Journal: Communications Biology

Article Title: Arabidopsis LSH10 transcription factor and OTLD1 histone deubiquitinase interact and transcriptionally regulate the same target genes

doi: 10.1038/s42003-023-04424-x

Figure Lengend Snippet: a Association of LSH10-His6 with the chromatin of the indicated target genes in the lsh10-1/LSH10-His6 plants. lsh10-1/LSH10-His6 , white bars; non-specific background signal obtained in the wild-type plants that do not express the His6 epitope, gray bars. The outside probe is the qChIP probe, depicted in Fig. , and located outside the EMSA probe locations. b Increase in H2B monoubiquitylation of the OSR2, WUS, ABI5 , and ARL promoter chromatin in the lsh10-1 mutant plants. Wild-type plants, dark gray bars; lsh10-1 , light gray bars; lsh10-2 , white bars. The chromatin association of LSH10-His6 and the degree of H2B monoubiquitylation were analyzed by qChIP with probes described in Fig. and listed in Supplementary Data . Error bars represent the SEM of n = 5 or n = 7 biological replicates with 3 technical repeats for each. The individual data points are indicated, and their numerical values are listed in Supplementary Data and Supplementary Data . Differences between mean values assessed by the two-tailed t-test are statistically significant for the p -values * p < 0.05 and ** p < 0.01; P ≥ 0.05 are not statistically significant (ns).

Article Snippet: 5 µl of the crude chromatin extracts were saved for use as an input control, and 100 µl were added into the microwell immobilized with the antibodies (non-specific rabbit IgG Isotype Control (Invitrogen, WB317638, 1 µg) as the negative control, specific rabbit anti-His-tag antibody (GenScript, A00174-40, 2.5 µg) or anti-monoubiquityl-histone H2B (Lys-120) (5546S, Cell Signaling Technology, Inc., 2.5 µg).

Techniques: Mutagenesis, Two Tailed Test

Journal: Communications Biology

Article Title: A maladaptive feedback mechanism between the extracellular matrix and cytoskeleton contributes to hypertrophic cardiomyopathy pathophysiology

doi: 10.1038/s42003-022-04278-9

Figure Lengend Snippet: Immunoblot analysis of L-type calcium channel ( a , b ) and β 1 integrin ( c , d ) protein expression performed on total heart homogenate pooled from groups of 5 pre- (10–15-wk-old) or post-cardiomyopathic (30–50-week-old) cTnI-G203S mice and age-matched wt counterparts. Representative immunoblots probed with L-type calcium channel α 1C subunit (Ca V 1.2, a ) or β 1 integrin ( c ) antibody, then GAPDH monoclonal antibody. Densitometry analysis of Ca V 1.2 ( b ) or β 1 integrin ( d ) protein expression, normalized to associated GAPDH expression. n = number of technical repeats. A Browne-Forsythe and Welch ANOVA ( b ) or Kruskal-Wallis test ( d ) determined statistical significance. Densitometry analysis of relative mTOR expression (calculated as Phospho-mTOR/Total mTOR) performed on cytoplasmic ( e ) and nuclear ( f ) fractions pooled from groups of five pre- or post-cardiomyopathic cTnI-G203S mice and age-matched wt counterparts. β-tubulin and histone H2B antibodies were used as loading controls for cytoplasmic and nuclear fractions respectively. n = number of technical repeats. A Browne-Forsythe and Welch ANOVA ( e ) or Kruskal-Wallis test ( f ) determined statistical significance. g Schematic indicating structural-functional link between the L-type calcium channel, cytoskeletal network, mitochondria, integrin and the extracellular matrix in wt and cTnI-G203S cardiac myocytes. A disrupted cytoskeletal architecture in cTnI-G203S cardiac myocytes may trigger a maladaptive feedback mechanism between increased cytoskeletal and extracellular matrix stiffness, resulting in a hypermetabolic mitochondrial state.

Article Snippet: Histone H2B (D2H6, rabbit mAb, Cell Signaling Technology, 12364): Species: H, M, R, Mk; Applications: WB, IHC, ChiP; Validated by Cell Signaling Technology.

