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Cell Signaling Technology Inc phosphorylated form
Effect of lead secosteroid–2-pyrazoline hybrids 3f , 3j , and 3k on signaling pathways in MCF-7 breast cancer cells. Antibodies against androgen receptor (AR), estrogen receptor α (ERα), GREB1, S6K, and its <t>phosphorylated</t> form (p-S6K), Bcl-2, and its phosphorylated form (p-Bcl-2), and α-tubulin were used for immunoblotting.

Effect of lead secosteroid–2-pyrazoline hybrids 3f , 3j , and 3k on signaling pathways in MCF-7 breast cancer cells. Antibodies against androgen receptor (AR), estrogen receptor α (ERα), GREB1, S6K, and its <t>phosphorylated</t> form (p-S6K), Bcl-2, and its phosphorylated form (p-Bcl-2), and α-tubulin were used for immunoblotting.

Phosphorylated Form, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 362 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/anti+hdac7+antibody/Phospho-HDAC4+(Ser632)%2FHDAC5+(Ser661)%2FHDAC7+(Ser486)+Antibody/pmc12731114-277-20-27
Average 94 stars, based on 362 article reviews

phosphorylated form - by Bioz Stars, 2026-10

94/100 stars

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1) Product Images from "Secosteroid–2-Pyrazoline Hybrids: Design, Synthesis, Biological Evaluation and Development of Therapeutic Combinations Against ERα-Positive Breast Cancer Cells"

Article Title: Secosteroid–2-Pyrazoline Hybrids: Design, Synthesis, Biological Evaluation and Development of Therapeutic Combinations Against ERα-Positive Breast Cancer Cells

Journal: Biomedicines

doi: 10.3390/biomedicines13123057

Figure Legend Snippet: Effect of lead secosteroid–2-pyrazoline hybrids 3f , 3j , and 3k on signaling pathways in MCF-7 breast cancer cells. Antibodies against androgen receptor (AR), estrogen receptor α (ERα), GREB1, S6K, and its phosphorylated form (p-S6K), Bcl-2, and its phosphorylated form (p-Bcl-2), and α-tubulin were used for immunoblotting.

Techniques Used: Protein-Protein interactions, Western Blot

Incubation:

Article Title: Activation of Tripartite Motif Containing 63 Expression by Transcription Factor EB and Transcription Factor Binding to Immunoglobulin Heavy Chain Enhancer 3 Is Regulated by Protein Kinase D and Class IIa Histone Deacetylases
Article Snippet: For Western blot analysis, 20 μg protein was separated by 10% SDS polyacrylamide gel electrophoresis and blotted onto polyvinylidene fluoride or nitrocellulose membranes (GE Healthcare, United Kingdom). .. Membranes were incubated with specific primary antibodies: anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, clone 6C5, Millipore, United States, 1:10,000), anti-HDAC4 antibody (Cell Signaling, United Kingdom, 1:1,000), anti-HDAC5 antibody (Cell Signaling, United Kingdom, 1:1,000), anti-HDAC7 antibody (Cell Signaling, United Kingdom, 1:1,000), anti-phospho-HDAC4 (Ser246)/HDAC5 (Ser259)/HDAC7 (Ser155) (Cell Signaling United Kingdom, 1:1,000), anti-phospho-HDAC4 (Ser632)/HDAC5 (Ser661)/HDAC7 (Ser486) (Cell Signaling United Kingdom, 1:1,000), anti-DYKDDDDK (FLAG-tag, Cell Signaling United Kingdom, 1:1,000), and anti-MYC (Millipore, 1:500) and secondary horseradish peroxidase-conjugated antibodies: anti-mouse IgG (Cell Signaling, United Kingdom, 1:3,000), anti-rabbit IgG (Cell Signaling, United Kingdom, 1:20,000), and anti-goat IgG (Abcam, United Kingdom, 1:3,000). .. The signal TM was visualized with Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, United States).

Membrane:

Article Title: Myeloid HDAC7 drives liver inflammation and systemic glucose dysregulation during diet-induced obesity
Article Snippet: Equal amounts of homogenates were boiled and subjected to electrophoresis using pre-cast 4-12% Bis-Tris NuPAGE gels in NuPAGE MOPS SDS running buffer (Invitrogen). .. After separation, proteins were transferred to a nitrocellulose membrane and immunoblotted for endogenous HDAC7 using an anti-HDAC7 antibody (Cell Signaling Technology, 1/1000) followed by an HRP-conjugated goat anti-rabbit IgG (Cell Signaling Technology, 0.026 μg/mL) and Tubulin as a loading control using a fluorescent Rhodamine anti-Tubulin antibody (Bio-Rad, 1/5000). ..

