EMBO/SourceData huggingface dataset:
Figure 9In RSPO3 rescue experiment, each mouse was treated with 5 μg RSPO3 in 100μL PBS by retro-orbital injection 30 min before ischemia. PBS treated mice were used as vehicle control. (C) Representative images of crypts immunostained with OLFM4 and β-catenin in PBS/RSPO3 treated EC-Foxc-DKO mice 24h after I/R. The accumulation of β-catenin in the nuclei of ISCs (dotted circles) was found in RSPO3 rescued mice. Paraffin sections (4 µm), (D) Quantification of relative fluorescent intensity (FI) of β-catenin immunostaining within ISC and (E) quantification of the number of OLFM4+ ISCs were performed based on Figure 9C. Data are box-and-whisker plots, Mann-Whitney U test, each symbol represents one mouse, N = 4~6, *P<0.05.In RSPO3 rescue experiment, each mouse was treated with 5 μg RSPO3 in 100μL PBS by retro-orbital injection 30 min before ischemia. PBS treated mice were used as vehicle control. (F) Representative images of immunostaining of CCND1 in intestines in PBS/RSPO3 treated EC-Foxc-DKO mice at I/R-24h. Scale bars = 100 µm. (G) Quantification of the number of CCND1+ epithelial cells per crypt at I/R-24h are based on Figure 9F. Data are box-and-whisker plots, Mann-Whitney U test, each symbol represents one mouse, N = 7, **P<0.01.In RSPO3 rescue experiment, each mouse was treated with 5 μg RSPO3 in 100μL PBS by retro-orbital injection 30 min before ischemia. PBS treated mice were used as vehicle control. (H) Relative mRNA expression of Rspo3 in sorted LECs and Cxcl12 in sorted BECs from intestines of PBS/RSPO3 treated EC-Foxc-DKO mice at I/R-18.5h. Data are box-and-whisker plots, Mann-Whitney U test, each symbol represents one mouse, N = 4~6, *P<0.05.In RSPO3 rescue experiment, each mouse was treated with 5 μg RSPO3 in 100μL PBS by retro-orbital injection 30 min before ischemia. PBS treated mice were used as vehicle control. Representative images of H&E staining show the rescue effects of RSPO3 in intestinal mucosa LEC-Foxc-DKO (I) mice as well as their control mice 24h after I/R. Red numbers indicate the Chiu scores. Quantification of Chiu Score for the intestines at I/R-24h are based H&E staining as shown in Figure 9A and 9I. Data are box-and-whisker plots, Mann-Whitney U test, each symbol represents one mouse, N = 6~13, **P<0.01.In RSPO3 rescue experiment, each mouse was treated with 5 μg RSPO3 in 100μL PBS by retro-orbital injection 30 min before ischemia. PBS treated mice were used as vehicle control. (K) Representative images of CD31/LYVE1 immunostaining in the intestines of PBS/RSPO3 treated LEC-Foxc-DKO mice at I/R-24h. (L, M) Quantification of the vessel density (L) (= vessel area/total intestinal tissue area x 100%) for the blood (B) and/or lymphatic (L) vessels (markers listed below the graph were used to identify B and L) as well as the measurement of lacteal length (M) was performed based on Figure 9K. The Data are box-and-whisker plots, Mann-Whitney U test, each symbol represents one mouse, N = 7, **P<0.01, n.s.=not significant.
Figure 10In CXCL12 rescue experiments, mice were treated with 50μg/kg CXCL12 in PBS by retro-orbital injection 30 min before ischemia. Mice treated with PBS were used as control. (H) Representative confocal images of CD31 immunostaining of distal jejuna in PBS- and CXCL12- treated EC-Foxc-DKO mice after I/R at 24h. Paraffin sections (15 µm), scale bars = 100 μm. L represents the lacteal length measured in Figure 10J. (I, J) (I) Quantification of CD31+ vessel density (% = total CD31+ vessel area/total intestinal tissue area x 100%) and (J) quantification of lacteal length were performed based on Figure 10H. Data are box-and-whisker plots, Mann-Whitney U test, each symbol represents one mouse, N = 4~6, **P<0.01.In CXCL12 rescue experiments, mice were treated with 50μg/kg CXCL12 in PBS by retro-orbital injection 30 min before ischemia. Mice treated with PBS were used as control. (K) Relative mRNA expression of Rspo3 in sorted intestinal LECs from PBS/CXCL12 treated EC-Foxc-DKO mice at I/R-18.5h. Data are box-and-whisker plots, Mann-Whitney U test, each symbol represents one mouse, N = 5~6, n.s.=not significant. The box-and-whisker plots in display the median value (central band in the box), second and third quartiles (bottom and top ends of the box, respectively) as well as minimum/maximum values (whiskers blow/above the box) of the data sets.
