Journal: Cell Death & Disease

Article Title: AnnexinA7 promotes epithelial–mesenchymal transition by interacting with Sorcin and contributes to aggressiveness in hepatocellular carcinoma

doi: 10.1038/s41419-021-04287-2

Figure Lengend Snippet: A , B The expression of ANXA7 and SRI in different cancers or specific cancer subtypes was analyzed through TIMER2. The expression of ANXA7 and SRI in HCC are higher ( n = 371) than normal liver tissues ( n = 50) (*** p < 0.001). C , D The expression of ANXA7 is higher in HCC ( n = 369) than in normal liver tissues ( n = 160) from the TCGA database. The expression of SRI is also higher in HCC ( n = 369) than in normal liver tissues ( n = 160) from the TCGA database ( * p < 0.05). E – G The expression of ANXA7 and SRI were higher in Huh-7/Hep3B cells than in normal human hepatocytes HL-7702 by qRT-PCR and western blotting. H Representative images of ANXA7 and SRI expression in normal human liver tissues and HCC tissues. I Quantification of the average means optical density for ANXA7 and SRI in normal liver tissues and HCC tissues (** p < 0.01, * p < 0.05). J ANXA7 expression level p ositively correlated with SRI expression in HCC cells ( r 2 = 0.882, p < 0.01). K ANXA7 expression level positively correlated with SRI expression in HCC tissues ( r 2 = 0.707, p < 0.01).

Article Snippet: The specific antibodies including ANXA7 (Abcam Cambridge, MA, USA), 1:500; SRI (Proteintech, Wuhan, China), 1:500; E-cadherin (Proteintech, Wuhan, China), 1:500; Cytokeratin (Proteintech, Wuhan, China), 1:500; N-cadherin (Proteintech, Wuhan, China), 1:500; Vimentin (Proteintech, Wuhan, China), 1:500; GAPDH (Proteintech, Wuhan, China) 1:1000.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Cell Death & Disease

Article Title: AnnexinA7 promotes epithelial–mesenchymal transition by interacting with Sorcin and contributes to aggressiveness in hepatocellular carcinoma

doi: 10.1038/s41419-021-04287-2

Figure Lengend Snippet: A There may be a potential interaction between ANXA7 and SRI, which was analyzed by the STRING database. B co-IP assay showed the interaction between ANXA7 and SRI in Huh-7/Hep3B cells. C Cellular immunofluorescence proved that ANXA7 and SRI are colocalized in the cytoplasm and cell membrane in Huh-7/Hep3B cells.

Article Snippet: The specific antibodies including ANXA7 (Abcam Cambridge, MA, USA), 1:500; SRI (Proteintech, Wuhan, China), 1:500; E-cadherin (Proteintech, Wuhan, China), 1:500; Cytokeratin (Proteintech, Wuhan, China), 1:500; N-cadherin (Proteintech, Wuhan, China), 1:500; Vimentin (Proteintech, Wuhan, China), 1:500; GAPDH (Proteintech, Wuhan, China) 1:1000.

Techniques: Co-Immunoprecipitation Assay, Immunofluorescence, Membrane

Journal: Cell Death & Disease

Article Title: AnnexinA7 promotes epithelial–mesenchymal transition by interacting with Sorcin and contributes to aggressiveness in hepatocellular carcinoma

doi: 10.1038/s41419-021-04287-2

Figure Lengend Snippet: A – D Upregulated ANXA7 increased SRI expression and downregulated ANXA7 decreased SRI expression at the mRNA level in Huh-7 and Hep3B cells (** p < 0.01,* p < 0.05). E – H Upregulated ANXA7 increased SRI expression at the protein level and promoted EMT, whereas downregulated ANXA7 decreased SRI expression at the protein level and inhibited EMT in Huh-7 and Hep3B cells (** p < 0.01,* p < 0.05). I – J IF was used to detect E-cadherin and cytokeratin. K , L IF assay was used to detect the N-cadherin and Vimentin in Huh-7/Hep3B cells.

