Figure 5A Knockdown of IRE1α or XBP - 1 leads to a decrease in cell survival . LNCaP cells were transfected with siRNA targeting either IRE1α or XBP - 1 ( 5 nM ) and starved in 2 % CT - FCS medium for 3 days before cell viability was measured using the CCK - 8 assay . The graph is representative of one experiment in triplicate and was repeated three times with similar results . Error bars represent SD with * P = 6 . 6 × 10 − 5 and 4 . 5 × 10 − 5 for comparison between Ctrl and siRNA against IRE1α and XBP - 1 , respectively , using paired Student ' s t - test . B XBP - 1 rescues the growth defect of siIRE1α - transfected LNCaP cells . LNCaP cells were transfected with 5 nM of indicated siRNA using Lipofectamine RNAiMax reagent . One day after siRNA transfection , the cells were transfected with either vector control ( Empty ) or Flag - XBP - 1S ( XBP - 1S ) . Three days after transfection , cells were harvested for western blot analysis or cultured for three more days before being applied to cell proliferation assay using the CCK - 8 reagent . The data are representative of two experiments in triplicate . Error bars represent SE . * P = 0 . 02 , * * P = 8 . 54 × 10 − 7 using paired Student ' s t - test . C , D IRE1α and XBP - 1 knockdown inhibits clonogenic capacity of LNCaP cells . Control LN - Scr ( Scr ) , LN - shIRE1 ( shIRE1 ) , or LN - shXBP1 ( shXBP - 1 ) cells were cultured for 3 weeks . The colonies formed were stained with crystal violet and photographed . The extent of IRE1α and XBP - 1 knockdown was determined by Western blot analysis . The area covered by colonies was quantified using the Gene Tools software ( SynGene ) . The data are representative of three experiments in triplicate . Error bars represent SEM . * P = 4 . 38 × 10 − 7 and * * P = 3 . 39 × 10 − 20 using paired Student ' s t - test . E Growth analysis of xenograftedLNCaPtumors in nude mice . LNCaP cells expressing shRNA against IRE1α ( LN - shIRE1 ) , XBP1 ( LN - shXBP - 1 ) , or control shRNA ( LN - Scr ) were subcutaneously implanted into both flanks of male nude mice ( 6 mice per group ) . Tumor size was measured at the indicated time points . Representative pictures of the tumors at harvest are shown . Error bars indicate SEM . * P = 0 . 03 for shIRE1 at week 7 , P = 0 . 02 for shXBP - 1 at week 7 , * * P = 0 . 01 for both shIRE1 and shXBP - 1 at week 8 using unpaired Student ' s t - test . F PCNA immunostaining in tumors from animals bearing LN - shIRE1 , LN - shXBP - 1 , or LN - Scr tumors . Scale bars : 100 μm .

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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 5A Knockdown of IRE1α or XBP-1 leads to a decrease in cell survival. LNCaP cells were transfected with siRNA targeting either IRE1α or XBP-1 (5 nM) and starved in 2% CT-FCS medium for 3 days before cell viability was measured using the CCK-8 assay. The graph is representative of one experiment in triplicate and was repeated three times with similar results. Error bars represent SD with *P = 6.6 × 10−5 and 4.5 × 10−5 for comparison between Ctrl and siRNA against IRE1α and XBP-1, respectively, using paired Student's t-test.B XBP-1 rescues the growth defect of siIRE1α-transfected LNCaP cells. LNCaP cells were transfected with 5 nM of indicated siRNA using Lipofectamine RNAiMax reagent. One day after siRNA transfection, the cells were transfected with either vector control (Empty) or Flag-XBP-1S (XBP-1S). Three days after transfection, cells were harvested for western blot analysis or cultured for three more days before being applied to cell proliferation assay using the CCK-8 reagent. The data are representative of two experiments in triplicate. Error bars represent SE. *P = 0.02, **P = 8.54 × 10−7 using paired Student's t-test.C, D IRE1α and XBP-1 knockdown inhibits clonogenic capacity of LNCaP cells. Control LN-Scr (Scr), LN-shIRE1 (shIRE1), or LN-shXBP1 (shXBP-1) cells were cultured for 3 weeks. The colonies formed were stained with crystal violet and photographed. The extent of IRE1α and XBP-1 knockdown was determined by Western blot analysis. The area covered by colonies was quantified using the Gene Tools software (SynGene). The data are representative of three experiments in triplicate. Error bars represent SEM. *P = 4.38 × 10−7 and **P = 3.39 × 10−20 using paired Student's t-test.E Growth analysis of xenograftedLNCaPtumors in nude mice. LNCaP cells expressing shRNA against IRE1α (LN-shIRE1), XBP1 (LN-shXBP-1), or control shRNA (LN-Scr) were subcutaneously implanted into both flanks of male nude mice (6 mice per group). Tumor size was measured at the indicated time points. Representative pictures of the tumors at harvest are shown. Error bars indicate SEM. *P = 0.03 for shIRE1 at week 7, P = 0.02 for shXBP-1 at week 7, **P = 0.01 for both shIRE1 and shXBP-1 at week 8 using unpaired Student's t-test.F PCNA immunostaining in tumors from animals bearing LN-shIRE1, LN-shXBP-1, or LN-Scr tumors. Scale bars: 100 μm.


Figure 6LNCaP xenografts were grown in nude mice until palpable . Mice were then intraperitoneally injected with 0 . 5 mg / kg toyocamycin or saline ( Vehicle ) ( tumor numbers : n = 15 , orn = 10 , respectively ) twice weekly . Tumor sizes were measured weekly with calipers . Error bars indicate SEM . * P = 0 . 04 ; * * P = 0 . 0004 ; * * * P = 0 . 002 using unpaired Student ' s t - test . Same procedure was repeated on VCaP xenografts , with similar findings as in LNCaP xenografts ( tumor numbers : n = 9 , n = 11 ) . Error bars indicate SEM . * P = 0 . 02 and * * P = 0 . 0006 using unpaired Student ' s t - test . XBP - 1S mRNA levels in LNCaP xenografts from animals treated with toyocamycin or vehicle . Tumors were harvested , RNA - extracted , and qPCR performed . Error bars indicate SEM . * P = 0 . 04 for LNCaP and * * P = 0 . 02 for VCaP using unpaired Student ' s t - test . PCNA staining in tumors from animals treated with either toyocamycin or saline . Scale bar : 100 μm .

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 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

Figure 6LNCaP xenografts were grown in nude mice until palpable. Mice were then intraperitoneally injected with 0.5 mg/kg toyocamycin or saline (Vehicle) (tumor numbers:n = 15, orn = 10, respectively) twice weekly.Tumor sizes were measured weekly with calipers. Error bars indicate SEM. *P = 0.04; **P = 0.0004; ***P = 0.002 using unpaired Student's t-test.Same procedure was repeated on VCaP xenografts, with similar findings as in LNCaP xenografts (tumor numbers: n = 9, n = 11). Error bars indicate SEM. *P = 0.02 and **P = 0.0006 using unpaired Student's t-test.XBP-1S mRNA levels in LNCaP xenografts from animals treated with toyocamycin or vehicle. Tumors were harvested, RNA-extracted, and qPCR performed. Error bars indicate SEM. *P = 0.04 for LNCaP and **P = 0.02 for VCaP using unpaired Student's t-test.PCNA staining in tumors from animals treated with either toyocamycin or saline. Scale bar: 100 μm.


Figure 7XBP - 1S expression was determined by IHC in two different cohorts of human prostatectomy samples . Representative pictures of benign and tumor samples . Tissue microarrays ( Wang et al , 2010 ) containing 25 benign and 283 tumor samples were stained with a XBP - 1S specific antiserum and scored by a pathologist . The P - value indicates the difference between XBP - 1S staining ( strong and moderate ) in normal vs cancer cells using Mann - Whitney test . XBP - 1S expression was determined by IHC of a neoadjuvant hormone therapy ( NHT ) tissue microarray containing samples from hormone naïve ( untreated ) ( n = 25 ) , patients that received NHT for 1 - 6 months ( n = 33 ) , and patients that received NHT for 7 - 12 months ( n = 50 ) , as indicated . Representative images are shown . Scale bars : 100 μm . Quantitative presentation of the data from ( C ) . * P = 0 . 006 in unpaired Student ' s t - test .

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Figure 7XBP-1S expression was determined by IHC in two different cohorts of human prostatectomy samples.Representative pictures of benign and tumor samples.Tissue microarrays (Wang et al, 2010) containing 25 benign and 283 tumor samples were stained with a XBP-1S specific antiserum and scored by a pathologist. The P-value indicates the difference between XBP-1S staining (strong and moderate) in normal vs cancer cells using Mann-Whitney test.XBP-1S expression was determined by IHC of a neoadjuvant hormone therapy (NHT) tissue microarray containing samples from hormone naïve (untreated) (n = 25), patients that received NHT for 1-6 months (n = 33), and patients that received NHT for 7-12 months (n = 50), as indicated. Representative images are shown. Scale bars: 100 μm.Quantitative presentation of the data from (C). *P = 0.006 in unpaired Student's t-test.