Figure 1A Immunohistochemical positive staining for NALCN in human prostate adenocarcinoma tissues, but not in normal or hyperplastic prostate tissues (tissue microarray: PR484, US Biomax, Inc). T2N0M0 - tumor invaded submucosa without any regional or distant metastases (stage II); T3aN0M0 - tumor had broken through the capsule of the prostate gland and invaded through muscularis propria (stage III); T3N1M0 - tumor invaded through muscularis propria with 1 to 3 regional lymph node metastases (stage IV). Data information: The images: scale bar is 100 µm.C Immunohistochemistry: significant correlation (P<0.0001) between positive staining of NALCN and Src in clinically localized prostate cancer, but not in non-cancerous prostate. Data information: The images: scale bar is 100 µm.D Immunohistochemistry: positive staining of NALCN in bone metastases produced by breast and colon cancers, but not in corresponding normal tissues. Data information: The images: scale bar is 100 µm.F NALCN knockdown suppresses invadopodia formation reported by Cy3-fluorescent gelatin degradation (arrows). Confocal images (left) of Cy3 (red) and DAPI (blue; nuclei) fluorescence in control (shCTL) and shNALCN 48 hours after plating the cells on gelatin. Bar diagram plot: NALCN suppression causes significant decrease of the number of gelatin degradation puncta counted within 48 h of incubation. Data information: The images: scale bar is 100 µm. Bar diagram plots show the degradation puncta per cell (F) mean±S.E.M. for N=5 (F) biological replicates per condition. **P<0.01, ***P<0.001, unpaired two-tailed Student's t-test.
Figure 2A Co-localisation of NALCN with invadopodia markers - Dynamin, Src and Tks: confocal images (left), 2D intensity histograms (middle) and statistical analysis (right). Scale bar: 5 µm.. Data information: The colocalization intensity (A) was measured using Pearson's correlation coefficient with Fiji ImageJ software. Bar diagram plots show mean±S.E.M.: (A) Pearson's correlation coefficient as a measure of co-localization with NALCN of Dynamin (n=4), Src (n=14) and Tks (n=4), n= number of cell ***P<0.001, unpaired two-tailed Student's t-test.C Top: temporal profiles of self-normalised fluorescence intensity (∆F/F0) of fluo-4 (green), averaged within outlined region (inset: left, green outline), and mCherry (red), averaged within 14 circles (inset: right, red outlines). Middle: galleries show confocal images of fluo-4 and mCherry fluorescence captured during two periods of interests, POIs (blue bars): for POI1- every image, for POI2 - every 100th image. Bottom left: confocal images of Dynamin2-mCherry fluorescence captured before and 3 min after stimulation with 10% FBS. Bottom middle and right: plots relate the dynamics of [Ca2+]c changes (Fluo-4) to formation of dynamin puncta (Dynamin2-mCherry) at two regions of interests (ROIs) (insets); the galleries (below) show every 40th image (after 90° rotation) during the POIs (plots: blue bars).D Top the overlay of NALCN-GFP and Cal-590 images is related to the gallery of self-normalized rainbow-coded Cal-590 images showing [Ca2+]c response to 10% FBS. Bottom: 3 rainbow-coded Cal-590 images are superimposed on grey-coded NALCN-GFP image and rotated by 90°. Arrows: NALCN-enriched structure.
Figure 3A FBS-induced transient [Ca2+]c elevations (Fluo-4, top left) are associated with transient decreases of Ca2+ concentration in endoplasmic reticulum ([Ca2+]ER) (Mag-Fluo-4, bottom left). Corresponding galleries (right) show every 100th image during the highlighted POIs (I and II, respectively). Visualization of the ER with pDsRed2-ER confirms the ER origin of Mag-Fluo-4 signal (inset bottom left: confocal images of cell fragment).C-D [Ca2+]c oscillations reported by Fluo-4 fluorescence in FBS-exposed PC-3 cells pre-treated with siCTL (C) or siNALCN (D) for 72 hours. Top: sample traces from 8 different cells. Middle: POIs (top: grey background) are presented on enlarged plots to emphasize that NALCN suppression abolishes "pacemaker events" observed in control (fitted red curves). Bottom: the galleries show every 3rd image during the POIs (middle: orange background). Scale bars: 10 μm.
Figure 4A-D Cytosolic Na+ concentration ([Na+]c) changes reported by ratiometric Na+ indicator SBFI following (A-C) switch of extracellular Na+ concentration ([Na+]o) from 0 to 130 mM or (D) application of 1 µM thapsigargin (TG). The effects of: (A) NALCN knockdown, (B) extracellular Ca2+ concentration ([Ca2+]o) and (C) Ca2+ store depletion. Data information: Data show mean±S.E.M. The cartoons highlight experimental design and the plots show mean±S.E.M. traces (n=45-501) of SBFI (olive) and Fura-2 (wine) fluorescence with the axes (∆R/R0) presented in corresponding colour.E-H Store-operated Ca2+ entry (SOCE)-induced changes of [Na+]c (SBFI) and [Ca2+]c (Fura-2). The effects of: NALCN knockdown (E), the reverse-mode plasmalemmal Na+/Ca2+ exchanger (RM-NCX) inhibitor KB-R7943 (1 µM) either at [Na+]o=130 mM (F) or upon [Na+]o switch from 0 to 130 mM following SOCE activation (G), and knockdown of the mitochondrial Na+/Ca2+ exchanger, siNCLX (H). Data information: Data show mean±S.E.M. The cartoons highlight experimental design and the plots show mean±S.E.M. traces (n=45-501) of SBFI (olive) and Fura-2 (wine) fluorescence with the axes (∆R/R0) presented in corresponding colour.I SOCE augments NALCN-mediated Na+ influx (top) by addressing NALCN to plasma membrane (bottom). Data information: Plots (I, top) compare mean rates of Na+ influx induced by [Na+]o-switch (n=283-360) and SOCE (n=180-216), and their shNALCN-sensitive fractions. Immunoblotting (I, bottom) compares NALCN expression in total cell lysates and biotinylated fractions before and after SOCE induction. Numbers show (in corresponding colour) mean values (n=3) of NALCN protein levels (band intensity) normalized to N-Cadherin, Calnexin and β-actin.J-K The effect of KB-R7943 (1 µM) on FBS-induced [Ca2+]c responses (Fura-2, ∆R/R0) and inward current (perforated patch, Vh=-80 mV), respectively. Data information: The bar diagram plots show mean signal temporal densities (J, n=20) and mean current densities (K, n=6-8) during initial transient (cyan) and oscillations (orange). n= number of cell **P<0.01, ***P<0.001, unpaired two-tailed Student's t-test.
Figure 5A Top: visualisation of mitochondria with MitoTracker Green confirms mitochondrial origin of Rhod-2 signal. Middle: FBS-induced [Ca2+]c oscillations (Fluo-4) are associated with [Ca2+]mito oscillations (Rhod-2). Bottom: every 2nd image from the highlighted (orange background) period. B Top: FBS-induced [Ca2+]c oscillations (Fluo-4) are associated with oscillations in O2- production by mitochondria (MitoSOX Red). Bottom: every 3rd image from the highlighted (orange background) period. Data information: Cartoons (left) highlight experimental design and assessed signaling pathways.C O2- metabolite, H2O2, suppresses FBS-induced [Ca2+]c oscillations (Fluo-4). Data information: In C, mean signal temporal density during POI2 was normalized to that during POI1 in control (n=58) and in 50 µM of H2O2 (n=74), and compared. Bar diagram plots: mean±S.E.M. n= number of cell, ***P<0.001, unpaired two-tailed Student's t-test. Cartoons (left) highlight experimental design and assessed signaling pathways.D Suppression of NCLX with siNCLX attenuates [Ca2+]c oscillations (Fluo-4). Data information: In D, mean signal temporal densities during highlighted periods were compared in control (n=18) and following 48 h pre-treatment with siNCLX (n=17). Bar diagram plots: mean±S.E.M. n= number of cell, ***P<0.001, unpaired two-tailed Student's t-test. Cartoons (left) highlight experimental design and assessed signaling pathways. Note that Na+ delivered to the cytosol by NALCN, on the one hand, activates RM-NCX, while on the other hand, is exchanged by NCLX to Ca2+ thus restricting elevation of [Ca2+]mito and, hence, production of superoxide (O2-) and H2O2, which is known to inhibit SERCA, RM-NCX and SOCE elements.
Figure 6D Corresponding traces of relative changes in total z-stack fluorescence, as indicated.E Plot: mean rates of secretion calculated as signal mass (cyan, left) per second. Data information: Data (E): mean±S.E.M. for siCTL (n=35), siNALCN (n=40), Control (n=45), EGTA-AM (n=35) and BAPTA-AM (n=62). n= number of cell, ***P<0.001; and n.s., not significant, unpaired two-tailed Student's t-test.
Figure 7D Left: Dorsal (top) and ventral (bottom) images of bioluminescence from mice 31 days after intra-tibial injection of control (shCTL: PC-3 Luc-shCTL) or NALCN depleted cells (shCTL: PC-3 Luc-shNALCN). Right: statistical comparison of total photon fluxes between the two groups of 10 mice (c.p.s.: count of photons per second±SD). Data information: *P<0.05, **P<0.01, and n.s., not significant, two-tailed Mann-Whitney U test.E Left: x-ray 3D images obtained from mice 8 weeks after intracardiac injections with control (PC-3 Luc) and cells stably overexpressing NALCN (+hNALCN). Right: box plots compare the incidence and total volume of bone lesions between the two groups of 11 mice: central band shows the mean, the boxes show lower and upper quartiles and whiskers show maximum data values. Data information: Red numbers and/or arrows in the images depict tumour take incidence/bone lesions. *P<0.05, **P<0.01, and n.s., not significant, two-tailed Mann-Whitney U test.
Figure 1(C) Immunoblot of WT, KO and A537T prostates probed with specific antibodies to detect ELAC2 and normalise its levels to Hsp60 that was used as a loading control (n=3). ***p= 1.09909E-07 for KO and ***p= 3.55091E-05 for A537T, Student's two-way t-test, values are means ± SD of biological replicates.(E) Survival data for ELAC2 WT (n=30), KO (n=30) and A537T (n=30) mice.(F) ELAC2 WT-TRAMP, KO-TRAMP and A537T-TRAMP prostates from 10- and 30-week-old mice. Bladder and seminal vesicles were removed in 10-week-old mice. Images show the typical differences between WT, KO and A537T prostates of at least 10 different mice per genotype.(G) Survival data for ELAC2 WT-TRAMP (n=30), KO-TRAMP (n=30) and A537T-TRAMP (n=30) mice (biological replicates). Gehan-Breslow-Wilcoxon test showed significant difference (*p=0.045) between the WT-TRAMP and KO-TRAMP or A537T-TRAMP survival curves.
Figure 2(A) Representative images of anterior, dorsal, ventral and lateral lobe sections from 30-week-old ELAC2 WT (n=7), KO (n=7) and A537T (n=5) mice. 5 µm sections of each lobe were cut and stained with haematoxylin and eosin.(B) Histological changes were scored according additional categories added to the scoring system (n=6-8 biological replicates). Values are means ± SD of biological replicates, ****p =0.001 compared with WT by two-tailed Student's t-test.
