Journal: Molecular Medicine

Article Title: Wnt5a-mediated autophagy contributes to the epithelial-mesenchymal transition of human bronchial epithelial cells during asthma

doi: 10.1186/s10020-024-00862-3

Figure Lengend Snippet: Primer sequences used in this study

Article Snippet: The antibodies used in the present research were as follows: rabbit anti-α-Tubulin antibody (1:2000, Servicebio, Wuhan, China); rabbit anti-α-SMA antibody (1:1000, Cell Signaling Technology, USA); rabbit anti-E-cadherin antibody (1:2000, Cell Signaling Technology); rabbit anti-Beclin1 antibody (1:1500, Cell Signaling Technology); rabbit anti-Collagen I antibody (1:1000, Cell Signaling Technology); rabbit anti-LC3 antibody (1:1500, Cell Signaling Technology); rabbit anti-Wnt5a antibody (1:1000, BOSTER, Wuhan, China); rabbit anti-CaMKII antibody (1:1000, BOSTER); rabbit anti-p-CaMKII antibody (1:1000, Cell Signaling Technology).

Techniques:

Journal: Molecular Medicine

Article Title: Wnt5a-mediated autophagy contributes to the epithelial-mesenchymal transition of human bronchial epithelial cells during asthma

doi: 10.1186/s10020-024-00862-3

Figure Lengend Snippet: HDM or IL-4 treatment increases Wnt5a and promotes EMT in 16HBE cells. ( A-C ) Microarray analysis examines patterns and correlations of WNT5A and ACTA2 in HBECs derived from asthma patients, utilizing data from the GSE18965 dataset, n = 9. ( D-F ) Microarray analysis scrutinizes expression patterns and conducts correlation analysis of WNT5A and N-CADHERIN in HBECs obtained from asthma patients, using data from the GSE20680 dataset, n = 9. ( G-H ) Microarray analysis was conducted to examine the expression of WNT5A in mouse asthmatic lung tissues from the GSE165969 and GSE71822 datasets, n = 3–6. ( I ) Rhodamine phalloidin staining was used to determine HDM-induced cytoskeleton morphology in 16HBE cells (bar = 20 μm). ( J-M ) Real-time PCR was employed to assess the mRNA expression levels of WNT5A, VIMENTIN, N-CADHERIN , and COL1A1 in addition to conducting a correlation analysis between EMT-related genes and Wnt5a in 16HBE cells subjected to HDM treatment. Data were normalized to β-ACTIN , n = 3. ( D-E ) The protein level of Wnt5a was detected by western blot, n = 3. ( N-O ) Western blot and quantification of E-cadherin, α-SMA, and Collagen I were performed on 16HBE cells exposed to HDM, n = 3. ( P-S ) Western blot and quantification of α-SMA, Collagen I, and Wnt5a were conducted on 16HBE cells treated with IL-4, n = 3. Bar graphs represent mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: The antibodies used in the present research were as follows: rabbit anti-α-Tubulin antibody (1:2000, Servicebio, Wuhan, China); rabbit anti-α-SMA antibody (1:1000, Cell Signaling Technology, USA); rabbit anti-E-cadherin antibody (1:2000, Cell Signaling Technology); rabbit anti-Beclin1 antibody (1:1500, Cell Signaling Technology); rabbit anti-Collagen I antibody (1:1000, Cell Signaling Technology); rabbit anti-LC3 antibody (1:1500, Cell Signaling Technology); rabbit anti-Wnt5a antibody (1:1000, BOSTER, Wuhan, China); rabbit anti-CaMKII antibody (1:1000, BOSTER); rabbit anti-p-CaMKII antibody (1:1000, Cell Signaling Technology).

Techniques: Microarray, Derivative Assay, Expressing, Staining, Real-time Polymerase Chain Reaction, Western Blot

Journal: Molecular Medicine

Article Title: Wnt5a-mediated autophagy contributes to the epithelial-mesenchymal transition of human bronchial epithelial cells during asthma

doi: 10.1186/s10020-024-00862-3

Figure Lengend Snippet: BOX5 alleviates IL-4-induced EMT in 16HBE cells. ( A-D ) Western blot and quantification were employed to measure Wnt5a, α-SMA, and E-cadherin, n = 3. ( E-F ) Real-time PCR was employed to assess the mRNA expression levels of VIMENTIN and N-CADHERIN in 16HBE cells subjected to IL-4 after the BOX5 treatment. Data were normalized to β-ACTIN , n = 3. ( G ) The wound healing assay was utilized to observe the migration of IL-4-treated 16HBE cells at different times after BOX5 intervention, and ( H ) migration rate was analyzed, n = 3. ( I ) Rhodamine phalloidin staining for IL-4-induced cytoskeleton morphology and BOX5 intervention. Data are shown as the mean ± SD. ** P < 0.01 and *** P < 0.001

Article Snippet: The antibodies used in the present research were as follows: rabbit anti-α-Tubulin antibody (1:2000, Servicebio, Wuhan, China); rabbit anti-α-SMA antibody (1:1000, Cell Signaling Technology, USA); rabbit anti-E-cadherin antibody (1:2000, Cell Signaling Technology); rabbit anti-Beclin1 antibody (1:1500, Cell Signaling Technology); rabbit anti-Collagen I antibody (1:1000, Cell Signaling Technology); rabbit anti-LC3 antibody (1:1500, Cell Signaling Technology); rabbit anti-Wnt5a antibody (1:1000, BOSTER, Wuhan, China); rabbit anti-CaMKII antibody (1:1000, BOSTER); rabbit anti-p-CaMKII antibody (1:1000, Cell Signaling Technology).

