Journal: Molecular and cellular biochemistry

Article Title: Early-life bisphenol A exposure causes neuronal pyroptosis in juvenile and adult male rats through the NF-κB/IL-1β/NLRP3/caspase-1 signaling pathway: exploration of age and dose as effective covariates using an in vivo and in silico modeling approach.

doi: 10.1007/s11010-024-05039-4

Figure Lengend Snippet: Fig. 8 The molecular docking analysis of BPA and neuro- inflammatory, autophagic as well as, pyroptotic molecules displaying 2D and 3D binding interactions of BPA [PubChem CID: 6623] against A NF-kB [PDB ID: 1NFK], B IL-1β [PDB ID: 2MIB], C IL-2 [PDB ID: 4YQX], D IL-12 [PDB ID: 3HMX], E COX-2 [PDB ID: 1PXX], F NLRP3 [PDB ID: 7vtq], G Beclin-1 [PDB ID: 2PON], H LC3A [PDB ID: 6TBE], I LC3B [PDB ID: 5XAC], J Caspase-1 [PDB ID: 6VIE]. The green dotted lines denote hydrogen bonds between ligand and aminoacids, whereas bink/purple dotted lines repre- sent hydrophobic interactions. Electrostatic interactions are shown as orange dotted lines. The red dotted line indicate an unfavorable donor-donor

Article Snippet: Fine Test (cat no. ER1965) provided the NLRP3 kit, LSBio (cat no. LS-F9917; LS-F19802) provided the LC3A and LC3B kits, respectively, and Cusabio (cat no. CSB-EL002658RA provided the beclin-1 kit.

Techniques: Binding Assay

Journal: Frontiers in Pharmacology

Article Title: NLRC5 Inhibits Inflammation of Secretory Phase Ectopic Endometrial Stromal Cells by Up-Regulating Autophagy in Ovarian Endometriosis

doi: 10.3389/fphar.2020.01281

Figure Lengend Snippet: NLRC5, LC3, Beclin1, p62, IL-6, TNF-α, and β-actin primers for qRT-PCR.

Article Snippet: The primary antibodies recognizing NLRC5 (ab105411, Abcam, Cambridge, MA, USA), LC3 (ab192890, Abcam, Cambridge, MA, USA), Beclin1 (ab207612, Abcam, Cambridge, MA, USA), p62 (18420-1-AP, Proteintech), IL-6 (ab6672, Abcam, Cambridge, MA, USA), TNF-α (ab6671, Abcam, Cambridge, MA, USA), and β-actin (GB12001, Servicebio, Wuhan, China) were used at 1:1,000, 1:1,000, 1:1,000, 1:1,000, 1:1,000, 1:1,000, 1:1,000, and 1:3000, respectively.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: NLRC5 Inhibits Inflammation of Secretory Phase Ectopic Endometrial Stromal Cells by Up-Regulating Autophagy in Ovarian Endometriosis

doi: 10.3389/fphar.2020.01281

Figure Lengend Snippet: The levels of NLRC5, inflammation, and autophagy in ESCs of patients with endometriosis (n = 5) and patients with leiomyoma (n = 5). (A) Representative image of immunofluorescence staining showing human ESCs displayed long-spindle with positively expressing vimentin and negatively expressing keratin. Photographs were taken at magnifications of 400×. (B , C) Representative western blotting and qRT-PCR showing NLRC5, IL-6, and TNF-α were up-regulated in endometriosis ectopic and eutopic ESCs of patients with endometriosis compared to the ESCs of patients with leiomyoma ( ** P < 0.01 vs. leiomyoma ESCs), and the levels of NLRC5, IL-6, and TNF-α in ectopic ESCs were also significantly higher than in the eutopic ESCs ( ## P < 0.01 vs. eutopic ESCs). Autophagy-related molecules LC3 and Beclin1 were down-regulated in ectopic and eutopic ESCs of patients with endometriosis compared to the ESCs of patients with leiomyoma ( ** P < 0.01 vs. leiomyoma ESCs), and the expression of LC3 and Beclin1 in ectopic ESCs was also significantly lower than in the eutopic ESCs ( ## P < 0.01 vs. eutopic ESCs). (D) Representative ELISA showing IL-6 and TNF-α were up-regulated in endometriosis ectopic and eutopic ESCs of patients with endometriosis compared to the ESCs of patients with leiomyoma ( ** P < 0.01 vs. leiomyoma ESCs), and the levels of IL-6 and TNF-α in ectopic ESCs were also significantly higher than in the eutopic ESCs ( ## P < 0.01 vs. eutopic ESCs). The expression levels of mRNA were normalized with respect to β-actin and were calculated using the 2 -ΔΔCt method. The protein expression levels were quantified by Image J software and normalized to β-actin protein levels. The results are represented as the mean ± SEM from at least three independent experiments.

