Journal: Oncogene

Article Title: Suppression of FOXO1 is responsible for a growth regulatory repressive transcriptional sub-signature of EWS-FLI1 in Ewing sarcoma

doi: 10.1038/onc.2013.361

Figure Lengend Snippet: FOX motifs are enriched in EWS-FLI1-repressed genes, and FOXO1 and FOXO3 are transcriptionally repressed by EWS-FLI1. ( a ) Result from the correlation analysis of gene expression with motif occurence. x axis: DNA motifs from TRANSFAC; y axis: Pearson correlation of gene expression change at time point 36 h (against 0 h) of conditional EWS-FLI1 suppression in A673sh cells with the number of motifs. Forkhead box motifs are marked by red squares. ( b ) Time-resolved expression of FOX genes upon knockdown of EWS-FLI1 in A673sh cells. Only genes with probe sets that passed quality filtering (see Materials and methods section) are shown. ( c ) Sequence of DNA motifs predicted to be specifically recognized by individual A673sh expressed FOX-factors. ( d ) Box plot of normalized gene expression values for FOXO1 and FOXO3 in primary tumor tissue. Data taken from. Blue: reference tissues from. Red: primary ES samples. The comparison of FOXO1 and FOXO3 shows that FOXO1 is ‘off' in comparison with a wide variety of reference tissues, whereas FOXO3 is ‘on'. ( e ) Representative chromatin immunopreciptation (ChIP)-PCRs in A673sh cells on two different FOXO1 promoter fragments. Fragments range from −961 to −736 and from −609 to −412 upstream of the transcription start site, including the ChIP-Seq hit at position −534 to −298, respectively and, for control, from −9071 to −8888 further upstream. ETS-binding sites (GGAA core motif) within these regions were identified using the ConSite tool. Specific ETS motifs responsible for FLI1 binding are ACGGAAG for fragment (−609/−412) and TAGGAAG/CGGGAAG for fragment (−961/−736), respectively. Signals for EWS-FLI1 binding were obtained exclusively for the two promoter fragments in the presence of EWS-FLI1, but were completely abrogated upon 48 h of doxycycline-induced knockdown of EWS-FLI1. Input DNA and ChIPs using an immunoglobulin G (IgG) control antibody were used for specificity control.

Article Snippet: The following antibodies were used: FOXO3 (H-144) and CDK2 (sc-163) (Santa Cruz, CA, USA), FOXO1 (C29H4), Phospho-FoxO1-Ser256 (9461), Phospho-FOXO3-Ser253 (9466), Lamin A/C (2032), AKT (9272) and p-AKT-Thr308 (244F9) antibodies from Cell Signaling Technology (New England Biolabs GmbH, Frankfurt, Germany).

Techniques: Gene Expression, Expressing, Knockdown, Sequencing, Comparison, ChIP-sequencing, Control, Binding Assay

Journal: Oncogene

Article Title: Suppression of FOXO1 is responsible for a growth regulatory repressive transcriptional sub-signature of EWS-FLI1 in Ewing sarcoma

doi: 10.1038/onc.2013.361

Figure Lengend Snippet: Subcellular localization of FOXO1 in A673sh cells is dependent on EWS-FLI1. ( a ) Immunofluorescence analysis of FOXO1 and FOXO3 subcellular localization in the presence or absence of EWS-FLI1. Flag-tagged FOXO1 or FOXO3 was transfected into A673sh cells in the absence and presence of doxycycline-induced EWS-FLI1 knockdown for 48 h post transfection, and subcellular localization was monitored by immunofluorescence using FLAG-tag-specific antibody (red) with 4',6-diamidino-2-phenylindole counterstain to delineate cell nuclei (blue). In the presence of EWS-FLI1, FOXO1 and FOXO3 stainings were confined exclusively to the cytoplasm. Upon EWS-FLI1 knockdown, only ectopically expressed FOXO1 but not FOXO3 translocated to the nucleus. ( b ) Immunoblot analysis of endogenous FOXO1 expression in cytoplasmic and nuclear cell fractions of A673sh cells. Basal expression of endogenous FOXO1 and FOXO3 in the presence of EWS-FLI1 was detectable exclusively in the cytoplasm. However, upon knockdown of EWS-FLI1 by doxycycline treatment for 48 h, FOXO1 expression was significantly increased and also expressed in the nucleus, whereas FOXO3 remained in the cytoplasm. In concordance with this observation, the levels of inactive phosphorylated FOXO1 and FOXO3 were reduced upon knockdown of EWS-FLI1. Note, that the double-band-appearance of FOXO1 is due to unspecific antibody recognition of both phosphorylated (upper) and unphosphorylated (lower) FOXO1 protein. ( c ) Kinetics of endogenous FOXO1 protein expression upon doxycycline-induced EWS-FLI1 knockdown in A673sh cells. Downregulation of EWS-FLI1 was already observed at 16 h, the time when cytoplasmic FOXO1 induction became first detectable, whereas significant nuclear FOXO1 expression was first observed at 36 h of doxycycline treatment with strongest expression at 48 h. Scale bars: 10 μ M.

Article Snippet: The following antibodies were used: FOXO3 (H-144) and CDK2 (sc-163) (Santa Cruz, CA, USA), FOXO1 (C29H4), Phospho-FoxO1-Ser256 (9461), Phospho-FOXO3-Ser253 (9466), Lamin A/C (2032), AKT (9272) and p-AKT-Thr308 (244F9) antibodies from Cell Signaling Technology (New England Biolabs GmbH, Frankfurt, Germany).

Techniques: Immunofluorescence, Transfection, Knockdown, FLAG-tag, Western Blot, Expressing

Journal: Oncogene

Article Title: Suppression of FOXO1 is responsible for a growth regulatory repressive transcriptional sub-signature of EWS-FLI1 in Ewing sarcoma

doi: 10.1038/onc.2013.361

Figure Lengend Snippet: Nuclear localization of FOXO1 but not FOXO3 is tightly regulated by AKT and CDK2. ( a ) Representative immunofluorescence analysis of A673sh cells transfected with Flag-FOXO1wt and treated or not with 40 μ M of the PI3K/AKT inhibitor LY294002 or 40 μ M of the canonical CDK2 inhibitor roscovitine or a combination of both 48 h post transfection for 24 h. Roscovitine and LY294002 treatments alone or in combination restore FOXO1 nuclear localization in the presence of EWS-FLI1, whereas FOXO3 remains in the cytoplasm . Results are representative of three experiments. ( b ) Quantification of exogenously expressed Flag-FOXO1 in nuclear cell fractions using LICOR Odyssey Infrared Imaging System ( c, d ) Knockdown of endogenous CDK2 by shRNA-mediated RNA interference is sufficient to restore nuclear FOXO1 localization. A673sh cells were transfected with FLAG-FOXO1 and either shCDK2 or a scrambled shRNA control, and immunofluorescence analysis ( c ) or cell fractionation followed by western blotting and quantification using LICOR Odyssey Infrared Imaging System ( d ) was conducted 48 h post transfection. ( e ) Efficient CDK2 protein knockdown was monitored on the immunoblot. Results for quantification of nuclear FOXO1 represent the mean±s.e.m. of three experiments performed. * P <0.05, ** P <0.01 and *** P <0.001. Scale bars: 10 μ M .

Article Snippet: The following antibodies were used: FOXO3 (H-144) and CDK2 (sc-163) (Santa Cruz, CA, USA), FOXO1 (C29H4), Phospho-FoxO1-Ser256 (9461), Phospho-FOXO3-Ser253 (9466), Lamin A/C (2032), AKT (9272) and p-AKT-Thr308 (244F9) antibodies from Cell Signaling Technology (New England Biolabs GmbH, Frankfurt, Germany).

Techniques: Immunofluorescence, Transfection, Imaging, Knockdown, shRNA, Control, Cell Fractionation, Western Blot

Journal: Cell Death & Disease

Article Title: Dual targeting of PI3Kδ and PPARα enhances antitumor activity via FoxO1 activation in follicular lymphoma

doi: 10.1038/s41419-026-08593-5

Figure Lengend Snippet: A Volcano plot of differentially expressed genes after combination treatment. B KEGG pathway enrichment highlighting apoptosis, cell-cycle, and FoxO signaling pathways. C GSEA showing enrichment of a FoxO1-related gene set (NES = 1.39812, p = 0.00244). D Heatmap of genes within the PI3K pathway (e.g., CDKN1A , CDKN1B , FoxO1 , FoxO3 , FoxO4 ). E – G Western blots for PI3K-p110δ, AKT, p-AKT, PPARα, and FoxO family proteins in Karpas-422, RL, and Sc-1 cells; the combination suppressed PI3K/AKT signaling and increased PPARα and FoxO1 (representative of three independent experiments). H Subcellular fractionation with Western blot showing increased nuclear FoxO1 after combination treatment (representative of three independent experiments). I Densitometric analysis of the nuclear/cytoplasmic ratio of FoxO1 ( n = 3). J qPCR showing increased FoxO1 mRNA after dual treatment ( n = 3). K Immunofluorescence images demonstrating increased FoxO1 expression and nuclear localization following combination treatment. Scale bars as indicated.

Article Snippet: The primary antibodies used were as follows: PI3K p110δ (A19742; ABclonal), pan-Akt (#4691; CST), phospho-Akt (Ser473, #4060; CST), PPARα (ab227074; Abcam), FoxO1 (#2880; CST), phospho-FoxO1 (Thr24, #9464; CST) and (Ser256, #9461; CST), FoxO3a (#12829; CST), FoxO4 (#9472; CST), Mcl-1 (#16225-1-AP; Proteintech), Bcl-2 (#12789-1-AP; Proteintech), Bim (A19702; ABclonal), Bax (#50599-2-Ig; Proteintech), Cleaved PARP(#5625; CST), P21(#2947; CST), p27 (#3686; CST), Cyclin E1 (#11935-1-AP; Proteintech), CDK2 (#10122-1-AP; Proteintech), phospho-CDK2 (#4539; CST), GLUT1(#73015; CST), PGK1(A12686; ABclonal), β-Tubulin (#10094-1-AP; Proteintech), Lamin B1 (#13435; CST), and β-Actin (#66009-1-Ig; Proteintech).

Techniques: Protein-Protein interactions, Western Blot, Fractionation, Immunofluorescence, Expressing

Journal: Cell Death & Disease

Article Title: Dual targeting of PI3Kδ and PPARα enhances antitumor activity via FoxO1 activation in follicular lymphoma

doi: 10.1038/s41419-026-08593-5

Figure Lengend Snippet: A Volcano plot of differential metabolites in RL cells (Chiglitazar vs control: 88 upregulated, 333 downregulated). B KEGG enrichment of altered metabolites highlighting FoxO signaling, carbon metabolism, and central carbon metabolism in cancer. C Integrated KEGG analysis combining transcriptomic and metabolomic data, identifying the FoxO pathway among the top-enriched pathways. D – F Functional assays showing reduced glucose uptake, lactate production, and pyruvate levels after Chiglitazar ( n = 3). G Western blots showing downregulation of HIF-1α, GLUT1, PGK1, p-AKT, and phosphorylated FoxO1 (T24, S256) (representative of three independent experiments).

Article Snippet: The primary antibodies used were as follows: PI3K p110δ (A19742; ABclonal), pan-Akt (#4691; CST), phospho-Akt (Ser473, #4060; CST), PPARα (ab227074; Abcam), FoxO1 (#2880; CST), phospho-FoxO1 (Thr24, #9464; CST) and (Ser256, #9461; CST), FoxO3a (#12829; CST), FoxO4 (#9472; CST), Mcl-1 (#16225-1-AP; Proteintech), Bcl-2 (#12789-1-AP; Proteintech), Bim (A19702; ABclonal), Bax (#50599-2-Ig; Proteintech), Cleaved PARP(#5625; CST), P21(#2947; CST), p27 (#3686; CST), Cyclin E1 (#11935-1-AP; Proteintech), CDK2 (#10122-1-AP; Proteintech), phospho-CDK2 (#4539; CST), GLUT1(#73015; CST), PGK1(A12686; ABclonal), β-Tubulin (#10094-1-AP; Proteintech), Lamin B1 (#13435; CST), and β-Actin (#66009-1-Ig; Proteintech).

Techniques: Control, Metabolomic, Functional Assay, Western Blot

Journal: Cell Death & Disease

Article Title: Dual targeting of PI3Kδ and PPARα enhances antitumor activity via FoxO1 activation in follicular lymphoma

doi: 10.1038/s41419-026-08593-5

Figure Lengend Snippet: A qPCR confirming FoxO1 mRNA knockdown in RL cells stably expressing sh FoxO1 #1 or sh FoxO1 #2 ( n = 3). B Western blot validation of FoxO1 protein reduction (representative of three independent experiments). C Densitometric analysis showing relative FoxO1 protein levels (~50% for sh FoxO1 #1; ~37% for sh FoxO1 #2; n = 3). D CCK-8 assays showing reduced drug sensitivity in FoxO1-knockdown cells treated with Linperlisib and Chiglitazar ( n = 3). E Annexin V/PI apoptosis assays showing decreased apoptosis upon combination treatment in FoxO1-deficient cells ( n = 3). F EdU assays showing increased DNA synthesis in knockdown cells under drug treatment ( n = 3). G Western blots of FoxO1 downstream targets (Bim, Bax, p21, p27) in control and FoxO1-knockdown cells (representative of three independent experiments).

Article Snippet: The primary antibodies used were as follows: PI3K p110δ (A19742; ABclonal), pan-Akt (#4691; CST), phospho-Akt (Ser473, #4060; CST), PPARα (ab227074; Abcam), FoxO1 (#2880; CST), phospho-FoxO1 (Thr24, #9464; CST) and (Ser256, #9461; CST), FoxO3a (#12829; CST), FoxO4 (#9472; CST), Mcl-1 (#16225-1-AP; Proteintech), Bcl-2 (#12789-1-AP; Proteintech), Bim (A19702; ABclonal), Bax (#50599-2-Ig; Proteintech), Cleaved PARP(#5625; CST), P21(#2947; CST), p27 (#3686; CST), Cyclin E1 (#11935-1-AP; Proteintech), CDK2 (#10122-1-AP; Proteintech), phospho-CDK2 (#4539; CST), GLUT1(#73015; CST), PGK1(A12686; ABclonal), β-Tubulin (#10094-1-AP; Proteintech), Lamin B1 (#13435; CST), and β-Actin (#66009-1-Ig; Proteintech).

