Journal: Journal of Biological Chemistry

Article Title: FoxO1 Deacetylation Regulates Thyroid Hormone-induced Transcription of Key Hepatic Gluconeogenic Genes

doi: 10.1074/jbc.m113.504845

Figure Lengend Snippet: FIGURE 3. T3 induced deacetylation of FoxO1 in a SirT1-dependent man- ner.A,T3decreasedacetylationofFoxO1inatime-dependentmanner.TR1- HepG2 cells were cultured with or without T3 (0.1 M) for the indicated time points. Protein was isolated, and Western blotting was performed to observe FoxO1 deacetylation. B, primary mouse hepatocytes were isolated using a standardtwo-stepcollagenaseperfusionmethodandculturedinDMEMcon- taining 10% Dowex-stripped FBS and 1 penicillin/streptomycin with or without T3 (0.1 M) treatment for 16 h. Protein was isolated, and Western blotting was performed to observe FoxO1 deacetylation. C, SirT1 KD signifi- cantly reduced T3-dependent deacetylation of FoxO1. Cells were cultured withcontrolsiRNAorSirT1siRNAfor48hfollowedbyT3treatment(0.1M)for 24 h. Protein was isolated, and Western blotting was done to observe FoxO1 deacetylation. Densitometric values of AcFoxO1 and FoxO1 were normalized with -actin, then the ratio (AcFoxO1/FoxO1) was plotted as relative density (n 3; *, p 0.05). Error bars represent mean S.D.

Article Snippet: B, to overexpress SirT1 in TR 1-HepG2 cells, FLAG-tagged human SirT1 and its deacetylase domain mutant (H363Y), cloned in pECE (Addgene plasmids 1791 and 1792, respectively) were transfected and confirmed at the protein level.

Techniques: Cell Culture, Isolation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: FoxO1 Deacetylation Regulates Thyroid Hormone-induced Transcription of Key Hepatic Gluconeogenic Genes

doi: 10.1074/jbc.m113.504845

Figure Lengend Snippet: FIGURE 4. T3 induced PCK1 and G6PC mRNA expression in a SirT1-depen- dent manner. A, SirT1 was knocked down using three independent siRNAs in TR1-HepG2 cells and confirmed at the protein level. Cells were cultured with controlsiRNAorSirT1siRNAfor48hfollowedbyreplacementofmediumwith T3-depleted medium as indicated under “Experimental Procedures.” Protein was harvested, and SirT1 KD was confirmed by Western blotting. B, to over- expressSirT1inTR1-HepG2cells,FLAG-taggedhumanSirT1anditsdeacety- lase domain mutant (H363Y), cloned in pECE (Addgene plasmids 1791 and 1792, respectively) were transfected and confirmed at the protein level. Cells were treated with empty vector as control or SirT1 vectors for 48 followed by replacement of medium with normal medium as indicated under “Experi- mental Procedures.” Protein was isolated, and SirT1 expression was con- firmed by Western blotting. C, SirT1 deficiency significantly reduced T3-de- pendent increase in PCK1 and G6PC expression. Cells were treated with control siRNA or SirT1 siRNA for 48 h followed by T3 treatment (0.1 M) for 24 h. 48 h after transfection cells were treated with T3 (0.1 M) for 24 h, total mRNAwascollected,andRT-qPCRanalysiswasperformedasindicatedunder “Experimental Procedures.” D, SirT1 overexpression significantly increased T3-induced PCK1 and G6PC expression. After 48 h of transfection cells were treated with T3 (0.1 M) for 24 h, total mRNA was collected, and RT-qPCR analysis was performed as indicated under “Experimental Procedures.” -Ac- tin was used as normalization control (n 3; *, p 0.05). Error bars represent mean S.D.

Article Snippet: B, to overexpress SirT1 in TR 1-HepG2 cells, FLAG-tagged human SirT1 and its deacetylase domain mutant (H363Y), cloned in pECE (Addgene plasmids 1791 and 1792, respectively) were transfected and confirmed at the protein level.

