EMBO/SourceData huggingface dataset:
Figure 5A. Cell lysates from HeLa cells KD or OE for either GOLPH3 or LCS were processed for SDS-PAGE and Western blotting with antibodies aimed at evaluating the activation status of the mTOR pathway. Data are representative of at least 3 independent experiments.B. Clonogenic assay of HeLa cells in adherence. 250 HeLa cells KD for either GOLPH3 or LCS were allowed to form colonies for 10 days. Afterward, colonies were fixed and stained as detailed in Materials and Methods. The graph on right reports the number of colonies per well in each indicated condition (data are means ± SEM of 3 independent experiments; *p <0.05, **p <0.01 [Student's t-test]).C. Soft agar colony formation assay of HeLa cells transfected with different concentration of either GOLPH3 (left upper graph) or LCS (left bottom graph) encoding plasmids. Soft agar colony formation assay of HeLa cells KD for either GOLPH3 or LCS (right upper graph). Soft agar colony formation assay of HeLa cells KD for LCS and GOLPH3 OE alone or in combination with LCS KD (right bottom graph). Colonies were stained and quantified as described in Materials and Methods; (individual data points are shown in the graphs; data are means ± SD of at least 3 independent experiments; *p <0.05, ***p <0.001, n.s. = not significant [Student's t-test]).D. Protein lysates from parental (left), GCS-KO (middle), and LCS-KO (right) HeLa cells, CTRL or GOLPH3-OE were processed for SDS-PAGE and WB with the indicated antibodies. Data are representative of at least 3 independent experiments.E. Soft agar colony formation assay of parental and LCS-KO HeLa cells expressing LCS-HA or GOLPH3. Colony number was quantified; (individual data points are shown in the graphs; data are means ± SD of at least 3 independent experiments; ***p <0.001 [Student's t-test]).F. Quantification of soft agar colony formation assay of LNCaP and DU145 prostatic cancer cell lines treated with increasing concentrations of siRNA targeting GOLPH3 or LCS (data are means ± SEM of at least 3 independent experiments; ***p <0.001 [Student's t-test]).
Figure 6D. Representative images of NSCLC sections stained for GOLPH3 and LCS. Scale bar, 100 µm.
Figure 2(B) Daughter cells of single aRGCs microinjected with either Cas9/control gRNA (top) or Cas9/gGFP (bottom) revealed by Dx-A555immunofluorescence (magenta); Cells 1, aRGC daughter; Cells 2, BP daughter. Dashed lines indicate ventricular surface. Images are maximum intensity projections of 20 (Control) and 24 (gGFP) optical sections. Scale bars, 20 µm.(C) Single optical sections of Cells 1 and Cells 2 depicted in (B), showing the effects of Cas9 and control gRNA (top) or gGFP (bottom) on GFP expression (as revealed by immunofluorescence, green). Cells 1 and Cells 2 are indicated by dotted lines; note the presence of GFP immunofluorescence in the daughter cells in control and its absence upon Cas9/gGFP microinjection. Scale bars, 5 µm.(D) Quantification of the proportion of daughter cells (Dx-A555+) of microinjected cells that show GFP expression 24 h after control (Con, white) or gGFP (black) microinjection. Control is set to 100% and the gGFP condition expressed relative to control (39%). Data are the mean of two independent experiments (3 embryos and 1 litter per experiment, total number of daughter cells scored: control 142, gGFP 88); bars indicate the variation of the two individual values from the mean (P<0.05).