Figure 12 - NBD - glucose uptake in murine pancreatic endothelial cells ( MS1 ) and primary human umbilical vein endothelial cells ( HUVEC ) treated for 2 h with VEGF - B167 ( B167 ) or VEGF - B186 ( B186 ) compared to control treated cells . Data presented as mean ± SEM from representative experiments performed in triplicates . Statistical evaluation using one - way ANOVA and Dunnett ´ s multiple comparisons test , p - value : * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ( compared to untreated control ) . Scale bar , 20 μm . Trans - cellular 2 - NBD - glucose transport kinetics in MS1 cells treated for 2 h with VEGF - B186 . Data presented as mean ± SEM from three individual experiments . Statistical evaluation using t - test , p - value : * * < 0 . 01 ( compared to untreated control ) . Cardiac glycogen content in 17 weeks old male Vegfb + / + ( n = 6 ) , Vegfb + / - ( n = 6 ) and Vegfb - / - mice ( n = 5 ) presented as mean ± SEM . Statistical evaluation using one - way ANOVA and Dunnett ´ s multiple comparisons test , p - value : * < 0 . 05 ( compared to Vegfb + / + ) . Glucose uptake and accumulation ( 18F - DG PET ) in hearts of HFD fed wild - type ( WT ) mice pre - treated for 1 week with 2H10 antibody ( n = 4 ) compared to control antibody treated mice ( n = 4 ) . Data presented as mean ± SEM . Statistical evaluation using two - way ANOVA , p - value : * * * * < 0 . 0001 . Linear regression analysis of the 18F - DG PET data in D assessing differences in initial ( T = 0 . 9 - 6 . 5 min ) glucose uptake rate . Data presented as mean curve slope ( m ) ± SEM . Statistical evaluation comparing slope differences using analysis of covariance ( ANCOVA ) , p - value : * < 0 . 05 .

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 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 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

Figure 12-NBD-glucose uptake in murine pancreatic endothelial cells (MS1) and primary human umbilical vein endothelial cells (HUVEC) treated for 2 h with VEGF-B167 (B167) or VEGF-B186 (B186) compared to control treated cells. Data presented as mean ± SEM from representative experiments performed in triplicates. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to untreated control). Scale bar, 20 μm.Trans-cellular 2-NBD-glucose transport kinetics in MS1 cells treated for 2 h with VEGF-B186. Data presented as mean ± SEM from three individual experiments. Statistical evaluation using t-test, p-value: ** <0.01 (compared to untreated control).Cardiac glycogen content in 17 weeks old male Vegfb+/+ (n=6), Vegfb+/- (n=6) and Vegfb-/- mice (n=5) presented as mean ± SEM. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05 (compared to Vegfb+/+).Glucose uptake and accumulation (18F-DG PET) in hearts of HFD fed wild-type (WT) mice pre-treated for 1 week with 2H10 antibody (n=4) compared to control antibody treated mice (n=4). Data presented as mean ± SEM. Statistical evaluation using two-way ANOVA, p-value: **** <0.0001.Linear regression analysis of the 18F-DG PET data in D assessing differences in initial (T=0.9-6.5 min) glucose uptake rate. Data presented as mean curve slope (m) ± SEM. Statistical evaluation comparing slope differences using analysis of covariance (ANCOVA), p-value: * <0.05.


Figure 2Relative mRNA expression of the Slc2a glucose transporter ( GLUT ) family members ( 1 - 13 ) in cardiac ECs derived from Vegfb + / + ( n = 4 ) and Vegfb - / - ( n = 4 ) mice . Data represent normalized PLIER values ( mean ± SEM ) from microarray analysis performed on n = 4 + 4 RNA samples ( one RNA sample / mouse ) . mRNA expression levels of the SLC2A transporter ( GLUT ) family members ( 1 - 14 ) in HUVECs . Data presented as mean ± StDev relative to RPL19 expression from 3 independent experiments . mRNA expression levels of VEGFB , SLC2A1 , NRP1 and FLT1 ( VEGFR1 ) in HUVECs . Data presented as mean ± StDev relative to RPL19 expression from 3 independent experiments . 2 - NBD - glucose uptake in response to 2 h treatment with VEGF - B186 ( B186 ) in HUVECs exhibiting siRNA - mediated knock - down of FLT1 , NRP1 , SCL2A1 , KDR ( VEGFR2 ) or control siRNA . Data presented as mean ± SEM from three individual experiments . Statistical evaluation using one - way ANOVA and Sidak ´ s multiple comparisons test , p - value : * < 0 . 05 ( comparisons made to respective untreated control ) . Representative images of GLUT1 ( left panels ) and CD31 ( right panels ) immunohistochemistry performed on consecutive sections from paraffin embedded mouse hearts . Scale bar , 100 μm . mRNA expression analysis from enriched cardiac endothelial cells ( open columns , n = 4 mice ) or whole heart ( closed columns , n = 3 mice ) . Data presented as mean ± StDev relative to Rpl19 expression . Enrichment factor is calculated as mean cardiac EC / mean whole heart and presented in the box .

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 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 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 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 2Relative mRNA expression of the Slc2a glucose transporter (GLUT) family members (1-13) in cardiac ECs derived from Vegfb+/+ (n=4) and Vegfb-/- (n=4) mice. Data represent normalized PLIER values (mean ± SEM) from microarray analysis performed on n=4+4 RNA samples (one RNA sample/mouse).mRNA expression levels of the SLC2A transporter (GLUT) family members (1-14) in HUVECs. Data presented as mean ± StDev relative to RPL19 expression from 3 independent experiments.mRNA expression levels of VEGFB, SLC2A1, NRP1 and FLT1 (VEGFR1) in HUVECs. Data presented as mean ± StDev relative to RPL19 expression from 3 independent experiments.2-NBD-glucose uptake in response to 2 h treatment with VEGF-B186 (B186) in HUVECs exhibiting siRNA-mediated knock-down of FLT1, NRP1, SCL2A1, KDR (VEGFR2) or control siRNA. Data presented as mean ± SEM from three individual experiments. Statistical evaluation using one-way ANOVA and Sidak´s multiple comparisons test, p-value: * <0.05 (comparisons made to respective untreated control).Representative images of GLUT1 (left panels) and CD31 (right panels) immunohistochemistry performed on consecutive sections from paraffin embedded mouse hearts. Scale bar, 100 μm.mRNA expression analysis from enriched cardiac endothelial cells (open columns, n=4 mice) or whole heart (closed columns, n= 3 mice). Data presented as mean ± StDev relative to Rpl19 expression. Enrichment factor is calculated as mean cardiac EC/mean whole heart and presented in the box.


Figure 3Immunofluorescence labeling of GLUT1 ( white ) , early endosomes ( EEA1 , green ) and nuclei ( blue ) in HUVECs treated with VEGF - B186 for 10 min , 30 min or 2 h . Scale bar , 10 µm . GLUT1 protein expression in HUVECs treated with VEGF - B186 for 15 min or 2 h . Data presented as mean ± StDev of 3 - 4 independent experiments relative to β - Actin expression . Statistical evaluation using t - test revealed no significant changes . Western blots shown in right panel . Sucrose gradient ( 15 - 50 % sucrose ) fractions of HUVEC cellular lysates after 2 h VEGF - B186 stimulation were analyzed with Western blots shown in Appendix Fig S2 . Different cellular sub - compartments were identified by accumulation of specific protein markers : EEA1 for early endosomes , LIMPII for late endosomes / lysosomes , flotillin for plasma membrane / lipid rafts and calnexin for endoplasmic reticulum ( ER ) ( left panels ) . Amount and sub - cellular distribution of VEGFR1 , NRP1 and GLUT1 protein in control or VEGF - B186 stimulated HUVECs shown in panels to the right . Data represent % protein relative to total protein of interest measured from a representative experiment . Quantification of GLUT1 protein in % relative to total measured protein in HUVECs after 2 h VEGF - B186 stimulation in different sucrose fractions ( upper panel ) . Data from two independent experiments are shown . Green arrows mark fractions with increased GLUT1 content and red arrows mark fractions with decreased GLUT1 content after VEGF - B186 stimulation . Changes in GLUT1 content in response to VEGF - B treatment are shown in the table ( bottom panel ) .

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 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 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 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

Figure 3Immunofluorescence labeling of GLUT1 (white), early endosomes (EEA1, green) and nuclei (blue) in HUVECs treated with VEGF-B186 for 10 min, 30 min or 2 h. Scale bar, 10 µm.GLUT1 protein expression in HUVECs treated with VEGF-B186 for 15 min or 2 h. Data presented as mean ± StDev of 3-4 independent experiments relative to β-Actin expression. Statistical evaluation using t-test revealed no significant changes. Western blots shown in right panel.Sucrose gradient (15-50% sucrose) fractions of HUVEC cellular lysates after 2 h VEGF-B186 stimulation were analyzed with Western blots shown in Appendix Fig S2. Different cellular sub-compartments were identified by accumulation of specific protein markers: EEA1 for early endosomes, LIMPII for late endosomes/lysosomes, flotillin for plasma membrane/lipid rafts and calnexin for endoplasmic reticulum (ER) (left panels). Amount and sub-cellular distribution of VEGFR1, NRP1 and GLUT1 protein in control or VEGF-B186 stimulated HUVECs shown in panels to the right. Data represent % protein relative to total protein of interest measured from a representative experiment.Quantification of GLUT1 protein in % relative to total measured protein in HUVECs after 2 h VEGF-B186 stimulation in different sucrose fractions (upper panel). Data from two independent experiments are shown. Green arrows mark fractions with increased GLUT1 content and red arrows mark fractions with decreased GLUT1 content after VEGF-B186 stimulation. Changes in GLUT1 content in response to VEGF-B treatment are shown in the table (bottom panel).


Figure 4Representative confocal ( grey ) and epifluorescence ( blue ) images of filipin staining in HUVECs in response to 2 h VEGF - B186 stimulation ( left panels ) . Cellular cholesterol content measured by quantification of fluorescent filipin staining . Data presented as mean ± SEM from a representative experiment performed in triplicates . Statistical evaluation using t - test , p - value : * < 0 . 05 . Scale bars , 10 µm . Measurement of cholesterol content ( B ) in HUVECs after VEGF - B186 stimulation for different time - points . Data presented as mean ± SEM from representative experiments performed in triplicates . Statistical evaluation using t - test , p - value : * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ( compared to respective control ) . Measurement of glucose uptake ( C ) and FA uptake ( D ) in HUVECs after VEGF - B186 stimulation for different time - points . Data presented as mean ± SEM from representative experiments performed in triplicates . Statistical evaluation using t - test , p - value : * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ( compared to respective control ) . Colorimetric quantification of non - esterified cholesterol extracted from HUVECs stimulated with VEGF - B186 for 20 min . Data are presented as mean ± SEM of three individual experiments . Statistical evaluation using t - test , p - value : * < 0 . 05 . Uptake of a fluorescent cholesterol tracer ( TopFluor ) in HUVECs after VEGF - B186 stimulation for 20 min or 2 h . Data presented as mean ± SEM from a representative experiment performed in triplicates . Statistical evaluation using one - way ANOVA and Dunnett ´ s multiple comparisons test , p - value : * * < 0 . 01 , * * * < 0 . 001 ( compared to respective untreated control ) . Quantification of the major cholesteryl ester ( CE ) species in HUVECs after VEGF - B186 stimulation for 20 min , analyzed by lipid mass spectrometry . Data presented as mean ± StDev of an experiment performed in triplicates .

