Journal: Clinical and Translational Science

Article Title: Identification of BAG2 and Cathepsin D as Plasma Biomarkers for Parkinson’s Disease

doi: 10.1111/cts.12920

Figure Lengend Snippet: Average protein levels of BAG2 and cathepsin D are decreased in the plasma of PD patients. ( a ) Preparation of the albumin/IgG‐depleted plasma samples from control and PD patients (20 subjects each) was verified by SDS‐PAGE. The plasma samples from the 20 control and patients with PD were each pooled for the quantitative analysis of the average levels of autophagy‐related proteins. The pooled samples were immunoblotted with antibodies specific to BAG2 ( b ), BAG3 ( c ), cathepsin B ( d ), cathepsin D ( e ), LAMP1 ( f ), LAMP2 ( g ), and WDFY3 ( h ). Each analysis was repeated as necessary. Arrows and asterisks indicate specific and cross‐reacting bands, respectively. ( i ) Intensities of specific bands of b–h were quantitatively analyzed, and the level of changes in the PD group was compared to that in the control group. Each blue dot indicates the result from each blot, and the grey bars represent mean values. ( j ) The fold change in BAG2 levels of the PD group. Values represent mean ± SEM of three independent experiments. ( k ) The fold change in cathepsin D levels of the PD group. Values represent mean ± SEM of two independent experiments. Molecular weight standards (in kDa) are shown to the left. ** P < 0.01 (Student t ‐test). BAG, BCL2‐associated athanogene; CBB, Coomassie Brilliant Blue; PD, Parkinson’s disease; SDS‐PAGE, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis.

Article Snippet: Primary antibodies specific to BCL2‐associated athanogene (BAG2; Bethyl Cat #A304‐751A), BAG3 (Bethyl Cat #A302‐807A), cathepsin B (Abcam Cat #ab58802), cathepsin D (Calbiochem Cat #IM‐03), LAMP1 (Abcam Cat #ab25630), LAMP2 (Proteintech Cat #66301‐1‐Ig), WDFY3 (Bethyl Cat #A301‐869A), and α‐Tubulin (Abcam Cat #ab18251) were purchased.

Techniques: Clinical Proteomics, Control, SDS Page, Molecular Weight, Polyacrylamide Gel Electrophoresis

Journal: Clinical and Translational Medicine

Article Title: Therapeutic induction of Bcl2‐associated athanogene 3‐mediated autophagy in idiopathic pulmonary fibrosis

doi: 10.1002/ctm2.935

Figure Lengend Snippet: BAG3‐mediated autophagy is insufficient in IPF fibroblasts. (A) Immunoblot analysis of BAG3, BAG1 or β‐actin from lung homogenates (LH) of IPF patients or healthy donors (HD) (left). The right part shows respective quantifications after normalizing their integrated density values (IDV) from n = 9 IPF patients and 5 HD. (B) Immunoblots from cell lysates of primary interstitial fibroblasts of IPF or HD for the indicated proteins. The right part shows respective IDVs that were normalized to β‐actin from n = 8 each for IPF and HD. p value summary: * p ≤0 .05, ** p ≤ 0.01, *** p ≤0.001. Abbreviation : ns, not significant.

Article Snippet: Nontargeting control siRNA and BAG3 siRNA were obtained from Santa Cruz Biotechnology. siRNA transfections were performed using Lipofectamine 2000 following manufacturer's protocols and as described before for 48 h followed by treatments with the indicated drugs for additional 24 h.