Techniques: Expressing, Cell Culture, Western Blot, Mutagenesis, Clone Assay, Transfection, Plasmid Preparation, Control, Isolation, Over Expression, Quantitative RT-PCR

Journal: Journal of Biological Chemistry

Article Title: FoxO1 Deacetylation Regulates Thyroid Hormone-induced Transcription of Key Hepatic Gluconeogenic Genes

doi: 10.1074/jbc.m113.504845

Figure Lengend Snippet: FIGURE 6. T3 induced FoxO1 recruitment to PCK1 and G6PC promoters in a SirT1-dependent manner. A, ChIP-qPCR analysis revealed that T3 increased FoxO1 recruitment on PCK1 and G6PC promoters at the IRE region (426 to 170 for PCK1 and 237 to 70 for G6PC gene promoter), which is known for FoxO1 binding in relation to gluconeogenesis. SirT1 deficiency significantly reduced T3-dependent binding of FoxO1. Cells were cultured with control siRNA or SirT1 siRNA for 48 h followed by replacement of medium with normal medium as indicated under “Experimental Procedures.” After 72 h of transfection, cells were treated with T3 for 1 h to analyze FoxO1 recruitment. B, ChIP-qPCR analysis of TRE on PCK1 promoter demonstrates that T3 (0.1 M) rapidly increased acetylation of histone H3 and H4 in this region. HDAC activity of SirT1 was also confirmed as evident from increased basal acetylation of histone H3 and H4 during SirT1 KD in TR-HepG2 cells. During SirT1 KD, T3 (0.1 M) further increased acetylation of these histones, which is significant. 2 l of immunoprecipitated DNA (1% input DNA) was used for qPCR analysis as indicated under “Experimental Procedures” (n 3; *, p 0.05). Error bars represent mean S.D.

Article Snippet: B, to overexpress SirT1 in TR 1-HepG2 cells, FLAG-tagged human SirT1 and its deacetylase domain mutant (H363Y), cloned in pECE (Addgene plasmids 1791 and 1792, respectively) were transfected and confirmed at the protein level.

Techniques: ChIP-qPCR, Binding Assay, Cell Culture, Control, Transfection, Activity Assay, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: FoxO1 Deacetylation Regulates Thyroid Hormone-induced Transcription of Key Hepatic Gluconeogenic Genes

doi: 10.1074/jbc.m113.504845

Figure Lengend Snippet: FIGURE 5. T3 increased PCK1, G6PC gene expression, and SirT1-depen- dentdeacetylationofFoxO1inmicewhichalsorequiredTR.A,hypothy- roidism and hyperthyroidism in male C57BL/6 mice (8–10 weeks old) were induced and confirmed as described under “Experimental Procedures.” B, after 14 h of TH injection, liver tissues were harvested, and total mRNA/protein was extracted. RT-qPCR and Western blot analysis were per- formed on EuTH, HypoTH, and HyperTH mice liver tissues as described under “Experimental Procedures.” An increase in PCK1 and G6PC expression in hyperthyroid mouse liver is significantly corroborated by FoxO1 deacetyla- tion.C,Ex527(0.8mg/day/100gofbodyweight,injectedintraperitoneallyfor 3 days) was used to inhibit SirT1 activity in vivo. After the first injection of Ex527, mice were injected subcutaneously with T3 (10 g/kg of body weight/ day for 2 days) along with Ex527. After 3 days, animals were euthanized, and the liver tissues were subjected to Western blot analysis as indicated under “Experimental Procedures.” Ex527-treated hyperthyroid mouse liver tissues showed a significant increase in FoxO1 acetylation. D, inhibition of TH-in- duced FoxO1 deacetylation in TR-null (TR/) mouse liver tissues showed that TR is essential for TH-dependent FoxO1 deacetylation. A description of TR/ mice is as indicated under “Experimental Procedures.” E and F, in vivo FoxO1 KD using hydrodynamic tail vein injection, as indicated under “Exper- imental Procedures,” significantly inhibited induction in PCK1 mRNA in HyperTH mouse livers when compared with EuTH. After TH treatment in FoxO1 KD mice, liver tissues were harvested, and total mRNA/protein was extracted. RT-qPCR and Western blot analysis were performed on EuTH and

Article Snippet: B, to overexpress SirT1 in TR 1-HepG2 cells, FLAG-tagged human SirT1 and its deacetylase domain mutant (H363Y), cloned in pECE (Addgene plasmids 1791 and 1792, respectively) were transfected and confirmed at the protein level.

Techniques: Gene Expression, Injection, Quantitative RT-PCR, Western Blot, Expressing, Activity Assay, In Vivo, Inhibition

Journal: Cell

Article Title: mTORC1 Activator SLC38A9 Is Required to Efflux Essential Amino Acids from Lysosomes and Use Protein as a Nutrient.

