Figure 6 ( A ) analysis using transcriptomic data from the liver of sepsisBIM mice ( 16h after intraperitoneal injection of live E . coli ) . ( B - C ) Enrichment plots from GSEA performed using LIVER - ID genes ( B ) or the response to ERS gene set ( GO : 0034976 ) ( C ) as the gene set and transcriptomic differences in sepsisBIM vs Control mouse liver as the ranked gene list . ( E ) Box plots showing Log2 FC for ERS UP , DOWN or unchanged genes in sepsisBIM vs Control mouse liver ( 6 mice per group ) . Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers . One - way ANOVA with Welch ' s correction and Dunnett ' s Modified Tukey - Kramer pairwise multiple comparison test was used to assess statistical significance , * P < 0 . 05 . ( F ) RT - qPCR analyses of selected ERS UP genes and LIVER - ID TFs monitoring expression changes in the liver of sepsisBIM vs Control mice ( 6 mice per group ) . The bar graph shows means ± SD ( standard deviations ) . One - sample t - test with BH correction for multiple testing was used to determine if the mean Log2 FC sepsisBIM / Control is statistically different from 0 , * P < 0 . 05 . ( G ) Nuclear extracts from livers of control or sepsisBIM mice were subjected to Western blot or Simple Western immunoassay with antibodies against HNF4A , NR1H4 or FOXA2 . LMNA was used as loading control . ( H ) Nuclear extracts from livers of sepsisBIM mice pre - treated for 4 consecutive days with vehicle or 500 mpk TUDCA were subjected to Western blot with antibodies against HNF4A , NR1H4 , FOXA2 or XBP1S . TFIIB was used as loading control . ( I ) Densitometric quantification of the protein expression data from 6 mice precondition shown in panel ( H ) ( 3 mice per condition ) The bar graph shows means ± SD ( standard deviations ) . One - sample t - test with BH correction for multiple testing was used to determine if the mean Log2 FC TUDCA / Vehicle is statistically different from 0 , * P < 0 . 05 . ( J ) Serum alanine aminotransferase ( ALT ) ( left ) and aspartate aminotransferase ( AST ) activities ( right ) from sepsisBIM mice pre - treated for 4 consecutive days with vehicle or 500 mpk TUDCA ( 10 mice per group ) . The bar graphs show means ± SD ( standard deviations ) . Student ' s t - test was used to assess statistical significance , * P < 0 . 05 .

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Figure 6(A) analysis using transcriptomic data from the liver of sepsisBIM mice (16h after intraperitoneal injection of live E. coli).(B-C) Enrichment plots from GSEA performed using LIVER-ID genes (B) or the response to ERS gene set (GO:0034976) (C) as the gene set and transcriptomic differences in sepsisBIM vs Control mouse liver as the ranked gene list.(E) Box plots showing Log2 FC for ERS UP, DOWN or unchanged genes in sepsisBIM vs Control mouse liver (6 mice per group). Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers. One-way ANOVA with Welch's correction and Dunnett's Modified Tukey-Kramer pairwise multiple comparison test was used to assess statistical significance, *P < 0.05.(F) RT-qPCR analyses of selected ERS UP genes and LIVER-ID TFs monitoring expression changes in the liver of sepsisBIM vs Control mice (6 mice per group). The bar graph shows means ± SD (standard deviations). One-sample t-test with BH correction for multiple testing was used to determine if the mean Log2 FC sepsisBIM/Control is statistically different from 0, *P < 0.05.(G) Nuclear extracts from livers of control or sepsisBIM mice were subjected to Western blot or Simple Western immunoassay with antibodies against HNF4A, NR1H4 or FOXA2. LMNA was used as loading control.(H) Nuclear extracts from livers of sepsisBIM mice pre-treated for 4 consecutive days with vehicle or 500 mpk TUDCA were subjected to Western blot with antibodies against HNF4A, NR1H4, FOXA2 or XBP1S. TFIIB was used as loading control. (I) Densitometric quantification of the protein expression data from 6 mice precondition shown in panel (H) (3 mice per condition) The bar graph shows means ± SD (standard deviations). One-sample t-test with BH correction for multiple testing was used to determine if the mean Log2 FC TUDCA/Vehicle is statistically different from 0, *P < 0.05.(J) Serum alanine aminotransferase (ALT) (left) and aspartate aminotransferase (AST) activities (right) from sepsisBIM mice pre-treated for 4 consecutive days with vehicle or 500 mpk TUDCA (10 mice per group). The bar graphs show means ± SD (standard deviations). Student's t-test was used to assess statistical significance, *P < 0.05.


Figure 7 ( A ) RT - qPCR analyses of selected ERS UP genes and LIVER - ID TFs in livers from sepsisCLP mice collected 10h , 30h or 3 days after CLP ( 15 mice per group ) vs livers from healthy pair - fed mice ( Control ) ( 15 mice per group ) . The bar graph shows means ± SD ( standard deviations ) . Wilcoxon test with BH correction for multiple testing was used to assess statistical significance , * P < 0 . 05 . ( B ) Box plots showing Log2 FC ERS / Control in mouse liver for ERS UP genes 8h and 34h after tunicamycin injection in WT or ATF6 KO mice ( 3 mice per experimental condition ) . Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers . One - sample t - test with BH correction for multiple testing was used to determine if the mean Log2 FC ERS / Control is statistically different from 0 , * P < 0 . 05 . NS , not significant . ( C ) Similar analyses to panel ( B ) for ERS DOWN and LIVER - ID genes ( 3 mice per experimental condition ) . Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers . One - sample t - test with BH correction for multiple testing was used to determine if the mean Log2 FC ERS / Control is statistically different from 0 , * P < 0 . 05 . ( D ) Main observations from ( Huck et al . , 2019 ) ( left ) and box plots showing Log2 FC HNF4A KO / Control in mouse liver ( 3 mice per group ) for LIVER - ID genes and TFs 5 days after PHx ( right ) . Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers . One - sample t - test with BH correction for multiple testing was used to determine if the mean Log2 FC HNF4A KO / Control is statistically different from 0 , * P < 0 . 05 . ( F ) Expression of LIVER - ID TF encoding genes was analyzed in the livers of deceased critically - ill patients with sepsis displaying agonal bilirubin levels below ( Bil < 2 ; n = 34 ) or above ( Bil > 2 ; n = 28 ) 2mg / dL . Median fold - change expression level of each group for the different genes has been used to generate the box plots . Expression levels in the critically - ill groups are expressed relative to those in the control group ( n = 18 ) . Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers . One - tailed t - test was used to assess whether expression of LIVER - ID TF encoding genes in the Bil > 2 group is significantly greater than in the Bil < 2 group , * P < 0 . 05 . ( G ) RT - qPCR analyses of indicated LIVER - ID TF encoding genes monitoring expression in the livers of Bil < 2 ( n = 34 ) or Bil > 2 ( n = 28 ) groups of deceased critically - ill patients with sepsis vs control donors ( n = 18 ) . Data are shown as box plots , with mRNA levels of the critically - ill groups expressed relative to those of the control group . Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers . Wilcoxon test was used to assess statistically significant differences with the Bil > 2 group , * P < 0 . 05 . ( H ) RT - qPCR analyses of indicated ERS UP genes performed Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers . Wilcoxon test was used to assess statistically significant differences with the Bil > 2 group , * P < 0 . 05 .

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Figure 7(A) RT-qPCR analyses of selected ERS UP genes and LIVER-ID TFs in livers from sepsisCLP mice collected 10h, 30h or 3 days after CLP (15 mice per group) vs livers from healthy pair-fed mice (Control) (15 mice per group). The bar graph shows means ± SD (standard deviations). Wilcoxon test with BH correction for multiple testing was used to assess statistical significance, *P < 0.05.(B) Box plots showing Log2 FC ERS/Control in mouse liver for ERS UP genes 8h and 34h after tunicamycin injection in WT or ATF6 KO mice (3 mice per experimental condition). Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers. One-sample t-test with BH correction for multiple testing was used to determine if the mean Log2 FC ERS/Control is statistically different from 0, *P < 0.05. NS, not significant. (C) Similar analyses to panel (B) for ERS DOWN and LIVER-ID genes (3 mice per experimental condition). Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers. One-sample t-test with BH correction for multiple testing was used to determine if the mean Log2 FC ERS/Control is statistically different from 0, *P < 0.05. (D) Main observations from (Huck et al., 2019) (left) and box plots showing Log2 FC HNF4A KO/Control in mouse liver (3 mice per group) for LIVER-ID genes and TFs 5 days after PHx (right). Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers. One-sample t-test with BH correction for multiple testing was used to determine if the mean Log2 FC HNF4A KO/Control is statistically different from 0, *P < 0.05.(F) Expression of LIVER-ID TF encoding genes was analyzed in the livers of deceased critically-ill patients with sepsis displaying agonal bilirubin levels below (Bil<2; n=34) or above (Bil>2; n=28) 2mg/dL. Median fold-change expression level of each group for the different genes has been used to generate the box plots. Expression levels in the critically-ill groups are expressed relative to those in the control group (n=18). Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers. One-tailed t-test was used to assess whether expression of LIVER-ID TF encoding genes in the Bil>2 group is significantly greater than in the Bil<2 group, *P < 0.05.(G) RT-qPCR analyses of indicated LIVER-ID TF encoding genes monitoring expression in the livers of Bil<2 (n=34) or Bil>2 (n=28) groups of deceased critically-ill patients with sepsis vs control donors (n=18). Data are shown as box plots, with mRNA levels of the critically-ill groups expressed relative to those of the control group. Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers. Wilcoxon test was used to assess statistically significant differences with the Bil>2 group, *P < 0.05.(H) RT-qPCR analyses of indicated ERS UP genes performed Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers. Wilcoxon test was used to assess statistically significant differences with the Bil>2 group, *P < 0.05.