Article Snippet: The specific antibodies including ANXA7 (Abcam Cambridge, MA, USA), 1:500; SRI (Proteintech, Wuhan, China), 1:500; E-cadherin (Proteintech, Wuhan, China), 1:500; Cytokeratin (Proteintech, Wuhan, China), 1:500; N-cadherin (Proteintech, Wuhan, China), 1:500; Vimentin (Proteintech, Wuhan, China), 1:500; GAPDH (Proteintech, Wuhan, China) 1:1000.

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: AnnexinA7 promotes epithelial–mesenchymal transition by interacting with Sorcin and contributes to aggressiveness in hepatocellular carcinoma

doi: 10.1038/s41419-021-04287-2

Figure Lengend Snippet: A , B Images of tumor xenografts and tumor weight (** p < 0.01). C , D The qRT-PCR analysis of ANXA7 and SRI in tumor xenografts (** p < 0.01). E , F Western blotting to detect ANXA7, SRI, E-cadherin, Cytokeratin, N-cadherin, and Vimentin in tumor xenografts (** p < 0.01, * p < 0.05). G , H Immunohistochemical analysis of ANXA7, SRI and EMT indicators in tumor xenografts (** p < 0.01, * p < 0.05).

Article Snippet: The specific antibodies including ANXA7 (Abcam Cambridge, MA, USA), 1:500; SRI (Proteintech, Wuhan, China), 1:500; E-cadherin (Proteintech, Wuhan, China), 1:500; Cytokeratin (Proteintech, Wuhan, China), 1:500; N-cadherin (Proteintech, Wuhan, China), 1:500; Vimentin (Proteintech, Wuhan, China), 1:500; GAPDH (Proteintech, Wuhan, China) 1:1000.

Techniques: Quantitative RT-PCR, Western Blot, Immunohistochemical staining

Journal: Cell Death & Disease

Article Title: AnnexinA7 promotes epithelial–mesenchymal transition by interacting with Sorcin and contributes to aggressiveness in hepatocellular carcinoma

doi: 10.1038/s41419-021-04287-2

Figure Lengend Snippet: A – D When SRI is upregulated, the expression of ANXA7 increased significantly, while when SRI is downregulated, the expression of ANXA7 also decreased in Huh-7 and Hep3B cells by qRT-PCR (** p < 0.01, * p < 0.05). E – H When SRI is upregulated, the expression of ANXA7 increased, and promoted EMT, whereas when SRI is downregulated, the expression of ANXA7 also decreased, and inhibited EMT in Huh-7 and Hep3B cells by western blotting (** p < 0.01, * p < 0.05). I , J IF was used to detect the E-cadherin and Cytokeratin after upregulating and downregulating SRI. K , L IF was used to detect the N-cadherin and Vimentin in Huh-7/Hep3B cells after upregulating and downregulating SRI.

Article Snippet: The specific antibodies including ANXA7 (Abcam Cambridge, MA, USA), 1:500; SRI (Proteintech, Wuhan, China), 1:500; E-cadherin (Proteintech, Wuhan, China), 1:500; Cytokeratin (Proteintech, Wuhan, China), 1:500; N-cadherin (Proteintech, Wuhan, China), 1:500; Vimentin (Proteintech, Wuhan, China), 1:500; GAPDH (Proteintech, Wuhan, China) 1:1000.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Cell Death & Disease

Article Title: AnnexinA7 promotes epithelial–mesenchymal transition by interacting with Sorcin and contributes to aggressiveness in hepatocellular carcinoma

doi: 10.1038/s41419-021-04287-2

Figure Lengend Snippet: Interaction between ANXA7 and SRI affects EMT progress and contributes to the aggressiveness in HCC.

Article Snippet: The specific antibodies including ANXA7 (Abcam Cambridge, MA, USA), 1:500; SRI (Proteintech, Wuhan, China), 1:500; E-cadherin (Proteintech, Wuhan, China), 1:500; Cytokeratin (Proteintech, Wuhan, China), 1:500; N-cadherin (Proteintech, Wuhan, China), 1:500; Vimentin (Proteintech, Wuhan, China), 1:500; GAPDH (Proteintech, Wuhan, China) 1:1000.

