Journal: Journal of Orthopaedic Surgery and Research

Article Title: PLXNC1 interference alleviates the inflammatory injury, apoptosis and extracellular matrix degradation of IL-1β-exposed chondrocytes via suppressing GRP78 expression

doi: 10.1186/s13018-023-04207-4

Figure Lengend Snippet: PLXNC1 interacts with GRP78 in IL-1β-treated CHON-001 cells. A The potential interacting partners of PLXNC1 were obtained via Biogrid database. B The possible interaction between PLXNC1 and GRP78 was predicted by HDOCK server. PLXNC1 and GRP78 were colored in brown and yellow, respectively. C CHON-001 cells were treated by ascending doses of IL-1β (2.5, 5 and 10 ng/ml) for 72 h. RT-qPCR and western blotting tested GRP78 expression in IL-1β-challenged CHON-001 cells. D and E Co-IP assay corroborated the binding between PLXNC1 and GRP78. F CHON-001 cells were treated by ascending doses of IL-1β (10 ng/ml) for 72 h and subsequently transfected with SiRNA-NC and SiRNA-PLXNC1. RT-qPCR and western blotting tested GRP78 expression following PLXNC1 absence. ** P < 0.01 and *** P < 0.001. PLXNC1 plexin C1, IL-1β interleukin-1beta, GRP78 glucose-regulating protein 78

Article Snippet: The small interfering RNAs (siRNAs) specific for PLXNC1 (SiRNA-PLXNC1-1/2) and the blank control (SiRNA-NC) were ordered from Guangzhou Ribobio Co., Ltd. GRP78 overexpression vector (Ov-GRP78) was developed by Sino Biological Inc. (Beijing, China) via inserting the prepared GRP78 cDNA into the pcDNA3.1(+) plasmid, referring to the empty vector as Ov-NC.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Co-Immunoprecipitation Assay, Binding Assay, Transfection

Journal: Journal of Orthopaedic Surgery and Research

Article Title: PLXNC1 interference alleviates the inflammatory injury, apoptosis and extracellular matrix degradation of IL-1β-exposed chondrocytes via suppressing GRP78 expression

doi: 10.1186/s13018-023-04207-4

Figure Lengend Snippet: GRP78 elevation counteracts the effects of PLXNC1 silencing on the viability and inflammation of IL-1β-exposed CHON-001 cells. A and B RT-qPCR and western blotting tested the transfection efficiency of GRP78 overexpression plasmids. CHON-001 cells were treated by ascending doses of IL-1β (10 ng/ml) for 72 h and subsequently co-transfected with SiRNA-PLXNC1 and Ov-GRP78. B CCK-8 assay judged CHON-001 cell viability. C RT-qPCR examined the levels of inflammatory factors including TNFα, IL-1β and IL-6. * P < 0.05; ** P < 0.01 and *** P < 0.001. PLXNC1 plexin C1, IL-1β interleukin-1beta, GRP78 glucose-regulating protein 78, TNFα tumor necrosis factor alpha, IL-6 interleukin-6

Article Snippet: The small interfering RNAs (siRNAs) specific for PLXNC1 (SiRNA-PLXNC1-1/2) and the blank control (SiRNA-NC) were ordered from Guangzhou Ribobio Co., Ltd. GRP78 overexpression vector (Ov-GRP78) was developed by Sino Biological Inc. (Beijing, China) via inserting the prepared GRP78 cDNA into the pcDNA3.1(+) plasmid, referring to the empty vector as Ov-NC.

Techniques: Quantitative RT-PCR, Western Blot, Transfection, Over Expression, CCK-8 Assay

Journal: Journal of Orthopaedic Surgery and Research

Article Title: PLXNC1 interference alleviates the inflammatory injury, apoptosis and extracellular matrix degradation of IL-1β-exposed chondrocytes via suppressing GRP78 expression

doi: 10.1186/s13018-023-04207-4

Figure Lengend Snippet: GRP78 elevation abolishes the impacts of PLXNC1 knockdown on the apoptosis and ECM degradation of IL-1β-exposed CHON-001 cells. CHON-001 cells were treated by ascending doses of IL-1β (10 ng/ml) for 72 h and subsequently co-transfected with SiRNA-PLXNC1 and Ov-GRP78. A TUNEL assay measured apoptosis. B Western blot analysis of the expression of apoptosis-associated proteins including Bcl-2, Bax, cleaved caspase 3 and caspase 3. C – F RT-qPCR and western blotting tested the expression of ECM degradation-associated proteins including MMP3, MMP-13, type II collagen, ADAMTS-4, ADAMTS-5 and aggrecan. * P < 0.05; ** P < 0.01 and *** P < 0.001. PLXNC1 plexin C1, IL-1β interleukin-1beta, GRP78 glucose-regulating protein 78, Bcl-2 B cell lymphoma-2, Bax Bcl-2-associated X, MMP3 matrix metallopeptidase 3, MMP-13 matrix metallopeptidase-13, ADAMTS-4 a disintegrin and metalloproteinase with thrombospondin motifs type 4, ADAMTS-5 a disintegrin and metalloproteinase with thrombospondin motifs type 5

Article Snippet: The small interfering RNAs (siRNAs) specific for PLXNC1 (SiRNA-PLXNC1-1/2) and the blank control (SiRNA-NC) were ordered from Guangzhou Ribobio Co., Ltd. GRP78 overexpression vector (Ov-GRP78) was developed by Sino Biological Inc. (Beijing, China) via inserting the prepared GRP78 cDNA into the pcDNA3.1(+) plasmid, referring to the empty vector as Ov-NC.