Techniques: Western Blot

Journal: Clinical and Translational Medicine

Article Title: Therapeutic induction of Bcl2‐associated athanogene 3‐mediated autophagy in idiopathic pulmonary fibrosis

doi: 10.1002/ctm2.935

Figure Lengend Snippet: Pirf, Aza and Ctd activate BAG3‐mediated autophagy and decrease IPF fibroblast proliferation. (A) IPF interstitial fibroblasts were treated with Veh, Pirf, Aza or Ctd alone or Pirf in combination with Aza or Ctd as indicated for 24 h. Cells were lysed, and immunoblotting was performed for the indicated proteins (left). Relative protein amounts from five IPF patients were normalized to β‐actin and their fold change was obtained after the values in Veh‐treated controls was set as one (right). (B) Representative immunofluorescence staining for PCNA (red) in IPF fibroblasts after treating with Veh or the indicated drugs for 24 h. Nuclei were stained with DAPI (blue), scale bar = 25 µm. In the right panel, fluorescence intensity of PCNA was quantified using ImageJ. Analysis was performed from n = 5 IPF patient fibroblasts. (C) Proliferation of IPF fibroblasts ( n = 5) as assessed by BrdU incorporation following indicated drug treatments for 24 h. Triplicates were measured, and absorbance values were set to one in Veh‐treated cells for the ease of understanding. (D) Analysis of BAG3 mRNA using qRT‐PCR in fibroblasts derived from IPF or HD lungs after treating them with the indicated drugs. Values were normalized to the house‐keeping gene β‐actin ( ACTB ). BAG3 mRNA expression in Veh‐treated IPF/HD fibroblasts was set as one. p ‐Value summary: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 show significant difference as compared to Veh‐treated cells and § p ≤ 0.05, §§ p ≤ 0.01, §§§ p ≤ 0.001 indicate significant difference as compared to Pirf‐treated cells

Article Snippet: Nontargeting control siRNA and BAG3 siRNA were obtained from Santa Cruz Biotechnology. siRNA transfections were performed using Lipofectamine 2000 following manufacturer's protocols and as described before for 48 h followed by treatments with the indicated drugs for additional 24 h.

Techniques: Western Blot, Immunofluorescence, Staining, Fluorescence, BrdU Incorporation Assay, Quantitative RT-PCR, Derivative Assay, Expressing

Journal: Clinical and Translational Medicine

Article Title: Therapeutic induction of Bcl2‐associated athanogene 3‐mediated autophagy in idiopathic pulmonary fibrosis

doi: 10.1002/ctm2.935

Figure Lengend Snippet: Therapeutic modulation but not siBAG3 per se affects autophagy in IPF fibroblasts. (A) IPF fibroblasts were transfected either with nontargeting siRNA (si‐NT) or with BAG3 siRNA (si‐BAG3) for 48 h followed by treatments with the indicated drugs for 24 h. Cell lysates were prepared, and western blots were performed for the indicated proteins. Bar graphs represent IDVs of BAG3, FLNC, LC3BII or α‐SMA that were normalized to β‐actin. Blots and analysis were performed from n = 3 IPF patient fibroblasts. p Value summary: *** p ≤ 0.001, significant difference between si‐NT and si‐BAG3 groups. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 show significant difference between Veh‐treated and the respective drug treated groups in cells transfected with si‐NT. § p ≤ 0.05, §§ p ≤ 0.01, §§§ p ≤ 0.001 show significant difference between si‐BAG3 transfected cells with the indicated drug treatments versus their respective drug‐treated counterparts in si‐NT transfected cells

Article Snippet: Nontargeting control siRNA and BAG3 siRNA were obtained from Santa Cruz Biotechnology. siRNA transfections were performed using Lipofectamine 2000 following manufacturer's protocols and as described before for 48 h followed by treatments with the indicated drugs for additional 24 h.

Techniques: Transfection, Western Blot

Journal: Clinical and Translational Medicine

Article Title: Therapeutic induction of Bcl2‐associated athanogene 3‐mediated autophagy in idiopathic pulmonary fibrosis

doi: 10.1002/ctm2.935

Figure Lengend Snippet: Therapeutic modulation of BAG3 decreases proliferation and COL1A1 in IPF PCLS. PCLS from explanted IPF patient lungs were treated with drugs, as indicated for 24 h. (A) PCLS lysates were prepared and tested for the indicated proteins via western blotting (top panel). Lower panels represent the quantifications of the indicated proteins after with β‐actin. Values in Veh‐treated PCLS were set as one. Treatment and representative analysis and blots from n = 3 IPF patients are shown. (B) Immunofluorescence staining for COL1A1 (red) on IPF PCLS upon Veh or drug treatments for 24 h has indicated. Nuclei were stained with DAPI (blue), scale bar = 200 µm. Lower panel indicates COL1A1 fluorescence intensity after drug treatments. Stainings and analysis were performed from n = 3 IPF PCLS per each group and about three technical replicates were performed. p Value summary: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 show significant difference as compared to Veh‐treated PCLS and § p ≤ 0.05, §§ p ≤ 0.01, §§§ p ≤ 0.001 indicate significant difference as compared to Pirf‐treated PCLS

