Journal: Cell Death & Disease

Article Title: BBC3 in macrophages promoted pulmonary fibrosis development through inducing autophagy during silicosis

doi: 10.1038/cddis.2017.78

Figure Lengend Snippet: BBC3-mediated autophagy processes in U937-differentiated macrophages exposed to silica. ( a and b ) Macrophages were pretreated with or without BafA1 (50 nM) ( b ) for 1 h followed by SiO 2 treatment for 12 h. Representative Western blot and densitometric analyses showing that bafilomycin A1 (BafA1) further increased accumulation of LC3BII and SQSTM1 induced by SiO 2 . Data are presented as the mean±SEM ( n =3); * P <0.05; ** P <0.01; *** P <0.001 versus the control group; # P <0.05 versus the SiO 2 group (Student's t -test). ( c and d ) Representative Western blot and densitometric analyses showing the effects of Bbc3 -specific siRNA on the expression of BECN1 and LC3B induced by SiO 2 . The results suggest that SiO 2 induced BECN1 and LC3BII expression, but reduced SQSTM1 expression, which were reversed by the Bbc3 -specific siRNA. Data are presented as the mean±SEM ( n =3); * P <0.05; ** P <0.01; *** P <0.001 versus the con-siRNA group; # P <0.05 versus the con-siRNA+SiO 2 group (two-way ANOVA). ( e ) U937-differentiated macrophages were transfected for 12 h with fluorescent mRFP-GFP-tagged LC3 plasmids and then treated with Bbc3 -specific siRNA or non-specific siRNA using Lipofectamine 2000, followed by treatment with SiO 2 for 24 h. Images were captured by confocal microscopy. The numbers of red, green, and yellow puncta were analyzed to evaluate autophagic flux, and more than 10 cells were quantified for each condition. SiO 2 significantly induced autophagy (yellow and red puncta), but Bbc3 -specific siRNA reversed this effect. Scale bar=10 μ m. Images are representative of five independent experiments

Article Snippet: Antibodies against BBC3 (SC374223), BECN1 (SC48341),VIM (SC7558), F4/80 (SC26642), LC3 (SC398822) and GAPDH (SC32233) were obtained from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA).

Techniques: Western Blot, Control, Expressing, Transfection, Confocal Microscopy

Journal: Cell Death & Disease

Article Title: BBC3 in macrophages promoted pulmonary fibrosis development through inducing autophagy during silicosis

doi: 10.1038/cddis.2017.78

Figure Lengend Snippet: BBC3-mediated autophagy processes in mouse silicosis model. ( a and b ) Representative Western blot and densitometric analyses showing that Bbc3 knockout decreased the LC3BII expression, but increased the SQSTM1 expression in bone marrow-derived macrophages (BMDMs) exposed to SiO 2 . Data are presented as the mean±S.E.M. ( n =3); * P <0.05; ** P <0.01 versus the WT group; ## P <0.01; ### P <0.001 versus the WT+SiO 2 group (two-way ANOVA). ( c and d ) Representative Western blot and densitometric analyses showing that BafA1 further increased accumulation of LC3BII induced by SiO 2 in BMDMs. Data are presented as the mean±SEM ( n =3); * P <0.05; ** P <0.01 versus the control group; ## P <0.01 versus the SiO 2 group (two-way ANOVA). ( e ) Immunohistochemical staining of WT and Bbc3 KO mouse lung tissue showing autophagy intensity. The results indicated that the loss of BBC3 reduced the number of macrophages and the expression of SQSTM1 in lung tissue sections. Scale bar=20 μ m. Images are representative of several individuals from each group ( n =4). ( f ) Representative western blot showing the expression of BBC3 in macrophages from healthy donors and silicosis patients. ( g ) Densitometric analyses of macrophage samples from five healthy donors and five silicosis patients suggested that BBC3 expression was elevated in macrophages from silicosis patients. Data are presented as the mean±S.E.M.; * P <0.05 versus the corresponding healthy control group (Student's t -test). ( h ) Representative western blot showing the expression of BECN1 and LC3B in macrophages from healthy donors and silicosis patients. ( i ) Densitometric analyses of macrophage samples from five healthy donors and five silicosis patients suggested that BECN1 and LC3BII expression was elevated in macrophages from silicosis patients. Data are presented as the mean±SEM; * P <0.05 versus the corresponding healthy control group (Student's t -test)

