Figure 3Interaction analysis of WT and mutated CtUba4 with GST - CtUrm1 by GST pull - down in the presence of 1mM ATP . CtUba4C202K covalently linked to GST - CtUrm1 is marked by an asterisk .

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Figure 3Interaction analysis of WT and mutated CtUba4 with GST-CtUrm1 by GST pull-down in the presence of 1mM ATP. CtUba4C202K covalently linked to GST-CtUrm1 is marked by an asterisk.


Figure 4Analysis of covalent adduct formation between CtUba4 WT or C202 mutants and carboxylated ( - OH ) or thiocarboxylated ( - SH ) CtUrm1 in the presence or absence of TBH , DTT , TCEP and HA , respectively . ATP : Adenosine triphosphate TBH : tert - Butyl hydroperoxide HA : Hydroxylamine ; DTT : 1 , 4 - Dithiothreitol ; TCEP : Tris ( 2 - carboxyethyl ) phosphine . Analysis of covalent adduct formation between CtUba4 WT / cysteine mutants and CtUrm1 - COSH in the presence of oxidizing agents . HP : Hydrogen peroxide ; D : Diamide . Phenotypic analysis of ScUba4 mutant yeast strains in response to rapamycin . MS / MS spectrum of the K202 - ε - GGH containing peptide from CtUba4C202K . A representative annotated fragmentation spectrum is shown with the b - and y - ions marked in red and blue , respectively . The precursor ion at m / z 591 , 98 is labelled in green . The peptide sequence is shown at the top with the collision - induced fragmentation pattern . The Urm1 conjugation site was identified by detection of the GGH remnant motif generated by chymotryptic digestion . Analysis of covalent adduct formation between CtUba4 WT or Cys202 mutants and thiocarboxylated ( - SH ) CtUrm1 in the presence or absence of TBH and HA , respectively .

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Figure 4Analysis of covalent adduct formation between CtUba4 WT or C202 mutants and carboxylated (-OH) or thiocarboxylated (-SH) CtUrm1 in the presence or absence of TBH, DTT, TCEP and HA, respectively. ATP: Adenosine triphosphate TBH: tert-Butyl hydroperoxide HA: Hydroxylamine; DTT: 1,4-Dithiothreitol; TCEP: Tris(2-carboxyethyl)phosphine.Analysis of covalent adduct formation between CtUba4 WT/cysteine mutants and CtUrm1-COSH in the presence of oxidizing agents. HP: Hydrogen peroxide; D: Diamide.Phenotypic analysis of ScUba4 mutant yeast strains in response to rapamycin.MS/MS spectrum of the K202-ε-GGH containing peptide from CtUba4C202K. A representative annotated fragmentation spectrum is shown with the b- and y-ions marked in red and blue, respectively. The precursor ion at m/z 591,98 is labelled in green. The peptide sequence is shown at the top with the collision-induced fragmentation pattern. The Urm1 conjugation site was identified by detection of the GGH remnant motif generated by chymotryptic digestion.Analysis of covalent adduct formation between CtUba4 WT or Cys202 mutants and thiocarboxylated (-SH) CtUrm1 in the presence or absence of TBH and HA, respectively.


Figure 1B . Quantification of time - course nucleosome H2A - Ub assays using the indicated BRCA1 / BARD1 truncations . Data are presented as the normalized fraction of H2A consumed during a time - course nucleosome ubiquitylation assay . Data points represent mean values and error bars are ± 1 - s . d . of n = 3 independent technical replicate experiments . The residue bounds of BRCA1RING are 1 - 112 , and BARD1RING are 26 - 140 . Additional details about all reagents used in these studies can be found in the methods section . C . Nucleosome binding curves from fluorescence - quenching - based measurements using the indicated BRCA1 / BARD1 constructs . Data show mean values and error bars are ± 1 - s . d . of n = 4 independent technical replicate experiments . The reported difference in affinities to BRCA1RING / BARD1RING is likely underestimated , as the minimal RING / RING heterodimer binding data was collected at a lower ionic strength than the other constructs to obtain well - behaved binding data ( 50 mM vs . 100 mM NaCl ) .

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Figure 1B. Quantification of time-course nucleosome H2A-Ub assays using the indicated BRCA1/BARD1 truncations. Data are presented as the normalized fraction of H2A consumed during a time-course nucleosome ubiquitylation assay. Data points represent mean values and error bars are ± 1-s.d. of n=3 independent technical replicate experiments. The residue bounds of BRCA1RING are 1-112, and BARD1RING are 26-140. Additional details about all reagents used in these studies can be found in the methods section.C. Nucleosome binding curves from fluorescence-quenching-based measurements using the indicated BRCA1/BARD1 constructs. Data show mean values and error bars are ± 1-s.d. of n=4 independent technical replicate experiments. The reported difference in affinities to BRCA1RING/BARD1RING is likely underestimated, as the minimal RING/RING heterodimer binding data was collected at a lower ionic strength than the other constructs to obtain well-behaved binding data (50 mM vs. 100 mM NaCl).


Figure 2B . 1H15N - HSQC NMR amide chemical shift perturbations ( CSPs ) observed for 15N - BARD1 141 - 216 signals when bound to a 36 - bp dsDNA fragment ( 1 : 1 molar equivalent complex ) . C . Representative SDS - PAGE gel monitoring in - gel fluorescence ( labelled DNA ) of UV - induced Bpa crosslinking of the E3 ligase fusion BRCA1 - ƒ - BARD1221 ( BCƒBD ) to nucleosomes ( left ) . In this construct , the C - terminus of BRCA11 - 104 is genetically fused to the N - terminus of BARD126 - 221 via a 12 - residue GlySer - linker . Quantification of Bpa crosslinking experiments with nucleosomes ( right ) . The intensity of each crosslinked band was normalized to the intensity of the L120Bpa crosslinked band for each replicate experiment . Graph bars show the mean ; error bars are ± 1 - s . d . and the open circles are the values of individual replicates for of n = 3 independent technical replicate experiments .

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Figure 2B. 1H15N-HSQC NMR amide chemical shift perturbations (CSPs) observed for 15N-BARD1 141-216 signals when bound to a 36-bp dsDNA fragment (1:1 molar equivalent complex).C. Representative SDS-PAGE gel monitoring in-gel fluorescence (labelled DNA) of UV-induced Bpa crosslinking of the E3 ligase fusion BRCA1-ƒ-BARD1221 (BCƒBD) to nucleosomes (left). In this construct, the C-terminus of BRCA11-104 is genetically fused to the N-terminus of BARD126-221 via a 12-residue GlySer-linker. Quantification of Bpa crosslinking experiments with nucleosomes (right). The intensity of each crosslinked band was normalized to the intensity of the L120Bpa crosslinked band for each replicate experiment. Graph bars show the mean; error bars are ± 1-s.d. and the open circles are the values of individual replicates for of n=3 independent technical replicate experiments.


Figure 3Quantification of H2A - Ub time - course assays using the indicated BRCA1 / BARD1 constructs . Data show the mean ; error bars are ± 1 - s . d . of n = 3 independent technical replicate experiments . Two single time - points from panel C from the BRCA1RING / BARD1RING curve were excluded from analysis due to their extremely low activities coupled with quenching / loading errors yielding normalized fraction of H2A consumed values of < 0 . Values for these excluded points are included as source data .

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Figure 3Quantification of H2A-Ub time-course assays using the indicated BRCA1/BARD1 constructs. Data show the mean; error bars are ± 1-s.d. of n=3 independent technical replicate experiments.Two single time-points from panel C from the BRCA1RING/BARD1RING curve were excluded from analysis due to their extremely low activities coupled with quenching/loading errors yielding normalized fraction of H2A consumed values of <0. Values for these excluded points are included as source data.


Figure 4C . Inhibition of UV - induced Bpa crosslinking between the indicated BRCA1 - f - BARD1221 Bpa - incorporated constructs and NCP185 substrates in the presence of increasing amounts of dsDNA or bubble - DNA competitor . Data show the mean ; error bars are ± 1 - s . d . of n = 3 ( W146Bpa ) or n = 4 ( A195Bpa ) independent technical replicate experiments . D . 1H15N - HSQC NMR CSPs observed to 15N - BARD1 141 - 216 signals when bound to a 36 - mer dsDNA ( blue bars ) or bubble - DNA ( gray bars with black outlines ) fragment ( 1 : 1 molar equivalent complex ) . Signals for residues 143 - 157 are broadened beyond detection in the bubble - DNA - bound spectrum ; for visualization purposes , gray bars are set equal to the CSP value for the W146Ne CSP that was observed ; the real CSP values are likely larger that this valueF . Single time - point H2A - Ub inhibition assays using heterodimers containing BRCA1RING / BARD1FL ( left , 12 min endpoint ) or the indicated BARD1 internal deletion mutant ( middle and right , 20 min endpoint ) and increasing amounts of dsDNA or bubble - DNA competitor . The same E3 concentration ( 50 nM ) was used for each BRCA1 / BARD1 truncation . A longer time point was used for the double - deletion mutant as the intrinsic H2A - Ub activity of this mutant was lower , likely due to the deletion of DNA binding regions .

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Figure 4C. Inhibition of UV-induced Bpa crosslinking between the indicated BRCA1-f-BARD1221 Bpa-incorporated constructs and NCP185 substrates in the presence of increasing amounts of dsDNA or bubble-DNA competitor. Data show the mean; error bars are ± 1-s.d. of n=3 (W146Bpa) or n=4 (A195Bpa) independent technical replicate experiments.D. 1H15N-HSQC NMR CSPs observed to 15N-BARD1 141-216 signals when bound to a 36-mer dsDNA (blue bars) or bubble-DNA (gray bars with black outlines) fragment (1:1 molar equivalent complex). Signals for residues 143-157 are broadened beyond detection in the bubble-DNA-bound spectrum; for visualization purposes, gray bars are set equal to the CSP value for the W146Ne CSP that was observed; the real CSP values are likely larger that this valueF. Single time-point H2A-Ub inhibition assays using heterodimers containing BRCA1RING/BARD1FL (left, 12 min endpoint) or the indicated BARD1 internal deletion mutant (middle and right, 20 min endpoint) and increasing amounts of dsDNA or bubble-DNA competitor. The same E3 concentration (50 nM) was used for each BRCA1/BARD1 truncation. A longer time point was used for the double-deletion mutant as the intrinsic H2A-Ub activity of this mutant was lower, likely due to the deletion of DNA binding regions.