Control:

Image Search Results

Journal: Biomedicines

Article Title: Secosteroid–2-Pyrazoline Hybrids: Design, Synthesis, Biological Evaluation and Development of Therapeutic Combinations Against ERα-Positive Breast Cancer Cells

doi: 10.3390/biomedicines13123057

Figure Lengend Snippet: Effect of lead secosteroid–2-pyrazoline hybrids 3f , 3j , and 3k on signaling pathways in MCF-7 breast cancer cells. Antibodies against androgen receptor (AR), estrogen receptor α (ERα), GREB1, S6K, and its phosphorylated form (p-S6K), Bcl-2, and its phosphorylated form (p-Bcl-2), and α-tubulin were used for immunoblotting.

Article Snippet: Primary antibodies against androgen receptor (AR), estrogen receptor α (ERα), GREB1, S6K, and its phosphorylated form (p-S6K), Bcl-2, and its phosphorylated form (p-Bcl-2), and α-tubulin were employed (Cell Signaling Technology, Danvers, MA, USA). α-Tubulin served as the internal loading control.

Techniques: Protein-Protein interactions, Western Blot

Myeloid Hdac7 overexpression promotes expression of inflammatory genes in the liver. mRNA levels of Hdac7 (A), Ccl2 (B), Il1b (C) and Mmp9 (D) in livers from 9-22 wk old MacHDAC7 and control MacBlue mice fed a chow diet. Data (normalised to Hprt ) represent mean ± SEM from n=12 mice (6 female and 6 male) for each group. Statistical significance was determined by Student’s t -test (ns, not significant; * p <0.05).

Journal: bioRxiv

Article Title: Myeloid HDAC7 drives liver inflammation and systemic glucose dysregulation during diet-induced obesity

doi: 10.64898/2025.12.04.691405

Figure Lengend Snippet: Myeloid Hdac7 overexpression promotes expression of inflammatory genes in the liver. mRNA levels of Hdac7 (A), Ccl2 (B), Il1b (C) and Mmp9 (D) in livers from 9-22 wk old MacHDAC7 and control MacBlue mice fed a chow diet. Data (normalised to Hprt ) represent mean ± SEM from n=12 mice (6 female and 6 male) for each group. Statistical significance was determined by Student’s t -test (ns, not significant; * p <0.05).

Article Snippet: After separation, proteins were transferred to a nitrocellulose membrane and immunoblotted for endogenous HDAC7 using an anti-HDAC7 antibody (Cell Signaling Technology, 1/1000) followed by an HRP-conjugated goat anti-rabbit IgG (Cell Signaling Technology, 0.026 μg/mL) and Tubulin as a loading control using a fluorescent Rhodamine anti-Tubulin antibody (Bio-Rad, 1/5000).

Techniques: Over Expression, Expressing, Control

Effect of myeloid Hdac7 overexpression on body weight gain in mice fed a chow versus HFHCHS diet. 8-10 wk old male MacBlue and MacHDAC7 mice were fed a chow diet (chow, n=6 per group) or a high fat, high cholesterol and high sucrose diet (HFHCHS, n=14-15 per group) for 24 wk. A. Weekly body weight. B. Average weight gain in mice fed a HFHCHS versus chow diet, calculated by substracting the average body weight of mice fed the chow diet from that of mice fed the HFHCHS. Data shown are mean ± SEM of n=6 (chow) or n=14-15 (HFHCHS) mice per group. Statistical analyses were perfomed using linear regression to compare the difference in body weight between MacBlue and MacHDAC7 mice fed on their respective diets or HFHCHS-induced weight gain (** p <0.01; **** p <0.0001). C. Fat and lean mass measured by EchoMRI at wk 23. D-G. Tissue weights of liver, epididymal white adipose tissue (WAT), kidney and spleen at wk 24. Data shown are mean ± SEM of n=6 (chow) or n=14 (HFHCHS) mice and were analysed using repeated measures (C) or ordinary (D-G) two-way ANOVA followed by Sidak’s multiple comparison (ns, not significant; * p <0.05; ** p <0.01; **** p <0.0001).

Journal: bioRxiv

Article Title: Myeloid HDAC7 drives liver inflammation and systemic glucose dysregulation during diet-induced obesity

doi: 10.64898/2025.12.04.691405

Figure Lengend Snippet: Effect of myeloid Hdac7 overexpression on body weight gain in mice fed a chow versus HFHCHS diet. 8-10 wk old male MacBlue and MacHDAC7 mice were fed a chow diet (chow, n=6 per group) or a high fat, high cholesterol and high sucrose diet (HFHCHS, n=14-15 per group) for 24 wk. A. Weekly body weight. B. Average weight gain in mice fed a HFHCHS versus chow diet, calculated by substracting the average body weight of mice fed the chow diet from that of mice fed the HFHCHS. Data shown are mean ± SEM of n=6 (chow) or n=14-15 (HFHCHS) mice per group. Statistical analyses were perfomed using linear regression to compare the difference in body weight between MacBlue and MacHDAC7 mice fed on their respective diets or HFHCHS-induced weight gain (** p <0.01; **** p <0.0001). C. Fat and lean mass measured by EchoMRI at wk 23. D-G. Tissue weights of liver, epididymal white adipose tissue (WAT), kidney and spleen at wk 24. Data shown are mean ± SEM of n=6 (chow) or n=14 (HFHCHS) mice and were analysed using repeated measures (C) or ordinary (D-G) two-way ANOVA followed by Sidak’s multiple comparison (ns, not significant; * p <0.05; ** p <0.01; **** p <0.0001).

