807/811) were from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Phospho-proteomics, Incubation, Western Blot, Immunostaining, SDS Page, Residue, Software

Journal: Free Radical Biology and Medicine

Article Title: CELL CYCLE ARREST BY MONOCHLORAMINE THROUGH THE OXIDATION OF RETINOBLASTOMA PROTEIN

doi: 10.1016/j.freeadbiomed.2003.10.013

Figure Lengend Snippet: Fig. 5. Effects of NH2Cl on protein phosphatase. (A) GST-pRB was pre-phosphorylated as described under Materials and Methods, then adsorbed to glutathione–agarose beads and used for phosphatase substrate. The beads were incubated with the soluble fraction of the control and NH2Cl-treated cell lysate for 90 min at 37jC, and the products were analyzed by Western blotting using phospho-specific antibody. The faint band indicates high phosphatase activity. The lower panel shows the equal load of the substrate protein. (B) Immunoblot of protein phosphatase 1. Jurkat cells were treated with NH2Cl and the cell lysate was prepared as described in the legend to Fig. 2. Expression of protein phosphatase 1, a major pRB phosphatase, was studied by SDS– PAGE and Western blotting.

Article Snippet: Antibodies specific to the phosphorylated pRB (Ser780, Ser795 and Ser807/811) were from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Incubation, Control, Western Blot, Activity Assay, Expressing, SDS Page

Journal: Scientific Reports

Article Title: Involvement of miR-15a in G0/G1 Phase Cell Cycle Arrest Induced by Porcine Circovirus Type 2 Replication

doi: 10.1038/srep27917

Figure Lengend Snippet: (A) Synchronized PK15 cells were mock infected (M) or infected with PCV2 (I) at an MOI of 1 and then collected at the indicated time points after infection. Cell lysates were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellular membranes, and immunoblotted with antibodies to cyclin D1, cyclin E, regulatory factor p21, and phosphorylated Rb (Ser795). Equal protein loads were verified with β-actin blots. p-, phosphorylated. ( B ) Amounts of the cell cycle regulator proteins in panel A were quantified by densitometric analysis and normalized against β-actin. Bars indicate the ratio of the cell cycle regulatory proteins in PCV2-infected samples to those in mock- infected samples. The results are presented as the means ± SD for three independent experiments. *P < 0.05.

Article Snippet: Rabbit, goat, or mouse antibodies against cyclin D1, cyclin E, phosphorylated-Rb (p-Rb) (Ser795), p21, β-actin were purchased from Santa Cruz Biotechnology.

Techniques: Infection, Polyacrylamide Gel Electrophoresis, SDS Page

Journal: Scientific Reports

Article Title: Involvement of miR-15a in G0/G1 Phase Cell Cycle Arrest Induced by Porcine Circovirus Type 2 Replication

doi: 10.1038/srep27917

Figure Lengend Snippet: ( A ) Predicted binding sequence is indicated between the seed sequence of miR-15a and its target sequence CCNE 3′UTR. ( B ) pGL3 Luciferase Reporter Vector containing porcine CCNE 3′UTR were co-transfected into synchronized PK15 cells with pRL-TK 24 hours after transfection of miR-15a inhibitors, miR-15a mimics and controls. 12 h after first transfection, the cells were infected with PCV2. 24 hours after the report plasmids tranfection, the cells were assayed using a dual luciferase assay. Firefly luciferase values were normalized to Renilla luciferase values and plotted as relative luciferase activity (means ± SD, n = 3). *Significantly different from wild-type reporter (p < 0.05). ( C ) Synchronized PK15 cells infected with PCV2 (24 h) after transfection with the miR-15a inhibitor or NC were assayed by FACS analysis. The histograms showed the percentage of cells in each phase of the cell cycles. The values are shown as the means of results from three independent experiments. *P < 0.05. ( D ) Total lysates from synchronized cells-infected with PCV2 (24 h) after transfection with the miR-15a inhibitor were subject to Western blotting for cyclin D1, cyclin E, and phosphorylated Rb (Ser795). β-actin was probed as the loading control. p-, phosphorylated.

Article Snippet: Rabbit, goat, or mouse antibodies against cyclin D1, cyclin E, phosphorylated-Rb (p-Rb) (Ser795), p21, β-actin were purchased from Santa Cruz Biotechnology.

Techniques: Binding Assay, Sequencing, Luciferase, Plasmid Preparation, Transfection, Infection, Activity Assay, Western Blot, Control