Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Dynamics of the lens basement membrane capsule and its interaction with connective tissue-like extracapsular matrix proteins.

doi: 10.1016/j.matbio.2020.12.005

Figure Lengend Snippet: Fig. 1. Development-state specific expression of basement membrane proteins in the chick embryo lens capsule. (A) Model of the lens capsule and its relationship to other compartments of the eye. The anterior capsule (red) associates with the aqueous humor and faces the cornea; the equatorial capsule (green) is directly linked to the ciliary zonules (CZ) which extend from the ciliary body (CB); and the posterior capsule (yellow) associates with the vitreous humor and faces the retina. The dia- gram represents the E15 eye, at which point the lens has matured and is the latest stage of development examined in these stud- ies. All four regions of fiber cell differentiation depicted here are present by E9. Whole embryonic chick eyes E5 (B-D), E9 (E-G), and E15 (H-J) were sectioned and stained for the three primary basement membrane proteins, laminin111 (B, E, and H, red), per- lecan (C, F, and I, green), and collagen IV (D, G, and J, purple). Overview images of the entire lens were acquired at each embry- onic stage examined, using the same settings to demonstrate the changes in expression/localization during development. Laminin111 and perlecan were present in all regions of the lens capsule at each developmental stage examined (B-I), their inten- sity lowest in the thinner anterior capsule region. While perlecan intensity and distribution appear to increase in the equatorial zone by E15, laminin localization is diminished in all regions of the capsule by this developmental time. Collagen IV developmen- tal expression and capsule distribution is distinct from laminin and perlecan, its expression limited to the posterior capsule at E5 and greatly increasing throughout all regions of the lens capsule by E15. Mag bar 50mm (B-D), 100mm (E-G) and 200mm (H-J). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Primary antibodies used in these studies are presented in Table 1 and include: Fibrillin-2 (JB3, Developmental Studies Hybridoma Bank (DSHB)), Tenascin-C (AB19013, Millipore), Fibronectin (PA1 23693, Thermofisher), Nidogen/ Entactin (1G12, DSHB), Perlecan (SC-33707, Santa Cruz), Laminin (L9393, Sigma Aldrich, Enhanced Validation), Laminin (31 or 31 2, DSHB), and Collagen IV (ab6586, Abcam).

Techniques: Expressing, Membrane, Cell Differentiation, Staining

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Dynamics of the lens basement membrane capsule and its interaction with connective tissue-like extracapsular matrix proteins.

doi: 10.1016/j.matbio.2020.12.005

Figure Lengend Snippet: Fig. 2. Dynamic basement protein remodeling in the equatorial lens capsule during development. Cryosections of embryonic chick eyes were immunolabeled for three primary basement membrane proteins, laminin111 (A, D, and G, red), perlecan (B, E, and H, green), and collagen IV (C, F, and I, purple) at E5 (A-C), E9 (D-F), and E15 (G-I). Each section was co-labeled with DAPI to detect lens equatorial epithelial cell nuclei (blue). Confocal z-stacks were acquired in the region of the equatorial lens capsule from which the images presented are a single optical plane (0.5 mm). While at E5 and E9 both laminin111 (A, D, open arrowheads) and perlecan (B,E, arrows) are distributed throughout the lens equatorial capsule, by E15 these basement membrane proteins are present in two distinct lamellae (G and H), one at the inner, lens cell-facing side of the capsule, the other at the superficial surface of the capsule where the perlecan lamella is wider than the laminin lamella. In contrast, at early stages of lens development collagen IV is primarily present only in a thin lamella along the cells of the lens equatorial epithelium (C, F, solid arrowheads) and has expanded throughout the equatorial cap- sule by E15 (I). Mag bar 20mm (A-I). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Primary antibodies used in these studies are presented in Table 1 and include: Fibrillin-2 (JB3, Developmental Studies Hybridoma Bank (DSHB)), Tenascin-C (AB19013, Millipore), Fibronectin (PA1 23693, Thermofisher), Nidogen/ Entactin (1G12, DSHB), Perlecan (SC-33707, Santa Cruz), Laminin (L9393, Sigma Aldrich, Enhanced Validation), Laminin (31 or 31 2, DSHB), and Collagen IV (ab6586, Abcam).