doi: 10.1016/j.cell.2017.09.046

Figure Lengend Snippet: Figure 1. A Mutant of SLC38A9 that Does Not Interact with Arginine Cannot Signal Arginine Sufficiency to mTORC1 (A) Schematic depicting domains of SLC38A9 and the location of the I68A and T133W point mutations. Transmembrane segment 1 of SLC38A9 shares sequence similarity with members of the APC superfamily of transporters. F13H10.3 is likely the C. elegans homolog of SLC38A9. (B) The T133W, but not the I68A, mutant of SLC38A9 is deficient in arginine transport in vitro. SDS-PAGE and Coomassie blue staining was used to analyze recombinant proteins purified from HEK293T cells. (C) Interaction of wild-type SLC38A9 and the T133W mutant, but not the Ragulator-Rag-binding mutant I68A or the control protein metap2, with endogenous Ragulator (p18 and p14) and Rag GTPases (RagA and RagC). HEK293T cells were transfected with the indicated cDNAs, and lysates were prepared and subjected to anti-FLAG immunoprecipitation and analyzed by immunoblotting. (D) Loss of SLC38A9 inhibits activation of mTORC1 by arginine, but not leucine. Cells starved of the indicated amino acid for 50 min were stimulated for 10 min with leucine or arginine, and cell lysates analyzed for the specified proteins and phosphorylation states. (E) For arginine to activate mTORC1 signaling, SLC38A9 must be able to interact with both arginine and Rag-Ragulator. Wild-type and SLC38A9-null cells stably expressing the indicated proteins were analyzed as in (D). See also Figure S1.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Glutathione Agarose Pierce 16100 Cholorquine Sigma C6628-25G Experimental Models: Cell Lines HEK293T ATCC CRL-3216 Mia-PaCa Gift from Rushika Perera N/A 8988T Gift from Rushika Perera N/A KP4 Gift from Rushika Perera N/A KRAS G12D/+ P53 / mouse PaCa cells Gift from Matthew G. Vander Heiden N/A HeLa ATCC ATCC CCL-2 Experimental Models: Organisms/Strains Male C57BL/6J mice 6-8 weeks Charles River 027 Oligonucleotides Primer sgSLC38A9 mouse Fwd: ATGCTATGTGTAT AGTCCAT This paper N/A Primer sgSLC38A9 mouse Rev: ATGGACTATACAC ATAGCAT This paper N/A Recombinant DNA pLJM1-FLAG-metap2 This Paper N/A pLJM60-FLAG-SLC38A9 Wang et al., 2015 Addgene 71858 pLJM60-FLAG-SLC38A9 I68A Wang et al., 2015 Addgene 71864 pLJM60-FLAG-SLC38A9 T133W This Paper #101833 pLJM60-FLAG-SLC38A9 delta110 Wang et al., 2015 Addgene 71861 pLJC5-FLAG-SLC38A9 This Paper N/A pLJC5-3XHA-TMEM192 This Paper #102930 pLJC5-2XFLAG-TMEM192 This Paper #102929 pLJC6-3XHA-TMEM192 This Paper N/A pLJC6-2XFLAG-TMEM192 This Paper N/A Software and Algorithms Prism version 6.0.1 GraphPad https://www.graphpad.com Other PD10 gel filtration Column General Electric 17085101 Extruder Set with Holder/Heating Block Avanti 610000

Techniques: Mutagenesis, Sequencing, In Vitro, SDS Page, Staining, Recombinant, Binding Assay, Control, Transfection, Immunoprecipitation, Western Blot, Activation Assay, Phospho-proteomics, Stable Transfection, Expressing

Journal: Cell

Article Title: mTORC1 Activator SLC38A9 Is Required to Efflux Essential Amino Acids from Lysosomes and Use Protein as a Nutrient.