Article Snippet: Nontargeting control siRNA and BAG3 siRNA were obtained from Santa Cruz Biotechnology. siRNA transfections were performed using Lipofectamine 2000 following manufacturer's protocols and as described before for 48 h followed by treatments with the indicated drugs for additional 24 h.

Techniques: Western Blot, Immunofluorescence, Staining, Fluorescence

Journal: Clinical and Translational Medicine

Article Title: Therapeutic induction of Bcl2‐associated athanogene 3‐mediated autophagy in idiopathic pulmonary fibrosis

doi: 10.1002/ctm2.935

Figure Lengend Snippet: Schematic representation of this study. Restoring insufficient BAG3‐mediated autophagy in IPF fibroblasts by the indicated drugs. The slope represents the synergistic effect of the indicated drugs on BAG3 inhibition. Small font indicates decreased expression or decreased autophagy flux and vice versa. To the left, highly proliferative IPF fibroblasts (green) are depicted, enwrapped in thick extracellular matrix (brown) with increased BAG3, FLNC and COL1A1 and insufficient BAG3‐mediated autophagy. To the right, decreased IPF fibroblast proliferation, decreased FLNC, COL1A1 and BAG3 protein levels and restored autophagy upon treatments with the indicated drugs is shown

Article Snippet: Nontargeting control siRNA and BAG3 siRNA were obtained from Santa Cruz Biotechnology. siRNA transfections were performed using Lipofectamine 2000 following manufacturer's protocols and as described before for 48 h followed by treatments with the indicated drugs for additional 24 h.

Techniques: Inhibition, Expressing

Journal: Virologica Sinica

Article Title: Autophagy induced by human adenovirus B7 structural protein VI inhibits viral replication.

doi: 10.1016/j.virs.2023.08.002

Figure Lengend Snippet: Fig. 5. pVI of HAdV-B7 interacted with the host protein BAG3. A HEK 293T cells were transfected with plasmids encoding BAG3-HA (2 μg) and pVI-Flag (2 μg) or vector-Flag (2 μg) for 24 h, followed by Co-IP with anti-Flag binding beads and immunoblot analysis with the indicated antibodies. B HEK 293T cells were transfected with plasmids encoding pVI-Flag (2 μg) and BAG3-HA (2 μg) or vector-HA (2 μg) for 24 h, followed by Co-IP with anti-HA binding beads and immunoblot analysis with the indicated antibodies. C A549 cells were transfected with plasmids encoding BAG3-HA (2 μg) and pVI-Flag (2 μg) or vector-Flag (2 μg) for 24 h, followed by Co-IP with anti-Flag binding beads and immunoblot analysis with the indicated antibodies. D A549 cells were transfected with plasmids encoding pVI-Flag (2 μg) and BAG3- HA (2 μg) or vector-HA (2 μg) for 24 h, followed by Co-IP with anti-HA binding beads and immunoblot analysis with the indicated antibodies. E HEK 293T cells were transfected with vector-Flag (2 μg) or pVI-Flag (2 μg). Cell lysates were evaluated by Western blotting using specific antibodies against BAG3 and LC3. Representative immunoblots were showed and densitometric analysis of the relative protein expression were from three independent experiments. *, P < 0.05. F HEK 293T cells were transfected with plasmids encoding pVI-Flag (2 μg) and BAG3-HA (2 μg), followed by labeling Flag, HA and LAMP1 with a specific primary antibody. The cell nucleus were stained with DAPI. Fluorescence signals were observed using confocal immunofluorescence microscopy. G Quantitative analysis of colocalized immunofluo- rescence intensity was using ImageJ, and approximately 25–30 cells in total for each condition were used for quantification. *, P < 0.05. Scale bars: 5 μm.