Article Snippet: The primary antibodies recognizing NLRC5 (ab105411, Abcam, Cambridge, MA, USA), LC3 (ab192890, Abcam, Cambridge, MA, USA), Beclin1 (ab207612, Abcam, Cambridge, MA, USA), p62 (18420-1-AP, Proteintech), IL-6 (ab6672, Abcam, Cambridge, MA, USA), TNF-α (ab6671, Abcam, Cambridge, MA, USA), and β-actin (GB12001, Servicebio, Wuhan, China) were used at 1:1,000, 1:1,000, 1:1,000, 1:1,000, 1:1,000, 1:1,000, 1:1,000, and 1:3000, respectively.

Techniques: Immunofluorescence, Staining, Expressing, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Software

Journal: Frontiers in Pharmacology

Article Title: NLRC5 Inhibits Inflammation of Secretory Phase Ectopic Endometrial Stromal Cells by Up-Regulating Autophagy in Ovarian Endometriosis

doi: 10.3389/fphar.2020.01281

Figure Lengend Snippet: Effect of NLRC5 over-expression on autophagy in EESCs (n = 7). (A) Representative image of immunofluorescence staining showing NLRC5 and LC3, Beclin1 are present both in the cytoplasm and nucleus, NLRC5 and LC3, Beclin1 were co-located in the nucleus. Photographs were taken at magnifications of 1600×. (B , C) Representative western blotting and qRT-PCR results showing over-expression of NLRC5 by transfection with NLRC5 plasmid significantly promoted NLRC5, LC3 Beclin1 expressions, and inhibited p62 expression when compared with vector group ( ** P < 0.01 vs. vector group). (D) Representative images showing LC3 staining in EESCs infected with GFP-RFP-LC3 adenovirus, over-expression of NLRC5 by transfection with NLRC5 plasmid significantly promoted yellow puncta when compared with vector group. Photographs were taken at magnifications of 1,600×, quantification of mean yellow puncta of 10–15 cells per condition is shown ( ** P < 0.01 vs. vector group). (E) Representative transmission electron microscopy (TEM) showing over-expression of NLRC5 by transfection with NLRC5 plasmid significantly promoted autophagosomes formation when compared with vector group, autophagosomes were highlighted by red arrows (left scale bar: 1μm; right scale bar: 2μm; ** P < 0.01 vs. vector group). The expression levels of mRNA were normalized with respect to β-actin and were calculated using the 2 -ΔΔCt method. The protein expression levels were quantified by Image J software and normalized to β-actin protein levels. The results are represented as the mean ± SEM from at least three independent experiments.

Article Snippet: The primary antibodies recognizing NLRC5 (ab105411, Abcam, Cambridge, MA, USA), LC3 (ab192890, Abcam, Cambridge, MA, USA), Beclin1 (ab207612, Abcam, Cambridge, MA, USA), p62 (18420-1-AP, Proteintech), IL-6 (ab6672, Abcam, Cambridge, MA, USA), TNF-α (ab6671, Abcam, Cambridge, MA, USA), and β-actin (GB12001, Servicebio, Wuhan, China) were used at 1:1,000, 1:1,000, 1:1,000, 1:1,000, 1:1,000, 1:1,000, 1:1,000, and 1:3000, respectively.

