Metamorph software version 7

For immunostaining, LGs were dissected from matured third instar larvae and fixed in 3.7% paraformaldehyde for 15 min. After repeated washing, the fixed samples were incubated with primary antibody at 4°C for overnight. The following anti-Mmp1 antibodies (#3A6B4, #3B8D12, and #5H7B11) were mixed and used (1:100 for each; DSHB, IA, USA). After extensive washing, specimens were incubated with Alexa 594 secondary antibody (1:400; Molecular Probe, USA). The LG specimens were observed under a fluorescence microscope (Olympus, Tokyo, Japan, model: IX81), outfitted with excitation, emission filter wheels (Olympus). The fluorescence signals were collected using a 10x dry objective lens. Specimens were illuminated with UV filtered and shuttered light using the appropriate filter wheel combinations through a GFP filter cube. GFP fluorescence images were captured with a CCD camera (Hamamatsu Photonics, Shizuoka, Japan). Image acquisition was controlled through the Metamorph software version 7.6 (Molecular Devices, Sunnyvale, CA, USA) and processed with Adobe Photoshop CS. The basement membrane of the LG cells was observed under a confocal microscope from the surface to the inside of the tissue (Fv10i, Olympus, Tokyo, Japan) by altering the focus along the z-axis. The confocal images obtained were then processed by the Fv10i software and Adobe photoshop CS (Adobe KK, Tokyo, Japan).

Immunostaining of imaginal discs and larval brains were carried out according to the method previously described in [27 (link)]. In this study, the following antibodies were used as primary antibodies: anti-GRP78 (StressMarq Biosciences, Cadboro Bay, Victoria, BC, Canada), rabbit anti-Cleaved Caspase-3 (Asp175) (9661, Cell Signaling, Danvers, MA, USA), and anti-phospho-JNK (pThr183, pTyr185) (Calbiochem Co., La Jolla, CA, USA). The samples were observed under a fluorescent microscope model IX81 (Olympus Co., Tokyo, Japan). Image acquisition was performed using the Metamorph software version 7.6 (Molecular Devices, San Jose, CA, USA), and processed with ImageJ or Adobe Photoshop CS3 (Adobe Japan, Tokyo, Japan). Quantification of immunofluorescence intensities in the wing discs, in which Bx-Gal4-dependent gene expression occurred, were performed using image-J software (NIH Image, Bethesda, MD, USA). The intensity values calculated were normalized as 1.0 [27 (link)]. Comparisons of the two groups were carried out using the Student’s t-test. A p value of less than 0.05 was considered to be significant.

In situ PLA that enables detection of protein interaction within a cell was performed according to the Duolink kit method (Nacalai Inc., Kyoto, Japan). We applied the in situ PLA method to examine a close association between three sets of proteins, Emb and CycB, Emb and Nup58, and Nup62 and CycB. For the detection of complexes containing the first protein set, we used anti-HA and anti-CycB antibodies to recognize complexes containing Emb-HA and Cyclin B. We used anti-HA and anti-GFP antibodies to detect complexes containing Emb-HA and Nup58-GFP. We used anti-HA and anti-CycB antibodies to recognize complexes containing Nup62-HA and CycB. We observed a positive control of in situ PLA signals indicating association between CycB and CDK1 using the relevant antibodies. Samples were observed with a fluorescent microscope (Olympus, Tokyo, Japan, model: IX81). Image acquisition was controlled through the Metamorph software version 7.6 (Molecular Devices) and processed with ImageJ version 1.51 or Adobe Photoshop CS4.