Techniques: Transfection, TUNEL Assay, Western Blot, Expressing, Quantitative RT-PCR

Journal: Journal of Orthopaedic Surgery and Research

Article Title: PLXNC1 interference alleviates the inflammatory injury, apoptosis and extracellular matrix degradation of IL-1β-exposed chondrocytes via suppressing GRP78 expression

doi: 10.1186/s13018-023-04207-4

Figure Lengend Snippet: PLXNC1 interference blocks IL-1β-activated ERS signaling in CHON-001 cells through declining GRP78 expression. CHON-001 cells were treated by ascending doses of IL-1β (10 ng/ml) for 72 h and subsequently co-transfected with SiRNA-PLXNC1 and Ov-GRP78. Western blotting tested the expression of ERS signaling-associated proteins including ATF4, p-IRE1α/IRE1α, TRAF2 and ASK1. *** P < 0.001. PLXNC1 plexin C1, IL-1β interleukin-1beta, GRP78 glucose-regulating protein 78, ATF4 activating transcription factor 4, p-IRE1α phosphorylated inositol requiring enzyme 1 alpha, IRE1α inositol requiring enzyme 1 alpha, TRAF2 tumor necrosis factor receptor-associated factor 2, ASK1 apoptosis signal-regulating kinase 1

Article Snippet: The small interfering RNAs (siRNAs) specific for PLXNC1 (SiRNA-PLXNC1-1/2) and the blank control (SiRNA-NC) were ordered from Guangzhou Ribobio Co., Ltd. GRP78 overexpression vector (Ov-GRP78) was developed by Sino Biological Inc. (Beijing, China) via inserting the prepared GRP78 cDNA into the pcDNA3.1(+) plasmid, referring to the empty vector as Ov-NC.

Techniques: Expressing, Transfection, Western Blot

Journal: Journal of cellular biochemistry

Article Title: Combined application of geranylgeranylacetone and amelogenin promotes angiogenesis and wound healing in human periodontal ligament cells.

doi: 10.1002/jcb.29903

Figure Lengend Snippet: FIGURE 1 GGA induces Grp78 expression in hPDLCs without affecting proliferation. (A) hPDLCs were stimulated with 200 μM GGA for the indicated intervals. The total RNA was isolated and analyzed to evaluate Grp78 mRNA expression using quantitative real‐time PCR. Data are represented as fold increases in the expression relative to that of GAPDH (n = 3). (B) After stimulation by GGA for the indicated intervals, whole‐cell lysates prepared from hPDLCs were subjected to immunoblot analysis with an anti‐Grp78 antibody. β‐actin was used as a control. Representative western blot analysis of total Grp78 is shown in the upper panel. Quantification of total Grp78 protein levels relative to β‐actin was performed using ImageJ (lower panel) (n = 3). (C) Diagram of the protocol for hPDLCs treatment: unstimulated cells, GGA, rM180, or GGA + rM180. Functional analysis with hPDLCs was performed at the indicated time points after the first medium exchange following 18 h of culture with media alone, with GGA, or rM180 (10 μg/ml). (D) WST‐8 assay was performed for the indicated intervals. Proliferation is expressed as the absorbance measured at 450–655 nm (n = 5). Data are represented as mean ± SD. GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; GGA, geranylgeranylacetone; Grp78, glucose‐regulated protein 78; hPDLC, human periodontal ligament cell; mRNA, messenger RNA; N.S., not significant; PCR, polymerase chain reaction. *p < .05; **p < .01; ***p < .001

Article Snippet: At approximately 60%–70% confluence, the cells were transfected with 2 μg of the Grp78‐ overexpression plasmid (pcDNA3.1 (+) Grp78/Bip, RRID: Addgene_32701; Addgene) using 2 μl of Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions.

Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction, Western Blot, Control, Functional Assay, Polymerase Chain Reaction

Journal: Journal of cellular biochemistry

Article Title: Combined application of geranylgeranylacetone and amelogenin promotes angiogenesis and wound healing in human periodontal ligament cells.

doi: 10.1002/jcb.29903

Figure Lengend Snippet: FIGURE 7 Proposed mechanism of wound healing in response to combined treatment with GGA and rM180 amelogenin in hPDLCs. GGA increases Grp78 expression and enhances the migration of hPDLCs. The increased Grp78 expression triggers the production of Angptl4 and Areg, which are involved in angiogenesis and wound healing, via the activation of HIF‐1α and PPARδ and the phosphorylation of CREB and PKA. Supplementation with rM180 amelogenin not only strongly enhances the migratory capacity of hPDLCs, but also accelerates their angiogenic activity owing to the upregulation of IL‐8, MCP‐1, and IL‐6. Angptl4, angiopoietin‐ like 4; Areg, amphiregulin; CREB, cAMP response element‐binding protein; GGA, geranylgeranylacetone; HIF‐1α, hypoxia‐inducible factor 1α; hPDLC, human periodontal ligament cell; IL‐8, interleukin‐8; MCP‐1, monocyte chemotactic protein 1; PKA, protein kinase A; PPARδ, peroxisome proliferator‐activated receptor δ

Article Snippet: At approximately 60%–70% confluence, the cells were transfected with 2 μg of the Grp78‐ overexpression plasmid (pcDNA3.1 (+) Grp78/Bip, RRID: Addgene_32701; Addgene) using 2 μl of Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions.