Techniques: Knockdown, Stable Transfection, Expressing, Western Blot, Biomarker Discovery, CCK-8 Assay, DNA Synthesis, Control

Journal: Cell Death & Disease

Article Title: Dual targeting of PI3Kδ and PPARα enhances antitumor activity via FoxO1 activation in follicular lymphoma

doi: 10.1038/s41419-026-08593-5

Figure Lengend Snippet: A PDX study design. B Representative tumor images at endpoint. C Tumor weights at endpoint ( n = 6 per group). D Tumor volume curves over time ( n = 6 per group). E Mouse body-weight curves; a mild reduction was observed in the Linperlisib group ( n = 6 per group). F Ki-67 IHC and G quantification ( n = 6 per group). Scale bars as indicated. H p-AKT IHC and I quantification showing reduced PI3K pathway activation in the combination group ( n = 6 per group). Scale bars as indicated. J FoxO1 IHC and K quantification showing enhanced nuclear FoxO1 in the combination group ( n = 6 per group). Scale bars as indicated.

Article Snippet: The primary antibodies used were as follows: PI3K p110δ (A19742; ABclonal), pan-Akt (#4691; CST), phospho-Akt (Ser473, #4060; CST), PPARα (ab227074; Abcam), FoxO1 (#2880; CST), phospho-FoxO1 (Thr24, #9464; CST) and (Ser256, #9461; CST), FoxO3a (#12829; CST), FoxO4 (#9472; CST), Mcl-1 (#16225-1-AP; Proteintech), Bcl-2 (#12789-1-AP; Proteintech), Bim (A19702; ABclonal), Bax (#50599-2-Ig; Proteintech), Cleaved PARP(#5625; CST), P21(#2947; CST), p27 (#3686; CST), Cyclin E1 (#11935-1-AP; Proteintech), CDK2 (#10122-1-AP; Proteintech), phospho-CDK2 (#4539; CST), GLUT1(#73015; CST), PGK1(A12686; ABclonal), β-Tubulin (#10094-1-AP; Proteintech), Lamin B1 (#13435; CST), and β-Actin (#66009-1-Ig; Proteintech).

Techniques: Activation Assay

Journal: Cancer science

Article Title: Foxo3a expression and acetylation regulate cancer cell growth and sensitivity to cisplatin.

doi: 10.1111/j.1349-7006.2010.01503.x

Figure Lengend Snippet: Fig. 1. Cisplatin-resistant cells are more sensitive to mithramycin than their parental cells and show decreased expression of Foxo3a transcription factor. (a) T24, T24 ⁄ DDP10, KK47, and KK47 ⁄ DDP20 cells were seeded into 96-well plates. The following day, various concentrations of mithramycin were applied. After incubation for 48 h, cell survival was analyzed by a cytotoxicity assay. Cell survival in the absence of mithramycin corresponds to 1. All values are representative of at least three independent experiments. Boxes, mean; bars, ±SD. (b) After extraction of total RNA from T24, T24 ⁄ DDP10, KK47, and KK47 ⁄ DDP20 cells and synthesis of cDNA, quantitative real-time PCR was performed using the primers and probes for Foxo3a and GAPDH. The Foxo3a transcript level was corrected for the corresponding GAPDH transcript level. All values represent at least three independent experiments. The level of Foxo3a transcript from T24 cells was defined as 1. Boxes, mean; bars, ±SD. Whole-cell extracts and nuclear extracts of T24, T24 ⁄ DDP10, KK47, and KK47 ⁄ DDP20 cells were subjected to SDS-PAGE, and western blotting was performed using the indicated antibodies. Immunoblots for b-actin and Lamin B1 are shown as loading controls.

Article Snippet: 101 | no. 5 | 1177–1185 C-terminally GFP-tagged Foxo3a protein was purchased from OriGene (Rockville, MD, USA).

Techniques: Expressing, Incubation, Cytotoxicity Assay, Extraction, Real-time Polymerase Chain Reaction, SDS Page, Western Blot

Journal: Cancer science

Article Title: Foxo3a expression and acetylation regulate cancer cell growth and sensitivity to cisplatin.

doi: 10.1111/j.1349-7006.2010.01503.x

Figure Lengend Snippet: Fig. 2. Foxo3a knockdown augments cancer cell growth and cisplatin resistance. (a) KK47 cells were transfected with 40 nM of control siRNA, Foxo3a siRNA #1 or Foxo3a siRNA #2. After extraction of total RNA and synthesis of cDNA, quantitative real-time PCR was performed using the primers and probes for Foxo3a and GAPDH. The Foxo3a transcript level was corrected for the corresponding GAPDH transcript level. All values represent at least three independent experiments. The level of Foxo3a transcript from KK47 cells transfected with control siRNA was defined as 1. Boxes, mean; bars, ±SD. Whole-cell extracts of KK47 cells transfected with 40 nM of control siRNA, Foxo3a siRNA #1, or Foxo3a siRNA #2 were subjected to SDS-PAGE, and western blotting was performed using the indicated antibodies. Immunoblot for b-actin is shown as a loading control. (b) KK47 cells transfected with 40 nM of control siRNA, Foxo3a siRNA #1, or Foxo3a siRNA #2 were seeded into 12-well plates and incubated. The number of cells was counted at the indicated times. The results were normalized to the number of cells at 0 h. All values represent at least three independent experiments. Boxes, mean; bars, ±SD. (c) KK47 cells transfected with 40 nM of control siRNA, Foxo3a siRNA #1, or Foxo3a siRNA #2 were seeded into 96-well plates. The following day, various concentrations of cisplatin were applied. After incubation for 48 h, cell survival was analyzed by a cytotoxicity assay. Cell survival in the absence of cisplatin corresponds to 1. All values are representative of at least three independent experiments. Boxes, mean; bars, ±SD.

Article Snippet: 101 | no. 5 | 1177–1185 C-terminally GFP-tagged Foxo3a protein was purchased from OriGene (Rockville, MD, USA).

Techniques: Knockdown, Transfection, Control, Extraction, Real-time Polymerase Chain Reaction, SDS Page, Western Blot, Incubation, Cytotoxicity Assay

Journal: Cancer science

Article Title: Foxo3a expression and acetylation regulate cancer cell growth and sensitivity to cisplatin.

doi: 10.1111/j.1349-7006.2010.01503.x

Figure Lengend Snippet: Fig. 3. Mithramycin increases sensitivity of cancer cells to cisplatin through Foxo3a induction by oxidative stress. (a) KK47 cells were seeded into 96-well plates and incubated. On the following day, the media were replaced with media containing vehicle, 2 nM of mithramycin, 2 nM of doxorubicin, 0.2 lM of cisplatin, or 2 lM of 5-fluorouracil (5-FU) and ⁄ or 5 mM of NAC. After 48 h, the cells were stained with CM-H2DCFDA and measured for their florescence intensities. All values are representative of at least three independent experiments. The fluorescence intensity of KK47 cells with vehicle was set as 1. Boxes, mean; bars, ±SD. (b) Whole-cell extracts of KK47 incubated with media containing vehicle, 2 lM of hydrogen peroxide, 2 nM of mithramycin, 2 nM of doxorubicin, 0.2 lM of cisplatin, or 2 lM of 5-FU and ⁄ or 5 mM of NAC for 48 h were subjected to SDS-PAGE, and western blotting was performed using the indicated antibodies. Immunoblot for b-actin is shown as a loading control. (c) T24 and KK47 cells were transfected with 40 nM of control siRNA or Sp1 siRNA. After extraction of total RNA and synthesis of cDNA, quantitative real-time PCR was performed using the primers and probes for Sp1, Foxo3a, b-actin, and GAPDH. The Sp1 and Foxo3a transcript levels were corrected for the corresponding b-actin and GAPDH transcript levels, respectively. All values represent at least three independent experiments. The level of Sp1 and Foxo3a transcript from T24 cells transfected with control siRNA was defined as 1. Boxes, mean; bars, ±SD. Whole-cell extracts of T24 and KK47 cells transfected with 40 nM of control siRNA or Sp1 siRNA were subjected to SDS-PAGE, and western blotting was performed using the indicated antibodies. Immunoblot for b-actin is shown as a loading control. (d) KK47 cells were seeded into 96-well plates with media containing vehicle, 2 nM of mithramycin, 2 nM of doxorubicin, or 2 lM of 5-FU. The following day, various concentrations of cisplatin were applied. After incubation for 48 h, cell survival was analyzed by a cytotoxicity assay. Cell survival in the absence of cisplatin corresponds to 1. All values are representative of at least three independent experiments. Boxes, mean; bars, ±SD. (e) KK47 cells transfected with 40 nM control siRNA, Foxo3a siRNA #1, or Foxo3a siRNA #2 were seeded into 96-well plates with media containing vehicle or 2 nM of mithramycin. The following day, various concentrations of cisplatin were applied. After incubation for 48 h, cell survival was analyzed by a cytotoxicity assay. Cell survival in the absence of cisplatin corresponds to 1. All values are representative of at least three independent experiments. Boxes, mean; bars, ±SD.

Article Snippet: 101 | no. 5 | 1177–1185 C-terminally GFP-tagged Foxo3a protein was purchased from OriGene (Rockville, MD, USA).

Techniques: Incubation, Staining, SDS Page, Western Blot, Control, Transfection, Extraction, Real-time Polymerase Chain Reaction, Cytotoxicity Assay

Journal: Cancer science

Article Title: Foxo3a expression and acetylation regulate cancer cell growth and sensitivity to cisplatin.

doi: 10.1111/j.1349-7006.2010.01503.x

Figure Lengend Snippet: Fig. 4. Deacetylation of Foxo3a is induced by cisplatin treatment and the acetylated-Foxo3a level is decreased in cisplatin-resistant cells. (a) KK47 cells were treated with 1 lM of cisplatin for the indicated times. The cells were then harvested and nuclear extracts were subjected to SDS-PAGE, and western blotting was performed using the indicated antibodies. Immunoblotting for Lamin B1 is shown as a loading control. (b) KK47-Foxo3a-GFP #1 cells were seeded into six-well plates. The following day, the cells were treated with 1 lM of cisplatin for the indicated times. Whole-cell extracts were immunoprecipitated with 2.0 lg of anti-GFP antibody and 20 lL of A ⁄ G agarose. After washing three times, the supernatants were subjected to SDS-PAGE, and western blotting was performed using the indicated antibodies. (c) Nuclear extracts of T24, T24 ⁄ DDP10, KK47, and KK47 ⁄ DDP20 cells were subjected to SDS-PAGE, and western blotting was performed using the indicated antibodies. Immunoblot for Lamin B1 is shown as a loading control. (d) Whole-cell extracts (500 lg) of T24, T24 ⁄ DDP10, KK47, and KK47 ⁄ DDP20 cells were immunoprecipitated with antibody against Foxo3a and A ⁄ G agarose. After washing three times, the supernatants were subjected to SDS-PAGE, and western blotting was performed using the indicated antibodies.

Article Snippet: 101 | no. 5 | 1177–1185 C-terminally GFP-tagged Foxo3a protein was purchased from OriGene (Rockville, MD, USA).

Techniques: SDS Page, Western Blot, Control, Immunoprecipitation

Journal: Cancer science

Article Title: Foxo3a expression and acetylation regulate cancer cell growth and sensitivity to cisplatin.

doi: 10.1111/j.1349-7006.2010.01503.x

Figure Lengend Snippet: Fig. 5. Silencing of the Foxo3a-associated acetyl- transferase p300 promotes cell growth and ren- ders cells resistant to cisplatin. (a, b) KK47 (a) and T24 (b) cells transfected with 40 nM control siRNA, p300 siRNA #1, or p300 siRNA #2 were seeded into 12-well plates and incubated. The number of cells was counted at the indicated times. The results were normalized to the number of cells at 0 h. All values represent at least three independent exper- iments. Boxes, mean; bars, ±SD. Whole-cell extracts of KK47 (a) and T24 (b) cells transfected with 40 nM of control siRNA, p300 siRNA #1 or p300 siRNA #2 were subjected to SDS-PAGE, and western blotting was performed using the indi- cated antibodies. Immunoblot for b-actin is shown as a loading control. (c,d) KK47 (c) and T24 (d) cells transfected with 40 nM control siRNA, p300 siRNA #1, or p300 siRNA #2 were seeded into 96- well plates. The following day, various concentra- tions of cisplatin or 5-fluorouracil (5-FU) were applied. After incubation for 48 h, cell survival was analyzed by a cytotoxicity assay. Cell survival in the absence of cisplatin or 5-FU corresponds to 1. All values are representative of at least three independent experiments. Boxes, mean; bars, ±SD.

Article Snippet: 101 | no. 5 | 1177–1185 C-terminally GFP-tagged Foxo3a protein was purchased from OriGene (Rockville, MD, USA).

Techniques: Transfection, Control, Incubation, SDS Page, Western Blot, Cytotoxicity Assay

Journal: Cancer science

Article Title: Foxo3a expression and acetylation regulate cancer cell growth and sensitivity to cisplatin.

doi: 10.1111/j.1349-7006.2010.01503.x

Figure Lengend Snippet: Fig. 6. Non-acetylation-mimicking Foxo3a overexpression suppresses cancer cell growth and cisplatin resistance to a lesser extent compared with wild-type Foxo3a overexpression. (a) Whole-cell extracts of the indicated stable transfectants of KK47 cells were subjected to SDS-PAGE, and western blotting was performed using the indicated antibodies. Immunoblot for b-actin is shown as a loading control. (b) Whole-cell extracts (500 lg) of the indicated stable transfectants of KK47 cells were immunoprecipitated with antibody against GFP and A ⁄ G agarose. After washing three times, the supernatants were subjected to SDS-PAGE, and western blotting was performed using the indicated antibodies. (c) The indicated stable transfectants of KK47 cells were seeded into 12-well plates and incubated. The number of cells was counted at the indicated times. The results were normalized to the number of cells at 0 h. All values represent at least three independent experiments. Boxes, mean; bars, ±SD. (d) The indicated stable transfectants of KK47 cells were seeded into 96-well plates. The following day, various concentrations of cisplatin were applied. After incubation for 48 h, cell survival was analyzed by a cytotoxicity assay. Cell survival in the absence of cisplatin corresponds to 1. All values are representative of at least three independent experiments. Boxes, mean; bars, ±SD.

