Fig 1: CSC-Like BPLER cells are associated with high HDAC1 and HDAC7 expression and sensitivity to pan-HDAC inhibitors. (a) The pan-HDAC inhibitor TSA preferentially inhibits BPLER proliferation (blue line), compared to HMLER (red line). The results are representative of at least three independent experiments, presented as a percentage of vehicle treated control, the error bars represent standard deviation of the mean (P<0.005). (b) Short-term (24 h) pretreatment with TSA (0.35 μM), preferentially inhibits BPLER sphere formation (3D) in drug-free medium with no effect on 2D proliferation in either BPLER or HMLER. In contrast, pretreatment with Taxol (50 nM) and 5-Fluorouracil (1.0 μM), preferentially inhibit 2D proliferation as compared to 3D sphere formation. The number of viable colonies from triplicate wells were determined by 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (INT) staining. Results are representative of at least three independent experiments presented as percentage of vehicle treated control (P<0.05). BPLER: 2D proliferation (white bars with blue outline) vs 3D sphere formation (blue bars). HMLER: 2D proliferation (white bars with red outline) vs 3D sphere formation (red bars). The error bars represent standard deviation of the mean. (c) BPLER cell lines express higher levels of HDAC1 and HDAC7 proteins compared to matched HMLER lines. Western blot of whole-cell lysates. β-Actin represents loading control. (d) Heatmap of the mRNA expression profile of HDAC1-11 does not reveal any consistent differences between BPLER and HMLER lines (red, increased expression; green, decreased expression). (e) Treatment of BPLER cells for 48 h with TSA (0.35 μM) leads to downregulation of HDAC1, HDAC7, CD44 and CD166 protein expression in BPLER cells. Western blot of whole-cell lysates. β-Actin represents loading control. (f) Double immunofluorescence staining of BPLER cells simultaneously with HDAC7 and CD44 antibodies demonstrate that HDAC7 and CD44 are co-expressed. DAPI (blue), HDAC7 (red) CD44 (green). Scale bar 25 μM.
Fig 2: Expression of HDAC7 and -8 in monoaminergic and neuropeptidergic neurons.The HDAC immunolocalizations (green) were visualized with a neuronal marker (magenta) and Hoechst stain (blue). A: HDAC7 was detected in the nucleus (arrow) of oxytocin neuron, with punctate immunoreactivity (arrowheads) in the PVN. B: HDAC7 was detected in the nuclei (arrows) of vasopressin neurons, with punctate immunoreactivity (arrowheads) in the PVN. C: HDAC7 was detected in the nuclei (arrows) of orexin neurons, with punctate immunoreactivity (arrowheads) in the LHA. D: HDAC7 was detected in the nuclei (arrows) of AgRP neurons, with punctate immunoreactivity (arrowheads) in the ARC. E: HDAC7 immunoreactivity was detected in the nuclei (arrows) of POMC neuron with dendritic structures (open arrows) and puncta in neuropil (arrowheads). F: HDAC7 was detected in the nuclei (arrows) of noradrenaline neurons, with punctate immunoreactivity (arrowheads) in the LC. G: HDAC7 was detected in the cytoplasm (open arrow) and the dendrites (arrowheads) of neurons of the PVN. H: Some HDAC7-immunoreactive puncta were overlapped with PSD95-immunoreactive puncta (open arrows) of the LHA. I: HDAC8 was detected in cells (open arrows) in the PVN, but not in vasopressin neurons (arrows). J: HDAC8 was detected in a cell in the LHA in a pericellular pattern (open arrow), with punctate immunoreactivity (arrowheads), but not in orexin neuron (arrow). K: HDAC8 was not observed in POMC neuron (arrows). HDAC8-immunoreactive puncta (arrowheads) were widely distributed in the ARC. L: HDAC8 was detected in histamine neurons in a pericellular pattern (open arrows), with punctate immunoreactivity (arrowheads) in the TMN. M: HDAC8 was not observed in dopamine neurons (arrows). N: HDAC8 was not observed in serotonin neurons (arrows). HDAC8-positive punctate immunoreactivities (arrowheads) were widely distributed in the DR. O: HDAC8 was not observed in noradrenaline neuron (arrow). HDAC8-positive punctate immunoreactivities (arrowheads) were widely distributed in the LC. P: HDAC8 was detected in the periphery of the cytoplasm (open arrow) and dendrites (arrowheads) of the TMN neuron. Scale bars indicate 10 µm (A–F, I–P) and 5 µm (G,H).