Techniques: Expressing, Migration, Activation Assay, Phospho-proteomics, Activity Assay, Binding Assay

Journal: Cell Proliferation

Article Title: Administration of Cripto in GRP 78 overexpressed human MSC s enhances stem cell viability and angiogenesis during human MSC transplantation therapy

doi: 10.1111/cpr.12463

Figure Lengend Snippet: Overexpression of GRP78 on human MSCs retains differentiation capabilities of stem cells. (A) Western blot analysis of GRP78 expression in hMSCs compared to that in overexpression GRP78‐hMSCs (GRP78‐hMSC). The right panel represents the expression levels of GRP78 that were normalized to β‐actin. Values represent mean ± SEM. **P < .01 vs. Control. (B) GRP78‐hMSC was investigated by immunohistochemistry for GRP78 (Green). Scale bar = 200 μM. (C) hMSCs and GRP78‐hMSC were differentiated into adipocytes, osteocytes and chondrocytes, and examined by Oli red O, Safranin O, and alkaline phosphatase staining respectively. Scale bar = 200 μM. (D) mRNA expression of fabp4, sox9 and opn in hMSC and GRP78‐hMSC. (E) Western blot analysis of SOX2, Nanog and OCT4 expressions in GRP78‐hMSC and the control hMSC

Article Snippet: hMSC cultured in 60 mm plates were overexpressed with 3 μg of pcDNA3.1 GRP78 vector (Invitrogen, Carlsbad, CA, USA), using lipofectamine 2000 (Invitrogen).

Techniques: Over Expression, Western Blot, Expressing, Immunohistochemistry, Staining

Journal: Cell Proliferation

Article Title: Administration of Cripto in GRP 78 overexpressed human MSC s enhances stem cell viability and angiogenesis during human MSC transplantation therapy

doi: 10.1111/cpr.12463

Figure Lengend Snippet: Binding of GRP78 with Cripto enhances cellular proliferation via JAK2‐STAT3 pathway. (A) Western blot analysis of p‐JAK2 and p‐STAT3 expressions in hMSCs, GRP78‐hMSC and hMSC pre‐treated with antibody GRP78 (Ab‐GRP78) exposed to Cripto for 0, 5, 10 and 20 minutes. The down panel represents the phosphorylation levels of JAK2 and STAT‐3 in control to β‐actin. Values represent the mean ± SEM. **P < .01 vs. control, ##P < .01 vs. GRP78‐hMSC. (B) hMSC, GRP78‐hMSC, and Ab‐GRP78 were treated with or without Cripto for 24 hour. Images of cellular proliferation measurement were shown using MTT assay. Values represent the mean ± SEM. *P < .05 vs control, **P < .01 vs. control, ##P < .01 vs treated Cripto, and $$P < .01 vs Cripto+GRP78‐hMSCs. (C) Image of single cell assay after 10 days in 96 well plate, stained with Giemsa stain. Scale bar = 100 μM. (D) The number of cells per field of view in each well of a 96 well plates is plotted (n = 3). Values represent the mean ± SEM. **P < .01 vs control, ##P < .01 vs treated Cripto, and $$P < .01 vs Cripto+GRP78‐hMSCs. (E) Western blot analysis of CDK 2, Cyclin E, CDK 4, and Cyclin D1 in hMSC, GRP78‐hMSC and Ab‐GRP78 were treated with or without Cripto for 24 hour. Values represent the mean ± SEM. *P < .05 vs control, **P < .01 vs control, #P < .05 vs treated Cripto, ##P < .01 vs treated Cripto and $$P < .01 vs Cripto+GRP78‐hMSCs. (F) hMSC, GRP78‐hMSC and Ab‐GRP78 were treated with or without Cripto for 24 hour. Measurement of CDK4/cyclin D1 kinase activation concentration in hMSCs using ELISA (n = 3). Values represent the mean ± SEM. **P < .01 vs untreated hMSCs. ##P < .01 vs treated Cripto, and $$P < .01 vs Cripto+GRP78‐hMSCs. (G) Images of flow cytometric analysis for PI staining to assess S phase populations in hMSC, GRP78‐hMSC and Ab‐GRP78 treated with Cripto. **P < .01 vs untreated hMSCs. ##P < 0.01 vs treated Cripto and $$P < .01 vs Cripto+GRP78‐hMSCs

Article Snippet: hMSC cultured in 60 mm plates were overexpressed with 3 μg of pcDNA3.1 GRP78 vector (Invitrogen, Carlsbad, CA, USA), using lipofectamine 2000 (Invitrogen).

Techniques: Binding Assay, Western Blot, MTT Assay, Staining, Giemsa Stain, Activation Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Cell Proliferation

Article Title: Administration of Cripto in GRP 78 overexpressed human MSC s enhances stem cell viability and angiogenesis during human MSC transplantation therapy

doi: 10.1111/cpr.12463

Figure Lengend Snippet: Overexpression of GRP78 on human MSCs increased migration and invasion through binding of surface GRP78 with Cripto. (A) Images of scratch wound‐healing migration assay of hMSCs, hMSCs with Cripto, GRP78+hMSC with Cripto and hMSCs with Cripto pre‐treated with Ab‐GRP78 for 0, and 24 hour. Scale bar = 100 μM. (B) The number of migrating cells is presented as the number of migrated cells per filed. Values represent mean ± SEM. **P < .01 vs control, ##P < .01 vs treated Cripto, and $$P < .01 vs Cripto+GRP78‐hMSC. (C) Invasion assay images of hMSCs, hMSCs with Cripto, hMSCs with Cripto pre‐treated with Ab‐GRP78, GRP78+hMSC with Cripto and GRP78+hMSCs with Cripto pre‐treated with Ab‐GRP78 for 24 hour. Scale bar = 100 μM. (D) Values represent the mean ± SEM. *P < .05 vs control, **P < .01 vs control, ##P < .01 vs treated Cripto, and $$P < .01 vs Ab‐GRP78‐hMSCs, &&P < .01 vs Cripto+GRP78‐hMSCs

Article Snippet: hMSC cultured in 60 mm plates were overexpressed with 3 μg of pcDNA3.1 GRP78 vector (Invitrogen, Carlsbad, CA, USA), using lipofectamine 2000 (Invitrogen).

