Journal: Cells

Article Title: Cancer Stem Cells in Metastatic Head and Neck Cutaneous Squamous Cell Carcinoma Express Components of the Renin-Angiotensin System

doi: 10.3390/cells10020243

Figure Lengend Snippet: Representative immunohistochemical-stained-sections of metastatic head and neck cutaneous squamous cell carcinoma tissue samples stained for components of the renin-angiotensin system. Angiotensinogen ( A , brown) was expressed on cells within the tumor nests (TNs, arrows ), and to a lesser degree in the peri-tumoral stroma (PTS, arrowheads ). Renin ( B , brown) was not present in the tissue samples. PRR ( C , brown), demonstrated cytoplasmic expression of cells within the TNs ( arrows ), and to a lesser extent cells within the PTS ( arrowheads ) in two cases. ACE ( D , brown) was expressed on the endothelium of the tumor microvessels within the PTS ( arrowheads ). ACE2 ( E , brown) was not present in the tissue samples. AT 2 R ( F , brown) was expressed by cells within the TNs ( arrows ) and the PTS ( arrowheads ). Nuclei were counter-stained with hematoxylin ( A – F , blue). Original magnification: 400×. n = 20.

Article Snippet: Immunohistochemical staining was performed on these sections using primary antibodies for angiotensinogen (1:50; cat#79299S, Cell Signaling, Danvers, MA, USA), renin (1:500; cat#14291-1-AP, Proteintech, Rosemont, IL, USA), PRR (1:500; cat#ab40790, Abcam, Cambridge, MA, USA), ACE (1:50; cat#PA5-83080, Invitrogen, Carlsbad, CA, USA), ACE2 (1:1000; cat#MAB933, R&D Systems, Minneapolis, MN, USA), and AT 2 R (1:2000; cat#NPBI-77368, Novus Biologicals, Littleton, CO, USA), with 3,3′-diaminobenzidine (ready-to-use; cat#DS9800, Leica, Wetzlar, Germany) as the chromogen.

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Cells

Article Title: Cancer Stem Cells in Metastatic Head and Neck Cutaneous Squamous Cell Carcinoma Express Components of the Renin-Angiotensin System

doi: 10.3390/cells10020243

Figure Lengend Snippet: Representative double immunohistochemical-stained section of metastatic head and neck cutaneous squamous cell carcinoma demonstrating cytoplasmic expression of angiotensinogen (red) in CSCs with nuclear expression of SOX2 (brown), within the TNs. Cell nuclei were counter-stained with hematoxylin (blue). Original magnification: 400×. n = 2.

Article Snippet: Immunohistochemical staining was performed on these sections using primary antibodies for angiotensinogen (1:50; cat#79299S, Cell Signaling, Danvers, MA, USA), renin (1:500; cat#14291-1-AP, Proteintech, Rosemont, IL, USA), PRR (1:500; cat#ab40790, Abcam, Cambridge, MA, USA), ACE (1:50; cat#PA5-83080, Invitrogen, Carlsbad, CA, USA), ACE2 (1:1000; cat#MAB933, R&D Systems, Minneapolis, MN, USA), and AT 2 R (1:2000; cat#NPBI-77368, Novus Biologicals, Littleton, CO, USA), with 3,3′-diaminobenzidine (ready-to-use; cat#DS9800, Leica, Wetzlar, Germany) as the chromogen.

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Cells

Article Title: Cancer Stem Cells in Metastatic Head and Neck Cutaneous Squamous Cell Carcinoma Express Components of the Renin-Angiotensin System

doi: 10.3390/cells10020243

Figure Lengend Snippet: Fold-change (2 ΔΔCt ) in transcript expression of components of the renin-angiotensin system (RAS): angiotensinogen (AGT), renin, PRR, ACE, ACE2, AT 1 R, and AT 2 R, as determined by RT-qPCR in four metastatic head and neck cutaneous squamous cell carcinoma (mHNcSCC) tissue samples ( A ) and four mHNcSCC-derived primary cell lines ( B ). CT values were normalized to the reference genes GAPDH and PUM1 to calculate ΔCT, and expression compared to that of UHR (y = 1). Error bars represent 95% confidence intervals of the mean. Graphs are shown with log2 scale.