Article Snippet: Antibodies against BBC3 (SC374223), BECN1 (SC48341),VIM (SC7558), F4/80 (SC26642), LC3 (SC398822) and GAPDH (SC32233) were obtained from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA).

Techniques: Western Blot, Knock-Out, Expressing, Derivative Assay, Control, Immunohistochemical staining, Staining

Journal: PLoS ONE

Article Title: Combination of LC3 shRNA Plasmid Transfection and Genistein Treatment Inhibited Autophagy and Increased Apoptosis in Malignant Neuroblastoma in Cell Culture and Animal Models

doi: 10.1371/journal.pone.0078958

Figure Lengend Snippet: Here, cells without any treatment were used as the untreated control (CTL). All experiments were performed in triplicates. (a) Detection of autophagic populations. Cells were treated with rapamycin and then stained with acridin orange (AO) for flow cytometric detection of the acidic vesicular organelles (AVO) in autophagic cells. (b) Determination of percentages of autophagic populations after AO staining. Significant difference between CTL and a treatment time was indicated by * P <0.05 whereas significant difference between a treatment time and another treatment time was indicated by # P <0.05. (c) Western blotting to show the protein levels of LC3 I and LC3 II forms in both cell lines. Expression of β-actin was used as a loading control in Western blotting.

Article Snippet: Both SK-N-BE2 and IMR-32 cell lines were allowed to grow till 90% confluency and then transfected with LC3 shRNA plasmid (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Control, Staining, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Combination of LC3 shRNA Plasmid Transfection and Genistein Treatment Inhibited Autophagy and Increased Apoptosis in Malignant Neuroblastoma in Cell Culture and Animal Models

doi: 10.1371/journal.pone.0078958

Figure Lengend Snippet: Here, cells treated with 200 nM rapamycin for 24 h were used as the treated control (CTL) SK-N-BE2 cells (red bars) and IMR-32 cells (blue bars). (a) Treatments (24 h): treated CTL cells, CTL shRNA plasmid, LC3 shRNA plasmid, GST, LC3 shRNA plasmid + GST, 10 µM cisplatin, and 10 µM cyclophosphamide. As presented in the bar diagrams, combination of 50 nM LC3 shRNA plasmid and 25 µM GST in SK-N-BE2 cells while combination of 100 nM LC3 shRNA plasmid and 25 µM GST in IMR-32 cells showed the best synergistic efficacy, as determined by the combination index (CI) value, for the highest decrease in residual cell viability. (b) Western blotting to show the effect of GST treatment on the expression of EGFR, a receptor tyrosine kinase, in both cell lines. Cells were treated with different doses (5, 10, 25 and 50 µM) of GST for 24 h. All experiments were performed in triplicates. Significant difference between CTL and a treatment was indicated by * P <0.05 while significant difference between single treatment and double treatment was indicated by # P <0.05.

Article Snippet: Both SK-N-BE2 and IMR-32 cell lines were allowed to grow till 90% confluency and then transfected with LC3 shRNA plasmid (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Control, shRNA, Plasmid Preparation, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Combination of LC3 shRNA Plasmid Transfection and Genistein Treatment Inhibited Autophagy and Increased Apoptosis in Malignant Neuroblastoma in Cell Culture and Animal Models

doi: 10.1371/journal.pone.0078958

Figure Lengend Snippet: Combination index (CI) values for the concentrations of LC3 shRNA plasmid and GST in human malignant neuroblastoma SK-N-BE2 and IMR-32 cells.