doi: 10.1016/j.cell.2017.09.046

Figure Lengend Snippet: Figure 2. Arginine, at Concentrations Found in Lysosomes, Promotes the Interaction of SLC38A9 with Rag-Ragulator (A) Whole-cell and lysosomal arginine and leucine concentrations. HEK293T cells were starved of the indicated amino acid for 50 min and re-stimulated with it for 10 min. The RPMI condition represents the non-starved state. Whole-cell and lysosomal arginine and leucine concentrations (mM) were measured using the LysoIP method described in the STAR Methods. Bar graphs show mean ± SEM (n = 3). (B) In vitro, arginine promotes the interaction of SLC38A9 with the Rag-Ragulator complex in a dose-dependent manner. Purified HA-GST-RagC/HA-RagB and HA-Ragulator were immobilized on glutathione affinity resin and incubated with FLAG-SLC38A9 in the presence of the indicated concentrations of arginine. HA- GST-Rap2A was used as a control. Proteins captured in the glutathione resin pull-down were analyzed by immunoblotting for the indicated proteins using anti- epitope tag antibodies. (C) Arginine and lysine, but not other amino acids, promote the interaction of SLC38A9 with Rag-Ragulator in vitro. Experiment was performed as in (B), except that all amino acids were at 1 mM. (D) Arginine does not promote the interaction of SLC38A9 T133W with Rag-Ragulator. The experiment was performed as in (B), except that arginine was used at 500 mM. See also Figure S2.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Glutathione Agarose Pierce 16100 Cholorquine Sigma C6628-25G Experimental Models: Cell Lines HEK293T ATCC CRL-3216 Mia-PaCa Gift from Rushika Perera N/A 8988T Gift from Rushika Perera N/A KP4 Gift from Rushika Perera N/A KRAS G12D/+ P53 / mouse PaCa cells Gift from Matthew G. Vander Heiden N/A HeLa ATCC ATCC CCL-2 Experimental Models: Organisms/Strains Male C57BL/6J mice 6-8 weeks Charles River 027 Oligonucleotides Primer sgSLC38A9 mouse Fwd: ATGCTATGTGTAT AGTCCAT This paper N/A Primer sgSLC38A9 mouse Rev: ATGGACTATACAC ATAGCAT This paper N/A Recombinant DNA pLJM1-FLAG-metap2 This Paper N/A pLJM60-FLAG-SLC38A9 Wang et al., 2015 Addgene 71858 pLJM60-FLAG-SLC38A9 I68A Wang et al., 2015 Addgene 71864 pLJM60-FLAG-SLC38A9 T133W This Paper #101833 pLJM60-FLAG-SLC38A9 delta110 Wang et al., 2015 Addgene 71861 pLJC5-FLAG-SLC38A9 This Paper N/A pLJC5-3XHA-TMEM192 This Paper #102930 pLJC5-2XFLAG-TMEM192 This Paper #102929 pLJC6-3XHA-TMEM192 This Paper N/A pLJC6-2XFLAG-TMEM192 This Paper N/A Software and Algorithms Prism version 6.0.1 GraphPad https://www.graphpad.com Other PD10 gel filtration Column General Electric 17085101 Extruder Set with Holder/Heating Block Avanti 610000

Techniques: In Vitro, Incubation, Control, Western Blot

Journal: Cell

Article Title: mTORC1 Activator SLC38A9 Is Required to Efflux Essential Amino Acids from Lysosomes and Use Protein as a Nutrient.

doi: 10.1016/j.cell.2017.09.046

Figure Lengend Snippet: Figure 6. SLC38A9 Is Required for Amino Acids Produced via Autophagy to Activate mTORC1 and to Support Cell Proliferation (A) Loss of ATG7 prevents the autophagy-mediated reactivation of mTORC1 that occurs after long-term leucine deprivation. Wild-type and ATG7-null HEK293T cells were deprived of leucine for either 50 min or the indicated time points and, where specified, re-stimulated for 10 min with leucine. Cell lysates were analyzed by immunoblotting for the total levels and phosphorylation states of the indicated proteins. (B) Loss of SLC38A9 prevents the autophagy-mediated reactivation of mTORC1 that occurs after long-term leucine deprivation. Wild-type or SLC38A9-null HEK293T cells were deprived of leucine for 50 min or the indicated time points and, where indicated, re-stimulated with leucine for 10 min. Cell lysates were analyzed by immunoblotting for the levels and phosphorylation states of the indicated proteins. (C) mTORC1 signaling does not reactivate after long-term leucine deprivation in cells expressing the T133W SLC38A9 mutant. Wild-type or SLC38A9-null HEK293T cells stably expressing the indicated proteins were starved for leucine for 50 min or 8 hr and, where indicated, re-stimulated with leucine for 10 min. Lysates were analyzed as in (A). (D) In cells lacking SLC38A9, lysosomal leucine concentrations do not drop upon starvation for leucine despite its depletion at the whole-cell level. Metabolite profiling of lysosomes from wild-type and SLC38A9-null cells deprived of leucine for the indicated times is shown. Fold changes are relative to concentrations of cells cultured in RPMI. Bar graphs show mean ± SEM (n = 3). See also Figure S6.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Glutathione Agarose Pierce 16100 Cholorquine Sigma C6628-25G Experimental Models: Cell Lines HEK293T ATCC CRL-3216 Mia-PaCa Gift from Rushika Perera N/A 8988T Gift from Rushika Perera N/A KP4 Gift from Rushika Perera N/A KRAS G12D/+ P53 / mouse PaCa cells Gift from Matthew G. Vander Heiden N/A HeLa ATCC ATCC CCL-2 Experimental Models: Organisms/Strains Male C57BL/6J mice 6-8 weeks Charles River 027 Oligonucleotides Primer sgSLC38A9 mouse Fwd: ATGCTATGTGTAT AGTCCAT This paper N/A Primer sgSLC38A9 mouse Rev: ATGGACTATACAC ATAGCAT This paper N/A Recombinant DNA pLJM1-FLAG-metap2 This Paper N/A pLJM60-FLAG-SLC38A9 Wang et al., 2015 Addgene 71858 pLJM60-FLAG-SLC38A9 I68A Wang et al., 2015 Addgene 71864 pLJM60-FLAG-SLC38A9 T133W This Paper #101833 pLJM60-FLAG-SLC38A9 delta110 Wang et al., 2015 Addgene 71861 pLJC5-FLAG-SLC38A9 This Paper N/A pLJC5-3XHA-TMEM192 This Paper #102930 pLJC5-2XFLAG-TMEM192 This Paper #102929 pLJC6-3XHA-TMEM192 This Paper N/A pLJC6-2XFLAG-TMEM192 This Paper N/A Software and Algorithms Prism version 6.0.1 GraphPad https://www.graphpad.com Other PD10 gel filtration Column General Electric 17085101 Extruder Set with Holder/Heating Block Avanti 610000