Techniques: Immunolabeling, Membrane, Labeling

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Dynamics of the lens basement membrane capsule and its interaction with connective tissue-like extracapsular matrix proteins.

doi: 10.1016/j.matbio.2020.12.005

Figure Lengend Snippet: Fig. 3. Bilaminar laminin/perlecan domains in the adult mouse equatorial zone. Cryosection of adult mouse eye immunolabeled for laminin111 (A,C, red) and perlecan (B,C, green), and co-labeled with DAPI to detect lens equatorial epithelial cell nuclei (blue) and imaged by confocal microscopy. The image was acquired as a single optical plane. Both laminin111 (A, arrows) and perlecan (B, arrowheads) are present as two distinct lamellae in the adult mouse lens equato- rial capsule, one at the inner, lens cell-facing side of the capsule, the other at the superficial surface of the capsule. Mag bar 20mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Primary antibodies used in these studies are presented in Table 1 and include: Fibrillin-2 (JB3, Developmental Studies Hybridoma Bank (DSHB)), Tenascin-C (AB19013, Millipore), Fibronectin (PA1 23693, Thermofisher), Nidogen/ Entactin (1G12, DSHB), Perlecan (SC-33707, Santa Cruz), Laminin (L9393, Sigma Aldrich, Enhanced Validation), Laminin (31 or 31 2, DSHB), and Collagen IV (ab6586, Abcam).

Techniques: Immunolabeling, Labeling, Confocal Microscopy

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Dynamics of the lens basement membrane capsule and its interaction with connective tissue-like extracapsular matrix proteins.

doi: 10.1016/j.matbio.2020.12.005

Figure Lengend Snippet: Fig. 4. The relationship between basement membrane proteins that comprise the anterior lens capsule. Cryo- sections of E15 chick embryo eyes were co-immunolabeled with antibodies to perlecan (green), laminin111 (teal), and collagen IV (purple), in the following combinations: (A) perlecan/laminin111, (D) collagen IV/perlecan, and (G) collagen IV/laminin111. Sections were co-labeled for F-actin (A,D,G; white) and nuclei (A-I; blue). Z-stacks were acquired of the anterior lens capsule by confocal microscopy imaging from which a single optical plane (0.5mm) is shown as an overlay of the co-immunolabeled basement membrane proteins (A,D,G), alongside of which is presented each fluorescent channel in greyscale (B,E-perlecan; C,H-laminin111; F,I-collagen IV). Collagen IV (F,I) extends throughout the anterior capsule and is detected in the anterior epithelium. Laminin (C,H) also spans this region of the lens capsule and is highly coincident with collagen IV (G). In this region of the capsule there is a perlecan gradient, its expression greatest along the inner region of the anterior capsule (E), where it is most highly coincident with collagen IV (D) and laminin (A). An additional per- lecan-rich layer is occasionally detected at the superficial surface of the anterior lens capsule (B). Mag bar 20mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Primary antibodies used in these studies are presented in Table 1 and include: Fibrillin-2 (JB3, Developmental Studies Hybridoma Bank (DSHB)), Tenascin-C (AB19013, Millipore), Fibronectin (PA1 23693, Thermofisher), Nidogen/ Entactin (1G12, DSHB), Perlecan (SC-33707, Santa Cruz), Laminin (L9393, Sigma Aldrich, Enhanced Validation), Laminin (31 or 31 2, DSHB), and Collagen IV (ab6586, Abcam).