Article Snippet: 101 | no. 5 | 1177–1185 C-terminally GFP-tagged Foxo3a protein was purchased from OriGene (Rockville, MD, USA).

Techniques: Over Expression, SDS Page, Western Blot, Control, Immunoprecipitation, Incubation, Cytotoxicity Assay

Journal: Oncotarget

Article Title: FOXO3 induces ubiquitylation of AKT through MUL1 regulation

doi: 10.18632/oncotarget.22793

Figure Lengend Snippet: (A) CDDP causes FOXO3 translocation into the nucleus. TPC1 cells were cultured on coverslips and treated with CDDP in the absence of serum for the indicated times. Immunofluorescent staining was then performed using the anti-FOXO3 ( red ), anti-Tubulin ( green ) antibody and DAPI ( blue ). Scale bar represents 50 μm. Tubulin and DAPI were also each applied to visualize the cytoplasm and nuclei, respectively. (B and C) FOXO3 knock-down inhibits CDDP-mediated MUL1 induction. TPC1 and BHP10-3 cells were transfected with scrambled RNAs or FOXO3 siRNA. At 24 hours after siRNA transfection, CDDP was treated for 12 or 24 hours in the absence of serum for Western blot and MUL1 RT-PCR. BINP3 or TNFSF10 was used as positive control against FOXO3. (D) CDDP induces increased activity of FOXO3 DNA binding. FOXO3 plasmids were transfected into TPC1 cells and then, cells were cultured with or without CDDP for 24 hours. FOXO3 DNA binding activity was determined with ChIP assay. (E) FOXO3 triple mutant (FOXO3 TM) or FOXO3 wild type (FOXO3 WT) plasmids were transfected to TPC1 and then, CDDP was treated for 24 hours. (F) CDDP induces increased level of FOXO3 reporter activity. The TPC1 cells were cotransfected with FOXO3 reporter plasmids (pGL4-FOXO3/Luc) and FOXO3. After 24 hours of incubation, cells were lysed and the reporter luciferase activities driven by the FOXO3 were determined.

Article Snippet: FOXO3-Flag, FOXO3 WT-HA and FOXO3 triple (T32A, S253A, S315A) mutant-HA (FOXO3 TM-HA) plasmid [ ] was obtained from Addgene (Cambridge, MA, USA).

Techniques: Translocation Assay, Cell Culture, Staining, Knockdown, Transfection, Western Blot, Reverse Transcription Polymerase Chain Reaction, Positive Control, Activity Assay, Binding Assay, Mutagenesis, Incubation, Luciferase

Journal: Oncotarget

Article Title: FOXO3 induces ubiquitylation of AKT through MUL1 regulation

doi: 10.18632/oncotarget.22793

Figure Lengend Snippet: (A and B) FOXO3 promotes CDDP-induced MUL1 expression and AKT ubiquitylation. (A) TPC1 cells were transfected with Mock or FOXO3-Flag plasmid and then, CDDP was treated for 24 hours in the absence of serum. The cells were subjected to Western blot assay with the indicated antibodies. (B) TPC1 cells were transfected with Mock or FOXO3-Flag together with the Myr-AKT/Myc-His and Ubi-HA plasmid. At 24 hours later, CDDP was treated for 24 hours with MG132 (1 μM) in the absence of serum. The cell lysates were subjected to denatured Ni-NTA His pull-down and Western blot. (C) FOXO3 knock-down inhibits CDDP-mediated AKT ubiquitylation. TPC1 cells were transfected with scrambled RNAs or FOXO3 siRNA and 24 hours later, Myr-AKT/Myc-His and Ubi-HA plasmid were transfected for 12 hours. CDDP was treated for 24 hours with MG132 (1 μM) in the absence of serum. The cell lysates were subjected to denatured Ni-NTA His pull-down and Western blot. (D) Mock or FOXO3-Flag plasmid transfected TPC1 cells were seeded in a 48-well plate and then, CDDP was treated for 24 hours in the absence of serum. Cell viability was measured by MTT assay (n = 6). Data are mean ± SD. Asterisks indicate statistically significant differences ( P < 0.05 ). (E) Scrambled RNAs or FOXO3 siRNA transfected TPC1 cells were seeded in a 48-well plate and then, CDDP was treated for 24 hours in the absence of serum. Cell viability was measured by MTT assay (n = 6). Data are mean ± SD. Asterisks indicate statistically significant differences ( P < 0.05 ).

Article Snippet: FOXO3-Flag, FOXO3 WT-HA and FOXO3 triple (T32A, S253A, S315A) mutant-HA (FOXO3 TM-HA) plasmid [ ] was obtained from Addgene (Cambridge, MA, USA).

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Knockdown, MTT Assay

Journal: Oncotarget

Article Title: FOXO3 induces ubiquitylation of AKT through MUL1 regulation

doi: 10.18632/oncotarget.22793

Figure Lengend Snippet: FOXO3 is inactivated in normal condition through phosphorylation by AKT or ubiquitylation, however, CDDP induces accumulation of MUL1 and leads to degradation of AKT through K48-linked ubiquitylation. Furthermore, CDDP-induced FOXO3 activation controls induction of MUL1.

Article Snippet: FOXO3-Flag, FOXO3 WT-HA and FOXO3 triple (T32A, S253A, S315A) mutant-HA (FOXO3 TM-HA) plasmid [ ] was obtained from Addgene (Cambridge, MA, USA).

Techniques: Phospho-proteomics, Activation Assay

Journal: Nucleic Acids Research

Article Title: MyoD- and FoxO3-mediated hotspot interaction orchestrates super-enhancer activity during myogenic differentiation

doi: 10.1093/nar/gkx488

Figure Lengend Snippet: FoxO3 and MyoD coordinate the genome-wide activation of SE hotspots during myoblast (MB) differentiation. ( A ) MyoD deletion decreased the activity at MyoD-bound hotspots within SEs compared to the wild-type (WT) control. H3K27ac ChIP-seq signal in WT or KO surrounding the aligned MyoD peak center in WT SE hotspots was calculated to measure the enhancer activity. ( B ) FoxO3 and some known co-factors of MyoD bind to MyoD peaks in SE hotspots in MT. ( C ) Genomic loci within SEs bound by both FoxO3 and MyoD displayed the highest H3K27ac density compared with FoxO3 or MyoD only sites in MT. *** P < 0.001. ( D ) PCC between H3K27ac or Pol II and TF ChIP-seq density revealed that the FoxO3 binding displayed the highest correlation with SE activity among the TFs. ( E ) A total of 96% of FoxO3 peaks within SE hotspots overlap with MyoD binding. ( F ) On the FoxO3-MyoD co-bound enhancer hotspots, a much higher percentage of MyoD binding E-box motif (5’-CAGGTG-3’) than FoxO3 binding Fork head motif (5’-GTAAACA-3’) was detected. ( G ) Schematic illustration of MyoD recruitment of FoxO3 to the hotspot region. ( H ) Snapshots of examples of MyoD/FoxO3 co-bound hotspots (red bar) and their associated genes. ( I ) Sequential ChIP showed both MyoD and FoxO3 bound on the same region in one hotspot associated with Myogenin , or two hotspots associated with Mb (Myoglobin) gene. ( J and K ) Decreased FoxO3 or H3K27ac binding on six selected examples of hotspots associated with Klf6, Medag, Ppp2r2a, Mb and Myogenin was detected in MyoD knockout (KO) cell lines #1 and #2 as compared to WT control. ( L and M ) Knockdown of FoxO3 by shRNA decreased MyoD or H3K27ac binding on the above SEs. ( N ) Knockdown of either MyoD or FoxO3 by siRNA reduced the expression of the above genes. ( O ) ChIP-PCR was performed to detect the enrichment of FoxO3 on the above hotspots in the differentiating myotubes (MTs) at −24, 24, 48 and 72 hr. ( P ) The expression of MyoD and FoxO3 in differentiating C2C12 was detected by western blotting using α-Tubulin as loading control. ( Q ) ChIP-PCR was performed to detect the enrichment of MyoD on the above hotspots at DM −24, 24, 48 and 72 hr. All qRT-PCR and ChIP-PCR data were normalized to GAPDH mRNA and ChIP input respectively and represent the average of three independent experiments ±s.d. * P < 0.05, ** P < 0.01 and *** P < 0.001.

Article Snippet: Antibodies for MyoD (Santa Cruz Biotechnology, sc-304X, rabbit polyclonal), FoxO3 (Santa Cruz Biotechnology, sc-48348X, rabbit polyclonal) and histone H3-K27 acetylation (Abcam, ab4729, rabbit polyclonal) were used for ChIP, or normal rabbit IgG (Santa Cruz Biotechnology, sc-2027) was used as negative control.

Techniques: Genome Wide, Activation Assay, Activity Assay, Control, ChIP-sequencing, Binding Assay, Knock-Out, Knockdown, shRNA, Expressing, Western Blot, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: MyoD- and FoxO3-mediated hotspot interaction orchestrates super-enhancer activity during myogenic differentiation

doi: 10.1093/nar/gkx488

Figure Lengend Snippet: Physical association of MyoD and FoxO3 at the hotspots in the differentiating MTs. ( A ) C2C12 was harvested in DM at 0, 24, 48 and 72 h and the lysates were immunoprecipitated with IgG or MyoD antibody and then detected for FoxO3 by western blotting. Numbers below each blot indicate the intensity of each band as measured by Image-Pro Plus. ( B ) The reciprocal IP was performed with IgG or FoxO3 antibody followed by WB detection of MyoD. ( C ) The binding between MyoD and FoxO3 at 48 h was abolished by either DNase I or ethidium bromide (EtBr) treatment. ( D ) When immuniprecipitated by Biotin, biotin-labeled Myogenin hotspot 3 (H3), but not biotin-labeled control, was able to retrieve MyoD and FoxO3. ( E ) Recombinant MyoD (rMyoD) protein retrieved FoxO3 from MT lysates and the addition of E-box motif DNA increased the retrieval. ( F ) Sucrose gradient of C2C12 nuclear extracts at DM −24, 48 and 72 h followed by western blotting detected the elution profiles of MyoD and FoxO3, which were found in the same fractions ( – and ) in DM 48 and 72 h. ( G and H ) The plasmid expressing various domains or the full-length ( -318) of HA-tagged MyoD was co-transfected with plasmid expressing Flag-tagged full length FoxO3. Lysates were pulled down by HA antibody and examined by western blotting using Flag antibody. For all the above panels, the representative images from two replicates are shown.

Article Snippet: Antibodies for MyoD (Santa Cruz Biotechnology, sc-304X, rabbit polyclonal), FoxO3 (Santa Cruz Biotechnology, sc-48348X, rabbit polyclonal) and histone H3-K27 acetylation (Abcam, ab4729, rabbit polyclonal) were used for ChIP, or normal rabbit IgG (Santa Cruz Biotechnology, sc-2027) was used as negative control.

Techniques: Immunoprecipitation, Western Blot, Binding Assay, Labeling, Control, Recombinant, Plasmid Preparation, Expressing, Transfection

Journal: Nucleic Acids Research

Article Title: MyoD- and FoxO3-mediated hotspot interaction orchestrates super-enhancer activity during myogenic differentiation

doi: 10.1093/nar/gkx488

Figure Lengend Snippet: MyoD and FoxO3 orchestrate the hotspot interactions. ( A ) Overexpression of MyoD in 10T1/2 cells induced the H3 or H4 hotspot reporter activities. ( B ) H3 or H4 reporter activity was decreased in MyoD KO cells. ( C ) Deletion of motif # 2 or both but not #1 diminished the induction of H3 reporter by MyoD overexpression in 10T1/2 cells. ( D ) The deletion of motif # 2 or both but not #1 diminished the H3 reporter activities in differentiating C2C12 MTs. ( E ) Deletion of the motif in H4 had no impact on H4 reporter activity in differentiating C2C12 MTs. ns, no significance. ( F ) The deletion of the motif had no impact on the induction of H4 activity by MyoD over-expression in 10T1/2 cells. ( G ) H2, H3 or H4 hotspot reporter activity was reduced in shFoxO3 MTs compared to the vector control. ( H ) H3K27ac ChIP-seq was performed in WT or MyoD KO cells to assess the impact of MyoD deletion on hotspot activity. ( I ) The interaction between H3 and H4 was decreased in MyoD KO cells as assessed by 3C-PCR. ( J and K ) The impact of each hotspot deletion on the enrichment of MyoD or FoxO3 on the hotspots and Myogenin promoter was examined by ChIP-PCR. All luciferase data were normalized to Renillia protein and represent the average of three independent experiments ± s.d. Relative Luciferase Unit (RLU) normalized to the vector or WT control is shown. All qRT-PCR and ChIP-PCR data were normalized to GAPDH mRNA and ChIP input respectively and represent the average of three independent experiments ±s.d. * P < 0.05, ** P < 0.01 and *** P < 0.001. ( L ) Schematic illustration of MyoD/FoxO3 mediated hotspot interaction that orchestrates Myogenin SE activation during MB differentiation. The dominant H3 hotspot is primed in the MB by MyoD binding; upon early differentiation, binding of FoxO3 further amplifies its activity and subsequent interaction with H4; H3 next interacts with H2 and H1 to activate their formation; H1 appears to repress the other three hotspots thus acting as a brake. Altogether the hierarchical activation of the four hotspots orchestrates the Myogenin gene expression.

Article Snippet: Antibodies for MyoD (Santa Cruz Biotechnology, sc-304X, rabbit polyclonal), FoxO3 (Santa Cruz Biotechnology, sc-48348X, rabbit polyclonal) and histone H3-K27 acetylation (Abcam, ab4729, rabbit polyclonal) were used for ChIP, or normal rabbit IgG (Santa Cruz Biotechnology, sc-2027) was used as negative control.