Techniques:

Journal: Experimental & Molecular Medicine

Article Title: Flightless-1 inhibits ER stress-induced apoptosis in colorectal cancer cells by regulating Ca 2+ homeostasis

doi: 10.1038/s12276-020-0448-3

Figure Lengend Snippet: a Sorcin expression levels were analyzed by western blot and quantitative real-time PCR. Data are shown as the mean ± SEM ( n = 3). *** p < 0.001, shRNA-Ctrl cells vs. shRNA-FliI cells. b After cotransfection of vector or sorcin with D1ER, FRET signals were measured. Data are shown as the mean ± SEM. *** p < 0.001, shRNA-Ctrl cells vs. shRNA-FliI cells. c Ctrl- and FliI-KD cells were treated with 50 nM TG for 6 h following expression of vector or sorcin. Cell lysates were analyzed by western blotting for phospho-PERK, phospho-IREα, GRP78/BiP, CHOP, sorcin, FliI, and tubulin.

Article Snippet: Mouse sorcin expression plasmids were purchased from Sino Biological (BDA, Beijing, China).

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, shRNA, Cotransfection, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: Sorcin Activates the Brain PMCA and Blocks the Inhibitory Effects of Molecular Markers of Alzheimer’s Disease on the Pump Activity

doi: 10.3390/ijms22116055

Figure Lengend Snippet: Sorcin activates the plasma membrane Ca 2+ -ATPase (PMCA) activity and prevents the inhibition of the pump by amyloid-β peptide (Aβ1-42) and tau. Purified PMCA (2.5 μg) was reconstituted with phosphatidylcholine (PC) (○) or phosphatidylserine (PS) (Δ), and incubated for 2 min at 37 °C, with Ca 2+ and increasing concentrations of sorcin, in the absence or presence of 30 µM Aβ1-42 (●■) or 300 nM tau (▲) in 25 µL. Afterward, samples were further diluted up to 1 mL in assay medium (final concentrations of Aβ and tau were 0.75 µM and 7.5 nM, respectively). The Ca 2+ -ATPase activity was measured as described in the Methods Section. Data are mean ± SE of eight experiments performed with three preparations.

Article Snippet: Recombinant Human SRI (sorcin) was obtained from Sino Biological US Inc, Eschborn, Germany.

Techniques: Membrane, Activity Assay, Inhibition, Purification, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Sorcin Activates the Brain PMCA and Blocks the Inhibitory Effects of Molecular Markers of Alzheimer’s Disease on the Pump Activity

doi: 10.3390/ijms22116055

Figure Lengend Snippet: Sorcin does not to protect PMCA activity from its inhibition by Aβ and tau in the presence of EGTA. Purified pig brain PMCA (2.5 μg) reconstituted in PC or PS was treated for 2 min at 37 °C, with 2 mM EGTA, in the absence (plain bars) or presence of 1 µM sorcin (stripped bars) and 30 µM Aβ or 300 nM tau (grey bars) in 25 μL. The mixture was further diluted up to 1 mL in the assay medium (without Ca 2+ ) and the reaction was started by addition of 1 mM ATP and 100 µM CaCl 2 . Data represent mean ± SE from three experiments performed in duplicate and with three different preparations.

Article Snippet: Recombinant Human SRI (sorcin) was obtained from Sino Biological US Inc, Eschborn, Germany.

Techniques: Activity Assay, Inhibition, Purification

Journal: International Journal of Molecular Sciences

Article Title: Sorcin Activates the Brain PMCA and Blocks the Inhibitory Effects of Molecular Markers of Alzheimer’s Disease on the Pump Activity

doi: 10.3390/ijms22116055

Figure Lengend Snippet: Sorcin activates all PMCA ( A ) and SERCA ( B ) isoforms. Twenty µg of membranes from COS cells overexpressing PMCA or SERCA isoforms were treated in the absence (plain bars) or presence of 1 μM sorcin, and 50 µM CaCl 2 (stripped bars) in 25 µL, and then diluted up to 1 mL with the assay medium plus 0.01% saponin. Activity was measured as indicated in the Methods Section after addition of 1 mM ATP. The 100% activities correspond to 0.124 ± 0.003, 0.107 ± 0.004, 0.093 ± 0.006 and 0.102 ± 0.01 µmol.min −1 .mg −1 for hPMCA1b, hPMCA2b, rPMCA3b and hPMCA4b respectively, and to 2.57 ± 0.018, 0.086 ± 0.015 and 0.216 ± 0.01 µmol.min −1 .mg −1 for SERCA1, SERCA2b and SERCA3, respectively. Data are mean ± SE of three experiments performed in triplicate with three preparations. p ≤ 0.001 vs. control.