Techniques: Over Expression, Comparison

Myeloid Hdac7 overexpression increases fasted blood glucose levels, glucose dysregulation and glycogen metabolism in mice fed a HFHCHS diet. 6 h fasted blood glucose levels of MacBlue and MacHDAC7 mice fed a chow ( A ) or HFHCHS ( C ) diet. Glucose tolerance test (GTT) was performed at wk 12 ( B and D ). Data shown are mean ± SEM of n=5-6 (chow) or n=15 (HFHCHS) mice for each group. AUC, area under curve analysis. E. Liver glycogen quantification. At termination of the study, mice were fasted for 6 h and glycogen levels were quantified in snap-frozen livers. Data (mean ± SEM) represent n=6 (chow) or n=14 (HFHCHS) mice for each group. F. Insulin levels in the circulation. Circulating insulin levels were measured before diet feeding (wk 0), at wk 4 and at end-point, after mice were fasted for 6 h. Data are mean ± SEM of n=5-6 (chow) or n=14 (HFHCHS) mice for each treatment group. Statistical analyses were performed using Student’s t -test ( A-D, F wk 0) or two-way ANOVA followed by Sidak’s multiple comparison ( B, D, E-F ) (ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001).

Journal: bioRxiv

Article Title: Myeloid HDAC7 drives liver inflammation and systemic glucose dysregulation during diet-induced obesity

doi: 10.64898/2025.12.04.691405

Figure Lengend Snippet: Myeloid Hdac7 overexpression increases fasted blood glucose levels, glucose dysregulation and glycogen metabolism in mice fed a HFHCHS diet. 6 h fasted blood glucose levels of MacBlue and MacHDAC7 mice fed a chow ( A ) or HFHCHS ( C ) diet. Glucose tolerance test (GTT) was performed at wk 12 ( B and D ). Data shown are mean ± SEM of n=5-6 (chow) or n=15 (HFHCHS) mice for each group. AUC, area under curve analysis. E. Liver glycogen quantification. At termination of the study, mice were fasted for 6 h and glycogen levels were quantified in snap-frozen livers. Data (mean ± SEM) represent n=6 (chow) or n=14 (HFHCHS) mice for each group. F. Insulin levels in the circulation. Circulating insulin levels were measured before diet feeding (wk 0), at wk 4 and at end-point, after mice were fasted for 6 h. Data are mean ± SEM of n=5-6 (chow) or n=14 (HFHCHS) mice for each treatment group. Statistical analyses were performed using Student’s t -test ( A-D, F wk 0) or two-way ANOVA followed by Sidak’s multiple comparison ( B, D, E-F ) (ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001).

Techniques: Over Expression, Comparison

Effect of myeloid Hdac7 deletion on body weight in mice fed a chow versus HFHCHS diet. 8-10 wk old male Hdac7 fl/fl ( Hdac7 +/+ ) and Hdac7 fl/fl LysM Cre ( Hdac7 -/- ) mice were fed a HFHCHS diet for 36 wk. A. Body weight was measured weekly. B. Fat and lean mass measured by EchoMRI at wk 35. C. Total organ weights for liver, spleen, epididymal white adipose tissue (WAT) and kidney were measured at the termination of the study. Data shown are mean ± SEM of 12 mice from each group. Statistical analysis was performed using linear regression (A), repeated measures two-way ANOVA followed by Sidak’s multiple comparison (B) or Student’s t -test (C) (ns, not significant).

Journal: bioRxiv

Article Title: Myeloid HDAC7 drives liver inflammation and systemic glucose dysregulation during diet-induced obesity

doi: 10.64898/2025.12.04.691405

Figure Lengend Snippet: Effect of myeloid Hdac7 deletion on body weight in mice fed a chow versus HFHCHS diet. 8-10 wk old male Hdac7 fl/fl ( Hdac7 +/+ ) and Hdac7 fl/fl LysM Cre ( Hdac7 -/- ) mice were fed a HFHCHS diet for 36 wk. A. Body weight was measured weekly. B. Fat and lean mass measured by EchoMRI at wk 35. C. Total organ weights for liver, spleen, epididymal white adipose tissue (WAT) and kidney were measured at the termination of the study. Data shown are mean ± SEM of 12 mice from each group. Statistical analysis was performed using linear regression (A), repeated measures two-way ANOVA followed by Sidak’s multiple comparison (B) or Student’s t -test (C) (ns, not significant).