Techniques: Over Expression, Immunofluorescence, Staining, Western Blot, Quantitative RT-PCR, Transfection, Plasmid Preparation, Expressing, Infection, Transmission Assay, Electron Microscopy, Software

Journal: Frontiers in Pharmacology

Article Title: NLRC5 Inhibits Inflammation of Secretory Phase Ectopic Endometrial Stromal Cells by Up-Regulating Autophagy in Ovarian Endometriosis

doi: 10.3389/fphar.2020.01281

Figure Lengend Snippet: Effects of NLRC5 inhibition on autophagy in EESCs (n = 7). (A , B) Representative western blotting and qRT-PCR results showing inhibition of NLRC5 by transfection with siRNA-NLRC5 significantly inhibited NLRC5, LC3 Beclin1 expressions, and promoted p62 expression when compared with those from the scrambled-RNAi group ( ** P < 0.01 vs. scrambled-RNAi group). (C) Representative images showing LC3 staining in EESCs infected with GFP-RFP-LC3 adenovirus; inhibition of NLRC5 by siRNA-NLRC5 transfection significantly inhibited yellow puncta when compared with scrambled-RNAi group. Photographs were taken at magnifications of 1600×, quantification of mean yellow puncta of 10-15 cells per condition is shown ( ** P < 0.01 vs. scrambled-RNAi group). (D) Representative TEM image showing inhibition of NLRC5 by siRNA-NLRC5 transfection significantly inhibited autophagosomes formation when compared with scrambled-RNAi group, autophagosomes were highlighted by red arrows(left scale bar: 1μm; right scale bar: 2μm; ** P < 0.01 vs. scrambled-RNAi group). The expression levels of mRNA were normalized with respect to β-actin and were calculated using the 2 -ΔΔCt method. The protein expression levels were quantified by Image J software and normalized to β-actin protein levels. The results are represented as the mean ± SEM from at least three independent experiments.

Article Snippet: The primary antibodies recognizing NLRC5 (ab105411, Abcam, Cambridge, MA, USA), LC3 (ab192890, Abcam, Cambridge, MA, USA), Beclin1 (ab207612, Abcam, Cambridge, MA, USA), p62 (18420-1-AP, Proteintech), IL-6 (ab6672, Abcam, Cambridge, MA, USA), TNF-α (ab6671, Abcam, Cambridge, MA, USA), and β-actin (GB12001, Servicebio, Wuhan, China) were used at 1:1,000, 1:1,000, 1:1,000, 1:1,000, 1:1,000, 1:1,000, 1:1,000, and 1:3000, respectively.

Techniques: Inhibition, Western Blot, Quantitative RT-PCR, Transfection, Expressing, Staining, Infection, Software

Journal: Oncology letters

Article Title: Suppression of autophagy facilitates hydrogen gas-mediated lung cancer cell apoptosis.

doi: 10.3892/ol.2020.11973

Figure Lengend Snippet: Figure 1. H2 treatment enhances lung cancer cell apoptosis and autophagy. After A549 and H1975 cells were treated with 20, 40 or 60% H2, the following assays were performed. (A) Cell Counting Kit‑8 assay was used to investigate cell growth. (B and C) Flow cytometry assay was used to detect cell apoptosis. (D and E) The protein levels of cleaved‑caspase 3, cleaved‑PARP, p62, Beclin1, LC3BII and LC3BI were detected using western blot analysis. *P<0.05 and **P<0.01 vs. control. PARP, poly ADP‑ribose polymerase; LC3, light chain 3; H2, hydrogen gas.

Article Snippet: Three short interfering (si)RNAs used to silence Beclin1 (si‐Beclin1‐1/2‐3; cat. no. SR322490) in the A549 and H1975 cells, and the nega‐ tive control vector (si‐NC, cat. no. SR322490) were purchased from OriGene Technologies, Inc..

Techniques: CCK-8 Assay, Flow Cytometry, Western Blot, Control

Journal: Oncology letters

Article Title: Suppression of autophagy facilitates hydrogen gas-mediated lung cancer cell apoptosis.