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Expressing, Wound Healing Assay, Migration, Staining

Journal: Molecular Medicine

Article Title: Wnt5a-mediated autophagy contributes to the epithelial-mesenchymal transition of human bronchial epithelial cells during asthma

doi: 10.1186/s10020-024-00862-3

Figure Lengend Snippet: Schematic illustration of the mechanisms of Wnt5a-induced EMT in HBECs. This hypothesis illustrates a molecular pathway in HBECs leading to airway remodeling, typically associated with classical autophagy pathway and EMT. In short, Wnt5a induced by HDM or IL-4 leads to the overactivation of autophagy in HBECs through the Ca 2+ /CaMKII signaling pathway, subsequently promoting bronchial epithelial EMT and triggering airway remodeling

Article Snippet: The antibodies used in the present research were as follows: rabbit anti-α-Tubulin antibody (1:2000, Servicebio, Wuhan, China); rabbit anti-α-SMA antibody (1:1000, Cell Signaling Technology, USA); rabbit anti-E-cadherin antibody (1:2000, Cell Signaling Technology); rabbit anti-Beclin1 antibody (1:1500, Cell Signaling Technology); rabbit anti-Collagen I antibody (1:1000, Cell Signaling Technology); rabbit anti-LC3 antibody (1:1500, Cell Signaling Technology); rabbit anti-Wnt5a antibody (1:1000, BOSTER, Wuhan, China); rabbit anti-CaMKII antibody (1:1000, BOSTER); rabbit anti-p-CaMKII antibody (1:1000, Cell Signaling Technology).

Techniques:

Journal: Toxics

Article Title: The Role of Autophagy in Copper-Induced Apoptosis and Developmental Neurotoxicity in SH-SY5Y Cells

doi: 10.3390/toxics13010063

Figure Lengend Snippet: Cu 2+ repressed the Wnt signaling pathway in SH-SY5Y cells. ( A ) The protein expression levels of Wnt1, Wnt5a and β-catenin. ( B – D ) Quantitative analysis for protein expression of Wnt1, Wnt5a and β-catenin. ( E ) The protein expression levels of PKC, p-GSK3β, and GSK3β. ( F – H ) Quantitative analysis for protein expression of PKC, p-GSK3β and GSK3β. * p < 0.05 and ** p < 0.01.

Article Snippet: The following antibodies were used: anti-βIII-tubulin (M0805-8), Wnt1 (ER65317), Wnt5a (ET1706-33), glycogen synthase kinase-3 (GSK3β, ET1607-71), p-GSK3β (ET1607-60), p62(HA721171), and Atg7 (ET1610-53), from Huabio Biotechnology (Hangzhou, China); anti-β-catenin (51067-2-AP) and PARP (13371-1-AP), from Proteintech (Wuhan, China); caspase 3 (A19654), Bax (A0207), p-PI3K (AP0427), β-actin (AC026), and H3 (A2348), from ABclonal Technology (Wuhan, China); anti-Bcl2 (15071), PI3K (4294), Akt (9272), p-Akt (9271), mTOR (2983), and p-mTOR (2974), from Cell Signaling Technology (Boston, MA, USA); anti-LC3B (EPR18709), from Abcam (Cambridge, MA, USA).

Techniques: Expressing

Journal: Toxics

Article Title: The Role of Autophagy in Copper-Induced Apoptosis and Developmental Neurotoxicity in SH-SY5Y Cells

doi: 10.3390/toxics13010063

Figure Lengend Snippet: Rapa rescued apoptosis and decreased the differentiation capacity induced by Cu 2+ in SH-SY5Y cells. ( A ) The protein expression levels and ( B ) quantitative analysis of mTOR and p-mTOR after Rapa pretreatment. ( C ) The protein expression levels and ( D , E ) quantitative analysis of p62 and LC3BII/LC3BI and after Rapa pretreatment. ( F , G ) The protein expression levels and quantitative analysis of PARP, caspase 3 and Bcl2 after Rapa pretreatment. ( H , I ) The protein expression levels and quantitative analysis of Wnt5a and β-catenin after Rapa pretreatment. ( J ) The proportions of differentiated cells after Rapa pretreatment. ( K ) The proportions of total branches of SH-SY5Y cells after Rapa pretreatment. ( L ) The immunostaining of βIII-tubulin of SH-SY5Y cells after Rapa pretreatment (scale bar-merge: 50 μm; magnification: 20 μm). (The hash symbol stands for the difference between the Rapa + 120 μM Cu 2+ group and the 120 μM Cu 2+ -treated group alone: * p < 0.05, ** p < 0.01 and ## p < 0.01).