Article Snippet: Both SK-N-BE2 and IMR-32 cell lines were allowed to grow till 90% confluency and then transfected with LC3 shRNA plasmid (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: shRNA, Plasmid Preparation

Journal: PLoS ONE

Article Title: Combination of LC3 shRNA Plasmid Transfection and Genistein Treatment Inhibited Autophagy and Increased Apoptosis in Malignant Neuroblastoma in Cell Culture and Animal Models

doi: 10.1371/journal.pone.0078958

Figure Lengend Snippet: Here, cells treated with 200 nM rapamycin for 24 h were used as the treated control (CTL). Then, single and combination therapies were performed for another 24 h. Treatment groups: treated CTL, CTL shRNA plasmid, LC3 shRNA plasmid, GST, LC3 shRNA plasmid + GST, and untreated CTL. We used 50 nM CTL or LC3 shRNA plasmid and 25 µM GST alone or in combination in SK-N-BE2 cells while 100 nM CTL or LC3 shRNA plasmid and 25 µM GST alone or in combination in IMR-32 cells. (a) Staining of cells with AO followed by fluorescence microscopy for detection of AVO in autophagic cells. (b) Flow cytometric analysis of the AO stained cells from all treatment groups for detection and determination of AVO in autophagic cells. (c) Presentation of amounts of autophagic cells in bar diagrams. Significant difference between treated CTL and another treatment or untreated CTL was indicated by * P <0.05 while significant difference between single treatment and double treatment was indicated by # P <0.05.

Article Snippet: Both SK-N-BE2 and IMR-32 cell lines were allowed to grow till 90% confluency and then transfected with LC3 shRNA plasmid (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Control, shRNA, Plasmid Preparation, Staining, Fluorescence, Microscopy

Journal: PLoS ONE

Article Title: Combination of LC3 shRNA Plasmid Transfection and Genistein Treatment Inhibited Autophagy and Increased Apoptosis in Malignant Neuroblastoma in Cell Culture and Animal Models

doi: 10.1371/journal.pone.0078958

Figure Lengend Snippet: (a) Time-dependent changes in expression of LC3. Cells were first treated with 200 nM rapamycin for 24 h and then transfected with LC3 shRNA plasmid for 0, 6, 12, and 24 h or treated with 10 µM 3MA for 24 h. (b) Changes in expression of key molecules involved in autophagy. Cells were first treated with 200 nM rapamycin for 24 h and then subjected to other treatments (24 h): CTL shRNA plasmid, LC3 shRNA plasmid, GST, and LC3 shRNA plasmid + GST. We used 50 nM CTL or LC3 shRNA plasmid and 25 µM GST alone or in combination in SK-N-BE2 cells while 100 nM CTL or LC3 shRNA plasmid and 25 µM GST alone or in combination in IMR-32 cells. Representative Western blots show changes in expression of LC3 I and LC3 II, Beclin 1, TLR-4, Myd88, p62, mTOR, and β-actin.

Article Snippet: Both SK-N-BE2 and IMR-32 cell lines were allowed to grow till 90% confluency and then transfected with LC3 shRNA plasmid (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing, Transfection, shRNA, Plasmid Preparation, Western Blot

Journal: PLoS ONE

Article Title: Combination of LC3 shRNA Plasmid Transfection and Genistein Treatment Inhibited Autophagy and Increased Apoptosis in Malignant Neuroblastoma in Cell Culture and Animal Models