Techniques: Over Expression, Activity Assay, Plasmid Preparation, Control, ChIP-sequencing, Luciferase, Quantitative RT-PCR, Activation Assay, Binding Assay, Gene Expression

Journal: The Journal of Biological Chemistry

Article Title: Forkhead Box O3A (FOXO3) and the Mitochondrial Disulfide Relay Carrier (CHCHD4) Regulate p53 Protein Nuclear Activity in Response to Exercise *

doi: 10.1074/jbc.M116.745737

Figure Lengend Snippet: Exercise-induced FOXO3 protein interacts with the FOXO3 DBE of the CHCHD4 gene. A, FOXO3 immunoblot of wild-type mouse skeletal muscle in the unexercised state (control, CTL) and post-exercise state at the indicated time points. Whole tissue and nuclear fraction samples are shown. GAPDH and lamin B1 served as total and nuclear protein loading controls, respectively. B, schematic of the three putative FOXO3 DBEs on the human CHCHD4 promoter relative to the start codon. Shown is the ChIP analysis of the interaction between FOXO3 and the DBEs of the CHCHD4 gene in HCT116 cells treated with CCCP for 6 h to induce FOXO3. Input DNA samples were diluted 1:20 for the agarose gel image. n = 3. C, luciferase activity of CHCHD4 promoter constructs containing wild-type or mutated (mut) DBE2 sequence. HCT116 cells were treated with CCCP to induce FOXO3 protein after transfection with the constructs. Control is without CCCP treatment. n = 4. D, effect of the constitutively active triple phosphorylation mutant FOXO3 (FOXO3-TM) on DBE2 promoter construct activity. HCT116 cells were co-transfected with FOXO3-TM cDNA 36 h prior to the luciferase assay. Control is without FOXO3-TM co-transfection. n = 4. Values are mean ± S.E. *, p < 0.05; **, p < 0.001.

Article Snippet: Antibodies and Immunoblot Analysis The following antibodies were used: CHCHD4 (polyclonal antibody H107, sc-98628, Santa Cruz Biotechnology), FOXO3 (2497, Cell Signaling Technology), GADPH (Ambion), histone H3 (ab1791, Abcam), lamin B1 (ab16048, Abcam), human p21 (OP64, Calbiochem), mouse p21 (OP76, Calbiochem), p53 (monoclonal antibody DO-1, sc-126, Santa Cruz Biotechnology), TIMM9 (11479-1-AP, Proteintech), tubulin ( {"type":"entrez-nucleotide","attrs":{"text":"A11126","term_id":"490968","term_text":"A11126"}} A11126 , Invitrogen), and VDAC (Rockland).

Techniques: Western Blot, Control, Agarose Gel Electrophoresis, Luciferase, Activity Assay, Construct, Sequencing, Transfection, Phospho-proteomics, Mutagenesis, Cotransfection

Journal: The Journal of Biological Chemistry

Article Title: Forkhead Box O3A (FOXO3) and the Mitochondrial Disulfide Relay Carrier (CHCHD4) Regulate p53 Protein Nuclear Activity in Response to Exercise *

doi: 10.1074/jbc.M116.745737

Figure Lengend Snippet: FOXO3 represses CHCHD4 expression. A, expression levels of CHCHD4 and p21 in HCT116 cells transfected with the FOXO3-TM plasmid. n = 3. B, p53 immunoblot of nuclear fraction and total cell extract of HCT116 cells transfected with the FOXO3-TM plasmid. C, representative confocal immunofluorescence images of p53 (red) in FOXO3-TM-transfected cells. DAPI stain (blue) shows nuclei. Values are mean ± S.E. *, p < 0.001. Scale bar = 10 μm.

Article Snippet: Antibodies and Immunoblot Analysis The following antibodies were used: CHCHD4 (polyclonal antibody H107, sc-98628, Santa Cruz Biotechnology), FOXO3 (2497, Cell Signaling Technology), GADPH (Ambion), histone H3 (ab1791, Abcam), lamin B1 (ab16048, Abcam), human p21 (OP64, Calbiochem), mouse p21 (OP76, Calbiochem), p53 (monoclonal antibody DO-1, sc-126, Santa Cruz Biotechnology), TIMM9 (11479-1-AP, Proteintech), tubulin ( {"type":"entrez-nucleotide","attrs":{"text":"A11126","term_id":"490968","term_text":"A11126"}} A11126 , Invitrogen), and VDAC (Rockland).

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Immunofluorescence, Staining

Journal: The Journal of Biological Chemistry

Article Title: Forkhead Box O3A (FOXO3) and the Mitochondrial Disulfide Relay Carrier (CHCHD4) Regulate p53 Protein Nuclear Activity in Response to Exercise *

doi: 10.1074/jbc.M116.745737

Figure Lengend Snippet: Knocking down FOXO3 increases CHCHD4 expression. A, CHCHD4 and p21 expression in HCT116 cells with knockdown of CCCP-induced FOXO3 with shRNA. NS, nonspecific shRNA. n = 3. B, CHCHD4 and p21 expression in HCT116 cells with knockdown of serum starvation-induced FOXO3 with shRNA. n = 3. C, p53 immunoblot of nuclear and total cell fractions of HCT116 cells after knockdown of FOXO3 induced by CCCP or serum starvation. D, representative confocal immunofluorescence image of p53 (red) in HCT116 cells after knockdown of FOXO3 induced by CCCP or serum starvation. DAPI stain (blue) shows nuclei. Values are mean ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Scale bar = 10 μm.

Article Snippet: Antibodies and Immunoblot Analysis The following antibodies were used: CHCHD4 (polyclonal antibody H107, sc-98628, Santa Cruz Biotechnology), FOXO3 (2497, Cell Signaling Technology), GADPH (Ambion), histone H3 (ab1791, Abcam), lamin B1 (ab16048, Abcam), human p21 (OP64, Calbiochem), mouse p21 (OP76, Calbiochem), p53 (monoclonal antibody DO-1, sc-126, Santa Cruz Biotechnology), TIMM9 (11479-1-AP, Proteintech), tubulin ( {"type":"entrez-nucleotide","attrs":{"text":"A11126","term_id":"490968","term_text":"A11126"}} A11126 , Invitrogen), and VDAC (Rockland).

Techniques: Expressing, Knockdown, shRNA, Western Blot, Immunofluorescence, Staining

Journal: The Journal of Biological Chemistry

Article Title: Forkhead Box O3A (FOXO3) and the Mitochondrial Disulfide Relay Carrier (CHCHD4) Regulate p53 Protein Nuclear Activity in Response to Exercise *

doi: 10.1074/jbc.M116.745737

Figure Lengend Snippet: Summary of exercise regulation of p53 subcellular partitioning. The exercise-induced transcription factor FOXO3 represses CHCHD4 expression, thereby preventing the translocation of p53 into the mitochondria and increasing its nuclear level and activity.

Article Snippet: Antibodies and Immunoblot Analysis The following antibodies were used: CHCHD4 (polyclonal antibody H107, sc-98628, Santa Cruz Biotechnology), FOXO3 (2497, Cell Signaling Technology), GADPH (Ambion), histone H3 (ab1791, Abcam), lamin B1 (ab16048, Abcam), human p21 (OP64, Calbiochem), mouse p21 (OP76, Calbiochem), p53 (monoclonal antibody DO-1, sc-126, Santa Cruz Biotechnology), TIMM9 (11479-1-AP, Proteintech), tubulin ( {"type":"entrez-nucleotide","attrs":{"text":"A11126","term_id":"490968","term_text":"A11126"}} A11126 , Invitrogen), and VDAC (Rockland).

Techniques: Expressing, Translocation Assay, Activity Assay

Journal: bioRxiv

Article Title: The AKT Forkhead box O transcription factor axis regulates human cytomegalovirus replication

doi: 10.1101/2022.04.14.488435

Figure Lengend Snippet: ( A ) Schematic denoting the influence of Akt activity on two key substrates, PRAS40 and FOXO transcription factors (FOXO TF). Left panel: when Akt is inactive, FOXO TF are able to enter the nucleus. Right panel: when Akt is recruited to membranes, such as during PI3K signaling, Akt is phosphorylated at activating residues Thr308 and Ser473. Once activated Akt in turn phosphorylates many downstream substrates, including PRAS40 and FOXO TF. Akt phosphorylation of FOXO TF recruits binding of 14-3-3 proteins, which sequester FOXO TF in the cytoplasm. ( B ) Fibroblasts were infected with HCMV strain TB40/E at MOI = 2. Lysates were harvested at the indicated times post infection (h post infection, hpi) and analyzed by Western blot for proteins immunoreactive to antibodies specific for pAkt; pAkt Ser473 and pAkt Thr308: Akt phosphorylated at Thr308 or Ser473, respectively; Akt: total Akt; PRAS40 Thr246: PRAS phosphorylated at Thr246, PRAS40: total PRAS40, IE1: the 72-kD HCMV immediate-early nuclear antigen, IE1. A section of nitrocellulose membrane was also subjected to Ponceau S staining (Ponceau) to assess total protein loading across lanes. ( C ) Fibroblasts were infected with HCMV strain TB40/E at MOI = 1. Cells were fixed in 4% PFA at the indicated times post-infection and stained with antibodies specific for FOXO3a (green) or HCMV IE1 antigen (blue). Scale bar: 25 µm.

Article Snippet: In , the same staining procedure was used except the ARPE-19 cells were not infected, and anti-HA antibody (Bethyl) and Alexa488 conjugated goat anti rabbit secondary antibody were used to detect FOXO3a-TM-ER by virtue of its 3× HA epitope tag.

Techniques: Activity Assay, Phospho-proteomics, Binding Assay, Infection, Western Blot, Membrane, Staining

Journal: bioRxiv

Article Title: The AKT Forkhead box O transcription factor axis regulates human cytomegalovirus replication

doi: 10.1101/2022.04.14.488435

Figure Lengend Snippet: (A-B) Fibroblasts carrying doxycycline inducible (“tet-on”) expression cassettes for either myristoylated AKT1 (myr-AKT) or a kinase dead control (K179M) were induced for 24 h using 100 ng/mL doxycycline hyclate and subsequently infected at MOI =1 with either HCMV strain AD169rv (A) or TB40/E (B). Supernatants were harvested at the indicated times post infection (days post infection, dpi) and measured for the infectious titer by TCID50 assay. (C) Representative confocal microscope images of formalin fixed cells at 24 h post infection, see for full time course. (D-E) At least 30 cells per condition were scored at 72 hpi for the effect of myr-AKT or myr-AKT-K179M expression (scored by HA epitope positivity in indirect immunofluorescent staining, the myr-AKT transgene carries an HA tag), or for the effect on HCMV infection (scored by IE1 antigen positive cells) on nuclear localization of endogenous FOXO3a, as indicated on the Y-axis.

Article Snippet: In , the same staining procedure was used except the ARPE-19 cells were not infected, and anti-HA antibody (Bethyl) and Alexa488 conjugated goat anti rabbit secondary antibody were used to detect FOXO3a-TM-ER by virtue of its 3× HA epitope tag.

Techniques: Expressing, Control, Infection, TCID50 Assay, Microscopy, Staining

Journal: bioRxiv

Article Title: The AKT Forkhead box O transcription factor axis regulates human cytomegalovirus replication

doi: 10.1101/2022.04.14.488435

Figure Lengend Snippet: siRNA complexes targeting FOXO1, FOXO3a or both FOXO1 and FOXO3a were reverse transfected into fibroblasts 24 h prior to infection with HCMV strain TB40/E at MOI = 1. (A) Supernatants were harvested at the indicated times post infection (days post infection, dpi). (B) Viral yield at day 5 post infection for 3 independent biological replicates of the experiment shown in panel A, asterisk indicates p=0.0276 using one-way ANOVA with Dunnet’s post-test to compare means of each siRNA treatment condition to NT control setting. (C) Viral DNA copies were enumerated by qPCR using a primer pair specific for HCMV UL69 and are shown normalized to copies of cellular 18S rDNA loci. Results are shown for samples collected at 2 h post infection (hpi) and 96 hpi to accurately indicate input levels of viral genomes against replicated viral DNA. (D-E) Protein lysates were obtained at the indicated times post infection and analyzed by Western blot to validate knockdown of FOXO3a or FOXO1, respectively, and to assess for effects on viral protein expression. Cellular GAPDH protein was detected as a loading control. NTC: non-targeting control siRNA.

Article Snippet: In , the same staining procedure was used except the ARPE-19 cells were not infected, and anti-HA antibody (Bethyl) and Alexa488 conjugated goat anti rabbit secondary antibody were used to detect FOXO3a-TM-ER by virtue of its 3× HA epitope tag.

Techniques: Transfection, Infection, Control, Western Blot, Knockdown, Expressing

Journal: bioRxiv

Article Title: The AKT Forkhead box O transcription factor axis regulates human cytomegalovirus replication

doi: 10.1101/2022.04.14.488435

Figure Lengend Snippet: (A) Cartoon depiction (created with Biorender) of chimeric FOXO3a “Triple AKT phosphoacceptor site Mutant” (TM), FOXO3a-TM-ER, in which FOXO3a-TM is fused to a mutant mouse estrogen receptor (ER) in which the anti-estrogen 4-hydroxy-tamoxifen (4-OHT) activates its nuclear localization. Therefore, the FOXO3a-TM-ER protein allows FOXO3a activity to be decoupled from AKT regulation. (B) Western blot analysis of ARPE-19 epithelial cells carrying a doxycycline-inducible myr-AKT expression cassette (HA-tagged) and constitutively expressing FOXO3a-TM-ER, also HA-tagged. (C) Confocal immunofluorescence microscopy was used to validate 4-OHT induced FOXO3a-TM-ER nuclear localization upon addition of 1 µM 4-OHT for 1 h. HA staining was used to detect the FOXO3a-TM-ER fusion protein; Hoesch 33342 signal is shown to counterstain nuclei the merged image. (D) One-step viral replication kinetics at MOI 2 for HCMV strains TB40/E and AD169 repaired for UL131 (AD169 r UL131 ) in ARPE-19 cells induced for myr-AKT expression (100 ng/mL doxycycline, ‘Dox’) and with or without 4-OHT treatment (1 µM) to induce nuclear localization of FOXO3a-TM-ER, as indicated. (E) Results for viral replication yield defect at day 8 post infection from 3 independent biological replicates of strain AD169 r UL131 in ARPE-19, asterisk indicates p=0.0457 by paired T-test. (F) Viral DNA synthesis results for the same time points shown in ( D ), comparing qPCR detected copies of the HCMV UL69 gene normalized to detection of cellular 18S rDNA copies. Note, the legend shown atop panel D applies to both D and F; dpi: days post infection. (G) Western blot analysis of viral protein expression following AD169 r UL131 infection (MOI 2 TCID50/mL) in ARPE-19 cells in the presence or absence of Dox and/or 4-OHT treatments, as described above.