doi: 10.3892/ol.2020.11973

Figure Lengend Snippet: Figure 2. Evaluation of autophagy effects on H2‑induced lung cancer cell apoptosis. (A) The protein expression levels of p62, Beclin1, LC3BII and LC3BI, following treatment of A549 and H1975 cells with RAPA were detected using western blot analysis. (B) Cell apoptosis was investigated using a flow cytometry assay. Western blot analysis of the expression of cleaved‑caspase 3 and cleaved‑PARP in (C) A549 and (D) H1975 cells treated with H2 or H2+RAPA. (E) The protein expression levels of p62, Beclin1, LC3BII and LC3BI were detected using western blot analysis following treatment of A549 and H1975 cells with 3‑MA. (F) Cell apoptosis was investigated using flow cytometry. Western blot analysis of the protein expression levels of cleaved‑caspase 3 and cleaved‑PARP following treatment of (G) A549 and (H) H1975 cells with H2 or H2+3‑MA. *P<0.05. **P<0.01. LC3, light chain 3; RAPA, rapamycin; PARP, poly ADP‑ribose polymerase; PI, propidium iodide; 3‑MA, 3‑methyladenine; H2, hydrogen gas.

Article Snippet: Three short interfering (si)RNAs used to silence Beclin1 (si‐Beclin1‐1/2‐3; cat. no. SR322490) in the A549 and H1975 cells, and the nega‐ tive control vector (si‐NC, cat. no. SR322490) were purchased from OriGene Technologies, Inc..

Techniques: Expressing, Western Blot, Flow Cytometry

Journal: Oncology letters

Article Title: Suppression of autophagy facilitates hydrogen gas-mediated lung cancer cell apoptosis.

doi: 10.3892/ol.2020.11973

Figure Lengend Snippet: Figure 3. Knockdown of Beclin1 enhances the role of H2 in promoting lung cancer cell apoptosis. (A) The knockdown efficiency of si‑Beclin1 in A549 and H1975 cells was detected using western blot analysis. (B) Western blot analysis was used to detect the protein expression levels of p62, LC3BII and LC3BI in A549 and H1975 cells and the results were subsequently (C) quantified. (D) Flow cytometry assay was used to investigate cell apoptosis. The protein expression levels of cleaved‑caspase 3 and cleaved‑PARP were determined using western blot analysis in (E) A549 and (F) H1975 cells. *P<0.05 and **P<0.01. si, short interfering; LC3, light chain 3; PARP, poly ADP‑ribose polymerase; PI, propidium iodide; H2, hydrogen gas.

Article Snippet: Three short interfering (si)RNAs used to silence Beclin1 (si‐Beclin1‐1/2‐3; cat. no. SR322490) in the A549 and H1975 cells, and the nega‐ tive control vector (si‐NC, cat. no. SR322490) were purchased from OriGene Technologies, Inc..

Techniques: Knockdown, Western Blot, Expressing, Flow Cytometry

Journal: Oncology letters

Article Title: Suppression of autophagy facilitates hydrogen gas-mediated lung cancer cell apoptosis.

doi: 10.3892/ol.2020.11973

Figure Lengend Snippet: Figure 4. H2 treatment increases lung cancer cell apoptosis and autophagy by repressing the activation of the STAT3/Bcl2 signaling pathway. The protein expression levels of STAT3, p‑STAT3 and Bcl2 were detected using western blot analysis in (A) A549 and (B) H1975 cells following treatment with different concentrations of H2. (C) STAT3 expression was detected using western blot analysis following transfection with OE‑STAT3 or OE‑NC. The protein expression levels of STAT3, p‑STAT3 and Bcl2 were detected using western blot analysis in (D) A549 and (E) H1975 cells following treatment with H2 and/or transfected with OE‑STAT3. Cell apoptosis was investigated using (F) flow cytometry and the results were subsequently (G) quantified. The protein expression levels of cleaved‑caspase 3, cleaved‑PARP, Beclin1, p62, LC3BII and LC3BI in (H) A549 and (I) H1975 cells were detected using western blot analysis following transfection with OE‑STAT3 and/or treated with H2. *P<0.05 and **P<0.01. STAT, signal transducer and activator of transcription; p‑, phosphorylated; PARP, poly ADP‑ribose polymerase; LC3, light chain 3; H2, hydrogen gas; OE, overexpression; NC, negative control

Article Snippet: Three short interfering (si)RNAs used to silence Beclin1 (si‐Beclin1‐1/2‐3; cat. no. SR322490) in the A549 and H1975 cells, and the nega‐ tive control vector (si‐NC, cat. no. SR322490) were purchased from OriGene Technologies, Inc..