Figure 1Immunoblot detection and its quantification of PDGFR - β in kidney cortex lysates of healthy and diseased human kidneys shows twice as much PDGFR - β abundance in diseased tissue . A stronger signal for phosphorylated PDGFR - β indicates the active status of the receptor . The high α - SMA content in the diseased kidneys shows that they are affected by fibrosis . Bar graphs show means ± SD , healthy n = 5 , diseased n = 6PDGFR - β staining is detectable in mesangium of healthy ( a ) and diseased ( b - d ) glomeruli of human kidneys . PDGFR - β positive mesangial cells expand in mesangioproliferating and scarring glomerular changes . Similarly , the population of PDGFR - β positive interstitial cells is discrete in healthy kidney cortex ( e ) but expands in fibrosis ( f - h ) . Arrows point to selected areas of positive PDGFR - β staining . Scale bar = 50 µmGFP expression under Pdgfrb - promoter in mice is present in mesangial cells of glomeruli and interstitial cells of cortex and medulla , but not in tubular cells . Nuclei are stained with DAPI ( blue ) . Scale bar = 50 µmTissue clearing with Scale and 3D reconstruction of Pdgfrb - GFP reporter mice in a healthy and fibrotic kidney ( UUO day 5 ) shows the expansion of Pdgfrb expressing cells in fibrosis .

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Figure 1Immunoblot detection and its quantification of PDGFR-β in kidney cortex lysates of healthy and diseased human kidneys shows twice as much PDGFR-β abundance in diseased tissue. A stronger signal for phosphorylated PDGFR-β indicates the active status of the receptor. The high α-SMA content in the diseased kidneys shows that they are affected by fibrosis. Bar graphs show means ± SD, healthy n = 5, diseased n = 6PDGFR-β staining is detectable in mesangium of healthy (a) and diseased (b-d) glomeruli of human kidneys. PDGFR-β positive mesangial cells expand in mesangioproliferating and scarring glomerular changes. Similarly, the population of PDGFR-β positive interstitial cells is discrete in healthy kidney cortex (e) but expands in fibrosis (f-h). Arrows point to selected areas of positive PDGFR-β staining. Scale bar = 50 µmGFP expression under Pdgfrb-promoter in mice is present in mesangial cells of glomeruli and interstitial cells of cortex and medulla, but not in tubular cells. Nuclei are stained with DAPI (blue). Scale bar = 50 µmTissue clearing with Scale and 3D reconstruction of Pdgfrb-GFP reporter mice in a healthy and fibrotic kidney (UUO day 5) shows the expansion of Pdgfrb expressing cells in fibrosis.


Figure 2Representative Ki67 immunofluorescence staining ( green ) in wt and Foxd1Cre : : Pdgfrb + / J mice , showing increased proliferation in the transgenic mice . Glomeruli are outlined with circles , arrowheads point to Ki67 positive interstitial cells . Nuclei are stained with DAPI ( blue ) . Scale bar = 50 µm . Quantification of proliferating Ki67 - positive cells specifically in glomeruli ( C ) , interstitium ( D ) and tubules ( E ) in Foxd1Cre : : Pdgfrb + / J mice ( black bars ) and wt mice ( white bars ) of 6 , 14 , 25 and 35 weeks of age . Foxd1Cre : : Pdgfrb + / J mice exhibited increased proliferation of glomerular and interstitial cells , whereas tubular epithelial cell proliferation was not altered . Data in C , D and E are shown as means ± SD of n = 3 animals per group . FoxD1 - reporter mice ( Foxd1 : : tdTomato ) confirmed the expansion of mesenchymal cells in 14 weeks old Foxd1Cre : : Pdgfrb + / J mice both in glomeruli and interstitium . Circles outline glomeruli . Scale bar = 50 µm . Quantification of FoxD1 - Tomato positive cells in the cortical interstitium in 14 weeks old Foxd1Cre : : Pdgfrb + / J and wt mice confirmed the significantly increased expansion of mesenchymal cells by 42 % in Foxd1Cre : : Pdgfrb + / J mice . Cells were counted in six viewfields at 40x magnification . Bar graphs show means ± SD of n = 3 mice per group . Isolated primary fibroblasts and mesangial cells from Foxd1Cre : : Pdgfrb + / J mice have higher proliferation rates assessed by bromodeoxyuridine ( BrdU ) incorporation assay compared to cells from wt mice . Bar graphs show means ± SD , n = 4 per group , * : p ≤ 0 . 05 compared to wt of same time point .

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Figure 2Representative Ki67 immunofluorescence staining (green) in wt and Foxd1Cre::Pdgfrb+/J mice, showing increased proliferation in the transgenic mice. Glomeruli are outlined with circles, arrowheads point to Ki67 positive interstitial cells. Nuclei are stained with DAPI (blue). Scale bar = 50 µm.Quantification of proliferating Ki67-positive cells specifically in glomeruli (C), interstitium (D) and tubules (E) in Foxd1Cre::Pdgfrb+/J mice (black bars) and wt mice (white bars) of 6, 14, 25 and 35 weeks of age. Foxd1Cre::Pdgfrb+/J mice exhibited increased proliferation of glomerular and interstitial cells, whereas tubular epithelial cell proliferation was not altered. Data in C, D and E are shown as means ± SD of n = 3 animals per group.FoxD1-reporter mice (Foxd1::tdTomato) confirmed the expansion of mesenchymal cells in 14 weeks old Foxd1Cre::Pdgfrb+/J mice both in glomeruli and interstitium. Circles outline glomeruli. Scale bar = 50 µm. Quantification of FoxD1-Tomato positive cells in the cortical interstitium in 14 weeks old Foxd1Cre::Pdgfrb+/J and wt mice confirmed the significantly increased expansion of mesenchymal cells by 42% in Foxd1Cre::Pdgfrb+/J mice. Cells were counted in six viewfields at 40x magnification. Bar graphs show means ± SD of n = 3 mice per group.Isolated primary fibroblasts and mesangial cells from Foxd1Cre::Pdgfrb+/J mice have higher proliferation rates assessed by bromodeoxyuridine (BrdU) incorporation assay compared to cells from wt mice. Bar graphs show means ± SD, n = 4 per group, *: p≤0.05 compared to wt of same time point.


Figure 3PAS staining of glomeruli of 25 weeks old wt and of 6 and 35 weeks old Foxd1Cre : : Pdgfrb + / J mice shows pathological changes of the glomeruli in Foxd1Cre : : Pdgfrb + / J mice . Glomerular tuft size of Foxd1Cre : : Pdgfrb + / J mice was significantly increased compared to wt mice during the time course . Immunhistological staining of α - SMA and its histomorphometric quantification ( D ) reveals expansion of activated mesangial cells in the glomerular tuft of in the transgenic mice in the time course . Immunhistological staining of collagen I and its histomorphometric quantification ( F ) shows that collagen I deposits in an increasing manner in Foxd1Cre : : Pdgfrb + / J mice during the time course .

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Figure 3PAS staining of glomeruli of 25 weeks old wt and of 6 and 35 weeks old Foxd1Cre::Pdgfrb+/J mice shows pathological changes of the glomeruli in Foxd1Cre::Pdgfrb+/J mice. Glomerular tuft size of Foxd1Cre::Pdgfrb+/J mice was significantly increased compared to wt mice during the time course.Immunhistological staining of α-SMA and its histomorphometric quantification (D) reveals expansion of activated mesangial cells in the glomerular tuft of in the transgenic mice in the time course.Immunhistological staining of collagen I and its histomorphometric quantification (F) shows that collagen I deposits in an increasing manner in Foxd1Cre::Pdgfrb+/J mice during the time course.


Figure 4Histological stainings and its histomorphometric quantification ( B ) of kidney cortex for α - SMA of wt ( 25 weeks ) and Foxd1Cre : : Pdgfrb + / J mice of an age of 6 and 35 weeks show expansion of myofibroblasts in the interstitium of the transgenic mice . Collagen I staining and its histomorphometric quantification ( D ) show the progression of interstitial fibrosis in the time course . The tubular stress marker Lipocalin - 2 ( LCN2 ) is not expressed in early time points but increases in time course in late stages when fibrosis is more prominent , as seen on histological stainingThe tubular stress marker Lipocalin - 2 ( LCN2 ) is not expressed in early time points but increases in time course in late stages when fibrosis is more prominent and mRNA levelA slight , but significant increase in interstitial ErHr3 positive stained macrophages was observed , particularly at later stages ( G ) . For evaluation , ErHr3 positive cells were counted on 20 view fields on 40x magnification ( H ) .

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Figure 4Histological stainings and its histomorphometric quantification (B) of kidney cortex for α-SMA of wt (25 weeks) and Foxd1Cre::Pdgfrb+/J mice of an age of 6 and 35 weeks show expansion of myofibroblasts in the interstitium of the transgenic mice.Collagen I staining and its histomorphometric quantification (D) show the progression of interstitial fibrosis in the time course.The tubular stress marker Lipocalin-2 (LCN2) is not expressed in early time points but increases in time course in late stages when fibrosis is more prominent, as seen on histological stainingThe tubular stress marker Lipocalin-2 (LCN2) is not expressed in early time points but increases in time course in late stages when fibrosis is more prominent and mRNA levelA slight, but significant increase in interstitial ErHr3 positive stained macrophages was observed, particularly at later stages (G). For evaluation, ErHr3 positive cells were counted on 20 view fields on 40x magnification (H).