Article Snippet: In , the same staining procedure was used except the ARPE-19 cells were not infected, and anti-HA antibody (Bethyl) and Alexa488 conjugated goat anti rabbit secondary antibody were used to detect FOXO3a-TM-ER by virtue of its 3× HA epitope tag.

Techniques: Mutagenesis, Activity Assay, Western Blot, Expressing, Immunofluorescence, Microscopy, Staining, Infection, DNA Synthesis

Journal: Frontiers in Immunology

Article Title: FOXO3 Transcription Factor Regulates IL-10 Expression in Mycobacteria-Infected Macrophages, Tuning Their Polarization and the Subsequent Adaptive Immune Response

doi: 10.3389/fimmu.2019.02922

Figure Lengend Snippet: The PI3K/Akt axis regulates BCG-induced IL-10 secretion in macrophages. Human TDMs and mice J774A.1 macrophages were mock-treated or infected with BCG at an MOI = 10 for 3 h, washed then treated or not with the PI3K inhibitor, Wortmannin (Wort, 100 nM), or with the Akt inhibitor, MK-2206 (5 μM) for 24 h. (A) Inactivation of PI3k/Akt axis was determined, 24 h post infection/treatment, in whole cell lysate of TDMs by western blotting using the indicated anti-phospho specific antibodies. Total Akt, FOXO3, and β-actin were used as loading controls. Shown are representative images of three independent experiments with similar results. (B) FOXO3 mRNA expression were quantified by qRT-PCR in both TDMs and J774A.1 macrophages. Levels of mRNA were normalized to GAPDH and fold induction was calculated relative to uninfected and untreated cells. (C) Assessment of IL-10 transcription and secretion levels, after the indicated treatments, were quantified by qRT-PCR and sandwich ELISA, respectively. For all panels, results are represented in box and whiskers plot format min to max and are representative of three independent experiments, each one carried out in triplicate. Asterisks indicate statistical significance (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: For immunoprecipitation, the sheared chromatin was diluted 10 times, precleared with Protein A/G agarose salmon sperm DNA (Millipore) for 30 min at 4°C, then incubated with rabbit anti-FKHRL1 (FOXO3) sc-11351X, Santa cruz Biotechnology) or an equal amount of rabbit anti-IgG (sc-2027, Santa cruz Biotechnology) at 4°C overnight.

Techniques: Infection, Western Blot, Expressing, Quantitative RT-PCR, Sandwich ELISA

Journal: Frontiers in Immunology

Article Title: FOXO3 Transcription Factor Regulates IL-10 Expression in Mycobacteria-Infected Macrophages, Tuning Their Polarization and the Subsequent Adaptive Immune Response

doi: 10.3389/fimmu.2019.02922

Figure Lengend Snippet: Knockdown of FOXO3 augments IL-10 expression in BCG-infected human primary macrophages. MDMs were transfected with FOXO3 specific siRNA (SiFOXO3) or with scramble control siRNA (siCT) for 48 h and then infected with BCG for 24 h. (A) Fold induction levels of FOXO3 and IL-10 mRNAs were assessed by RT-qPCR in BCG-infected cells. (B) IL-10 secretion by the indicated treated cells was assessed in the culture supernatants by ELISA. Knocking down of FOXO3 expression was further verified by immunoblotting. Data are shown as box and whiskers (min to max values) and are representative of three independent experiments, each one carried out in triplicate ( n = 3, ** p < 0.01).

Article Snippet: For immunoprecipitation, the sheared chromatin was diluted 10 times, precleared with Protein A/G agarose salmon sperm DNA (Millipore) for 30 min at 4°C, then incubated with rabbit anti-FKHRL1 (FOXO3) sc-11351X, Santa cruz Biotechnology) or an equal amount of rabbit anti-IgG (sc-2027, Santa cruz Biotechnology) at 4°C overnight.

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

Journal: Frontiers in Immunology

Article Title: FOXO3 Transcription Factor Regulates IL-10 Expression in Mycobacteria-Infected Macrophages, Tuning Their Polarization and the Subsequent Adaptive Immune Response

doi: 10.3389/fimmu.2019.02922

Figure Lengend Snippet: FOXO3 inhibits IL-10 promoter activity in BCG-infected macrophages. (A) Schematic representation of the four putative Forkhead DNA-binding elements (FKHR-DBEs) in the proximal region of the human IL-10 promoter (-588), identified by Genomatix algorithm. The sequence in red is a typical FOXO3 binding motif. (B) RAW 264.7 cells were co-transfected with pGL3 plasmid containing the −588 bp IL-10 promoter region fragments plus either the GFP or GFP-FOXO3-TM-encoding vectors. Twenty-four hours later, cells were infected or not with BCG (MOI ~10) for 6 h before measuring the Luciferase activity in GFP positive alive cells. The data correspond to the mean ± SEM and correspond to a one representative of two independent experiments, each one conducted in triplicates ( n = 2, ** p < 0.01).

Article Snippet: For immunoprecipitation, the sheared chromatin was diluted 10 times, precleared with Protein A/G agarose salmon sperm DNA (Millipore) for 30 min at 4°C, then incubated with rabbit anti-FKHRL1 (FOXO3) sc-11351X, Santa cruz Biotechnology) or an equal amount of rabbit anti-IgG (sc-2027, Santa cruz Biotechnology) at 4°C overnight.

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

Journal: Frontiers in Immunology

Article Title: FOXO3 Transcription Factor Regulates IL-10 Expression in Mycobacteria-Infected Macrophages, Tuning Their Polarization and the Subsequent Adaptive Immune Response

doi: 10.3389/fimmu.2019.02922

Figure Lengend Snippet: FOXO3 binds to IL-10 promoter, inhibiting its transcriptional activity in BCG-infected cells. In order to determine the contribution of each Forkhead DNA-binding element identified in silico , four mutants of IL-10 promoter were generated as indicated in (A) . (B) representation of gene reporter assay performed on the wild type IL-10 promoter and the four mutants (Mut1, Mut2, Mut3, Mut4). RAW 264.7 macrophages were transfected with the indicated constructs. Twenty-four hours later, cells were infected or not with BCG and luciferase activity was measured 6 h later in GFP positive alive cells. The data correspond to the mean ± SEM of three different experiments conducted in triplicates. Asterisks indicate statistical significance ( n = 3, ** p < 0.01, *** p < 001). (C) TDMs were infected with BCG for 3 h and treated with Wort for additional 2 h. Next, CHIP assay was conducted to verify FOXO3 recruitment to the site −203 of the IL-10 promoter with either anti-FOXO3 Ab (lane 1 and 2) or anti-IgG isotype Ab (lane 3 and 4). Total input of BCG infected and non-infected cells are shown in lanes 5 and 6.

Article Snippet: For immunoprecipitation, the sheared chromatin was diluted 10 times, precleared with Protein A/G agarose salmon sperm DNA (Millipore) for 30 min at 4°C, then incubated with rabbit anti-FKHRL1 (FOXO3) sc-11351X, Santa cruz Biotechnology) or an equal amount of rabbit anti-IgG (sc-2027, Santa cruz Biotechnology) at 4°C overnight.

Techniques: Activity Assay, Infection, Binding Assay, In Silico, Generated, Reporter Assay, Transfection, Construct, Luciferase

Journal: Frontiers in Immunology

Article Title: FOXO3 Transcription Factor Regulates IL-10 Expression in Mycobacteria-Infected Macrophages, Tuning Their Polarization and the Subsequent Adaptive Immune Response

doi: 10.3389/fimmu.2019.02922

Figure Lengend Snippet: FOXO3 tunes phenotypic maturation of mycobacteria-infected macrophages. (A) Human monocytes were transfected either with SiCT or SiFOXO3 for 48 h followed by PPD stimulation and incubation with the autologous lymphocytes for 4 days. Whole cells were then collected and tested for the expression levels of CD80 and CD86 by qRT-PCR. Data are presented as box and whiskers graphs (min to max) from three independent experiments (i.e., MDMs from 3 different donors), each one carried out in triplicate ( n = 3, ** p < 0.001, *** p < 0.0001). (B) Non-infected or BCG-infected BMDMs were treated or not with MK-2206 (5 μM) for 24 h. The expression levels of the M1 phenotype surface markers CD86, MHC-MHC-II, and MHC-I, in CD11b + cells, were determined by FACS analysis using specific antibodies. FACS analysis was performed on two independent experiments and each sample was run in triplicates ( n = 2, * p < 0.05, ** p < 0.001, *** p < 0.0001). (C) J774A.1 were mock or infected with BCG at an MOI = 10 for 3 h, washed then treated or not with the Akt inhibitor MK (5 μM) for 24 h. The transcript levels of M2 marker, Arg1, was assessed by RT-PCR. Data are presented as box and whiskers (min to max) and are from two separate experiments, each one carried out in triplicate ( n = 2, * p < 0.05). (D) TDMs were infected with BCG at an MOI = 10 for 3 h. Following incubation, cells were treated with Amikacin (200 μg/ml) for 1 h to remove extracellular bacteria. The infected cells were then washed and treated with MK (5 μM) for 4 days. The antibiotic Isonicotinylhydrazide (INH) was used as positive control. Cells were lysed with sterile PBS containing 0.1% triton X100 and 10-fold serial dilutions of lysates were plated on MB7H11 for CFU Enumeration. The Intracellular colony forming units were determined at the indicated time points. UT-D0, untreated at Day 0; UT-D4, untreated at Day 4. Data are shown as box and whiskers (min to max values) and representative of two separate experiments, each one carried out in quadruplets. Asterisks indicate statistical significance ( n = 2, *** p < 0.0001).

Article Snippet: For immunoprecipitation, the sheared chromatin was diluted 10 times, precleared with Protein A/G agarose salmon sperm DNA (Millipore) for 30 min at 4°C, then incubated with rabbit anti-FKHRL1 (FOXO3) sc-11351X, Santa cruz Biotechnology) or an equal amount of rabbit anti-IgG (sc-2027, Santa cruz Biotechnology) at 4°C overnight.

Techniques: Infection, Transfection, Incubation, Expressing, Quantitative RT-PCR, Marker, Reverse Transcription Polymerase Chain Reaction, Bacteria, Positive Control, Sterility

Journal: Frontiers in Immunology

Article Title: FOXO3 Transcription Factor Regulates IL-10 Expression in Mycobacteria-Infected Macrophages, Tuning Their Polarization and the Subsequent Adaptive Immune Response

doi: 10.3389/fimmu.2019.02922

Figure Lengend Snippet: Silencing of FOXO3 in macrophages modulates Th1 immune response against mycobacterial infection. Human monocytes were transfected either with SiCT or SiFOXO3 for 48 h followed by PPD stimulation and incubation with the autologous lymphocytes for 4 days. (A) Cells were then collected and tested for the expression levels of the indicated genes by qRT-PCR. (B) Supernatants were collected and analyzed by ELISA for IFN-γ, TNF-α, IL-10, and IL-6 secretion. The results are shown in box and whiskers (min to max values) from three independent experiments (i.e., MDMs from 3 different donors), each one carried out in triplicate. Asterisks indicate statistical significance (* p < 0.05, ** p < 0.001, *** p < 0.0001).

Article Snippet: For immunoprecipitation, the sheared chromatin was diluted 10 times, precleared with Protein A/G agarose salmon sperm DNA (Millipore) for 30 min at 4°C, then incubated with rabbit anti-FKHRL1 (FOXO3) sc-11351X, Santa cruz Biotechnology) or an equal amount of rabbit anti-IgG (sc-2027, Santa cruz Biotechnology) at 4°C overnight.

Techniques: Infection, Transfection, Incubation, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: FOXO3 Transcription Factor Regulates IL-10 Expression in Mycobacteria-Infected Macrophages, Tuning Their Polarization and the Subsequent Adaptive Immune Response

doi: 10.3389/fimmu.2019.02922

Figure Lengend Snippet: FOXO3, a signaling hub for a protective immune response against mycobacterial infection. In order to compromise host resistance, M.tb predominantly activates the pro-survival pathway PI3K/Akt to inhibit apoptosis and induce IL-10 through FOXO3 inhibition. Activated Akt will then phosphorylate the pro-apoptotic transcription factor FOXO3 at three residues, Thr32, Ser253, and Ser315, leading to its translocation to the cytosol where it is stored as a complex FOXO3/chaperone protein 14-3-3 and/or degraded by the proteasome. However, PI3K/Akt inhibition leads to activation of FOXO3, which will accumulate inside the nucleus and restrict IL-10 secretion through direct binding to its proximal promoter. Inhibition of IL-10 signaling is necessary to restore the anti-mycobacterial capacity of the host and increase the polarization of macrophage toward a proinflammatory M1 phenotype, which is crucial for the shaping of an effective Th1 adaptive response against M.tb pathogenesis. Activation of FOXO3 would also induce the expression of PUMA and NOXA, the two effectors of macrophage apoptosis. Apoptotic bodies charged with mycobacterial antigens will be ingested by the bystander dendritic cells (DC) to better cross-prime T cells. In conclusion, activated FOXO3 in infected macrophages benefit the protection against TB in many ways. (i) Induces apoptosis of infected-macrophages and disrupts the intracellular replication of M.tb within the host. (ii) Inhibits IL-10 and restore the antimicrobial host responses. (iii) Boost the adaptive immune response to M.tb .