Techniques: Produced, Western Blot, Phospho-proteomics, Expressing, Mutagenesis, Stable Transfection, Cell Culture

Journal: The FASEB Journal

Article Title: Sirtuin 6 protects against hepatic fibrogenesis by suppressing the YAP and TAZ function

doi: 10.1096/fj.202200522r

Figure Lengend Snippet: FIGURE 9 SIRT6 deacetylates YAP1 at multiple lysine residues. (A) A diagram of human YAP1 domain structure and several known acetylated lysine residues. (B) Real-time PCR analysis of CYR61, CTGF, and ANKRD1 mRNAs in the LX-2 cells transfected with either WT or mutant human YAP1 plasmids in the presence of 5 ng/ml TGF-β1 (n = 3). (C) YAP1 acetylation analysis in LX-2 cells transfected with WT or mutant human YAP1 plasmids together with vector or SIRT6 plasmids in the presence of 5 ng/ml TGF-β1. Data are presented as mean ± SEM. *p < .05, **p < .01, ***p < .001 versus vector control, and #p < .05, ##p < .001, ###p < .001 versus WT YAP.

Article Snippet: Human YAP1 was a gift from Yosef Shaul (Addgene, Cambridge, MA; plasmid # 18881; http:// n2t.net/addge ne:18881; RRID:Addgene_18881).

Techniques: Real-time Polymerase Chain Reaction, Transfection, Mutagenesis, Plasmid Preparation, Control

Journal: Nature Communications

Article Title: A FAK/HDAC5 signaling axis controls osteocyte mechanotransduction

doi: 10.1038/s41467-020-17099-3

Figure Lengend Snippet: a WT, HDAC4-cKO ( HDAC4 flfl ;DMP1-cre ), HDAC5-KO, and H4H5-DKO mice were subjected in vivo cantilever bending of the right tibia. Each mouse underwent a 3-week regimen (3 days/week, 100 cycles/day, 2500 µε peak normal strain), and dynamic histomorphometry was performed on the tibia mid-shaft. Calcein labeling was performed at 2 days and 11 days prior to sacrifice. Exogenous loading significantly increased p.BFR in WT, HDAC4-cKO, and HDAC5 KO compared with contralateral tibiae. No significant p.BFR elevation observed in H4H5-DKO mice compared with contralateral tibiae ( n = 5–9 mice per group). p.SL (periosteal single-labeling surface), p.DL (periosteal double-labeling surface), pMAR (periosteal mineral apposition rate), p.MS (periosteal mineralizing surface), p.BFR (periosteal bone-mineral formation rate), WT (wild-type mice), H5KO (HDAC5 −/− ), H4KO (HDAC4 flfl ;DMP1-cre), DKO (H4H5-DKO, HDAC5 −/− ; HDAC4 fl/fl ;DMP1-cre). b Sclerostin immunohistochemistry (IHC) was performed in WT and H4H5-DKO mice ( n = 3). High-magnification images show representative images of sclerostin-positive and -negative cells in cortical bones. c All transverse sections were counted by ImageJ. Sclerostin-positive cells numbers are normalized by entire osteocyte number. ( n = 3 mice per group) P -values vs control (contralateral tibia). d qRT-PCR analyses from bone marrow-flushed tibias of WT and H4H5-DKO mice. Exogenous loading significantly reduced Sost mRNA expression in WT, but not H4H5-DKO mice. ( n = 4) P -values vs contralateral tibia. e , f Non-phospho (active) β-catenin IHC in WT and H4H5-DKO mice. Exogenous loading increased active β-catenin staining in periosteal cells of WT, but not in H4H5-DKO mice. Each experiment was repeated three times. ( n = 3 mice per group) P -values vs control (contralateral tibia) are shown in the figure. Two-sided unpaired t test was used ( a, c, d, f ). Data are expressed as mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: HDAC5 S259/498A mutant cDNA was obtained from Addgene (plasmid 32216), HDAC5 Y642F construct was synthesized de novo (VectorBuilder).

