Journal: The FASEB Journal

Article Title: Identification of the novel role of sterol regulatory element binding proteins (SREBPs) in mechanotransduction and intraocular pressure regulation

doi: 10.1096/fj.202301185r

Figure Lengend Snippet: FIGURE 2 Constitutive induction of SREBPs activation modulates actin and focal adhesion dynamics. (A and B) Immunofluorescence (IF) shows the distribution of SREBP1, SREBP2, filamentous actin (F-actin) fibers, and vinculin in HTM cells under AdMT and Ad5-N- SREBPs treatments. (A) Ad5-N-SREBP1a (second-row third column), and Ad5-N-SREBP1c (third-row third column) induced strong staining of SREBP1 in the nucleus in HTM cells compared to AdMT (first-row third column). Similarly, (B) Ad5-N-SREBP2 (second-row third column) induced strong staining of SREBP2 in the nucleus in HTM cells compared to AdMT (first-row third column). Compared to AdMT (first-row fifth column), (A) Ad5-N-SREBP1a (second-row fifth column) and Ad5-N-SREBP1c (third-row fifth column), and (B) Ad5-N- SREBP2 (second-row fifth column) caused the increased distribution of F-actin fibers stained by phalloidin (purple/grayscale) in HTM cells and induced increased lamellipodia and filopodia formation (indicated by yellow arrows). (A) Ad5-N-SREBP1a (second-row fourth column), Ad5-N-SREBP1c (third-row fourth column), and (B) Ad5-N-SREBP2 (second-row fourth column) also induced more distribution of vinculin (green/grayscale) at the edges of F-actin fibers (indicated by white arrows) in HTM cells compared to the AdMT (first-row fourth column). The nucleus was stained with DAPI in blue. Images were captured in z-stack in a confocal microscope, and stacks were orthogonally projected. Scale bar 20 microns. (C) Immunofluorescence (IF) shows the distribution of Arp2/3 and RAPH1 in HTM cells under AdMT and Ad5-N-SREBPs treatment. Compared to AdMT, Ad5-N-SREBPs induced the distribution of Arp2/3 and RAPH1 at the cell membrane and near the filopodia structures. Images were captured in z-stack in a confocal microscope, and stacks were orthogonally projected. Scale bar 5 microns. (D) Transmission electron microscope (TEM) image of HTM cells under AdMT and Ad5-N-SREBPs treatment. Compared to AdMT, Ad5-N-SREBP1a, Ad5-N-SREBP1c, and Ad5-N-SREBP2 induced membrane bending and filopodia formation. Scale bar as shown in the figure.

Article Snippet: Generation of replication- defective recombinant adenovirus expressing N- SREBPs under CMV promoter was performed using ViraPower Adenoviral Expression System (#K4930- 00, Invitrogen) as described earlier.57 N- SREBP1a (1470 bps), N- SREBP1c (1398 bps), and NSREBP2 (1440 bps) were amplified using high- fidelity PCR (Advantage- HF 2 PCR kit, #639123; Clontech) from human pcDNA3.1- 2xFLAG- SREBP1a (#26801, Addgene), pcDNA3.1- 2xFLAG- SREBP1c (#26802, Addgene) and pcDNA3.1- 2xFLAG- SREBP- 2 (#26807, Addgene).

Techniques: Activation Assay, Immunofluorescence, Staining, Microscopy, Membrane, Transmission Assay

Journal: The FASEB Journal

Article Title: Identification of the novel role of sterol regulatory element binding proteins (SREBPs) in mechanotransduction and intraocular pressure regulation

doi: 10.1096/fj.202301185r

Figure Lengend Snippet: FIGURE 7 SREBP activation is a critical regulator of ECM engagement to the matrix sites. (A) Regulation of ECM mRNA expression under fatostatin treatment. Fatostatin treatment significantly decreased mRNA expression in fibronectin (FN), collagen 1α (COL1α), collagen 6α (COL6α), collagen 4α (COL4α), tenascin C (TNC), transforming growth factor β2 (TGFβ2), and sarcospan (SSPN) in HTM cells compared to DMSO-treated control HTM cells. (B) Immunoblotting for whole cell lysate (CL) shows that fatostatin significantly decreased FN, and COL1A protein expression compared to DMSO-treated control HTM cells. GAPDH was used as a loading control. (C) Immunoblotting for conditioned media (CM) shows that fatostatin significantly decreased FN and COL1A protein expression in HTM CM compared to DMSO-treated control HTM CM. Ponceau S was used as a loading control. (D and E) Immunofluorescence (IF) shows the FN and COL1A expression and distribution in HTM cells after DMSO or fatostatin treatment. Fatostatin reduced FN (green staining) and COL1A (green staining) distribution compared to the control. The nucleus was stained with DAPI in blue. (F and G) Immunofluorescence (IF) shows that compared to fatostatin combined with AdMT treatment (first-row third column), there was increased FN and COL1A expression and distribution in HTM cells in fatostatin combined with Ad5-N-SREBP1a (second-row third column), Ad5-N-SREBP1c (third-row third column) and Ad5-N-SREBP2 (fourth-row third column) treatments. The nucleus was stained with DAPI in blue. Images were captured in z-stack in a confocal microscope, and stacks were orthogonally projected. Scale bar 20 microns. The lower magnification images of the cells can be visualized in the Figure S7. Values represent the mean ± SEM, where n = 4 (biological replicates). *p < .05 was considered statistically significant.

Article Snippet: Generation of replication- defective recombinant adenovirus expressing N- SREBPs under CMV promoter was performed using ViraPower Adenoviral Expression System (#K4930- 00, Invitrogen) as described earlier.57 N- SREBP1a (1470 bps), N- SREBP1c (1398 bps), and NSREBP2 (1440 bps) were amplified using high- fidelity PCR (Advantage- HF 2 PCR kit, #639123; Clontech) from human pcDNA3.1- 2xFLAG- SREBP1a (#26801, Addgene), pcDNA3.1- 2xFLAG- SREBP1c (#26802, Addgene) and pcDNA3.1- 2xFLAG- SREBP- 2 (#26807, Addgene).

Techniques: Activation Assay, Expressing, Control, Western Blot, Immunofluorescence, Staining, Microscopy