Journal: Theranostics

Article Title: Targeting the self-amplifying loop between ATF6 and SNAI1 to inhibit epithelial‒mesenchymal transition in fibrotic lesions

doi: 10.7150/thno.109442

Figure Lengend Snippet: The ATF6 arm of the UPR pathway is activated in a lens EMT model. (A) Experimental design. (B) Images under a slit-lamp microscope of adult control mice, lens-punctured mice at 6 h, and lens-punctured mice at 7 d. Scale bars, 200 μm. (C) Representative images of HE-stained lens sections from adult control mice 6 h after lens puncture and 7 d after lens puncture. Scale bars, 50 μm. (D) Results of GSEA enrichment. (E) Western blotting and quantification of the EMT markers fibronectin (FN), N-cadherin (N-cad), and SNAI1; the epithelial cell marker E-cadherin (E-cad); and the UPR markers p-PERK, p-IRE1α, full-length ATF6 (ATF6), cleaved ATF6 (c-ATF6) (n = 5). Data are presented as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001. (F) Whole-mount immunostaining and 3D cross-sectional images of ATF6 and SNAI1. Quantitative colocalization analysis of immunostaining for ATF6 and SNAI1 by Pearson's correlation coefficient (n = 5). Scale bars, 100 μm. Data are presented as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001. (G) Representative images of immunostained lens sections of EMT markers, including FN, α-SMA, SNAI1, and ATF6, from adult control mice 6 h after lens puncture and 7 d after lens puncture. Scale bars, 50 μm.

Article Snippet: Male Atf6 +/- mice were purchased from Cyagen Biosciences (Guangdong, China).

Techniques: Microscopy, Control, Staining, Western Blot, Marker, Immunostaining

Journal: Theranostics

Article Title: Targeting the self-amplifying loop between ATF6 and SNAI1 to inhibit epithelial‒mesenchymal transition in fibrotic lesions

doi: 10.7150/thno.109442

Figure Lengend Snippet: Activation of the UPR drives EMT in human lens epithelial cells. (A) Western blotting and quantification of the UPR markers BIP, CHOP, ATF6, and c-ATF6; the EMT markers FN, N-cadherin, and SNAI1; and the epithelial cell marker E-cadherin (n = 5). The asterisk denotes an unglycosylated form of full-length ATF6 due to TM treatment. Data are presented as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001. (B) qPCR and quantification of HSPA5, ATF6, FN, and SNAI1 expression (n = 5). Data are presented as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001. (C) Immunofluorescence staining of ATF6 and SNAI1. Scale bars, 50 μm. (D) Nuclear protein fraction analysis (n = 5). Data are presented as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001. (E) Migration of HLE-B3 cells stimulated with TM was assessed using wound healing and transwell assays. Scale bars, 50 μm. (F) Quantification of wound closure and the number of migrating cells (n = 5). Data are presented as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Male Atf6 +/- mice were purchased from Cyagen Biosciences (Guangdong, China).

Techniques: Activation Assay, Western Blot, Marker, Expressing, Immunofluorescence, Staining, Migration

Journal: Theranostics

Article Title: Targeting the self-amplifying loop between ATF6 and SNAI1 to inhibit epithelial‒mesenchymal transition in fibrotic lesions

doi: 10.7150/thno.109442

Figure Lengend Snippet: ATF6 regulates lens epithelial cell EMT in vivo . (A) Images of AAV-mCherry-infected mouse lenses and AAV-ATF6-infected mouse lenses under a slit-lamp microscope. Scale bars, 200 μm. (B) Representative images of HE-stained sections of AAV-mCherry-infected mouse lenses and AAV-ATF6-infected mouse lenses. Scale bars, 200 μm and 50 μm. (C) Quantification of the area of subcapsular plaque (n = 5). Data are presented as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001. (D) Immunofluorescence staining of the EMT marker SNAI1 and colocalization of mCherry and SNAI1 in lens sections. Scale bars, 25 μm. (E) Quantification of SNAI1-positive area (n = 5). Quantitative colocalization analysis of immunostaining for ATF6 and SNAI1 by Pearson's correlation coefficient (n = 5). Data are presented as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001. (F) Experimental design. (G) Images of lenses at 7 d after injury in wild-type (WT) mice and Atf6 +/- mice under a slit-lamp microscope. Scale bars, 200 μm. (H) Representative images of HE-stained sections 7 d after lens puncture from WT and Atf6 +/- mice. Scale bars, 50 μm. (I) Immunofluorescence staining of the EMT markers α-SMA, FN, SNAI1, and ATF6 in murine lens sections. Scale bars, 50 μm.