Figure 1A Levels of ULK1 in MEFs exposed to hypoxia (1% O2) for the indicated times.actin, Western blotB Quantification of the ratio of ULK1 to actin in (A).C Levels of ULK1 in MEFs treated with FCCP (20 μM) for the indicated times.actinD Quantification of the ratio of ULK1 to actin in (C).E MEFs were exposed to hypoxia (1% O2) for 12 h or FCCP (20 μM) for 6 h and then fixed by 4% paraformaldehyde. Cells were stained with anti‐TIM23 (mouse) and anti‐ULK1 (rabbit) primary antibodies, then Alexa Fluor‐488‐labeled donkey anti‐rabbit IgG and Alexa Fluor‐555‐labeled donkey anti‐mouse IgG secondary antibodies before analysis by immunofluorescence microscopy. Scale bar, 10 μm.F Immunoblots of subcellular fractions from control MEFs or MEFs that were exposed to hypoxia (1% O2) for 12 h. PNS, post‐nuclear supernatant; Cyto, cytosol; Mito, mitochondria.ULK1G Immunoblots of subcellular fractions from control MEFs or MEFs that were treated with FCCP (20 μM) for 6 h. PNS, post‐nuclear supernatant; Cyto, cytosol; Mito, mitochondria.ULK1
Figure 2A HeLa cells were co−transfected with FUNDC1−Myc and FLAG−ULK1. Cells were lysed and immunoprecipitated with anti−Myc antibody 24 h after transfection.B FLAG−FUNDC1 was co−transfected with GFP−vector or GFP−ULK1 for 24 h in HeLa cells, and then, cell lysates were immunoprecipitated with anti−GFP antibody.CMEFs were exposed to hypoxia (1% O2) for 12 h or FCCP (20 μM) for 3 h, followed by immunoprecipitation using anti‐ULK1 antibody.D The expression of FUNDC1 is induced by 10 ng/ml tetracycline (Tet) in FUNDC1‐inducible HeLa cells. Cells were transfected with HA‐ULK1 or HA‐ULK1 (K46N) for 24 h. Then, the cell lysates were prepared for immunoblotting using indicated antibodies.EULK1+/+ cells were transfected with FUNDC1‐Myc, and ULK1-/- cells were transfected with FUNDC1‐Myc alone, FUNDC1‐Myc and HA‐ULK1, or FUNDC1‐Myc and HA‐ULK1 (K46N). Cells were harvested and lysed for immunoblots24 h after transfection.F Purified GST‐FUNDC1 and GST‐FUNDC1 (S17A) were subjected to an in vitro kinase assay with Myc‐ULK1 immunoprecipitated by anti‐Myc antibody from Myc‐ULK1‐transfected cells about 24 h post‐transfection. Phosphorylated FUNDC1 was detected by anti‐p‐S17‐FUNDC1 antibody. The red asterisks mark the target bands.G Western blotULK1, FUNDC1, and the phosphorylated FUNDC1 in MEFs exposed to hypoxia (1% O2) for the indicated times.HWestern blot analysis of the kinetics of ULK1, FUNDC1, and the phosphorylated in MEFs treated with FCCP(20 μM) for the indicated times.actinI, J The single mutant which can abolish the ULK1 and FUNDC1 interaction was identified. HeLa cells were co‐transfected with Flag‐ULK1 and the indicated constructs. 24 h after transfection, cells were lysed for immunoprecipitation by anti‐Flag antibody.KMEFs were transfected with FUNDC1‐Myc and its mutants after the endogenous FUNDC1 was knocked down by siRNA. 36 h after transfection, cells were harvested in the absence or presence of 50 nM bafilomycin A1 (BAF1) and then lysed for immunoblotting with the indicated antibodies.L MEFs were co‐transfected with FUNDC1‐Myc or its mutants and MitoDsred after the endogenous FUNDC1 was knocked down by siRNA. 24 h post‐transfection, cells were treated with hypoxic (1% O2) conditions for 12 h or FCCP (20 μM) for 6 h before fixed by 4% paraformaldehyde and stained with indicated antibodies. Scale bar, 10 μm.
Figure 3AULK1+/+ and ULK1−/− cells were cultured under hypoxic (1% O2) condition for 12 h or 24 h in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cell lysates were immunoblotted.B ULK1+/+ and ULK1−/− cells were treated with FCCP (20 μM) for the indicated times in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cell lysates were immunoblotted.C ULK1+/+ and ULK1−/− cells were transfected with FUNDC1−Myc for 24 h and then harvested in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cell lysates were immunoblotted.D ULK1−/− cells were transfected with HA‐ULK1 or HA‐ULK1 (K46N), then cultured under hypoxic (1% O2) conditions for 24 h in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cells lysates were immunoblotted.EULK1−/− cells were transfected with HA−ULK1 or HA−ULK1 and then treated with FCCP (20 μM) for 24 h. Cells were harvested in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cell lysates were immunoblotted.F FUNDC1‐knockdown MEFs were transfected with FUNDC1‐Myc or FUNDC1-Myc (S17A) for the indicated times. Cell lysates were used for immunoblotting.G ULK1+/+ cells were transfected with FUNDC1‐Myc, and ULK1−/− cells were transfected with FUNDC1‐Myc or FUNDC1-Myc (S17D). 24 h after transfection, cells were fixed with 4% paraformaldehyde and stained with anti‐Myc (mouse) and anti‐LC3 (rabbit) primary antibodies for immunofluorescence microscopy. Scale bar, 10 μm.H ULK1−/− cells were transfected with FUNDC1‐Myc or FUNDC1-Myc (S17D) for 24 h in the absence or presence of 50 nm bafilomycin A1 (BAF1) for an additional 6 h before harvesting. Cell lysates were immunoblotted.I ULK1−/− cells were transfected with FUNDC1‐Myc or FUNDC1-Myc (S17D) for 24 h in the absence or presence of 50 nm bafilomycin A1 (BAF1) for an additional 6 h before harvesting. Cell lysates were detected with the citrate synthase activity kit.J HeLa cells were transfected with FUNDC1‐Myc, FUNDC1-Myc (S17A), or FUNDC1-Myc (S17D). 24 h after transfection, cell lysates were immunoprecipitated with anti‐LC3 antibody.
Figure 4A ULK1+/+ or ULK1−/− cells were cultured under hypoxic (1% O2) or normoxic conditions in the absence or presence of 50 nM bafilomycin A1 (BAF1). ULK1−/− cells were transfected with HA‐ULK1, and ULK1−/− FUNDC1‐KD cells were transfected with HA‐ULK1 and FUNDC1‐Myc, then cultured under hypoxic (1% O2) conditions for 24 h in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cells were harvested and lysed for immunoblotting assays.B ULK1+/+ or ULK1−/− cells were treated with or without FCCP (20 μM) in the absence or presence of 50 nM bafilomycin A1 (BAF1). ULK1−/− cells were transfected with HA‐ULK1 and ULK1−/− FUNDC1‐KD cells were transfected with HA‐ULK1 and FUNDC1‐Myc and then treated with FCCP (20 μM) for 24 h in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cells were harvested and lysed for immunoblotting assays.C ULK1+/+, ULK1−/− FUNDC1‐KD, or ULK1−/− FUNDC1‐KD cells rescued by introduction of HA‐ULK1 and FUNDC1‐Myc were cultured under hypoxic (1% O2) conditions for 24 h or with FCCP (20 μM) for 24 h. Samples were analyzed by electron microscopy. Scale bar, 2 μm. The yellow asterisks mark mitochondria. The red arrows indicate double‐membraned autophagic structures. The yellow arrow denotes autolysosomes.D-E The mitochondrial coverage and mitochondrial−containing AVs shown in (C) were quantified. Recon, reconstitution.
Figure 1A Representative immunofluorescence microscopy staining of GFP of small intestinal tissue of LGR5‐GFP knock‐in (LGR5‐GFPki) mice. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm.B Quantification of GFP staining intensity of intestinal stem and progenitor cells (ISPCs) at indicated positions in basal crypts of LGR5‐GFPkimice (n = 80 crypts from four mice). Each dot presents one cell. Red lines: mean values ± SEM. Unpaired two‐tailed Student's t‐test.C Representative immunofluorescence microscopy staining of Msi1 of small intestinal tissue of young mTerc+/+mice. Dashed line outlines a crypt. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm.D Quantification of Msi1 staining intensity of ISPCs at indicated positions in basal crypts of young mTerc+/+mice (n = 150 crypts from three mice). Each dot presents one cell. Red lines: mean values ± SEM. Unpaired two‐tailed Student's t‐test.E Representative FACS plots showing gating of LGR5hi and LGR5lo populations within the LGR5+ (GFP‐positive) cell gating.F Representative FACS plots showing gating of LGR5hi‐high, LGR5hi‐low, LGR5lo‐high, and LGR5lo‐low populations within the LGR5hi gating.G-I mRNA expression of indicated genes relative to GAPDH in LGR5hi‐high, LGR5hi‐low, LGR5lo‐high, and LGR5lo‐low populations (n = 3 mice). Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.
Figure 2A mRNA expression of indicated components of the canonical Wnt/β‐catenin pathway in freshly isolated small intestinal crypts of 12‐ to 16‐month‐old G3 mTerc−/−mice and age‐matched mTerc+/+mice (n = 5 mice per group).B Axin2 mRNA expression in LGR5+ cells of 12‐ to 16‐month‐old G3 mTerc−/−mice and mTerc+/+mice (n = 3 mice per group).C mRNA expression of indicated components of the canonical Wnt/β‐catenin pathway and of the p53 target gene p21 in cultured crypts of 2‐month‐old G3 mTerc−/−mice and mTerc+/+mice (n = 3 mice per group).
Figure 3A-J Flow cytometry analysis of freshly isolated crypt cells from the small intestine of young (2-3 month) and old (12-16 month) LGR5‐GFPki, mTerc+/+mice and LGR5‐GFPki, G3 mTerc−/−mice (n = 4 mice per group). (A, B, D, E, H, I) Representative FACS plots depicting the analysis of LGR5+ cells. Note the reduction in LGR5+ cells in G3 mTerc−/−mice with the remaining cells showing almost exclusively weak expression of GFP (LGR5lo) and that within the fraction of LGR5hi cells, the cells with high LGR5‐reporter activity (LGR5hi‐high) are preferentially depleted in response to IR. (C, F) Quantification of (C) the number of LGR5+ cells and (F) the number of LGR5hi and LGR5lo cells. (G) Quantification of the percentage of LGR5hi cells and LGR5lo cells within the fraction of LGR5+ cells. (J) Quantification of the survival rate of LGR5hi‐high cells and LGR5hi‐low cells within the fraction of LGR5hi cells comparing old G3 mTerc−/−mice to mTerc+/+mice.K-O Immunofluorescence microscopy staining of GFP in basal crypts of 9‐month‐old LGR5‐GFPki, G3 mTerc−/−mice and age‐matched LGR5‐GFPki, mTerc+/+mice (n = 2 mice per group). (K, L) Representative pictures are given. Dashed lines outline the crypts. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm. (M) Quantification of absolute number of GFP+ISPCs at indicated positions at the crypt base per 50 crypts. (N) The histogram depicts the depletion rate (%) of LGR5+ cells in LGR5‐GFPki, G3 mTerc−/−mice compared to age‐matched LGR5‐GFPki, mTerc+/+mice at indicated positions in the basal crypts. (O) Quantification of GFP staining intensity of ISPCs at indicated positions in positively stained basal crypts. Note that the staining intensity of position 4 cells is equal in mTerc+/+mice and G3 mTerc−/−mice and is significantly lower than position 1 and 2 cells of mTerc+/+mice.
Figure 4A-I Staining of small intestinal tissue sections of 12‐ to 16‐month‐old G3 mTerc−/− mice and age‐matched mTerc+/+ mice (n = 4-5 mice per group). Representative pictures of (A, B) H&amp;amp;E staining, (C, F, I) Quantification of the absolute numbers of ISPCs at the indicated positions in the crypt base per 50 crypts as determined by counting of (C) spindle‐shaped cells in between and adjacent to the Paneth cells. Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.A-I Staining of small intestinal tissue sections of 12‐ to 16‐month‐old G3 mTerc−/−mice and age‐matched mTerc+/+mice (n = 4-5 mice per group). Representative pictures of (D, E) PCNA staining. Arrowheads and numbers indicate the position of ISPCs in the basal crypts. Scale bars: 20 μm. (C, F, I) Quantification of the absolute numbers of ISPCs at the indicated positions in the crypt base per 50 crypts as determined by counting of cells staining positive for (F) PCNA . Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.A-I Staining of small intestinal tissue sections of 12‐ to 16‐month‐old G3 mTerc−/− mice and age‐matched mTerc+/+ mice (n = 4-5 mice per group). Representative pictures of ((G, H) Msi1 staining are given. Arrowheads and numbers indicate the position of ISPCs in the basal crypts. Scale bars: 20 μm. (C, F, I) Quantification of the absolute numbers of ISPCs at the indicated positions in the crypt base per 50 crypts as determined by counting of (I) Msi1. Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.J, K Representative pictures of Olfm4 in situ hybridization on small intestinal sections of 9‐month‐old G3 mTerc−/− mice (K) and age‐matched mTerc+/+ (J) mice. Arrowheads point to positive cells. Dashed lines outline the crypts. Note that the remaining ISPCs in G3 mTerc−/− mice were mostly located in position 4 above the Paneth cells. Scale bar: 20 μm.
Figure 5A-I Three‐month‐old mTerc+/+ mice were exposed to 12 Gy γ‐irradiation. Small intestinal tissue was collected at indicated time points after IR (n = 5 mice per group). (A-F) Representative pictures of PCNA staining. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm. Note the depletion of PCNA+ CBC cells in position 1 and 2 in between the Paneth cells at 3-48 h after IR and the regeneration of these cells at 4-6 days after IR. (G) Quantification of PCNA+ ISPCs at the indicated positions in basal crypts at the indicated time points after IR. Mean values ± SEM are given.A-I Three‐month‐old mTerc+/+ mice were exposed to 12 Gy γ‐irradiation. Small intestinal tissue was collected at indicated time points after IR (n = 5 mice per group). (H, I) Representative pictures of Olfm4 in situ hybridization. Arrowheads point to positive cells. Dashed lines outline the crypts in irradiated samples. Scale bar: 20 μm. Note the selective survival of ISPCs above the Paneth cells at 24 h after IR.J-X Three‐month‐old LGR5‐GFPki, mTerc+/+ mice were exposed to 12 Gy γ‐irradiation. Small intestinal tissue was collected at indicated time points after IR. (J-N) Representative pictures of GFP staining at indicated time points after irradiation. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm.J-X Three‐month‐old LGR5‐GFPki, mTerc+/+mice were exposed to 12 Gy γ‐irradiation. Small intestinal tissue was collected at indicated time points after IR. (O-X) Flow cytometry analysis of freshly isolated basal crypt cells at the indicated time points after IR (n = 4-10 mice per group). (O) The histogram depicts the number of LGR5+ cells in freshly isolated basal crypts at the indicated time point after IR. (P, Q) Flow cytometry analysis was used to determine the percentage of (P) LGR5hi cells and (Q) LGR5lo cells within the fraction of LGR5+ cells. (R‐W) Representative FACS plots of small intestinal crypt cells of non‐irradiated mice, 24 h after irradiation. Note the reduction in LGR5+ cells in irradiated mice with the remaining cells showing almost exclusively weak expression of GFP (LGR5lo). (X) Quantification of the survival rate of LGR5hi‐high cells and LGR5hi‐low cells within the fraction of LGR5hi cells comparing irradiated mice to non‐irradiated mice. Note the preferential depletion of LGR5hi‐high cells within LGR5hi cell population upon IR. Mean values ± SEM are given. NIR, non‐irradiated; IR, irradiated.