Techniques: In Vivo, Labeling, Immunohistochemistry, Control, Quantitative RT-PCR, Expressing, Staining

Journal: Nature Communications

Article Title: A FAK/HDAC5 signaling axis controls osteocyte mechanotransduction

doi: 10.1038/s41467-020-17099-3

Figure Lengend Snippet: a WT, HDAC4 KO, HDAC5-KO, and HDAC4/HDAC5 (H4H5) DKO Ocy454 cells were treated plus/minus FFSS for 3 h, followed by RT-qPCR for Sost . FFSS for 3 h reduced Sost expression in WT cells to a greater degree than in H4H5-DKO cells. P -values vs STATIC condition. Absolute SOST expression data are shown in the left panel ( n = 4 cells per group for control and DKO, n = 3 cells per group for H4KO and H5KO). The right panel shows the ratio of SOST comparing FFSS versus static-treatment within each cell line. P -values adjusted for multiple comparisons (versus control) are shown in the right panel. b , c HDAC5-deficient Ocy454 cells infected with FLAG-tagged HDAC5 were subjected to FFSS (3 h) followed by immunocytochemistry staining for HDAC4/5 and then confocal microscopy. Pictures show a representative image of each condition. HDAC5 (green, localization determined by anti-FLAG immunostaining), endogenous HDAC4 (red), and DAPI (blue). The nuclear densitometric intensity of FLAG-HDAC5 and endogenous HDAC4 were measured by ImageJ. P -values vs STATIC condition ( n = 15 cells for STATIC, n = 21 cells for HDAC5 FFSS, n = 29 cells for HDAC4 FFSS). Each experiment was repeated three times. d Ocy454 cells were subjected to FFSS for the indicated times and subjected to subcellular fractionation followed by immunoblotting. The nuclear fraction of endogenous HDAC5 and HDAC4 was quantified by densitometry over time. ( n = 3 biologic cell replicates were performed) P -values for each time point versus 0 min are shown in the right panel. e Volcano plot showing up- and downregulated genes by RNA-seq by FFSS treatment for 3 h in Ocy454 cells. Blue dots represent significantly regulated genes (log2 fold change (FFSS/STATIC) < −1 or >1, FDR < 0.05), black dots represent genes whose expression is not significantly regulated by FFSS. Sost gene was confirmed as a downregulated gene. f Control and HDAC4/5-deficient Ocy454 cells were treated plus/minus FFSS for 3 h followed by RNA-seq. Volcano plots as in e are shown. The majority of FFSS-induced DEGs are not regulated in cells lacking HDAC4/5. g Gene ontology analysis of FFSS-regulated genes showed enrichment of pathways linked to integrin/FAK signaling (matrix cellular components). In this graph, the x axis corresponds to the gene ontology enrichment score (adjusted P -value) for genes differentially expressed in response to FFSS. h Upstream analysis suggests that FAK and PYK2 are potential upstream candidate regulators of the coordinated gene expression changes seen by RNA-seq in response to FFSS. In this graph, the x axis corresponds to the upstream analysis score (adjusted P -value) for genes that were differentially expressed in response to FFSS. Listed in the figure are the FFSS-induced DEGs found in the upstream FAK-dependent “signature”. One-sided a, d and two-sided c unpaired t test, and one-way analysis of variance (ANOVA) followed by Tukey–Kramer post hoc test ( a in the right panel) were used. Data are expressed as mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: HDAC5 S259/498A mutant cDNA was obtained from Addgene (plasmid 32216), HDAC5 Y642F construct was synthesized de novo (VectorBuilder).