Techniques: Comparison

Myeloid Hdac7 deficiency reduced fasting blood glucose levels in mice fed a HFHCHS diet. 8-10 wk old male Hdac7 +/+ and Hdac7 -/- mice were fed a HFHCHS diet for 36 wk. A. Fasted blood glucose levels were measured at wk 0, 8, 14, 16 and 24. Data are mean ± SEM of n=12 mice for each group and were analysed using Student’s t -test (ns, not significant; * p < 0.05; **** p < 0.0001). Glucose tolerance test (GTT) at wk 14 and wk 32 ( B-C ) and insulin tolerance test (ITT) at wk 19 and wk 34 ( D-E ). Data shown are mean ± SEM of n=12 mice/ group. Statistical analyses were performed using repeated measures two-way ANOVA followed by Sidak’s multiple comparison. Area under curve (AUC) of GTTs and ITTs were analysed using Student’s t -test (ns, not significant; * p < 0.05; ** p < 0.01).

Journal: bioRxiv

Article Title: Myeloid HDAC7 drives liver inflammation and systemic glucose dysregulation during diet-induced obesity

doi: 10.64898/2025.12.04.691405

Figure Lengend Snippet: Myeloid Hdac7 deficiency reduced fasting blood glucose levels in mice fed a HFHCHS diet. 8-10 wk old male Hdac7 +/+ and Hdac7 -/- mice were fed a HFHCHS diet for 36 wk. A. Fasted blood glucose levels were measured at wk 0, 8, 14, 16 and 24. Data are mean ± SEM of n=12 mice for each group and were analysed using Student’s t -test (ns, not significant; * p < 0.05; **** p < 0.0001). Glucose tolerance test (GTT) at wk 14 and wk 32 ( B-C ) and insulin tolerance test (ITT) at wk 19 and wk 34 ( D-E ). Data shown are mean ± SEM of n=12 mice/ group. Statistical analyses were performed using repeated measures two-way ANOVA followed by Sidak’s multiple comparison. Area under curve (AUC) of GTTs and ITTs were analysed using Student’s t -test (ns, not significant; * p < 0.05; ** p < 0.01).

Techniques: Comparison

Myeloid Hdac7 deletion reduces inflammatory responses in macrophages but does not affect hepatic inflammatory gene expression or serum CCL2 in mice fed a HFHCHS diet. A. Bone marrow cells from Hdac7 +/+ and Hdac7 -/- mice on the HFHCHS diet were collected and differentiated in the presence of CSF-1 for 7 days and lysed, after which protein levels of HDAC7 were determined by immunoblotting, with Tubulin as a loading control (n=6 per group). BMMs from a pair of 12 wk old Hdac7 +/+ and Hdac7 -/- mice on a chow diet were used as a positive control. B. Differentiated BMMs from mice of indicated genotypes on a HFHCHS diet were stimulated with LPS (0.5 ng/ml) for 4 h. CCL2 (left) and nigericin-induced IL-1β secretion (right) in culture supernatants were measured by ELISA (left hand side of graphs). BMMs from 12 wk old mice on a chow diet were used as inter-assay control (right hand side of graphs). Relative CCL2 and IL-1β production was normalised to the LPS-inducible production from BMMs of control Hdac7 +/+ mice on the chow diet. Data shown are mean ± SEM of 12 mice for each group and were analysed using repeated measures two-way ANOVA followed by Bonferroni’s correction (* p < 0.05; *** p < 0.001; **** p < 0.0001). C-E. Quantification of inflammatory mediators in the liver and circulation. mRNA levels of Ccl2 (C) and Il1b (D) (relative to Hprt ) were measured from freshly homogenised frozen livers. CCL2 levels in mouse sera was measured by ELISA (E). Data shown are mean ± SEM from 12 mice for each group and were analysed using Student’s t -test (ns, not significant).

Journal: bioRxiv

Article Title: Myeloid HDAC7 drives liver inflammation and systemic glucose dysregulation during diet-induced obesity

doi: 10.64898/2025.12.04.691405

Figure Lengend Snippet: Myeloid Hdac7 deletion reduces inflammatory responses in macrophages but does not affect hepatic inflammatory gene expression or serum CCL2 in mice fed a HFHCHS diet. A. Bone marrow cells from Hdac7 +/+ and Hdac7 -/- mice on the HFHCHS diet were collected and differentiated in the presence of CSF-1 for 7 days and lysed, after which protein levels of HDAC7 were determined by immunoblotting, with Tubulin as a loading control (n=6 per group). BMMs from a pair of 12 wk old Hdac7 +/+ and Hdac7 -/- mice on a chow diet were used as a positive control. B. Differentiated BMMs from mice of indicated genotypes on a HFHCHS diet were stimulated with LPS (0.5 ng/ml) for 4 h. CCL2 (left) and nigericin-induced IL-1β secretion (right) in culture supernatants were measured by ELISA (left hand side of graphs). BMMs from 12 wk old mice on a chow diet were used as inter-assay control (right hand side of graphs). Relative CCL2 and IL-1β production was normalised to the LPS-inducible production from BMMs of control Hdac7 +/+ mice on the chow diet. Data shown are mean ± SEM of 12 mice for each group and were analysed using repeated measures two-way ANOVA followed by Bonferroni’s correction (* p < 0.05; *** p < 0.001; **** p < 0.0001). C-E. Quantification of inflammatory mediators in the liver and circulation. mRNA levels of Ccl2 (C) and Il1b (D) (relative to Hprt ) were measured from freshly homogenised frozen livers. CCL2 levels in mouse sera was measured by ELISA (E). Data shown are mean ± SEM from 12 mice for each group and were analysed using Student’s t -test (ns, not significant).