Article Snippet: Male Atf6 +/- mice were purchased from Cyagen Biosciences (Guangdong, China).

Techniques: In Vivo, Infection, Microscopy, Staining, Immunofluorescence, Marker, Immunostaining

Journal: Theranostics

Article Title: Targeting the self-amplifying loop between ATF6 and SNAI1 to inhibit epithelial‒mesenchymal transition in fibrotic lesions

doi: 10.7150/thno.109442

Figure Lengend Snippet: The ATF6-SNAI1 self-amplifying loop drives EMT in vitro . (A) FN, ATF6, c-ATF6, and SNAI1 expression in the control, LV-mCherry, LV-ATF6 and LV-ATF6+LV-shSNAI1 groups, were subjected to western blot analyses. Protein expression was quantified (n = 5). Data are presented as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001. (B, C) Quantification of the ATF6 and SNAI1 expression by qPCR and western blotting. Data are presented as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001. (D) Luciferase assay experimental design and expression in 293T cells. No. 1 refers to [-2000/+100] SNAI1 , No. 2 refers to [-1500/-600] SNAI1 , No. 3 refers to [-600/-200] SNAI1 , No. 4 refers to [-200/+100] SNAI1 , No. 5 refers to [-200/+100 Delate] SNAI1 and No. 6 refers to [-200/+100 Mutant] SNAI1 (n = 3) . Data are presented as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001. (E) Luciferase assay experimental design and expression in 293T cells. No. 1 indicates [-2000/+130] ATF6 , No. 2 indicates [-1300/+130] ATF6 , No. 3 indicates [-800/+130] ATF6 , No. 4 indicates [-500/+130] ATF6 (n = 3) . No. 5 refers to [-500/+100 Delate] ATF6 and No. 6 refers to [-500/+100 Mutant] ATF6 (n = 3) . Data are presented as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001 (F) Study design for human lens capsule explant culture. (G) Immunofluorescence staining of SNAI1 in AAV-mCherry-infected human lens capsules and AAV-ATF6-infected human lens capsules. Arrow indicates nuclear fluorescent signals of SNAI1. Scale bars, 25 μm.

Article Snippet: Male Atf6 +/- mice were purchased from Cyagen Biosciences (Guangdong, China).

Techniques: In Vitro, Expressing, Control, Western Blot, Luciferase, Mutagenesis, Immunofluorescence, Staining, Infection, Capsules

Journal: Theranostics

Article Title: Targeting the self-amplifying loop between ATF6 and SNAI1 to inhibit epithelial‒mesenchymal transition in fibrotic lesions

doi: 10.7150/thno.109442

Figure Lengend Snippet: Effects of inhibiting ATF6 activation on EMT-like cell behaviors in vitro . (A) qPCR and quantification of ATF6, FN and SNAI1 expression (n = 5). Data are presented as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001. (B) Western blotting and analysis of the EMT markers FN, SNAI1, ATF6 and c-ATF6 (n = 5). The asterisk denotes an unglycosylated form of full-length ATF6 due to TM treatment. Data are presented as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001. (C) Migration ability of HLE-B3 cells in the TM, TM+LV-shATF6, and TM+MLT groups, as detected by wound healing and transwell assays. (n = 5). Scale bars, 50 μm. Quantification of wound closure and the number of migrating cells (n = 5). Data are presented as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001. (D) Immunofluorescence staining of SNAI1 in HLE-B3 cells in the TM, TM+LV-shATF6, and TM+MLT groups. Scale bars, 50 μm. Quantitative colocalization analysis of immunostaining for DAPI and SNAI1 by Pearson's correlation coefficients (n = 5). Data are presented as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001. (E) qPCR and quantification of FN and SNAI1 expression (n = 5). Data are presented as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001. (F) Western blotting and analysis of the EMT markers FN, SNAI1 and c-ATF6 (n = 5). Data are presented as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001. (G) Migration ability of HLE-B3 cells in the TM, TM+Ceapin-A7 groups, as detected by wound healing. (n = 5). Scale bars, 50 μm. Quantification of wound closure (n = 5). Data are presented as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Male Atf6 +/- mice were purchased from Cyagen Biosciences (Guangdong, China).

