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. 2023 Nov 15;623(7989):1062–1069. doi: 10.1038/s41586-023-06726-w

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© The Author(s) 2023, corrected publication 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 6 — a, Scheme showing the experimental workflow targeting G3BP1 and G3BP2 using CRISPR/Cas9 system delivered as a ribonucleoprotein (RNP) complex by nucleofection. b, immunoblot showing G3BP1 and G3BP2 protein levels in iPSDM WT and iPSDM G3BP^nf, ACTB was used as a loading control (n = 3). c, representative images of proteolytic activity (magma colormap) in iPSDM WT and iPSDM G3BP^nf incubated with the pan-cathepsin activity-based probe (iABP). Scale bar: 20 μm. d,e, Bar plots show iABP intensity quantification (d) and lysosomal area per cell evaluation (e) in iPSDM WT and iPSDM G3BP^nf, n = 3 independent experiments. f, high-content imaging SG evaluation in iPSDM WT and iPSDM G3BP^nf left untreated or treated with LLOMe (1 mM, 30 min). Bar plots represent mean values ± SEM of one out of three independent experiments (n = 3), ***P < 0.001, two-way ANOVA, Šídák’s multiple comparisons test. g, LysoTracker basal intensity quantification of iPSDM WT and iPSDM G3BP^nf, n = 3 independent experiments. h-i, LysoTracker basal intensity quantification and immunoblot showing G3BP1 and G3BP2 protein levels in HeLa WT and HeLa G3BP1/2 knockdown (KD) cells (h), and in hMDM WT and hMDM G3BP^nf (i), ACTB was used as a loading control, n = 3 independent experiments. j, Image sequence of iPSDM WT and iPSDM G3BP^nf incubated with LysoTracker (seen as black puncta) before adding LLOMe (left panel), 2 min after (central panel) and 10 min after washout (left panel). Scale bar: 10 μm. k-l, Show quantification of LTR puncta intensity relative to basal values (pre-LLOMe) for the indicated conditions in HeLa WT (k) and HeLa KD (l), n = 3 independent experiments, ***P < 0.001, two-tailed t-test. m, Shows the LTR intensity at 10 min after washout in HeLa WT compared to HeLa KD, n = 3 independent experiments. ***P < 0.001, two-tailed t-test. n, Image sequence of hMDM WT and hMDM G3BP^nf incubated with LysoTracker (seen as black puncta) before adding LLOMe (left panel), 2 min after (central panel) and 10 min after washout (left panel). Scale bar: 10 μm. o-p, Show quantification of LTR puncta intensity relative to basal values (pre-LLOMe) for the indicated conditions in hMDM WT (o) and hMDM G3BP^nf (p), n = 3 independent experiments. ***P < 0.001, two-tailed t-test. q, Shows the LTR intensity at 10 min after washout in hMDM WT compared to hMDM G3BP^nf, n = 3 independent experiments. ***P < 0.001, two-tailed t-test. Bar plots show mean ± SEM. For gel source data, see Supplementary Fig. 1.

Source Data