Figure 5Creatinine clearance was measured in mice of an age of 10 , 14 , 25 and 35 weeks and decreased continuously in Foxd1Cre : : Pdgfrb + / J mice , with a predicted complete loss of renal function 62 weeks in transgenic mice using linear regression analyses as described previously ( Steiger et al , 2018 ) . 10 weeks n = 5 wt and n = 4 Foxd1Cre : : Pdgfrb + / J , 14 weeks n = 2 per group , 25 weeks n = 8 wt and n = 8 Foxd1Cre : : Pdgfrb + / J , 35 weeks n = 3 per group The blood urea nitrogen ( BUN ) concentrations were higher in Foxd1Cre : : Pdgfrb + / J mice compared to wt mice , apart from 25 weeks old group . Bar graphs show means ± SD , 10 weeks n = 7 wt and n = 5 Foxd1Cre : : Pdgfrb + / J , 14 weeks n = 3 per group , 25 weeks n = 11 wt and n = 12 Foxd1Cre : : Pdgfrb + / J , 35 weeks n = 3 per

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Figure 5Creatinine clearance was measured in mice of an age of 10, 14, 25 and 35 weeks and decreased continuously in Foxd1Cre::Pdgfrb+/J mice, with a predicted complete loss of renal function 62 weeks in transgenic mice using linear regression analyses as described previously (Steiger et al, 2018). 10 weeks n = 5 wt and n = 4 Foxd1Cre::Pdgfrb+/J, 14 weeks n = 2 per group, 25 weeks n = 8 wt and n = 8 Foxd1Cre::Pdgfrb+/J, 35 weeks n = 3 per group The blood urea nitrogen (BUN) concentrations were higher in Foxd1Cre::Pdgfrb+/J mice compared to wt mice, apart from 25 weeks old group. Bar graphs show means ± SD, 10 weeks n = 7 wt and n = 5 Foxd1Cre::Pdgfrb+/J, 14 weeks n = 3 per group, 25 weeks n = 11 wt and n = 12 Foxd1Cre::Pdgfrb+/J, 35 weeks n = 3 per group


Figure 6Erythrocyte numbers in Foxd1Cre : : Pdgfrb + / J mice continuously . Horizontal lines are means ± SD of n = 3 per group . * : p ≤ 0 . 05 compared to same wt of same time point . hematocrit in Foxd1Cre : : Pdgfrb + / J mice continuously . Horizontal lines are means ± SD of n = 3 per group . * : p ≤ 0 . 05 compared to same wt of same time point . hemoglobin content ( C ) decrease in Foxd1Cre : : Pdgfrb + / J mice continuously . Horizontal lines are means ± SD of n = 3 per group . * : p ≤ 0 . 05 compared to same wt of same time point . EPO levels shown as delta from respective wt littermates . Horizontal lines are means ± SD of n = 3 per group . * : p ≤ 0 . 05 compared to same wt of same time point . RNA in - situ hybridization ( RNAScope ) of Epo ( green ) and Pdgfrb ( purple ) in renal kidney cortex of 35 week old mice shows that EPO - producing cells co - express Pdgfrb . Foxd1Cre : : Pdgfrb + / J mice have less EPO - producing cells ( arrows ) than wt mice . After induction of anemia by blood taking , the erythrocyte numbers recovered after one week in wt mice , whereas Foxd1Cre : : Pdgfrb + / J mice were not able to recover their erythrocytes numbers at all . Data are means ± SD of n = 5 per 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 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 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 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 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

Figure 6Erythrocyte numbers in Foxd1Cre::Pdgfrb+/J mice continuously. Horizontal lines are means ± SD of n = 3 per group. *: p≤0.05 compared to same wt of same time point.hematocrit in Foxd1Cre::Pdgfrb+/J mice continuously. Horizontal lines are means ± SD of n = 3 per group. *: p≤0.05 compared to same wt of same time point.hemoglobin content (C) decrease in Foxd1Cre::Pdgfrb+/J mice continuously. Horizontal lines are means ± SD of n = 3 per group. *: p≤0.05 compared to same wt of same time point.EPO levels shown as delta from respective wt littermates. Horizontal lines are means ± SD of n = 3 per group. *: p≤0.05 compared to same wt of same time point.RNA in-situ hybridization (RNAScope) of Epo (green) and Pdgfrb (purple) in renal kidney cortex of 35 week old mice shows that EPO-producing cells co-express Pdgfrb. Foxd1Cre::Pdgfrb+/J mice have less EPO-producing cells (arrows) than wt mice.After induction of anemia by blood taking, the erythrocyte numbers recovered after one week in wt mice, whereas Foxd1Cre::Pdgfrb+/J mice were not able to recover their erythrocytes numbers at all. Data are means ± SD of n = 5 per group.