Techniques: Gene Expression, Western Blot, Control, Positive Control, Enzyme-linked Immunosorbent Assay, Inter Assay

Histological assessment of livers from Hdac7 +/+ and Hdac7 -/- mice. At termination of the study, mice were fasted for 12 h and euthanised. A. Liver pathology was assessed after H&E (left) and Siriud Red staining (right). Arrows indicate microvesicular steatosis (black), macrovesicular steatosis (blue), and a focus of inflammation within the lobule (red), scale bar: 100 μm. B-F. Quantification of lobular inflammation (B), lipid droplet counts (C), steatosis (D), NAFLD activity score (NAS, E) and fibrosis stage (F) as per the NASH clinical research network scoring system after H&E staining. Data shown represent mean ± SEM of 12 mice per group and were analysed using Student’s t -test (B, D-E) or two-way ANOVA followed by Sidak’s multiple comparison (C) (ns, not significant).

Journal: bioRxiv

Article Title: Myeloid HDAC7 drives liver inflammation and systemic glucose dysregulation during diet-induced obesity

doi: 10.64898/2025.12.04.691405

Figure Lengend Snippet: Histological assessment of livers from Hdac7 +/+ and Hdac7 -/- mice. At termination of the study, mice were fasted for 12 h and euthanised. A. Liver pathology was assessed after H&E (left) and Siriud Red staining (right). Arrows indicate microvesicular steatosis (black), macrovesicular steatosis (blue), and a focus of inflammation within the lobule (red), scale bar: 100 μm. B-F. Quantification of lobular inflammation (B), lipid droplet counts (C), steatosis (D), NAFLD activity score (NAS, E) and fibrosis stage (F) as per the NASH clinical research network scoring system after H&E staining. Data shown represent mean ± SEM of 12 mice per group and were analysed using Student’s t -test (B, D-E) or two-way ANOVA followed by Sidak’s multiple comparison (C) (ns, not significant).

Techniques: Staining, Activity Assay, Comparison

Association of HDAC7 mRNA expression with signatures of human CLD. A. mRNA levels of HDAC7 in liver biopsies from patients with early (stages 0-2) versus advanced (stages 3-4) of CLD was determined by RNAseq. Differentially expressed genes were identified using edgeR with TMM normalisation and Benjamini-Hochberg method for fdr correction. Data indicate mean ± SEM, n = 69 (circles = early stages and triangles = advanced stages). B-F. Spatial transcriptomic profiling of liver tissues from MacBlue and MacHDAC7 mice was performed using the 10x Genomics Xenium platform. Expression of eCFP and HDAC7 mRNAs was used to identify hepatic myeloid cells expressing HDAC7 (CFP⁺/HDAC7⁺ cells). B. A heatmap showing the 53 differentially expressed genes in CFP⁺/HDAC7⁺ macrophages from MacBlue versus MacHDAC7 livers. C-F. Spatial co-expression analysis of VCAN ( C ), THBS1 ( D ), MMP9 ( E ) and RAB8B ( F ) in MacBlue and MacHDAC7 livers. Data are representative of two independent tissue samples from litter-matched male MacBlue and MacHDAC7 mice fed a chow diet.

Journal: bioRxiv

Article Title: Myeloid HDAC7 drives liver inflammation and systemic glucose dysregulation during diet-induced obesity

doi: 10.64898/2025.12.04.691405

Figure Lengend Snippet: Association of HDAC7 mRNA expression with signatures of human CLD. A. mRNA levels of HDAC7 in liver biopsies from patients with early (stages 0-2) versus advanced (stages 3-4) of CLD was determined by RNAseq. Differentially expressed genes were identified using edgeR with TMM normalisation and Benjamini-Hochberg method for fdr correction. Data indicate mean ± SEM, n = 69 (circles = early stages and triangles = advanced stages). B-F. Spatial transcriptomic profiling of liver tissues from MacBlue and MacHDAC7 mice was performed using the 10x Genomics Xenium platform. Expression of eCFP and HDAC7 mRNAs was used to identify hepatic myeloid cells expressing HDAC7 (CFP⁺/HDAC7⁺ cells). B. A heatmap showing the 53 differentially expressed genes in CFP⁺/HDAC7⁺ macrophages from MacBlue versus MacHDAC7 livers. C-F. Spatial co-expression analysis of VCAN ( C ), THBS1 ( D ), MMP9 ( E ) and RAB8B ( F ) in MacBlue and MacHDAC7 livers. Data are representative of two independent tissue samples from litter-matched male MacBlue and MacHDAC7 mice fed a chow diet.