Figure 5A . Western blot analysis to detect HA - BARD1 in nuclei isolated from HeLa - shBARD1 cells stably expressing wild - type or Δ194 - 216 mutant of HA - BARD1 , where endogenous BARD1 was depleted by doxycycline - induced shBARD1 expression . The nuclei were salt - fractionated into NS100 , NS300 , NS420 , and NP420 to assess the amount of HA - BARD1 associated with chromatin . Data are representative of n = 2 biological replicate experiments . B . Quantification of cells with > 5 HA - BARD1 foci ( left panel ) and number of HA - BARD1 foci per nucleus ( right panel ) with and without olaparib treatment . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , by Student ' s t - test . Data show the individual points from n = 3 biological replicate experiments . C . Clonogenic survival of HeLa - shBARD1 cells stably expressing wild type or Δ194 - 216 mutant of HA - BARD1 upon treatment with indicated amount of olaparib and cisplatin . Data points show the mean ; error bars represent SEM of n = 3 biological replicate experiments .

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Figure 5A. Western blot analysis to detect HA-BARD1 in nuclei isolated from HeLa-shBARD1 cells stably expressing wild-type or Δ194-216 mutant of HA-BARD1, where endogenous BARD1 was depleted by doxycycline-induced shBARD1 expression. The nuclei were salt-fractionated into NS100, NS300, NS420, and NP420 to assess the amount of HA-BARD1 associated with chromatin. Data are representative of n=2 biological replicate experiments.B. Quantification of cells with >5 HA-BARD1 foci (left panel) and number of HA-BARD1 foci per nucleus (right panel) with and without olaparib treatment. *p<0.05, **p<0.01, ***p<0.001, by Student's t-test. Data show the individual points from n=3 biological replicate experiments.C. Clonogenic survival of HeLa-shBARD1 cells stably expressing wild type or Δ194-216 mutant of HA-BARD1 upon treatment with indicated amount of olaparib and cisplatin. Data points show the mean; error bars represent SEM of n=3 biological replicate experiments.


Figure 6B , C . Quantification of time - course H2A - Ub assays using the indicated BRCA1 / BARD1 constructs and chromatin substrates . data show the mean ; error bars are ± 1 - s . d . of n = 3 independent technical replicate experiments . D . Quantification of time - course H2A - Ub assays using homogenously modified H2A K15 - Ub mono - nucleosome substrates and indicated BRCA1 / BARD1 constructs . data show the mean ; error bars are ± 1 - s . d . of n = 3 independent technical replicate experiments . One time - point from Panel D was determined to be an outlier due to a reaction quenching error and excluded from analysis . The excluded value is reported as source data . G , H . Quantification of time - course H2A - Ub assays using the indicated combinations of di - NCP substrate and BRCA1 / BARD1 heterodimer . In each case , the y - axis reports on the fraction of H2Aobserve ( as shown in Panel F ) . Lower E3 concentrations were used in these reactions than in those presented in Figure 1 using unmodified mono - nucleosome substrates ( 15 nM vs . 100 nM ) , accounting for the slower observed H2A - Ub kinetics for the unmodified di - nucleosome substrate . data show the mean ; error bars are ± 1 - s . d . of n = 3 independent technical replicate experiments .

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Figure 6B, C. Quantification of time-course H2A-Ub assays using the indicated BRCA1/BARD1 constructs and chromatin substrates. data show the mean; error bars are ± 1-s.d. of n=3 independent technical replicate experiments.D. Quantification of time-course H2A-Ub assays using homogenously modified H2A K15-Ub mono-nucleosome substrates and indicated BRCA1/BARD1 constructs. data show the mean; error bars are ± 1-s.d. of n=3 independent technical replicate experiments. One time-point from Panel D was determined to be an outlier due to a reaction quenching error and excluded from analysis. The excluded value is reported as source data.G, H. Quantification of time-course H2A-Ub assays using the indicated combinations of di-NCP substrate and BRCA1/BARD1 heterodimer. In each case, the y-axis reports on the fraction of H2Aobserve (as shown in Panel F). Lower E3 concentrations were used in these reactions than in those presented in Figure 1 using unmodified mono-nucleosome substrates (15 nM vs. 100 nM), accounting for the slower observed H2A-Ub kinetics for the unmodified di-nucleosome substrate. data show the mean; error bars are ± 1-s.d. of n=3 independent technical replicate experiments.


Figure 1A - I . Correlative light electron microscopy ( CLEM ) showing Prom1 at the plasma membrane of primary hepatocytes . Primary hepatocytes were isolated from 12 - week - old male WT ( Prom1 + / + ) or KO ( Prom1cre - ert2 - nlacz ) mice . The boxed areas in panel ( A , D , G ) and ( B , E , H ) were magnified and presented as panel ( B , E , H ) and ( C , F , I ) , respectively . Scale bar ; 5 μm ( A , D and G ) , 1 μm ( B , E and H ) , and 0 . 2 μm ( C , F and I ) . J . Immunofluorescent staining of Prom1 , CK19 and HNF4α in two serial cryosections of mouse liver . The boxed area in the top panel was magnified and presented as the bottom panel . Asterisks ( * ) in the top panel indicate cholangiocytes of the bile bucts and triangles ( Γ ) in the bottom panel indicate hepatocytes . Scale bar , 100 μm . Primary hepatocytes were isolated from 12 - week - old male Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice and serum - starved . ( K ) Differentially expressed genes showing more than a 2 - fold change with P < 0 . 05 ( 118 DEGs ) induced ( n = 55 ) or repressed ( n = 63 ) by Prom1 knockout ( n = 2 / group ) . G6pc , glucose - 6 - phosphatase catalytic subunit ; Pck1 , phosphoenolpyruvate carboxykinase 1 . Primary hepatocytes were isolated from 12 - week - old male Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice and serum - starved . ( L ) Relative expression levels of G6pc and Pck1 mRNAs after serum starvation for 16 hours were determined by qRT - PCR ( n = 3 / group ) . Primary hepatocytes were isolated from 12 - week - old male Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice and serum - starved . ( M ) Levels of the Prom1 , PCK , G6PC and GAPDH proteins were determined by immunoblotting . Primary hepatocytes were isolated from 12 - week - old male Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice and serum - starved . ( N ) Prom1 + / + or Prom1 - / - hepatocytes serum - starved for 8 hours were incubated with 2 - [ 3H ] deoxyglucose for 10 minutes . Glucose uptake was assessed by calculating the amount of cell - associated radioactivity normalized to the amount of protein ( n = 3 mice / group ) .

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Figure 1A-I. Correlative light electron microscopy (CLEM) showing Prom1 at the plasma membrane of primary hepatocytes. Primary hepatocytes were isolated from 12-week-old male WT (Prom1+/+) or KO ( Prom1cre-ert2-nlacz) mice. The boxed areas in panel (A, D, G) and (B, E, H) were magnified and presented as panel (B, E, H) and (C, F, I), respectively. Scale bar; 5 μm (A, D and G), 1 μm (B, E and H), and 0.2 μm (C, F and I).J. Immunofluorescent staining of Prom1, CK19 and HNF4α in two serial cryosections of mouse liver. The boxed area in the top panel was magnified and presented as the bottom panel. Asterisks (*) in the top panel indicate cholangiocytes of the bile bucts and triangles (Γ) in the bottom panel indicate hepatocytes. Scale bar, 100 μm.Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice and serum-starved. (K) Differentially expressed genes showing more than a 2-fold change with P < 0.05 (118 DEGs) induced (n=55) or repressed (n=63) by Prom1 knockout (n = 2/group). G6pc, glucose-6-phosphatase catalytic subunit; Pck1, phosphoenolpyruvate carboxykinase 1.Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice and serum-starved. (L) Relative expression levels of G6pc and Pck1 mRNAs after serum starvation for 16 hours were determined by qRT-PCR (n = 3/group).Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice and serum-starved. (M) Levels of the Prom1, PCK, G6PC and GAPDH proteins were determined by immunoblotting.Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice and serum-starved. (N) Prom1+/+ or Prom1-/- hepatocytes serum-starved for 8 hours were incubated with 2-[3H]deoxyglucose for 10 minutes. Glucose uptake was assessed by calculating the amount of cell-associated radioactivity normalized to the amount of protein (n = 3 mice/group).