Figure 7Five days after the induction of unilateral ureteral obstruction ( UUO ) , a model of obstructive nephropathy and interstitial fibrosis , we assessed fibrosis ( collagen I , α - SMA ) and inflammation ( F4 / 80 ) , the classical read - out parameters in this model . Compared to wt mice with UUO , Foxd1Cre : : Pdgfrb + / J mice developed significantly stronger fibrosis and inflammation ( A ) . Representative pictures of the histological stainings are shown in ( B ) . Scale bar = 50 µm All data in A and B show means ± SD , wt n = 4 , Foxd1Cre : : Pdgfrb + / J n = 3 . * : p ≤ 0 . 05 compared to wtAngiotensin II infusion via an osmotic pump induced hypertension similarly in wt and Foxd1Cre : : Pdgfrb + / J mice ( C ) . In this model of hypertensive injury , the kidney function and even the histopathology was largely unaffected in wt mice , whereas the Foxd1Cre : : Pdgfrb + / J mice showed a significantly increased BUN values and prominent glomerulopathy after 28 days compared to the starting values ( day 0 ) , to wt animals after 28 days and to age - matched Foxd1Cre : : Pdgfrb + / J mice without Angiotensin II infusion ( D ) . Consistently the creatinine clearance per g bodyweight decreased in the hypertensive Foxd1Cre : : Pdgfrb + / J mice ( E ) . PAS staining showed more prominent mesangial expansion and intratubular protein casts indicative of proteinuria only in hypertensive Foxd1Cre : : Pdgfrb + / J mice . Hypertension did not induce mesangial expansion and activation in wt mice , but led to a significant increase in hypertensive Foxd1Cre : : Pdgfrb + / J mice as visualized by α - SMA staining . ( H ) Histomorphometric analysis of α - SMA positive percentage of the glomerular tuft area . 22 weeks old Foxd1Cre : : Pdgfrb + / J mice were treated with imatinib ( daily gavage , 50mg / kg bodyweight for 21 days ; Foxd1Cre : : Pdgfrb + / J + imatinib ) and were compared to control Foxd1Cre : : Pdgfrb + / J mice receiving vehicle ( water ; Foxd1Cre : : Pdgfrb + / J + water ) . Aged - matched wt mice are indicated as dashed lines . Histomorphometric quantifications showed a significant reduction of α - SMA abundance in the glomerular tufts of imatinib treated Foxd1Cre : : Pdgfrb + / J mice . 22 weeks old Foxd1Cre : : Pdgfrb + / J mice were treated with imatinib ( daily gavage , 50mg / kg bodyweight for 21 days ; Foxd1Cre : : Pdgfrb + / J + imatinib ) and were compared to control Foxd1Cre : : Pdgfrb + / J mice receiving vehicle ( water ; Foxd1Cre : : Pdgfrb + / J + water ) . Aged - matched wt mice are indicated as dashed lines . Glomerular cellularity evaluated by counting the total number of cells per glomerular tuft , normalized to the area , were significantly reduced in imatinib treated Foxd1Cre : : Pdgfrb + / J mice compared to control mice . 22 weeks old Foxd1Cre : : Pdgfrb + / J mice were treated with imatinib ( daily gavage , 50mg / kg bodyweight for 21 days ; Foxd1Cre : : Pdgfrb + / J + imatinib ) and were compared to control Foxd1Cre : : Pdgfrb + / J mice receiving vehicle ( water ; Foxd1Cre : : Pdgfrb + / J + water ) . Aged - matched wt mice are indicated as dashed lines . Histomorphometric quantifications of collagen I showed no effect of imatinib treatment on mesangial sclerosis in the glomeruli ( K ) , whereas reduced interstitial fibrosis in imatinib treated Foxd1Cre : : Pdgfrb + / J mice compared to controls was found ( L ) . 22 weeks old Foxd1Cre : : Pdgfrb + / J mice were treated with imatinib ( daily gavage , 50mg / kg bodyweight for 21 days ; Foxd1Cre : : Pdgfrb + / J + imatinib ) and were compared to control Foxd1Cre : : Pdgfrb + / J mice receiving vehicle ( water ; Foxd1Cre : : Pdgfrb + / J + water ) . Aged - matched wt mice are indicated as dashed lines . Hemoglobin serum levels were significantly improved in imatinib treated mice . 22 weeks old Foxd1Cre : : Pdgfrb + / J mice were treated with imatinib ( daily gavage , 50mg / kg bodyweight for 21 days ; Foxd1Cre : : Pdgfrb + / J + imatinib ) and were compared to control Foxd1Cre : : Pdgfrb + / J mice receiving vehicle ( water ; Foxd1Cre : : Pdgfrb + / J + water ) . Aged - matched wt mice are indicated as dashed lines . Representative pictures of the histological stainings of α - SMA ( left ) and collagen I ( middle : glomeruli , right : cortical interstitium ) . Scale bar = 50 µm22 weeks old Foxd1Cre : : Pdgfrb + / J mice were treated with imatinib ( daily gavage , 50mg / kg bodyweight for 21 days ; Foxd1Cre : : Pdgfrb + / J + imatinib ) and were compared to control Foxd1Cre : : Pdgfrb + / J mice receiving vehicle ( water ; Foxd1Cre : : Pdgfrb + / J + water ) . Aged - matched wt mice are indicated as dashed lines . Total PDGFR - β protein levels were decreased in Foxd1Cre : : Pdgfrb + / J mice after imatinib treatment , as shown using western blot ( O ) and its densitometric evaluation ( P ) .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 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 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 7Five days after the induction of unilateral ureteral obstruction (UUO), a model of obstructive nephropathy and interstitial fibrosis, we assessed fibrosis (collagen I, α-SMA) and inflammation (F4/80), the classical read-out parameters in this model. Compared to wt mice with UUO, Foxd1Cre::Pdgfrb+/J mice developed significantly stronger fibrosis and inflammation (A). Representative pictures of the histological stainings are shown in (B). Scale bar = 50 µm All data in A and B show means ±SD, wt n = 4, Foxd1Cre::Pdgfrb+/J n = 3. *: p≤0.05 compared to wtAngiotensin II infusion via an osmotic pump induced hypertension similarly in wt and Foxd1Cre::Pdgfrb+/J mice (C).In this model of hypertensive injury, the kidney function and even the histopathology was largely unaffected in wt mice, whereas the Foxd1Cre::Pdgfrb+/J mice showed a significantly increased BUN values and prominent glomerulopathy after 28 days compared to the starting values (day 0), to wt animals after 28 days and to age-matched Foxd1Cre::Pdgfrb+/J mice without Angiotensin II infusion (D).Consistently the creatinine clearance per g bodyweight decreased in the hypertensive Foxd1Cre::Pdgfrb+/J mice (E).PAS staining showed more prominent mesangial expansion and intratubular protein casts indicative of proteinuria only in hypertensive Foxd1Cre::Pdgfrb+/J mice.Hypertension did not induce mesangial expansion and activation in wt mice, but led to a significant increase in hypertensive Foxd1Cre::Pdgfrb+/J mice as visualized by α-SMA staining. (H) Histomorphometric analysis of α-SMA positive percentage of the glomerular tuft area.22 weeks old Foxd1Cre::Pdgfrb+/J mice were treated with imatinib (daily gavage, 50mg/kg bodyweight for 21 days; Foxd1Cre::Pdgfrb+/J + imatinib) and were compared to control Foxd1Cre::Pdgfrb+/J mice receiving vehicle (water; Foxd1Cre::Pdgfrb+/J + water). Aged-matched wt mice are indicated as dashed lines. Histomorphometric quantifications showed a significant reduction of α-SMA abundance in the glomerular tufts of imatinib treated Foxd1Cre::Pdgfrb+/J mice.22 weeks old Foxd1Cre::Pdgfrb+/J mice were treated with imatinib (daily gavage, 50mg/kg bodyweight for 21 days; Foxd1Cre::Pdgfrb+/J + imatinib) and were compared to control Foxd1Cre::Pdgfrb+/J mice receiving vehicle (water; Foxd1Cre::Pdgfrb+/J + water). Aged-matched wt mice are indicated as dashed lines. Glomerular cellularity evaluated by counting the total number of cells per glomerular tuft, normalized to the area, were significantly reduced in imatinib treated Foxd1Cre::Pdgfrb+/J mice compared to control mice.22 weeks old Foxd1Cre::Pdgfrb+/J mice were treated with imatinib (daily gavage, 50mg/kg bodyweight for 21 days; Foxd1Cre::Pdgfrb+/J + imatinib) and were compared to control Foxd1Cre::Pdgfrb+/J mice receiving vehicle (water; Foxd1Cre::Pdgfrb+/J + water). Aged-matched wt mice are indicated as dashed lines. Histomorphometric quantifications of collagen I showed no effect of imatinib treatment on mesangial sclerosis in the glomeruli (K), whereas reduced interstitial fibrosis in imatinib treated Foxd1Cre::Pdgfrb+/J mice compared to controls was found (L).22 weeks old Foxd1Cre::Pdgfrb+/J mice were treated with imatinib (daily gavage, 50mg/kg bodyweight for 21 days; Foxd1Cre::Pdgfrb+/J + imatinib) and were compared to control Foxd1Cre::Pdgfrb+/J mice receiving vehicle (water; Foxd1Cre::Pdgfrb+/J + water). Aged-matched wt mice are indicated as dashed lines. Hemoglobin serum levels were significantly improved in imatinib treated mice.22 weeks old Foxd1Cre::Pdgfrb+/J mice were treated with imatinib (daily gavage, 50mg/kg bodyweight for 21 days; Foxd1Cre::Pdgfrb+/J + imatinib) and were compared to control Foxd1Cre::Pdgfrb+/J mice receiving vehicle (water; Foxd1Cre::Pdgfrb+/J + water). Aged-matched wt mice are indicated as dashed lines. Representative pictures of the histological stainings of α-SMA (left) and collagen I (middle: glomeruli, right: cortical interstitium). Scale bar = 50 µm22 weeks old Foxd1Cre::Pdgfrb+/J mice were treated with imatinib (daily gavage, 50mg/kg bodyweight for 21 days; Foxd1Cre::Pdgfrb+/J + imatinib) and were compared to control Foxd1Cre::Pdgfrb+/J mice receiving vehicle (water; Foxd1Cre::Pdgfrb+/J + water). Aged-matched wt mice are indicated as dashed lines. Total PDGFR-β protein levels were decreased in Foxd1Cre::Pdgfrb+/J mice after imatinib treatment, as shown using western blot (O) and its densitometric evaluation (P).


Figure 8Gene expression arrays were performed in 6 week old Foxd1Cre : : Pdgfrb + / J mice compared to wt mice . Listed are genes with log2 fold changes higher or lower than 0 . 6 . Out of these 46 genes , 17 are IFN - regulated genes , 5 are involved in autophagy and 5 in matrix remodeling and fibrosis . Same genes were analysed in microdissected glomeruli ( G ) and tubulointerstitium ( T ) of patients with diabetic ( DN , G : n = 14 , T : n = 18 ) , hypertensive ( HT , G : n = 15 , T : n = 21 ) nephropathy and living donors ( LD , G : n = 42 , T : n = 42 ) as controls , revealing a similar expression profile . Heatmap depicting pathway activities in Foxd1Cre : : Pdgfrb + / J mice and human CKD patients according to PROGENy . Based on the gene array datasets transcription factor activities were estimated by using DoRothEA . Depicted are the most regulated ones regulated both in Foxd1Cre : : Pdgfrb + / J mice and in human CKD patients .

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 B-PANEL_START 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

Figure 8Gene expression arrays were performed in 6 week old Foxd1Cre::Pdgfrb+/J mice compared to wt mice. Listed are genes with log2 fold changes higher or lower than 0.6. Out of these 46 genes, 17 are IFN-regulated genes, 5 are involved in autophagy and 5 in matrix remodeling and fibrosis. Same genes were analysed in microdissected glomeruli (G) and tubulointerstitium (T) of patients with diabetic (DN, G: n=14, T: n=18), hypertensive (HT, G: n=15, T: n=21) nephropathy and living donors (LD, G: n=42, T: n=42) as controls, revealing a similar expression profile.Heatmap depicting pathway activities in Foxd1Cre::Pdgfrb+/J mice and human CKD patients according to PROGENy.Based on the gene array datasets transcription factor activities were estimated by using DoRothEA. Depicted are the most regulated ones regulated both in Foxd1Cre::Pdgfrb+/J mice and in human CKD patients.


Figure 11 . SARS - CoV - 2 activates delayed innate immune responses in lung epithelial cells Measurements of replication and innate immune induction in Calu - 3 lung epithelial cells infected with SARS - CoV - 2 at MOIs 0 . 08 , 0 . 4 and 2 TCID50VERO / cell . ( A ) Replication of SARS - CoV - 2 genomic and subgenomic E RNAs ( qRT - PCR ) . ( B ) Quantification of N staining from cells in ( A ) by flow cytometry . Mean percentage of N - positive of all live - gated cells is shown + / - SEM , n = 2 . ( C ) Representative example of immunofluorescence staining of N protein ( green ) after SARS - CoV - 2 infection of Calu - 3 at MOI 0 . 4 TCID50VERO / cell , at time points shown . Nuclei ( DAPI , blue ) , cell mask ( red ) . Scale bar represents 50µM . Fold induction of ( F ) interferons ( IFNβ , IFNλ1 and IFNλ3 ) pro - inflammatory mediators ( IL - 6 and CCL5 ) each overlaid with SARS - CoV - 2 E ( qRT - PCR ) . Fold induction of IFN stimulated genes ( CXCL10 and IFIT2 ) each overlaid with SARS - CoV - 2 E ( qRT - PCR ) .

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 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

Figure 11.SARS-CoV-2 activates delayed innate immune responses in lung epithelial cells Measurements of replication and innate immune induction in Calu-3 lung epithelial cells infected with SARS-CoV-2 at MOIs 0.08, 0.4 and 2 TCID50VERO/cell. (A) Replication of SARS-CoV-2 genomic and subgenomic E RNAs (qRT-PCR). (B) Quantification of N staining from cells in (A) by flow cytometry. Mean percentage of N-positive of all live-gated cells is shown +/- SEM, n=2.(C) Representative example of immunofluorescence staining of N protein (green) after SARS-CoV-2 infection of Calu-3 at MOI 0.4 TCID50VERO/cell, at time points shown. Nuclei (DAPI, blue), cell mask (red). Scale bar represents 50µM.Fold induction of (F) interferons (IFNβ, IFNλ1 and IFNλ3) pro-inflammatory mediators (IL-6 and CCL5) each overlaid with SARS-CoV-2 E (qRT-PCR).Fold induction of IFN stimulated genes (CXCL10 and IFIT2) each overlaid with SARS-CoV-2 E (qRT-PCR).


Figure 2Peak SARS - CoV - 2 replication precedes innate immune activation . ( A - I ) ( A , C ) Representative images of NF - κB p65 ( A ) ( red ) and IRF3 ( C ) ( red ) nuclear localisation in mock or SARS - CoV - 2 infected ( MOI 0 . 4 TCID50VERO / cell ) Calu - 3 cells at 24 hpi . SARS - CoV - 2 N protein ( green . ( E and G ) Representative images of IL - 6 mRNA ( E ) detected by FISH ( red ) and N protein ( green ) , or IFIT1 mRNA ( G ) ( green ) with N protein ( red ) , both with nuclei ( DAPI , blue ) in mock or SARS - CoV - 2 infected ( MOI 0 . 4 TCID50VERO / cell ) Calu - 3 cells at 24 hpi . ( B , D , F , H , I ) Single cell analysis time course quantifying the Integrated Nuclear Intensity of NF - κB p65 ( B ) , IRF3 ( D ) , or overall integrated intensity for IL - 6 ( F ) or IFIT1 ( H ) mRNA over time in N protein positive cells and N protein negative cells ( I ) . n = 2 . Kruskal - Wallis test with Dunn ' s multiple comparison . * ( p < 0 . 05 ) , * * * * ( p < 0 . 0001 ) . Scale bar represents 50µM .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2Peak SARS-CoV-2 replication precedes innate immune activation. (A-I) (A,C) Representative images of NF-κB p65 (A) (red) and IRF3 (C) (red) nuclear localisation in mock or SARS-CoV-2 infected (MOI 0.4 TCID50VERO/cell) Calu-3 cells at 24 hpi. SARS-CoV-2 N protein (green. (E and G) Representative images of IL-6 mRNA (E) detected by FISH (red) and N protein (green) , or IFIT1 mRNA (G) (green) with N protein (red), both with nuclei (DAPI, blue) in mock or SARS-CoV-2 infected (MOI 0.4 TCID50VERO/cell) Calu-3 cells at 24 hpi. (B, D, F, H, I) Single cell analysis time course quantifying the Integrated Nuclear Intensity of NF-κB p65 (B), IRF3 (D) , or overall integrated intensity for IL-6 (F) or IFIT1 (H) mRNA over time in N protein positive cells and N protein negative cells (I). n=2. Kruskal-Wallis test with Dunn's multiple comparison. * (p<0.05), **** (p<0.0001). Scale bar represents 50µM.


Figure 3 ( E - H ) Measurement of ( E ) viral genomic and subgenomic E RNA ( F ) fold induction of CXCL10 , ( G ) IFIT2 and ( H ) and IL - 6 at 24 hpi , of Calu - 3 cells with SARS - CoV - 2 ( MOI 0 . 04 TCID50VERO / cell ) with Remdesivir treatment ( 5μM ) prior to , at the time of , or 8 h post - infection . Mean + / - SEM , n = 3 , One way ANOVA with Dunnett ' s multiple comparisons test to compare to untreated infected condition ( ' mock ' ) , * * ( p < 0 . 01 ) , * * * ( p < 0 . 001 ) , * * * * ( p < 0 . 0001 ) . ( I ) Representative example of immunofluorescence staining of dsRNA ( red ) and N protein ( green ) after SARS - CoV - 2 infection of Calu - 3 at MOI 0 . 4 TCID50VERO / cell , at time points shown . Nuclei ( DAPI , blue ) . Scale bar represents 50µM . ( Q - R ) ( Q ) Viral E RNA and ( R ) released infectious virus ( TCID50VERO / cell ) at 24 hpi of infected Calu - 3 cells depleted for MAVs , RIG - I or MDA5 . Mean + / - SEM , n = 3 . Each group compared to siCtrl by One Way ANOVA with Dunnett ' s multiple comparisons test , * , p > 0 . 05 , * * ( p < 0 . 01 ) , n . s : non significant .

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Figure 3(E-H) Measurement of (E) viral genomic and subgenomic E RNA (F) fold induction of CXCL10, (G) IFIT2 and (H) and IL-6 at 24 hpi, of Calu-3 cells with SARS-CoV-2 (MOI 0.04 TCID50VERO/cell) with Remdesivir treatment (5μM) prior to, at the time of, or 8 h post-infection. Mean +/- SEM, n=3, One way ANOVA with Dunnett's multiple comparisons test to compare to untreated infected condition ('mock'), ** (p<0.01), *** (p<0.001), **** (p<0.0001).(I) Representative example of immunofluorescence staining of dsRNA (red) and N protein (green) after SARS-CoV-2 infection of Calu-3 at MOI 0.4 TCID50VERO/cell, at time points shown. Nuclei (DAPI, blue). Scale bar represents 50µM.(Q-R) (Q) Viral E RNA and (R) released infectious virus (TCID50VERO/cell) at 24 hpi of infected Calu-3 cells depleted for MAVs, RIG-I or MDA5. Mean +/- SEM, n=3. Each group compared to siCtrl by One Way ANOVA with Dunnett's multiple comparisons test, *, p>0.05, ** (p<0.01), n.s : non significant.


Figure 4 ( G - H ) Viral genomic and subgenomic E RNA at 24 hpi ( RT - qPCR ) with DMSO or TPCA ( G ) or Rux ( H ) treatment . Data information : ( A - H ) Mean + / - SEM , n = 3 , statistical comparisons are made by unpaired t test comparing inhibitor - treated to mock - treated SARS - CoV - 2 infected conditions at each MOI and each timepoint . * ( p < 0 . 05 ) , * * ( p < 0 . 01 ) , * * * ( p < 0 . 001 ) , * * * * ( p < 0 . 0001 ) .

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Figure 4(G-H) Viral genomic and subgenomic E RNA at 24 hpi (RT-qPCR) with DMSO or TPCA (G) or Rux (H) treatment. Data information: (A-H) Mean +/- SEM, n=3, statistical comparisons are made by unpaired t test comparing inhibitor-treated to mock-treated SARS-CoV-2 infected conditions at each MOI and each timepoint. * (p<0.05), ** (p<0.01), *** (p<0.001), **** (p<0.0001).


Figure 5 ( B - J ) Calu - 3 cells were transfected with siRNA targeting MAVS or non - targeting control ( siCtrl ) ( B - D ) or treated with DMSO vehicle or inhibitors 10 μM TPCA - 1 ( E - G ) or 2 μM Ruxolitinib ( Rux ) ( H - J ) as shown , and were mock - infected or infected with SARS - CoV - 2 at MOI 0 . 04 TCID50VERO / cell . Virus containing conditioned media ( CoM ) was harvested at 48 hpi . MDM were treated with Calu - 3 virus containing CoM for 6 hpi , before washing and measuring MDM gene expression ( B , E , H ) , and MDM activation markers by flowcytometry 48 h later ( C , D , F , G , I , J ) , plotting relative median fluorescent intensity ( MFI ) compared to mock - infected siCtrl ( C , D ) or mock - infected DMSO control ( F , G , I , J ) . Legends in ( B ) , ( E ) and ( H ) apply to ( C , D ) , ( F , G ) and ( I , J ) respectively . The inhibitors in ( E ) and ( H ) were tested side - by - side with the same mock condition . Mean + / - SEM shown , data from 4 - 6 independent MDM donors is shown . Statistical comparison by two - tailed paired t - test comparing MDM exposed to control infected CoM to siMAVS / inhibitor treated infected CoM . * ( p < 0 . 05 ) , * * ( p < 0 . 01 ) , * * * ( p < 0 . 001 ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-J) Calu-3 cells were transfected with siRNA targeting MAVS or non-targeting control (siCtrl) (B-D) or treated with DMSO vehicle or inhibitors 10 μM TPCA-1 (E-G) or 2 μM Ruxolitinib (Rux) (H-J) as shown, and were mock-infected or infected with SARS-CoV-2 at MOI 0.04 TCID50VERO/cell. Virus containing conditioned media (CoM) was harvested at 48 hpi. MDM were treated with Calu-3 virus containing CoM for 6 hpi, before washing and measuring MDM gene expression (B, E, H), and MDM activation markers by flowcytometry 48 h later (C,D,F,G,I,J) , plotting relative median fluorescent intensity (MFI) compared to mock-infected siCtrl (C, D) or mock-infected DMSO control (F, G, I, J). Legends in (B), (E) and (H) apply to (C,D), (F,G) and (I,J) respectively. The inhibitors in (E) and (H) were tested side-by-side with the same mock condition. Mean +/- SEM shown, data from 4-6 independent MDM donors is shown. Statistical comparison by two-tailed paired t-test comparing MDM exposed to control infected CoM to siMAVS/inhibitor treated infected CoM. * (p<0.05), ** (p<0.01), *** (p<0.001).


Figure 6Calu - 3 cells were infected with SARS - CoV - 2 ( MOI 0 . 04 TCID50VERO / cell ) in the presence or absence of 10ng / ml IL - 1b . ( U ) Non - adherent cells were collected at 48h post infection and live / dead stained . Cells were acquired by flowcytometry and cell counts determined by time - gating . For statistical comparison , total cell numbers were compared . ( V ) Viral release into culture supernatants at 24 h was measured by TCID50 on VeroE6 cells . ( Q - V ) Mock and SARS - CoV - 2 infected conditions were compared with or without IL1b - treatment , respectively , by unpaired T test ( n = 3 ) . * , p < 0 . 05 ; n . s . , non - significant . Mean + / - SEM shown .