Techniques: Membrane, Immunolabeling, Labeling, Confocal Microscopy, Imaging, Expressing

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Dynamics of the lens basement membrane capsule and its interaction with connective tissue-like extracapsular matrix proteins.

doi: 10.1016/j.matbio.2020.12.005

Figure Lengend Snippet: Fig. 5. Structural imaging of the relationship between basement membrane proteins in the anterior lens cap- sule. 3D surface structure renderings were created with Imaris software from confocal z-stacks in the region of the ante- rior lens capsule (denoted by the area within the red rectangle in the model in A) of cryosections of the E15 chick embryo eye immunolabeled for basement membrane proteins laminin (B,C,J,K), perlecan (F,G,L,M) and collagen IV (D,H). The sections were co-immunolabeled for (B-D) laminin (teal)/collagen IV (purple), (F-H) perlecan (green)/collagen IV (purple), or (J-M) laminin (teal)/perlecan (green). Each was co-labeled for F-actin (white) and nuclei (dark blue). (C,G,K,M) are zoomed in structural images of (B,F,J,L), respectively. E and I are presented to demonstrate the surface structure of the basal surface of the anterior epithelial cells shown in C and G, respectively. These structural renderings reveal that lami- nin has a typical sheet-like structure that is contacted by the basal surfaces of the lens epithelium while perlecan and col- lagen IV have a basolateral distribution, with expression of collagen IV by the lens epithelium also observed (modeled in N). Z-stack sizes (B-E) 39 slices; (F-I) 36 slices; (J-M) 36 slices. LC lens capsule. Mag bar 0.5mm (C,E,G,I,K,M), 1mm (B,D,F,H), and 3mm (J,L). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Primary antibodies used in these studies are presented in Table 1 and include: Fibrillin-2 (JB3, Developmental Studies Hybridoma Bank (DSHB)), Tenascin-C (AB19013, Millipore), Fibronectin (PA1 23693, Thermofisher), Nidogen/ Entactin (1G12, DSHB), Perlecan (SC-33707, Santa Cruz), Laminin (L9393, Sigma Aldrich, Enhanced Validation), Laminin (31 or 31 2, DSHB), and Collagen IV (ab6586, Abcam).

Techniques: Imaging, Membrane, Software, Immunolabeling, Labeling, Expressing

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Dynamics of the lens basement membrane capsule and its interaction with connective tissue-like extracapsular matrix proteins.

doi: 10.1016/j.matbio.2020.12.005

Figure Lengend Snippet: Fig. 6. The relationship between basement membrane proteins that comprise the equatorial lens capsule. Cryo- sections of E15 chick embryo eyes were co-immunolabeled with antibodies to perlecan (green), laminin111 (teal), and collagen IV (purple), in the following combinations: (A) perlecan/laminin111, (D) collagen IV/perlecan, and (G) collagen IV/laminin111. Sections were co-labeled for F-actin (D,G; white) and nuclei (A-I; blue). Z-stacks were acquired of the equatorial lens capsule by confocal microscopy imaging from which a single optical plane (0.5mm) is shown as an overlay of the co-immunolabeled basement membrane proteins (A,D,G), alongside of which is presented each fluorescent chan- nel in greyscale (B,E-perlecan; C,H-laminin111; F,I-collagen IV). In the equatorial region of the lens capsule there are two distinct laminin and perlecan lamella that are located at the inner and outer surfaces of the capsule and are highly coinci- dent (A), with perlecan (B,E) extending farther toward the center of the capsule than laminin (C,H). Collagen IV is expressed throughout the equatorial capsule (F,I), co-distributing with the perlecan/laminin lamella at the inner and outer capsule surfaces that sandwich the collagen IV-rich region between them (D,G). Collagen IV immunolabeling was most intense in a thin basement membrane zone along the capsule’s innermost surface (F,I), and at times, distinct layers of col- lagen IV were resolved across this capsule zone (F). Mag bar 20mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Primary antibodies used in these studies are presented in Table 1 and include: Fibrillin-2 (JB3, Developmental Studies Hybridoma Bank (DSHB)), Tenascin-C (AB19013, Millipore), Fibronectin (PA1 23693, Thermofisher), Nidogen/ Entactin (1G12, DSHB), Perlecan (SC-33707, Santa Cruz), Laminin (L9393, Sigma Aldrich, Enhanced Validation), Laminin (31 or 31 2, DSHB), and Collagen IV (ab6586, Abcam).