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 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 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 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

Figure 4Representative confocal (grey) and epifluorescence (blue) images of filipin staining in HUVECs in response to 2 h VEGF-B186 stimulation (left panels). Cellular cholesterol content measured by quantification of fluorescent filipin staining. Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using t-test, p-value: * <0.05. Scale bars, 10 µm.Measurement of cholesterol content (B) in HUVECs after VEGF-B186 stimulation for different time-points. Data presented as mean ± SEM from representative experiments performed in triplicates. Statistical evaluation using t-test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to respective control).Measurement of glucose uptake (C) and FA uptake (D) in HUVECs after VEGF-B186 stimulation for different time-points. Data presented as mean ± SEM from representative experiments performed in triplicates. Statistical evaluation using t-test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to respective control).Colorimetric quantification of non-esterified cholesterol extracted from HUVECs stimulated with VEGF-B186 for 20 min. Data are presented as mean ± SEM of three individual experiments. Statistical evaluation using t-test, p-value: * <0.05.Uptake of a fluorescent cholesterol tracer (TopFluor) in HUVECs after VEGF-B186 stimulation for 20 min or 2 h. Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: ** <0.01, *** <0.001 (compared to respective untreated control).Quantification of the major cholesteryl ester (CE) species in HUVECs after VEGF-B186 stimulation for 20 min, analyzed by lipid mass spectrometry. Data presented as mean ± StDev of an experiment performed in triplicates.


Figure 5Measurement of cellular cholesterol content ( upper panel ) and glucose uptake ( lower panel ) in HUVECs treated with VEGF - B186 for 1 h or after 15 min treatment with cholesterol - extracting methyl - beta - cyclodextrin ( MbCD ) . Data presented as mean ± SEM from a representative experiment performed in triplicates . Statistical evaluation using one - way ANOVA and Dunnett ´ s multiple comparisons test , p - value : * * * < 0 . 001 ( compared to untreated control ) . Measurement of cellular cholesterol content ( upper panel ) and glucose uptake ( lower panel ) in HUVECs treated with VEGF - B186 for 15 min alone or supplemented with soluble methyl - beta - cyclodextrin complexed with cholesterol ( 5 , 10 or 20 μg / ml cholesterol ) . Data presented as mean ± SEM from a representative experiment performed in triplicates . Statistical evaluation using one - way ANOVA and Dunnett ´ s multiple comparisons test , p - value : * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ( compared to control w / o cholesterol ) .

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 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

Figure 5Measurement of cellular cholesterol content (upper panel) and glucose uptake (lower panel) in HUVECs treated with VEGF-B186 for 1 h or after 15 min treatment with cholesterol-extracting methyl-beta-cyclodextrin (MbCD). Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: *** <0.001 (compared to untreated control).Measurement of cellular cholesterol content (upper panel) and glucose uptake (lower panel) in HUVECs treated with VEGF-B186 for 15 min alone or supplemented with soluble methyl-beta-cyclodextrin complexed with cholesterol (5, 10 or 20 μg/ml cholesterol). Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to control w/o cholesterol).


Figure 6mRNA expression analysis of lipoprotein receptors in HUVECs . Data presented as mean ± StDev relative to RPL19 expression from 3 independent experiments . Cellular cholesterol content in HUVECs treated with control siRNA or siRNA targeting LDLR or Scavenger receptor B1 ( SCARB1 , SRBI ) followed by 15 min stimulation with VEGF - B186 . Data presented as mean ± SEM from representative experiments performed in triplicates . Statistical evaluation using one - way ANOVA and Dunnett ´ s multiple comparisons test , p - value : * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ( compared to respective control ) . glucose uptake ( C ) in HUVECs treated with control siRNA or siRNA targeting LDLR or Scavenger receptor B1 ( SCARB1 , SRBI ) followed by 15 min stimulation with VEGF - B186 . Data presented as mean ± SEM from representative experiments performed in triplicates . Statistical evaluation using one - way ANOVA and Dunnett ´ s multiple comparisons test , p - value : * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ( compared to respective control ) . Cellular cholesterol content of ECs freshly isolated from Ldlr + / + and Ldlr - / - mice stimulated for 15 min with VEGF - B186 . Data presented as mean ± SEM from a representative experiment performed in triplicates . Statistical evaluation using one - way ANOVA and Tukey ´ s multiple comparisons test , p - value : * * * < 0 . 001 ( compared to Ldlr + / + control ) . Cellular cholesterol content ( left panel ) and glucose uptake ( right panel ) of HUVECs treated with de novo cholesterol synthesis inhibitor simvastatin . Data presented as mean ± SEM from a representative experiment performed in triplicates . Statistical evaluation using one - way ANOVA and Tukey ´ s multiple comparisons test , p - value : * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ( compared to untreated control ) . p - value : # < 0 . 05 , # # # < 0 . 001 ( compared to simvastatin control ) . Cell surface biotinylated LDLR protein levels in HUVECs after 20 min of VEGF - B167 or VEGF - B186 stimulation . Data presented as mean ± SEM from three independent experiments . Western blot shown below . Statistical evaluation using t - test , p - value : * < 0 . 05 ( compared to control ) . Representative images of cell surface distribution of the LDLR by immunofluorescence staining of non - permeabilized cells ( white , left panels ) , Dil - LDL cell surface binding ( evaluated at 4 ° C , middle panels ) and Dil - LDL uptake ( evaluated at 37 ° C , right panels ) in HUVECs in response to VEGF - B186 stimulation for 10 min . Scale bar , 10 µm .

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 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 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 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 O O O O O O O O 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 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

Figure 6mRNA expression analysis of lipoprotein receptors in HUVECs. Data presented as mean ± StDev relative to RPL19 expression from 3 independent experiments.Cellular cholesterol content in HUVECs treated with control siRNA or siRNA targeting LDLR or Scavenger receptor B1 (SCARB1, SRBI) followed by 15 min stimulation with VEGF-B186. Data presented as mean ± SEM from representative experiments performed in triplicates. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to respective control).glucose uptake (C) in HUVECs treated with control siRNA or siRNA targeting LDLR or Scavenger receptor B1 (SCARB1, SRBI) followed by 15 min stimulation with VEGF-B186. Data presented as mean ± SEM from representative experiments performed in triplicates. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to respective control).Cellular cholesterol content of ECs freshly isolated from Ldlr+/+ and Ldlr-/- mice stimulated for 15 min with VEGF-B186. Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using one-way ANOVA and Tukey´s multiple comparisons test, p-value: *** <0.001 (compared to Ldlr+/+ control).Cellular cholesterol content (left panel) and glucose uptake (right panel) of HUVECs treated with de novo cholesterol synthesis inhibitor simvastatin. Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using one-way ANOVA and Tukey´s multiple comparisons test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to untreated control). p-value: # <0.05, ### <0.001 (compared to simvastatin control).Cell surface biotinylated LDLR protein levels in HUVECs after 20 min of VEGF-B167 or VEGF-B186 stimulation. Data presented as mean ± SEM from three independent experiments. Western blot shown below. Statistical evaluation using t-test, p-value: * <0.05 (compared to control).Representative images of cell surface distribution of the LDLR by immunofluorescence staining of non-permeabilized cells (white, left panels), Dil-LDL cell surface binding (evaluated at 4 °C, middle panels) and Dil-LDL uptake (evaluated at 37 °C, right panels) in HUVECs in response to VEGF-B186 stimulation for 10 min. Scale bar, 10 µm.


Figure 7Quantification of non - esterified cholesterol content by means of filipin staining in the blood vessel compartment ( podocalyxin + ) in hearts of 15 - week old male C57BL / 6 Vegfb + / - ( n = 5 ) and Vegfb - / - mice ( n = 7 ) , normalized to wildtype ( Vegfb + / + ) mice ( n = 8 ) . Data presented as box - and - whisker plots . Boxes represents lower / upper quartiles with the median values indicated with a horizontal line . Whiskers represent min - max values . Statistical evaluation using one - way ANOVA and Dunnett ´ s multiple comparisons test , p - value : * < 0 . 05 , * * < 0 . 01 ( compared to Vegfb + / + mice ) . Quantification of non - esterified cholesterol content by means of filipin staining in hearts of 15 - week old male C57BL / 6 Vegfb + / - ( n = 5 ) and Vegfb - / - ( n = 7 ) mice , normalized to wildtype ( Vegfb + / + ) mice ( n = 8 ) . Data presented as box - and - whisker plots . Boxes represents lower / upper quartiles with the median values indicated with a horizontal line . Whiskers represent min - max values . Statistical evaluation using one - way ANOVA and Dunnett ´ s multiple comparisons test , p - value : * * < 0 . 01 ( compared to Vegfb + / + mice ) . Quantification of non - esterified cholesterol content by means of filipin staining in hearts of 25 - week old male CD1 Flt1 + / lacz ( n = 20 ) mice , normalized to wildtype CD1 ( Flt1 + / + ) mice ( n = 16 ) . Data presented as box - and - whisker plots . Boxes represents lower / upper quartiles with the median values indicated with a horizontal line . Whiskers represent min - max values . Statistical evaluation using t - test , p - value : * * < 0 . 01 . Quantification of non - esterified cholesterol content by means of filipin staining in hearts of 12 to 16 - week old male C57BL / 6 wildtype ( Vegfb + / + ) ( n = 4 ) , BKS . C57BL / 6 db / + Vegfb + / + ( n = 8 ) , db / db Vegfb + / - ( n = 10 ) and db / db Vegfb - / - ( n = 4 ) mice , normalized to db / db Vegfb + / + ( n = 6 ) mice . Data presented as box - and - whisker plots . Boxes represents lower / upper quartiles with the median values indicated with a horizontal line . Whiskers represent min - max values . Statistical evaluation using one - way ANOVA and Dunnett ´ s multiple comparisons test , p - value : * < 0 . 05 , * * * < 0 . 001 ( compared to db / db Vegfb + / + mice ) . Left panel : Quantification of non - esterified cholesterol content by means of filipin staining in hearts of C57BL / 6 male mice after 25 weeks on HFD , long - term treated with isotype control ( n = 17 ) or VEGF - B blocking antibody ( 2H10 , n = 17 ) initiated after 5 weeks on HFD , normalized to age - matched mice on chow diet ( n = 10 ) . Data presented as box - and - whisker plots . Boxes represents lower / upper quartiles with the median values indicated with a horizontal line . Whiskers represent min - max values . Right panel : Body weights ( mean ± SEM ) of HFD ( n = 17 + 17 ) and chow fed littermate mice ( n = 10 ) . Statistical evaluation using one - way ANOVA and Dunnett ´ s multiple comparisons test , p - value : * < 0 . 05 , * * * < 0 . 001 ( compared to HFD control mice ) . Quantification of non - esterified cholesterol content by means of filipin staining in hearts of mice on HFD for 17 weeks followed by treatment for 1 week with 2H10 antibody ( n = 5 ) compared to control antibody treated mice ( n = 5 ) . Data presented as box - and - whisker plots . Boxes represents lower / upper quartiles with the median values indicated with a horizontal line . Whiskers represent min - max values . Statistical evaluation using t - test , p - value : * * < 0 . 01 .

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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O 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

Figure 7Quantification of non-esterified cholesterol content by means of filipin staining in the blood vessel compartment (podocalyxin+) in hearts of 15-week old male C57BL/6 Vegfb+/- (n=5) and Vegfb-/- mice (n=7), normalized to wildtype (Vegfb+/+) mice (n=8). Data presented as box-and-whisker plots. Boxes represents lower/upper quartiles with the median values indicated with a horizontal line. Whiskers represent min-max values. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, ** <0.01 (compared to Vegfb+/+ mice).Quantification of non-esterified cholesterol content by means of filipin staining in hearts of 15-week old male C57BL/6 Vegfb+/- (n=5) and Vegfb-/- (n=7) mice, normalized to wildtype (Vegfb+/+) mice (n=8). Data presented as box-and-whisker plots. Boxes represents lower/upper quartiles with the median values indicated with a horizontal line. Whiskers represent min-max values. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: ** <0.01 (compared to Vegfb+/+ mice).Quantification of non-esterified cholesterol content by means of filipin staining in hearts of 25-week old male CD1 Flt1+/lacz (n=20) mice, normalized to wildtype CD1 (Flt1+/+) mice (n=16). Data presented as box-and-whisker plots. Boxes represents lower/upper quartiles with the median values indicated with a horizontal line. Whiskers represent min-max values. Statistical evaluation using t-test, p-value: ** <0.01.Quantification of non-esterified cholesterol content by means of filipin staining in hearts of 12 to 16-week old male C57BL/6 wildtype (Vegfb+/+) (n=4), BKS.C57BL/6 db/+ Vegfb+/+ (n=8), db/db Vegfb+/- (n=10) and db/db Vegfb-/- (n=4) mice, normalized to db/db Vegfb+/+ (n=6) mice. Data presented as box-and-whisker plots. Boxes represents lower/upper quartiles with the median values indicated with a horizontal line. Whiskers represent min-max values. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, *** <0.001 (compared to db/db Vegfb+/+ mice).Left panel: Quantification of non-esterified cholesterol content by means of filipin staining in hearts of C57BL/6 male mice after 25 weeks on HFD, long-term treated with isotype control (n=17) or VEGF-B blocking antibody (2H10, n=17) initiated after 5 weeks on HFD, normalized to age-matched mice on chow diet (n=10). Data presented as box-and-whisker plots. Boxes represents lower/upper quartiles with the median values indicated with a horizontal line. Whiskers represent min-max values. Right panel: Body weights (mean ± SEM) of HFD (n=17+17) and chow fed littermate mice (n=10). Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, *** <0.001 (compared to HFD control mice).Quantification of non-esterified cholesterol content by means of filipin staining in hearts of mice on HFD for 17 weeks followed by treatment for 1 week with 2H10 antibody (n=5) compared to control antibody treated mice (n=5). Data presented as box-and-whisker plots. Boxes represents lower/upper quartiles with the median values indicated with a horizontal line. Whiskers represent min-max values.Statistical evaluation using t-test, p-value: ** <0.01.


Figure 1At 72 h after transfection with siRNA duplexes for BAG6 or control siRNA ( 10 nM each ) , the intracellular localization of TfnR in HeLa cells was examined ( shown as green ) . Nuclear DNA was stained with Hoechst 33342 ( shown as blue ) . Control knockdown ( left panel ) , Rab8a knockdown ( center panel ) , and BAG6 knockdown ( right panel ) . Intracellular localization of Ptc1 ( green ) in HeLa cells . Nuclei were stained with Hoechst 33342 ( shown as blue ) . Knockdown of Rab8a ( with Rab8a siRNA # 1 , # 2 , and # 3 ) stimulated the accumulation and stabilization of Ptc1 protein in HEK293 cells . Knockdown of BAG6 ( with BAG6 siRNA # 1 ) stimulated the accumulation and stabilization of Ptc1 protein in HEK293 cells .

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 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

Figure 1At 72 h after transfection with siRNA duplexes for BAG6 or control siRNA (10 nM each), the intracellular localization of TfnR in HeLa cells was examined (shown as green). Nuclear DNA was stained with Hoechst 33342 (shown as blue). Control knockdown (left panel), Rab8a knockdown (center panel), and BAG6 knockdown (right panel).Intracellular localization of Ptc1 (green) in HeLa cells. Nuclei were stained with Hoechst 33342 (shown as blue).Knockdown of Rab8a (with Rab8a siRNA#1, #2, and #3) stimulated the accumulation and stabilization of Ptc1 protein in HEK293 cells.Knockdown of BAG6 (with BAG6 siRNA#1) stimulated the accumulation and stabilization of Ptc1 protein in HEK293 cells.


Figure 2BAG6 protein co - precipitated Rab8a , while neither Rab7 nor Rab11a were co - precipitated with BAG6 . Flag - tagged Rab8a , Rab7 , Rab11a , and luciferase - CL1 ( Lc - CL1 ; a positive control ) were expressed in HeLa cells and the cells were treated with 10 μM MG - 132 for 4 h . Flag - immunoprecipitates were blotted with anti - BAG6 and anti - Flag antibodies , respectively . Note that all cells used were treated with 10 μM MG - 132 for 4 h . S - tagged BAG6 pull - down efficiently co - precipitated Rab8a ( T22N ) , a GDP - bound mutant , while BAG6 scarcely co - precipitated Rab8a ( Q67L ) , a constitutively active mutant . Co - precipitation of Rab8a WT with BAG6 was used as a standard . MG - 132 ( 10 μM ) was included in the cell culture for 4 h , as indicated . Note that the T22N mutant protein was expressed at lower levels than either WT or Q67L , and that this was partly due to increased degradation , as will be shown later . S - BAG6 stands for N - terminally S - tagged BAG6 proteinAnti - Flag signals in ( C ) were quantified , and relative signal intensities are presented . The value of the WT Rab8a signal with MG - 132 was defined as 1 . 0 . Note that all signal intensities of Flag - tag were normalized by that of the Rab8a input signals . A series of Flag - Rab8a mutants were immunoprecipitated and quantified the amount of endogenous BAG6 that were coprecipitated with Flag - Rab8a . Note that all cells used were treated with 10 μM MG - 132 for 4 h . Deficiency of Rabin8 , a GEF for Rab8a , enhanced the physical interaction between BAG6 and WT Rab8a proteins . Flag - tagged WT Rab8a was expressed in Rabin8 siRNA - treated cells , and Flag immunoprecipitates were probed with an anti - BAG6 antibody . Note that all cells used were treated with 10 μM MG - 132 for 4 h .

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Figure 2BAG6 protein co-precipitated Rab8a, while neither Rab7 nor Rab11a were co-precipitated with BAG6. Flag-tagged Rab8a, Rab7, Rab11a, and luciferase-CL1 (Lc-CL1; a positive control) were expressed in HeLa cells and the cells were treated with 10 μM MG-132 for 4 h. Flag-immunoprecipitates were blotted with anti-BAG6 and anti-Flag antibodies, respectively. Note that all cells used were treated with 10 μM MG-132 for 4 h.S-tagged BAG6 pull-down efficiently co-precipitated Rab8a (T22N), a GDP-bound mutant, while BAG6 scarcely co-precipitated Rab8a (Q67L), a constitutively active mutant. Co-precipitation of Rab8a WT with BAG6 was used as a standard. MG-132 (10 μM) was included in the cell culture for 4 h, as indicated. Note that the T22N mutant protein was expressed at lower levels than either WT or Q67L, and that this was partly due to increased degradation, as will be shown later. S-BAG6 stands for N-terminally S-tagged BAG6 proteinAnti-Flag signals in (C) were quantified, and relative signal intensities are presented. The value of the WT Rab8a signal with MG-132 was defined as 1.0. Note that all signal intensities of Flag-tag were normalized by that of the Rab8a input signals.A series of Flag-Rab8a mutants were immunoprecipitated and quantified the amount of endogenous BAG6 that were coprecipitated with Flag-Rab8a. Note that all cells used were treated with 10 μM MG-132 for 4 h.Deficiency of Rabin8, a GEF for Rab8a, enhanced the physical interaction between BAG6 and WT Rab8a proteins. Flag-tagged WT Rab8a was expressed in Rabin8 siRNA-treated cells, and Flag immunoprecipitates were probed with an anti-BAG6 antibody. Note that all cells used were treated with 10 μM MG-132 for 4 h.


Figure 3C - terminal geranylgeranylation of Rab8a was dispensable for BAG6 recognition . The N - terminal GTPase domain of Rab8a was essential for BAG6 recognition . A series of Flag - tagged truncated fragments of WT Rab8a were expressed in HeLa cells with S - tagged BAG6 and treated with ( + ) or without ( - ) protease inhibitors for 4 h . Flag - Rab8a substrates were immunoprecipitated and probed with an anti - BAG6 antibody . Hydrophobicity recognition domain of BAG6 ( N465 ) was critical for Rab8a recognition . A schematic representation of the BAG6 - truncated proteins used in this experiment is shown in the upper panel . Numbers denote the corresponding amino acids of mammalian BAG6 . Positions of UBL , BUILD , and DUF3538 domains , which are all linked to hydrophobicity recognition by BAG6 [ 37 ] , are indicated . WT , wild - type ; ∆ N465 , N - terminal 465 residues - deleted mutant ; and Ν465 , C - terminal 689 residues - deleted mutant . CHX chase experiments show that GDP - bound Rab8a ( T22N ) was a highly labile protein , while the GTP - bound active mutant ( Q67L ) was a stable protein ( E ) . Instability of the T22N mutant was not perturbed by the C204S mutation ( F ) . Actin was used as a loading control . N - terminal 100 residue fragment of the Rab8a GTPase domain was sensitive to the proteasome inhibitor , while the ∆ N100 fragment was not . Rab8a ( T22N ) was polyubiquitinated in the presence of the proteasome inhibitor . Asterisks indicate non - specific bands .

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Figure 3C-terminal geranylgeranylation of Rab8a was dispensable for BAG6 recognition.The N-terminal GTPase domain of Rab8a was essential for BAG6 recognition. A series of Flag-tagged truncated fragments of WT Rab8a were expressed in HeLa cells with S-tagged BAG6 and treated with (+) or without (-) protease inhibitors for 4 h. Flag-Rab8a substrates were immunoprecipitated and probed with an anti-BAG6 antibody.Hydrophobicity recognition domain of BAG6 (N465) was critical for Rab8a recognition. A schematic representation of the BAG6-truncated proteins used in this experiment is shown in the upper panel. Numbers denote the corresponding amino acids of mammalian BAG6. Positions of UBL, BUILD, and DUF3538 domains, which are all linked to hydrophobicity recognition by BAG6 [37], are indicated. WT, wild-type; ∆N465, N-terminal 465 residues-deleted mutant; and Ν465, C-terminal 689 residues-deleted mutant.CHX chase experiments show that GDP-bound Rab8a (T22N) was a highly labile protein, while the GTP-bound active mutant (Q67L) was a stable protein (E). Instability of the T22N mutant was not perturbed by the C204S mutation (F). Actin was used as a loading control.N-terminal 100 residue fragment of the Rab8a GTPase domain was sensitive to the proteasome inhibitor, while the ∆N100 fragment was not.Rab8a (T22N) was polyubiquitinated in the presence of the proteasome inhibitor. Asterisks indicate non-specific bands.


Figure 4Kyte - Doolittle hydrophobicity plots of the complete amino acid sequence of human WT Rab8a and T22N - 3IS mutant . The hydrophobicity peak within WT Switch I was abolished in the 3IS mutant ( indicated within the red box ) . The numbers on the horizontal axis denote the corresponding amino acid positions in these proteins . Substitution of the hydrophobic residues of Switch I with hydrophilic residues ( T22N 3IS ) abolished its binding to BAG6 protein . Rab8a ( T22N - 3IS ) mutant protein was highly stable , while Rab8a ( T22N ) protein was quite unstable in HeLa cells . Actin was used as a loading control . Rab8a ( T22N - 3IS ) mutant was not subject to polyubiquitin co - precipitation , while Rab8a ( T22N ) protein was .

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Figure 4Kyte-Doolittle hydrophobicity plots of the complete amino acid sequence of human WT Rab8a and T22N-3IS mutant. The hydrophobicity peak within WT Switch I was abolished in the 3IS mutant (indicated within the red box). The numbers on the horizontal axis denote the corresponding amino acid positions in these proteins.Substitution of the hydrophobic residues of Switch I with hydrophilic residues (T22N 3IS) abolished its binding to BAG6 protein.Rab8a (T22N-3IS) mutant protein was highly stable, while Rab8a (T22N) protein was quite unstable in HeLa cells. Actin was used as a loading control.Rab8a (T22N-3IS) mutant was not subject to polyubiquitin co-precipitation, while Rab8a (T22N) protein was.


Figure 5Rab8a ( T22N ) protein accumulated in BAG6 - knockdown cells . HeLa cells were transfected with siRNA duplexes for BAG6 or control siRNA . At 48 h after siRNA transfection , Flag tagged - Rab8a ( T22N ) was expressed in the cells . At 24 h after Rab8a ( T22N ) transfection , the cells were chased with 50 μg / mL CHX and harvested at the indicated time after CHX addition . Actin was used as a loading control . Anti - Flag blot signals in the control or BAG6 siRNA - treated cells were quantified , and relative signal intensities after CHX addition were calculated . The value of the Flag signal at 0 h was defined as 1 . 0 . Note that all signal intensities of the Flag - tag were normalized by that of actin , a loading control , in each sample . Polyubiquitin modification of Rab8a was abolished in BAG6 - knockdown cells . Flag - Rab8a ( T22N ) immunoprecipitates were blotted with an anti - polyubiquitin antibody ( FK2 , left panel ) . As a negative control , siRNA # 1scr was used . Anti - polyubiquitin signals co - precipitated with Rab8a ( T22N ) ( a representative example is shown in the left panel ) were quantified ( right panel ) . Note that the intensities of the co - precipitated polyubiquitin signal were normalized both by the input ubiquitin - signal and bait Flag - signal . Defective distribution of the endosomal protein TfnR in HeLa cells with the excess accumulation of the inactive form of Rab8a . Right panel shows a merged image of TfnR staining ( shown as green ) , Rab8a ( T22N ) staining ( magenta ) , and Hoechst 33342 nuclear staining ( blue ) .

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Figure 5Rab8a (T22N) protein accumulated in BAG6-knockdown cells. HeLa cells were transfected with siRNA duplexes for BAG6 or control siRNA. At 48 h after siRNA transfection, Flag tagged-Rab8a (T22N) was expressed in the cells. At 24 h after Rab8a (T22N) transfection, the cells were chased with 50 μg/mL CHX and harvested at the indicated time after CHX addition. Actin was used as a loading control.Anti-Flag blot signals in the control or BAG6 siRNA-treated cells were quantified, and relative signal intensities after CHX addition were calculated. The value of the Flag signal at 0 h was defined as 1.0. Note that all signal intensities of the Flag-tag were normalized by that of actin, a loading control, in each sample.Polyubiquitin modification of Rab8a was abolished in BAG6-knockdown cells. Flag-Rab8a (T22N) immunoprecipitates were blotted with an anti-polyubiquitin antibody (FK2, left panel). As a negative control, siRNA#1scr was used. Anti-polyubiquitin signals co-precipitated with Rab8a (T22N) (a representative example is shown in the left panel) were quantified (right panel). Note that the intensities of the co-precipitated polyubiquitin signal were normalized both by the input ubiquitin-signal and bait Flag-signal.Defective distribution of the endosomal protein TfnR in HeLa cells with the excess accumulation of the inactive form of Rab8a. Right panel shows a merged image of TfnR staining (shown as green), Rab8a (T22N) staining (magenta), and Hoechst 33342 nuclear staining (blue).


Figure 6A series of major Rab family proteins were immunoprecipitated from HeLa cell extracts and then probed with an anti - BAG6 antibody . Co - precipitation of Rab8a ( T22N ) with BAG6 was used as a positive control for this experiment . Quantification of BAG6 co - precipitation with Rab family proteins . The graph shows the quantities of BAG6 protein in the respective anti - Flag precipitates that were normalized by the amount of Flag - Rab bait proteins . The value of BAG6 co - precipitation with Rab8a ( T22N ) was defined as 1 . 0 . The quantities of the respective immunosignals were also normalized to the actin signal of each sample , and represent the median calculated from at least 3 independent biological replicates . The number of independent biological replicates was as follows : Rab1a , n = 4 ; Rab1b , n = 5 ; Rab2b , n = 3 ; Rab3a , n = 4 ; Rab4 , n = 4 ; Rab5a , n = 3 ; Rab5b , n = 4 ; Rab6a , n = 5 ; Rab6b , n = 4 ; Rab7a , n = 3 ; Rab8a , n = 14 ; Rab9b , n = 4 ; Rab10 , n = 6 ; Rab11a , n = 3 ; Rab11b , n = 3 ; Rab12 , n = 2 ; Rab13 , n = 6 ; Rab14 , n = 3 ; Rab15 , n = 5 ; Rab35 , n = 3 .

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Figure 6A series of major Rab family proteins were immunoprecipitated from HeLa cell extracts and then probed with an anti-BAG6 antibody. Co-precipitation of Rab8a (T22N) with BAG6 was used as a positive control for this experiment.Quantification of BAG6 co-precipitation with Rab family proteins. The graph shows the quantities of BAG6 protein in the respective anti-Flag precipitates that were normalized by the amount of Flag-Rab bait proteins. The value of BAG6 co-precipitation with Rab8a (T22N) was defined as 1.0. The quantities of the respective immunosignals were also normalized to the actin signal of each sample, and represent the median calculated from at least 3 independent biological replicates. The number of independent biological replicates was as follows: Rab1a, n = 4; Rab1b, n = 5; Rab2b, n = 3; Rab3a, n = 4; Rab4, n = 4; Rab5a, n = 3; Rab5b, n = 4; Rab6a, n = 5; Rab6b, n = 4; Rab7a, n = 3; Rab8a, n = 14; Rab9b, n = 4; Rab10, n = 6; Rab11a, n = 3; Rab11b, n = 3; Rab12, n = 2; Rab13, n = 6; Rab14, n = 3; Rab15, n = 5; Rab35, n = 3.