Techniques: Activation Assay, In Vitro, Expressing, Western Blot, Migration, Immunofluorescence, Staining, Immunostaining

Journal: Theranostics

Article Title: Targeting the self-amplifying loop between ATF6 and SNAI1 to inhibit epithelial‒mesenchymal transition in fibrotic lesions

doi: 10.7150/thno.109442

Figure Lengend Snippet: Melatonin effectively mitigated EMT-like alterations in parallel with Atf6 knockdown in vivo . (A) Images of the lenses of LP mice, LP+LV-shATF6-treated mice, and LP+MLT mice under a slit lamp microscope. Scale bars, 200 & 500 μm. Quantification of the subcapsular plaque area (n = 6). Data are presented as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001. (B) Representative images of lens sections with immunofluorescence staining of α‑SMA and FN. Scale bars, 10 μm. (C) Whole-mount immunofluorescence staining for ATF6 and SNAI1 in the above groups. Scale bars, 50 μm. (D) Western blotting and quantification of the EMT markers SNAI1 and c-ATF6 (n = 5). Data are presented as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001. (E) Images of laser-induced lens-punctured mice treated with MLT for 7 or 14 d under a slit‒lamp microscope. Scale bars, 200 μm. Quantification of opacity area (n = 5). Data are presented as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001. (F) Images of the lenses of LP mice and LP+Ceapin-A7-treated mice under a slit lamp microscope. Scale bars, 200 μm. Quantification of opacity area (n = 13). Data are presented as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Male Atf6 +/- mice were purchased from Cyagen Biosciences (Guangdong, China).

Techniques: Knockdown, In Vivo, Microscopy, Immunofluorescence, Staining, Western Blot

Journal: BMC Research Notes

Article Title: Dengue 2 infection of HepG2 liver cells results in endoplasmic reticulum stress and induction of multiple pathways of cell death

doi: 10.1186/1756-0500-6-372

Figure Lengend Snippet: Activation of the UPR in DENV 2 infected HepG2 cells. HepG2 cells were either infected with DENV 2 at m.o.i. 10 or mock infected and examined at 24 hours post infection by confocal microscopy for the expression of (a) GRP78 and PERK or GRP78 and ATF6 or (b) GRP78 alone or (c) GRP78 and DENV E protein. For (a) and (c) representative merged images are shown.

Article Snippet: Antibodies used were a 1:10 dilution of rabbit polyclonal anti GRP78 antibody (sc-13968; Santa Cruz Biotechnology Inc., Santa Cruz, CA.) followed by either a 1:50 dilution of a Rhodamine Red X conjugated goat anti rabbit IgG antibody (111-295-144; Jackson, West Grove, PA) or a 1:300 dilution of a FITC conjugated donkey anti rabbit IgG antibody (sc-2090; Santa Cruz Biotechnology Inc.), a 1:10 dilution of a goat polyclonal anti GRP78 antibody (sc-1050; Santa Cruz Biotechnology, Inc) followed by a 1:100 dilution of a Cy5 conjugated rabbit anti goat IgG antibody (81–1616; Invitrogen, Grand Island NY), a 1:200 dilution of mouse monoclonal anti dengue complex (MAB8705, Chemicon, EMD Millipore Corporation, Billerica, MA CA) followed by a 1:10 dilution of a FITC conjugated goat anti mouse IgG antibody (02-18-06; KPL, Guilford, UK), a 1:50 dilution of a mouse monoclonal anti ATF6 antibody (IMG-273; Imgenex, San Diego, CA) followed by a 1:10 dilution of a FITC conjugated goat anti mouse IgG (02-18-06; KPL, Gaithersburg, MD), a 1:50 dilution of a goat polyclonal anti PERK antibody (sc-9481; Santa Cruz Biotechnology, Inc.) followed by a 1:100 dilution of a Cy5 conjugated rabbit anti-goat IgG (81–1616; Invitrogen).

Techniques: Activation Assay, Infection, Confocal Microscopy, Expressing