Article Snippet: Recombinant Human SRI (sorcin) was obtained from Sino Biological US Inc, Eschborn, Germany.

Techniques: Activity Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Sorcin Activates the Brain PMCA and Blocks the Inhibitory Effects of Molecular Markers of Alzheimer’s Disease on the Pump Activity

doi: 10.3390/ijms22116055

Figure Lengend Snippet: Sorcin activates Ca 2+ -ATPase activities of overexpressed full hPMCA4b, their truncated variants and SERCA2b isoforms in a concentration-dependent manner and prevents their inhibition by Aβ and tau. Twenty µg of COS cells’ membranes overexpressing full hPMCA4b, or truncated hPMCA4b-L1086* and hPMCA4b-R1052* and SERCA2b isoforms were treated with increasing concentrations of sorcin, without (○) or with 30 μM Aβ1-42 (●) or 300 nM tau (▲), in 25 µL, and then diluted up to 1 mL with the assay medium plus 0.01% saponin, resulting in the indicated final concentrations of sorcin. Activities were measured as indicated in the Methods Section, after addition of 1 mM ATP. The 100% activities correspond to 0.226 ± 0.02, 0.235 ± 0.03, 0.242 ± 0.02 and 0.190 ± 0.003 µmol. min −1 . mg −1 for hPMCA4b, hPMCA-L1086*, hPMCA-L1052* and SERCA2b, respectively. Data represent mean ± SE of three experiments performed with three preparations.

Article Snippet: Recombinant Human SRI (sorcin) was obtained from Sino Biological US Inc, Eschborn, Germany.

Techniques: Concentration Assay, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: Sorcin Activates the Brain PMCA and Blocks the Inhibitory Effects of Molecular Markers of Alzheimer’s Disease on the Pump Activity

doi: 10.3390/ijms22116055

Figure Lengend Snippet: ( A ) Sorcin prevents the inhibition of PMCA activity by Aβ and/or tau in membranes from human control (HC) and AD (HAD) brain samples. Ten µg of membranes from HC (Braak stages I and II) and from HAD (Braak stages V and VI) were incubated without (plain bars) or with (stripped bars) 1 µM sorcin and 30 μM Aβ1-42 or 300 nM tau (grey bars) in 25 µL, and then diluted up to 1 mL with assay medium plus 0.01% saponin. PMCA activity was assayed after triggering the reaction with 1 mM ATP, as indicated in the Methods Section. Data represent mean ± SE values of three experiments performed with four preparations of each Braak stage. * p ≤ 0.001 vs. MV without sorcin. ( B ) Expression levels of sorcin in human brain samples at increasing Braak stages of AD. Twenty µg of human brain samples (SN1) of Braak stages I, II, III, IV, V and VI, were electrophoresed into 12% SDS-PAGE gel, electro-transferred to PVDF and immuno-stained with the anti-sorcin antibody, as indicated in the Methods Section. The immunoblot is representative of three assays with at least three samples from each stage. Relative expression of sorcin vs. GAPDH is shown as mean ± SE values (arbitrary units). * p ≤ 0.001 vs. Braak stage I.

Article Snippet: Recombinant Human SRI (sorcin) was obtained from Sino Biological US Inc, Eschborn, Germany.

