Figure 2.
TNIP1 is degraded by autophagy. (A) U2OS cells were treated with 1 μM Torin-1 for 4 h; proteasomal or lysosomal degradation were inhibited by 10 μM MG132 or 2 nM ConA, respectively. Under fed conditions (DMSO) and in Torin-1 treated cells blockage of lysosomal acidification led to a significant increase of TNIP1 protein abundance. Shown are representative blots of three biological replicates. (B) Quantification of blots shown in A (n = 3). * = P < 0.05, ** = P < 0.01, *** = P < 0.001 unpaired, two-sided t test compared to DMSO treated samples. Error bars indicate SEM. (C) Confocal images showing colocalization between endogenous TNIP1, LAMP1 and LC3 in U2OS cells treated for 5 h with either BafA1 or vehicle DMSO. Cells were immunostained against endogenous TNIP1 (yellow), LAMP1 (cyan), and LC3 (purple) and imaged by Airyscan using the Zeiss LSM880 confocal microscope. Inserts highlight TNIP1 localized in LAMP1- and LC3-positive structures. Due to BafA1 treatment leading to accumulation of the immunostained proteins, signal intesities in the DMSO image have been increased relative to the BafA1 treated image during post-processing. Scale bars are 5 µm for the airyscan images and 1 µm for the inserts. (D) U2OS cells were transiently transfected with either mCherry-EYFP or mCherry-EYFP-TNIP1. 24 h after transfection, cells were either left untreated or treated with BafA1 for 4 h. BF = bright field. Scale bars, 20 µm. Quantification of red-only TNIP1 dots over total TNIP1 dots was done using Volocity software (PerkinElmer), with intensity cut-offs based on BafA1 intensity of red and green dots (n = 3). Around 2,000–3,000 dots were counted for each condition within each replicate. * = P < 0.05, unpaired, two-sided t test. Error bars indicates SD. In B and D, data distribution was assumed to be normal, but this was not formally tested. Source data are available for this figure: SourceData F2.

TNIP1 is degraded by autophagy. (A) U2OS cells were treated with 1 μM Torin-1 for 4 h; proteasomal or lysosomal degradation were inhibited by 10 μM MG132 or 2 nM ConA, respectively. Under fed conditions (DMSO) and in Torin-1 treated cells blockage of lysosomal acidification led to a significant increase of TNIP1 protein abundance. Shown are representative blots of three biological replicates. (B) Quantification of blots shown in A (n = 3). * = P < 0.05, ** = P < 0.01, *** = P < 0.001 unpaired, two-sided t test compared to DMSO treated samples. Error bars indicate SEM. (C) Confocal images showing colocalization between endogenous TNIP1, LAMP1 and LC3 in U2OS cells treated for 5 h with either BafA1 or vehicle DMSO. Cells were immunostained against endogenous TNIP1 (yellow), LAMP1 (cyan), and LC3 (purple) and imaged by Airyscan using the Zeiss LSM880 confocal microscope. Inserts highlight TNIP1 localized in LAMP1- and LC3-positive structures. Due to BafA1 treatment leading to accumulation of the immunostained proteins, signal intesities in the DMSO image have been increased relative to the BafA1 treated image during post-processing. Scale bars are 5 µm for the airyscan images and 1 µm for the inserts. (D) U2OS cells were transiently transfected with either mCherry-EYFP or mCherry-EYFP-TNIP1. 24 h after transfection, cells were either left untreated or treated with BafA1 for 4 h. BF = bright field. Scale bars, 20 µm. Quantification of red-only TNIP1 dots over total TNIP1 dots was done using Volocity software (PerkinElmer), with intensity cut-offs based on BafA1 intensity of red and green dots (n = 3). Around 2,000–3,000 dots were counted for each condition within each replicate. * = P < 0.05, unpaired, two-sided t test. Error bars indicates SD. In B and D, data distribution was assumed to be normal, but this was not formally tested. Source data are available for this figure: SourceData F2.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal