EMBO/soda-nel huggingface dataset:
Metabolic remodeling maintains a reducing environment for rapid activation of replication checkpoint (2021)
Figure 2

Figure 2. The kinase activity of SNF1 is critical for the timely activation of Mec1 and Rad53.

(A) The snf1Δ and snf4Δ mutant cells are hypersensitive to HU. Five-fold serial dilutions of the overnight cultures were assayed on normal growth media (YPD) with or without 200 mM HU. A plate in which 2% glucose was replaced by 2% non-fermentable carbon (galactose) was applied as a control. All plates were cultured at 30°C for 48 h before photography.

(B) Rad53 hyperphosphorylation of WT and snf1Δ in response to HU. Cells were synchronized in G1 and released into the fresh medium containing 200 mM HU for the indicated time. Cell extracts were prepared for immunoblotting against anti-Rad53 antibodies. Tubulin was probed as a loading control.

(C) Rad53 phosphorylation in various snf1 alleles in response to HU. The plasmids expressing the indicated snf1 alleles were transformed into snf1Δ. Rad53 phosphorylation was detected as above.

(D) The snf1 kinase-defective mutants are sensitive to HU. Yeast spot assays were performed as in Fig 2A. The phospho-mimetic snf1T210D (a constitutive active mutant) was applied as a control for nonphosphrylable snf1T210A.

(E) H2A S129 phosphorylation (namely γ-H2A) of WT and snf1Δ in response to HU. γ-H2A was detected by immunoblotting.

(F, G) HU induces a rapid Snf1 T210 phosphorylation. Exponential cells were treated by 200 mM HU for the indicated time. Protein extracts were prepared as described in the Methods and Materials. Three independent experiments were done. Quantitation of Snf1 T210 and Snf1 signals was performed by Image J. The relative ratio of Snf1 T210/Snf1 was shown in (H). Error bars represent standard deviations (SD) from three biological repeats.
Metabolic remodeling maintains a reducing environment for rapid activation of replication checkpoint (2021)
Figure 3

Figure 3. SNF1 affects RPA recruitment on ssDNA.

(A) A significant delay in RPA foci formation in the absence of Snf1. The percentage of cells with RPA foci was calculated and plotted against the incubation time in 200 mM HU. Error bars represent SD from three biological repeats. At least 200 cells were analyzed for each experiment of three independent experiments. Statistical significance was evaluated based on Student's t-test (*0.01 < P-values < 0.05; **0.001 < P-values < 0.01).

(B) The experimental scheme for the Rfa1-13MYC ChIP in the indicated yeast strains. (C) Snap shot of Rfa1 ChIP-Seq at Chromosome VI. Cells were synchronized in G1 and released into the fresh medium containing 200 mM HU for 30 min. The sequencing reads were mapped to the yeast reference genome. The red dashed box indicated the relative read intensity of ARS607.

(D) The average Rfa1 ChIP-seq read density from cells released into HU medium around ACS sites. ACS, ARS consensus sequence.

(E, F) SNF1 is required for efficient recruitment of Rfa1, but not Psf2, to HU-stalled replication forks. Cells were grown and synchronized in G1 by α-factor before release into the fresh medium supplemented with 200 mM HU for the indicated time. Cell extracts were prepared and subjected to MYC-ChIP of Rfa1-13MYC (E) or Psf2-13MYC (F). The amounts of DNA in the precipitates were quantified by qPCR. Error bars represent SD from three biological repeats.
Metabolic remodeling maintains a reducing environment for rapid activation of replication checkpoint (2021)
Figure 4

Figure 4. mig1Δ rescues the checkpoint defects of snf1Δ.

(A, B) Rfa1-GFP foci and Rfa1-13MYC ChIP assays were performed as in Fig 3. At least 200 cells were analyzed for each experiment of three independent experiments. Statistical significance was evaluated based on Student's t-test (*0.01 < P-values < 0.05; **0.001 < P-values < 0.01) (A). Error bars represent SD from three biological repeats (B).

(C, D) mig1Δ restores Rad53 phosphorylation and growth of the snf1 mutants in the presence of HU. The experiments were done as in Fig 2.