doi: 10.1371/journal.pone.0078958

Figure Lengend Snippet: Cells were first treated with 200 nM rapamycin for 24 h and then subjected to other treatments (24 h): CTL shRNA plasmid, LC3 shRNA plasmid, GST, LC3 shRNA plasmid + GST, 3MA (10 µM) + GST, and WM (5 µM) + GST. We used 50 nM CTL or LC3 shRNA plasmid and 25 μM GST alone or in combination in SK-N-BE2 cells while 100 nM CTL or LC3 shRNA plasmid and 25 μM GST alone or in combination in IMR-32 cells. (a) In situ Wright staining was performed to show morphological features of apoptosis in both cell lines. (b) Annexin V-FITC/PI binding assay followed by flow cytometry of the cells to show accumulation of apoptotic population (quadrant A4). (c) Determination of amounts of apoptosis based on flow cytometry. All experiments were performed in triplicates. Significant difference between CTL shRNA plasmid and another treatment was indicated by * P <0.05 while significant difference between single treatment and double treatment was indicated by # P <0.05.

Article Snippet: Both SK-N-BE2 and IMR-32 cell lines were allowed to grow till 90% confluency and then transfected with LC3 shRNA plasmid (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: shRNA, Plasmid Preparation, In Situ, Wright Stain, Binding Assay, Flow Cytometry

Journal: PLoS ONE

Article Title: Combination of LC3 shRNA Plasmid Transfection and Genistein Treatment Inhibited Autophagy and Increased Apoptosis in Malignant Neuroblastoma in Cell Culture and Animal Models

doi: 10.1371/journal.pone.0078958

Figure Lengend Snippet: Cells were first treated with 200 nM rapamycin for 24 h and then subjected to other treatments (24 h): CTL shRNA plasmid, LC3 shRNA plasmid, GST, and LC3 shRNA plasmid + GST. We used 50 nM CTL or LC3 shRNA plasmid and 25 µM GST alone or in combination in SK-N-BE2 cells while 100 nM CTL or LC3 shRNA plasmid and 25 µM GST alone or in combination in IMR-32 cells. Representative Western blots show levels of expression of Bax, Bcl-2, cytochrome c, COX-4, active caspase-3, PARP fragment, and β-actin. Expression of COX-4 (an internal control in mitochondria) was used for monitoring mitochondrial release of cytochrome c into the cytosol.

Article Snippet: Both SK-N-BE2 and IMR-32 cell lines were allowed to grow till 90% confluency and then transfected with LC3 shRNA plasmid (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: shRNA, Plasmid Preparation, Western Blot, Expressing, Control

Journal: PLoS ONE

Article Title: Combination of LC3 shRNA Plasmid Transfection and Genistein Treatment Inhibited Autophagy and Increased Apoptosis in Malignant Neuroblastoma in Cell Culture and Animal Models

doi: 10.1371/journal.pone.0078958

Figure Lengend Snippet: Both SK-N-BE2 and IMR32 cells were harvested, counted, and suspended in an equal volume of highly-concentrated Matrigel and then cell suspension (100 µl) was injected in nude mice for development of xenografts during 3 weeks. Xenograft bearing animals were pre-treated with rapamycin (2 mg/kg body weight/day) for 7 days and then randomly assigned to four therapeutic treatment groups: CTL shRNA plasmid, LC3 shRNA plasmid, GST, and LC3 shRNA plasmid + GST. Animals were daily injected with CTL shRNA plasmid (50 µg/mouse), LC3 shRNA plasmid (50 µg/mouse), or/and GST (2 mg/kg body weight) for 15 days. The data are representative of at least 3 independent experiments using 4 mice in each therapeutic treatment group. (a) Mice with SK-N-BE2 and IMR-32 xenografts. (b) Tumors after surgical removal from the animals. (c) Estimation of tumor volumes in both neuroblastoma xenografts. Significant difference between CTL shRNA plasmid and another treatment was indicated by * P <0.05 while significant difference between single treatment and double treatment was indicated by # P <0.05. (d) H&E staining to show hisopathological alterations in tumor sections after the therapeutic treatments. (e) Representative Western blots to show changes in expression of LC3 II, Beclin 1, Bax, Bcl-2, active caspase-3, PARP fragment, andβ-actin.

