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1.
Figure 3

Figure 3. KFERQ-PS-CFP2 colocalizes with lysosomes after induction of CMA. From: A photoconvertible fluorescent reporter to track chaperone-mediated autophagy.

(a) Localization of photoconverted KFERQ-PS-CFP2 (green) in LysoTracker-stained compartments (red) in mouse fibroblasts maintained in the presence or absence of serum. (b) Percentage of colocalization of KFERQ-PS-CFP2 with the indicated proteins. Values are mean + SE of 3 different experiments with >50 cells counted per experiment. (c–f) Co-localization of photoconverted KFERQ-PS-CFP2 (green) with LAMP-1, LAMP-2 or LAMP-2A (b), LC3, CD-M6PR, Rab5 (d) and ubiquitin (e) in mouse fibroblasts maintained in serum-free media. d. Immunofluorescence with antibodies that recognize PS-CFP2 (green) and hsc70 (red) in cells maintained in the absence of serum and fixed with methanol to eliminate the soluble cytosolic fraction. Scale bars: 5μm

Hiroshi Koga, et al. Nat Commun. ;2:386-386.
2.
Figure 4

Figure 4. Association of KFERQ-PS-CFP2 with lysosomes requires functional CMA. From: A photoconvertible fluorescent reporter to track chaperone-mediated autophagy.

(a) Mouse fibroblasts knocked-down for LAMP-2A and transfected with KFERQ-PS-CFP2 were photoconverted and maintained in media supplemented (+) or not (−) with serum for 16h. Representative images of cells also immunostained for LAMP-1 are shown. (b,c) Mouse fibroblasts stably expressing KFERQ-PS-CFP2 were photoconverted and maintained for 16h in media supplemented (+) or not (−) with serum in the presence of 3-methyladenine (3-MA) to inhibit macroautophagy. (b) Representative images of the 3-MA-treated cells. (c) Quantification of the number of green fluorescence puncta per cell in 3-MA treated or not cells maintained in the presence (white bars) or absence (black bars) of serum. Values are mean + SE of 3 different experiments with >50 cells counted per experiment. No significant differences were detectable between samples supplemented or not with 3MA. Scale bars: 5μm.

Hiroshi Koga, et al. Nat Commun. ;2:386-386.
3.
Figure 2

Figure 2. Temporal course of the changes in KFERQ-PS-CFP2 fluorescence during starvation. From: A photoconvertible fluorescent reporter to track chaperone-mediated autophagy.

(a–c) Temporal changes in the green fluorescence pattern of photoactivated NIH3T3 cells stably expressing KFERQ-PS-CFP2 maintained in the presence (black circles) or absence (white squares) of serum for the indicated times. (a) Representative images of cells maintained in serum-free media. Quantification of the number of puncta/cell (b) and green fluorescence intensity (c). Values are expressed as percentage of fluorescence at time 0 and half-lives (t1/2) are indicated. Differences in the number of puncta and fluorescence intensity between cells maintained in the presence and absence of serum (*) were significant for p=0.048 (16h) and p=0.04 (24h) (t-test). (d) The same cells imaged in the cyan channel, before and at the indicated times after photoconversion (arrow) by exposure to 405nm light. Top: Representative images of the cyan fluorescence. Bottom: Quantification of the intensity of the cyan fluorescence. Values are expressed as times the intensity before photoconversion that was given an arbitrary value of 1. Values are mean + SE of 3 different experiments with >10 cells counted per experiment. Scale bars: 5μm

Hiroshi Koga, et al. Nat Commun. ;2:386-386.
4.
Figure 7

Figure 7. A photoactivable fluorescent CMA reporter. From: A photoconvertible fluorescent reporter to track chaperone-mediated autophagy.

(a) Scheme of the experimental design to monitor CMA activity in cultured cells using the KFERQ-PA-mCherry1 reporter. (b) Mouse fibroblasts (NIH3T3) stably expressing KFERQ-PA-mCherry1 were photoactivated and maintained in media supplemented (+) or not (−) with serum for 16h. Left: Representative images. Nuclei are staining with DAPI. Bottom: Quantification of the number of red fluorescent puncta per cell. Values are mean + SE of three different experiments with >20 cells counted per experiment. *; p=0.001, ANOVA-Bonferroni. (c) Temporal changes in the mCherry fluorescent intensity in cells stably expressing KFERQ-PA-mCherry1 at the indicated times after photoactivation using FACS analysis. Cells were maintained in media supplemented (white squares) or not (black squares) with serum, and fluorescence by the MFI was calculated on events gated on living cells. Values are expressed as percentage of the intensity at time 0 and are mean + SE of 3–4 individual experiments. (d) Representative FACS experiment showing the effect of serum removal at 6h (top) and 42h (bottom). PS: photoswitching. Scale bars: 5μm.

Hiroshi Koga, et al. Nat Commun. ;2:386-386.
5.
Figure 5

Figure 5. KFERQ-PS-CFP2 proteins reach the lysosomal lumen. From: A photoconvertible fluorescent reporter to track chaperone-mediated autophagy.