Article Snippet: For immunoprecipitation, the sheared chromatin was diluted 10 times, precleared with Protein A/G agarose salmon sperm DNA (Millipore) for 30 min at 4°C, then incubated with rabbit anti-FKHRL1 (FOXO3) sc-11351X, Santa cruz Biotechnology) or an equal amount of rabbit anti-IgG (sc-2027, Santa cruz Biotechnology) at 4°C overnight.

Techniques: Infection, Inhibition, Translocation Assay, Activation Assay, Binding Assay, Expressing

Journal: Genes & Development

Article Title: Initiation and maintenance of pluripotency gene expression in the absence of cohesin

doi: 10.1101/gad.251835.114

Figure Lengend Snippet: Cohesin-deficient ES cells have increased reprogramming ability. ( A ) The fusion between ES cells and somatic cells results in heterokaryons, where the two nuclei remain discrete within a shared cytoplasm. The presence of separate ES cell and somatic nuclei shows that cell division has not yet occurred. After 3–4 d, nuclear fusion occurs and gives rise to proliferating hybrid cells. ( B ) Genetic depletion of Rad21 mRNA ( left ) and protein ( right ) from mES cells. ( Left ) Quantitative RT–PCR analysis of Rad21 mRNA expression in control (black) and Rad21 −/− (red) ES cells after 4-OHT-mediated activation of ERt2Cre for 0 or 24 h (normalized to Ubc and Ywhaz ; mean ± SD; n = 3). ( Right ) Immunoblot analysis showed substantially reduced Rad21 protein expression but no significant induction of the DNA damage response marker γ-H2AX in control or Rad21 −/− ES cells 24 h after ERt2Cre induction. Irradiation of Rad21 WT ES cells served as a positive control for γ-H2AX induction. Tubulin was used as the loading control. ( C ) The cell cycle profile of Rad21 −/− ES cells as assessed by propidium iodide staining for DNA content and flow cytometry was unchanged 24 h after ERt2Cre induction, but later time points saw an accumulation of cells in the G2/M phase of the cell cycle. ( D ) Quantitative RT–PCR was used to monitor the expression of the p53 target genes Cdkn1a (p21), Cdkn2a (p16), and Mdm2 at the indicated times after the 4-OHT-induced deletion of Rad21 in ES cells. Twenty-four hours after ERt2Cre activation, ES control (black) and Rad21 −/− (red) ES cells were either fused with human B (hB) somatic cells to halt their proliferation (fused) or allowed to continue proliferating (unfused) (normalized to Ubc and Ywhaz and shown relative to control ES cells; mean ± SD; n = 3). (*) P < 0.05 ( t -test). ( E ) Experimental outline of heterokaryons between mES cells and hB cells. ES cells used were Rad21 wild type ( Rad21 +/+ ) treated with 4-OHT, Rad21 lox/lox (undeleted Rad21 conditional) treated with ethanol, or Rad21 −/− (ERt2Cre Rad21 lox/lox ) treated with 4-OHT. Quantitative RT–PCR with primers that selectively amplify human transcripts was used to monitor the activation of the key pluripotency-associated factors POU5F1 (OCT4), NANOG , SOX2 , CRIPTO , and REX1 ( top row) and the B-cell-specific genes CD45 , CD37 , and CD19 ( bottom row) on days 1–3 (normalized to GAPDH ; mean ± SD; n = 3). (*) P < 0.05 ( t -test).

Article Snippet: Cells or heterokaryons were plated on poly-L-lysine-coated coverslips (Sigma), fixed with 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.5% Triton X-100 (Sigma) for 5 min, treated with blocking solution (3% goat serum, 0.1% Triton X-100), and incubated with primary antibodies to Rad21 (1:500; ab154769, Abcam), Oct-3/4 (1:200; mouse monoclonal BD611203, BD Biosciences), Nanog (1:200; rabbit polyclonal REC-RCAB0001P, Cosmo Bio), or actin (1:500; mouse monoclonal MAB1501, Millipore) for 1 h at room temperature or overnight at 4°C and with secondary antibodies (Molecular Probes) for 1 h at room temperature.

Techniques: Quantitative RT-PCR, Expressing, Control, Activation Assay, Western Blot, Marker, Irradiation, Positive Control, Staining, Flow Cytometry

Journal: Genes & Development

Article Title: Initiation and maintenance of pluripotency gene expression in the absence of cohesin

doi: 10.1101/gad.251835.114

Figure Lengend Snippet: Acute depletion of Rad21 triggers selective changes in gene expression and chromatin conformation but not a global collapse of pluripotency gene expression. ( A ) Genes related to DNA damage and chromosome missegregation are preferentially induced by prolonged knockdown of cohesin subunits but not by acute cohesin deletion. P -values (log 10 ) are indicated by a color scale from black to red. ( B ) Most pluripotency genes remain expressed normally in ERt2Cre Rad21 lox/lox ES cells 24 h after ERt2Cre induction. Pluripotency genes that do change expression are listed along with the log 2 fold change (F-C). ( C ) Rad21 ChIP ( left ; normalized to input; mean ± SD; n = 3), mRNA expression ( middle ; normalized to Ubc and Ywhaz ; mean ± SD; n = 3), and chromatin conformation ( right ; relative interaction frequency; mean ± SD; n = 3) of Nanog in control (black) and acutely cohesin-depleted (red) ES cells. Differentiating ES cells (green) provide a reference for the state of the Nanog locus during differentiation. ( D ) Analysis of the Lefty1 locus as detailed in C for Nanog . Note the persistence of Lefty1 promoter–enhancer interactions in cohesin-depleted ES cells. ( E ) Analysis of the Klf4 locus as detailed in C for Nanog . Note the persistence of Klf4 promoter–enhancer interactions in cohesin-depleted ES cells.

Article Snippet: Cells or heterokaryons were plated on poly-L-lysine-coated coverslips (Sigma), fixed with 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.5% Triton X-100 (Sigma) for 5 min, treated with blocking solution (3% goat serum, 0.1% Triton X-100), and incubated with primary antibodies to Rad21 (1:500; ab154769, Abcam), Oct-3/4 (1:200; mouse monoclonal BD611203, BD Biosciences), Nanog (1:200; rabbit polyclonal REC-RCAB0001P, Cosmo Bio), or actin (1:500; mouse monoclonal MAB1501, Millipore) for 1 h at room temperature or overnight at 4°C and with secondary antibodies (Molecular Probes) for 1 h at room temperature.

Techniques: Gene Expression, Knockdown, Expressing, Control

Journal: Genes & Development

Article Title: Initiation and maintenance of pluripotency gene expression in the absence of cohesin

doi: 10.1101/gad.251835.114

Figure Lengend Snippet: Myc drives reprogramming of somatic nuclei in ES cell heterokaryons. ( A ) Increased Myc mRNA ( left ) and protein expression ( right ) by cohesin-deficient ES cells shown by quantitative RT–PCR and Western blotting, respectively. Histone H3 (H3) was used as a loading control. Data are representative of three independent experiments. ( B ) Schematic of the fusion of puromycin-resistant hB cells with control mES cells, cohesin-deficient mES cells, or cohesin-deficient mES cells pretreated with Myc inhibitor for 6 h prior to the fusion. ( C ) Pretreatment of cohesin-deficient ES cells with Myc inhibitor compromised the expression of Myc itself by disrupting autoregulation by Myc but did not affect the expression of Rad21 . ( D ) Pharmacological inhibition of Myc restrains the reprogramming potential of cohesin-deficient ES cells. Quantitative RT–PCR was used to monitor the ability of cohesin-deficient ES cells pretreated with Myc inhibitor to induce the expression of POU5F1 (OCT4), NANOG , CRIPTO , and REX1 in heterokaryons with hB cells (normalized to GAPDH ; mean ± SD; n = 5). (*) P < 0.05; (**) P < 0.001 ( t -test). ( E ) Schematic of the fusion of hB cells with control ES cells or ES transduced with Myc expression vector. ( F ) Retroviral expression of Myc mRNA ( left ) and protein ( right ) in mES cells shown by RT–PCR and Western blotting, respectively. Histone H3 (H3) was used as a loading control. Data are representative of five independent experiments. ( G ) Myc expression is sufficient to confer enhanced reprogramming activity to ES cells. Quantitative RT–PCR was used to monitor the ability of control mES cells (IRES-GFP) and mES cells transduced with Myc expression vector (Myc-IRES-GFP) to induce the expression of POU5F1 (OCT4), NANOG , CRIPTO , and REX1 in heterokaryons with hB cells (normalized to GAPDH ; mean ± SD; n = 5). (*) P < 0.05; (**) P < 0.001 ( t -test).

Article Snippet: Cells or heterokaryons were plated on poly-L-lysine-coated coverslips (Sigma), fixed with 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.5% Triton X-100 (Sigma) for 5 min, treated with blocking solution (3% goat serum, 0.1% Triton X-100), and incubated with primary antibodies to Rad21 (1:500; ab154769, Abcam), Oct-3/4 (1:200; mouse monoclonal BD611203, BD Biosciences), Nanog (1:200; rabbit polyclonal REC-RCAB0001P, Cosmo Bio), or actin (1:500; mouse monoclonal MAB1501, Millipore) for 1 h at room temperature or overnight at 4°C and with secondary antibodies (Molecular Probes) for 1 h at room temperature.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Inhibition, Transduction, Plasmid Preparation, Retroviral, Reverse Transcription Polymerase Chain Reaction, Activity Assay

Journal: Genes & Development

Article Title: Initiation and maintenance of pluripotency gene expression in the absence of cohesin

doi: 10.1101/gad.251835.114

Figure Lengend Snippet: Myc drives heterokaryon-mediated reprogramming by promoting DNA replication in somatic nuclei. ( A ) Efficient induction of DNA replication in somatic nuclei fused with cohesin-deficient ES cells. Confocal analysis of DNA replication in heterokaryons between hB cells (encircled) and mES cells (45-min EdU pulse on day 1 after fusion). (Green) EdU; (red) actin; (blue) DAPI. Bar, 10 μm. ( B ) Summary of percentages and numbers (BrdU + /total; 45-min BrdU pulse on day 1 after fusion) of BrdU + hB cells fused with Rad21 wild-type ( Rad21 wt ), undeleted Rad21 lox/lox ( Rad21 conditional), or Rad21 −/− (cohesin-deficient) ES cells from three independent experiments. Representative images of BrdU-positive ( top ) and BrdU-negative ( bottom ) hB-cell nuclei in the indicated heterokaryons (45-min BrdU pulse on day 1 after fusion) are shown. (Green) BrdU; (red) actin; (blue) DAPI. Bar, 10 μm. ( C ) Pharmacological inhibition of Myc restrains the ability of cohesin-deficient ES cells to trigger DNA replication in somatic nuclei. DNA replication in heterokaryons between hB cells and Rad21 wild-type ( Rad21 WT ) ES cells, Rad21 -deleted (cohesin-deficient) ES cells, or Rad21 -deleted ES cells pretreated with Myc inhibitor for 6 h prior to fusion. Myc expression enhances the ability of ES cells to trigger DNA replication in somatic nuclei. DNA replication in heterokaryons between hB cells and wild-type ES cells transduced with control IRES-GFP vector (Control) or Myc -IRES-GFP ( Myc -GFP). Representative images of EdU-positive ( top ) and EdU-negative ( bottom ) hB-cell nuclei in the indicated heterokaryons (45-min EdU pulse on day 1 after fusion) are shown. (Green) EdU; (red) actin; (blue) DAPI. Bar, 10 μm.

Article Snippet: Cells or heterokaryons were plated on poly-L-lysine-coated coverslips (Sigma), fixed with 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.5% Triton X-100 (Sigma) for 5 min, treated with blocking solution (3% goat serum, 0.1% Triton X-100), and incubated with primary antibodies to Rad21 (1:500; ab154769, Abcam), Oct-3/4 (1:200; mouse monoclonal BD611203, BD Biosciences), Nanog (1:200; rabbit polyclonal REC-RCAB0001P, Cosmo Bio), or actin (1:500; mouse monoclonal MAB1501, Millipore) for 1 h at room temperature or overnight at 4°C and with secondary antibodies (Molecular Probes) for 1 h at room temperature.

Techniques: Inhibition, Expressing, Transduction, Control, Plasmid Preparation

Journal: Genes & Development

Article Title: Initiation and maintenance of pluripotency gene expression in the absence of cohesin

doi: 10.1101/gad.251835.114

Figure Lengend Snippet: Heterokaryon-mediated reprogramming requires cohesin expression by somatic cells. ( A ) Genetic depletion of Rad21 mRNA ( left ) and protein ( right ) from nonproliferating thymocytes was achieved by combining developmentally regulated CD4Cre with a conditional Rad21 allele. ( B ) Experimental outline and analysis of reprogramming in heterokaryons between hES cells and control (CD4Cre Rad21 +/lox or CD4Cre Rad21 +/+ ) or cohesin-deficient (CD4Cre Rad21 lox/lox ) mouse thymocytes. ( C ) Quantitative RT–PCR with primers that selectively amplify mouse transcripts was used to monitor the expression of the key pluripotency-associated factors Pou5f1 (Oct4), Nanog , Sox2 , Cripto , and Rex1 ( top row) and the thymocyte-specific genes Cd4 and Cd28 ( bottom row) on days 1–3 (normalized to Gapdh ; mean ± SD; n = 3). (*) P < 0.05 ( t -test). ( D ) Experimental outline of heterokaryons between hES cells and control (Ert2Cre Rad21 +/lox ) or cohesin-deficient (Ert2Cre Rad21 lox/lox ) pre-B cells. ( E ) Genetic depletion of Rad21 mRNA ( left ) and protein ( right ) from mouse pre-B cells. ( F ) Quantitative RT–PCR with primers that selectively amplify mouse transcripts was used to monitor the activation of key pluripotency markers as in C ( top row) and the pre-B cell-specific genes Cd10 , Cd19 , and Pax5 ( bottom row) (normalized to Gapdh ; mean ± SD; n = 3). (*) P < 0.05.