(E, F) snf1Δ shows less RPA on chromatin, which can be restored by mig1Δ. Cells were collected and fractionated into chromatin-bound (Chromatin) and non-chromatin-bound fractions (non-Chromatin) followed by immunoblotting. Tubulin and Orc2 were applied as controls for non-chromatin and chromatin fractions, respectively. The signals were quantified by Image J and quantifications were shown in (F). Error bars represent SD from three biological repeats. Statistical significance was evaluated based on Student's t-test (*0.01 < P-values < 0.05).
Metabolic remodeling maintains a reducing environment for rapid activation of replication checkpoint (2021)
Figure 5

Figure 5. Mig1 phosphorylation promotes RPA recruitment and HU resistance by attenuating its interaction with Ssn6.

(A) T489 and S495, within Mig1C, are predicted to be the phosphorylation sites of SNF1 by global phosphorylation sites.

(B) Mig1 T489 and S495 are the main phosphorylation sites of SNF1 in response to HU. The SNF1 kinase complex was partially purified from HU-treated Snf1-5FLAG or snf1-KD-5FLAG yeast cells. Recombinant Mig1 or its mutant proteins (mig1-2A, T489AS495A) were purified from E. coli. In vitro kinase assays were performed as described in Methods and Materials. Proteins were immunoblotted using the indiated antibodies. The anti-AMPK substrate antibodies were used to probe the phosphorylation of the substrate Mig1. Less signals were observed when Mig1 was replaced by mig1-2A. Phosphorylation of Snf1 Thr 172 indicates the activation status of the Snf1 kinase.

(C, D) Phospho-mimetic mig1-2D bypasses the role of SNF1 in HU resistance (C) and RPA recruitment (D). HU sensitivity and Rfa1-13MYC ChIP assays were conducted as described in Fig 2A and Fig 3D. Error bars represent SD from three biological repeats.

(E) The diagram of the strategy to reinforce the Mig1-Ssn6 interaction via a GFP and GBP pair (left panel). The persistent Mig1-Ssn6 association abolishes the phospho-mimicking effect of mig1-2D (right panel). Yeast spot assays were performed as in Fig 2A.
Metabolic remodeling maintains a reducing environment for rapid activation of replication checkpoint (2021)
Figure 6

Figure 6. Zwf1 is the principal target of Mig1 in replication stress response.

(A) ZWF1 is required for snf1Δmig1Δ growing in HU. Overnight cultures were spotted onto plates containing HU, and cultivated at 30°C for 48 h before photograph. Yeast spot assays were performed as in Fig 2A.

(B) Rfa1-GFP foci were measured as in Fig 3. At least 200 cells were analyzed for each experiment of three independent experiments. Error bars represent SD from three biological repeats. Statistical significance was evaluated based on Student's t-test (*0.01 < P-values < 0.05).

(C) Rfa1-13MYC ChIP assays after HU treatment. Strains were synchronized in G1 phase by α-factor prior to release into fresh media containing 200 mM HU for the indicated time points. Cell extracts were prepared as in Fig 3. Error bars represent SD from three biological repeats.

(D) Phosphorylation of Mig1 T489 S495 completely restores the ZWF1 mRNA levels in snf1Δ. The experiments were done as in Fig 1. Error bars represent SD from three biological repeats. Statistical significance was evaluated based on Student's t-test (*0.01 < P-values < 0.05; **0.001 < P-values < 0.01).
Metabolic remodeling maintains a reducing environment for rapid activation of replication checkpoint (2021)
Figure 7

Figure 7. α-KG addition restores the defects of RPA recruitment and Rad53 activation in snf1Δ.

(A) The SNF1-Mig1 axis induces NADPH upon HU treatment. The relative ratios of cellular NADPH/NADP before or after HU treatment were measured as described in Methods and Materials. Error bars represent SD from three biological repeats. Statistical significance was evaluated based on Student's t-test (*0.01< P-values < 0.05; **0.001 < P-values < 0.01).

(B) The effects of α-KG on the HU sensitivity of snf1Δ and other checkpoint mutants. Yeast spot assays were performed as in Fig 2A.

(C) α-KG restores the levels of Rfa1-GFP foci in snf1Δ. Rfa1-GFP foci were measured as in Fig 3. At least 200 cells were analyzed for each experiment. Error bars represent SD from three biological repeats. Statistical significance was evaluated based on Student's t-test (*0.01 < P-values < 0.05).

(D) α-KG restores Rad53 phosphorylation in snf1Δ. CBB indicates coomassie bright blue (CBB) staining of the membrane as the loading control.

(E) The effects of α-KG on the HU sensitivity of Rfa1 zinc finger mutants (C486S, C491S, C505S, C508S). Yeast spot assays were performed as in Fig 2A.

(F) The proposed working model. In brief, we uncover that sugar metabolism is rewired to protect genome stability by means of a redox mechanism. Mechanistically, an SNF1-Mig1-ZWF1-NADPH axis stimulates the ssDNA-binding of RPA, thereby promoting a fast activation mode of Mec1-Rad53 kinase cascade to fight against replication threats.

Expanded View Figure legends
Antisense-mediated exon skipping: a therapeutic strategy for titin-based dilated cardiomyopathy. (2015)
Figure 1

Exon skipping strategy and evaluation of 2OMePS antisense oligonucleotides (AONs) in HL-1 mouse cardiomyocytes

Schematic of the study design.

RT–PCR analysis (top) and representative direct sequencing (bottom) of Ttn exon 326 transcripts from HL-1 cardiomyocytes transiently transfected with different 2OMePS AONs. Only 2OMePS-AON1 and 2OMePS-AON1 + 3 (*) lead to a correct excision of exon 326 as confirmed by direct sequencing.

Immunofluorescence images of sarcomeric α-actinin in untransfected and 2OMePS-AON1 + 3-transfected HL-1 cardiomyocytes. Scale bars, 10 μm.
Antisense-mediated exon skipping: a therapeutic strategy for titin-based dilated cardiomyopathy. (2015)
Figure 2

AON-mediated skipping of exon 326 in TTN Ser14450fsX4 induced pluripotent stem cell (iPSC)-derived cardiomyocytes

RT–PCR analysis of TTN exon 326 transcripts from DCM cardiomyocytes transiently transfected with 2OMePS-AON1, 2OMePS-AON3, and 2OMePS-AON1 + 3.

RT–PCR analysis (left) and representative direct sequencing (right) of TTN exon 326 transcripts from DCM cardiomyocytes infected with the U7snRNA-TTNAONs-IRES-GFP lentiviral vector carrying the AON1 and 3 sequences (TTN-AON) or with a control vector (U7snRNA-ScrAONs-IRES-GFP).

Mass spectrometry-based analysis of titin peptides in cells infected with the U7snRNA-ScrAONs-IRES-GFP (Scr-AON) and U7snRNA-TTNAONs-IRES-GFP (TTN-AON) vectors. Unsupervised hierarchical clustering identified a cluster significantly enriched in peptides mapping to exon 326 that was down-regulated in DCM Scr-AON cardiomyocytes compared to CTR Scr-AON cardiomyocytes (n = 3, P = 9.03E−8, Fisher's exact test, FDR = 0.04, top). Down-regulation of exon 326 was also detected in DCM TTN-AON cells when compared to DCM Scr-AON cells (n = 3, P = 0.02, Fisher's exact test, bottom).
Antisense-mediated exon skipping: a therapeutic strategy for titin-based dilated cardiomyopathy. (2015)
Figure 3

Exon skipping-based rescue of sarcomeric defects in TTN Ser14450fsX4 iPSC-derived cardiomyocytes

Experimental design.

Percentage of iPSC-derived cardiomyocytes with cross-striated myofibrils occupying a fraction (partially organized, dark gray) or the whole cytoplasm (fully organized, light gray) from two control and two patient clones. Statistical difference was tested using the two-sided chi-squared test (CTR1 basal: n = 283, CTR2 basal: n = 250, DCM1 basal: n = 255, DCM2 basal: n = 236; ***P = 1.93E−16).

Immunofluorescence images of titin (Z-disk portion) and cardiac troponin T (cTNT, A-band) in well-organized sarcomeres from CTR and DCM single cardiomyocytes under basal conditions. Scale bars, 5 μm.

Immunofluorescence images (left) of α-actinin and cTNT in CTR and DCM single cardiomyocytes, illustrating 3 different levels of sarcomeric organization (perinuclear, fully and peripherally organized). Percentage of cells with different levels of sarcomeric organization (right) under basal and stress conditions. Statistical difference was tested using the chi-squared test (CTR1 basal: n = 283, CTR2 basal: n = 250, DCM1 basal: n = 255, DCM2 basal: n = 236, CTR1 Iso: n = 245, CTR2 Iso: n = 230; DCM1 Iso: n = 242 and DCM2 Iso: n = 269; ***P = 1.93E−16 CTR basal versus DCM basal; ***P = 9.61E−34 CTR Iso versus DCM Iso, **P = 0.001 DCM basal versus DCM Iso). No significant differences were observed comparing CTR basal and CTR Iso groups. Scale bars, 25 μm.