Article Snippet: Rabbit antiSQSTM1/p62 antibody (5114S), rabbit anti-BAG3 antibody (23842), mouse anti-LAMP1 antibody (15665), rabbit anti-DYKDDDDK/FLAG tag antibody (14793), and rabbit anti-Atg5 antibody (12994) were purchased from Cell Signaling Technology (Massachusetts, USA).

Techniques: Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Binding Assay, Western Blot, Expressing, Labeling, Staining, Fluorescence, Microscopy

Journal: Virologica Sinica

Article Title: Autophagy induced by human adenovirus B7 structural protein VI inhibits viral replication.

doi: 10.1016/j.virs.2023.08.002

Figure Lengend Snippet: Fig. 6. BAG3 interacted with pVI in a WW domain-dependent manner. A Schematic diagram of BAG3-WT and the truncation mutants BAG3-ΔN and BAG3-ΔC, highlighting the locations of the functional domains, including the N-terminal WW domain (blue) and the C-terminal BAG domain (red). All three proteins contained HA tags. B HEK 293T cells transfected with the indicated plasmid (2 μg) and BAG3-WT or truncation mutant proteins were detected by Western blotting. C Extracts from HEK 293T cells transfected with the indicated plasmid combinations were first immunoprecipitated with anti-Flag binding beads. BAG3-WT or truncation mutant proteins were detected in the precipitates by Western blotting using an anti-HA-specific primary antibody. D Extracts from HEK 293T cells transfected with the indicated plasmid combinations were first immunoprecipitated with anti-HA binding beads. pVI was detected in the precipitates by Western blotting using an anti- Flag-specific primary antibody.

Article Snippet: Rabbit antiSQSTM1/p62 antibody (5114S), rabbit anti-BAG3 antibody (23842), mouse anti-LAMP1 antibody (15665), rabbit anti-DYKDDDDK/FLAG tag antibody (14793), and rabbit anti-Atg5 antibody (12994) were purchased from Cell Signaling Technology (Massachusetts, USA).

Techniques: Functional Assay, Transfection, Plasmid Preparation, Mutagenesis, Western Blot, Immunoprecipitation, Binding Assay

Journal: Virologica Sinica

Article Title: Autophagy induced by human adenovirus B7 structural protein VI inhibits viral replication.

doi: 10.1016/j.virs.2023.08.002

Figure Lengend Snippet: Fig. 7. BAG3 interacted with the PPSY structural domain of pVI through its WW structural domain. A Schematic diagram of pVI-WT, pVI truncation mutants, and PPSY mutants (PAGG). All five proteins contained Flag tags. B HEK 293T cells transfected with the indicated plasmid (2 μg) and pVI-WT or truncation mutant proteins were detected by Western blotting. C Extracts from HEK 293T cells transfected with the indicated plasmid combinations were first immunoprecipitated with anti-Flag binding beads. BAG3-ΔC was detected in the precipitates by Western blotting using an anti-HA-specific primary antibody. D Extracts from HEK 293T cells transfected with the indicated plasmid combinations were first immunoprecipitated with anti-HA binding beads. pVI-WT or mutant proteins were detected in the precipitates by Western blotting using an anti-Flag-specific primary antibody. E Extracts from HEK 293T cells transfected with the indicated plasmid combinations were first immunoprecipitated with anti-Flag binding beads. BAG3-WT or mutant proteins were detected in the precipitates by Western blotting using an anti-HA-specific primary antibody. F Extracts from HEK 293T cells transfected with the indicated plasmid combinations were first immunoprecipitated with anti-HA binding beads. pVI 1–170 was detected in the precipitates by Western blotting using an anti-Flag-specific primary antibody.

Article Snippet: Rabbit antiSQSTM1/p62 antibody (5114S), rabbit anti-BAG3 antibody (23842), mouse anti-LAMP1 antibody (15665), rabbit anti-DYKDDDDK/FLAG tag antibody (14793), and rabbit anti-Atg5 antibody (12994) were purchased from Cell Signaling Technology (Massachusetts, USA).