Techniques: Membrane, Immunolabeling, Labeling, Confocal Microscopy, Imaging, Capsules

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Dynamics of the lens basement membrane capsule and its interaction with connective tissue-like extracapsular matrix proteins.

doi: 10.1016/j.matbio.2020.12.005

Figure Lengend Snippet: Fig. 7. Super-resolution images of the laminin/perlecan bilateral lamella in the equatorial lens capsule. Super- resolution microscopy of cryosections of E15 chick embryo eyes co-immunolabeled for (A,C,D,F) perlecan (green) and (B,C,E,F) laminin111 (red), or immunolabeled for (G,H) nidogen (teal) imaged at the lens equatorial capsule. D,E,F,H are zoomed in regions of A,B,C,G, respectively. Images are a 0.21 mm optical slice from a confocal z-stack. There is signifi- cant co-localization of the laminin and perlecan lamella at the inner and outer surfaces of the lens (C,F). In contrast, nido- gen spans the equatorial capsule region (G,H). Mag bar: (A,B,C,G) 20mm; (D,E,F,H) 10mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Primary antibodies used in these studies are presented in Table 1 and include: Fibrillin-2 (JB3, Developmental Studies Hybridoma Bank (DSHB)), Tenascin-C (AB19013, Millipore), Fibronectin (PA1 23693, Thermofisher), Nidogen/ Entactin (1G12, DSHB), Perlecan (SC-33707, Santa Cruz), Laminin (L9393, Sigma Aldrich, Enhanced Validation), Laminin (31 or 31 2, DSHB), and Collagen IV (ab6586, Abcam).

Techniques: Super-Resolution Microscopy, Immunolabeling

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Dynamics of the lens basement membrane capsule and its interaction with connective tissue-like extracapsular matrix proteins.

doi: 10.1016/j.matbio.2020.12.005

Figure Lengend Snippet: Fig. 9. Structural imaging of the relationship between basement membrane proteins in the equatorial lens cap- sule. 3D surface structure renderings were created with Imaris software from confocal z-stacks in the region of the equa- torial lens capsule (denoted by the area within the red rectangle in the model in A) of cryosections of the E15 chick embryo eye immunolabeled for basement membrane proteins perlecan (B,C,H, green), laminin111 (D,E,I, teal) and colla- gen IV (F, purple). The sections were immunolabeled for (B,C) perlecan (green), or represent sections co-immunolabeled for (D-G) laminin (teal)/collagen IV (purple), or (H,I) perlecan (green)/laminin (teal). (B-G) were co-labeled for F-actin (white) and nuclei (dark blue), with G presented to show the structure of the basal surface of the equatorial epithelial cells depicted in E. (C,E) are zoomed in structural images of (B,D), respectively. The structural images of laminin and perlecan provide the first look of the organization of the bilateral localization of the inner and outer laminin/perlecan basement mem- brane lamellae at the inner and outer surfaces of the lens equatorial capsule (modeled in J). While collagen IV is present throughout the entire width of the equatorial lens capsule (F), the structural organization of collagen IV at the inner and outer domains of this capsular zone parallel that of laminin (D,E). Z-stack sizes (D-G) 44 slices; (B,C) 58 slices; (H,I) 54 slices. LC- lens capsule. Mag bar 0.7mm (E, G-I), 0.8mm (C), and 5mm (B, D, F). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Primary antibodies used in these studies are presented in Table 1 and include: Fibrillin-2 (JB3, Developmental Studies Hybridoma Bank (DSHB)), Tenascin-C (AB19013, Millipore), Fibronectin (PA1 23693, Thermofisher), Nidogen/ Entactin (1G12, DSHB), Perlecan (SC-33707, Santa Cruz), Laminin (L9393, Sigma Aldrich, Enhanced Validation), Laminin (31 or 31 2, DSHB), and Collagen IV (ab6586, Abcam).