Article Snippet: Cells or heterokaryons were plated on poly-L-lysine-coated coverslips (Sigma), fixed with 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.5% Triton X-100 (Sigma) for 5 min, treated with blocking solution (3% goat serum, 0.1% Triton X-100), and incubated with primary antibodies to Rad21 (1:500; ab154769, Abcam), Oct-3/4 (1:200; mouse monoclonal BD611203, BD Biosciences), Nanog (1:200; rabbit polyclonal REC-RCAB0001P, Cosmo Bio), or actin (1:500; mouse monoclonal MAB1501, Millipore) for 1 h at room temperature or overnight at 4°C and with secondary antibodies (Molecular Probes) for 1 h at room temperature.

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

Journal: Genes & Development

Article Title: Initiation and maintenance of pluripotency gene expression in the absence of cohesin

doi: 10.1101/gad.251835.114

Figure Lengend Snippet: Compromised DNA replication in cohesin-deficient somatic nuclei and rescue by Myc. ( A ) Transfer of ES cell-derived factors to somatic nuclei in heterokaryons. ( Left ) Confocal analysis of control ( top row) and cohesin-deficient (rows 2–4) thymocytes 1 d after fusion with ES cells shows efficient transfer of ES cell-derived pluripotency factors, but not of cohesin, to somatic cell nuclei in experimental heterokaryons. Thymocyte nuclei were distinguished from ES cell nuclei by size and DAPI pattern (as described in Supplemental Fig. 3B) and are outlined. (Blue) DAPI; (green) actin; (red) Rad21, Oct4, or Nanog. Bar, 10 μm. ( Right ) Oct4 and Nanog, but not Rad21, are efficiently transferred from ES cell to somatic nuclei 1 d after fusion. ( B ) Compromised DNA replication in cohesin-deficient pre-B cells fused with mES cells. mES cells were pre-labeled with 10 μM EdU overnight prior to fusion with mouse pre-B cells to discriminate ES cell and somatic nuclei. Heterokaryons were pulsed with BrdU for 45 min at 6 or 24 h after fusion or overnight (o/n), and BrdU labeling was scored in (EdU-negative) pre-B-cell nuclei. Quantitative evaluation shows compromised DNA replication by cohesin-deficient pre-B-cell nuclei in ES cell heterokaryons at all time points analyzed. Data are from five independent experiments. ( C ) DNA replication is compromised in cohesin-deficient thymocytes stimulated to undergo DNA replication in vitro. Results of confocal analysis of activation-induced DNA synthesis (EdU incorporation from two independent experiments) are shown. ( D , left ) Confocal analysis of sequential EdU incorporation patterns during the progression from early (stage I) to late (stage V) S phase in thymocytes stimulated to undergo DNA replication in vitro. (Green) EdU; (blue) DAPI:. Bar, 5 μm. Control ( top ) and cohesin-deficient ( bottom ) thymocytes show comparable patterns of EdU incorporation, indicating that the global organization of DNA replication is preserved in cohesin-deficient thymocytes (no stage V nuclei were observed for cohesin-deficient thymocytes). ( Right ) Cumulative frequencies of S-phase stages in control and cohesin-deficient thymocytes. ( E ) Myc rescues impaired DNA replication in cohesin-deficient somatic cells. Efficient induction of DNA replication in cohesin-deficient somatic nuclei fused with Myc -expressing ES cells. Confocal analysis of DNA replication in heterokaryons between control ( Rad21 lox/lox ) or cohesin-deficient ( Rad21 −/− ) mouse thymocytes (encircled) and ZHBTc4 ES cells transduced with either Myc ( Myc- IRES-GFP) or control vector (IRES-GFP; 45-min EdU pulse on day 1 after fusion). (Green) EdU; (red) actin; (blue) DAPI. Bar, 10 μm. ( F ) Numbers and percentages of EdU-positive control ( Rad21 lox/lox ) or cohesin-deficient ( Rad21 −/− ) thymocytes fused with ZHBTc4 ES cells transduced with either Myc ( Myc- IRES-GFP) or control vector (IRES-GFP). Data are from three independent experiments. ( G ) Myc rescues reprogramming of cohesin-deficient somatic cells in ES cell heterokaryons. Expression of thymocyte-derived Pou5f1 in heterokaryons between control ( Rad21 lox/lox ) or cohesin-deficient ( Rad21 −/− ) thymocytes and ZHBTc4 ES cells transduced with either Myc ( Myc- IRES-GFP) or control vector (IRES-GFP; normalized to Gapdh ; mean ± SD; n = 3).

Article Snippet: Cells or heterokaryons were plated on poly-L-lysine-coated coverslips (Sigma), fixed with 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.5% Triton X-100 (Sigma) for 5 min, treated with blocking solution (3% goat serum, 0.1% Triton X-100), and incubated with primary antibodies to Rad21 (1:500; ab154769, Abcam), Oct-3/4 (1:200; mouse monoclonal BD611203, BD Biosciences), Nanog (1:200; rabbit polyclonal REC-RCAB0001P, Cosmo Bio), or actin (1:500; mouse monoclonal MAB1501, Millipore) for 1 h at room temperature or overnight at 4°C and with secondary antibodies (Molecular Probes) for 1 h at room temperature.

Techniques: Derivative Assay, Control, Labeling, In Vitro, Activation Assay, DNA Synthesis, Expressing, Transduction, Plasmid Preparation, Positive Control

Journal: Redox Biology

Article Title: Dietary salt initiates redox signaling between endothelium and vascular smooth muscle through NADPH oxidase 4

doi: 10.1016/j.redox.2022.102296

Figure Lengend Snippet: Western analyses of content of Akt, pAkt, FoxO1, and pFoxO1 in aortic lysates from endothelial Nox4 −/− and littermate mice fed the 0.3% and 4.0% NaCl diets. A , While on the 0.3% NaCl diet, the endothelial Nox4 −/− mice demonstrated less Akt activity (pAkt(S473)) ( P < 0.05) than the littermates. When fed the 4.0% NaCl diet, activation of Akt increased ( P < 0.05) in the littermate controls. There were significant effects of Nox4 ( P < 0.05) and dietary salt ( P < 0.05) on Akt activation and an interaction effect ( P < 0.05) between Nox4 and dietary salt. (n = 4 mice in each group; ns, not significant; * P < 0.05; Two-way ANOVA). B , Activation of FoxO1 (pFoxO1(S256)) and FoxO3a (pFoxO3a(S253)), two downstream phosphorylation targets of Akt [ , ], increased during the high salt intake. There were significant effects of Nox4 ( P < 0.05) and dietary salt ( P < 0.05) on FoxO1 and FoxO3a activation as well as an interaction effect ( P < 0.05) between Nox4 and dietary salt on FoxO1 activation. (n = 4 mice in each group; ns, not significant; * P < 0.05; Two-way ANOVA).

Article Snippet: RNA interference was accomplished using Catalase Human siRNA Oligo Duplex (Locus ID 847, OriGene Technologies Inc., Rockvile, MD), SignalSilence® FoxO1 siRNA I (Cat# 6242, Cell Signaling Technology, Danvers, MA), and SignalSilence® FoxO3a siRNA I (Cat# 6302, Cell Signaling Technology, Danvers, MA); Trilencer-27 universal scrambled negative siRNA (Cat# SR30004, OriGene Technologies Inc.) served as a control in these experiments.

Techniques: Western Blot, Activity Assay, Activation Assay, Phospho-proteomics

Journal: Redox Biology

Article Title: Dietary salt initiates redox signaling between endothelium and vascular smooth muscle through NADPH oxidase 4

doi: 10.1016/j.redox.2022.102296

Figure Lengend Snippet: Inhibition of Akt activation prevented H 2 O 2 -induced increases in Runx2 and decreases in catalase in vascular smooth muscle cells (VSMC). A , Addition of LY294002, a pharmacological inhibitor of phosphatidylinositol 3-kinase (PI3K) , inhibited the activation of Akt during overnight incubation of VSMC in medium containing H 2 O 2 , 0.4 mM. Both H 2 O 2 ( P < 0.05) and LY294002 ( P < 0.05) affected Akt activation and an interaction effect ( P < 0.05) between H 2 O 2 and LY294002 on Akt activation was observed. (n = 4 samples in each group; ns, not significant; * P < 0.05; Two-way ANOVA) B and C , Phosphorylated FoxO1 (pFoxO1(S256)) and FoxO3a (pFoxO3a(S253)), two downstream phosphorylation targets of Akt [ , ], increased during incubation of VSMC in medium containing 0.4 mM H 2 O 2 . The addition of LY294002 inhibited these responses. There were significant effects of H 2 O 2 ( P < 0.05) and LY294002 ( P < 0.05) on FoxO1 and FoxO3a activation as well as an interaction effect ( P < 0.05) between H 2 O 2 and LY294002 on FoxO1 activation (n = 4 samples in each group; ns, not significant; * P < 0.05; Two-way ANOVA). D , Addition of H 2 O 2 , 0.4 mM, into medium increased VSMC Runx2, which was prevented by LY294002. Both H 2 O 2 ( P < 0.05) and LY294002 ( P < 0.05) affected Runx2 levels and an interaction effect ( P < 0.05) between H 2 O 2 and LY294002 on Runx2 levels was observed (n = 4 samples in each group; ns, not significant; * P < 0.05; Two-way ANOVA). E , Addition of H 2 O 2 , 0.4 mM, into medium of VSMC decreased catalase; the addition of LY294002 mitigated this decrease in protein levels. Both H 2 O 2 ( P < 0.05) and LY294002 ( P < 0.05) affected catalase levels in VSMC and an interaction effect ( P < 0.05) between H 2 O 2 and LY294002 on catalase levels was observed (n = 4 samples in each group; ns, not significant; * P < 0.05; Two-way ANOVA). F , Consistent with the western analysis data, H 2 O 2 , 0.4 mM, decreased catalase activity and this effect was mitigated by the addition of LY294002. Both H 2 O 2 ( P < 0.05) and LY294002 ( P < 0.05) affected catalase activity, but there was no interaction effect ( P = 0.1355) between H 2 O 2 and LY294002 (n = 8 samples in each group; * P < 0.05; Two-way ANOVA).

Article Snippet: RNA interference was accomplished using Catalase Human siRNA Oligo Duplex (Locus ID 847, OriGene Technologies Inc., Rockvile, MD), SignalSilence® FoxO1 siRNA I (Cat# 6242, Cell Signaling Technology, Danvers, MA), and SignalSilence® FoxO3a siRNA I (Cat# 6302, Cell Signaling Technology, Danvers, MA); Trilencer-27 universal scrambled negative siRNA (Cat# SR30004, OriGene Technologies Inc.) served as a control in these experiments.

Techniques: Inhibition, Activation Assay, Incubation, Phospho-proteomics, Western Blot, Activity Assay

Journal: Redox Biology

Article Title: Dietary salt initiates redox signaling between endothelium and vascular smooth muscle through NADPH oxidase 4

doi: 10.1016/j.redox.2022.102296

Figure Lengend Snippet: siRNA-mediated knockdown of FoxO1 or FoxO3a increased basal Runx2 levels and H 2 O 2 -induced increases in Runx2 in VSMC. A, Western blot showed successful knockdown of FoxO1 by FoxO1-specific siRNA. Addition of H 2 O 2 into the medium induced the inhibition of FoxO1 (indicated by the increase in pFoxO1(S256)). In these studies, FoxO1 siRNA ( P < 0.05), but not H 2 O 2 ( P = 0.07), affected levels of pFoxO1(S253) in VSMC, but there was an interaction effect ( P < 0.05) between H 2 O 2 and FoxO1 siRNA on pFoxO1(S256). (n = 4 samples in each group; ns, not significant; * P < 0.05; Two-way ANOVA) B, Knockdown of FoxO1 increased basal expression of Runx2 and increased H 2 O 2 -mediated increases in Runx2. H 2 O 2 ( P < 0.05) and FoxO1 siRNA ( P < 0.05) affected levels of Runx2 in VSMC and an interaction effect ( P < 0.05) between H 2 O 2 and FoxO1-specific siRNA on levels of pFoxO1(S256) was observed. (n = 4 samples in each group; * P < 0.05; Two-way ANOVA) C , Western blot showed successful knockdown of FoxO3a by FoxO3a-specific siRNA. Addition of H 2 O 2 into the medium induced the inhibition of FoxO3a (indicated by the increase in pFoxO3a(S253)). In these studies, H 2 O 2 ( P < 0.05) and FoxO3a siRNA ( P < 0.05) affected levels of pFoxO3a(S253) in VSMC and an interaction effect ( P < 0.05) between H 2 O 2 and FoxO3a siRNA on pFoxO3a(S253) was observed (n = 4 samples in each group; ns, not significant; * P < 0.05; Two-way ANOVA). D , Knockdown of FoxO3a increased basal expression of Runx2 and increased H 2 O 2 -mediated increases in Runx2. H 2 O 2 ( P < 0.05) and FoxO3a siRNA ( P < 0.05) affected levels of Runx2 in VSMC, but an interaction effect ( P > 0.05) between H 2 O 2 and FoxO3a siRNA on levels of pFoxO3a(S253) was not observed (n = 4 samples in each group; ns, not significant; * P < 0.05; Two-way ANOVA).

Article Snippet: RNA interference was accomplished using Catalase Human siRNA Oligo Duplex (Locus ID 847, OriGene Technologies Inc., Rockvile, MD), SignalSilence® FoxO1 siRNA I (Cat# 6242, Cell Signaling Technology, Danvers, MA), and SignalSilence® FoxO3a siRNA I (Cat# 6302, Cell Signaling Technology, Danvers, MA); Trilencer-27 universal scrambled negative siRNA (Cat# SR30004, OriGene Technologies Inc.) served as a control in these experiments.

Techniques: Knockdown, Western Blot, Inhibition, Expressing

Journal: Redox Biology

Article Title: Dietary salt initiates redox signaling between endothelium and vascular smooth muscle through NADPH oxidase 4

doi: 10.1016/j.redox.2022.102296

Figure Lengend Snippet: Proposed redox-mediated endothelial cell-aortic smooth muscle cell communication . When fed a diet containing 4.0% NaCl, endothelial activity of NOX4 produced amounts of H 2 O 2 sufficient to generate an endothelial cell-aortic smooth muscle cell communication that directly altered smooth muscle function. Specifically, reduction in PP2a activity promoted Akt activation and downstream inactivation of FoxO1 and FoxO3a. Stimulation of this important signaling pathway increased Runx2 and reduced catalase, which facilitated the activity of H 2 O 2 and increased further Runx2 in aortic smooth muscle.