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Western Blot, Immunoprecipitation, Binding Assay

Journal: Virologica Sinica

Article Title: Autophagy induced by human adenovirus B7 structural protein VI inhibits viral replication.

doi: 10.1016/j.virs.2023.08.002

Figure Lengend Snippet: Fig. 8. BAG3-induced autophagy inhibited viral replication. A549 cells (A) and 16HBE cells (B) were transfected with si BAG3 or siNC. Cell lysates were subjected to Western blotting analysis 24 h after transfection. A549 cells (C) and 16HBE cells (D) were transfected with plasmids encoding BAG3-HA or vector-HA for 24 h. Cell lysates were subjected to Western blotting analysis at 24 h after transfection to detect the expression levels of BAG3, p62, LC3, DBP by using specific antibodies. Representative immunoblots were showed and densitometric analysis of the relative protein expression were from three independent experiments. Statistical sig- nificance was analyzed by Student's t-test, *, P < 0.05; **, P < 0.01. A549 cells (E) and 16HBE cells (F) were transfected with si BAG3 or siNC. Cell lysates were subjected to qPCR analysis using E1A-specific primers. A549 cells (G) and 16HBE cells (H) were transfected with plasmids encoding BAG3-HA or vector-HA for 24 h. Cell lysates were subjected to qPCR analysis using E1A-specific primers. Quantification of the relative mRNA levels of E1A from three independent experiments. Statistical significance was analyzed by Student's t-test, **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Rabbit antiSQSTM1/p62 antibody (5114S), rabbit anti-BAG3 antibody (23842), mouse anti-LAMP1 antibody (15665), rabbit anti-DYKDDDDK/FLAG tag antibody (14793), and rabbit anti-Atg5 antibody (12994) were purchased from Cell Signaling Technology (Massachusetts, USA).

Techniques: Transfection, Western Blot, Plasmid Preparation, Expressing

Journal: Chemmedchem

Article Title: Identification of a New Interesting BAG3 Modulator Able to Disrupt Cancer‐Related Pathways

doi: 10.1002/cmdc.202500310

Figure Lengend Snippet: Schematic illustration of the BAG3 protein sequence. Gray boxes highlight specific domains: WW (with two conserved tryptophan residues), IPV (a tripeptide motif isoleucine‐proline‐valine), PxxP (a proline‐rich motif). Black bars indicate LiP (limited proteolysis) peptides identified as involved in binding interactions, located from amino acids 231 to 249, 432 to 445, and 461 to 473, respectively.

Article Snippet: Recombinant human BAG3 protein (Bcl2‐associated athanogene 3) was obtained from Novus Biologicals (Littleton, Colorado), while the BAG3 domain (BAG3‐BD) was sourced from ARETA International S.r.l. (Gerenzano, Italy).

Techniques: Sequencing, Binding Assay

Journal: Chemmedchem

Article Title: Identification of a New Interesting BAG3 Modulator Able to Disrupt Cancer‐Related Pathways

doi: 10.1002/cmdc.202500310

Figure Lengend Snippet: Binding mode of a) SP18 (colored by atom type: C green, N blue, O red), b) compound 1 (colored by atom type: C faded magenta, N blue, O red), c) compound 2 (colored by atom type: C orange, N blue, O red), d) compound 5 (colored by atom type: C azure, N blue, O red), e) compound 6 (colored by atom type: C violet, N blue, O red, Cl green), f) compound 7 (colored by atom type: C yellow, N blue, O red, Br dark red), g) compound 8 (colored by atom type: C cyan, N blue, O red, I magenta). H bonds, halogen bonds, and π interactions are reported in yellow, violet, and green dotted lines, respectively. Residues corresponding to the sequence of murine BAG3 BD as renumbered in the Protein Data Bank (PDB code: 1UK5) and corresponding residues belonging to human BAG3 BD are reported in black and red, respectively.