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Figure 6Calu-3 cells were infected with SARS-CoV-2 (MOI 0.04 TCID50VERO/cell) in the presence or absence of 10ng/ml IL-1b. (U) Non-adherent cells were collected at 48h post infection and live/dead stained. Cells were acquired by flowcytometry and cell counts determined by time-gating. For statistical comparison, total cell numbers were compared. (V) Viral release into culture supernatants at 24 h was measured by TCID50 on VeroE6 cells. (Q-V) Mock and SARS-CoV-2 infected conditions were compared with or without IL1b-treatment, respectively, by unpaired T test (n=3). *, p<0.05; n.s., non-significant. Mean +/- SEM shown.


Figure 1D . Validation of selected CRISPR screening hits by targeted gene inactivation . Genes are plotted on the x - axis from lowest FDR value ( IFIT1 ) to highest FDR value ( CD163L1 ) as determined by MAGeCK analysis of the genome - wide screen . All samples were normalized to non - targeting controls ( " Non - Targ " ) for graphing . Graphs represent the mean with error bars representing standard deviation . One - way ANOVA with Dunnett ' s test comparing each group to the non - targeting control was performed on non - normalized data , n = 3 biological replicates .

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Figure 1D. Validation of selected CRISPR screening hits by targeted gene inactivation. Genes are plotted on the x-axis from lowest FDR value (IFIT1) to highest FDR value (CD163L1) as determined by MAGeCK analysis of the genome-wide screen. All samples were normalized to non-targeting controls ("Non-Targ") for graphing. Graphs represent the mean with error bars representing standard deviation. One-way ANOVA with Dunnett's test comparing each group to the non-targeting control was performed on non-normalized data, n=3 biological replicates.


Figure 2D . Heat maps of FPKM ( Left ) and log2FC ( Right ) values ( from RNA - Seq ) of IFN - induced CRISPR screening hits at 6 and 24 h post IFN - treatment . Heatmap intensity represents the mean of 3 independent biological replicates . E . Western Blots from lysates of U - 2 OS cells treated with IFNβ ( 20 U / mL ) for 6 and 24 h . Antibodies were used to probe for IFN - induced CRISPR screening hits .

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 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 2D. Heat maps of FPKM (Left) and log2FC (Right) values (from RNA-Seq) of IFN-induced CRISPR screening hits at 6 and 24 h post IFN-treatment. Heatmap intensity represents the mean of 3 independent biological replicates.E. Western Blots from lysates of U-2 OS cells treated with IFNβ (20 U/mL) for 6 and 24 h. Antibodies were used to probe for IFN-induced CRISPR screening hits.


Figure 3A . Effects of CRISPR - based gene inactivation of IRF9 , STAT1 , and STAT2 or ZC3HAV1 , IFIT3 , and IFIT1 on VEEV infection ( MOI of 10 for 5 h ) in U - 2 OS cells with IFNβ pretreatment ( 20 U / mL for 24 h ) . B . Effects of CRISPR - based gene inactivation of IRF9 , STAT1 , and STAT2 or ZC3HAV1 , IFIT3 , and IFIT1 on VEEV infection ( MOI of 10 for 4 . 5 h ) in Huh7 . 5 cells with ( + symbols ) and without ( - symbols ) IFNβ pretreatment ( 100 U / mL for 24 h ) . Data information : data are presented as mean + / - standard deviation . n = 3 independent biological replicates , one - way ANOVA with Dunnett ' s test . ns p > 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , and * * * * p < 0 . 0001 . D . Dose response curve of IFNβ pretreatment ( Left , 2 - fold dilutions from 200 U / mL to 1 . 6 U / mL for 24 h ) or IFNλ pretreatment ( Right , 2 - fold dilutions from 500 ng / mL to 3 . 9 ng / mL for 24 h ) demonstrating inhibition of VEEV infection ( MOI of 5 for 5 h ) in control ( circles ) , IRF9 / STAT1 / STAT2 - targeted ( squares ) , or ZAP / IFIT3 / IFIT1 - targeted ( triangles ) U - 2 OS cells . Graphs represent the mean of normalized and transformed ( from % Infection to % Inhibition ) data for 3 biological replicates with error bars representing standard deviation .

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 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

Figure 3A. Effects of CRISPR-based gene inactivation of IRF9, STAT1, and STAT2 or ZC3HAV1, IFIT3, and IFIT1 on VEEV infection (MOI of 10 for 5 h) in U-2 OS cells with IFNβ pretreatment (20 U/mL for 24 h). B. Effects of CRISPR-based gene inactivation of IRF9, STAT1, and STAT2 or ZC3HAV1, IFIT3, and IFIT1 on VEEV infection (MOI of 10 for 4.5 h) in Huh7.5 cells with (+ symbols) and without (- symbols) IFNβ pretreatment (100 U/mL for 24 h). Data information: data are presented as mean +/- standard deviation. n=3 independent biological replicates, one-way ANOVA with Dunnett's test. ns p > 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.D. Dose response curve of IFNβ pretreatment (Left, 2-fold dilutions from 200 U/mL to 1.6 U/mL for 24 h) or IFNλ pretreatment (Right, 2-fold dilutions from 500 ng/mL to 3.9 ng/mL for 24 h) demonstrating inhibition of VEEV infection (MOI of 5 for 5 h) in control (circles), IRF9/STAT1/STAT2-targeted (squares), or ZAP/IFIT3/IFIT1-targeted (triangles) U-2 OS cells. Graphs represent the mean of normalized and transformed (from %Infection to %Inhibition) data for 3 biological replicates with error bars representing standard deviation.


Figure 4A . U - 2 OS control , IRF9 / STAT1 / STAT2 - targeted , and ZAP / IFIT3 / IFIT1 - targeted cells were infected with GFP - expressing ONNV virus ( left ) or ZIKV ( right ) after IFNβ pretreatment ( 20 U / mL ) . Data information : In data are presented as mean + / - standard deviation . n = 3 independent biological replicates , one - way ANOVA with Dunnett ' s test . ns p > 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , and * * * * p < 0 . 0001 . B . Huh7 . 5 control , IRF9 / STAT1 / STAT2 - targeted , and ZAP / IFIT3 / IFIT1 - targeted cells were infected with GFP - expressing Sindbis virus ( AR86 strain , left ) or vesicular stomatitis virus ( right ) after IFNβ pretreatment ( 100 U / mL ) . The percentage of infected ( GFP + ) cells was quantified by flow cytometry . Data information : In data are presented as mean + / - standard deviation . n = 3 independent biological replicates , one - way ANOVA with Dunnett ' s test . ns p > 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , and * * * * p < 0 . 0001 .

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 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

Figure 4A. U-2 OS control, IRF9/STAT1/STAT2-targeted, and ZAP/IFIT3/IFIT1-targeted cells were infected with GFP-expressing ONNV virus (left) or ZIKV (right) after IFNβ pretreatment (20 U/mL). Data information: In data are presented as mean +/- standard deviation. n=3 independent biological replicates, one-way ANOVA with Dunnett's test. ns p > 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.B. Huh7.5 control, IRF9/STAT1/STAT2-targeted, and ZAP/IFIT3/IFIT1-targeted cells were infected with GFP-expressing Sindbis virus (AR86 strain, left) or vesicular stomatitis virus (right) after IFNβ pretreatment (100 U/mL). The percentage of infected (GFP+) cells was quantified by flow cytometry. Data information: In data are presented as mean +/- standard deviation. n=3 independent biological replicates, one-way ANOVA with Dunnett's test. ns p > 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.


Figure 5A . Heatmaps showing the ratio of infected cells at 6 , 9 , and 12 h of infection with VEEV - GFP . U - 2 OS control , IRF9 / STAT1 / STAT2 - targeted , or ZAP / IFIT3 / IFIT1 - targeted cells were treated with or without IFNβ ( 20 U / mL for 24 h ) and infected with an MOI of 0 . 5 ( left ) , 1 ( middle ) , or 5 ( right ) of VEEV - GFP . Infection was quantified by flow cytometry . Heatmap intensity represents the mean of 3 independent biological replicates . B . ( Top ) Quantification of VEEV production ( 12 h post - infection , MOI of 2 , titered in BHK - 21J cells ) by plaque assay of control ( far left : without IFN - pretreatment ) , IRF9 / STAT1 / STAT2 - targeted , or ZAP / IFIT3 / IFIT1 - targeted cells after IFNβ pretreatment ( IFNβ 20 U / mL for 24 h ) . ( Bottom ) Western blot shows IFN - mediated induction and CRISPR - based targeting of IRF9 , STAT1 , STAT2 , ZAP , IFIT3 , and IFIT1 . Red asterisks denote long ( * * ) and short ( * ) isoform of ZAP . Data information : data are presented as mean + / - standard deviation . n = 3 independent biological replicates , one - way ANOVA with Dunnett ' s test on log transformed data . ns p > 0 . 05 , * * * p < 0 . 001 , and * * * * p < 0 . 0001 . C . ( Top ) Quantification of VEEV production ( 12 h post - infection , MOI of 2 , titered in BHK - 21J cells ) by plaque assay in control ( far left : without IFN - pretreatment ) , IRF9 / STAT1 / STAT2 - targeted , ZAP / IFIT3 / IFIT1 - targeted , or ZAP / IFIT3 / IFIT1 - targeted cells that were subsequently CRISPR targeted with a guide targeting BST2 , RSAD2 , ISG20 , or IFITM3 after IFNβ pretreatment ( IFNβ 20 U / mL for 24 h ) . ( Bottom ) Western blot shows IFN - mediated induction and CRISPR - based targeting of BST2 , RSAD2 , ISG20 , and IFITM3 . Black squares denote non - specific bands . Data information data are presented as mean + / - standard deviation . n = 3 independent biological replicates , one - way ANOVA with Dunnett ' s test on log transformed data . ns p > 0 . 05 , * * * p < 0 . 001 , and * * * * p < 0 . 0001 .