Techniques: Over Expression, Migration, Binding Assay, Invasion Assay

Journal: Cell Proliferation

Article Title: Administration of Cripto in GRP 78 overexpressed human MSC s enhances stem cell viability and angiogenesis during human MSC transplantation therapy

doi: 10.1111/cpr.12463

Figure Lengend Snippet: In vivo, Cripto is overexpressed in hindlimb ischaemia and overexpression of GRP78 on human MSCs increased recruitment of Cripto to MSCs via GRP78 receptor of ischaemia injury site. (A) Images of immunohistochemistry results for Cripto (green) in the ischaemia injury tissue 3 days after the murine hindlimb ischaemia operation. Scale bar = 100 μm. (B) Western blot analysis showing the expression of Cripto in ischaemia injury tissue at post‐operative for 0, 4, 12, 24 and 72 hour. The down panel represents the expression levels of Cripto which were normalized to α‐tubulin. Values represent the mean ± SEM. **P < .01 vs normal tissues. (C) At post‐operative of 3 days, immunohistochemistry assay for HNA (green) and Cripto (red) was performed in the ischaemia injury tissue of each group. Scale bar = 100 μm. (D) Existence of transplanted hMSCs were quantified based on the number of HNA and Cripto double positive cells. Values represent the mean ± SEM. **P < .01 vs. transplanted hMSCs, ##P < .01 vs transplanted overexpression GRP78 on hMSCs

Article Snippet: hMSC cultured in 60 mm plates were overexpressed with 3 μg of pcDNA3.1 GRP78 vector (Invitrogen, Carlsbad, CA, USA), using lipofectamine 2000 (Invitrogen).

Techniques: In Vivo, Over Expression, Immunohistochemistry, Western Blot, Expressing

Journal: Cell Proliferation

Article Title: Administration of Cripto in GRP 78 overexpressed human MSC s enhances stem cell viability and angiogenesis during human MSC transplantation therapy

doi: 10.1111/cpr.12463

Figure Lengend Snippet: Overexpression GRP78 on human MSCs decreased cell apoptosis in murine hindlimb ischaemia. (A) At post‐operative of 3 days, immunohistochemistry assay for PCNA (green) was performed in the ischaemia injury tissue of each group. Scale bar = 100 μm. (B) Cell proliferation was quantified as the number of PCNA positive cells. Values represent the mean ± SEM. **P < .01 vs transplanted hMSCs, ##P < .01 vs transplanted overexpression GRP78 on hMSCs. (C) At post‐operative of 3 days, immunohistochemistry assay for caspase‐3 (red) was performed in the ischaemia injury tissue of each group. Scale bar = 100 μm. (D) Cell apoptosis was quantified as the number of caspase‐3 positive cells. Values represent the mean ± SEM. **P < .01 vs injection of PBS, #P < .05, ##P < .01 vs transplanted hMSCs, and $$P < .01 vs transplanted overexpression GRP78 on hMSCs. (E) Western blot analysis showing the expression of BCL‐2, BAX, cleaved caspase‐3 and cleaved PARP‐1 in ischaemia injury tissue of each group at post‐operative for 3 days. The down panel represents the expression levels of GRP78 were normalized to α‐tubulin. Values represent the mean ± SEM. **P < .01 vs injection of PBS, #P < .05, ##P < .01 vs transplanted hMSCs, and $$P < .01 vs transplanted overexpression GRP78 on hMSCs

Article Snippet: hMSC cultured in 60 mm plates were overexpressed with 3 μg of pcDNA3.1 GRP78 vector (Invitrogen, Carlsbad, CA, USA), using lipofectamine 2000 (Invitrogen).

Techniques: Over Expression, Immunohistochemistry, Injection, Western Blot, Expressing

Journal: Cell Proliferation

Article Title: Administration of Cripto in GRP 78 overexpressed human MSC s enhances stem cell viability and angiogenesis during human MSC transplantation therapy

doi: 10.1111/cpr.12463

Figure Lengend Snippet: Overexpression GRP78 on human MSCs increased secretion of angiogenic cytokines in murine hindlimb ischaemia. (A) Measurements of human VEGF, HGF and FGF concentrations in ischaemia injury tissue of each group at post‐operative for 3 days using ELISA. Ischaemia injury tissue of each group at post‐operative for 3 days. Values represent the mean ± SEM. **P < .01 vs injection of PBS, ##P < .01 vs transplanted hMSCs, and $$P < .01 vs transplanted overexpression GRP78 on hMSCs

Article Snippet: hMSC cultured in 60 mm plates were overexpressed with 3 μg of pcDNA3.1 GRP78 vector (Invitrogen, Carlsbad, CA, USA), using lipofectamine 2000 (Invitrogen).

Techniques: Over Expression, Enzyme-linked Immunosorbent Assay, Injection

Journal: Journal of Dental Sciences

Article Title: Heat stress promotes osteogenic and odontogenic differentiation of stem cells from apical papilla via glucose-regulated protein 78-mediated autophagy

doi: 10.1016/j.jds.2024.05.007

Figure Lengend Snippet: Overexpression of GRP78 promoted osteogenic and odontogenic differentiation of SCAPs. (A) Western blot analysis of DSPP, GRP78, ALP, RUNX2, and OSX in the NC-over group and GRP78-over group. (B) Quantitative analysis of Western blot in (A). (C) qRT-PCR analysis of DSPP, GRP78, ALP, RUNX2, and OSX in the NC-over group and GRP78-over group. (D–E) ALP staining and ALP activity assay on day 7 after osteogenic induction. Scale bar = 100 μm. (F–G) Image and quantification of Alizarin red S staining on day 14 after osteogenic induction. Scale bar = 100 μm. (H) Immunofluorescence staining of ALP, RUNX2, and GRP78. Scale bar = 50 μm ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: GRP78 small interfering RNAs (siGRP78) and overexpression plasmid were designed (Ribobio, Guangzhou, China).