Techniques: Imaging, Membrane, Software, Immunolabeling, Labeling

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Dynamics of the lens basement membrane capsule and its interaction with connective tissue-like extracapsular matrix proteins.

doi: 10.1016/j.matbio.2020.12.005

Figure Lengend Snippet: Fig. 10. The relationship between basement membrane proteins that comprise the posterior lens capsule. Cry- osections of E15 chick embryo eyes were co-immunolabeled with antibodies to perlecan (green), laminin111 (teal), and collagen IV (purple), in the following combinations: (A) perlecan/laminin111, (D) collagen IV/perlecan, and (G) collagen IV/laminin111. Sections were co-labeled for F-actin (D,G; white). Z-stacks were acquired of the posterior lens capsule by confocal microscopy imaging from which a single optical plane (0.5mm) is shown as an overlay of the co-immunolabeled basement membrane proteins (A,D,G), alongside of which is presented each fluorescent channel in greyscale (B,E-perle- can; C,H-laminin111; F,I-collagen IV). Perlecan is present as two distinct lamellae in the posterior capsule (B,E), overlap- ping with both laminin (A) and collagen IV (D), both basement membrane proteins that span this capsule zone (G-I). Mag bar 20mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Primary antibodies used in these studies are presented in Table 1 and include: Fibrillin-2 (JB3, Developmental Studies Hybridoma Bank (DSHB)), Tenascin-C (AB19013, Millipore), Fibronectin (PA1 23693, Thermofisher), Nidogen/ Entactin (1G12, DSHB), Perlecan (SC-33707, Santa Cruz), Laminin (L9393, Sigma Aldrich, Enhanced Validation), Laminin (31 or 31 2, DSHB), and Collagen IV (ab6586, Abcam).

Techniques: Membrane, Immunolabeling, Labeling, Confocal Microscopy, Imaging

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Dynamics of the lens basement membrane capsule and its interaction with connective tissue-like extracapsular matrix proteins.

doi: 10.1016/j.matbio.2020.12.005

Figure Lengend Snippet: Fig. 11. Structural imaging of the relationship between basement membrane proteins in the posterior lens cap- sule. 3D surface structure renderings were created with Imaris software from confocal z-stacks in the region of the poste- rior lens capsule (denoted by the area within the red rectangle in the model in A) of cryosections of the E15 chick embryo eye immunolabeled for basement membrane proteins laminin (B,C,H,I), perlecan (D,E,J,K), and collagen IV (F). The sec- tions were immunolabeled for (B,C) laminin, or co-immunolabeled for (D-G) perlecan (green)/collagen IV (purple), or (H- K) laminin (teal)/perlecan (green). (B-G) were co-labeled for F-actin (white), with G presented to show the structure of the basal surface of the lens fiber cells depicted in E. (C,E,I,K) are zoomed in structural images of (B,D,H,J), respectively. These structural renderings show that the bilateral organization of perlecan continues into the posterior region of the lens capsule, a wide outer lamella and a thin inner lamella (D,E,J,K), both of which are located within the laminin (H,I) and col- lagen IV (F) domains that span the posterior capsule (modeled in L). Z-stack sizes (B,C) 39 slices; (D-G) 44 slices; (H- K) 47 slices. LC lens capsule. Mag bar 1mm (C, E, G, I, and K) and 3mm (B, D, F, H, and J). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Primary antibodies used in these studies are presented in Table 1 and include: Fibrillin-2 (JB3, Developmental Studies Hybridoma Bank (DSHB)), Tenascin-C (AB19013, Millipore), Fibronectin (PA1 23693, Thermofisher), Nidogen/ Entactin (1G12, DSHB), Perlecan (SC-33707, Santa Cruz), Laminin (L9393, Sigma Aldrich, Enhanced Validation), Laminin (31 or 31 2, DSHB), and Collagen IV (ab6586, Abcam).