Figure 3(A) Representative images of anterior, dorsal, ventral and lateral lobe sections from 30-week-old WT-TRAMP (n=5), KO-TRAMP (n=5) and A537T-TRAMP mice (n=5). 5 µm sections of each lobe were cut and stained with haematoxylin and eosin. (B) Histological changes scored according to Suttie et al. with additional categories (n=6-7 biological replicates), values are means ± SD of biological replicates. Anterior lobe * p =0.013, Dorsal lobe ** p = 0.003, * p = 0.025, Ventral lobe * p =0.031 and Lateral lobe ** p = 0.0038, * p = 0.032, compared with WT-TRAMP by two-tailed Student's t-testing.(C) Representative histological images of complete prostate sections from 30-week-old WT-TRAMP, KO-TRAMP and A537T-TRAMP showing the prominent tumours (black arrows) in the mutant strains compared to the WT-TRAMP mice. Images show the typical differences between WT-TRAMP, KO-TRAMP and A537T-TRAMP prostates of at least 10 different mice per genotype.
Figure 5Specific protein changes that were in common between the KO and A537T lines at 30 weeks are shown in volcano plots (B) increased proteins are shown in red and reduced proteins are shown in blue.
Figure 6(E-F) Dot plots showing expression change of mRNA/miRNA pairs in the 30-week mutant lines compared to their respective controls. mRNA log2 fold change compared to controls is on the y-axis while the colour and x-axis denote the log2 fold change observed in the miRNA compared to controls.
Figure 7WT ELAC2-EGFP or A537T ELAC2-EGFP were expressed in LNCaP cells and the associated proteins that immunoprecipitated with the EGFP antibody were detected by mass spectrometry. EGFP-only protein was used as a control. Volcano plots are showing the enriched proteins with proteins and (C) shows the proteins that have reduced or increased association with A537T ELAC2. Volcano plots comparing Log2 fold change against the -log10 p-value and significant (p < 0.05) enriched proteins are shown in red and proteins with reduced association are shown in blue for three biologically independent experiments.
Figure 1B HCT116 and SW480 cells with GDH1 knockdown were incubated under normoxia and hypoxia, respectively. Then, cell viability was assessed by trypan blue staining. (n=3). Data information: Data are mean ± SD from the biological replicates (B Statistics: two-way ANOVA with Tukey's HSD post hoc test (B)D The cells in Figure 1B were collected after incubation under normoxia or hypoxia induction for 6 hours, and the HIF1α protein level was measured via immunoblot analysis.F HIF1A gene transcription was slightly impaired after GDH1 depletion. The cells in Figure 1B were collected after incubation under normoxia or hypoxia, and the HIF1A mRNA level was analysed via quantitative real-time PCR (qRT‒PCR). (n=3; ns, not significant). Data information: Data are mean ± SD from the biological replicates Statistics: unpaired two-tailed student's t-testH Tumour formation study. HCT116-shNT and HCT116-shGDH1 cells were injected into the left groin of nude mice. Solid tumours were stained with an antibody against Ki67 (Scale bars: 50 μm). (n=3) (H). Data information: Data are mean ± SD from the biological replicates Statistics: unpaired two-tailed student's t-test
Figure 2(E) H&E staining of colon cancer tumours from GDH1IEC+/+ and GDH1IEC-/- mice (scale bar, 200 μm). F F4/80 staining in a noninflamed portion of the colon after AMO/DSS treatment (scale bar, 50 μm). G ZO-1 staining in colon sections of mice treated with sequential AMO/DSS treatment (scale bar, 200 μm).I Survival percentages of GDH1IEC+/+ mice (n=11) and GDH1IEC-/- mice (n=9) during progression of the inflammatory cancer switch. Data information: Statistics: log-rank test (I).K Both HIF1α protein levels and AMPK activity were assessed in the intestine of GDH1IEC-/- mice after AOM/DSS treatment using the indicated antibodies.
Figure 3C GDH1-FLAG in (B) was examined using antibodies against pan-lysine acetylation and methylation and pan-serine/threonine and tyrosine phosphorylation.E HCT116 or SW480 cells containing WT or GDH1 with K503/K527R mutations were incubated under normoxia and hypoxia, respectively. GDH1-FLAG was enriched, and an antibody against pan-lysine acetylation was used to test GDH1-lysine acetylation.H GDH1-depleted cell lines were constructed and then rescued with the expression of wild-type GDH1 or K503/K527R mutant GDH1.