Figure 7BAG6 knockdown induced the abnormal distribution of Golgi apparatus markers . Representative images of the trans - Golgi membrane protein Stx6 ( green ) in BAG6 - suppressed CHO cells with a Chinese hamster - specific siRNA ( cBAG6 siRNA # 2 ) . Scale bar : 10 μm ( A ) . Fluorescent signals were detected using a laser scanning confocal microscopy system . Nuclei were stained by Hoechst ( blue ) . BAG6 knockdown induced the abnormal distribution of Golgi apparatus markers . Images of the cis - Golgi membrane protein GS28 and the cis - Golgi matrix protein GM130 in BAG6 - suppressed CHO cells with another Chinese hamster - specific siRNA ( cBAG6 siRNA # 5 ) . GS28 ( green ) and GM130 ( red ) are indicated by arrowheads . Scale bar : 10 μm . ( B ) . Fluorescent signals were detected using a laser scanning confocal microscopy system . Nuclei were stained by Hoechst ( blue ) . Glycosylation of the IL - 2Rα transmembrane protein was not reduced by BAG6 knockdown . Flag - tagged WT IL - 2Rα protein was expressed in HeLa cells with ( + ) or without ( - ) BAG6 siRNA , and was immunoprecipitated with an anti - Flag antibody . The precipitates were incubated with ( + ) or without ( - ) 10 unit of the deglycosylation enzyme PNGase F , and subjected to Western blot analysis with an anti - Flag antibody . Low - mobility ( indicated as glycosylated ) and high mobility ( indicated as non - glycosylated ) signals of WT IL - 2Rα are indicated . Defects in the distribution of cell surface glycoproteins in BAG6 - suppressed cells . The graph quantitatively displays the number of fluorescence counts per cell as the mean ± S . D . calculated from 10 independent biological replicates ( E ) . Defects in the distribution of cell surface glycoproteins in BAG6 - suppressed cells . Lectin GS - II - derived cell surface signals were counted using ImageJ software . SRP54 knockdown was used as a positive control for this experiment t - test ) . Cell surface fluorescent signals were detected by confocal microscopy without plasma membrane permeabilization . BAG6 siRNA down - regulated the cell surface expression of glycoproteins ( F ) .

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Figure 7BAG6 knockdown induced the abnormal distribution of Golgi apparatus markers. Representative images of the trans-Golgi membrane protein Stx6 (green) in BAG6-suppressed CHO cells with a Chinese hamster-specific siRNA (cBAG6 siRNA#2). Scale bar: 10 μm (A). Fluorescent signals were detected using a laser scanning confocal microscopy system. Nuclei were stained by Hoechst (blue).BAG6 knockdown induced the abnormal distribution of Golgi apparatus markers. Images of the cis-Golgi membrane protein GS28 and the cis-Golgi matrix protein GM130 in BAG6-suppressed CHO cells with another Chinese hamster-specific siRNA (cBAG6 siRNA#5). GS28 (green) and GM130 (red) are indicated by arrowheads. Scale bar: 10 μm. (B). Fluorescent signals were detected using a laser scanning confocal microscopy system. Nuclei were stained by Hoechst (blue).Glycosylation of the IL-2Rα transmembrane protein was not reduced by BAG6 knockdown. Flag-tagged WT IL-2Rα protein was expressed in HeLa cells with (+) or without (-) BAG6 siRNA, and was immunoprecipitated with an anti-Flag antibody. The precipitates were incubated with (+) or without (-) 10 unit of the deglycosylation enzyme PNGase F, and subjected to Western blot analysis with an anti-Flag antibody. Low-mobility (indicated as glycosylated) and high mobility (indicated as non-glycosylated) signals of WT IL-2Rα are indicated.Defects in the distribution of cell surface glycoproteins in BAG6-suppressed cells. The graph quantitatively displays the number of fluorescence counts per cell as the mean ± S.D. calculated from 10 independent biological replicates (E).Defects in the distribution of cell surface glycoproteins in BAG6-suppressed cells. Lectin GS-II-derived cell surface signals were counted using ImageJ software. SRP54 knockdown was used as a positive control for this experiment t-test). Cell surface fluorescent signals were detected by confocal microscopy without plasma membrane permeabilization. BAG6 siRNA down-regulated the cell surface expression of glycoproteins (F).


Figure 1C Immunostaining for the naive pluripotency marker KLF17 and the shared pluripotency marker OCT4 . Scale bar : 50μm . Data information : Representative images of n = 3 biological replicates are shown . D Gene - expression analysis by qPCR of KiPS primed - iPSCs , KiPS - chemical resetting at day 14 , and KiPS naive - iPSCs . Data information : Bars indicate the mean ± SEM of n = 3 biological replicates shown as dots . ( D ) Expression was normalised to the mean of HPD06 naive - iPSCs samples . I Barplots showing the absolute expression ( CPM ) of twelve trophoblast genes found to be significantly UP - regulated in KiPS - chemical resetting vs KiPS primed - iPSCs comparison . Data information : Bars indicate the mean ± SEM of n = 3 biological replicates shown as dots . * * * P < 0 . 001 , * * * * P < 0 . 0001 , ns no significance ( Student ' s t - test ) .

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Figure 1C Immunostaining for the naive pluripotency marker KLF17 and the shared pluripotency marker OCT4. Scale bar: 50μm. Data information: Representative images of n=3 biological replicates are shown.D Gene-expression analysis by qPCR of KiPS primed-iPSCs, KiPS-chemical resetting at day 14, and KiPS naive-iPSCs. Data information: Bars indicate the mean ± SEM of n=3 biological replicates shown as dots. (D) Expression was normalised to the mean of HPD06 naive-iPSCs samples.I Barplots showing the absolute expression (CPM) of twelve trophoblast genes found to be significantly UP-regulated in KiPS-chemical resetting vs KiPS primed-iPSCs comparison. Data information: Bars indicate the mean ± SEM of n=3 biological replicates shown as dots. ***P < 0.001, ****P < 0.0001, ns no significance (Student's t-test).


Figure 2B Gene expression analysis by RT - qPCR for naive ( KLF17 , TFCP2L1 , and NANOG ) and trophoblast ( GATA3 , GATA2 , KRT7 , ELF5 , TP63 , and TFAP2A ) marker genes in naive - iPSCs and TSC cells derived from two different naive - iPSC lines ( HPD06 and H9 ) . I Gene - expression analysis by qPCR of KiPS primed - iPSCs , KiPS in PXGL medium at day 14 , KiPS in TSC medium at day 14 , H9 naive - TSCs on ColIV , H9 naive - TSCs on MEF and HPD06 naive - iPSCs . Data information : ( I ) Dots indicate biological replicates and bars indicate the mean ± SEM of n = 3 biological replicates . Top : naive , primed , and general pluripotency markers , expression was normalised to HPD06 naive iPSCs ; bottom : trophoblast markers , expression was normalised to H9 naive - TSCs . J Immunostaining for the pluripotency markers OCT4 and KLF17 , and the TSC markers GATA3 and KRT7 of HPD06 naive - iPSC and ccTSC . 01 cultured on MEF and on collagen IV ( ColIV ) after 3 passages . Scale bars : 30 μm . Data information : Representative images of n = 2 biological replicates are shown .

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Figure 2B Gene expression analysis by RT-qPCR for naive (KLF17, TFCP2L1, and NANOG) and trophoblast (GATA3, GATA2, KRT7, ELF5, TP63, and TFAP2A) marker genes in naive-iPSCs and TSC cells derived from two different naive-iPSC lines (HPD06 and H9).I Gene-expression analysis by qPCR of KiPS primed-iPSCs, KiPS in PXGL medium at day 14, KiPS in TSC medium at day 14, H9 naive-TSCs on ColIV, H9 naive-TSCs on MEF and HPD06 naive-iPSCs. Data information: (I) Dots indicate biological replicates and bars indicate the mean ± SEM of n=3 biological replicates. Top: naive, primed, and general pluripotency markers, expression was normalised to HPD06 naive iPSCs; bottom: trophoblast markers, expression was normalised to H9 naive-TSCs.J Immunostaining for the pluripotency markers OCT4 and KLF17, and the TSC markers GATA3 and KRT7 of HPD06 naive-iPSC and ccTSC.01 cultured on MEF and on collagen IV (ColIV) after 3 passages. Scale bars: 30 μm. Data information: Representative images of n=2 biological replicates are shown.


Figure 3B Immunostaining for the pluripotency markers OCT4 , and the TSC markers GATA3 and KRT7 of KiPS primed - iPSCs , HPD06 naive - iPSCs , ccTSC . 02 at passage 4 , ccTSC . 01 at passage 4 and HPD06 naive - TSCs . Scale bars : 30 μm . Data information : ( B ) Representative images of n = 3 biological replicates are shown . F Barplots showing the absolute expression as log ( CPM + 1 ) of ADAP2 , HAVCR1 , SLC28A3 , ARID5B , and GATA2 in the reported conditions . Data information : ( F ) Bars indicate the mean ± SEM of at least n = 5 biological replicates from at least 2 independent studies shown with different symbols . * * * P < 0 . 001 , ns no significance ( Student ' s t - test ) .

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Figure 3B Immunostaining for the pluripotency markers OCT4, and the TSC markers GATA3 and KRT7 of KiPS primed-iPSCs, HPD06 naive-iPSCs, ccTSC.02 at passage 4, ccTSC.01 at passage 4 and HPD06 naive-TSCs. Scale bars: 30 μm. Data information: (B) Representative images of n=3 biological replicates are shown.F Barplots showing the absolute expression as log(CPM+1) of ADAP2, HAVCR1, SLC28A3, ARID5B, and GATA2 in the reported conditions. Data information: (F) Bars indicate the mean ± SEM of at least n=5 biological replicates from at least 2 independent studies shown with different symbols. ***P < 0.001, ns no significance (Student's t-test).