Techniques: Over Expression, Western Blot, Quantitative RT-PCR, Staining, ALP Activity Assay, Immunofluorescence

Journal: Journal of Dental Sciences

Article Title: Heat stress promotes osteogenic and odontogenic differentiation of stem cells from apical papilla via glucose-regulated protein 78-mediated autophagy

doi: 10.1016/j.jds.2024.05.007

Figure Lengend Snippet: Primer sequences for qRT-PCR analysis of gene expression. DSPP, dentin sialophosphoprotein; GRP78, glucose-regulated protein 78; ALP, alkaline phosphatase; RUNX2, RUNX family transcription factor 2; OSX, osterix.

Article Snippet: GRP78 small interfering RNAs (siGRP78) and overexpression plasmid were designed (Ribobio, Guangzhou, China).

Techniques: Gene Expression

Journal: Journal of Dental Sciences

Article Title: Heat stress promotes osteogenic and odontogenic differentiation of stem cells from apical papilla via glucose-regulated protein 78-mediated autophagy

doi: 10.1016/j.jds.2024.05.007

Figure Lengend Snippet: Heat stress enhanced osteogenic and odontogenic differentiation of SCAPs. (A) Western blot analysis of DSPP, RUNX2, and OSX in the NC group and HS group. (B) Quantitative analysis of Western blot in (A). (C) qRT-PCR analysis of DSPP, GRP78, RUNX2, and OSX in the NC group and HS group. (D) Image of Alizarin red S staining after being cultured in OM for 14 days. Scale bar = 100 μm. (E) Quantitative analysis of ARS staining in (D). (F–G) Immunofluorescence staining and quantification of ALP, RUNX2. Scale bar = 50 μm ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: GRP78 small interfering RNAs (siGRP78) and overexpression plasmid were designed (Ribobio, Guangzhou, China).

Techniques: Western Blot, Quantitative RT-PCR, Staining, Cell Culture, Immunofluorescence

Journal: Journal of Dental Sciences

Article Title: Heat stress promotes osteogenic and odontogenic differentiation of stem cells from apical papilla via glucose-regulated protein 78-mediated autophagy

doi: 10.1016/j.jds.2024.05.007

Figure Lengend Snippet: Heat stress upregulated the expression of GRP78. (A) Western blot analysis of GRP78 in the NC group and HS group. (B) Quantitative analysis of Western blot in (A). (C) qRT-PCR analysis of GRP78 in the NC group and HS group. (D) Immunofluorescence staining of GRP78 in the NC group and HS group. Scale bar = 50 μm ∗∗ P < 0.01.

Article Snippet: GRP78 small interfering RNAs (siGRP78) and overexpression plasmid were designed (Ribobio, Guangzhou, China).

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

Journal: Journal of Dental Sciences

Article Title: Heat stress promotes osteogenic and odontogenic differentiation of stem cells from apical papilla via glucose-regulated protein 78-mediated autophagy

doi: 10.1016/j.jds.2024.05.007

Figure Lengend Snippet: Knockdown of GRP78 suppressed osteogenic and odontogenic differentiation of SCAPs. (A) Western blot analysis of DSPP, GRP78, ALP, RUNX2, and OSX in SCAPs transfected with or without GRP78 siRNA. (B) Quantitative analysis of Western blot in (A). (C) qRT-PCR analysis of DSPP, GRP78, ALP, RUNX2, and OSX in SCAPs transfected with or without GRP78 siRNA. (D–E) ALP staining and ALP activity assay after 7 days of osteogenic induction. Scale bar = 100 μm. (F–G) Image and quantification of Alizarin red S staining after 14 days of osteogenic induction. Scale bar = 100 μm. (H) Immunofluorescence staining of ALP and RUNX2. Scale bar = 50 μm ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: GRP78 small interfering RNAs (siGRP78) and overexpression plasmid were designed (Ribobio, Guangzhou, China).

Techniques: Knockdown, Western Blot, Transfection, Quantitative RT-PCR, Staining, ALP Activity Assay, Immunofluorescence

Journal: Journal of Dental Sciences

Article Title: Heat stress promotes osteogenic and odontogenic differentiation of stem cells from apical papilla via glucose-regulated protein 78-mediated autophagy

doi: 10.1016/j.jds.2024.05.007

Figure Lengend Snippet: Overexpression of GRP78 promoted osteogenic and odontogenic differentiation of SCAPs. (A) Western blot analysis of DSPP, GRP78, ALP, RUNX2, and OSX in the NC-over group and GRP78-over group. (B) Quantitative analysis of Western blot in (A). (C) qRT-PCR analysis of DSPP, GRP78, ALP, RUNX2, and OSX in the NC-over group and GRP78-over group. (D–E) ALP staining and ALP activity assay on day 7 after osteogenic induction. Scale bar = 100 μm. (F–G) Image and quantification of Alizarin red S staining on day 14 after osteogenic induction. Scale bar = 100 μm. (H) Immunofluorescence staining of ALP, RUNX2, and GRP78. Scale bar = 50 μm ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: GRP78 small interfering RNAs (siGRP78) and overexpression plasmid were designed (Ribobio, Guangzhou, China).