(a) Immunoblot with the antibody that recognizes PS-CFP2 of homogenates (Hom), cytosol (Cyt) and lysosomal membranes (MB) and matrices (Mtx) isolated from mouse fibroblasts stably expressing PS-CFP2 or KFERQ-PS-CFP2 proteins 16h after serum removal. (b) Immunoblot for the indicated proteins of homogenates (Hom), mitochondria (Mit) and lysosomal membranes and matrices isolated from cells stably expressing KFERQ-PS-CFP2 maintained for 16h in media supplemented (+) or not (−) with serum. (c) Quantification of the amount of KFERQ-PS-CFP2 associated to lysosomes (matrix and membrane) performed in blots as the one shown in b. Values are expressed as percentage of the total KFERQ-PS-CFP2 in the cell and are mean + SE of three different experiments. Difference in the lysosomal associated KFERQ-PS-CFP2 between serum+ and serum− condition (*) were significant for p=0.033 (t-test). (d) Changes in the intracellular content of KFERQ-PS-CFP2 upon serum removal. Values are expressed as percentage of KFERQ-PS-CFP2 present in serum-supplemented cells and area mean + SE of three different experiments. Values are mean+ SE of three different experiments and differences are significant (*) for p=0.007 (t-test). (e–g) Effect of inhibition of macroautophagy by treatment with 3-methyladenine (3-MA) on the lysosomal content of KFERQ-PS-CFP2 in cells maintained in serum-free media for 16h. (e) Immunoblot for the indicated proteins of the same fractions as described in b. The amount of KFERQ-PS-CFP2 associated to lysosomes (f) and the changes in the intracellular content of KFERQ-PS-CFP2 were calculated as in c and d. Values are mean+ SE of three different experiments and differences are significant (*) for p=0.032 (t-test).

Hiroshi Koga, et al. Nat Commun. ;2:386-386.
6.
Figure 1

Figure 1. The fluorescence pattern of the KFERQ-PS-CFP2 reporter changes during starvation. From: A photoconvertible fluorescent reporter to track chaperone-mediated autophagy.

(a) Scheme of the insertion of the CMA-targeting motif in PS-CFP2. (b) Map of pKFERQ-PS-CFP2. (c) Scheme of the experimental design to monitor CMA activity in cultured cells using the KFERQ-PS-CFP2 reporter. (d) NIH3T3 cells stably expressing PS-CFP2 proteins were exposed to a 405nm light for the indicated times and photoconverting was monitored by imaging cells in the green and blue channels. (e,f) Mouse fibroblasts (NIH3T3) stably expressing PS-CFP2 or KFERQ-PS-CFP2 were photoconverted and maintained in media supplemented (+) or not (−) with serum for 16h. (e) Representative images in both channels. (f) Quantification of the number of green fluorescent puncta per cell in cells maintained in the presence (white bars) or absence (gray bars) of serum. Values are mean + SE of four different experiments with >50 cells counted per experiment. (*) Differences with PS-CFP2 in the green channel (p=0.005, ANOVA-Bonferroni). (§) Differences with samples in the presence of serum (p=0.03, ANOVA-Bonferroni). (g) Green fluorescence intensity at excitation 450nm and emission 510–550nm. Fluorescence intensity in each sample was normalized by the number of cells present in the sample and the percentage of intensity at time 0 still remaining after 16h of incubation in the presence (white bars) or absence (gray bars) of serum was calculated for each condition. Values are expressed as times the decay observed in cells maintained in the presence of serum and are means + SE of 3 independent experiments. Differences with serum (*) were significant for p=0.04 (t-test). Scale bars: 5μm

Hiroshi Koga, et al. Nat Commun. ;2:386-386.
7.
Figure 6

Figure 6. Practical application of the KFERQ-PS-CFP2 reporter protein to monitor changes in CMA activity. From: A photoconvertible fluorescent reporter to track chaperone-mediated autophagy.

(a,b) Human neuroblastoma (SH-SY5Y), hepatoma (HuH-7) and breast adenocarcinoma (MCF-7) cell lines were transiently transfected with KFERQ-PS-CFP2, photoconverted and maintained in media supplemented (+) or not (−) with serum for 16h. Images show representative cells after immunostaining for LAMP-2A (red). (b) Quantification of the number of green fluorescent puncta per cell under basal conditions (black bars) and after serum removal (gray bars). Values of NIH3T3 are included as reference. Values are mean + SE of three to five different experiments with >50 cells counted per experiment. Differences with (*) NIH3T3 or (§) with cell supplemented with serum are significant for p<0.05 (range 0.002–0.03, ANOVA-Bonferroni). (c) Primary mouse skin fibroblasts were transduced with a lentiviral vector for expression of KFERQ-PS-CFP2, photoconverted and maintained in media supplemented (+) or not (−) with serum for 16h. Left: representative image in the green channel. Right: Quantification of the number of green fluorescent puncta per cell in cells maintained in the presence (black bars) or absence (gray bars) of serum. Values are mean + SE of three different experiments with >50 cells counted per experiment. (p=0.002, ANOVA-Bonferroni). (d) Mouse fibroblasts stably expressing KFERQ-PS-CFP2 were plated in 384-well plates, maintained in the presence (white bar) or absence (black bars) of serum and subjected to treatment with 3-methyladenine (3-MA), the proteasome inhibitor MG-132 or H2O2. (e) Mouse fibroblasts stably expressing KFERQ-PS-CFP2 were plated in 384-well plates, maintained in the presence of serum supplemented media and leave untreated (white bar) or subjected to the indicated treatments (black bars). In both d and e, after fixation, images were subjected to high content image analysis and the percent of responders was calculated in >200 cells/condition. Value are mean + SE of results in three different experiments. * p<0.001 (range 0.0002–0.0007, t-test). Scale bars: 5μm.

Hiroshi Koga, et al. Nat Commun. ;2:386-386.

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