Figure 6B, C LGR5hi and LGR5lo cells were freshly isolated from 3‐month‐old LGR5‐GFPki mice 6 h after 12 Gy γ‐irradiation (IR) or from non‐irradiated (NIR) mice (n = 3 mice per group). (B) qPCR analysis of relative mRNA expression of apoptosis‐related genes compared to GAPDH. Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.B, C LGR5hi and LGR5lo cells were freshly isolated from 3‐month‐old LGR5‐GFPki mice 6 h after 12 Gy γ‐irradiation (IR) or from non‐irradiated (NIR) mice (n = 3 mice per group). (C) Representative Western blot analysis of cell lysates for the expression of phospho‐p53 and cleaved caspase‐3.D, E Three‐month‐old mTerc+/+ mice were γ‐irradiated with 12 Gy. Small intestinal tissue was collected at 3 h after IR (n = 3 mice per group). (D) Representative picture of TUNEL staining. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm. (E) Percentage of TUNEL‐positive ISPCs at indicated positions in basal crypts. Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.F-P Two‐month‐old LGR5‐GFPki, p53+/+ mice and LGR5‐GFPki, p53−/− mice were exposed to 12 Gy γ‐irradiation (n = 4 mice per group). Basal crypts were isolated at 24 h after IR. (F-H) Flow cytometry analysis of the survival rate of LGR5+ cells (F), LGR5hi and LGR5lo cells (G), and LGR5hi‐high and LGR5hi‐low cells (H) of irradiated mice (24 h after IR) compared to non‐irradiated mice (NIR). Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test. (I-P) Representative FACS plots of LGR5+ cells in freshly isolated basal crypts from irradiated and non‐irradiated mice of indicated genotypes. Note the stronger rescue of survival rate of LGR5hi‐high than the LGR5hi‐low cells upon p53 deletion. NIR, non‐irradiated; IR, irradiated.
Figure 7Crypts of 2‐ to 3‐month‐old LGR5ki mice were cultured and γ‐irradiated with 6 Gy on day 10 of culture. Sixteen hours before irradiation, cells were exposed to the indicated treatment (n = 4 independent experiments per group).A-J Representative FACS plots of indicated treatments are given. NIR, non‐irradiated; IR, irradiated.K-P Flow cytometry analysis of the percentage of LGR5+, LGR5hi, and LGR5lo cells of irradiated cultures (24 h after IR) compared to the percentage in non‐irradiated controls (NIR) set to 100%. (K-M) Note that inhibition of Wnt signaling by DKK1 treatment or R‐spondin reduction (50% Rspo) partially rescued the decrease in LGR5+ cells in irradiated cultures. (N-P) Note that activation of Wnt signaling by 6‐BIO treatment aggravated the decrease in LGR5+ cells in irradiated cultures. Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.
Figure 8A-G Two‐month‐old LGR5‐GFPki, mTerc+/+mice were i.v. injected with LRP6 neutralization antibody, and IgG serves as a negative control. Thirty‐six hours after injection, small intestinal tissue was collected for GFPimmunofluorescence microscopy staining. (A-F) Representative pictures are given. Dashed lines outline the crypts. Arrowheads and numbers indicate cell positions in the crypts. Scale bar: 20 μm. (G) Quantification of GFP staining intensity of cells at indicated positions in basal crypts (n = 3 mice per group, fifty positively stained cells were measured per mouse). Note the GFP staining intensity was substantially reduced in anti‐LRP6 antibody‐injected group with intact maintenance of ISPCs at the crypt bottom as shown by DAPI staining.H-L Two‐month‐old mTerc+/+ mice were i.v. injected with anti‐LRP6 antibody 12 h before γ‐irradiation. Small intestinal tissue was collected at 24 h after IR (n = 3-4 mice per group). IgG serves as a negative control. (H-K) Representative pictures of PCNA staining of small intestine of non‐irradiated mice (H, I) or 6 Gy‐irradiated mice (J, K). Note the anti‐LRP6 antibody injection did not induce loss of CBC cells at position 1 and 2 in non‐irradiated mice. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm. (L) Quantification of PCNA+ ISPCs at the indicated positions in basal crypts 24 h after IR with indicated doses or non‐irradiated mice.M-R Two‐month‐old LGR5‐GFPki, mTerc+/+ mice were i.v. injected with LRP6 neutralization antibody 12 h before 12 Gy γ‐irradiation. IgG serves as a negative control. Small intestinal tissue was collected at 24 h after IR (n = 4 mice per group). (M-P) Representative FACS plots of indicated treatments. (Q) Flow cytometry analysis of the number of LGR5+ ISPCs of irradiated mice compared to the number in non‐irradiated mice (NIR) set to 100%. (R) Quantification of the number of LGR5+, LGR5hi, and LGR5lo cells of mice with indicated treatment by flow cytometry. Note that the total LGR5+cell frequency was not significantly different between anti‐LRP6 antibody‐injected and control IgG‐injected mice, though the LGR5hi fraction was reduced in the LRP6 antibody‐injected group.