Techniques: Over Expression, Western Blot, Quantitative RT-PCR, Staining, ALP Activity Assay, Immunofluorescence

Journal: Journal of Dental Sciences

Article Title: Heat stress promotes osteogenic and odontogenic differentiation of stem cells from apical papilla via glucose-regulated protein 78-mediated autophagy

doi: 10.1016/j.jds.2024.05.007

Figure Lengend Snippet: GRP78 siRNA reversed the role of heat stress on committed differentiation of SCAPs. (A) Western blot analysis of DSPP, GRP78, ALP, RUNX2, and OSX in SCAPs with or without heat stress and GRP78 siRNA. (B) Quantitative analysis of Western blot in (A). (C, E) ALP staining and ALP activity assay in SCAPs. Scale bar = 100 μm. (D, F) Image and quantification of Alizarin red S staining. Scale bar = 100 μm ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: GRP78 small interfering RNAs (siGRP78) and overexpression plasmid were designed (Ribobio, Guangzhou, China).

Techniques: Western Blot, Staining, ALP Activity Assay

Journal: Journal of Dental Sciences

Article Title: Heat stress promotes osteogenic and odontogenic differentiation of stem cells from apical papilla via glucose-regulated protein 78-mediated autophagy

doi: 10.1016/j.jds.2024.05.007

Figure Lengend Snippet: Heat stress-induced GRP78 increased the committed differentiation of SCAPs by activating autophagy. (A) Western blot analysis of Beclin-1 and LC3 II/I in SCAPs exposed to heat stress, followed by recovery at 37 °C for 0, 3, 6, or 12 h. (B) Quantitative analysis of Western blot in (A). (C) Western blot analysis of Beclin-1 and LC3 II/I in SCAPs exposed to heat stress with or without 3-MA pretreatment. (D) Quantitative analysis of Western blot in (C). (E) Western blot analysis of GRP78, Beclin-1, and LC3 II/I in SCAPs exposed to heat stress with or without GRP78 siRNA pretreatment. (F) Quantitative analysis of Western blot in (E). (G) TEM analysis of autophagosomes in SCAPs exposed to heat stress after pretreatment with GRP78 siRNA or 3-MA. Autophagosomes were indicated by black arrows. Scale bar = 2 μm; (H) Western blot analysis of DSPP, ALP, RUNX2, and OSX in SCAPs exposed to heat stress with or without 3-MA pretreatment. (I) Quantitative analysis of Western blot in (H). (J–K) ALP staining and ALP activity assay on day 7 after osteogenic induction. Scale bar = 100 μm. (L–M) Image and quantification of Alizarin red S staining on day 14 after osteogenic induction. Scale bar = 100 μm. NS, no significance, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: GRP78 small interfering RNAs (siGRP78) and overexpression plasmid were designed (Ribobio, Guangzhou, China).

Techniques: Western Blot, Staining, ALP Activity Assay

Journal: Neurobiology of aging

Article Title: The Loss of Glucose Regulated Protein 78 (GRP78) during Normal Aging or from siRNA Knockdown Augments Human Alpha-Synuclein (α-syn) Toxicity to Rat Nigral Neurons

doi: 10.1016/j.neurobiolaging.2015.02.018

Figure Lengend Snippet: Aged nigral cells have a reduced capacity to upregulate endogenous GRP78 in response to human α-syn overexpression. Graphs show the level of TH, GRP78 or total α-syn expression in rAAV-human α-syn injected side compared to control rAAV-BV administered in young (2 month) and old (24 month) animals (see immunoblot images in Supplementary Figure 4). Each graph shows the change induced in each of the three proteins in response to human α-syn neurotoxicity. The amount of detected proteins was normalized to GAPDH. Two-way ANOVA analysis. Tukey’ multiple comparisons test are indicated as *, **, *** = P < 0.05, 0.01 and 0.001; N = 4 (for TH and GRP78) and N = 3 (for α-syn) per group.

Article Snippet: Sustained overexpression of GRP78 protects aging nigral neurons against α-syn neurotoxicity To test the hypothesis that a prolonged increase in GRP78 could be therapeutically beneficial for aging nigral DA cells, we injected rAAV vectors in the SNc of 12-month old female Sprague-Dawley rats to overexpress GRP78 and human α-syn simultaneously.

Techniques: Over Expression, Expressing, Injection, Control, Western Blot

Journal: Neurobiology of aging

Article Title: The Loss of Glucose Regulated Protein 78 (GRP78) during Normal Aging or from siRNA Knockdown Augments Human Alpha-Synuclein (α-syn) Toxicity to Rat Nigral Neurons

doi: 10.1016/j.neurobiolaging.2015.02.018

Figure Lengend Snippet: GRP78 expression can be augmented with specific shRNAs. Expression of GRP78 shRNA (A) and human wt α-syn alone (B) and together (C) in the SNc at one month after rAAV injection. (A) Comparison of three different shRNAs (si-1, si-2, si-3) against rat GRP78 and a scrambled control shRNA (si-C) in the SNc of rats at one month after vector injection. All four vectors also express GFP. Pooled (N=3 for each group) nigral tissue extracts were western blotted with GRP78, TH and GAPDH antibodies. The amount of GRP78 was normalized to GAPDH and the ratio of the injected (R) vs uninjected (L) sides was calculated (I/U). Note there was no loss of TH-positive staining in any of the groups at this time point. (B) α-Syn protein level in the SNc at one month after vector injection. Nigral tissue was excised at one month post injection from uninjected (L) and injected (R) sides of individual animals (13–18) that had been treated with rAAV-human α-syn. Nigral tissue extract was western blotted with anti-α-syn antibody that equally recognizes rat and human α-syn (Gorbatyuk et al., 2012). The ratio of α-syn on the injected and uninjected sides (I/U) was calculated for each animal. Note there was no loss of TH-positive staining in any of the animal groups at this time point. (C) Confocal images demonstrate co-expression of both GFP (green) (expressed by the GRP78 shRNA vectors) and human wt α-syn (blue) in TH-positive (red) neurons of the SNc injected with both viruses simultaneously (bar = 150 μm).