Techniques: Expressing

SST m/m mice show higher light sensitivity at dusk in DD condition. ( A ), 200-lx light pulse at CT12 induced larger phase delays in SST m/m mice. Left , Representative double-plotted actograms of SST +/+ ( Upper ) or SST m/m mice ( Lower) illuminated with a light pulse (200 lx, 30 min) at CT12. Yellow boxes indicate the time of light illumination. Right , Amounts of phase delay (mean ± s.e.m; n = 10 ( SST +/+ ) and 9 ( SST m/m ); ** P < 0.01, unpaired t -test). ( B ), Effect of dim light pulse (2 lx, 30 min) at CT12. Left , Representative double-plotted actograms of SST +/+ ( Upper ) or SST m/m mice ( Lower ). Yellow boxes indicate the time of light illumination. Middle, Magnified presentations of the activities shown in left panels. Right, Amounts of phase delay (mean ± s.e.m; n = 10 ( SST +/+ ) and 11 ( SST m/m ); *** P < 0.001, unpaired t -test). ( C) , Dim light pulse (2 lx, 30 min) at CT12 induced higher cFos mRNA expression and ERK phosphorylation in the SCN of SST m/m mice. Expression of cFos mRNA was examined by in situ hybridization using digoxigenin-labelled probe. Phosphorylated ERK was immunohistochemically stained with pERK antibody. Representative micrographs of cFos mRNA expression ( Left panels ) and phosphorylated ERK ( Right panels ). Arrows indicate the cFos mRNA positive cells and phosphorylated ERK-positive cells. Scale bars, 100 μm. ( D ), Confocal microscopy revealed abundant cells (DAPI, blue) in the VL SCN surrounded by both RHT (green) and SST (red) terminals in SST +/+ mice. Note the dense distribution of SST-terminals proximal to the RHT-terminals in the VL SCN of SST +/+ mice, but none in SST m/m mice. Scale bars, 100 μm ( Upper ) and 10 μm ( Lower ). ( E ), Exposure to a dim light pulse (2 lx, 30 min) at CT12 induced cFOS protein expression in SSTR1 -positive cells in the VL SCN of SST m/m mice. Left , Expression of SSTR1 mRNA (blue) and cFOS protein (brown) was examined. The lower panels show a magnified image of the area indicated by the red rectangle displayed in the upper panels. Arrowheads indicate the cells expressing both SSTR1 mRNA and cFOS protein. Right , Ratio of cFOS-positive cells in SSTR1 -positive cells (mean ± s.e.m; n = 4 (both genotypes); ** P < 0.01, unpaired t -test). Scale bars, 100 μm ( Upper ) and 10 μm ( Lower ). ( F ), Electron microscopy revealed that SST neurons innervate cells in the VL SCN. Left , asymmetrical axo-dendritic input (red arrow) from SST-negative axon to SST-positive dendrite. N, nucleus. Right , symmetrical axo-somatic output (innervation: blue arrows) from SST-positive axon to SST-negative soma. Scale bars, 500 nm. ( G ), Increased SSTR1 mRNA expression in the SCN of SST m/m mice. Quantitative RT-PCR analysis of SSTR1 mRNA was performed using SCN tissue samples precisely collected from SST m/m and SST +/+ mice using a laser-microdissection technique. ( H ), CH-275, an SSTR1 agonist, ameliorated the abnormal delay in locomotor activity in SST m/m mice under long photoperiod. Representative double-plotted actograms of vehicle- ( upper ) or CH-275-treated SST m/m mice ( lower ) entrained for more than three weeks in LD18h:6 h and released to DD at ZT12. CH-275 was administered into the SCN of SST m/m mice using an osmotic mini pump. The time of the surgery is indicated by the stars. Amounts of phase advance (mean ± s.e.m; n = 6 (vehicle) and 7 (CH-275); * P < 0.05, unpaired t -test). The phase was determined by fitting a regression line across several days in DD and extrapolating it back to the first day.