Article Snippet: Immunohistochemical staining was performed on these sections using primary antibodies for angiotensinogen (1:50; cat#79299S, Cell Signaling, Danvers, MA, USA), renin (1:500; cat#14291-1-AP, Proteintech, Rosemont, IL, USA), PRR (1:500; cat#ab40790, Abcam, Cambridge, MA, USA), ACE (1:50; cat#PA5-83080, Invitrogen, Carlsbad, CA, USA), ACE2 (1:1000; cat#MAB933, R&D Systems, Minneapolis, MN, USA), and AT 2 R (1:2000; cat#NPBI-77368, Novus Biologicals, Littleton, CO, USA), with 3,3′-diaminobenzidine (ready-to-use; cat#DS9800, Leica, Wetzlar, Germany) as the chromogen.

Techniques: Expressing, Quantitative RT-PCR, Derivative Assay

Journal: Cells

Article Title: Cancer Stem Cells in Metastatic Head and Neck Cutaneous Squamous Cell Carcinoma Express Components of the Renin-Angiotensin System

doi: 10.3390/cells10020243

Figure Lengend Snippet: Western blotting of protein extracted from six metastatic head and neck cutaneous squamous cell carcinoma (mHNcSCC) tissue samples (T1–T6) and four mHNcSCC-derived primary cell lines (C1–C4) detected the expression angiotensinogen ( A ) in five of the six tissue samples at the appropriate molecular weight of 55 kDa, but not in any of the mHNcSCC-derived primary cell lines. PRR ( B ) was detected in all of the six tissue samples and four primary cell lines at the expected molecular weight at 35 kDa for the full-length transmembrane isoform and the shorter secreted isoform. ACE ( C ) was detected in all 6 tissue samples, but only three of the four cell lines, at the appropriate weight of 195 kDa. ACE2 ( D ) and AT 2 R ( E ) were not detected in any of the six tissue samples or four primary cell line samples investigated, but were present in the positive controls. Lanes 1–6 indicate six tissue samples used, lanes 7–10 indicate cell lines. +ve indicates positive control: plasma for angiotensinogen; tonsil for PRR; mouse lung for ACE; kidney for ACE2; and mouse heart for AT 2 R. The molecular weight ladder (kDa) is labeled for each blot. Blots for α-tubulin are presented in .

Article Snippet: Immunohistochemical staining was performed on these sections using primary antibodies for angiotensinogen (1:50; cat#79299S, Cell Signaling, Danvers, MA, USA), renin (1:500; cat#14291-1-AP, Proteintech, Rosemont, IL, USA), PRR (1:500; cat#ab40790, Abcam, Cambridge, MA, USA), ACE (1:50; cat#PA5-83080, Invitrogen, Carlsbad, CA, USA), ACE2 (1:1000; cat#MAB933, R&D Systems, Minneapolis, MN, USA), and AT 2 R (1:2000; cat#NPBI-77368, Novus Biologicals, Littleton, CO, USA), with 3,3′-diaminobenzidine (ready-to-use; cat#DS9800, Leica, Wetzlar, Germany) as the chromogen.

Techniques: Western Blot, Derivative Assay, Expressing, Molecular Weight, Positive Control, Labeling

Journal: Journal of Cellular and Molecular Medicine

Article Title: Salidroside ameliorates Adriamycin nephropathy in mice by inhibiting β‐catenin activity

doi: 10.1111/jcmm.14340

Figure Lengend Snippet: Salidroside administration blocks renal β‐catenin activation induced by ADR. (A) Immunohistochemical analysis showing β‐catenin protein expression and localization in the kidney. Representative micrographs were shown. Scale bar = 20 µm. (B) Western blot analysis showing renal β‐catenin protein abundance in the nucleus and cytoplasm. Nucleus and cytoplasm lysates were distinguished using antibodies against Lamin A/C and Tubulin respectively (left panel). Lamin A/C and Actin were used as loading controls. (C) Quantitative analysis of β‐catenin protein levels as shown in (B). (D) Axin2 and Cyclin D1 expressions were examined. (E) Western blot analysis showing the effects of salidroside on β‐catenin downstream target gene expression. (F) Quantitative analysis of protein levels as shown in (E). Lamin A/C and Actin were used as loading controls. The results are the means ± SEM of three independent experiments. ** P < 0.01; *** P < 0.001 vs CON; # P < 0.05; ## P < 0.01, ### P < 0.001 vs ADR alone. n = 5. ADR, adriamycin; AGT, angiotensinogen; CON, control; MMP7, matrix metalloproteinase 7; PAI‐1, plasminogen activator inhibitor‐1; Sal, salidroside