Article Snippet: Sustained overexpression of GRP78 protects aging nigral neurons against α-syn neurotoxicity To test the hypothesis that a prolonged increase in GRP78 could be therapeutically beneficial for aging nigral DA cells, we injected rAAV vectors in the SNc of 12-month old female Sprague-Dawley rats to overexpress GRP78 and human α-syn simultaneously.

Techniques: Expressing, shRNA, Injection, Comparison, Control, Plasmid Preparation, Western Blot, Staining

Journal: Neurobiology of aging

Article Title: The Loss of Glucose Regulated Protein 78 (GRP78) during Normal Aging or from siRNA Knockdown Augments Human Alpha-Synuclein (α-syn) Toxicity to Rat Nigral Neurons

doi: 10.1016/j.neurobiolaging.2015.02.018

Figure Lengend Snippet: Degree of decline in GRP78 expression correlates with striatal TH protein levels, nigral DA cell survival and amplitude of behavioral asymmetry in response to human α-syn (αS) overexpression. (A) GRP78 protein expression level in DA neurons of the SNc at 3 months after rAAV-GRP78 shRNA injection. Confocal images show GRP78 (red) and TH (blue) as well as native GFP (green) in nigral neurons at 3 month after combined injection of viruses expressing si-3 and BV. TH is used as a marker for DA cells. Images illustrate protein expression levels of GRP78 (red) in nigral TH+ neurons (blue) on non-injected (left panel) and injected (right panel) sides. (B) The effect of GRP78 decline and human wt α-syn expression on striatal TH protein levels (upper graph), unbiased estimation of nigral TH+ neuron survival as well as striatal DA level and rotational behavior (bottom graph) at 3 months after rAAV injection. Striatal TH-protein level and the number of nigral TH+ cells remaining on the injected side shown as a percentage of the uninjected side ± SE. Amount of striatal TH protein, DA and number of nigral TH+ neurons were most dramatically reduced in si-3 + αS injected rats compared to control si-3 + BV as well as to si-C + αS (and si-2 + αS for TH cells count) injected animals. The amphetamine induced rotation test revealed a significant number of ipsilateral rotations only in rats injected with si-3 + αS versus control si-3 + BV animals. Two-way ANOVA analysis. Tukey’ multiple comparisons test are indicated as *, **, ***, **** = P < 0.05, 0.01, 0,001 and 0.0001, respectively vs. appropriate control rats; N = 6 (for control groups) and 10 (for experimental groups). (C) Bright-field photomicrographs showing remaining TH-positive cells in the SNc of representative animals from different experimental groups. Bar = 250 μm.

Article Snippet: Sustained overexpression of GRP78 protects aging nigral neurons against α-syn neurotoxicity To test the hypothesis that a prolonged increase in GRP78 could be therapeutically beneficial for aging nigral DA cells, we injected rAAV vectors in the SNc of 12-month old female Sprague-Dawley rats to overexpress GRP78 and human α-syn simultaneously.

Techniques: Expressing, Over Expression, shRNA, Injection, Marker, Control

Journal: Neurobiology of aging

Article Title: The Loss of Glucose Regulated Protein 78 (GRP78) during Normal Aging or from siRNA Knockdown Augments Human Alpha-Synuclein (α-syn) Toxicity to Rat Nigral Neurons

doi: 10.1016/j.neurobiolaging.2015.02.018

Figure Lengend Snippet: Overexpression of GRP78 in aging nigral DA neurons diminishes α-syn induced neurodegeneration. (A) Amphetamine-induced rotation test vs time post rAAV injections. Starting at 3 month a significant number of rotations toward the injected side (positive, ipsilateral rotation) was revealed only in α-syn injected rats compared to all other animal groups. Amphetamine induced rotation was measured for 90 min and the average rotation + SE is shown. Two-way ANOVA analysis. Tukey’s multiple comparison test is indicated as +, ## and +++ (### or ***) = P < 0.05, 0.01 and 0.001 vs. BV (#), GRP78 (+) and α-syn plus GRP78 (*); N= 9 per group. (B) GRP78 protein expression in DA neurons of the SNc at 9 months after AAV-GRP78 shRNA injection. Confocal Images illustrate protein expression level of GRP78 (red) in nigral TH-positive (green) neurons on non-injected (left panel) and injected (right panel) sides. (C) Quantitate analysis of striatal TH protein revealed by western blot (left graph, see Supplementary Figure 5). The graph shows the level of TH protein remaining in the striatum on the injected side as a percentage of the uninjected side ± SE at 9 months post injection. Two-way ANOVA analysis. Tukey’s multiple comparison test is indicated as ** and *** (### or +++) = P < 0.01 and 0.001, respectively vs. BV (#), GRP78 (+) and α-syn plus GRP78 (*); N = 8–9 per group. Middle graph shows loss of DA in the striatum injected with a vector expressing α-syn. Overexpression of GRP78 protein along with α-syn significantly (by 49%) preserved DA levels as compared with α-syn injected alone. This was also significantly different from the BV control. Overexpression of GRP78 protein alone did not have an effect on DA in injected SNcs compared to control BV injection. Two-way ANOVA analysis. Tukey’s multiple comparison test is indicated as ** and *** (### or +++) = P < 0.01 and 0.001, respectively vs. BV (#), GRP78 (+) and α-syn plus GRP78 (*); N = 5 (GRP78 control) – 7 (all others) per group. Right graph shows unbiased estimation of TH positive cells remaining in SNc on the injected side as a percentage of the uninjected side ± SE at 9 months post injection. The number of TH-positive neurons counted in the α-syn injected SNc was dramatically reduced compared to control BV rats. Co-expression of α-syn and GRP78 proteins led to significant rescue of TH-positive cells preventing cell death compared to a single human α-syn injection. However, these animals demonstrate a 28% and 24% loss of TH-positive cells compared to BV and GRP78 animals respectively, which is significant in both cases. GRP78 overexpression alone did not reduce the number of TH-positive cells. Two-way ANOVA analysis. Tukey’s multiple comparison test is indicated as ** and *** (### or +++) = P < 0.01 and 0.001, respectively vs. BV (#), GRP78 (+) and α-syn plus GRP78 (*); N = 8–9 per group. (D) Bright-field photomicrographs showing remaining TH-positive cells and human α-syn immunostaining in the SNc of representative animals from different experimental groups.