Techniques: Western Blot, Expressing, Functional Assay

Journal: Science Advances

Article Title: ZBTB18 restricts chromatin accessibility and prevents transcriptional adaptations that drive metastasis

doi: 10.1126/sciadv.abq3951

Figure Lengend Snippet: ( A ) PCA of RNA-seq data indicates that E0771GFP, M11GFP, and M12GFP have distinct gene expression patterns ( n = 3 biological replicates per cell line). ( B ) Differential gene expression for M11GFP versus M12GFP and M11GFP versus E0771GFP. Only genes with significant differential expression ( P < 0.05) for both comparisons are shown. ( C ) Numbers of transcription factors with increased target gene expression ( P < 0.05) when comparing RNA-seq data obtained from E0771GFP, M11GFP, and M12GFP cell lines, as determined using GSEA analysis with the MSigDB C3 TFT (transcription factor target) collection. Genes with an FPKM of >1 were included in this analysis. ( D ) GSEA enrichment plot for ZBTB18 target gene expression in M11GFP cells compared with E0771GFP cells (left) and M12GFP cells (right). NES, normalized enrichment score. ( E ) Relative Zbtb18 mRNA levels in E0771GFP versus M11GFP (left; n = 3 biological replicates) and 67NR versus 4T1 (right; n = 4 biological replicates) cells, as determined by RT-qPCR. ( F ) ZBTB18 protein levels, detected by immunoblotting. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as loading control. Two biological replicates are shown. Right: Relative ZBTB18 expression levels calculated after normalization with GAPDH ( n = 4 biological replicates). ( G and H ) Relative expression levels of selected ZBTB18 target genes in E0771GFP and M11GFP (G) ( n = 3 biological replicates) or 67NR and 4T1 (H) ( n = 4 biological replicates) cells, as determined by RT-qPCR. ( I ) ZBTB18 protein expression levels, in whole-cell lysates and nuclear fractions, detected by immunoblotting. Nuclear histone H2B (H2B) and cytoplasmic GAPDH were used as controls. Relative ZBTB18 expression levels calculated after normalization with H2B are shown. ( J ) Immunofluorescence staining for ZBTB18 in E0771GFP and M11GFP cells. Cell nuclei are stained with DAPI (4′,6-diamidino-2-phenylindole). Scale bar, 50 μm. (E to H) Means ± SEM, unpaired two-sided t test. ns P > 0.05, * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: The primary antibodies used were anti-ZBTB18 (ZNF238; Proteintech, #12714-1-AP; RRID:AB_2218388; Atlas Antibodies, HPA074019), anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Millipore, #MAB374; RRID:AB_2107445), anti–histone H2B (H2B; Cell Signaling Technology, #12364;; RRID:AB_2714167), anti–phospho-Smad2 (Cell Signaling Technology, #3108, RRID:AB_490941), anti-Smad2/3 (Cell Signaling Technology, #8685; RRID:AB_10889933), and anti-TGFBR2 (Cell Signaling Technology, #79424; RRID:AB_2799933).

Techniques: RNA Sequencing Assay, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: The SAGA HAT module is tethered by its SWIRM domain and modulates activity of the SAGA DUB module

doi: 10.1101/2022.12.29.522244

Figure Lengend Snippet: SAGA’s HAT module regulates SAGA DUB activity independent of enzymatic activity. A: Quantitation of western blots of SAGA DUB activity on recombinant H2B-Ub nucleosomes (400 nM) under conditions of limiting SAGA enzyme (25 nM) with and without the SWIRM domain. B: Quantitation of western blots of SAGA DUB activity on HeLa nucleosomes with ~50 nM H2B ubiquitinated nucleosome. SAGA DUB activity is compared with and without the SWIRM domain, with excess SAGA, 100 nM. C: WT SAGA DUB (25 nM) activity on recombinant H2B-Ub nucleosomes (400 nM) in absence and presence of acetyl CoA (10 μ M). D: Quantitation of C and replicate.

Article Snippet: Samples were visualized by western blot with an anti H2B-K120Ub antibody (Cell Signaling #5546).

Techniques: Activity Assay, Quantitation Assay, Western Blot, Recombinant

Journal: PLoS ONE

Article Title: Immunomodulatory Effects of Bone Marrow-Derived Mesenchymal Stem Cells on Pro-Inflammatory Cytokine-Stimulated Human Corneal Epithelial Cells

doi: 10.1371/journal.pone.0101841

Figure Lengend Snippet: Antibodies, conjugates and immunoreagents.

Article Snippet: Rabbit anti-histone H2B , 1∶1000 , WB , Cell Signaling, MA, USA.