Techniques: Quantitative RT-PCR, Expressing, Control, Infection, Immunocytochemistry, Staining, Confocal Microscopy, Immunostaining, Fractionation, Western Blot, RNA Sequencing, Gene Expression

Journal: Nature Communications

Article Title: A FAK/HDAC5 signaling axis controls osteocyte mechanotransduction

doi: 10.1038/s41467-020-17099-3

Figure Lengend Snippet: a Immunoprecipitation by anti-phosphotyrosine (referred to as “p-Y-1000” throughout) antibody. FFSS was performed for 10 min with Ocy454 cells. Phosphotyrosine immunoprecipitation was performed on cell lysates treated as indicated, and protein expression was determined by western blotting. FFSS reduced HDAC4 and HDAC5 with p-Y-1000 immunoprecipitation. b FLAG immunoprecipitation was performed in HDAC5-deficient cells stably expressing FLAG-HDAC5, subjected to FFSS for times as indicated. phospho-HDAC5 was decreased in a time-dependent manner. c HEK293T cells were transfected with FLAG-HDAC4, then treated for one hour as indicated with PF562271 (10 µM) or vanadate (1 mM) followed by phosphotyrosine immunoprecipitation and then immunoblotting. Tyrosine-phosphorylated HDAC4 was increased by vanadate treatment and decreased by FAK inhibitor treatment. P -values adjusted for multiple comparisons vs control are shown. d FAK kinase activity was measured by ADP-Glo kinase assay. E4Y1 (poly(Glu)/poly(Tyr) ratio of 4:1) polypeptides were used as a control substrate of FAK tyrosine kinase. Increased FAK kinase activity was detected when recombinant human HDAC5 protein was used as a substrate. e Recombinant human HDAC5 and E4Y1 (a control substrate) were incubated with ɣ-32P-ATP and FAK tyrosine kinase for 1 h, then separate by SDS-PAGE followed by autoradiography. FAK treatment showed ɣ-32P-ATP-positive band at the expected HDAC5 recombinant protein size. f Kinase assay reactions as in e were separated by SDS-PAGE followed by immunoblotting as indicated. CBB indicates Coomassie Brilliant Blue stain. One-way ANOVA followed by Tukey–Kramer post hoc test was used ( d ). Data are expressed as mean ± SEM. Each experiment was repeated three times ( a–c, e, f ). Source data are provided as a Source Data file.

Article Snippet: HDAC5 S259/498A mutant cDNA was obtained from Addgene (plasmid 32216), HDAC5 Y642F construct was synthesized de novo (VectorBuilder).

Techniques: Immunoprecipitation, Expressing, Western Blot, Stable Transfection, Transfection, Control, Activity Assay, Kinase Assay, Recombinant, Incubation, SDS Page, Autoradiography, Staining

Journal: Nature Communications

Article Title: A FAK/HDAC5 signaling axis controls osteocyte mechanotransduction

doi: 10.1038/s41467-020-17099-3

Figure Lengend Snippet: a , b Recombinant HDAC5 was phosphorylated in vitro by FAK followed by phospho-modification analysis by mass spectrometry. Tyrosine 642 on HDAC5 was phosphorylated in a FAK-dependent manner. Top panel: the total base peak chromatogram of the chymotrypsin digested HDAC5 in the absence of FAK treatment ( a ) or presence of FAK treatment ( b ). Bottom panel: extracted ion chromatogram for the m/z value (788.75) of the phosphorylated peptide KKLFSDAQPLQPLQVY#QAPL (# symbol represents phosphorylation) demonstrating the ability to detect the phosphorylated peptide upon FAK treatment ( b ). c 293T cells were transfected with FLAG-tagged WT and Y642F HDAC5 cDNAs followed by anti-FLAG immunoprecipitation and then elution with FLAG peptide. Eluted protein was then used as a substrate for in vitro kinase assays plus/minus recombinant FAK followed by immunoblotting as indicated. HDAC5 WT, but not Y642F mutant, is recognized by HDAC5 pY642 antibody. Each experiment was repeated three times. d Control and single-cell FAK-knockout cells were treated plus/minus FFSS (10 min) followed by immunoblotting as indicated. HDAC5 Y642 phosphorylation is reduced by FFSS, and dramatically reduced in FAK-mutant cells, as quantified in the bottom panel. ( n = 3 biologic replicates for protein were performed) P -values adjusted for multiple comparisons vs control in STATIC conditions are shown. e HDAC5-deficient Ocy454 cells were reconstituted with FLAG-tagged lentiviral constructs followed by immunoblotting as indicated. f Lentiviral reconstituted HDAC5-deficient cells were subjected to subcellular fractionation followed by immunoblotting as indicated. The percentage of the total FLAG-HDAC5 (WT or Y642F) in the nuclear fraction was measured by densitometry. ( n = 3 biologic replicates for protein were performed) P -values vs HDAC5 WT. g Cells as in f were subjected to anti-FLAG immunocytochemistry. Nuclear FLAG intensity was quantified ( n = 20 cells were analyzed). HDAC5 Y642F shows increased nuclear localization compared to HDAC5 WT grown under identical conditions. h Cells as in f were grown at 37 °C for 14 days. RNA was isolated for RT-qPCR. HDAC5-deficient cells show increased SOST expression which is reduced to a greater extent by HDAC5 Y642F than HDAC5 WT reconstitution. ( n = 4 biologic replicates for RNA for WT + eGFP and H5KO + eGFP) P -values (red) vs WT + eGFP. ( n = 5 biologic replicates for RNA for H5KO + H5WT and H5KO + H5Y642F) P -values (blue) vs H5KO + H5WT. Two-sided unpaired t test ( f , h ) and one-way ANOVA followed by Tukey–Kramer post hoc test ( d ) were used. Data are expressed as mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: HDAC5 S259/498A mutant cDNA was obtained from Addgene (plasmid 32216), HDAC5 Y642F construct was synthesized de novo (VectorBuilder).