Article Snippet: The antibodies anti‐α‐SMA (smooth muscle actin) (catalog no. #19245), anti‐Lamin A/C (catalog no. #4777), anti‐MMP7 (matrix metalloproteinase 7) (catalog no. #3801), anti‐AGT (angiotensinogen) (catalog no. #79299), anti‐PAI‐1 (plasminogen activator inhibitor‐1) (catalog no. #11907), anti‐Axin2 (catalog no. #2151), anti‐Cyclin D1 (catalog no. 2978) and anti‐Snail (catalog no. #3879) were from Cell Signaling Technology (Beverley, MA, USA).

Techniques: Activation Assay, Immunohistochemical staining, Expressing, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Salidroside ameliorates Adriamycin nephropathy in mice by inhibiting β‐catenin activity

doi: 10.1111/jcmm.14340

Figure Lengend Snippet: Activation of β‐catenin by Wnt1 abolishes the protective effects of salidroside against ADR injury in podocytes. (A‐B) The transfection of Wnt1 in cultured podocytes successfully activates β‐catenin activity. (C) Quantitative analysis of Axin2 and Cyclin D1 as shown in (B). (D) The repression of salidroside on β‐catenin activity was counteracted by Wnt1 in ADR‐treated podocytes. (E) Quantitative analysis of Western blots as shown in (D). (F) The inhibition of β‐catenin downstream target gene expressions by salidroside was re‐activated by Wnt1 in ADR‐treated podocytes. (G) Quantitative analysis of Western blots as shown in (F). (H) Activation of β‐catenin by Wnt1 reverses the effects of salidroside on podocin and nephrin expressions in ADR‐injured podocytes. (I) Quantitative analysis of Western blots as shown in (H). (J) Wnt1 has no effects on podocin and nephrin expressions. (K) Quantitative analysis of Western blots as shown in (J). (L) Effects of salidroside on Axin2 and Cyclin D1 expressions in podocytes transfected with the plasmid bearing Wnt1. Lamin A/C and Actin were used as loading controls. The results are the means ± SEM of three independent experiments. EV, empty vector; CON, control; ADR, adriamycin; Sal, salidroside; MMP7, matrix metalloproteinase 7; AGT, angiotensinogen; PAI‐1, plasminogen activator inhibitor‐1. * P < 0.05 and *** P < 0.001 vs CON; # P < 0.05; ## P < 0.01 and ### P < 0.001 vs ADR; ^^ P < 0.01 and ^^^ P < 0.001 vs ADR + Sal

Article Snippet: The antibodies anti‐α‐SMA (smooth muscle actin) (catalog no. #19245), anti‐Lamin A/C (catalog no. #4777), anti‐MMP7 (matrix metalloproteinase 7) (catalog no. #3801), anti‐AGT (angiotensinogen) (catalog no. #79299), anti‐PAI‐1 (plasminogen activator inhibitor‐1) (catalog no. #11907), anti‐Axin2 (catalog no. #2151), anti‐Cyclin D1 (catalog no. 2978) and anti‐Snail (catalog no. #3879) were from Cell Signaling Technology (Beverley, MA, USA).

Techniques: Activation Assay, Transfection, Cell Culture, Activity Assay, Western Blot, Inhibition, Plasmid Preparation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Salidroside ameliorates Adriamycin nephropathy in mice by inhibiting β‐catenin activity