Article Snippet: Both SK-N-BE2 and IMR-32 cell lines were allowed to grow till 90% confluency and then transfected with LC3 shRNA plasmid (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Suspension, Injection, shRNA, Plasmid Preparation, Staining, Western Blot, Expressing

Journal: Journal of Dental Sciences

Article Title: Inactivation of PI3K/Akt promotes the odontoblastic differentiation and suppresses the stemness with autophagic flux in dental pulp cells

doi: 10.1016/j.jds.2021.05.013

Figure Lengend Snippet: Effect of autophagy inhibition on odontogenic differentiation and mineralization in human dental pulp cells (HDPCs). (A) Cells were cultured in differentiation media (DM) without or with 1 mM 3-methyladenine (3MA) for 3, 7, or 14 days. Cell lysates were subjected to Western blot analysis using anti-dentin matrix protein-1 (DMP-1), dentin sialoprotein (DSP), LC3 and p62 antibodies. (B) Cells cultured in DM with 1 mM 3MA for 14 days were stained with BCIP®/NBT Liquid Substrate System (ALP staining) and with Alizarin red S. Stained cells were dissolved with 10% (w/v) cetylpyridinium chloride and quantified using an absorbance microplate reader at 570 nm. (C) Cells were transduced with lentivirus containing either control shRNA lentiviral particles (shNTC) or LC3 shRNA lentiviral particles (shLC3) and then subjected to odontogenic induction for 3 or 7 days. Cell lysates were subjected to Western blot analysis to determine protein levels of DMP-1 and LC3. (D) Cells were transduced with lentivirus containing either shNTC or shLC3. The degree of differentiation was determined based on ALP activity and Alizarin red S staining at 7 days. Data are presented as mean ± SD from triplicate independent experiments. ∗∗ p < 0.01 vs. control cells or shNTC group; # p < 0.05, ## p < 0.01, vs. 3MA-untreated cells or shNTC group.

Article Snippet: After culturing for 24 h, control shRNA lentiviral particles (shNTC, Santa Cruz Biotechnology) and LC3 shRNA lentiviral particles (shLC3, Santa Cruz Biotechnology) were supplemented with 5 μg/ml polybrene (Santa Cruz Biotechnology).

Techniques: Inhibition, Cell Culture, Western Blot, Staining, Microplate Reader Absorbance Measurement, Transduction, Control, shRNA, Activity Assay

Journal: Journal of Dental Sciences

Article Title: Inactivation of PI3K/Akt promotes the odontoblastic differentiation and suppresses the stemness with autophagic flux in dental pulp cells

doi: 10.1016/j.jds.2021.05.013

Figure Lengend Snippet: Effect of autophagy inhibition on expression of stem cell multi/pluri-potency markers during odontogenic differentiation in human dental pulp cells (HDPCs). (A–C) Cells were grown in differentiation media (DM) without or with 3-methyladenine (3MA) (1 mM), LC3 shRNA lentiviral particles (shLC3) and Akt activator SC79 (10 μg/ml) for 3, 7, or 14 days and Western blot analysis was performed with CD146, Oct4, and Nanog antibodies. Densitometry analysis is presented as relative ratio compared with β-Actin. Data are presented as mean ± SD from triplicate independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. 0 day; # p < 0.05, ## p < 0.01, vs. 3MA or SC79-untreated cells.

Article Snippet: After culturing for 24 h, control shRNA lentiviral particles (shNTC, Santa Cruz Biotechnology) and LC3 shRNA lentiviral particles (shLC3, Santa Cruz Biotechnology) were supplemented with 5 μg/ml polybrene (Santa Cruz Biotechnology).