Percentage of perinuclear, fully, and peripherally organized single cardiomyocytes from two CTR and two DCM iPSC clones after infection with the U7snRNA-ScrAONs-IRES-GFP and U7snRNA-TTNAONs-IRES-GFP lentiviruses. Statistical difference was tested using the two-sided chi-squared test (CTR1 Scr-AON: n = 190, CTR2 Scr-AON: n = 200, DCM1 Scr-AON: n = 221, DCM2 Scr-AON: n = 115, CTR1 TTN-AON: n = 223, CTR2 TTN-AON: n = 187, DCM1 TTN-AON: n = 171, DCM2 TTN-AON: n = 243; ***P = 4.22E−15 CTR Scr-AON versus DCM Scr-AON; ***P = 4.61E−02 DCM Scr-AON versus DCM TTN-AON). No significant differences were observed comparing CTR Scr-AON and CTR TTN-AON groups.
Antisense-mediated exon skipping: a therapeutic strategy for titin-based dilated cardiomyopathy. (2015)
Figure 4

Exon skipping-based rescue of SRF target gene expression and Nbr1/p62/SQSTM1/MURF2 subcellular distribution in TTN Ser14450fsX4 iPSC-derived cardiomyocytes

qRT–PCR analysis of SRF target genes (MYH6, MYH7 and ACTC1) in CTR and DCM single cardiomyocytes under basal condition (no infection, NI) and after infection with control U7snRNA-ScrAONs-IRES-GFP (Scr-AON) and the U7snRNA-TTNAONs-IRES-GFP (TTN-AON) lentiviruses. Statistical difference was tested using the two-sided Student's t-test (**P = 0.009, CTR Scr-AON versus DCM Scr-AON; *P = 0.04, DCM Scr-AON versus DCM TTN-AON for MYH6; **P = 0.002, CTR Scr-AON versus DCM Scr-AON; **P = 0.002, DCM Scr-AON versus DCM TTN-AON for MYH7; **P = 0.004, CTR Scr-AON versus DCM Scr-AON; *P = 0.02, DCM Scr-AON versus DCM TTN-AON for ACTC1). No significant differences were observed comparing the CTR NI, CTR Scr-AON and CTR TTN-AON groups and comparing the DCM NI and DCM Scr-AON groups. Expression values were relative to CTR Scr-AON, normalized to GAPDH, and presented as mean ± SEM, n = 3.

Immunofluorescence images showing normal (a, b, e, f, i, l, o, p) and altered (c, d, g, h, m, n, q, r) intracellular distribution of SRF (a and b, nuclear; c and d, cytoplasmic), MURF2 (e and f, sarcomeric; g and h, nuclear), Nbr1 (i and l, sarcomeric; m and n, diffused), and SQSTM1/p62 (o and p, sarcomeric; q and r, diffused) in representative single cardiomyocytes (left). Sarcomeres are marked by α-actinin. On the right, percentage of CTR and DCM cardiomyocytes showing cytoplasmic expression of SRF, nuclear accumulation of MURF2, and diffused expression of Nbr1 and of SQSTM1/p62 after infection with control U7snRNA-ScrambleAONs-IRES-GFP (Scr-AON) and the U7snRNA-TTNAONs-IRES-GFP (TTN-AON) lentiviruses (right). Data represent mean values ± SEM from two control and two DCM clones. Statistical difference was tested using the two-sided chi-squared test (CTR Scr-AON: n = 874, n = 874, n = 882 and n = 890, CTR TTN-AON: n = 880, n = 990, n = 878 and n = 890, DCM Scr-AON: n = 890, n = 887, n = 884 and n = 886, DCM TTN-AON: n = 900, n = 875, n = 899 and n = 891 for SRF, MURF2, Nbr1 and SQSTM1/p62, respectively; *P = 0.01, CTR Scr-AON versus DCM Scr-AON; *P = 0.04, DCM Scr-AON versus DCM TTN-AON for SRF; *P = 0.02, CTR Scr-AON versus DCM Scr-AON; *P = 0.04, DCM Scr-AON versus DCM TTN-AON for MURF2; *P = 0.03, CTR Scr-AON versus DCM Scr-AON; *P = 0.03, DCM Scr-AON versus DCM TTN-AON for Nbr1; **P = 0.009, CTR Scr-AON versus DCM Scr-AON; *P = 0.01, DCM Scr-AON versus DCM TTN-AON for SQSTM1/p62). No significant differences were observed comparing CTR Scr-AON and CTR TTN-AON groups. Scale bars, 50 μm.
Antisense-mediated exon skipping: a therapeutic strategy for titin-based dilated cardiomyopathy. (2015)
Figure 5

Exon skipping-based rescue of the DCM phenotype in Ttn Ser14450fsX4 knock-in embryos

A Experimental design.