Figure 2A . Levels of G6pc and Pck1 mRNAs in the liver of mice after 18 hours fasting were determined by qRT - PCR . The level of each mRNA was normalized to the level of 18s rRNA ( n = 3 / fed , 7 or 8 / fasted ) . B . Levels of phospho - PKA substrates ( p - PKA substrates ) , phospho - CREB ( p - CREB ) , CREB , G6pc Pck , Prom1 and GAPDH were determined after 18 hours fasting . Band intensity for p - CREB was quantified using Photoshop software . C . Blood glucose levels ( mg / dL ) of mice after fasted for 24 hours ( n = 12 or 14 / group ) . D . Serum glucagon level in the fed or fasted mice ( n = 3 / fed , 7 or 8 / fasted ) . E . Glycogen contents in the liver of mouse in each condition was shown by PAS ( periodic acid schiff ) staining . Scale bar , 500 μm . Primary hepatocytes were isolated from 12 - week - old male Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice , serum - starved and stimulated with glucagon . F . Levels of the G6pc and Pck1 mRNAs 2 hours after glucagon stimulation ( 10 nM ) were determined by qRT - PCR . The level of each mRNA was normalized to the level of the 18S rRNA . For these experiments , primary hepatocytes were cultured under 3D conditions using a Matrigel matrix ( n = 3 mice / group ) . Primary hepatocytes were isolated from 12 - week - old male Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice , serum - starved and stimulated with glucagon . G . Levels of phospho - PKA substrates ( p - PKA substrates ) , phospho - CREB ( p - CREB ) , CREB , phospho - Inositol trisphosphate receptor ( p - IP3R ) , IP3R , phospho - Hormone - Sensitive Lipase ( p - HSL ) , HSL , Prom1 and Gapdh 10 minutes after glucagon stimulation ( 10 nM ) were determined by immunoblotting . Primary hepatocytes were isolated from 12 - week - old male Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice , serum - starved and stimulated with glucagon . H . The nuclear localization of p - CREB 10 minutes after glucagon stimulation ( 10 nM ) was determined by immunofluorescence staining . Scale bar , 20 μm . Primary hepatocytes were isolated from 12 - week - old male Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice , serum - starved and stimulated with glucagon . I . Relative PKA activities 10 minutes after glucagon stimulation ( 10 nM ) were determined using the PKA assay kit in the absence or presence of 10 μM IBMX ( n = 3 mice / group ) . Primary hepatocytes were isolated from 12 - week - old male Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice , serum - starved and stimulated with glucagon . J . The cAMP concentration 10 minutes after glucagon stimulation ( 10 nM ) was determined using the cAMP assay kit in the absence or presence of 10 μM IBMX ( n = 3 mice / group ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. Levels of G6pc and Pck1 mRNAs in the liver of mice after 18 hours fasting were determined by qRT-PCR. The level of each mRNA was normalized to the level of 18s rRNA (n = 3/fed, 7 or 8/fasted).B. Levels of phospho-PKA substrates (p-PKA substrates), phospho-CREB (p-CREB), CREB, G6pc Pck, Prom1 and GAPDH were determined after 18 hours fasting. Band intensity for p-CREB was quantified using Photoshop software.C. Blood glucose levels (mg/dL) of mice after fasted for 24 hours (n = 12 or 14/group).D. Serum glucagon level in the fed or fasted mice (n = 3/fed, 7 or 8/fasted).E. Glycogen contents in the liver of mouse in each condition was shown by PAS (periodic acid schiff) staining. Scale bar, 500 μm.Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice, serum-starved and stimulated with glucagon. F. Levels of the G6pc and Pck1 mRNAs 2 hours after glucagon stimulation (10 nM) were determined by qRT-PCR. The level of each mRNA was normalized to the level of the 18S rRNA. For these experiments, primary hepatocytes were cultured under 3D conditions using a Matrigel matrix (n = 3 mice/group).Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice, serum-starved and stimulated with glucagon. G. Levels of phospho-PKA substrates (p-PKA substrates), phospho-CREB (p-CREB), CREB, phospho-Inositol trisphosphate receptor (p-IP3R), IP3R, phospho-Hormone-Sensitive Lipase (p-HSL), HSL, Prom1 and Gapdh 10 minutes after glucagon stimulation (10 nM) were determined by immunoblotting.Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice, serum-starved and stimulated with glucagon. H. The nuclear localization of p-CREB 10 minutes after glucagon stimulation (10 nM) was determined by immunofluorescence staining. Scale bar, 20 μm.Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice, serum-starved and stimulated with glucagon. I. Relative PKA activities 10 minutes after glucagon stimulation (10 nM) were determined using the PKA assay kit in the absence or presence of 10 μM IBMX (n = 3 mice/group).Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice, serum-starved and stimulated with glucagon. J. The cAMP concentration 10 minutes after glucagon stimulation (10 nM) was determined using the cAMP assay kit in the absence or presence of 10 μM IBMX (n = 3 mice/group).


Figure 312 - week - old male WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were fasted for 4 hours and intraperitoneally injected with glucagon . ( A ) Blood glucose levels ( mg / dL ) were measured 0 , 15 , 30 , 60 and 120 minutes after glucagon stimulation ( 200 µg / kg body weight , n = 10 / group ) . 12 - week - old male WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were fasted for 4 hours and intraperitoneally injected with glucagon . ( B ) Levels of p - PKA substrates , p - CREB , CREB and GAPDH in the liver after a 5 minutes glucagon treatment were determined by immunoblotting ( 2 mg / kg body weight ) . 12 - week - old male WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were fasted for 4 hours and intraperitoneally injected with glucagon . ( C ) Relative PKA activities in the liver after a 5 minutes glucagon treatment were determined using the PKA assay kit ( 2 mg / kg body weight , n = 3 / group ) . 12 - week - old male WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were fasted for 4 hours and intraperitoneally injected with glucagon . ( D ) The cAMP concentration in the liver after a 5 minutes glucagon treatment was determined using the cAMP assay kit ( 2 mg / kg body weight , n = 3 / group ) . Glucose disposal rates were measured in 12 - week - old male mice using glucose , pyruvate ( E ) tolerance tests ( n = 10 mice / group ) . Glucose disposal rates were measured in 12 - week - old male mice using insulin ( F ) tolerance tests ( n = 10 mice / group ) . G . Levels of p - AKT , AKT , p - ERK , ERK , IRβ and GAPDH were determined by immunoblotting after insulin stimulation ( 10 nM ) . Primary hepatocytes were isolated from 12 - week - old male WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice , serum - starved for 16 hours and stimulated with isoprenaline . ( H ) The nuclear localization of p - CREB after isoprenaline stimulation ( 10 μM ) for 10 minutes was determined by immunofluorescence staining . Scale bar , 20 μm . Primary hepatocytes were isolated from 12 - week - old male WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice , serum - starved for 16 hours and stimulated with isoprenaline . ( I ) Levels of p - PKA substrates , p - CREB , CREB , Prom1 and GAPDH after isoprenaline stimulation ( 10 μM ) for 0 , 10 or 20 minutes were determined by immunoblotting . Primary hepatocytes were isolated from 12 - week - old male WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice , serum - starved for 16 hours and stimulated with isoprenaline . ( J ) The cAMP concentration after isoprenaline stimulation ( 10 μM ) for 10 minutes was determined using the cAMP assay kit in the presence of 10 μM IBMX ( n = 3 / group ) . WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were fasted for 4 hours and intraperitoneally injected with epinephrine ( 3µg / 10g ) . ( K ) Blood glucose levels ( mg / dL ) were measured 0 , 15 , 30 , 60 and 120 minutes after epinephrine stimulationWT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were fasted for 4 hours and intraperitoneally injected with epinephrine ( 3µg / 10g ) . ( L ) Levels of p - PKA substrates , p - CREB , CREB and GAPDH in the liver 15 minutes after epinephrine treatment were determined by immunoblotting . M - O . WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were fasted for 4 hours and subjected to immobilization test . ( M ) Blood glucose levels ( mg / dL ) were measured 0 , 60 and 120 minutes after immobilization ( n = 10 / group ) . WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were fasted for 4 hours and subjected to immobilization test . ( N ) Levels of p - PKA substrates , p - CREB , CREB and GAPDH in the liver after a 30 minutes - immobilization were determined by immunoblotting . WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were fasted for 4 hours and subjected to immobilization test . ( O ) Serum epinephrine level of mice after a 30 minutes - immobilization was determined ( n = 3 / control group , 7 or 8 / immobilized group ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 312-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with glucagon. (A) Blood glucose levels (mg/dL) were measured 0, 15, 30, 60 and 120 minutes after glucagon stimulation (200 µg/kg body weight, n = 10/group).12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with glucagon. (B) Levels of p-PKA substrates, p-CREB, CREB and GAPDH in the liver after a 5 minutes glucagon treatment were determined by immunoblotting (2 mg/kg body weight).12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with glucagon. (C) Relative PKA activities in the liver after a 5 minutes glucagon treatment were determined using the PKA assay kit (2 mg/kg body weight, n = 3/group).12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with glucagon. (D) The cAMP concentration in the liver after a 5 minutes glucagon treatment was determined using the cAMP assay kit (2 mg/kg body weight, n = 3/group).Glucose disposal rates were measured in 12-week-old male mice using glucose, pyruvate (E) tolerance tests (n = 10 mice/group).Glucose disposal rates were measured in 12-week-old male mice using insulin (F) tolerance tests (n = 10 mice/group).G. Levels of p-AKT, AKT, p-ERK, ERK, IRβ and GAPDH were determined by immunoblotting after insulin stimulation (10 nM).Primary hepatocytes were isolated from 12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice, serum-starved for 16 hours and stimulated with isoprenaline. (H) The nuclear localization of p-CREB after isoprenaline stimulation (10 μM) for 10 minutes was determined by immunofluorescence staining. Scale bar, 20 μm.Primary hepatocytes were isolated from 12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice, serum-starved for 16 hours and stimulated with isoprenaline. (I) Levels of p-PKA substrates, p-CREB, CREB, Prom1 and GAPDH after isoprenaline stimulation (10 μM) for 0, 10 or 20 minutes were determined by immunoblotting.Primary hepatocytes were isolated from 12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice, serum-starved for 16 hours and stimulated with isoprenaline. (J) The cAMP concentration after isoprenaline stimulation (10 μM) for 10 minutes was determined using the cAMP assay kit in the presence of 10 μM IBMX (n = 3/group).WT (Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with epinephrine (3µg/10g). (K) Blood glucose levels (mg/dL) were measured 0, 15, 30, 60 and 120 minutes after epinephrine stimulationWT (Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with epinephrine (3µg/10g). (L) Levels of p-PKA substrates, p-CREB, CREB and GAPDH in the liver 15 minutes after epinephrine treatment were determined by immunoblotting.M-O. WT (Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and subjected to immobilization test. (M) Blood glucose levels (mg/dL) were measured 0, 60 and 120 minutes after immobilization (n = 10/group).WT (Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and subjected to immobilization test. (N) Levels of p-PKA substrates, p-CREB, CREB and GAPDH in the liver after a 30 minutes-immobilization were determined by immunoblotting.WT (Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and subjected to immobilization test. (O) Serum epinephrine level of mice after a 30 minutes-immobilization was determined (n = 3/control group, 7 or 8/immobilized group).