Techniques: Inhibition, Activity Assay, Control, Incubation, Expressing, SDS Page, Staining, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Sorcin Activates the Brain PMCA and Blocks the Inhibitory Effects of Molecular Markers of Alzheimer’s Disease on the Pump Activity

doi: 10.3390/ijms22116055

Figure Lengend Snippet: Protective effects of sorcin on Ca 2+ -ATPase activity ( A , B ), cell viability ( C ), ROS production ( D ) and apoptosis ( E , F ), in human neuroblastoma cells. SH-SY5Y cells were treated without or with 5 μM Aβ1-42 or 10 nM tau in the absence and presence of 1 μM sorcin for 24 h, as detailed in the Methods Section. ( A ) Ca 2+ -ATPase activity was assayed in 10 μg of membranes, previously treated as indicated above. ( B ) Ca 2+ -ATPase activity was measured by incubating non-treated cells with 30 μM Aβ1-42 or 300 nM tau in the absence and presence of 1 µM sorcin, in 25 µL, and then diluted up to 1 mL with assay medium plus 0.01% saponin. Activity data are mean ± SE of three independent experiments. Cells treated with Aβ or tau. ( C ) Cell viability was assayed in control and treated cells after 1 h incubation with 150 µg/mL MTT (white bars) or stained with 0.4% trypan blue (black bars). Data are expressed as percentage of untreated cells, as mean ± SE of five independent experiments. ( D ) The production of ROS was determined by incubating non-treated and treated cells with 40 μM H 2 DCFDA, as indicated in the Methods Section. Data are expressed as mean ± SE of fluorescence intensity of three independent experiments. ( E ) DAPI staining in non-treated and treated cells. Representative fluorescent microscopy images show apoptotic cells with condensed and fragmented nuclei (white arrows). Scale bar: 10 µm. Apoptotic nuclei were quantified with respect to the total number of seeded cells. Values are mean ± SE of ten images per coverslip, obtained from three cell cultures. ( F ) Caspase-3 activity was measured in non-treated and treated cell lysates by detecting cleavage of the caspase substrate for 1 h at 37 °C. Data are represented as a percentage relative to non-treated cells (0.206 ± 0.005 fluorescence intensity/µg protein). Values are mean ± SE of three experiments. * p ≤ 0.001 vs. cells treated with Aβ or tau.

Article Snippet: Recombinant Human SRI (sorcin) was obtained from Sino Biological US Inc, Eschborn, Germany.

Techniques: Activity Assay, Control, Incubation, Staining, Fluorescence, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Sorcin Activates the Brain PMCA and Blocks the Inhibitory Effects of Molecular Markers of Alzheimer’s Disease on the Pump Activity

doi: 10.3390/ijms22116055

Figure Lengend Snippet: Blot overlay assays of the interaction between sorcin and PMCA, Aβ and tau. ( A ) PMCA (0.5 μg) and sorcin (1 μg) were run in 10% SDS gel, transferred to PVDF membranes and incubated sequentially with 0.5 µg PMCA and the a-PMCA antibody 5F10. The panel shows binding of PMCA to sorcin. ( B ) Sorcin (1 ug), Aβ (3 µg) and tau (3 µg) were run in three gradient 10–20% gels, transferred to PVDF and incubated sequentially with 3 µg Aβ or 3 µg tau, and with a-Aβ1-42 or a-tau antibodies, as indicated. The panel shows binding of both Aβ and tau to sorcin. It also shows a Western blot of pure sorcin with the a-sorcin antibody, as control. Immunoblots are representatives from three different assays.

Article Snippet: Recombinant Human SRI (sorcin) was obtained from Sino Biological US Inc, Eschborn, Germany.

Techniques: SDS-Gel, Incubation, Binding Assay, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Sorcin Activates the Brain PMCA and Blocks the Inhibitory Effects of Molecular Markers of Alzheimer’s Disease on the Pump Activity

doi: 10.3390/ijms22116055

Figure Lengend Snippet: The scheme summarizes the interactions of sorcin with PMCA (red) and SERCA (blue), in the presence of Ca 2+ that lead to Ca 2+ -ATPase activation. Sorcin also interacts with AD markers, such as tau and Aβ, preventing their binding to PMCA and their inhibitory effects on PMCA activity.

Article Snippet: Recombinant Human SRI (sorcin) was obtained from Sino Biological US Inc, Eschborn, Germany.

Techniques: Activation Assay, Binding Assay, Activity Assay