Techniques: Imaging, Membrane, Software, Immunolabeling, Labeling

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Dynamics of the lens basement membrane capsule and its interaction with connective tissue-like extracapsular matrix proteins.

doi: 10.1016/j.matbio.2020.12.005

Figure Lengend Snippet: Fig. 16. Fibrillin-2 extracapsular matrix is closely associated with the lens basement membrane capsule. Cryo- sections of E15 chick embryo eyes were co-labeled for (A,C,E) fibrillin-2 (green), laminin (red) and nuclei (blue), or (B,D, F) fibrillin-2 (green) and perlecan (red). Images are a single optical plane (0.5 mm) of confocal z-stacks acquired in (A,B) the upper equatorial zone at its border with the lens anterior, (C,D) at the lens equator (EQ), and (E,F) in the lens posterior region. The extracapsular fibrillin-2 matrix zone is closely associated with laminin and perlecan along the superficial layer of the lens basement membrane capsule (arrowheads). Arrows point to regions labeling for the basement membrane pro- teins alone, with the arrow on the right in C indicating the very close apposition of fibrillin-2 with the outer laminin base- ment membrane lamella. CB ciliary body, CZ ciliary zonules. Mag bar 20mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Primary antibodies used in these studies are presented in Table 1 and include: Fibrillin-2 (JB3, Developmental Studies Hybridoma Bank (DSHB)), Tenascin-C (AB19013, Millipore), Fibronectin (PA1 23693, Thermofisher), Nidogen/ Entactin (1G12, DSHB), Perlecan (SC-33707, Santa Cruz), Laminin (L9393, Sigma Aldrich, Enhanced Validation), Laminin (31 or 31 2, DSHB), and Collagen IV (ab6586, Abcam).

Techniques: Membrane, Labeling

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Dynamics of the lens basement membrane capsule and its interaction with connective tissue-like extracapsular matrix proteins.

doi: 10.1016/j.matbio.2020.12.005

Figure Lengend Snippet: Fig. 17. Super-resolution images of the laminin/fibrillin-2 and perlecan/fibrillin-2 lamellae in the equatorial lens capsule. Super-resolution microscopy of cryosections of E15 chick embryo eyes co-immunolabeled for (A-D) fibrillin-2 (green) and either (A,B) laminin111 (red), or (C,D) perlecan (red) and imaged in the upper region of the lens equatorial zone near its border with the anterior of the lens (A,C) or directly at the lens equator (B,D). Each image is a 0.21 mm opti- cal slice. Fibrillin-2 is closely associated with the superficial surface of the outer laminin/perlecan lamella of the lens cap- sule. Mag bar 10 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Primary antibodies used in these studies are presented in Table 1 and include: Fibrillin-2 (JB3, Developmental Studies Hybridoma Bank (DSHB)), Tenascin-C (AB19013, Millipore), Fibronectin (PA1 23693, Thermofisher), Nidogen/ Entactin (1G12, DSHB), Perlecan (SC-33707, Santa Cruz), Laminin (L9393, Sigma Aldrich, Enhanced Validation), Laminin (31 or 31 2, DSHB), and Collagen IV (ab6586, Abcam).

Techniques: Super-Resolution Microscopy, Immunolabeling

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 1. miR-206 targets cyclin D1. (A) Sequence alignment between miR-206 and the 3′UTRs of cyclin D1 from different species. In brackets the 3′UTR size. (B) Diagram of the luciferase reporter construct with the putative miR-206 binding site (WT 3′UTR) and mutations (3′UTR MUT). (C) Relative lucif- erase activity was measured in HeLa cells after transfection of reporter constructs along with pSP65-U1 (CTR) or pSP65–206 (miR-206). Relative Firefly luciferase values were determined by a ratio of Firefly to Renilla luciferase with the control set to 1.00. Values are the means ± SD of 3 separate experi- ments. **A Student t test performed between control and miR-206 transfected cells yielded P values < 0.01.