Journal: Scientific Reports

Article Title: Somatostatin regulates the clock sensitivity to evening light

doi: 10.1038/s41598-025-26904-2

Figure Lengend Snippet: SST m/m mice show higher light sensitivity at dusk in DD condition. ( A ), 200-lx light pulse at CT12 induced larger phase delays in SST m/m mice. Left , Representative double-plotted actograms of SST +/+ ( Upper ) or SST m/m mice ( Lower) illuminated with a light pulse (200 lx, 30 min) at CT12. Yellow boxes indicate the time of light illumination. Right , Amounts of phase delay (mean ± s.e.m; n = 10 ( SST +/+ ) and 9 ( SST m/m ); ** P < 0.01, unpaired t -test). ( B ), Effect of dim light pulse (2 lx, 30 min) at CT12. Left , Representative double-plotted actograms of SST +/+ ( Upper ) or SST m/m mice ( Lower ). Yellow boxes indicate the time of light illumination. Middle, Magnified presentations of the activities shown in left panels. Right, Amounts of phase delay (mean ± s.e.m; n = 10 ( SST +/+ ) and 11 ( SST m/m ); *** P < 0.001, unpaired t -test). ( C) , Dim light pulse (2 lx, 30 min) at CT12 induced higher cFos mRNA expression and ERK phosphorylation in the SCN of SST m/m mice. Expression of cFos mRNA was examined by in situ hybridization using digoxigenin-labelled probe. Phosphorylated ERK was immunohistochemically stained with pERK antibody. Representative micrographs of cFos mRNA expression ( Left panels ) and phosphorylated ERK ( Right panels ). Arrows indicate the cFos mRNA positive cells and phosphorylated ERK-positive cells. Scale bars, 100 μm. ( D ), Confocal microscopy revealed abundant cells (DAPI, blue) in the VL SCN surrounded by both RHT (green) and SST (red) terminals in SST +/+ mice. Note the dense distribution of SST-terminals proximal to the RHT-terminals in the VL SCN of SST +/+ mice, but none in SST m/m mice. Scale bars, 100 μm ( Upper ) and 10 μm ( Lower ). ( E ), Exposure to a dim light pulse (2 lx, 30 min) at CT12 induced cFOS protein expression in SSTR1 -positive cells in the VL SCN of SST m/m mice. Left , Expression of SSTR1 mRNA (blue) and cFOS protein (brown) was examined. The lower panels show a magnified image of the area indicated by the red rectangle displayed in the upper panels. Arrowheads indicate the cells expressing both SSTR1 mRNA and cFOS protein. Right , Ratio of cFOS-positive cells in SSTR1 -positive cells (mean ± s.e.m; n = 4 (both genotypes); ** P < 0.01, unpaired t -test). Scale bars, 100 μm ( Upper ) and 10 μm ( Lower ). ( F ), Electron microscopy revealed that SST neurons innervate cells in the VL SCN. Left , asymmetrical axo-dendritic input (red arrow) from SST-negative axon to SST-positive dendrite. N, nucleus. Right , symmetrical axo-somatic output (innervation: blue arrows) from SST-positive axon to SST-negative soma. Scale bars, 500 nm. ( G ), Increased SSTR1 mRNA expression in the SCN of SST m/m mice. Quantitative RT-PCR analysis of SSTR1 mRNA was performed using SCN tissue samples precisely collected from SST m/m and SST +/+ mice using a laser-microdissection technique. ( H ), CH-275, an SSTR1 agonist, ameliorated the abnormal delay in locomotor activity in SST m/m mice under long photoperiod. Representative double-plotted actograms of vehicle- ( upper ) or CH-275-treated SST m/m mice ( lower ) entrained for more than three weeks in LD18h:6 h and released to DD at ZT12. CH-275 was administered into the SCN of SST m/m mice using an osmotic mini pump. The time of the surgery is indicated by the stars. Amounts of phase advance (mean ± s.e.m; n = 6 (vehicle) and 7 (CH-275); * P < 0.05, unpaired t -test). The phase was determined by fitting a regression line across several days in DD and extrapolating it back to the first day.

Article Snippet: We used a rabbit polyclonal antibody specific for the phosphorylated forms of ERK 1 and 2 (Cell Signaling; catalog code 9101) and a rabbit antibody for cFOS protein identification (Abcam; catalog code 7963).

Techniques: Expressing, Phospho-proteomics, In Situ Hybridization, Staining, Confocal Microscopy, Electron Microscopy, Quantitative RT-PCR, Laser Capture Microdissection, Activity Assay

A, Confluent ECs were incubated with PKD inhibitor CRT0066101 at 2.5 μM (CRT) for 1h prior to stimulation with 5μM Yoda1 for 10 min. The cultures were lysed with 2X SDS-PAGE sample buffer and analyzed by immunoblotting with antibodies that detect HDAC7 phoshorylated at Ser 486 , PKD1 phosphorylated at Ser 910 , HDAC7 and GAPDH (as a loading control). B , Bars display the quantification of HDAC7 phosphorylation. Mean ± SEM; n=3 independent experiments, **p<0.001 by Student’s t-test. C , Confluent ECs were treated without or with 2.5 μM CRT0066101 (CRT) for 1 h and then stimulated with Yoda1 for 60 min, as indicated. ECs were fixed and stained with an antibody that detects HDAC7 and images obtained by confocal microscopy. Scale bar = 15μm. D, Bars represent the percentage of ECs treated with Yoda1 (Y1), CRT0066101 (CRT) or their combination (Y1+CRT) with nuclear HDAC7 obtained from 5–6 fields with a total of ~30 individual cells in each field, mean ± SEM; n= 5 fields, **p<0.001 control vs Y1 and ## p<0.001 Y1 vs Y1 +CRT. E, Ratio of nuclear/cytoplasmic HDAC7 fluorescence in 5–6 fields with a total of ~30 individual cells in each field, mean ± SEM; n= 5 fields, **p<0.001 control vs Y1 and ## p<0.001 Y1 vs Y1 +CRT. The treatments were as described in panel D. F, Confluent ECs were incubated with 10 μg/ml mitomycin C for 2 h. A scratch wound was then created with a sterile 200-μl pipette tip and after washing, wounded cells were stimulated with Yoda1 or Yoda2 at 5μM for 18 h. The cultures were fixed with 4% paraformaldehyde and stained with Giemsa stain. Representative microscopy fields are shown. Images were captured at original magnification 10X. G, Bars represent relative migration (average of 5 fields per experiment) of ECs stimulated with Yoda1 or Yoda2 in the absence or presence of CRT0066101 (1 μM and 2.5 μM). Bars represent the means ± SEM n= 4 for Yoda1 and n=3 for Yoda2 independent experiments., *p<0.02, **p,0.01 by Student’s t-test.