Article Snippet: The following antibodies were used: anti-βIII-tubulin (M0805-8), Wnt1 (ER65317), Wnt5a (ET1706-33), glycogen synthase kinase-3 (GSK3β, ET1607-71), p-GSK3β (ET1607-60), p62(HA721171), and Atg7 (ET1610-53), from Huabio Biotechnology (Hangzhou, China); anti-β-catenin (51067-2-AP) and PARP (13371-1-AP), from Proteintech (Wuhan, China); caspase 3 (A19654), Bax (A0207), p-PI3K (AP0427), β-actin (AC026), and H3 (A2348), from ABclonal Technology (Wuhan, China); anti-Bcl2 (15071), PI3K (4294), Akt (9272), p-Akt (9271), mTOR (2983), and p-mTOR (2974), from Cell Signaling Technology (Boston, MA, USA); anti-LC3B (EPR18709), from Abcam (Cambridge, MA, USA).

Techniques: Expressing, Immunostaining

Journal: Toxics

Article Title: The Role of Autophagy in Copper-Induced Apoptosis and Developmental Neurotoxicity in SH-SY5Y Cells

doi: 10.3390/toxics13010063

Figure Lengend Snippet: CQ exacerbated apoptosis and decreased the differentiation capacity induced by Cu 2+ in SH-SY5Y cells. ( A , B ) The protein expression levels and quantitative analysis of p62 and LC3BII/LC3BI after CQ pretreatment. ( C – E ) The protein expression levels and quantitative analysis of PARP, caspase 3, Bax and Bcl2 after CQ pretreatment. ( F – H ) The protein expressions of Wnt5a, nucleus β-catenin and cytoplasm β-catenin were assessed by Western blot after CQ pretreatment. ( I ) The proportions of differentiated cells after CQ pretreatment. ( J ) The proportions of total branches of SH-SY5Y cells after CQ pretreatment. ( K ) The immunostaining of βIII-tubulin of SH-SY5Y cells after CQ pretreatment (scale bar-merge: 50 μm; magnification: 20 μm). * p < 0.05, ** p < 0.01, # p < 0.05, and ## p < 0.01.

Article Snippet: The following antibodies were used: anti-βIII-tubulin (M0805-8), Wnt1 (ER65317), Wnt5a (ET1706-33), glycogen synthase kinase-3 (GSK3β, ET1607-71), p-GSK3β (ET1607-60), p62(HA721171), and Atg7 (ET1610-53), from Huabio Biotechnology (Hangzhou, China); anti-β-catenin (51067-2-AP) and PARP (13371-1-AP), from Proteintech (Wuhan, China); caspase 3 (A19654), Bax (A0207), p-PI3K (AP0427), β-actin (AC026), and H3 (A2348), from ABclonal Technology (Wuhan, China); anti-Bcl2 (15071), PI3K (4294), Akt (9272), p-Akt (9271), mTOR (2983), and p-mTOR (2974), from Cell Signaling Technology (Boston, MA, USA); anti-LC3B (EPR18709), from Abcam (Cambridge, MA, USA).

Techniques: Expressing, Western Blot, Immunostaining

Journal: Pharmaceutical biology

Article Title: Yiguanjian decoction inhibits macrophage M1 polarization and attenuates hepatic fibrosis induced by CCl 4 /2-AAF.

doi: 10.1080/13880209.2021.1961820

Figure Lengend Snippet: Figure 3. YGJ inhibits the activation of M1 macrophages and non-canonical Wnt signalling pathways and inhibits the differentiation of hepatic progenitor cells into myofibroblasts in vivo. (a) STAT1, IRF3, IRF5, IRF8, and SOCS3 protein bands were depicted in the immunoblot images, and (b) the densitometric quantification of the protein bands presented as a histogram (n ¼ 5 per group). (c) The mRNA expressions of Wnt-4, -5 A, -5B, FZD-2, -3, and -6 in liver were measured by RT-PCR and nor- malized to GAPDH mRNA (n ¼ 5 per group). (d) Wnt5A, Wnt5B, and FZD2 protein bands were depicted in the immunoblot images, and (e) the densitometric quantifi- cation of the protein bands presented as a histogram (n ¼ 5 per group). (f) OV6, SOX9, EpCAM, CK19, and Hep mRNA expressions were measured by RT-PCR and normalized to GAPDH mRNA (n ¼ 5 per group). (g) EpCAM protein bands were depicted in the immunoblot images, and (h) the densitometric quantification of the protein bands presented as a histogram (n ¼ 5 per group). p < 0.05 and p < 0.01. N: untreated group (control); 2-AAF/CCl4: 2-acetylaminofluorene/carbon tetra- chloride-treated group; YGJ: 2-AAF/CCl4 þ Yiguanjian decoction-treated group; SORA: 2-AAF/CCl4 þ sorafenib-treated group.

Article Snippet: Rabbit polyclonal antibodies against Wnt5A and Wnt5B were purchased from Signalway Antibody LLC (MD, USA).

Techniques: Activation Assay, In Vivo, Western Blot, Reverse Transcription Polymerase Chain Reaction, Control