Figure 4A Gene - expression analysis by qPCR of syncytiotrophoblast ( left ) and extravillous ( right ) markers . Data information : ( A ) Dots indicate biological replicates . Left : expression was normalised to HPD06 naive - STBs and ccTSC . 02 - STBs ; Right : expression was normalised to HPD06 naive - EVTs and ccTSC . 02 - EVTs . B Top : Immunostaining for the TSC marker KRT7 and the STB marker SDC1 of ccTSC . 02 - STBs , HPD06 naive - STBs , ccTSC . 02 , and HPD06 naive - TSCs . Scale bars : 50 μm . Bottom : Immunostaining for the TSC marker KRT7 and the EVT marker HLA - G of ccTSC . 02 - EVTs , HPD06 naive - EVTs , ccTSC . 02 , and HPD06 naive - TSCs . Scale bars : 25 μm . Data information : ( B ) Representative images of n = 2 biological replicates are shown .

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 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

Figure 4A Gene-expression analysis by qPCR of syncytiotrophoblast (left) and extravillous (right) markers. Data information: (A) Dots indicate biological replicates. Left: expression was normalised to HPD06 naive-STBs and ccTSC.02-STBs; Right: expression was normalised to HPD06 naive-EVTs and ccTSC.02-EVTs.B Top: Immunostaining for the TSC marker KRT7 and the STB marker SDC1 of ccTSC.02-STBs, HPD06 naive-STBs, ccTSC.02, and HPD06 naive-TSCs. Scale bars: 50 μm. Bottom: Immunostaining for the TSC marker KRT7 and the EVT marker HLA-G of ccTSC.02-EVTs, HPD06 naive-EVTs, ccTSC.02, and HPD06 naive-TSCs. Scale bars: 25 μm. Data information: (B) Representative images of n=2 biological replicates are shown.


Figure 5D Barplots showing the absolute expression as log ( CPM + 1 ) of general pluripotency ( OCT4 and NANOG ) , naive ( SUSD2 ) , and TSC - specific genes ( GATA2 , GATA3 , KRT7 , and ADAP2 ) in the reported conditions highlighted in different colours . Data information : ( D ) Dots indicate biological replicates and bars indicate the mean ± SEM of n = 3 biological replicates .

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 5D Barplots showing the absolute expression as log(CPM+1) of general pluripotency (OCT4 and NANOG), naive (SUSD2), and TSC-specific genes (GATA2, GATA3, KRT7, and ADAP2) in the reported conditions highlighted in different colours. Data information: (D) Dots indicate biological replicates and bars indicate the mean ± SEM of n=3 biological replicates.


Figure 6B Immunostaining for the TSC markers GATA3 and KRT7 , the naive pluripotency marker SUSD2 and general pluripotency markers OCT4 of KiPS primed - iPSCs , KiPS at day 3 of the conversion , KiPS at day 7 in TSC medium or PXGL , KiPS at day 14 in TSC medium or PXGL , HPD06 naive - TSCs and HPD06 naive - iPSCs . Scale bars : 25 μm . Data information : ( B ) Representative images of n = 2 biological replicates are shown . C Single - cell scatter plots showing the log ( CPM + 1 ) integrated intensity of TSC markers GATA3 and KRT7 , naive pluripotency marker SUSD2 , and general pluripotency marker OCT4 of KiPS primed - iPSCs , KiPS at day 3 of the conversion , KiPS at day 7 in TSC medium or PXGL , KiPS at day 14 in TSCs medium or PXGL , HPD06 naive - TSCs and HPD06 naive - iPSCs . Black lines represent the threshold used to define positivity to a given marker . Data information : ( C ) Fluorescence intensity was measured by immunofluorescence in 5 to 12 independent fields from 2 independent experiments .

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 O O O O O O O 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 6B Immunostaining for the TSC markers GATA3 and KRT7, the naive pluripotency marker SUSD2 and general pluripotency markers OCT4 of KiPS primed-iPSCs, KiPS at day 3 of the conversion, KiPS at day 7 in TSC medium or PXGL, KiPS at day 14 in TSC medium or PXGL, HPD06 naive-TSCs and HPD06 naive-iPSCs. Scale bars: 25 μm. Data information: (B) Representative images of n=2 biological replicates are shown.C Single-cell scatter plots showing the log(CPM+1) integrated intensity of TSC markers GATA3 and KRT7, naive pluripotency marker SUSD2, and general pluripotency marker OCT4 of KiPS primed-iPSCs, KiPS at day 3 of the conversion, KiPS at day 7 in TSC medium or PXGL, KiPS at day 14 in TSCs medium or PXGL, HPD06 naive-TSCs and HPD06 naive-iPSCs. Black lines represent the threshold used to define positivity to a given marker. Data information: (C) Fluorescence intensity was measured by immunofluorescence in 5 to 12 independent fields from 2 independent experiments.


f1a , Immunoprecipitation of Flag - beclin 1 constructs with Nef - HA in HeLa cells 24 h post - transfection . b , GFP - LC3 - positive dots ( autophagosomes ) in MCF7 cells expressing GFP - LC3 and Flag - beclin 1 constructs grown in either normal medium or starved in EBSS for 2 h . d , Biochemical assessment of autophagy ( p62 and LC3 immunoblots ) in peptide - treated HeLa cells ( 3 h ) . e , f , Representative images ( e ) and quantification of GFP - LC3 - positive dots ( f ) in peptide - treated HeLa / GFP - LC3 cells ( 30 µM , 3 h ) . Scale bars , 20 µm . g , GFP - LC3 - positive dots in siRNA - transfected peptide - treated HeLa / GFP - LC3 cells ( 30 µM , 3 h ) . h , Model of Tat - beclin 1 peptide ( left ) based on corresponding elements of the beclin 1 evolutionarily conserved domain ( ECD ) structure ( centre ) . Essential phenylalanine side chains , magenta ; positions of solubility mutations , pink ; lipid interaction site , yellow . ECD surface representation ( right ) illustrates exposure of corresponding peptide ( cyan ) . i , p62 and LC3 immunoblots in peptide - treated HeLa cells ( 3 h ) . In b , f , g , bars represent mean ± s . e . m . of triplicate samples ( 50 - 100 cells per sample ) . Similar results were observed in three independent experiments . * P 0 . 05 , * * P 0 . 01 ; t - test .

B-PANEL_START 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 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 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 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 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

f1a, Immunoprecipitation of Flag-beclin 1 constructs with Nef-HA in HeLa cells 24 h post-transfection.b, GFP-LC3-positive dots (autophagosomes) in MCF7 cells expressing GFP-LC3 and Flag-beclin 1 constructs grown in either normal medium or starved in EBSS for 2 h.d, Biochemical assessment of autophagy (p62 and LC3 immunoblots) in peptide-treated HeLa cells (3 h).e, f, Representative images (e) and quantification of GFP-LC3-positive dots (f) in peptide-treated HeLa/GFP-LC3 cells (30 µM, 3 h). Scale bars, 20 µm.g, GFP-LC3-positive dots in siRNA-transfected peptide-treated HeLa/GFP-LC3 cells (30 µM, 3 h).h, Model of Tat-beclin 1 peptide (left) based on corresponding elements of the beclin 1 evolutionarily conserved domain (ECD) structure (centre). Essential phenylalanine side chains, magenta; positions of solubility mutations, pink; lipid interaction site, yellow. ECD surface representation (right) illustrates exposure of corresponding peptide (cyan).i, p62 and LC3 immunoblots in peptide-treated HeLa cells (3 h). In b, f, g, bars represent mean ± s.e.m. of triplicate samples (50-100 cells per sample). Similar results were observed in three independent experiments. *P 0.05, **P 0.01; t-test.


f2a , HeLa cells were treated with biotin - conjugated peptides ( 30 μM , 3 h ) and proteins bound to peptides were analysed by immunoblot with anti - GAPR - 1 . b - T - B , biotin - Tat - beclin 1 ; b - T - S , biotin - Tat - scrambled . b , Immunoprecipitation of Flag - beclin 1 with GAPR - 1 - Myc in HeLa cells 24 h post - transfection . c , GFP - LC3 - positive dots in GAPR - 1 siRNA - transfected peptide - treated HeLa / GFP - LC3 cells ( 20 & amp ; amp ; mgr ; M , 3 h ) with or without 100 nM bafilomycin A1 . Bars represent mean ± s . e . m . of triplicate samples ( 50 - 100 cells per sample ) . Similar results were observed in three independent experiments . NC , non - silencing control . d , Localization of beclin 1 and GM130 ( a Golgi marker ) in HeLa cells stably transduced with empty vector ( left panel ) or GAPR - 1 - Myc ( middle panel ) or transfected with GAPR - 1 siRNA ( right panel ) and treated with peptide ( 20 μM , 1 h ) . Scale bar , 20 μm . e , Immunoblot of beclin 1 in post - nuclear supernatant ( PNS ) and Golgi - enriched fractions in HeLa cells stably transduced with empty vector or GAPR - 1 - Myc after peptide treatment ( 20 μM , 2 h ) . f , WIPI2 dots in cells in the experimental conditions shown in d . Bars represent mean ± s . e . m . for 100 - 150 cells . * P 0 . 05 , * * * P 0 . 001 ; t - test . † † † P 0 . 001 ; two - way ANOVA for comparison of magnitude of changes between groups .