Article Snippet: Sustained overexpression of GRP78 protects aging nigral neurons against α-syn neurotoxicity To test the hypothesis that a prolonged increase in GRP78 could be therapeutically beneficial for aging nigral DA cells, we injected rAAV vectors in the SNc of 12-month old female Sprague-Dawley rats to overexpress GRP78 and human α-syn simultaneously.

Techniques: Over Expression, Injection, Comparison, Expressing, shRNA, Western Blot, Plasmid Preparation, Control, Immunostaining

Journal: Journal of Clinical and Translational Hepatology

Article Title: Roles of SET7/9 and LSD1 in the Pathogenesis of Arsenic-induced Hepatocyte Apoptosis

doi: 10.14218/jcth.2020.00185

Figure Lengend Snippet: Fig. 2. Effect of arsenic on ERS-related proteins in L02 hepatocytes. Cells were exposed to arsenic at 100 µmol/L for 24 h. Proteins, prepared from whole cell extracts, were analyzed by western blotting. The activities of GRP78, CHOP, and LSD1 were determined with respective specific antibodies. (A) SET7/9 protein knock-down efficiency. CHOP expression was measured in control, model and SET7/9 knock-down in L02 cells. (B) Data shown are mean±standard deviation of three independent experiments, *p<0.05 vs. control group; #p<0.05 vs. negative transfection group. (C) LSD1 protein knock-down and overexpression efficiency. GRP78, CHOP, and LSD1 expression was measured in control, model and LSD1 knock-down and OG-L002 group of L02 cells. (D) Data shown are mean±standard deviation of three independent experiments, *p<0.05 vs. control group; #p<0.05 vs. negative transfection group.

Article Snippet: Fetal bovine serum, Dulbecco’s modified Eagle’s medium (GIBCO, New York, NY, USA), antibodies against GRP78, C/EBP-homologous protein (CHOP), LSD1, H3 and H3K4me1 (Abcam, Cambridge, UK), antibodies against H3K4me2 (Active Motif, Carlsbad, CA, USA), GAPDH (Bioprimacy Biotechnology, Wuhan, China), secondary antibodies (Boster Biological Engineering, Wuhan, China), SET7/9-specific small interfering RNAs (siRNAs) were purchased from Shanghai Jima Gene (China), negative small hairpin (sh)RNA and LSD1 overexpressed plasmids were obtained from Shanghai Jima Gene.

Techniques: Western Blot, Knockdown, Expressing, Control, Standard Deviation, Transfection, Over Expression

Journal: Journal of Clinical and Translational Hepatology

Article Title: Roles of SET7/9 and LSD1 in the Pathogenesis of Arsenic-induced Hepatocyte Apoptosis

doi: 10.14218/jcth.2020.00185

Figure Lengend Snippet: Fig. 5. Effect of H3K4me1 and H3K4me2 on GRP78, ATF4, and CHOP promoter activity in L02 hepatocytes. (A) Changes in enrichment of H3K4me1 and H3K4me2 in the GRP78 promoter region. Data shown are mean±standard deviation of three independent experiments; *p<0.05 vs. control group, **p<0.01 vs. control group. (B) Changes of the enrichment of H3K4me1 and H3K4me2 in the ATF4 promoter region. Data shown are mean±standard deviation of three independent experi- ments; * p<0.05 vs. control group, **p<0.01 vs. control group. (C) Changes of the enrichment of H3K4me1 and H3K4me2 in the CHOP promoter region. Data shown are mean±standard deviation of three independent experiments; *p<0.05 vs. control group, **p<0.01 vs. control group.

Article Snippet: Fetal bovine serum, Dulbecco’s modified Eagle’s medium (GIBCO, New York, NY, USA), antibodies against GRP78, C/EBP-homologous protein (CHOP), LSD1, H3 and H3K4me1 (Abcam, Cambridge, UK), antibodies against H3K4me2 (Active Motif, Carlsbad, CA, USA), GAPDH (Bioprimacy Biotechnology, Wuhan, China), secondary antibodies (Boster Biological Engineering, Wuhan, China), SET7/9-specific small interfering RNAs (siRNAs) were purchased from Shanghai Jima Gene (China), negative small hairpin (sh)RNA and LSD1 overexpressed plasmids were obtained from Shanghai Jima Gene.

Techniques: Activity Assay, Standard Deviation, Control