B, C Immunofluorescence images of sarcomeric α-actinin and myosin (B) and assessment of cardiac function (C) in homozygous Ttn knock-in embryos cultured for 24 h after mScrAON and mTtnAON transient transfection (n = 3 per group) or lentivirus delivery (n = 7 per group). Scale bars, 10 μm.

D Electron microscopy analysis in Ttn knock-in embryos cultured for 24 h after mScrAON and mTtnAON transient transfection. mTtnAON treatment of homozygous embryos rescued myofibril formation (left, black arrows), resulting in thicker filaments (right).

Data information: Statistical difference was tested using the two-sided Student's t-test (**P = 0.007 and *P = 0.01 for fractional area change and **P = 0.009 for filament width differences). Data represent mean values ± SEM. Scale bars, 0.2 μm.
Antisense-mediated exon skipping: a therapeutic strategy for titin-based dilated cardiomyopathy. (2015)
Figure 6

Prevention of DCM phenotype development in adult Ttn Ser14450fsX4 knock-in mice by injection of vivo-morpholino-modified antisense oligonucleotides

Experimental design.

Echocardiographic analysis of adult knock-in mice. Statistical difference was tested using the two-sided Student's t-test (WT: n = 6, HET: n = 6; HET + vPMO-mScrAON: n = 10 and HET + vPMO-mTtnAON: n = 14; *P = 0.013, HET versus HET + vPMO-mTtnAON and *P = 0.02, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON for LVEDD differences; **P = 0.006, HET versus HET + vPMO-mTtnAON and *P = 0.02, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON for LV-EF differences; *P = 0.03, HET versus HET + vPMO-mTtnAON and *P = 0.04, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON for IVSd differences; **P = 0.001, HET versus HET + vPMO-mTtnAON and *P = 0.03, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON and for PWd differences). No significant differences were observed among groups at day 0 and day 7. Data represent mean values ± SEM.

Masson's trichrome staining of heart sections from HET animals injected with vPMO-mScrAONs and vPMO-mTtnAON. Scale bars, 1 mm.
Antisense-mediated exon skipping: a therapeutic strategy for titin-based dilated cardiomyopathy. (2015)
Figure 7

Efficient skipping of exon 326 in vivo-morpholino-modified antisense oligonucleotide-treated adult Ttn Ser14450fsX4 knock-in mice

Fluorescence in situ hybridization (FISH) of heart muscle tissue from adult knock-in mice with a probe complementary to vPMO-mTtnAON. Scale bars, 250 and 50 μm (magnification).

RT–PCR analysis of Ttn exon 326 transcripts from heart tissue of untreated WT and HET animals and vPMO-mScrAON- and vPMO-mTtnAON-treated mice (left). Representative direct sequencing of Ttn exon 326 transcripts from vPMO-mTtnAON-treated HET heart tissue (right).

Mass spectrometry-based analysis of titin peptides in adult knock-in mice injected with vPMO-mScrAONs and vPMO-mTtnAONs. Unsupervised hierarchical clustering identified a cluster enriched in exon 326 peptides that was down-regulated in vPMO-mTtnAON-treated animals compared to vPMO-mScrAON-treated littermates. Another cluster enriched in C-terminal peptides was up-regulated in the vPMO-mTtnAON group compared to the vPMO-mScrAON group (n = 3, P = 0.02, Fisher's exact test, FDR = 0.04).
Autolysosomal β‐catenin degradation regulates Wnt‐autophagy‐p62 crosstalk (2013)
Figure 1