Article Snippet: Recombinant human BAG3 protein (Bcl2‐associated athanogene 3) was obtained from Novus Biologicals (Littleton, Colorado), while the BAG3 domain (BAG3‐BD) was sourced from ARETA International S.r.l. (Gerenzano, Italy).

Techniques: Binding Assay, Sequencing

Journal: BMC Cancer

Article Title: Bcl-2-associated athanogene 3(BAG3) is associated with tumor cell proliferation, migration, invasion and chemoresistance in colorectal cancer

doi: 10.1186/s12885-018-4657-2

Figure Lengend Snippet: BAG3 protein expression in colorectal cancer tissues and adjacent non-tumor tissues

Article Snippet: The slides were probed with BAG3 primary antibody (1:100, Cat # NBPI-866442, Novus Biologicals, USA) at 4 °C overnight and then incubated with secondary antibody (1:5000, rabbit, Amersham Biosciences) followed by DAB kit (Life Technologies, USA).The PBS solution was used as negative control.

Techniques: Expressing

Journal: BMC Cancer

Article Title: Bcl-2-associated athanogene 3(BAG3) is associated with tumor cell proliferation, migration, invasion and chemoresistance in colorectal cancer

doi: 10.1186/s12885-018-4657-2

Figure Lengend Snippet: Kaplan-Meier analysis of overall survival(months) in 90 patients with high and low BAG3 expression. BAG3 protein expression in tumor tissue is not associated with colorectal cancer patient prognosis ( P = 0.069 > 0.05)

Article Snippet: The slides were probed with BAG3 primary antibody (1:100, Cat # NBPI-866442, Novus Biologicals, USA) at 4 °C overnight and then incubated with secondary antibody (1:5000, rabbit, Amersham Biosciences) followed by DAB kit (Life Technologies, USA).The PBS solution was used as negative control.

Techniques: Expressing

Journal: BMC Cancer

Article Title: Bcl-2-associated athanogene 3(BAG3) is associated with tumor cell proliferation, migration, invasion and chemoresistance in colorectal cancer

doi: 10.1186/s12885-018-4657-2

Figure Lengend Snippet: Univariate and multivariate Cox regression proportional hazards analysis

Article Snippet: The slides were probed with BAG3 primary antibody (1:100, Cat # NBPI-866442, Novus Biologicals, USA) at 4 °C overnight and then incubated with secondary antibody (1:5000, rabbit, Amersham Biosciences) followed by DAB kit (Life Technologies, USA).The PBS solution was used as negative control.

Techniques: Expressing

Journal: BMC Cancer

Article Title: Bcl-2-associated athanogene 3(BAG3) is associated with tumor cell proliferation, migration, invasion and chemoresistance in colorectal cancer

doi: 10.1186/s12885-018-4657-2

Figure Lengend Snippet: BAG3 stable overexpression in HCT-116 cells. a The relative expression of BAG3 mRNA in cells. b Western blot analysis of BAG3 overexpression in HCT-116 cells. Data represent the mean ± S.D. from three independent experiments

Article Snippet: The slides were probed with BAG3 primary antibody (1:100, Cat # NBPI-866442, Novus Biologicals, USA) at 4 °C overnight and then incubated with secondary antibody (1:5000, rabbit, Amersham Biosciences) followed by DAB kit (Life Technologies, USA).The PBS solution was used as negative control.

Techniques: Over Expression, Expressing, Western Blot

Journal: BMC Cancer

Article Title: Bcl-2-associated athanogene 3(BAG3) is associated with tumor cell proliferation, migration, invasion and chemoresistance in colorectal cancer

doi: 10.1186/s12885-018-4657-2

Figure Lengend Snippet: Overexpression of BAG3 promotes HCT-116 cell growth in vitro. a Cell counting was used to analyze cell proliferation. b RTCA assay was performed to record the cell survival curves. c Colony formation assay was performed to investigate colony formation ability in HCT-116 cells. d Quantitative results of colony formation analyzed with Image J. e EdU assay were used to examine cell viability. f Quantitative results of EdU assay analyzed with Image J. Data represent the mean ± S.D. from three independent experiments. * P < 0.05; ** P < 0.01

Article Snippet: The slides were probed with BAG3 primary antibody (1:100, Cat # NBPI-866442, Novus Biologicals, USA) at 4 °C overnight and then incubated with secondary antibody (1:5000, rabbit, Amersham Biosciences) followed by DAB kit (Life Technologies, USA).The PBS solution was used as negative control.