Techniques: Inhibition, Expressing, shRNA, Western Blot

Journal: Journal of Dental Sciences

Article Title: Inactivation of PI3K/Akt promotes the odontoblastic differentiation and suppresses the stemness with autophagic flux in dental pulp cells

doi: 10.1016/j.jds.2021.05.013

Figure Lengend Snippet: Effect of autophagy inhibition on odontogenic differentiation in rat tooth crown-pulp lysates after tooth cavity preparation. (A, B) Localization of proteins was visualized after fluorescence immunohistochemistry staining in sagittal tooth sections from rats without or with 3-methyladenine (3MA) intraperitoneal (IP) injection (10 mg/kg weight) at 10 days after tooth cavity preparation (n = 4). Green fluorescence indicates LC3B-positive and blue fluorescence indicates DMP-1-positive cells. Yellow dotted curves indicate tooth cavity preparation sites. Scale bars, 500 μm for 50× magnification and 50 μm for 400× magnification. (C) Pooled samples of tooth crown-pulp lysates isolated from rats with or without 3MA IP injection were analyzed by Western blot analysis (n = 4). β-Actin was used as an internal control.

Article Snippet: After culturing for 24 h, control shRNA lentiviral particles (shNTC, Santa Cruz Biotechnology) and LC3 shRNA lentiviral particles (shLC3, Santa Cruz Biotechnology) were supplemented with 5 μg/ml polybrene (Santa Cruz Biotechnology).

Techniques: Inhibition, Fluorescence, Immunohistochemistry, Staining, Injection, Isolation, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: miR-29a-3p Regulates Autophagy by Targeting Akt3-Mediated mTOR in SiO 2 -Induced Lung Fibrosis

doi: 10.3390/ijms241411440

Figure Lengend Snippet: Autophagy regulates silica-induced pulmonary fibrosis. ( A , B ) Representative electron microscopy images from mouse lung tissues exposed to 50 mg/kg silica for 0, 7, 14, and 28 days and TC-1 cells treated with different concentrations (0, 1, 5, and 10 ng/mL) of TGF-β1 for 48 h; yellow arrows indicate autophagosomes. ( C ) Western blot analysis of proteins associated with autophagy Beclin1 and LC3-α/β level in mouse lung tissues exposed to silica. ( D ) Western blot analysis of proteins associated with autophagy Beclin1 and LC3-α/β level in TGF-β1-treated TC-1 cells. ( E ) Western blot results of Beclin1 and LC3α/β as well as fibrosis markers, α-SMA and collagen I, in TC-1 cells treated with autophagy inhibitor (3-MA) and inducer (rapamycin) together with 10 ng/mL TGF-β1. Data are presented as the mean ± SD from at least three independent experiments; * indicates p < 0.05 vs. the control group, ** indicates p < 0.01 vs. the control group, # indicates p < 0.05 vs. the TGF-β1 group, and ## indicates p < 0.01 vs. the TGF-β1 group.

Article Snippet: TC-1 cells transfected with miR-29a-3p were treated with 10 ng/mL of TFG-β1 for 24 h and then fixed with 4% paraformaldehyde for 30 min, washed 3 times with PBS, and serum blocked for 10 min. Then the cell was incubated overnight at 4 °C with the following specific primary antibody: anti-alpha-SMA (1:100; Affinity, Cincinnati, OH, USA), anti-LC3α/β pAb (1:100; MBL, Nagoya, Japan), washed 3 times with PBS and then incubated with fluorescence secondary antibodies corresponding to the host species of the primary antibodies donkey anti-rabbit IgG (Invitrogen, Waltham, MA, USA) or goat anti-mouse IgG (ABclonal, Wuhan, China) at a dilution ratio of 1:100 for 1.5 h at 37 °C.