Journal: American journal of physiology. Cell physiology

Article Title: Activation of the Piezo1 channel stimulates protein kinase D and migration in human aortic endothelial cells

doi: 10.1152/ajpcell.00457.2025

Figure Lengend Snippet: A, Confluent ECs were incubated with PKD inhibitor CRT0066101 at 2.5 μM (CRT) for 1h prior to stimulation with 5μM Yoda1 for 10 min. The cultures were lysed with 2X SDS-PAGE sample buffer and analyzed by immunoblotting with antibodies that detect HDAC7 phoshorylated at Ser 486 , PKD1 phosphorylated at Ser 910 , HDAC7 and GAPDH (as a loading control). B , Bars display the quantification of HDAC7 phosphorylation. Mean ± SEM; n=3 independent experiments, **p<0.001 by Student’s t-test. C , Confluent ECs were treated without or with 2.5 μM CRT0066101 (CRT) for 1 h and then stimulated with Yoda1 for 60 min, as indicated. ECs were fixed and stained with an antibody that detects HDAC7 and images obtained by confocal microscopy. Scale bar = 15μm. D, Bars represent the percentage of ECs treated with Yoda1 (Y1), CRT0066101 (CRT) or their combination (Y1+CRT) with nuclear HDAC7 obtained from 5–6 fields with a total of ~30 individual cells in each field, mean ± SEM; n= 5 fields, **p<0.001 control vs Y1 and ## p<0.001 Y1 vs Y1 +CRT. E, Ratio of nuclear/cytoplasmic HDAC7 fluorescence in 5–6 fields with a total of ~30 individual cells in each field, mean ± SEM; n= 5 fields, **p<0.001 control vs Y1 and ## p<0.001 Y1 vs Y1 +CRT. The treatments were as described in panel D. F, Confluent ECs were incubated with 10 μg/ml mitomycin C for 2 h. A scratch wound was then created with a sterile 200-μl pipette tip and after washing, wounded cells were stimulated with Yoda1 or Yoda2 at 5μM for 18 h. The cultures were fixed with 4% paraformaldehyde and stained with Giemsa stain. Representative microscopy fields are shown. Images were captured at original magnification 10X. G, Bars represent relative migration (average of 5 fields per experiment) of ECs stimulated with Yoda1 or Yoda2 in the absence or presence of CRT0066101 (1 μM and 2.5 μM). Bars represent the means ± SEM n= 4 for Yoda1 and n=3 for Yoda2 independent experiments., *p<0.02, **p,0.01 by Student’s t-test.

Article Snippet: Primary antibodies used were as follows: Phospho-PKD Ser 916 (Cell Signaling Technology Cat# 2051, RRID:AB_330841; final dilution 1:1000); phospho-PKD Ser 744/748 (Cell Signaling Technology Cat#2054; RRID:AB_2172539; final dilution 1:1000), PKD1 (Cell Signaling Technology Cat#2052; RRID:AB_2268946; final dilution 1:1000), phospho-HDAC4 (Ser632)/HDAC5 (Ser661)/HDAC7 (Ser486) (Cell Signaling Technology Cat#3424; RRID:AB_2118727; final dilution 1:1000), HDAC7 (E7O8V) (Cell Signaling Technology Cat#10831; RRID:AB_2797731; final dilution 1:150 for immunofluorescence) and HDAC7 (D4E1L) (Cell Signaling Technology Cat#33418; RRID:AB_2716756; final dilution 1:1000 for western blots) were all from Cell Signaling Technology (Danvers, MA).

Techniques: Activation Assay, Phospho-proteomics, Migration, Incubation, SDS Page, Western Blot, Control, Staining, Confocal Microscopy, Fluorescence, Sterility, Transferring, Giemsa Stain, Microscopy

Journal: American journal of physiology. Cell physiology

Article Title: Activation of the Piezo1 channel stimulates protein kinase D and migration in human aortic endothelial cells

doi: 10.1152/ajpcell.00457.2025

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phosphorylated form (Cell Signaling Technology Inc)

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