Figure 4A . The expression of Prom1 , radixin , AKAP7 , AKAP8 , AKAP8I , AKAP9 , AKAP10 , AKAP12 or AKAP13 was silenced in WT hepatocytes using siRNAs . Hepatocytes were then serum - starved for 18 hours and stimulated with glucagon ( 10 nM ) for 10 minutes . The phosphorylation of PKA substrates was determined by immunoblotting . Prom1 and radixin expression were silenced in WT ( Prom1 + / + ) and KO ( Prom1 - / - ) hepatocytes by infection with an adenovirus harboring sh - control ( sh - Con ) , sh - radixin ( sh - Rdx ) or sh - Prom1 for 24 hours . Hepatocytes were further serum - starved for 18 hours and stimulated with glucagon ( 10 nM ) for 10 minutes . ( B ) The nuclear localization of p - CREB after glucagon stimulation was determined by immunofluorescence staining . Blue ; DAPI , Red ; p - CREB . The percentage of cells with nuclear p - CREB staining among more than 300 cells in each group was also statistically analyzed ( Figure EV3G ) . Scale bar , 20 μm . Prom1 and radixin expression were silenced in WT ( Prom1 + / + ) and KO ( Prom1 - / - ) hepatocytes by infection with an adenovirus harboring sh - control ( sh - Con ) , sh - radixin ( sh - Rdx ) or sh - Prom1 for 24 hours . Hepatocytes were further serum - starved for 18 hours and stimulated with glucagon ( 10 nM ) for 10 minutes . ( C ) Levels of p - PKA substrates , p - CREB , CREB , p - IP3R , IP3R , Prom1 , radixin and GAPDH after glucagon stimulation ( 10 minutes ) were determined by immunoblotting . D . Immunofluorescence staining of Prom1 in mouse liver tranduced with adenoviral particles . Confocal microscope images of WT mouse liver infected with adenovirus harboring sh - Con or sh - Rdx linked with IRES - GFP . Scale bar , 20 μm . 12 - week - old male wild type mice were infected with an adenovirus harboring sh - control ( sh - Con ) or sh - radixin ( sh - Rdx ) for 3 days , fasted for 4 hours and intraperitoneally injected with glucagon . ( E ) Blood glucose levels ( mg / dL ) were measured 0 , 15 , 30 , 60 and 120 minutes after glucagon administration ( 200 µg / kg body weight , n = 10 mice / group ) . 12 - week - old male wild type mice were infected with an adenovirus harboring sh - control ( sh - Con ) or sh - radixin ( sh - Rdx ) for 3 days , fasted for 4 hours and intraperitoneally injected with glucagon . ( F ) Levels of p - PKA , p - CREB , CREB , radixin and GAPDH in the liver after a 10 - minutes glucagon stimulation ( 2 mg / kg body weight ) were determined by immunoblotting . 12 - week - old male wild type mice were infected with an adenovirus harboring sh - control ( sh - Con ) or sh - radixin ( sh - Rdx ) for 3 days , fasted for 4 hours and intraperitoneally injected with glucagon . ( G ) Glucose disposal rates in sh - Con or sh - Rdx ( radixin ) mice were measured using glucose , insulin and pyruvate tolerance tests ( n = 10 mice / group ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. The expression of Prom1, radixin, AKAP7, AKAP8, AKAP8I, AKAP9, AKAP10, AKAP12 or AKAP13 was silenced in WT hepatocytes using siRNAs. Hepatocytes were then serum-starved for 18 hours and stimulated with glucagon (10 nM) for 10 minutes. The phosphorylation of PKA substrates was determined by immunoblotting.Prom1 and radixin expression were silenced in WT (Prom1+/+) and KO (Prom1-/-) hepatocytes by infection with an adenovirus harboring sh-control (sh-Con), sh-radixin (sh-Rdx) or sh-Prom1 for 24 hours. Hepatocytes were further serum-starved for 18 hours and stimulated with glucagon (10 nM) for 10 minutes. (B) The nuclear localization of p-CREB after glucagon stimulation was determined by immunofluorescence staining. Blue; DAPI, Red; p-CREB. The percentage of cells with nuclear p-CREB staining among more than 300 cells in each group was also statistically analyzed (Figure EV3G). Scale bar, 20 μm.Prom1 and radixin expression were silenced in WT (Prom1+/+) and KO (Prom1-/-) hepatocytes by infection with an adenovirus harboring sh-control (sh-Con), sh-radixin (sh-Rdx) or sh-Prom1 for 24 hours. Hepatocytes were further serum-starved for 18 hours and stimulated with glucagon (10 nM) for 10 minutes. (C) Levels of p-PKA substrates, p-CREB, CREB, p-IP3R, IP3R, Prom1, radixin and GAPDH after glucagon stimulation (10 minutes) were determined by immunoblotting.D. Immunofluorescence staining of Prom1 in mouse liver tranduced with adenoviral particles. Confocal microscope images of WT mouse liver infected with adenovirus harboring sh-Con or sh-Rdx linked with IRES-GFP. Scale bar, 20 μm.12-week-old male wild type mice were infected with an adenovirus harboring sh-control (sh-Con) or sh-radixin (sh-Rdx) for 3 days, fasted for 4 hours and intraperitoneally injected with glucagon. (E) Blood glucose levels (mg/dL) were measured 0, 15, 30, 60 and 120 minutes after glucagon administration (200 µg/kg body weight, n = 10 mice/ group).12-week-old male wild type mice were infected with an adenovirus harboring sh-control (sh-Con) or sh-radixin (sh-Rdx) for 3 days, fasted for 4 hours and intraperitoneally injected with glucagon. (F) Levels of p-PKA, p-CREB, CREB, radixin and GAPDH in the liver after a 10-minutes glucagon stimulation (2 mg/kg body weight) were determined by immunoblotting.12-week-old male wild type mice were infected with an adenovirus harboring sh-control (sh-Con) or sh-radixin (sh-Rdx) for 3 days, fasted for 4 hours and intraperitoneally injected with glucagon. (G) Glucose disposal rates in sh-Con or sh-Rdx (radixin) mice were measured using glucose, insulin and pyruvate tolerance tests (n = 10 mice/group).


Figure 5Primary hepatocytes were obtained from 12 - week - old male WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice or KO ( Prom1 - / - ) hepatocytes infected with an adenovirus harboring LacZ ( Ad - LacZ ) or Prom1 ( Ad - Prom1 ) for 24 hours . The molecular interaction between Prom1 and radixin ( A ) in these hepatocytes was determined by a proximity ligation assay using anti - Prom1 and anti - radixin antibodies ( A ) Primary hepatocytes were obtained from 12 - week - old male WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice or KO ( Prom1 - / - ) hepatocytes infected with an adenovirus harboring LacZ ( Ad - LacZ ) or Prom1 ( Ad - Prom1 ) for 24 hours . The molecular interaction between Prom1 and PKA regulatory subunits ( B ) in these hepatocytes was determined by a proximity ligation assay using anti - Prom1 and anti - PKA regulatory subunits ( B ) , respectively . Scale bar , 20 μm . C . Human liver specimens were analyzed by double immunostaining for PROM1 ( red ) and radixin ( green ) . The lower panels represent the magnification of the square frame in the upper panels . Scale bar , 20 μm . D . The molecular interaction between Prom1 , radixin ( RDX ) and actin in WT mouse primary hepatocytes was determined by co - immunoprecipitation with an anti - Prom1 antibody or isotype control IgG . WCL ; whole cell lysate . HEK293 cells were transiently transfected with the combination of Prom1 - FLAG and Myc - radixin plasmids for 24 hours . The molecular interaction between Prom1 - FLAG and Myc - radixin was determined by co - immunoprecipitation using isotype control IgG , anti - Myc ( E ) antibodies . HEK293 cells were transiently transfected with the combination of Prom1 - FLAG and Myc - radixin plasmids for 24 hours . The molecular interaction between Prom1 - FLAG and Myc - radixin was determined by co - immunoprecipitation using isotype control IgG , anti - FLAG ( F ) antibodies . G . The cellular localization of radixin and actin in WT ( Prom1 + / + ) and KO ( Prom1 - / - ) hepatocytes was determined by immunofluorescence staining for radixin and phalloidin staining . Scale bar , 20 μm . H . Detergent - resistant lipid rafts were isolated from WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mouse liver samples . Levels of the Prom1 , radixin ( RDX ) , flotillin and transferrin receptor ( TfR ) proteins were determined in each fraction after sucrose gradient ultracentrifugation using immunoblotting . I . Detergent - resistant lipid rafts were isolated from WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mouse primary hepatocytes after cholesterol depletion by methyl - β - cyclodextrin ( mβCD , 30 mM ) for 30 minutes at 4 oC . Levels of the Prom1 , radixin , flotillin and transferrin receptor proteins were determined in each fraction after sucrose gradient ultracentrifugation using immunoblotting .

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Figure 5Primary hepatocytes were obtained from 12-week-old male WT (Prom1+/+) and KO (Prom1-/-) mice or KO (Prom1-/-) hepatocytes infected with an adenovirus harboring LacZ (Ad-LacZ) or Prom1 (Ad-Prom1) for 24 hours. The molecular interaction between Prom1 and radixin (A) in these hepatocytes was determined by a proximity ligation assay using anti-Prom1 and anti-radixin antibodies (A)Primary hepatocytes were obtained from 12-week-old male WT (Prom1+/+) and KO (Prom1-/-) mice or KO (Prom1-/-) hepatocytes infected with an adenovirus harboring LacZ (Ad-LacZ) or Prom1 (Ad-Prom1) for 24 hours. The molecular interaction between Prom1 and PKA regulatory subunits (B) in these hepatocytes was determined by a proximity ligation assay using anti-Prom1 and anti-PKA regulatory subunits (B), respectively. Scale bar, 20 μm.C. Human liver specimens were analyzed by double immunostaining for PROM1 (red) and radixin (green). The lower panels represent the magnification of the square frame in the upper panels. Scale bar, 20 μm.D. The molecular interaction between Prom1, radixin (RDX) and actin in WT mouse primary hepatocytes was determined by co-immunoprecipitation with an anti-Prom1 antibody or isotype control IgG. WCL; whole cell lysate.HEK293 cells were transiently transfected with the combination of Prom1-FLAG and Myc-radixin plasmids for 24 hours. The molecular interaction between Prom1-FLAG and Myc-radixin was determined by co-immunoprecipitation using isotype control IgG, anti-Myc (E) antibodies.HEK293 cells were transiently transfected with the combination of Prom1-FLAG and Myc-radixin plasmids for 24 hours. The molecular interaction between Prom1-FLAG and Myc-radixin was determined by co-immunoprecipitation using isotype control IgG, anti-FLAG (F) antibodies.G. The cellular localization of radixin and actin in WT (Prom1+/+) and KO (Prom1-/-) hepatocytes was determined by immunofluorescence staining for radixin and phalloidin staining. Scale bar, 20 μm.H. Detergent-resistant lipid rafts were isolated from WT (Prom1+/+) and KO (Prom1-/-) mouse liver samples. Levels of the Prom1, radixin (RDX), flotillin and transferrin receptor (TfR) proteins were determined in each fraction after sucrose gradient ultracentrifugation using immunoblotting.I. Detergent-resistant lipid rafts were isolated from WT (Prom1+/+) and KO (Prom1-/-) mouse primary hepatocytes after cholesterol depletion by methyl-β-cyclodextrin (mβCD, 30 mM) for 30 minutes at 4 oC. Levels of the Prom1, radixin, flotillin and transferrin receptor proteins were determined in each fraction after sucrose gradient ultracentrifugation using immunoblotting.