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Sequencing, Luciferase, Construct, Binding Assay, Activity Assay, Transfection, Control

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 2. Expression kinetics of miR-206 and cyclin D1 in differentiating C2C12 cells. C2C12 myoblasts were seeded in GM at 1.5 × 104/cm2. Cells were shifted in DM 24 h after plating and left to differentiate for further 72 h. (A) Northern blot analysis of miR-206 expression in C2C12 cells after 24 h in GM (0) and at different time points upon shift to DM. (B) Western blot analysis of cyclin D1 and MyHC expression in C2C12 cells cultured as in (A). Equal RNA and protein loading was confirmed by detecting, snRNA U2 and β-tubulin, respectively. (C) MyHC immunofluorescence staining (green) of C2C12 cells after 24 h in GM (DM 0 h) and after 72 h in DM (DM 72 h). Nuclei were counterstained in blue (DAPI) and individual pictures of the same field, taken with a DC camera, were merged using a LEICA Microsystems Imaging Equipment. Bar = 20 μm.

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Expressing, Northern Blot, Western Blot, Cell Culture, Immunofluorescence, Staining, Imaging

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 3. miR-206 controls cyclin D1 accumulation in C2C12 cells. C2C12 myoblasts were seeded in GM at 2.5 × 103/cm2. Cells were transfected 24 h after plaiting. (A) Northern blot analysis of miR-206 expression (upper) and western blot analysis of cyclin D1 expression (lower) in C2C12 cells 48 h after transfection with a control vector (CTR) or with a miR-206 expression vector (miR-206). Cells were kept in GM throughout the experiment. (B) The effect of miR-206 overexpression on C2C12 cell proliferation and differentiation was evaluated 48 h after transfection by 1 h BrdU incorporation and MyHC staining, respectively. Results are represented relative to the BrdU+ nuclei or nuclei in MyHC+ cells in CTR (set to 1.00), as individually assessed in each independent experiment. Values are the means ± SD of 3 separate experiments. *A Student t test performed between control and miR-206 transfected cells yielded P values < 0.05. (C) Immunofluorescence staining of cyclin D1 (pink) and MyHC (green) 48 h after transfection. Nuclei were counterstained in blue with DAPI. Individual pictures of the same field, taken with a DC camera, were merged using a LEICA Microsystems Imaging Equipment. To obtain cyclin D1 images, before merging, individual pictures were pseudocol- ored using a LEICA Microsystems Imaging software. Bar = 10 μm. (D) C2C12 myoblasts were seeded at low (LD) and high (HD) density in GM. Cells were shifted to DM the day after plating and analyzed after further 3 d. The panels show a northern blot analysis of miR-206 expression (left panel) and a western blot analysis of cyclin D1 and differentiation associ- ated marker expression (right panel) after 24 h in GM and 72 h after shift- ing to DM. Equal RNA and protein loading was confirmed by detecting, snRNA U2 and β-tubulin, respectively.

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Transfection, Northern Blot, Expressing, Western Blot, Control, Plasmid Preparation, Over Expression, BrdU Incorporation Assay, Staining, Immunofluorescence, Imaging, Software, Marker