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

Figure 5A. Heatmaps showing the ratio of infected cells at 6, 9, and 12 h of infection with VEEV-GFP. U-2 OS control, IRF9/STAT1/STAT2-targeted, or ZAP/IFIT3/IFIT1-targeted cells were treated with or without IFNβ (20 U/mL for 24 h) and infected with an MOI of 0.5 (left), 1 (middle), or 5 (right) of VEEV-GFP. Infection was quantified by flow cytometry. Heatmap intensity represents the mean of 3 independent biological replicates.B. (Top) Quantification of VEEV production (12 h post-infection, MOI of 2, titered in BHK-21J cells) by plaque assay of control (far left: without IFN-pretreatment), IRF9/STAT1/STAT2-targeted, or ZAP/IFIT3/IFIT1-targeted cells after IFNβ pretreatment (IFNβ 20 U/mL for 24 h). (Bottom) Western blot shows IFN-mediated induction and CRISPR-based targeting of IRF9, STAT1, STAT2, ZAP, IFIT3, and IFIT1. Red asterisks denote long (**) and short (*) isoform of ZAP. Data information: data are presented as mean +/- standard deviation. n=3 independent biological replicates, one-way ANOVA with Dunnett's test on log transformed data. ns p > 0.05, ***p < 0.001, and ****p < 0.0001.C. (Top) Quantification of VEEV production (12 h post-infection, MOI of 2, titered in BHK-21J cells) by plaque assay in control (far left: without IFN-pretreatment), IRF9/STAT1/STAT2-targeted, ZAP/IFIT3/IFIT1-targeted, or ZAP/IFIT3/IFIT1-targeted cells that were subsequently CRISPR targeted with a guide targeting BST2, RSAD2, ISG20, or IFITM3 after IFNβ pretreatment (IFNβ 20 U/mL for 24 h). (Bottom) Western blot shows IFN-mediated induction and CRISPR-based targeting of BST2, RSAD2, ISG20, and IFITM3. Black squares denote non-specific bands. Data information data are presented as mean +/- standard deviation. n=3 independent biological replicates, one-way ANOVA with Dunnett's test on log transformed data. ns p > 0.05, ***p < 0.001, and ****p < 0.0001.


Figure 1B , C MCF - 7 cells expressing GFP - INPP4B or GFP - vector were cultured in growth media or EBSS for the indicated times , then lysed and immunblotted with LC3B antibodies and GAPDH antibodies as a loading control ( B ) . Data represent the relative LC3B - II levels normalized to GAPDH , and expressed relative to growth media - treated GFP - vector cells which were assigned an arbitrary value of 1 ( n = 3 experiments ) ( C ) . Data information : Data is presented as mean ± SD . p values determined by one - way ANOVA with Tukey post hoc test in C , D , E MCF - 7 cells expressing NT , INPP4B # 1 or INPP4B # 2 shRNA were cultured in growth media or EBSS for the indicated times , then lysed and immunblotted with LC3B antibodies and GAPDH antibodies as a loading control ( D ) . Data represent the relative LC3B - II levels normalized to GAPDH , and expressed relative to growth media - treated NT shRNA cells which were assigned an arbitrary value of 1 ( n = 3 experiments ) ( E ) . Data information : Data is presented as mean ± SD . p values determined by one - way ANOVA in E . F , G MCF - 7 cells expressing GFP - INPP4B or GFP - vector were cultured in growth media or EBSS for 4 hours , then fixed and immunostained with p62 antibodies , and co - stained with DAPI and phalloidin ( F ) . Data represent the number of p62 + puncta relative to cell area ( µm2 ) ( n = 3 experiments , > 50 cells / experiment ) ( G ) . Data information : Data is presented as mean ± SD . The insets at the lower right of each image are higher power regions of the boxed areas . Scale bar is 10 µm in F p values determined by one - way ANOVA with Tukey post hoc test in G , H , I MCF - 7 cells expressing GFP - INPP4B or GFP - vector were treated with 100 nM bafilomycin A1 or DMSO as a vehicle control for 4 hours , then lysed and immunblotted with LC3B antibodies and GAPDH antibodies as a loading control ( H ) . Data represent the relative LC3B - II levels normalized to GAPDH , and expressed relative to DMSO - treated GFP - vector cells which were assigned an arbitrary value of 1 ( I ) ( n = 3 experiments ) . Data information : Data is presented as mean ± SD . p values determined by one - way ANOVA with Tukey post hoc test in , I , J , K MCF - 7 cells expressing GFP - INPP4B or GFP - vector were treated with 100 nM bafilomycin A1 or DMSO as a vehicle control for 4 hours , then fixed and immunostained with p62 antibodies , and co - stained with DAPI and phalloidin ( J ) . Data represent the number of p62 + puncta relative to cell area ( µm2 ) ( n = 3 experiments , > 50 cells / experiment ) ( K ) . Data information : Data is presented as mean ± SD . The insets at the lower right of each image are higher power regions of the boxed areas . Scale bar is 10 µm in J . p values determined by one - way ANOVA with Tukey post hoc test in K , L , M MCF - 7 cells expressing GFP - INPP4B or GFP - vector were treated with 2 µM BYL719 or DMSO as a vehicle control for the indicated times . Cells were lysed and immunblotted with LC3B , pAKTS473 , AKT ( pan ) , pS6KT389 or S6K antibodies and GAPDH antibodies as a loading control ( L ) . Data represent the relative LC3B - II levels normalized to GAPDH , and expressed relative to DMSO - treated GFP - vector cells which were assigned an arbitrary value of 1 ( n = 3 experiments ) ( M ) . Data information : Data is presented as mean ± SD . p values determined by one - way ANOVA with Tukey post hoc test in M , N , O MCF - 7 cells expressing GFP - INPP4B or GFP - vector were treated with 1 µM or 10 µM SAR405 or DMSO as a vehicle control for 4 hours . Cells were lysed and immunblotted with LC3B antibodies and GAPDH antibodies as a loading control ( N ) . Data represent the relative LC3B - II levels normalized to GAPDH , and expressed relative to DMSO - treated GFP - vector cells which were assigned an arbitrary value of 1 ( n = 3 experiments ) ( O ) . Data information : Data is presented as mean ± SD . p values determined by one - way ANOVA with Tukey post hoc test in 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 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

Figure 1B, C MCF-7 cells expressing GFP-INPP4B or GFP-vector were cultured in growth media or EBSS for the indicated times, then lysed and immunblotted with LC3B antibodies and GAPDH antibodies as a loading control (B). Data represent the relative LC3B-II levels normalized to GAPDH, and expressed relative to growth media-treated GFP-vector cells which were assigned an arbitrary value of 1 (n=3 experiments) (C). Data information: Data is presented as mean ± SD. p values determined by one-way ANOVA with Tukey post hoc test in C,D, E MCF-7 cells expressing NT, INPP4B #1 or INPP4B #2 shRNA were cultured in growth media or EBSS for the indicated times, then lysed and immunblotted with LC3B antibodies and GAPDH antibodies as a loading control (D). Data represent the relative LC3B-II levels normalized to GAPDH, and expressed relative to growth media-treated NT shRNA cells which were assigned an arbitrary value of 1 (n=3 experiments) (E). Data information: Data is presented as mean ± SD. p values determined by one-way ANOVA in E.F, G MCF-7 cells expressing GFP-INPP4B or GFP-vector were cultured in growth media or EBSS for 4 hours, then fixed and immunostained with p62 antibodies, and co-stained with DAPI and phalloidin (F). Data represent the number of p62+ puncta relative to cell area (µm2) (n=3 experiments, >50 cells/experiment) (G). Data information: Data is presented as mean ± SD. The insets at the lower right of each image are higher power regions of the boxed areas. Scale bar is 10 µm in F p values determined by one-way ANOVA with Tukey post hoc test in G,H, I MCF-7 cells expressing GFP-INPP4B or GFP-vector were treated with 100 nM bafilomycin A1 or DMSO as a vehicle control for 4 hours, then lysed and immunblotted with LC3B antibodies and GAPDH antibodies as a loading control (H). Data represent the relative LC3B-II levels normalized to GAPDH, and expressed relative to DMSO-treated GFP-vector cells which were assigned an arbitrary value of 1 (I) (n=3 experiments). Data information: Data is presented as mean ± SD. p values determined by one-way ANOVA with Tukey post hoc test in , I,J, K MCF-7 cells expressing GFP-INPP4B or GFP-vector were treated with 100 nM bafilomycin A1 or DMSO as a vehicle control for 4 hours, then fixed and immunostained with p62 antibodies, and co-stained with DAPI and phalloidin (J). Data represent the number of p62+ puncta relative to cell area (µm2) (n=3 experiments, >50 cells/experiment) (K). Data information: Data is presented as mean ± SD. The insets at the lower right of each image are higher power regions of the boxed areas. Scale bar is 10 µm in J. p values determined by one-way ANOVA with Tukey post hoc test in K,L, M MCF-7 cells expressing GFP-INPP4B or GFP-vector were treated with 2 µM BYL719 or DMSO as a vehicle control for the indicated times. Cells were lysed and immunblotted with LC3B, pAKTS473, AKT(pan), pS6KT389 or S6K antibodies and GAPDH antibodies as a loading control (L). Data represent the relative LC3B-II levels normalized to GAPDH, and expressed relative to DMSO-treated GFP-vector cells which were assigned an arbitrary value of 1 (n=3 experiments) (M). Data information: Data is presented as mean ± SD. p values determined by one-way ANOVA with Tukey post hoc test in M,N, O MCF-7 cells expressing GFP-INPP4B or GFP-vector were treated with 1 µM or 10 µM SAR405 or DMSO as a vehicle control for 4 hours. Cells were lysed and immunblotted with LC3B antibodies and GAPDH antibodies as a loading control (N). Data represent the relative LC3B-II levels normalized to GAPDH, and expressed relative to DMSO-treated GFP-vector cells which were assigned an arbitrary value of 1 (n=3 experiments) (O). Data information: Data is presented as mean ± SD. p values determined by one-way ANOVA with Tukey post hoc test in O,