Figure 6The molecular interactions between PROM1 and radixin mutants were determined by co - immunoprecipitation with an anti - FLAG antibody . HEK293 cells were co - transfected with plasmids expressing Myc - radixin and various deletion mutants of PROM1 - FLAG ( A ) for 24 hours . The molecular interactions between PROM1 and radixin mutants were determined by co - immunoprecipitation with an anti - FLAG antibody . HEK293 cells were co - transfected with plasmids expressing PROM1 - FLAG and various deletion mutants of Myc - radixin ( B ) for 24 hours . C . The molecular interaction between PROM1 - FLAG and Myc - radixin in the presence of increasing amounts of Myc - FERM was determined by co - immunoprecipitation with an anti - FLAG antibody . WT primary hepatocytes were infected with an adenovirus harboring FERM - Myc ( Ad - FERM - Myc ) or LacZ ( Ad - LacZ ) for 48 hours . The cellular localization of radixin was determined by immunofluorescence staining . Scale bar , 20 μm . WT primary hepatocytes were infected with an adenovirus harboring FERM - Myc ( Ad - FERM - Myc ) or LacZ ( Ad - LacZ ) for 48 hours . the nuclear localization of p - CREB after glucagon stimulation for 10 minutes ( E ) was determined by immunofluorescence staining . Scale bar , 20 μm . WT primary hepatocytes were infected with an adenovirus harboring FERM - Myc ( Ad - FERM - Myc ) or LacZ ( Ad - LacZ ) for 48 hours . ( F ) Ad - FERM - Myc - or Ad - LacZ - expressing WT hepatocytes were serum - starved for 16 hours and stimulated with glucagon ( 10 nM ) for 10 minutes . Levels of p - PKA substrates , p - CREB , CREB , FERM - Myc and GAPDH were determined by immunoblotting . Twelve - week - old male wild type mice were infected with an adenovirus harboring GFP or FERM for 3 days , fasted for 4 hours and intraperitoneally injected with glucagon . ( G ) Blood glucose levels ( mg / dL ) were measured 0 , 15 , 30 , 60 and 120 minutes after glucagon stimulation ( 0 . 2 mg / kg body weight , n = 10 mice / group ) . Twelve - week - old male wild type mice were infected with an adenovirus harboring GFP or FERM for 3 days , fasted for 4 hours and intraperitoneally injected with glucagon . ( H ) Levels of p - CREB , CREB , p - PKA substrates , and FERM in the liver 10 minutes after glucagon stimulation ( 2 mg / kg body weight ) were determined by immunoblotting ( n = 3 mice / group ) . Twelve - week - old male wild type mice were infected with an adenovirus harboring GFP or FERM for 3 days , fasted for 4 hours and intraperitoneally injected with glucagon . ( I ) Glucose disposal rates in GFP - or FERM - overexpressing mice were measured using glucose , pyruvate and insulin tolerance tests ( n = 10 mice / group ) .

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Figure 6The molecular interactions between PROM1 and radixin mutants were determined by co-immunoprecipitation with an anti-FLAG antibody. HEK293 cells were co-transfected with plasmids expressing Myc-radixin and various deletion mutants of PROM1-FLAG (A) for 24 hours.The molecular interactions between PROM1 and radixin mutants were determined by co-immunoprecipitation with an anti-FLAG antibody. HEK293 cells were co-transfected with plasmids expressing PROM1-FLAG and various deletion mutants of Myc-radixin (B) for 24 hours.C. The molecular interaction between PROM1-FLAG and Myc-radixin in the presence of increasing amounts of Myc-FERM was determined by co-immunoprecipitation with an anti-FLAG antibody.WT primary hepatocytes were infected with an adenovirus harboring FERM-Myc (Ad-FERM-Myc) or LacZ (Ad-LacZ) for 48 hours. The cellular localization of radixin was determined by immunofluorescence staining. Scale bar, 20 μm.WT primary hepatocytes were infected with an adenovirus harboring FERM-Myc (Ad-FERM-Myc) or LacZ (Ad-LacZ) for 48 hours. the nuclear localization of p-CREB after glucagon stimulation for 10 minutes (E) was determined by immunofluorescence staining. Scale bar, 20 μm.WT primary hepatocytes were infected with an adenovirus harboring FERM-Myc (Ad-FERM-Myc) or LacZ (Ad-LacZ) for 48 hours. (F) Ad-FERM-Myc- or Ad-LacZ-expressing WT hepatocytes were serum-starved for 16 hours and stimulated with glucagon (10 nM) for 10 minutes. Levels of p-PKA substrates, p-CREB, CREB, FERM-Myc and GAPDH were determined by immunoblotting.Twelve-week-old male wild type mice were infected with an adenovirus harboring GFP or FERM for 3 days, fasted for 4 hours and intraperitoneally injected with glucagon. (G) Blood glucose levels (mg/dL) were measured 0, 15, 30, 60 and 120 minutes after glucagon stimulation (0.2 mg/kg body weight, n = 10 mice/ group).Twelve-week-old male wild type mice were infected with an adenovirus harboring GFP or FERM for 3 days, fasted for 4 hours and intraperitoneally injected with glucagon. (H) Levels of p-CREB, CREB, p-PKA substrates, and FERM in the liver 10 minutes after glucagon stimulation (2 mg/kg body weight) were determined by immunoblotting (n = 3 mice/ group).Twelve-week-old male wild type mice were infected with an adenovirus harboring GFP or FERM for 3 days, fasted for 4 hours and intraperitoneally injected with glucagon. (I) Glucose disposal rates in GFP- or FERM-overexpressing mice were measured using glucose, pyruvate and insulin tolerance tests (n = 10 mice/group).


Figure 7 ( A ) Wild type primary hepatocytes were infected with an adenovirus harboring sh - control ( sh - Con ) , sh - radixin ( sh - Rdx ) , GFP ( Ad - GFP ) , and / or sh - resistant wildtype radixin ( Ad - RdxR ) or AKAP - dead LPTD mutant of radixin ( Ad - LPTDR ) for 24 hours . ( B ) Increasing amount of Ad - RdxR or Ad - LPTDR were infected to WT primary hepatocytes for 24 hours . Hepatocytes were further serum - starved for 18 hours and stimulated with glucagon ( 10 nM ) for 10 minutes . Levels of p - PKA substrates , p - CREB , CREB , radixin , Myc - radixin and GAPDH were determined by immunoblotting . Four - week - old male WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were fed a high - fat diet for 8 weeks ( Diet - Induced Obesity ; DIO ) , and fasted for 4 hours before experiments . ( C ) DIO WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were intraperitoneally injected with glucagon . Blood glucose levels ( mg / dL ) were measured 0 , 15 , 30 , 60 and 120 minutes after glucagon ( 100 µg / kg body weight ) stimulation ( n = 10 mice / group ) . Four - week - old male WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were fed a high - fat diet for 8 weeks ( Diet - Induced Obesity ; DIO ) , and fasted for 4 hours before experiments . ( D ) Levels of p - PKA substrates , p - CREB , CREB , Prom1 and GAPDH were determined by immunoblotting 10 minutes after glucagon ( 2 mg / kg body weight ) stimulation ( n = 3 mice / group ) . Four - week - old male WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were fed a high - fat diet for 8 weeks ( Diet - Induced Obesity ; DIO ) , and fasted for 4 hours before experiments . ( E ) Glucose disposal rates in DIO WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were measured using glucose and insulin tolerance tests ( n = 10 mice / group ) . Four - week - old male WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were fed a high - fat diet for 8 weeks ( Diet - Induced Obesity ; DIO ) , and fasted for 4 hours before experiments . ( F ) DIO KO ( Prom1 - / - ) mice were infected with an adenovirus harboring LacZ or Prom1 for 24 hours , fasted for 4 hours and intraperitoneally injected with glucagon ( 100 µg / kg body weight ) . Blood glucose levels ( mg / dL ) were measured 0 , 15 , 30 , 60 and 120 minutes after glucagon stimulation ( GST ) . Glucose disposal rates were measured using the pyruvate tolerance test ( PTT ) ( n = 10 mice / group ) .

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Figure 7(A) Wild type primary hepatocytes were infected with an adenovirus harboring sh-control (sh-Con), sh-radixin (sh-Rdx), GFP (Ad-GFP), and/or sh-resistant wildtype radixin (Ad-RdxR) or AKAP-dead LPTD mutant of radixin (Ad-LPTDR) for 24 hours.(B) Increasing amount of Ad-RdxR or Ad-LPTDR were infected to WT primary hepatocytes for 24 hours. Hepatocytes were further serum-starved for 18 hours and stimulated with glucagon (10 nM) for 10 minutes. Levels of p-PKA substrates, p-CREB, CREB, radixin, Myc-radixin and GAPDH were determined by immunoblotting.Four-week-old male WT (Prom1+/+) and KO (Prom1-/-) mice were fed a high-fat diet for 8 weeks (Diet-Induced Obesity; DIO), and fasted for 4 hours before experiments. (C) DIO WT (Prom1+/+) and KO (Prom1-/-) mice were intraperitoneally injected with glucagon. Blood glucose levels (mg/dL) were measured 0, 15, 30, 60 and 120 minutes after glucagon (100 µg/kg body weight) stimulation (n = 10 mice/ group).Four-week-old male WT (Prom1+/+) and KO (Prom1-/-) mice were fed a high-fat diet for 8 weeks (Diet-Induced Obesity; DIO), and fasted for 4 hours before experiments. (D) Levels of p-PKA substrates, p-CREB, CREB, Prom1 and GAPDH were determined by immunoblotting 10 minutes after glucagon (2 mg/kg body weight) stimulation (n = 3 mice/group).Four-week-old male WT (Prom1+/+) and KO (Prom1-/-) mice were fed a high-fat diet for 8 weeks (Diet-Induced Obesity; DIO), and fasted for 4 hours before experiments. (E) Glucose disposal rates in DIO WT (Prom1+/+) and KO (Prom1-/-) mice were measured using glucose and insulin tolerance tests (n = 10 mice/group).Four-week-old male WT (Prom1+/+) and KO (Prom1-/-) mice were fed a high-fat diet for 8 weeks (Diet-Induced Obesity; DIO), and fasted for 4 hours before experiments. (F) DIO KO (Prom1-/-) mice were infected with an adenovirus harboring LacZ or Prom1 for 24 hours, fasted for 4 hours and intraperitoneally injected with glucagon (100 µg/kg body weight). Blood glucose levels (mg/dL) were measured 0, 15, 30, 60 and 120 minutes after glucagon stimulation (GST). Glucose disposal rates were measured using the pyruvate tolerance test (PTT) (n = 10 mice/group).