Techniques: Recombinant, In Vitro, Modification, Mass Spectrometry, Phospho-proteomics, Transfection, Immunoprecipitation, Western Blot, Mutagenesis, Control, Knock-Out, Construct, Fractionation, Immunocytochemistry, Isolation, Quantitative RT-PCR, Expressing

Journal: Nature Communications

Article Title: A FAK/HDAC5 signaling axis controls osteocyte mechanotransduction

doi: 10.1038/s41467-020-17099-3

Figure Lengend Snippet: a Ocy454 cells were treated with cilengitide (10 µM and 50 µM) or PF562271 (10 µM) for 4 h, followed by RT-qPCR for FFSS-regulated genes as indicated. Both cilengitide and PF562271 regulate expression of FFSS-responsive genes. ( n = 4 biologic replicates for RNA) P -values adjusted for multiple comparisons vs controls are shown. b Cells were treated with cilengitide (50 µM) for the indicated times followed by immunoblotting. c Cells were treated with the indicated doses of cilengitide (1 h) followed by immunoblotting. pHDAC4/5 immunoblotting was performed using an antibody that recognizes HDAC4 pS246 and HDAC5 pS259. d Saos2 cells were treated with cilengitide (100 µM) for 60 min followed by immunoblotting. e WT and HDAC4/5 double-knockout (H4H5-DKO) cells were treated with vehicle or cilengitide (50 µM) for 4 h followed by Sost RT-qPCR. Cilengitide treatment decreased Sost expression in control cells, but not in H4H5-DKO cells. ( n = 4 biologic replicates for RNA) P -values vs control in the left and right panels. Two-sided unpaired t test e and one-way ANOVA followed by Tukey–Kramer post hoc test ( a ) were used. Data are expressed as mean ± SEM. Each experiment was repeated three times ( b – d ). Source data are provided as a Source Data file.

Article Snippet: HDAC5 S259/498A mutant cDNA was obtained from Addgene (plasmid 32216), HDAC5 Y642F construct was synthesized de novo (VectorBuilder).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Double Knockout, Control

Journal: Technology in Cancer Research & Treatment

Article Title: Nipped-B-like Protein Sensitizes Esophageal Squamous Cell Carcinoma Cells to Cisplatin via Upregulation of PUMA

doi: 10.1177/1533033820960726

Figure Lengend Snippet: NIPBL is relevant to the growth of ESCC cells. A, Western blotting analysis of NIPBL expression in ESCC cell lines. GAPDH is shown as loading control. Normal esophageal squamous epithelial tissue from 2 patients, N1 and N2, were used as the control. B, Western blotting analysis of NIPBL expression in COLO-680N cells transfected with the NIPBL overexpressing vector. GAPDH is shown as loading control. C, Relative cell proliferation of COLO-680N with NIPBL overexpression was determined by the MTS assay. Cells were transfected with pEGFP-N1-FLAG vector or NIPBL recombinant vector respectively, and the relative cell proliferation was determined by MTS assay after transfection for 72 h. All experiments were repeated thrice and the representative results are shown. The statistical significance is p < 0.001 (Student’s t -test, *** represents p < 0.001). NIPBL expression in EC9706 cells transfected with NIPBL siRNA was determined by quantitative real-time PCR (D) and western blotting analysis (E). siRNA 1 and siRNA 2 are 2 different NIPBL siRNAs, whereas control is a non-targeting scrambled control siRNA. F, Relative cell proliferation in Eca-109 and EC9706 cells with NIPBL depletion was determined by MTS assay after transfection with NIPBL siRNA for 72 h. All experiments were repeated thrice and the representative results are shown. The statistical significance is p < 0.001 (Student’s t -test, *** represents p < 0.001).

Article Snippet: NIPBL ORF (1-8,094 bp) was cloned into the pEGFP-N1-FLAG vector (Addgene, Watertown, Massachusetts, USA).

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Over Expression, MTS Assay, Recombinant, Real-time Polymerase Chain Reaction