Article Snippet: RNA interference was accomplished using Catalase Human siRNA Oligo Duplex (Locus ID 847, OriGene Technologies Inc., Rockvile, MD), SignalSilence® FoxO1 siRNA I (Cat# 6242, Cell Signaling Technology, Danvers, MA), and SignalSilence® FoxO3a siRNA I (Cat# 6302, Cell Signaling Technology, Danvers, MA); Trilencer-27 universal scrambled negative siRNA (Cat# SR30004, OriGene Technologies Inc.) served as a control in these experiments.

Techniques: Activity Assay, Produced, Activation Assay

Journal: mSphere

Article Title: B Cell Receptor-Responsive miR-141 Enhances Epstein-Barr Virus Lytic Cycle via FOXO3 Inhibition

doi: 10.1128/mSphere.00093-21

Figure Lengend Snippet: miR-141-3p and EBV miR-BART9-3p regulate 3′ UTRs through common seed interactions. (A) Alignment of miR-141-3p and miR-200a-3p with ebv-miR-BART9-3p. The sequence homology within the seed regions (nt 1 to 7) of these miRNAs is highlighted. (B) miRNA expression vectors (pLCE or BART9 or miR-141) were cotransfected into HEK293T cells with reporters harboring perfect binding sites in the luciferase 3′ UTR for either miR-BART9-3p (BART9i) or miR-141-3p (miR-141i). At 48 h posttransfection, cells were lysed in 1× passive lysis buffer. The luciferase activity was measured using a dual luciferase reporter kit. Values are reported relative to empty reporter in control (pLCE) cells. Shown are the averages from three independent experiments performed in triplicate. *, P < 0.05 (Student t test). RLU, relative light units. (C) Luciferase reporter assays confirm common 3′ UTRs targeted by miR-141 and miR-BART9. HEK293T cells were cotransfected with psiCheck2 vector harboring selected 3′ UTR luciferase reporters and EBV miRNA expression vectors (pLCE-based). At 48 to 72 h posttransfection, cells were lysed and assayed for dual luciferase activity. Shown are the averages from at least three experiments performed in triplicate. *, P < 0.05 (Student t test). RLU, relative light units. The type of seed match between each individual miRNA and 3′ UTR are labeled in different colors. (D and E) Representative immunoblots and quantification of immunoblots for Foxo3a, RanBP9, and Zeb2 in HEK293T cells transfected with miRNA expression vectors. Gapdh is shown as a loading control. Band intensities were quantified using ImageJ, normalized to loading controls, and reported relative to empty vector (pLCE) control cells. Reported are the results of three (RanBP9 and Zeb2) or four (Foxo3a) independent experiments.

Article Snippet: Blots were probed with primary antibodies to Zeb2 (sc-271984; Santa Cruz), Foxo3a (2497S; Cell Signaling), phospho-Foxo3a Ser294 (5538S; Cell Signaling), RanBP9 (17755-1-AP; Proteintech), EBV Ea-D (BMRF1, sc-0261; Santa Cruz), EBV Zebra (BZLF1, sc-53904; Santa Cruz), BHRF1 (biorbyt orb518144), GAPDH (sc-47724; Santa Cruz), or beta-actin (sc-47778; Santa Cruz), followed by horseradish peroxidase-conjugated secondary antibodies (anti-rabbit IgG or anti-mouse IgG).

Techniques: Sequencing, Expressing, Binding Assay, Luciferase, Lysis, Activity Assay, Control, Plasmid Preparation, Labeling, Western Blot, Transfection

Journal: mSphere

Article Title: B Cell Receptor-Responsive miR-141 Enhances Epstein-Barr Virus Lytic Cycle via FOXO3 Inhibition

doi: 10.1128/mSphere.00093-21

Figure Lengend Snippet: miR-141 regulates Foxo3a levels in BL cells. (A and B) TaqMan qRT-PCR analysis of miR-141 in Ramos-iCas9 and Akata-iCas9 confirms knockdown of miR-141. Values are normalized to miR-16 and reported relative to levels of anti-IgM-treated empty gRNA control cells. *, P < 0.05 (Student t test). (C and D) Immunoblot for Foxo3a in Ramos-iCas9 and Akata-iCas9 cells stably transduced with either empty gRNA (Control) or gRNA against miR-141 (miR-141 mt). Gapdh levels are shown as loading controls. Band intensities were quantified using ImageJ, normalized to loading controls, and reported relative to mock-treated empty gRNA control cells. Shown are representative results for one of two independent experiments.

Article Snippet: Blots were probed with primary antibodies to Zeb2 (sc-271984; Santa Cruz), Foxo3a (2497S; Cell Signaling), phospho-Foxo3a Ser294 (5538S; Cell Signaling), RanBP9 (17755-1-AP; Proteintech), EBV Ea-D (BMRF1, sc-0261; Santa Cruz), EBV Zebra (BZLF1, sc-53904; Santa Cruz), BHRF1 (biorbyt orb518144), GAPDH (sc-47724; Santa Cruz), or beta-actin (sc-47778; Santa Cruz), followed by horseradish peroxidase-conjugated secondary antibodies (anti-rabbit IgG or anti-mouse IgG).

Techniques: Quantitative RT-PCR, Knockdown, Control, Western Blot, Stable Transfection, Transduction

Journal: mSphere

Article Title: B Cell Receptor-Responsive miR-141 Enhances Epstein-Barr Virus Lytic Cycle via FOXO3 Inhibition

doi: 10.1128/mSphere.00093-21

Figure Lengend Snippet: Foxo3a was phosphorylated and subjected to degradation in response to BCR cross-linking. (A and C) Time course of Ser294 phospho-Foxo3a and total Foxo3a protein levels in EBV-positive Akata cells treated with anti-IgG (A) or MutuI cells treated with anti-IgM (C). Gapdh levels are shown as loading controls. Shown are representative results of two to three independent experiments. (B and D) The band intensities in panels A and C were quantified using ImageJ, normalized to loading controls, and reported relative to levels at 0 min ( n = 2). (E and F) Time course of Foxo3a protein levels in EBV-positive Akata cells treated with anti-IgG (E) or MutuI cells treated with anti-IgM (F). Significant reduction of total Foxo3a protein occurs at between 24 and 48 h. Shown are representative results of three independent experiments. (G and H) The band intensities in panels E and F were quantified using ImageJ, normalized to loading controls, and reported relative to levels at 0 min ( n = 3).

Article Snippet: Blots were probed with primary antibodies to Zeb2 (sc-271984; Santa Cruz), Foxo3a (2497S; Cell Signaling), phospho-Foxo3a Ser294 (5538S; Cell Signaling), RanBP9 (17755-1-AP; Proteintech), EBV Ea-D (BMRF1, sc-0261; Santa Cruz), EBV Zebra (BZLF1, sc-53904; Santa Cruz), BHRF1 (biorbyt orb518144), GAPDH (sc-47724; Santa Cruz), or beta-actin (sc-47778; Santa Cruz), followed by horseradish peroxidase-conjugated secondary antibodies (anti-rabbit IgG or anti-mouse IgG).

Techniques:

Journal: mSphere

Article Title: B Cell Receptor-Responsive miR-141 Enhances Epstein-Barr Virus Lytic Cycle via FOXO3 Inhibition

doi: 10.1128/mSphere.00093-21

Figure Lengend Snippet: EBV miR-BART9 enhances Foxo3a inhibition. (A) miR-BART9 enhances Foxo3a suppression after surface Ig cross-linking. Akata-EBV Neg cells transduced with pLCE-miR-BART9 were treated with anti-IgG at 5 days postransduction. After 48 h of treatment, lysates were collected and subjected to immunoblotting as described above. Shown are representative results of three independent experiments. (B) TaqMan qRT-PCR analysis of miR-BART9 expression. EBV-negative Akata (Akata-EBV Neg) cells were transduced with pLCE-based miR-BART9 expression vectors. miR-BART9 expression values are normalized to cellular miR-16 and reported relative to levels in MutuI cells treated with anti-IgM for 48 h ( n = 3). (C) Immunoblot of Foxo3a and lytic proteins in mock-treated or anti-IgM-treated MutuI-iCas9 single clones stably transduced with either empty gRNA (Control) or gRNA against miR-BART9 (BART9 mt). Beta-actin levels are shown as loading controls. Shown are the results of one of three representative and independent experiments. (D) TaqMan qRT-PCR analysis of miR-BART9 in MutuI-iCas9 confirms significant knockdown of miR-BART9, while homolog miRNAs miR-141 and miR-200c are not significantly impacted. Values are normalized to miR-16 and reported relative to levels of mock-treated empty gRNA control cells. **, P < 0.01 (Student t test). (E) qRT-PCR analysis of EBV viral gene expression. Expression levels are normalized to GAPDH and reported relative to anti-IgM-treated empty gRNA control cells. Student t tests were performed to compare expression of all eight genes in anti-IgM treated control and BART9 mt show significant differences ( P < 0.01). (F and G) Genomic DNA (F) or supernatant (G) was collected from mock-treated or anti-IgM-treated MutuI-iCas9 single clones stably transduced with either empty gRNA or gRNA against miR-BART9. Viral loads were determined by qPCR assay using primers to the LMP1 region. For cell-associated viral loads (F), values are normalized to GAPDH. For viral loads in supernatant (G), values are determined by LMP1 standard curve. In both cases, values are reported relative to viral levels in anti-IgM-treated empty gRNA control cells. Shown are the averages from three independent experiments. *, P < 0.05; **, P < 0.01 (Student t test).

Article Snippet: Blots were probed with primary antibodies to Zeb2 (sc-271984; Santa Cruz), Foxo3a (2497S; Cell Signaling), phospho-Foxo3a Ser294 (5538S; Cell Signaling), RanBP9 (17755-1-AP; Proteintech), EBV Ea-D (BMRF1, sc-0261; Santa Cruz), EBV Zebra (BZLF1, sc-53904; Santa Cruz), BHRF1 (biorbyt orb518144), GAPDH (sc-47724; Santa Cruz), or beta-actin (sc-47778; Santa Cruz), followed by horseradish peroxidase-conjugated secondary antibodies (anti-rabbit IgG or anti-mouse IgG).

Techniques: Inhibition, Transduction, Western Blot, Quantitative RT-PCR, Expressing, Clone Assay, Stable Transfection, Control, Knockdown, Gene Expression

Journal: mSphere

Article Title: B Cell Receptor-Responsive miR-141 Enhances Epstein-Barr Virus Lytic Cycle via FOXO3 Inhibition

doi: 10.1128/mSphere.00093-21

Figure Lengend Snippet: Posttranscriptional inhibition of FOXO3 augments the EBV lytic cycle. (A) MutuI cells were stably transduced with empty vector control (pLCE) or shRNA against FOXO3 and treated with anti-IgM for 48 h. Total RNA was collected and FOXO3 expression levels were determined by qRT-PCR. Values are normalized to GAPDH and shown relative to pLCE control cells. Shown are the averages from six independent experiments. *, P < 0.05 (Student t test). (B) MutuI cells stably transduced with vector control (pLCE) or shFOXO3 were treated with anti-IgM for 48 h, and lysates from mock-treated or anti-IgM-treated cells were collected. EBV BMRF1 and Zta proteins were detected by immunoblotting. Gapdh levels are shown as loading controls. Band intensities were quantified using ImageJ, normalized to loading controls, and reported relative to anti-IgM-treated empty vector cells. (C) Genomic DNA was isolated from MutuI cells stably transduced with pLCE or shFOXO3 and assayed by qPCR for viral loads. Values are normalized to GAPDH and reported relative to the levels of 48 h anti-IgM-treated empty vector cells. Shown are the averages from three independent experiments. *, P < 0.05 (Student t test). (D) Akata-tet-Z cells were stably transduced with empty vector control (pLCE) or shRNA against FOXO3 and treated with doxycycline for 48 h. Immunoblots were performed on lysates collected from mock- or doxycycline-treated cells for Foxo3a and BMRF1 lytic gene product. Shown are the results of one of three representative independent experiments. Gapdh levels are shown as loading controls. Band intensities were quantified using ImageJ, normalized to loading controls, and reported relative to mock-treated empty vector cells (Foxo3a) or doxycycline-treated empty vector cells (BMRF1). (E) Quantification of BMRF1 immunoblots. Reported are the averages from three independent experiments. *, P < 0.05 (Student t test). (F) BMRF1 target genes are upregulated upon FOXO3 inhibition. RNA was isolated from Akata-tet-Z cells at 48 h after Zta induction, and gene expression was assayed by qRT-PCR. Values are normalized to GAPDH and reported relative to levels of pLCE empty vector cells. Shown are the averages from five independent experiments. *, P < 0.05 (Student t test). (G) Proposed model of how BCR-responsive miR-141 and EBV miR-BART9 promote progression of the lytic cycle. (Panel 1) BCR-cross-linking induces phosphorylation of Foxo3a and expression of miR-141 and EBV miRNAs. (Panel 2) miR-141 and miR-BART9 suppress common host targets that include FOXO3. Posttranscriptional inhibition of Foxo3a enhances progression of EBV lytic reactivation.

Article Snippet: Blots were probed with primary antibodies to Zeb2 (sc-271984; Santa Cruz), Foxo3a (2497S; Cell Signaling), phospho-Foxo3a Ser294 (5538S; Cell Signaling), RanBP9 (17755-1-AP; Proteintech), EBV Ea-D (BMRF1, sc-0261; Santa Cruz), EBV Zebra (BZLF1, sc-53904; Santa Cruz), BHRF1 (biorbyt orb518144), GAPDH (sc-47724; Santa Cruz), or beta-actin (sc-47778; Santa Cruz), followed by horseradish peroxidase-conjugated secondary antibodies (anti-rabbit IgG or anti-mouse IgG).

Techniques: Inhibition, Stable Transfection, Transduction, Plasmid Preparation, Control, shRNA, Expressing, Quantitative RT-PCR, Western Blot, Isolation, Gene Expression, Phospho-proteomics