Figure 2 ( D and E ) Growth comparisons of bir1 ∆ - ad2 strains that contain ( 18 strains ) or do not contain ( 28 strains ) confirmed suppressor mutations as measured by area of colony growth after serial dilution . The mean ( red line ) is shown . 10 - fold serial dilutions were made on YPAD plates containing either 0 . 1 % DMSO ( D ) or 10µg / mL of benomyl ( E ) .

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Figure 2(D and E) Growth comparisons of bir1∆-ad2 strains that contain (18 strains) or do not contain (28 strains) confirmed suppressor mutations as measured by area of colony growth after serial dilution. The mean (red line) is shown. 10-fold serial dilutions were made on YPAD plates containing either 0.1% DMSO (D) or 10µg/mL of benomyl (E).


Figure 3 ( A ) Serial dilutions on YPAD media at the permissive ( 25 ˚ C ) or restrictive ( 35 ˚ C ) temperatures for the ipl1 - 321 mutation . The suppressor mutations from the Dam1c ( blue ) were the only mutants that rescued viability at the restrictive temperature . ( B ) Top : representative images showing Bub3 - mNeonGreen localization to kinetochores ( Nuf2 - mRuby3 ) , indicating spindle assembly checkpoint activation in wild - type yeast . Scale bar is 2µm . Bottom : quantification of the proportion of prometaphase cells with Bub3 - mNeonGreen foci . Only the mutations in Dam1 complex ( blue ) show a significant increase in SAC activity . Data are from at least 5 independent experiments . Means and standard deviations are shown .

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Figure 3(A) Serial dilutions on YPAD media at the permissive (25˚C) or restrictive (35˚C) temperatures for the ipl1-321 mutation. The suppressor mutations from the Dam1c (blue) were the only mutants that rescued viability at the restrictive temperature.(B) Top: representative images showing Bub3-mNeonGreen localization to kinetochores (Nuf2-mRuby3), indicating spindle assembly checkpoint activation in wild-type yeast. Scale bar is 2µm. Bottom: quantification of the proportion of prometaphase cells with Bub3-mNeonGreen foci. Only the mutations in Dam1 complex (blue) show a significant increase in SAC activity. Data are from at least 5 independent experiments. Means and standard deviations are shown.


Figure 4 ( B ) Serial dilutions of strains engineered with the indicated mutations were tested for rescue of BIR1 deletion . 10 - fold serial dilutions on the indicated media are shown . dam1 ( S20D ) rescues loss of CPC activity to a similar extent as a suppressor mutant in Duo1 .

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Figure 4(B) Serial dilutions of strains engineered with the indicated mutations were tested for rescue of BIR1 deletion. 10-fold serial dilutions on the indicated media are shown. dam1(S20D) rescues loss of CPC activity to a similar extent as a suppressor mutant in Duo1.


Figure 5 ( B ) Left : representative images showing the localization of Dad3 - mNeonGreen ( Dam1c member ) and Nuf2 - mRuby3 ( kinetochore marker ) in prometaphase cells . Scale bar is 2µm . Right : quantification of the intensity of Dad3 - mNeonGreen at kinetochores / spindles in prometaphase cells . Each individual point on the graph represents a single measurement of Dad3 - mNeonGreen intensity ; up to 10 measurements per replicate . Data are from 5 independent experiments . ( C ) Left : representative images showing the localization of Dad3 - mNeonGreen ( Dam1c member ) and Nuf2 - mRuby3 ( kinetochore marker ) in anaphase cells . Scale bar is 2µm . Right : quantification of the intensity of Dad3 - mNeonGreen at the spindle in anaphase cells . The intensity was measured in the middle of the spindle using a perpendicular line - scan . Each individual point on the graph represents a single measurement of Dad3 - mNeonGreen intensity ; up to 10 measurements per replicate . Data are from 5 independent experiments . ( D ) Quantification of the intensity of Dad3 - mNeonGreen at kinetochores in anaphase cells . Kinetochore position was based on Nuf2 - mRuby3 localization . The differences in localization at the anaphase kinetochores are much weaker than those measured along the spindle ( Fig 5C ) . Each individual point on the graph represents a single measurement of Dad3 - mNeonGreen intensity ; up to 10 measurements per replicate . Data are from 5 independent experiments . The mean ( red line ) is shown .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(B) Left: representative images showing the localization of Dad3-mNeonGreen (Dam1c member) and Nuf2-mRuby3 (kinetochore marker) in prometaphase cells. Scale bar is 2µm. Right: quantification of the intensity of Dad3-mNeonGreen at kinetochores/spindles in prometaphase cells. Each individual point on the graph represents a single measurement of Dad3-mNeonGreen intensity; up to 10 measurements per replicate. Data are from 5 independent experiments.(C) Left: representative images showing the localization of Dad3-mNeonGreen (Dam1c member) and Nuf2-mRuby3 (kinetochore marker) in anaphase cells. Scale bar is 2µm. Right: quantification of the intensity of Dad3-mNeonGreen at the spindle in anaphase cells. The intensity was measured in the middle of the spindle using a perpendicular line-scan. Each individual point on the graph represents a single measurement of Dad3-mNeonGreen intensity; up to 10 measurements per replicate. Data are from 5 independent experiments.(D) Quantification of the intensity of Dad3-mNeonGreen at kinetochores in anaphase cells. Kinetochore position was based on Nuf2-mRuby3 localization. The differences in localization at the anaphase kinetochores are much weaker than those measured along the spindle (Fig 5C). Each individual point on the graph represents a single measurement of Dad3-mNeonGreen intensity; up to 10 measurements per replicate. Data are from 5 independent experiments. The mean (red line) is shown.


Figure 6 ( A ) Serial dilutions of strains engineered with the indicated mutations were tested for rescue of BIR1 deletion . 10 - fold serial dilutions on the indicated media are shown . The met30 - 6 and cdc4 - 1 alleles are temperature sensitive . Two independent clones of each of these alleles are shown . The serial dilutions were performed at the permissive temperature ( 20 ˚ C ) . ( B ) The amount of mNeonGreen - Sli15 ( CPC member ) located at kinetochores ( Nuf2 - mCherry ) was measured in prometaphase . Sgo1 was depleted by placing it under a galactose - inducible promoter and switching to glucose - containing media ( YPAD ) . Representative images are on the left . Scale bar is 2µm . Averages from 3 independent experiments are shown . ( C ) Measurements of Aurora B intensity at the inner centromere of prometaphase kinetochores . Inner centromeres were identified as the regions directly between two centromeres ( ACA staining ) . Images from an example WT cell are shown to the right . Scale bar is 5µm long . Each data point is an individual chromosome . More than 15 cells for each cell line were measured across two independent experiments . The mean ( red line ) is shown .

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Figure 6(A) Serial dilutions of strains engineered with the indicated mutations were tested for rescue of BIR1 deletion. 10-fold serial dilutions on the indicated media are shown. The met30-6 and cdc4-1 alleles are temperature sensitive. Two independent clones of each of these alleles are shown. The serial dilutions were performed at the permissive temperature (20˚C).(B) The amount of mNeonGreen-Sli15 (CPC member) located at kinetochores (Nuf2-mCherry) was measured in prometaphase. Sgo1 was depleted by placing it under a galactose-inducible promoter and switching to glucose-containing media (YPAD). Representative images are on the left. Scale bar is 2µm. Averages from 3 independent experiments are shown.(C) Measurements of Aurora B intensity at the inner centromere of prometaphase kinetochores. Inner centromeres were identified as the regions directly between two centromeres (ACA staining). Images from an example WT cell are shown to the right. Scale bar is 5µm long. Each data point is an individual chromosome. More than 15 cells for each cell line were measured across two independent experiments. The mean (red line) is shown.


Figure 1A CAFs isolation from human pancreatic tumour resections ( left panel ) and in vitro primo - culture ( right panel ) . B Isolated CAFs and PaSC characterization in vitro by immunofluorescence using anti - vimentin ( left panel ) or anti - α - SMA ( right panel ) antibody ( one representative field of n = 3 ) . D , E Panc - 1 cell viability was assessed by MTT . Results ( mean ± SD ) are presented for each treatment ( CM from PaSC , or from CAF , or from CAF ± SOM230 , SOM230 ) as a percentage of the respective gemcitabine - untreated cells ( = 100 % ) ( n = 4 ; from left to right : * * P = 0 . 007 , # # P = 0 . 006 in D ; * P = 0 . 032 , # P = 0 . 041 , # P = 0 . 047 in E ) . F Apoptosis induced by gemcitabine was evaluated by Western blot using an anti - cleaved caspase - 3 or anti - PARP antibody ( representative of n = 3 ) . G Panc - 1 was analysed by flow cytometry . Percentages ( mean ± SD ) of annexin V - positive cells are indicated ( n = 3 ; from left to right : * * P = 0 . 005 , # # P = 0 . 004 , * * P = 0 . 006 ) .