Techniques: Activation Assay, Expressing, Western Blot, Transfection, Flow Cytometry, Over Expression, Negative Control

Journal: BMC Plant Biology

Article Title: 14-3-3 proteins inhibit autophagy by regulating SINAT-mediated proteolysis of ATG6 in Arabidopsis

doi: 10.1186/s12870-024-05854-3

Figure Lengend Snippet: 14-3-3λ and 14-3-3κ interact with ATG6. ( A ) Y2H assay of the interaction between 14-3-3λ and 14-3-3κ with ATG6. Full-length 14-3-3λ or 14-3-3κ was fused to the BD domain and coexpressed with ATG6-AD in yeast strain YH109. The positive clones were selected on the SD medium lacking Trp, Leu, His, and Ade (-LWH) containing 5 mM 3-amino-1,2,4- triazole. AD indicates the empty AD plasmid. ( B ) In vivo Co-IP assay of the association between ATG6 and 14-3-3s (14-3-3λ and 14-3-3κ) proteins. FLAG-tagged 14-3-3s (14-3-3λ-GFP-FLAG and 14-3-3κ-GFP-FLAG) and ATG6-HA were transiently coexpressed in protoplasts separated from 4-week-old WT plants under light conditions for 16 h, and immunoprecipitated with FLAG magnetic beads. The numbers on the left indicate the molecular weight (kD) of each size marker. ( C ) BiFC assay of ATG6 and 14-3-3λ and 14-3-3κ in Arabidopsis protoplasts. The split nYFP and cYFP fusions ATG6-cYFP and 14-3-3λ-nYFP or 14-3-3κ-nYFP were coexpressed in leaf protoplasts for 16 h under light conditions. The ATG6-cYFP + nYFP vectors were similarly coexpressed as a negative control. Confocal images obtained from YFP, chlorophyll autofluorescence, brightfield, and merged images are shown. Bars = 10 μm

Article Snippet: For the in vivo ubiquitination assay, ATG6-HA and SINAT1-FLAG or 14-3-3λ-GFP-FLAG were co-expressed in Arabidopsis protoplasts for 16 h. The total protein was immunoprecipitated with HA magnetic beads (cat. no. HY-K0201, MCE, USA), and the immunoprecipitated products were separated via 8% SDS-PAGE.

Techniques: Y2H Assay, Clone Assay, Plasmid Preparation, In Vivo, Co-Immunoprecipitation Assay, Immunoprecipitation, Magnetic Beads, Molecular Weight, Marker, Bimolecular Fluorescence Complementation Assay, Negative Control

Journal: BMC Plant Biology

Article Title: 14-3-3 proteins inhibit autophagy by regulating SINAT-mediated proteolysis of ATG6 in Arabidopsis

doi: 10.1186/s12870-024-05854-3

Figure Lengend Snippet: 14-3-3 proteins regulate the ubiquitination and degradation of ATG6. ( A ) 14-3-3 proteins enhance the ubiquitination of ATG6. ATG6-HA and 14-3-3λ-GFP-FLAG were transiently cotransfected in protoplasts isolated from WT plants, and the ubiquitination of ATG6 was detected by immunoprecipitation and immunoblot analysis using anti-Ub antibodies. ( B ) Stability of ATG6 protein in the 14-3-3λ 14-3-3k double mutant ( 14-3-3λ/k ) and WT. ATG6-HA was transiently expressed in protoplasts separated from 4-week-old WT and 14-3-3λ/k double mutant plants for 16 h. Specific anti-HA antibodies were used for immunoblot analysis. ( C ) Degradation of ATG6 in 14-3-3λ/k double mutant and WT seedlings upon CHX treatment. ATG6-HA was transiently expressed in WT and 14-3-3λ/k double mutant protoplast for 16 h, followed by treatment with 10 µM CHX for indicated times. Specific anti-HA antibodies were used for immunoblot analysis. Relative intensity of each protein normalized to the loading control is shown below. Numbers on the left indicate the molecular weight (kD) of each band. GFP-HA/FLAG was used as the control for transfection efficiency. Ponceau S-stained membranes are shown below the blots to indicate the amount of protein loaded per lane

Article Snippet: For the in vivo ubiquitination assay, ATG6-HA and SINAT1-FLAG or 14-3-3λ-GFP-FLAG were co-expressed in Arabidopsis protoplasts for 16 h. The total protein was immunoprecipitated with HA magnetic beads (cat. no. HY-K0201, MCE, USA), and the immunoprecipitated products were separated via 8% SDS-PAGE.