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 4. Inhibition of miR-206 rescues cyclin D1 in myotubes (A) Experimental scheme. C2C12 myoblasts were induced to differ- entiate in DM in the presence of AraC. After 3 d, AraC was washed out and cells left to recover in DM for further 24 h. Finally, pure myotubes were transfected with LNA against miR-206 and analyzed 48 h later. (B) Northern blot analysis of miR-206 and miR-1 expression (left panel) and western blot analysis of cyclin D1 expression (right panel) in pure myotubes transfected with a control LNA (LNA C) or anti-miR-206 LNA (LNA 206). Cyclin D1 expression in proliferating myoblasts is also shown (GM). Equal RNA and protein loading was confirmed by detecting, snRNA U2 and β-tubulin, respectively. (C) Double immunofluorescence staining of MyHC and cyclin D1 of pure myotubes transfected with a control LNA (LNA C) or anti-miR-206 LNA (LNA 206). Individual pictures of the same field, taken with a DC camera, were merged using a LEICA Microsystems Imaging Equipment. Bar = 10 μm.

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Inhibition, Transfection, Northern Blot, Expressing, Western Blot, Control, Double Immunofluorescence Staining, Imaging

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 5. miR-206 inhibits cell proliferation in Ras-transformed fibro- blasts. (A) Expression levels of cyclin D1 in NIH3T3(Ras) cells as compared with NIH3T3(BN) cells. (B) Real-time PCR analysis of miR-206 expres- sion in NIH3T3(Ras) cells. Results are shown relative to untransformed NIH3T3(BN) cells set to value 1.00. Each sample was analyzed in tripli- cate, and values are the means ± SD of 3 independent experiments. **A Student t test performed between untransformed and transformed cells yielded P values < 0.01. (C) NIH3T3(Ras) cells were transfected with a control vector (CTR) or with a miR-206 expression vector (miR-206) and analyzed 24 h later. Upper, northern blot analysis of miR-206 expression; lower, western blot analysis of cyclin D1 expression. (D) Effect of miR-206 forced expression on cell proliferation as determined by 1 h BrdU incor- poration. Data are reported relative to BrdU+ nuclei in CTR (set to 1.00), as individually assessed in each independent experiment. Values are the means ± SD of 3 separate experiments. *A Student t test performed between control and miR-206 transfected cells yielded P values < 0.05. Equal RNA and protein loading was confirmed by detecting, snRNA U2, and β-tubulin, respectively.

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Transformation Assay, Expressing, Real-time Polymerase Chain Reaction, Transfection, Control, Plasmid Preparation, Northern Blot, Western Blot

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 6. Relationship between miR-206 downregulation and cyclin D1 expression in NSCLCs. (A) Northern blot analysis of miR-206 in different murine tissues. snRNA U2 levels were used as a loading control. (B) Real-time PCR analysis of miR-206 expression in human NSCLC tissues. Results are shown relative to the matched normal lung tissues set to value 1.00. Each sample was analyzed in triplicate, and values are the means ± SD of three independent experiments. **A Student t test performed between normal and tumor tissues yielded P values < 0.01. (C) Western blot analysis of cyclin D1 expression in normal and neoplastic lung tissues. Equal protein loading was confirmed by detecting actin. n, normal tissue; t = tumor tissue

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Expressing, Northern Blot, Control, Real-time Polymerase Chain Reaction, Western Blot

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 7. miR-206 inhibits cancer cell proliferation through repression of cyclin D1. (A) A549 and HeLa cells were transfected with a control vec- tor (CTR) or with a miR-206 expression vector (miR-206) and analyzed 72 h later. Top panel: northern blot analysis of miR-206 expression; lower panel, western blot analysis of cyclin D1 expression. Equal RNA and protein loading was confirmed by detecting, snRNA U2 and β-tubulin, respectively. (B) Effect of miR-206 forced expression on cell prolifera- tion as determined by 1 h BrdU incorporation and immunofluorescence staining. Data are reported relative to BrdU+ nuclei in CTR (set to 1.00), as individually assessed in each independent experiment. Values are the means ± SD of 3 separate experiments. *A Student t test performed between control and miR-206 transfected cells yielded P values <0.05.

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Transfection, Control, Expressing, Plasmid Preparation, Northern Blot, Western Blot, BrdU Incorporation Assay, Immunofluorescence, Staining