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Figure 1A CAFs isolation from human pancreatic tumour resections (left panel) and in vitro primo-culture (right panel).B Isolated CAFs and PaSC characterization in vitro by immunofluorescence using anti-vimentin (left panel) or anti-α-SMA (right panel) antibody (one representative field of n = 3).D, E Panc-1 cell viability was assessed by MTT. Results (mean ± SD) are presented for each treatment (CM from PaSC, or from CAF, or from CAF ± SOM230, SOM230) as a percentage of the respective gemcitabine-untreated cells (= 100%) (n = 4; from left to right: **P = 0.007, ##P = 0.006 in D; *P = 0.032, #P = 0.041, #P = 0.047 in E).F Apoptosis induced by gemcitabine was evaluated by Western blot using an anti-cleaved caspase-3 or anti-PARP antibody (representative of n = 3).G Panc-1 was analysed by flow cytometry. Percentages (mean ± SD) of annexin V-positive cells are indicated (n = 3; from left to right: **P = 0.005, ##P = 0.004, **P = 0.006).


Figure 2Immunoblotting of protein extracts from PaSCs or CAFs treated ( + ) or not with SOM230 ( 10 − 7 M ) for 48 h , using the anti - puromycin antibody ( representative of n = 3 ) . Polysomes profiles of CAFs treated or not with SOM230 for 48 h ( representative of n = 3 ) . Immunoblotting using an anti - P - Akt , anti - P - S6 or anti - β - actin ( loading control ) antibody of protein extracts from PaSCs or CAFs treated ( + ) with SOM230 for 30 min ( representative of n = 3 ) . Immunoblotting of protein extracts from CAFs treated ( + ) or not with SOM230 ( 10 − 7 M ) for the indicated times , using anti - P - Akt , anti - P - S6 or anti - 4E - BP1 antibody ( representative of n = 3 ) . Protein concentration in untreated or SOM230 - treated CAF - CM or extracts normalized per 1 × 106 cells . Results ( mean ± SD ) are presented as a percentage of the untreated CAFs ( = 100 % ) ( n = 3 ; from left to right : # # P = 0 . 008 , # # P = 0 . 007 ) . Immunoblotting using an anti - 4E - BP1 or anti - GAPDH ( loading control ) antibody of protein extracts from siCTR - or si4E - BP1 - transfected CAFs ( representative of n = 3 ) . Immunoblotting of equal amounts of protein from siCTR - or si4E - BP1 - transfected CAFs treated ( + ) or not with SOM230 for 48 h , using the anti - puromycin antibody ( representative of n = 3 ) . Panc - 1 cell viability was assessed by MTT . Panc - 1 cells were incubated with gemcitabine in the presence of CM from untreated or SOM230 - treated CAFs transfected with the siCTR or si4E - BP - 1 . Results ( mean ± SD ) are presented as a percentage of the untreated CAFs ( = 100 % ) ( n = 3 ; * * P = 0 . 003 , § § P = 0 . 002 ) . Caspase - 3 and PARP cleavage induced by gemcitabine in Panc - 1 was evaluated by Western blot using the respective antibodies ( representative of n = 3 ) . Arrow indicates cleaved forms of PARP .

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Figure 2Immunoblotting of protein extracts from PaSCs or CAFs treated (+) or not with SOM230 (10−7 M) for 48 h, using the anti-puromycin antibody (representative of n = 3).Polysomes profiles of CAFs treated or not with SOM230 for 48 h (representative of n = 3).Immunoblotting using an anti-P-Akt, anti-P-S6 or anti-β-actin (loading control) antibody of protein extracts from PaSCs or CAFs treated (+) with SOM230 for 30 min (representative of n = 3).Immunoblotting of protein extracts from CAFs treated (+) or not with SOM230 (10−7 M) for the indicated times, using anti-P-Akt, anti-P-S6 or anti-4E-BP1 antibody (representative of n = 3).Protein concentration in untreated or SOM230-treated CAF-CM or extracts normalized per 1 × 106 cells. Results (mean ± SD) are presented as a percentage of the untreated CAFs (= 100%) (n = 3; from left to right: ##P = 0.008, ##P = 0.007).Immunoblotting using an anti-4E-BP1 or anti-GAPDH (loading control) antibody of protein extracts from siCTR- or si4E-BP1-transfected CAFs (representative of n = 3).Immunoblotting of equal amounts of protein from siCTR- or si4E-BP1-transfected CAFs treated (+) or not with SOM230 for 48 h, using the anti-puromycin antibody (representative of n = 3).Panc-1 cell viability was assessed by MTT. Panc-1 cells were incubated with gemcitabine in the presence of CM from untreated or SOM230-treated CAFs transfected with the siCTR or si4E-BP-1. Results (mean ± SD) are presented as a percentage of the untreated CAFs (= 100%) (n = 3; **P = 0.003, §§P = 0.002).Caspase-3 and PARP cleavage induced by gemcitabine in Panc-1 was evaluated by Western blot using the respective antibodies (representative of n = 3). Arrow indicates cleaved forms of PARP.


Figure 3A Expression of the somatostatin receptor subtype 1 ( sst1 ) analysed by RT - qPCR in the human pancreatic endocrine cell line BON and in CAFs isolated from 11 different patients ( mean ± SD ) . B Immunoblotting of protein extracts from PaSCs and CAFs , using anti - sst1 , anti - α - SMA , anti - P - S6 or anti - GAPDH ( loading control ) antibody ( representative of n = 3 ) . C Immunoblotting of protein extracts from BON , CAFs and indicated pancreatic cancer cell lines , using anti - sst1 or anti - GAPDH ( loading control ) antibody ( representative of n = 3 ) . D Immunofluorescence confocal microscopy analyses of α - SMA and sst1 in CAFs . Co - localization of α - SMA and sst1 is shown ( merge ) ( one representative field of n = 3 ) ( scale bar = 100 μm ) . E - G Representative immunofluorescence confocal microscopy analyses of sst1 co - localization with cytokeratin - 19 ( n = 42 PDAC samples ) ( E ) , α - SMA ( n = 42 PDAC samples ) ( F ) or with P - 4E - BP1 ( n = 25 PDAC samples ) ( G ) in serial slides ( scale bar = 100 μm ) . Dashed white lines represent the limits between stroma and tumour epithelial glands . H , I Immunoblotting of protein extracts from siCTR - or sisst1 - transfected CAFs treated ( + ) or not with SOM230 for 48 h ( sst1 , puromycin and GAPDH as loading control , H ) or for 30 min ( P - Akt , P - S6 , 4E - BP1 and GAPDH as loading control , I ) ( representative of n = 3 ) . J Panc - 1 cell viability was assessed by MTT . Panc - 1 cells were incubated with gemcitabine in the presence of CM from untreated or SOM230 - treated CAFs transfected with siCTR or si - sst1 . Results ( mean ± SD ) are presented as the percentage of the untreated cells ( = 100 % ) ( n = 3 ; * * P = 0 . 003 , § § P = 0 . 002 ) .

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Figure 3A Expression of the somatostatin receptor subtype 1 (sst1) analysed by RT-qPCR in the human pancreatic endocrine cell line BON and in CAFs isolated from 11 different patients (mean ± SD).B Immunoblotting of protein extracts from PaSCs and CAFs, using anti-sst1, anti-α-SMA, anti-P-S6 or anti-GAPDH (loading control) antibody (representative of n = 3).C Immunoblotting of protein extracts from BON, CAFs and indicated pancreatic cancer cell lines, using anti-sst1 or anti-GAPDH (loading control) antibody (representative of n = 3).D Immunofluorescence confocal microscopy analyses of α-SMA and sst1 in CAFs. Co-localization of α-SMA and sst1 is shown (merge) (one representative field of n = 3) (scale bar = 100 μm).E-G Representative immunofluorescence confocal microscopy analyses of sst1 co-localization with cytokeratin-19 (n = 42 PDAC samples) (E), α-SMA (n = 42 PDAC samples) (F) or with P-4E-BP1 (n = 25 PDAC samples) (G) in serial slides (scale bar = 100 μm). Dashed white lines represent the limits between stroma and tumour epithelial glands.H, I Immunoblotting of protein extracts from siCTR- or sisst1-transfected CAFs treated (+) or not with SOM230 for 48 h (sst1, puromycin and GAPDH as loading control, H) or for 30 min (P-Akt, P-S6, 4E-BP1 and GAPDH as loading control, I) (representative of n = 3).J Panc-1 cell viability was assessed by MTT. Panc-1 cells were incubated with gemcitabine in the presence of CM from untreated or SOM230-treated CAFs transfected with siCTR or si-sst1. Results (mean ± SD) are presented as the percentage of the untreated cells (= 100%) (n = 3; **P = 0.003, §§P = 0.002).


Figure 4A - D Immunoblotting using an anti - P - Akt , anti - P - S6 , anti - 4EB - P1 or anti - GAPDH ( loading control ) antibody of protein extracts from CAFs pre - treated ( + ) or not overnight with PTX ( 100 ng / ml ) ( A ) , gallein ( 10 μM ) ( B ) , NSC87877 ( 10 μM ) ( C ) , Src inhibitor ( 5 μM ) ( D ) , treated ( + ) or not with SOM230 ( 10 − 7 M ) for 30 min ( representative of n = 3 ) . E , F Immunoblotting using anti - P - Akt , anti - P - S6 , anti - 4EB - P1 or anti - GAPDH ( loading control ) antibody of protein extracts from CAF siCTR - or siSHP2 - ( E ) or siPTPε - ( F ) transfected CAF treated or not with SOM230 for 30 min ( representative of n = 3 ) . G , H Immunoblotting using an anti - P - Akt , anti - P - S6 , anti - 4EB - P1 or anti - GAPDH ( loading control ) antibody of protein extracts from CAFs treated with the indicated molecules ( PDGF receptor inhibitor , 5 - 10 μM ; Jak1 / 2 inhibitor ruxolitinib , 5 or 10 μM ; EGFR inhibitor , 150 or 300 nM and recombinant PDGF , 5 μg / ml ) ( representative of n = 3 ) .

