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: 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, 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, Infection, Immunocytochemistry, Staining, Confocal Microscopy, Immunostaining, Fractionation, Western Blot, RNA Sequencing Assay

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, 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, Transfection, Immunoprecipitation, Western Blot, Mutagenesis, 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