Fig 3: Hdac7 KO bones show increased osteoclasts and bone resorption.(A) TRAP staining reveals increased osteoclasts in trabecular bone at the proximal tibia from KO mice compared to WT controls at 3 months age. Upper row at 10x magnification, scale bar 200 μm. Lower row at 40x magnification, scale bar 50 μm (B) Measurement of C-telopeptide of collagen (CTX) in serum of WT and KO mice at 3 months. Data represent the mean of 7 animals of each genotype. Samples were compared using an unpaired Student’s t-test * p<0.05 vs. WT.
Fig 4: Nuclear-cytoplasmic trafficking of class IIa HDACs in reprogramming. (A) Immunofluorescence for HDAC7 in fibroblasts, iPSCs, and ESCs. Nuclei stained with DAPI are in blue. Scale = 50 μm. (B) Immunoblot for HDAC7 in the cytoplasmic (C) fraction versus the nuclear (N) fraction of fibroblasts, iPSCs, and ESCs. HDAC1 and GAPDH are loading controls for the nuclear fraction and cytoplasmic fractions, respectively. (C) Double immunostaining for HDAC7 or the indicated proteins in fibroblasts transduced with OSKM and HDAC7wt at the indicated time points. Nuclei stained with DAPI are in blue. E-cad = E-cadherin. Scale = 50 μm. (D) Immunoblot for total HDAC7 in the cytoplasmic fraction versus the nuclear fraction of fibroblasts transduced with OSKM and HDAC7wt at different indicated time points. Histone H3 and GAPDH are loading controls for nuclear fraction and cytoplasmic fraction, respectively. (E) Schematic of HDAC7wt and HDAC7sa (top). Immunofluorescence for total HDAC7 of HEK293T cells transduced with HDAC7sa (bottom). (F) Phase contrast and Oct4-GFP+ images of fibroblasts transduced with OSKM and either empty vector (control), HDAC7wt, or HDAC7sa in serum medium + Vc. Scale = 100 μm. (G) Number of GFP+ colonies in fibroblasts transduced with OSKM and either empty vector, HDAC7wt, or HDAC7sa in serum medium (day 18) and serum medium + Vc (day 16). Data is the mean ± standard error of the mean (SEM) (n = 3). The P value was calculated using two-tailed unpaired Student's t-test, **P < 0.01. (also hereafter). (H) Phase contrast and Oct4-GFP+ images of fibroblasts transduced with OSKM and either empty vector, HDAC4wt, or HDAC4sa in serum medium + Vc. Scale = 100 μm. (I) Number of GFP+ colonies in fibroblasts transduced with OSKM and either empty vector, HDAC4wt, or HDAC4sa in serum medium (day 18) and serum medium + Vc (day 16). Data is the mean ± SEM (n = 3).
Fig 5: Nuclear HDAC7 binds to OSKM and recruits NCoR/SMRT co-repressors to derail reprogramming (A) Immunoprecipitation of nuclear extracts from HEK293T cells individually transduced with HA-tagged OCT4, SOX2, KLF4, or c-MYC in combination with FLAG-tagged GFP or HDAC7sa, followed by immunoblot. IP = immunoprecipitation and IB = immunoblot. (B) Immunoprecipitation of nuclear extracts from fibroblasts transduced with OSKM and FLAG-tagged GFP or HDAC7sa at day 8, followed by immunoblot. (C) ChIP of HDAC7 (FLAG) and OCT4 (HA) in fibroblasts transduced with HA-OCT4, SOX2, KLF4, c-MYC, and FLAG-HDAC7sa at day 9, followed by qPCR analysis for the indicated pluripotency genes. Data is the mean ± SEM (n = 4). (D) Schematic depicting HDAC7sa-Del and HDAC7sa-H717F. (E) Immunoprecipitation of nuclear extracts from HEK293T cells transduced with HA-tagged HDAC7sa, HDAC7sa-H717F and HDAC7sa-Del in combination with FLAG-tagged GFP, NCoR or SMRT, followed by immunoblot. (F) Phase contrast and Oct4-GFP+ images of fibroblasts transduced with OSKM and HDAC7sa, HDAC7sa-Del, or HDAC7sa-H717F in serum medium + Vc. Scale = 100 μm. (G) Number of GFP+ colonies in fibroblasts transduced with OSKM and HDAC7wt, HDAC7sa, HDAC7sa-Del, or HDAC7sa-H717F in serum medium (day 18) and serum medium + Vc (day 16). Data is the mean ± SEM (n = 3). (H) Number of GFP+ colonies in fibroblasts transduced with OSKM and HDAC7sa, together with shRNAs against Luciferase (shLuc), Ncor1 (shNcor1), or Ncor2 (shNcor2) in serum medium (day 18) and serum medium + Vc (day 16). Data is the mean ± SEM (n = 3) (I) Number of GFP+ colonies in fibroblasts transduced with HDAC7sa and the indicated variants of OSKM in serum medium (day 18) and serum medium + Vc (day 16). Data is the mean ± SEM (n = 3).
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