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Figure 4A-D Immunoblotting using an anti-P-Akt, anti-P-S6, anti-4EB-P1 or anti-GAPDH (loading control) antibody of protein extracts from CAFs pre-treated (+) or not overnight with PTX (100 ng/ml) (A), gallein (10 μM) (B), NSC87877 (10 μM) (C), Src inhibitor (5 μM) (D), treated (+) or not with SOM230 (10−7 M) for 30 min (representative of n = 3).E, F Immunoblotting using anti-P-Akt, anti-P-S6, anti-4EB-P1 or anti-GAPDH (loading control) antibody of protein extracts from CAF siCTR- or siSHP2- (E) or siPTPε- (F) transfected CAF treated or not with SOM230 for 30 min (representative of n = 3).G, H Immunoblotting using an anti-P-Akt, anti-P-S6, anti-4EB-P1 or anti-GAPDH (loading control) antibody of protein extracts from CAFs treated with the indicated molecules (PDGF receptor inhibitor, 5-10 μM; Jak1/2 inhibitor ruxolitinib, 5 or 10 μM; EGFR inhibitor, 150 or 300 nM and recombinant PDGF, 5 μg/ml) (representative of n = 3).


Figure 5A - C MIA PaCa - 2 - GLuc cells were injected with or without CAFs into the pancreas of nude mice . Mice were treated with each indicated treatment ( SOM230 - LAR at day 7 and gemcitabine at days 7 , 10 , 14 and 17 ) , and the plasmatic luciferase activity was measured ( mean ± SD ) at days 7 , 14 and 21 ( n = 5 ) ( P = 0 . 003 for gemcitabine + SOM230LAR versus untreated ) ( A ) . Tumours were excised , photographed ( scale bars are 1 cm ) ( B ) , weighted ( P = 0 . 0008 ) ( C ) and paraffin - embedded for immunohistofluorescence analyses using an anti - cleaved caspase - 3 antibody ( E ) . D Human tumours were subcutaneously xenografted in nude mice and tumour volumes ( mean ± SD ) measured ( at day 37 , P = 0 . 0009 for gemcitabine + SOM230LAR versus untreated and P = 0 . 021 for gemcitabine + SOM230LAR versus gemcitabine ) . Mice were treated with the indicated treatments ( SOM230 - LAR at days 3 and 31 , and gemcitabine at day 3 and twice a week thereafter ) . E , F Tumours excised from mice as in ( A - C ) were paraffin - embedded for immunohistofluorescence analyses using an anti - cleaved caspase - 3 antibody ( scale bar = 100 μm ) ( E ) . Cleaved caspase - 3 quantification was performed by counting the mean number of positive cells per field in five independent tumours ( mean ± SD ) ( F ) ( * P = 0 . 036 , * * P = 0 . 009 ) . G , H Tumours excised from mice as in ( D ) were paraffin - embedded for immunohistofluorescence analyses using an anti - cleaved caspase - 3 antibody ( scale bar = 100 μm ) ( G ) . Cleaved caspase - 3 quantification was performed by counting the mean number of positive cells per field in five independent tumours ( mean ± SD ) ( H ) ( * P = 0 . 027 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A-C MIA PaCa-2-GLuc cells were injected with or without CAFs into the pancreas of nude mice. Mice were treated with each indicated treatment (SOM230-LAR at day 7 and gemcitabine at days 7, 10, 14 and 17), and the plasmatic luciferase activity was measured (mean ± SD) at days 7, 14 and 21 (n = 5) (P = 0.003 for gemcitabine + SOM230LAR versus untreated) (A). Tumours were excised, photographed (scale bars are 1 cm) (B), weighted (P = 0.0008) (C) and paraffin-embedded for immunohistofluorescence analyses using an anti-cleaved caspase-3 antibody (E).D Human tumours were subcutaneously xenografted in nude mice and tumour volumes (mean ± SD) measured (at day 37, P = 0.0009 for gemcitabine + SOM230LAR versus untreated and P = 0.021 for gemcitabine + SOM230LAR versus gemcitabine). Mice were treated with the indicated treatments (SOM230-LAR at days 3 and 31, and gemcitabine at day 3 and twice a week thereafter).E, F Tumours excised from mice as in (A-C) were paraffin-embedded for immunohistofluorescence analyses using an anti-cleaved caspase-3 antibody (scale bar = 100 μm) (E). Cleaved caspase-3 quantification was performed by counting the mean number of positive cells per field in five independent tumours (mean ± SD) (F) (*P = 0.036, **P = 0.009).G, H Tumours excised from mice as in (D) were paraffin-embedded for immunohistofluorescence analyses using an anti-cleaved caspase-3 antibody (scale bar = 100 μm) (G). Cleaved caspase-3 quantification was performed by counting the mean number of positive cells per field in five independent tumours (mean ± SD) (H) (*P = 0.027).


Figure 6A Membrane antibody array assay using CM from SOM230 - treated or not CAFs ( representative of n = 3 ) . Controls are circled in blue dashed line and IL - 6 in red square . B , C Anti - IL - 6 ELISA assay ( mean ± SD ) using CM or protein extracts from SOM230 - treated or not CAF ( from left to right : § § § P = 0 . 0002 , § § § P = 0 . 0002 ) ( B ) , or CM from siCTR - or si4E - BP1 - transfected CAFs treated or not with SOM230 ( from left to right : * * * P = 0 . 0003 , $ $ P = 0 . 004 ) ( C ) ( n = 3 ) . B , C Anti - IL - 6 ELISA assay ( mean ± SD ) using CM or protein extracts from SOM230 - treated or not CAF ( from left to right : § § § P = 0 . 0002 , § § § P = 0 . 0002 ) ( B ) , or CM from siCTR - or si4E - BP1 - transfected CAFs treated or not with SOM230 ( from left to right : * * * P = 0 . 0003 , $ $ P = 0 . 004 ) ( C ) ( n = 3 ) . D Panc - 1 cell viability was assessed by MTT . Results ( mean ± SD ) are presented for each treatment as the percentage of the respective untreated cells ( = 100 % ) ( n = 3 ; from left to right : * * P = 0 . 004 , # # P = 0 . 001 , § P = 0 . 023 , § P = 0 . 019 ) . E , F Immunoblotting using an anti - cleaved caspase - 3 , anti - PARP or anti - GAPDH ( loading control ) antibody of protein extracts from Panc - 1 cells incubated with CAF - CM pre - incubated or not with an anti - IL - 6 neutralizing antibody ( E ) , or with human recombinant IL - 6 ( representative of n = 3 ) ( F ) . G , H Immunoblotting using an anti - survivin or anti - GAPDH ( loading control ) antibody of protein extracts from Panc - 1 cells stimulated or not with CAF - CM pre - incubated or not with anti - IL - 6 neutralizing antibody ( G ) , or with human recombinant IL - 6 ( representative of n = 3 ) ( H ) . I Immunoblotting using an anti - P - Akt , anti - P - S6 or anti - β - actin ( loading control ) antibody of protein extracts from SOM230 - treated or SOM230 - untreated CAF incubated or not with IL - 6 neutralizing antibody ( representative of n = 3 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A Membrane antibody array assay using CM from SOM230-treated or not CAFs (representative of n = 3). Controls are circled in blue dashed line and IL-6 in red square.B, C Anti-IL-6 ELISA assay (mean ± SD) using CM or protein extracts from SOM230-treated or not CAF (from left to right: §§§P = 0.0002, §§§P = 0.0002) (B), or CM from siCTR- or si4E-BP1-transfected CAFs treated or not with SOM230 (from left to right: ***P = 0.0003, $$P = 0.004) (C) (n = 3).B, C Anti-IL-6 ELISA assay (mean ± SD) using CM or protein extracts from SOM230-treated or not CAF (from left to right: §§§P = 0.0002, §§§P = 0.0002) (B), or CM from siCTR- or si4E-BP1-transfected CAFs treated or not with SOM230 (from left to right: ***P = 0.0003, $$P = 0.004) (C) (n = 3).D Panc-1 cell viability was assessed by MTT. Results (mean ± SD) are presented for each treatment as the percentage of the respective untreated cells (= 100%) (n = 3; from left to right: **P = 0.004, ##P = 0.001, §P = 0.023, §P = 0.019).E, F Immunoblotting using an anti-cleaved caspase-3, anti-PARP or anti-GAPDH (loading control) antibody of protein extracts from Panc-1 cells incubated with CAF-CM pre-incubated or not with an anti-IL-6 neutralizing antibody (E), or with human recombinant IL-6 (representative of n = 3) (F).G, H Immunoblotting using an anti-survivin or anti-GAPDH (loading control) antibody of protein extracts from Panc-1 cells stimulated or not with CAF-CM pre-incubated or not with anti-IL-6 neutralizing antibody (G), or with human recombinant IL-6 (representative of n = 3) (H).I Immunoblotting using an anti-P-Akt, anti-P-S6 or anti-β-actin (loading control) antibody of protein extracts from SOM230-treated or SOM230-untreated CAF incubated or not with IL-6 neutralizing antibody (representative of n = 3).


Figure 7A - C Immunohistofluorescence using an anti - α - SMA or anti - IL - 6 antibody in paraffin - embedded sections from MIA PaCa - 2 - Gluc and CAF cells orthotopic co - xenografted tumours from Fig5A ( A ) or from human tumour subcutaneously xenografted from Fig5D ( B ) in nude mice treated or not with gemcitabine + SOM230 ( representative of five different tumours ) . Representative co - localization of α - SMA with IL - 6 in human PDAC samples ( C ) ( n = 15 ) . Scale bar = 100 μm . D , E Plasma was collected from MIA PaCa - 2GLuc and CAFs ( * P = 0 . 034 , * * P = 0 . 007 ) ( D ) , or human pancreatic tumour ( * P = 0 . 028 , * * P = 0 . 004 ) ( E ) , xenografts in nude mice and human IL - 6 plasmatic concentrations ( mean ± SD ) were quantified by ELISA ( n = 5 mice / group ) . * treated versus untreated mice .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A-C Immunohistofluorescence using an anti-α-SMA or anti-IL-6 antibody in paraffin-embedded sections from MIA PaCa-2-Gluc and CAF cells orthotopic co-xenografted tumours from Fig5A (A) or from human tumour subcutaneously xenografted from Fig5D (B) in nude mice treated or not with gemcitabine + SOM230 (representative of five different tumours). Representative co-localization of α-SMA with IL-6 in human PDAC samples (C) (n = 15). Scale bar = 100 μm.D, E Plasma was collected from MIA PaCa-2GLuc and CAFs (*P = 0.034, **P = 0.007) (D), or human pancreatic tumour (*P = 0.028, **P = 0.004) (E), xenografts in nude mice and human IL-6 plasmatic concentrations (mean ± SD) were quantified by ELISA (n = 5 mice / group). * treated versus untreated mice.