doi: 10.1111/jcmm.14340

Figure Lengend Snippet: Activation of β‐catenin by β‐catenin S33Y diminishes the protective roles of salidroside against ADR injury in podocytes. (A‐B) The transfection of β‐catenin S33Y in cultured podocytes successfully activates β‐catenin activity. (C) Quantitative analysis of Axin2 and Cyclin D1 as shown in (B). (D) The repression of salidroside on β‐catenin activity was counteracted by β‐catenin S33Y in ADR‐treated podocytes. (E) Quantitative analysis of Western blots as shown in (D). (F) The inhibition of β‐catenin downstream target gene expressions by salidroside was re‐activated by β‐catenin S33Y in ADR‐treated podocytes. (G) Quantitative analysis of Western blots as shown in (F). (H) Activation of β‐catenin by β‐catenin S33Y reverses the effects of salidroside on podocin and nephrin expressions in ADR‐injured podocytes. (I) Quantitative analysis of Western blots as shown in (H). (J) β‐catenin S33Y has no effects on podocin and nephrin expressions. (K) Quantitative analysis of Western blots as shown in (J). (L) Effects of salidroside on Axin2 and Cyclin D1 expressions in podocytes transfected with the plasmid bearing β‐catenin S33Y. Lamin A/C and Actin were used as loading controls. The results are the means ± SEM of three independent experiments. EV, empty vector; CON, control; ADR, adriamycin; Sal, salidroside; MMP7, matrix metalloproteinase 7; AGT, angiotensinogen; PAI‐1, plasminogen activator inhibitor‐1. * P < 0.05; ** P < 0.01 and *** P < 0.001 vs CON; # P < 0.05; ## P < 0.01 and ### P < 0.001 vs ADR; ^ P < 0.05; ^^ P < 0.01 and ^^^ P < 0.001 vs ADR + Sal

Article Snippet: The antibodies anti‐α‐SMA (smooth muscle actin) (catalog no. #19245), anti‐Lamin A/C (catalog no. #4777), anti‐MMP7 (matrix metalloproteinase 7) (catalog no. #3801), anti‐AGT (angiotensinogen) (catalog no. #79299), anti‐PAI‐1 (plasminogen activator inhibitor‐1) (catalog no. #11907), anti‐Axin2 (catalog no. #2151), anti‐Cyclin D1 (catalog no. 2978) and anti‐Snail (catalog no. #3879) were from Cell Signaling Technology (Beverley, MA, USA).

Techniques: Activation Assay, Transfection, Cell Culture, Activity Assay, Western Blot, Inhibition, Plasmid Preparation

Journal: Biology of Sex Differences

Article Title: HDAC9 is an epigenetic repressor of kidney angiotensinogen establishing a sex difference

doi: 10.1186/s13293-017-0140-z

Figure Lengend Snippet: Contribution of HDAC9 to AGT expression in PTC. PTC were treated by HDAC9 inhibitor ( a ) or siRNA ( b , c ). Thereafter, AGT mRNA levels in the cells and AGT protein levels in the cultured medium were measured by qRT-PCR and AGT ELISA, respectively. Total AGT protein amount secreted from the cells to the culture medium were calculated based on the volume of medium. Nega-si negative control siRNA-transfected group, HDAC9-si HDAC9 siRNA-transfected group. Data are expressed as mean ± SE. Asterisk ( P < 0.05) indicates significant difference compared with the negative siRNA transfected group

Article Snippet: A rabbit anti-histone deacetylase 9 (HDAC9) antibody from Abcam (ab109446), rabbit anti-AGT antibody from IBL (JP28101), rabbit anti acetyl-Histone H3 (Lys5, #9675), and rabbit anti-acetyl-Histone 4 (Lys18, #8647) from Cell Signaling Technology were used.

Techniques: Expressing, Cell Culture, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Negative Control, Transfection

Journal: Biology of Sex Differences

Article Title: HDAC9 is an epigenetic repressor of kidney angiotensinogen establishing a sex difference

doi: 10.1186/s13293-017-0140-z

Figure Lengend Snippet: Association of HDAC9 with AGT promoter. ChiP assays were performed to test interaction between HDAC9 and AGT promoter region. Two primer sets for a proximal region (from −437 to −951 bp) and a distal region (from −1,427 to −1,879 bp) of AGT promoter were designed and used in the assay (summarized in panel a ). An anti-HDAC9 antibody ( b ) and anti-histone H3 and H4 antibodies ( c ) were used in the assays. Rabbit IgG was used as a negative control

Article Snippet: A rabbit anti-histone deacetylase 9 (HDAC9) antibody from Abcam (ab109446), rabbit anti-AGT antibody from IBL (JP28101), rabbit anti acetyl-Histone H3 (Lys5, #9675), and rabbit anti-acetyl-Histone 4 (Lys18, #8647) from Cell Signaling Technology were used.

Techniques: Negative Control