Techniques: Ubiquitin Proteomics, Isolation, Immunoprecipitation, Western Blot, Mutagenesis, Control, Molecular Weight, Transfection, Staining

Journal: BMC Plant Biology

Article Title: 14-3-3 proteins inhibit autophagy by regulating SINAT-mediated proteolysis of ATG6 in Arabidopsis

doi: 10.1186/s12870-024-05854-3

Figure Lengend Snippet: 14-3-3λ is required for SINAT1-mediated ubiquitination and degradation of ATG6. ( A ) 14-3-3 proteins increase the interaction of SINAT1 and ATG6. In vivo Co-IP assay showing the interaction between ATG6-HA and SINAT1-FLAG in the presence of 14-3-3λ-HA. Constructs encoding ATG6-HA and SINAT1-FLAG with or without 14-3-3λ-HA were transiently co-transfected into WT protoplasts and incubated under light conditions for 16 h before immunoprecipitation with FLAG magnetic beads. GFP-FLAG was co-transfected with ATG6-HA as a negative control. Numbers on the left indicate the molecular weight (kD) of each band. The intensity of the immunoprecipitated ATG6-HA relative to the expression of SINAT1-FLAG is shown below. ( B ) 14-3-3λ is essential for SINAT1-mediated ubiquitination of ATG6 in vivo. ATG6-HA and SINAT1-FLAG constructs were transiently co-transfected into Arabidopsis protoplasts prepared from WT plants with or without 14-3-3λ-GFP-FLAG and incubated for 16 h under constant light conditions. Ubiquitination of ATG6-HA was determined with anti-Ub and anti-HA antibodies. Numbers on the left indicate the molecular weight (kD) of each band. Relative intensity of each protein normalized to the GFP-FLAG is shown below

Article Snippet: For the in vivo ubiquitination assay, ATG6-HA and SINAT1-FLAG or 14-3-3λ-GFP-FLAG were co-expressed in Arabidopsis protoplasts for 16 h. The total protein was immunoprecipitated with HA magnetic beads (cat. no. HY-K0201, MCE, USA), and the immunoprecipitated products were separated via 8% SDS-PAGE.

Techniques: Ubiquitin Proteomics, In Vivo, Co-Immunoprecipitation Assay, Construct, Transfection, Incubation, Immunoprecipitation, Magnetic Beads, Negative Control, Molecular Weight, Expressing

Journal: BMC Plant Biology

Article Title: 14-3-3 proteins inhibit autophagy by regulating SINAT-mediated proteolysis of ATG6 in Arabidopsis

doi: 10.1186/s12870-024-05854-3

Figure Lengend Snippet: ATG6 is a target of autophagy pathway. ( A ) Degradation of ATG6 is attenuated by ConA treatment. ATG6-HA was transiently transferred into WT protoplasts for 16 h, followed by application of CHX with or without ConA for indicated times. The stability of ATG6-HA was detected using anti-HA antibodies. ( B ) Half-life of ATG6 in WT and autophagy defective mutant atg5-1 . ATG6-HA was transiently transferred into protoplasts isolated from WT and atg5-1 mutants. After incubation for 16 h, the cells were treated with 10 µM CHX for indicated times. The degradation of ATG6-HA was analyzed using anti-HA antibodies. Numbers on the left indicate the molecular weight (kD) of each band. Relative intensity of each protein normalized to the GFP-HA is shown below

Article Snippet: For the in vivo ubiquitination assay, ATG6-HA and SINAT1-FLAG or 14-3-3λ-GFP-FLAG were co-expressed in Arabidopsis protoplasts for 16 h. The total protein was immunoprecipitated with HA magnetic beads (cat. no. HY-K0201, MCE, USA), and the immunoprecipitated products were separated via 8% SDS-PAGE.

Techniques: Mutagenesis, Isolation, Incubation, Molecular Weight