Modulation of β‐catenin expression regulates autophagosome number. (A–C) Measurement of autophagy during nutrient starvation in HT29 cells stably expressing YFP–LC3. (A) Increased LC3 puncta per cell following β‐catenin knockdown with siRNA compared with a non‐targeting (NT) control siRNA. Columns show autophagosome numbers (mean±s.e.m., n>200 cells in >20 fields of view per experiment of three independent treatments) assessed under normal and starved (2 h) conditions with β‐catenin or non‐targeting (NT) siRNA. Representative images (B) of YFP–LC3 puncta (green) and DAPI nuclei staining (blue) are shown. (C) Upper panel: western blotting showed increased LC3‐II expression in HT29 cells with β‐catenin knockdown compared to control cells after 2 h starvation. Lower panel: quantification of the LC3‐II/β‐actin ratio by densitometry (mean±s.e.m. of four independent treatments, *P=0.011 in nutrient conditions, *P=0.035 under starvation). (D and E) HCT116 β‐cateninWT/− cells overexpressing a control or β‐cateninS33Y plasmid nutrient starved for 24 h. (D) Immunostaining of LC3 (green) with DAPI nuclei staining (blue) showed decreased LC3 puncta with β‐cateninS33Y overexpression. (E) By western blotting, LC3‐II decreased with β‐cateninS33Y overexpression (upper panel). Lower panel: quantification of the LC3‐II/β‐actin ratio by densitometry (mean±s.e.m. of four independent treatments, *P=0.015). (F) Comparison of isogenic β‐cateninWT/− and β‐catenin−/ΔS45 HCT116 cells. Upper panel: western blotting showed decreased LC3‐II in HCT116 β‐catenin−/ΔS45 cells. Lower panel: western blotting confirmed by quantification using densitometry of the LC3‐II/β‐actin ratio (mean±s.e.m. of three independent treatments, *P=0.046).Source data for this figure is available on the online supplementary information page.
Autolysosomal β‐catenin degradation regulates Wnt‐autophagy‐p62 crosstalk (2013)
Figure 2

β‐Catenin negatively regulates p62 expression but does not block autophagic flux. (A) Protein expression of p62 increased following β‐catenin knockdown under normal and starved conditions in HT29 cells. (B) Comparison of p62 expression in isogenic HCT116 β‐cateninWT/− and HCT116 β‐catenin−/ΔS45 cells by western blotting showed decreased p62 expression in β‐catenin−/ΔS45 cells. (C) LC3 puncta number per cell during starvation and nutrient addback post starvation. Left panel: LC3 puncta number reduces in non‐targeting (NT) siRNA control with nutrient addback. Right panel: LC3 puncta number reduces in β‐catenin knockdown cells with nutrient addback (mean±s.e.m. of three independent treatments; n>200 cells in >20 fields of view per experiment). Western blotting for β‐catenin confirmed knockdown. (D) Western blotting of LC3 and p62 in HT29 cells following β‐catenin knockdown and starvation with lysosomal inhibitors. LC3‐II and p62 protein expression showed an increase with β‐catenin siRNA in the presence of autophagy flux inhibitors chloroquine (10 μM) or bafilomycin A1 (100 nM; applied for the final 30 min of the 8 h) compared to β‐catenin knockdown alone. Quantification by densitometry of the LC3‐II/β‐actin ratio (mean±s.e.m. of four independent experiments). See also Supplementary Figure S1A.Source data for this figure is available on the online supplementary information page.
Autolysosomal β‐catenin degradation regulates Wnt‐autophagy‐p62 crosstalk (2013)
Figure 3

β‐Catenin deletion increases LC3 puncta and p62 expression in mouse intestinal epithelium in vivo. (A–D) LC3 staining (red) in mouse intestinal crypt and villus epithelium increased (white arrowheads) 2 days post β‐catenin deletion in β‐catenin−/lox‐villin‐creERT2 mice (C and D) compared to control β‐catenin+/lox‐villin‐creERT2 mice (A and B). Further magnification of the areas marked by white squares is shown (lower panels). (E–H) p62 staining (green) increased in the crypt and villus epithelium 2 days after β‐catenin deletion in β‐catenin−/lox‐villin‐creERT2 mice (G and H) compared to control in β‐catenin+/lox‐villin‐creERT2 mice (E and F). DAPI staining (blue) identifies nuclei. β‐Catenin deletion was confirmed by immunofluorescence (Supplementary Figure S1B). Red and green channel levels were adjusted post acquisition for clarity (equal level changes applied across the entire figure). Further magnification of the areas marked by white squares is shown (lower panels). (I and J) Western blotting on extracts from mouse intestinal epithelial tissue demonstrated increased p62 and LC3‐II protein expression 2 (I) and 4 (J) days post β‐catenin deletion in β‐catenin−/lox‐villin‐creERT2 mice compared to control β‐catenin+/lox‐villin‐creERT2 mice.Source data for this figure is available on the online supplementary information page.