Figure 4B Immunoblot analysis of GDH1-FLAG levels at different time points.D The K503Q mutation reduces GDH1 activity leading to αKG production while enhancing the enzymatic activity leading to Glu production. His-tagged GDH1 was purified from E. coli. (n=3). Data information: Data are mean ± SD from the biological replicates (C-G Statistics: one-way ANOVA with Tukey's HSD post hoc testF HCT116 or SW480 cells were depleted of endogenous GDH1 and rescued with SFB-tagged rGDH1 WT, K503Q or K503R. rGDH1-SFB proteins were enriched using M2-FLAG beads to determine the Vmax of αKG production (F). (n=3). Data information: Data are mean ± SD from the biological replicates Statistics: one-way ANOVA with Tukey's HSD post hoc testITC assays were performed with precipitated His-GDH1 proteins and αKG (I). (n=3). Data information: Data are mean ± SD from the biological replicates I). Statistics: one-way ANOVA with Tukey's HSD post hoc test I).
Figure 5A, B Immunoblot of the HIF1α protein level under hypoxia for 6 hours when enzymes related to the αKG level were overexpressed in cells, including GDH1 (A). Relative αKG levels in contrast to the control (B). (n=3). Data information: Data are mean ± SD from the biological replicates (B Statistics: one-way ANOVA with Tukey's HSD post hoc test (BE Immunofluorescence analysis of GDH1-FLAG and HA-EGLN1 was performed to assess their colocalization under normoxia and hypoxia.I EGLN1 activity test. His-GDH1 with K503 or K527 acetylation (AcK503 or AcK527) and mimic acetylation at K503 or K527 (K503Q or K527Q) were purified from E. coli. Then, His-GDH1 was incubated for 1 hour with HA-EGLN1(I). EGLN1 activity was tested by converting αKG to succinate. (n=3). Data information: Data are mean ± SD from the biological replicates Statistics: one-way ANOVA with Tukey's HSD post hoc testDetection of HIF1α protein stability (L) in the indicated cell lines under hypoxia. (n=3).the mRNA expression levels of the HIF1α target genes GLUT1, HK1, PGK1 and CCND1 (M) in the indicated cell lines under hypoxia. (n=3). Data information: Data are mean ± SD from the biological replicates Statistics: one-way ANOVA with Tukey's HSD post hoc testO Detection of HIF1α protein stability in the indicated cell lines under normoxia.P HIF1α transactivation was examined in the indicated cell lines under hypoxia with or without treatment with the HIF1α transactivation inhibitor 2-MeOE2 (1 μM) for 12 hours. (n=3). Data information: Data are mean ± SD from the biological replicates P). Statistics: one-way ANOVA with Tukey's HSD post hoc test , P)
Figure 6C GDH1-K503 or GDH1-K527 acetylation occurred in both the cytosol and nucleus.D, The levels of both AcK503 and AcK527 were analysed when cells were transfected with small interfering RNA (siRNA) targeting TIP60, PCAF, CBP or p300 (D)F HCT116 and SW480 cells were transfected with or without FLAG-p300. The p300-associated proteins were enriched with M2-FLAG beads and subjected to immunoblot analysis using antibodies against GDH1, GDH1-K503 or GDH1-K527 acetylation.K The HIF1A gene was transiently knocked down, and HCT116 cells were cotransfected with FLAG-p300 and HA-GDH1 for a Co-IP assay.
Figure 7A, Cell viability in the indicated cell lines under hypoxia were analysed by trypan blue (A) (n=3). Data information: Data are mean ± SD from the biological replicates (A, Statistics: two-way ANOVA with Tukey's HSD post hoc test (AI Detection of AcK503/527-GDH1 expression in CRC grades I+Ⅱ and III+Ⅳ. (grade I+II, n=21; grade III+IV, n=12). J Detection of AcK503/527-GDH1 expression in patients with (＋) or without (－) recurrence. (－, n=17; ＋, n=17). Data information: Data are mean ± SD from the biological replicates I, J Statistics: Mann-Whitney U-test (I, J)K Patient survival corresponding to different levels of AcK503/527-GDH1 expression (High, n=38; Low, n=32). Data information: Statistics: log-rank test (K).