laser confocal microscope imaging analysis  (Evident Corporation)

Journal: Plant & cell physiology

Article Title: Identification of New ATG8s-Binding Proteins with Canonical LC3-Interacting Region in Autophagosomes of Barley Callus.

doi: 10.1093/pcp/pcac015

Figure Lengend Snippet: Fig. 5 RPP3 is recruited to autophagosomes under TM-induced ER stress. (A) RPP3 showed the punctate structure under TM-induced ER stress. GFP-RPP3 was expressed in protoplasts before TM treatment as control. The protoplasts then were incubated in tunicamycin (TM, 50 µg ml−1), Concanamycin (ConA, 1 µM), or tunicamycin (TM, 50 µg ml−1) + Concanamycin (ConA, 1 µM) containing media for 24 h. Bar = 10 µm. (B) Quantification of the autophagosomes (APG) per cell. Bars represent the mean (±SD) of at least 30 cells from three biological replicates. (C) RPP3-mCherry puncta colocalized with GFP-ATG8 on autophagosomes. The white arrowheads denote the autophagosomes induced by 50 µg ml−1 TM and/or 1 µM ConA treatment. Bar = 10 µm. The right panel shows the Pearson’s coefficient analysis of the colocalization between RPP3- mCherry and GFP-ATG8 under different treatments. Bars represent the mean (±SD) of at least 30 cells from three biological replicates. (D–E) Autophagic flux analysis of p35S::GFP-ATG8b and p35S::RPP3-GFP by TM treatment. GFP-ATG8b was introduced as the positive control. Each group was incubated in media with or without 50 µg ml−1 TM for 12 h. In addition, the CK group and TM treatment group were supplied with 1 µM ConA.

Article Snippet: Confocal microscopy and colocalization analysis Confocal imaging was performed using a model FV3000 laser scanning confocal microscope (Olympus).

Techniques: Control, Incubation, Positive Control