Techniques: Over Expression, In Vitro, Cell Counting, Colony Assay, EdU Assay

Journal: BMC Cancer

Article Title: Bcl-2-associated athanogene 3(BAG3) is associated with tumor cell proliferation, migration, invasion and chemoresistance in colorectal cancer

doi: 10.1186/s12885-018-4657-2

Figure Lengend Snippet: Knockout of BAG3 expression in HCT-116 cells. a A fluorescence microscope was used to confirm the transfection efficiency in HCT-116 cells after 72 h of lentiviral transfection. b DNA agarose gel electrophoresis was performed to confirm the knockout of the target gene. c Western blot analysis was used to examine the expression of BAG3 protein in knockout cells. Data represent mean ± S.D. from three independent experiments

Article Snippet: The slides were probed with BAG3 primary antibody (1:100, Cat # NBPI-866442, Novus Biologicals, USA) at 4 °C overnight and then incubated with secondary antibody (1:5000, rabbit, Amersham Biosciences) followed by DAB kit (Life Technologies, USA).The PBS solution was used as negative control.

Techniques: Knock-Out, Expressing, Fluorescence, Microscopy, Transfection, Agarose Gel Electrophoresis, Western Blot

Journal: BMC Cancer

Article Title: Bcl-2-associated athanogene 3(BAG3) is associated with tumor cell proliferation, migration, invasion and chemoresistance in colorectal cancer

doi: 10.1186/s12885-018-4657-2

Figure Lengend Snippet: BAG3 knockout inhibits colorectal cancer cells growth in vitro . a Cell counting was used to analyze cell proliferation. b RTCA assay was performed to record the cell survival curves. c Colony formation assay was performed to investigate the colony formation ability of HCT-116 cells with BAG3 knockout. d Quantitative results of colony formation were analyzed with Image J. e EdU assay was used to examine cell viability. f The quantitative results of the EdU assay were analyzed with Image J. Data represent the mean ± S.D. from three independent experiments. * P < 0.05; ** P < 0.01

Article Snippet: The slides were probed with BAG3 primary antibody (1:100, Cat # NBPI-866442, Novus Biologicals, USA) at 4 °C overnight and then incubated with secondary antibody (1:5000, rabbit, Amersham Biosciences) followed by DAB kit (Life Technologies, USA).The PBS solution was used as negative control.

Techniques: Knock-Out, In Vitro, Cell Counting, Colony Assay, EdU Assay

Journal: BMC Cancer

Article Title: Bcl-2-associated athanogene 3(BAG3) is associated with tumor cell proliferation, migration, invasion and chemoresistance in colorectal cancer

doi: 10.1186/s12885-018-4657-2

Figure Lengend Snippet: BAG3 overexpression promotes the invasion and migration of HCT-116 cells in vitro. a A wound-healing assay was performed to investigate the migration ability of HCT-116 cells with BAG3 overexpression. b A transwell invasion assay was performed to investigate the invasive ability of HCT-116 cells with BAG3 overexpression. c The quantitative result of the transwell invasion assay was analyzed with Image J. Data represent the mean ± S.D. from three independent experiments. * P < 0.05; ** P < 0.01

Article Snippet: The slides were probed with BAG3 primary antibody (1:100, Cat # NBPI-866442, Novus Biologicals, USA) at 4 °C overnight and then incubated with secondary antibody (1:5000, rabbit, Amersham Biosciences) followed by DAB kit (Life Technologies, USA).The PBS solution was used as negative control.