Techniques: Purification

Journal: PLoS ONE

Article Title: Immunomodulatory Effects of Bone Marrow-Derived Mesenchymal Stem Cells on Pro-Inflammatory Cytokine-Stimulated Human Corneal Epithelial Cells

doi: 10.1371/journal.pone.0101841

Figure Lengend Snippet: A to C shows FCM analysis of HCE-T cells treated with BMS-345541 (5 µM), a NF-κB pathway inhibitor, significantly inhibited IFN-γ (100 U/ml) and TNF (100 U/ml) induced ICAM-1 (A), HLA-ABC (B), and HLA-DR (C) expression. Data represents mean ± SEM of four separate experiments (n = 4). *: p<0.05, **: p<0.01. D and E (upper panel) show representative bands of western blots for NF-κB p65 (65 kDa) in nuclear extracts (NE, D) and cytoplasmic extracts (CE, E). Histone H2B (15 kDa) and GAPDH (37 kDa) were used as the loading controls for nuclear and cytoplasmic extracts, respectively. Protein was prepared from HCE-T cells with or without IFN-γ/TNF stimulation, or HCE-T/MSC co-cultured with 24 h and 48 h IFN-γ/TNF treatment. The lower panel shows the fold changes of NF-κB within nuclear extracts and cytoplasmic extracts compared with the untreated group, using Histone H2B and GAPDH as loading controls. Data represents mean ± SEM of four separate experiments (n = 4). *: p<0.05. F to I show representative immunocytochemistry images. Untreated HCE-T cells displayed weak NF-κB nuclear and cytoplasmic staining (F); NF-κB expression showed marked nuclear and perinuclear translocation after 24 h IFN-γ/TNF stimulation of HCE-T monocultures (G); NF-κB perinuclear and nuclear translocation decreased at 24 h in IFN-γ/TNF in stimulated HCECs/MSC co-cultures (H); HCE-T monolayer showed no obvious staining with rabbit IgG (I). The “+” or “−“ symbol denotes the presence or absence of the denoted treatment or cell type at the left.

Article Snippet: Rabbit anti-histone H2B , 1∶1000 , WB , Cell Signaling, MA, USA.

Techniques: Expressing, Western Blot, Cell Culture, Immunocytochemistry, Staining, Translocation Assay

Journal: BMC Complementary and Alternative Medicine

Article Title: Danning tablets attenuates α-naphthylisothiocyanate-induced cholestasis by modulating the expression of transporters and metabolic enzymes

doi: 10.1186/1472-6882-14-249

Figure Lengend Snippet: The effects of DNts on mRNA expressions of transporters and metabolic enzymes in the liver of ANIT-induced cholestasis rats. Relative hepatic mRNA levels of (A) basolateral uptake transporters (Oatp1, Ntcp) and basolateral efflux transporters (Mrp3), (B) basolateral efflux transporters (Mrp4, Ostα and Ostβ) (C) canalicular efflux transporters (Mrp2, Mdr2 and Bsep), and (D) bile acid-metabolizing enzymes (Cyp2b1, Ugt1a1 and Sult2a1) were determined by qRT-PCR in liver samples isolated from rats treated with vehicle, DNts, ANIT or DNts + ANIT (n = 3 for each group). Expression was normalized to Gapdh, and each bar represents the mean ± SD of three independent experiments. Significant difference between two groups. * p < 0.05, ** p < 0.01 vs. control group; # p < 0.05, ## p < 0.01 vs. the ANIT-treated group.

Article Snippet: Membranes were blocked in 5% skim milk and incubated with antibodies against Bsep (sc-25571, 1:200, Santa Cruz), Ntcp (#6522-1, 1:1000, epitomics), Oatp1 (LS-C113034-50, 1:1000, LifeSpan BioSciences), Mrp3(ab3375, 1:50, abcam), Mdr2 (SAB2100008, 1:1000, sigma) and Na + /K + -ATPase (#3010, 1:1000, Cell Signaling), respectively.

Techniques: Quantitative RT-PCR, Isolation, Expressing

Journal: BMC Complementary and Alternative Medicine

Article Title: Danning tablets attenuates α-naphthylisothiocyanate-induced cholestasis by modulating the expression of transporters and metabolic enzymes

doi: 10.1186/1472-6882-14-249

Figure Lengend Snippet: The hepatic protein levels of transporters Oatp1, Ntcp, Mrp3, Mdr2 and Bsep were determined by Western blot (n = 3 for each group). Quantification of the protein expressions was performed by densitometric analysis of the blots following normalization to Na + /K + -ATPase expression. Each bar represents the mean ± SD of three independent experiments. Significant difference between two groups. * p < 0.05, ** p < 0.01 vs. control group; # p < 0.05, ## p < 0.01 vs. the ANIT-treated group.

Article Snippet: Membranes were blocked in 5% skim milk and incubated with antibodies against Bsep (sc-25571, 1:200, Santa Cruz), Ntcp (#6522-1, 1:1000, epitomics), Oatp1 (LS-C113034-50, 1:1000, LifeSpan BioSciences), Mrp3(ab3375, 1:50, abcam), Mdr2 (SAB2100008, 1:1000, sigma) and Na + /K + -ATPase (#3010, 1:1000, Cell Signaling), respectively.

Techniques: Western Blot, Expressing