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f2a, HeLa cells were treated with biotin-conjugated peptides (30 μM, 3 h) and proteins bound to peptides were analysed by immunoblot with anti-GAPR-1. b-T-B, biotin-Tat-beclin 1; b-T-S, biotin-Tat-scrambled.b, Immunoprecipitation of Flag-beclin 1 with GAPR-1-Myc in HeLa cells 24 h post-transfection.c, GFP-LC3-positive dots in GAPR-1 siRNA-transfected peptide-treated HeLa/GFP-LC3 cells (20 &amp;mgr;M, 3 h) with or without 100 nM bafilomycin A1. Bars represent mean ± s.e.m. of triplicate samples (50-100 cells per sample). Similar results were observed in three independent experiments. NC, non-silencing control.d, Localization of beclin 1 and GM130 (a Golgi marker) in HeLa cells stably transduced with empty vector (left panel) or GAPR-1-Myc (middle panel) or transfected with GAPR-1 siRNA (right panel) and treated with peptide (20 μM, 1 h). Scale bar, 20 μm.e, Immunoblot of beclin 1 in post-nuclear supernatant (PNS) and Golgi-enriched fractions in HeLa cells stably transduced with empty vector or GAPR-1-Myc after peptide treatment (20 μM, 2 h).f, WIPI2 dots in cells in the experimental conditions shown in d. Bars represent mean ± s.e.m. for 100-150 cells. *P 0.05, ***P 0.001; t-test. †††P 0.001; two-way ANOVA for comparison of magnitude of changes between groups.


f3a , Percentage of cells with small htt103Q aggregates ( left ) and number of aggregates per cell ( right ) in HeLa cells expressing doxycycline ( Dox ) - repressible CFP - htt103Q after daily treatment with doxycycline or peptide ( 20 μM , 4 h per day ) for 2 days . Bars represent mean ± s . e . m . of triplicate samples ( 60 - 120 cells per sample ) . Similar results were observed in three independent experiments . b , Filter trap assays for htt103Q large and small aggregates in HeLa / htt103Q cells . c , Viral titres in HeLa cells infected with 0 . 1 plaque - forming units ( p . f . u . ) per cell of SINV , CHIKV or WNV ( strain TX02 ) and treated with peptide ( 10 μM , 4 - 8 h post - infection ) . Values represent geometric mean ± s . e . m . for triplicate samples of supernatants collected 18 h post - infection ( SINV ) or 24 h post - infection ( CHIKV and WNV ) . Similar results were observed in three independent experiments . d , Bacteriacolony - forming units ( c . f . u . ) in primary BMDMs infected with L . monocytogenes ΔactA mutant strain DPL - 4029 for 30 min and treated with peptide ( 10 µM from 0 to 2 h post - infection ) . Bars represent mean ± s . e . m . of triplicate samples . Similar results were observed in three independent experiments . e , HIV - 1 p24 antigen release in primary human MDMs infected with HIV - 1 24 h after initiation of daily peptide treatment . Values represent mean ± s . e . m . of triplicate samples . Similar results were observed in MDMs from three independent donors . f , HIV - 1p24 antigen release in MDMs transduced with nonspecific scrambled shRNA ( shNS ) or ATG5 shRNA ( shATG5 ) and treated daily with peptide ( 5 µM ) . Values represent mean ± s . e . m . of triplicate wells . Similar results were observed in MDMs from two independent donors . g , LC3 immunoblot of MDMs transduced with the indicated shRNA at day 0 and day 10 after HIV - 1 infection . * P 0 . 05 ; * * P 0 . 01 ; * * * P 0 . 001 ; t - test .

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 B-PANEL_START 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 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 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

f3a, Percentage of cells with small htt103Q aggregates (left) and number of aggregates per cell (right) in HeLa cells expressing doxycycline (Dox)-repressible CFP-htt103Q after daily treatment with doxycycline or peptide (20 μM, 4 h per day) for 2 days. Bars represent mean ± s.e.m. of triplicate samples (60-120 cells per sample). Similar results were observed in three independent experiments.b, Filter trap assays for htt103Q large and small aggregates in HeLa/htt103Q cells.c, Viral titres in HeLa cells infected with 0.1 plaque-forming units (p.f.u.) per cell of SINV, CHIKV or WNV (strain TX02) and treated with peptide (10 μM, 4-8 h post-infection). Values represent geometric mean ± s.e.m. for triplicate samples of supernatants collected 18 h post-infection (SINV) or 24 h post-infection (CHIKV and WNV). Similar results were observed in three independent experiments.d, Bacteriacolony-forming units (c.f.u.) in primary BMDMs infected with L. monocytogenes ΔactA mutant strain DPL-4029 for 30 min and treated with peptide (10 µM from 0 to 2 h post-infection). Bars represent mean ± s.e.m. of triplicate samples. Similar results were observed in three independent experiments.e, HIV-1 p24 antigen release in primary human MDMs infected with HIV-1 24 h after initiation of daily peptide treatment. Values represent mean ± s.e.m. of triplicate samples. Similar results were observed in MDMs from three independent donors.f, HIV-1p24 antigen release in MDMs transduced with nonspecific scrambled shRNA (shNS) or ATG5 shRNA (shATG5) and treated daily with peptide (5 µM). Values represent mean ± s.e.m. of triplicate wells. Similar results were observed in MDMs from two independent donors.g, LC3 immunoblot of MDMs transduced with the indicated shRNA at day 0 and day 10 after HIV-1 infection. *P  0.05; **P  0.01; ***P  0.001; t-test.


f4a , GFP - LC3 - positive dots in tissues of 6 - week - old GFP - LC3 mice treated with the indicated peptide ( 20 mg kg − 1 i . p . , 6 h ) . A minimum of ten fields was counted per tissue section . Bars represent mean ± s . e . m . for three mice . Similar results were observed in three independent experiments . b , p62 immunoblot of brains of 5 - day - old GFP - LC3 mice treated with the indicated peptide ( 20 mg kg − 1 i . p . , 6 h ) . c , Survival curves of 5 - day - old C57BL / 6J mice infected with CHIKV ( 106 p . f . u . s . c . ) and treated daily with peptide ( 15 mg kg − 1 i . p . beginning 1 day post - infection ) . d - f , Representative images of WNV envelope antigen and TdT - mediated dUTP nick end labelling ( TUNEL ) staining ( d ) ( T , Tat alone ) , quantification of cell death in brain ( e ) and survival curves ( f ) for 5 - day - oldC57BL / 6Jmice infected with WNV ( Egypt strain 101 , 1 p . f . u . intracerebral ( i . c . ) ) and treated daily with peptide ( D - amino acid forms , 20mgkg − 1 i . p . beginning 1day post - infection ) . Images in d are from cerebral cortex day 6 post - infection . Similar results were observed in all regions of the brain for three mice per group . Scale bar , 20μm . Bars in e represent mean ± s . e . m . TUNEL - positive cells per unit area of brain for three mice . gd - f , Representative images of WNV envelope antigen and TdT - mediated dUTP nick end labelling ( TUNEL ) staining ( d ) ( T , Tat alone ) , quantification of cell death in brain ( e ) , and survival curves ( f ) for 5 - day - oldC57BL / 6Jmice infected with WNV ( Egypt strain 101 , 1 p . f . u . intracerebral ( i . c . ) ) and treated daily with peptide ( D - amino acid forms , 20mgkg − 1 i . p . beginning 1day post - infection ) . Images in d are from cerebral cortex day 6 post - infection . Similar results were observed in all regions of the brain for three mice per group . Scale bar , 20μm . Bars in e represent mean ± s . e . m . TUNEL - positive cells per unit area of brain for three mice . gg , Geometric mean + s . e . m . viral titres of WNV - infected mousebrains day 6 post - infection . Values represent combined data for 12 - 20 mice per treatment group from 10 to 12 litters . Data in c and f represent combined survival probabilities for three and four independent litters , respectively , in each group . Similar results were observed in each independent experiment . * P 0 . 05 ; * * P 0 . 01 ; * * * P 0 . 001 ; NS , not significant ; t - test .

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 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 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 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 O O O 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

f4a, GFP-LC3-positive dots in tissues of 6-week-old GFP-LC3 mice treated with the indicated peptide (20 mg kg−1 i.p., 6 h). A minimum of ten fields was counted per tissue section. Bars represent mean ± s.e.m. for three mice. Similar results were observed in three independent experiments.b, p62 immunoblot of brains of 5-day-old GFP-LC3 mice treated with the indicated peptide (20 mg kg−1 i.p., 6 h).c, Survival curves of 5-day-old C57BL/6J mice infected with CHIKV (106 p.f.u. s.c.) and treated daily with peptide (15 mg kg−1 i.p. beginning 1 day post-infection).d-f, Representative images of WNV envelope antigen and TdT-mediated dUTP nick end labelling (TUNEL) staining (d) (T, Tat alone), quantification of cell death in brain (e) and survival curves (f) for 5-day-oldC57BL/6Jmice infected with WNV (Egypt strain 101, 1 p.f.u. intracerebral (i.c.)) and treated daily with peptide (D-amino acid forms, 20mgkg−1 i.p. beginning 1day post-infection). Images in d are from cerebral cortex day 6 post-infection. Similar results were observed in all regions of the brain for three mice per group. Scale bar, 20μm. Bars in e represent mean±s.e.m. TUNEL-positive cells per unit area of brain for three mice. gd-f, Representative images of WNV envelope antigen and TdT-mediated dUTP nick end labelling (TUNEL) staining (d) (T, Tat alone), quantification of cell death in brain (e), and survival curves (f) for 5-day-oldC57BL/6Jmice infected with WNV (Egypt strain 101, 1 p.f.u. intracerebral (i.c.)) and treated daily with peptide (D-amino acid forms, 20mgkg−1 i.p. beginning 1day post-infection). Images in d are from cerebral cortex day 6 post-infection. Similar results were observed in all regions of the brain for three mice per group. Scale bar, 20μm. Bars in e represent mean±s.e.m. TUNEL-positive cells per unit area of brain for three mice. gg, Geometric mean + s.e.m. viral titres of WNV-infected mousebrains day 6 post-infection. Values represent combined data for 12-20 mice per treatment group from 10 to 12 litters. Data in c and f represent combined survival probabilities for three and four independent litters, respectively, in each group. Similar results were observed in each independent experiment. *P 0.05; **P 0.01; ***P 0.001; NS, not significant; t-test.