Techniques: Over Expression, Migration, In Vitro, Wound Healing Assay, Transwell Invasion Assay

Journal: BMC Cancer

Article Title: Bcl-2-associated athanogene 3(BAG3) is associated with tumor cell proliferation, migration, invasion and chemoresistance in colorectal cancer

doi: 10.1186/s12885-018-4657-2

Figure Lengend Snippet: BAG3 knockout inhibits the invasion and migration of HCT-116 cells in vitro. a A wound-healing assay was performed to investigate the invasive ability of HCT-116 cells. b A transwell assay was performed to investigate the invasive ability of HCT-116 cells. c The quantitative result of the transwell assay was analyzed with Image J. Data represent the mean ± S.D. from three independent experiments. * P < 0.05; ** P < 0.01

Article Snippet: The slides were probed with BAG3 primary antibody (1:100, Cat # NBPI-866442, Novus Biologicals, USA) at 4 °C overnight and then incubated with secondary antibody (1:5000, rabbit, Amersham Biosciences) followed by DAB kit (Life Technologies, USA).The PBS solution was used as negative control.

Techniques: Knock-Out, Migration, In Vitro, Wound Healing Assay, Transwell Assay

Journal: BMC Cancer

Article Title: Bcl-2-associated athanogene 3(BAG3) is associated with tumor cell proliferation, migration, invasion and chemoresistance in colorectal cancer

doi: 10.1186/s12885-018-4657-2

Figure Lengend Snippet: Influence of BAG3 expression levels on HCT-116 chemoresistance to 5-FU treatment. HCT-116 cells with BAG3 overexpression ( a ) or BAG3 knokout ( b ) treated with 5-FU in different concentrations and duration. MTT assay was used to detect cell viability. The apoptosis level in HCT-116 cells with BAG3 overexpression ( c ) or knockout ( d ) and control treated with 5-FU in different concentrations and duration. * P < 0.05

Article Snippet: The slides were probed with BAG3 primary antibody (1:100, Cat # NBPI-866442, Novus Biologicals, USA) at 4 °C overnight and then incubated with secondary antibody (1:5000, rabbit, Amersham Biosciences) followed by DAB kit (Life Technologies, USA).The PBS solution was used as negative control.

Techniques: Expressing, Over Expression, MTT Assay, Knock-Out, Control

Journal: BMC Cancer

Article Title: Bcl-2-associated athanogene 3(BAG3) is associated with tumor cell proliferation, migration, invasion and chemoresistance in colorectal cancer

doi: 10.1186/s12885-018-4657-2

Figure Lengend Snippet: Microarray analysis of changes in the gene expression and signaling pathways affected by BAG3 knockout in HCT-116 cells. a Heat map representation of 1224 genes with significant differential expression in HCT-116 cells with or without BAG3 knockout. b Functional pathway enrichment analysis using IPA. c Microarray data were further confirmed by qRT-PCR analysis of selected genes. d Gene interaction network in HCT-116 cells after BAG3 knockout. Green represents downregulated genes, while red represents upregulated genes. The solid lines represent direct interactions, while the dotted lines represent indirect interactions

Article Snippet: The slides were probed with BAG3 primary antibody (1:100, Cat # NBPI-866442, Novus Biologicals, USA) at 4 °C overnight and then incubated with secondary antibody (1:5000, rabbit, Amersham Biosciences) followed by DAB kit (Life Technologies, USA).The PBS solution was used as negative control.

Techniques: Microarray, Gene Expression, Protein-Protein interactions, Knock-Out, Quantitative Proteomics, Functional Assay, Quantitative RT-PCR

Journal: BMC Cancer

Article Title: Bcl-2-associated athanogene 3(BAG3) is associated with tumor cell proliferation, migration, invasion and chemoresistance in colorectal cancer

doi: 10.1186/s12885-018-4657-2

Figure Lengend Snippet: BAG3 protein expression and clinicopathological features of colorectal cancer patients

Article Snippet: The slides were probed with BAG3 primary antibody (1:100, Cat # NBPI-866442, Novus Biologicals, USA) at 4 °C overnight and then incubated with secondary antibody (1:5000, rabbit, Amersham Biosciences) followed by DAB kit (Life Technologies, USA).The PBS solution was used as negative control.

Techniques: Expressing