Techniques: Electron Microscopy, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: miR-29a-3p Regulates Autophagy by Targeting Akt3-Mediated mTOR in SiO 2 -Induced Lung Fibrosis

doi: 10.3390/ijms241411440

Figure Lengend Snippet: Inhibition of Akt3 expression promotes autophagy in lung epithelial cells and ameliorates fibrosis by activating mTOR. ( A ) Representative images of Akt3 immunohistochemistry in lung tissue of mice with silicosis; Yellow arrows indicate positive expression of Akt3. ( B ) Western blot analysis of Akt3 and phospho-mTOR levels in lung tissues of mice with silicosis. ( C ) qRT-PCR analysis of Akt3 levels from TC-1 cells treated with various doses of TGF-β1. ( D ) The expression of phospho-mTOR, autophagy-related proteins LC3α/β and Beclin1, and fibrosis markers, α-SMA and collagen I, were verified using Western blot in TC-1 cells transfected with 50 nM of siR-Akt3 or siR-NC before treatment with 10 ng/mL of TGF-β1. Data are presented as the mean ± SD from at least three independent experiments; * indicates p < 0.05 vs. the control group, ** indicates p < 0.01 vs. the control group, # indicates p < 0.05 vs. the siR-NC plus TGF-β1 group, and ## indicates p < 0.01 vs. the siR-NC plus TGF-β1 group.

Article Snippet: TC-1 cells transfected with miR-29a-3p were treated with 10 ng/mL of TFG-β1 for 24 h and then fixed with 4% paraformaldehyde for 30 min, washed 3 times with PBS, and serum blocked for 10 min. Then the cell was incubated overnight at 4 °C with the following specific primary antibody: anti-alpha-SMA (1:100; Affinity, Cincinnati, OH, USA), anti-LC3α/β pAb (1:100; MBL, Nagoya, Japan), washed 3 times with PBS and then incubated with fluorescence secondary antibodies corresponding to the host species of the primary antibodies donkey anti-rabbit IgG (Invitrogen, Waltham, MA, USA) or goat anti-mouse IgG (ABclonal, Wuhan, China) at a dilution ratio of 1:100 for 1.5 h at 37 °C.

Techniques: Inhibition, Expressing, Immunohistochemistry, Western Blot, Quantitative RT-PCR, Transfection

Journal: International Journal of Molecular Sciences

Article Title: miR-29a-3p Regulates Autophagy by Targeting Akt3-Mediated mTOR in SiO 2 -Induced Lung Fibrosis

doi: 10.3390/ijms241411440

Figure Lengend Snippet: miR-29a-3p regulates autophagy via the Akt3/mTOR axis to attenuate TGF-β1–induced fibrosis in vitro. ( A ) Transfection of miR-29a-3p mimics into TC-1 cells followed by treatment with 10 ng/mL of TGF-β1. The expression of phospho-mTOR, autophagy-associated proteins LC3α/β and Beclin1, as well as fibrosis markers α-SMA and collagen I in TC-1 cells, were detected via Western blot. ( B ) Immunofluorescence results of LC3α/β and α-SMA in TC-1 cells transfected with miR-29a-3p mimics before treatment with 10 ng/mL TGF-β1. Data are presented as the mean ± SD from at least three independent experiments; * indicates p < 0.05 vs. the control group, ** indicates p < 0.01 vs. the control group, # indicates p < 0.05 vs. the TGF-β1 plus NC mimics group, and ## indicates p < 0.01 vs. the TGF-β1 plus NC mimics group.

Article Snippet: TC-1 cells transfected with miR-29a-3p were treated with 10 ng/mL of TFG-β1 for 24 h and then fixed with 4% paraformaldehyde for 30 min, washed 3 times with PBS, and serum blocked for 10 min. Then the cell was incubated overnight at 4 °C with the following specific primary antibody: anti-alpha-SMA (1:100; Affinity, Cincinnati, OH, USA), anti-LC3α/β pAb (1:100; MBL, Nagoya, Japan), washed 3 times with PBS and then incubated with fluorescence secondary antibodies corresponding to the host species of the primary antibodies donkey anti-rabbit IgG (Invitrogen, Waltham, MA, USA) or goat anti-mouse IgG (ABclonal, Wuhan, China) at a dilution ratio of 1:100 for 1.5 h at 37 °C.

Techniques: In Vitro, Transfection, Expressing, Western Blot, Immunofluorescence