Figure 2A , B MCF - 7 cells expressing NT , INPP4B # 1 or INPP4B # 2 shRNA were fixed and immunostained with recombinant GST - 2xFYVE and LAMP2 antibodies , and co - stained with DAPI ( A ) . Data represent the proportion of 2xFYVE + lysosomes ( n = 3 experiments , > 40 cells / experiment ) ( B ) . Arrows indicate co - localization between 2xFYVE and LAMP2 . Data information : Data is presented as mean ± SD . The insets at the lower right or bottom of each image are higher power regions of the boxed areas . Scale bar is 10 µm in A , p values determined by one - way ANOVA with Tukey post hoc test in BC MCF - 7 cells were treated with 0 . 02 % ( w / v ) saponin for 30 seconds , then fixed and immunostained with INPP4B and either LAMP1 or CD63 antibodies , and co - stained with DAPI . Arrows indicate co - localization between INPP4B and CD63 . Data information : The insets at the lower right or bottom of each image are higher power regions of the boxed areas . Scale bar is 10 µm in C , D , E MCF - 7 cells co - expressing GFP - INPP4B or GFP - vector , and Hrs or NT shRNA , were fixed and immunostained with recombinant GST - 2xFYVE and LAMP2 antibodies , and co - stained with DAPI ( D ) . Data represent the proportion of 2xFYVE + lysosomes ( n = 3 experiments , > 30 cells / experiment ) ( E ) . Arrows indicate co - localization between 2xFYVE and LAMP2 . Data information : Data is presented as mean ± SD . The insets at the lower right or bottom of each image are higher power regions of the boxed areas . Scale bar is 10 µm in D , p values determined by one - way ANOVA with Tukey post hoc test inF , G MCF - 7 cells co - expressing GFP - INPP4B or GFP - vector , and Hrs or NT shRNA , were fixed and immunostained with recombinant GST - 2xFYVE and CD63 antibodies , and co - stained with DAPI ( F ) . Data represent the proportion of 2xFYVE + late endosomes ( n = 3 experiments , > 30 cells / experiment ) ( G ) . Arrows indicate co - localization between 2xFYVE and LAMP2 . Data information : Data is presented as mean ± SD . The insets at the lower right or bottom of each image are higher power regions of the boxed areas . Scale bar is 10 µm in F . p values determined by one - way ANOVA with Tukey post hoc test in G , H , I MCF - 7 cells co - expressing GFP - INPP4B or GFP - vector , and Hrs or NT shRNA , were lysed and immunoblotted with LC3B antibodies , and GAPDH antibodies as a loading control ( H ) . Data represent the relative LC3B - II levels normalized to GAPDH , and expressed relative to GFP - vector ; NT shRNA cells which were assigned an arbitrary value of 1 ( n = 3 experiments ) ( I ) . Data information : Data is presented as mean ± SD . p values determined by one - way ANOVA in I .

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 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 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

Figure 2A, B MCF-7 cells expressing NT, INPP4B #1 or INPP4B #2 shRNA were fixed and immunostained with recombinant GST-2xFYVE and LAMP2 antibodies, and co-stained with DAPI (A). Data represent the proportion of 2xFYVE+ lysosomes (n=3 experiments, >40 cells/experiment) (B). Arrows indicate co-localization between 2xFYVE and LAMP2. Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in A, p values determined by one-way ANOVA with Tukey post hoc test in BC MCF-7 cells were treated with 0.02% (w/v) saponin for 30 seconds, then fixed and immunostained with INPP4B and either LAMP1 or CD63 antibodies, and co-stained with DAPI. Arrows indicate co-localization between INPP4B and CD63. Data information: The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in C,D, E MCF-7 cells co-expressing GFP-INPP4B or GFP-vector, and Hrs or NT shRNA, were fixed and immunostained with recombinant GST-2xFYVE and LAMP2 antibodies, and co-stained with DAPI (D). Data represent the proportion of 2xFYVE+ lysosomes (n=3 experiments, >30 cells/experiment) (E). Arrows indicate co-localization between 2xFYVE and LAMP2. Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in D, p values determined by one-way ANOVA with Tukey post hoc test inF, G MCF-7 cells co-expressing GFP-INPP4B or GFP-vector, and Hrs or NT shRNA, were fixed and immunostained with recombinant GST-2xFYVE and CD63 antibodies, and co-stained with DAPI (F). Data represent the proportion of 2xFYVE+ late endosomes (n=3 experiments, >30 cells/experiment) (G). Arrows indicate co-localization between 2xFYVE and LAMP2. Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in F. p values determined by one-way ANOVA with Tukey post hoc test in G,H, I MCF-7 cells co-expressing GFP-INPP4B or GFP-vector, and Hrs or NT shRNA, were lysed and immunoblotted with LC3B antibodies, and GAPDH antibodies as a loading control (H). Data represent the relative LC3B-II levels normalized to GAPDH, and expressed relative to GFP-vector;NT shRNA cells which were assigned an arbitrary value of 1 (n=3 experiments) (I). Data information: Data is presented as mean ± SD. p values determined by one-way ANOVA in I.


Figure 3A , B MCF - 7 cells expressing GFP - INPP4B or GFP - vector were fixed and immunostained with LAMP1 or LAMP2 antibodies , and co - stained with DAPI and phalloidin ( A ) . Data represent the number of LAMP1 + or LAMP2 + puncta relative to cell area ( µm2 ) ( n = 3 experiments , > 50 cells / experiment ) ( B ) . Data information : Data is presented as mean ± SD . Scale bar is 10 µm in A , p values determined by two - tailed unpaired t test in B , C , D Snapshots of Magic Red cathepsin B substrate and Hoechst 33342 captured in live MCF - 7 cells expressing GFP - INPP4B or GFP - vector ( C ) . Data represent the number of Magic Red cathepsin B + puncta per cell ( n = 3 experiments , > 50 cells / experiment ) ( D ) . Data information : Data is presented as mean ± SD . Scale bar is 10 µm in C , p values determined by two - tailed unpaired t test in D , E , F MCF - 7 cells expressing NT or INPP4B # 1 shRNA were cultured in growth media or EBSS for the indicated times , then fixed and immunostained with LAMP1 antibodies and co - stained with DAPI and phalloidin ( E ) . Data represent the number of LAMP1 + puncta relative to cell area ( µm2 ) ( n = 3 experiments , > 30 cells per experiment ) ( F ) . Data information : Data is presented as mean ± SD . Scale bar is 10 µm in p values determined by one - way ANOVA with Tukey post hoc test in FG , H MCF - 7 cells expressing GFP - INPP4B or GFP - vector were transfected with NT , PIK3CA # 1 or PIK3CA # 2 siRNA . After 24 hours , cells were fixed and immunostained with LAMP1 antibodies , and co - stained with DAPI and phalloidin ( G ) . Data represent the number of LAMP1 + puncta relative to cell area ( µm2 ) ( n = 3 experiments , > 40 cells per experiment ) ( H ) . Data information : Data is presented as mean ± SD . Scale bar is 10 µm in G . p values determined by one - way ANOVA in H .

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 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 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

Figure 3A, B MCF-7 cells expressing GFP-INPP4B or GFP-vector were fixed and immunostained with LAMP1 or LAMP2 antibodies, and co-stained with DAPI and phalloidin (A). Data represent the number of LAMP1+ or LAMP2+ puncta relative to cell area (µm2) (n=3 experiments, >50 cells/experiment) (B). Data information: Data is presented as mean ± SD. Scale bar is 10 µm in A, p values determined by two-tailed unpaired t test in B,C, D Snapshots of Magic Red cathepsin B substrate and Hoechst 33342 captured in live MCF-7 cells expressing GFP-INPP4B or GFP-vector (C). Data represent the number of Magic Red cathepsin B+ puncta per cell (n=3 experiments, >50 cells/experiment) (D). Data information: Data is presented as mean ± SD. Scale bar is 10 µm in C, p values determined by two-tailed unpaired t test in D,E, F MCF-7 cells expressing NT or INPP4B #1 shRNA were cultured in growth media or EBSS for the indicated times, then fixed and immunostained with LAMP1 antibodies and co-stained with DAPI and phalloidin (E). Data represent the number of LAMP1+ puncta relative to cell area (µm2) (n=3 experiments, >30 cells per experiment) (F). Data information: Data is presented as mean ± SD. Scale bar is 10 µm in p values determined by one-way ANOVA with Tukey post hoc test in FG, H MCF-7 cells expressing GFP-INPP4B or GFP-vector were transfected with NT, PIK3CA #1 or PIK3CA #2 siRNA. After 24 hours, cells were fixed and immunostained with LAMP1 antibodies, and co-stained with DAPI and phalloidin (G). Data represent the number of LAMP1+ puncta relative to cell area (µm2) (n=3 experiments, >40 cells per experiment) (H). Data information: Data is presented as mean ± SD. Scale bar is 10 µm in G. p values determined by one-way ANOVA in H.


Figure 4A , B Snapshots of LAMP1 - GFP , Magic Red cathepsin B substrate and Hoechst 33342 captured in live HeLa cells transfected with INPP4B or NT siRNA ( A ) . Data represent the number of LAMP1 + / Magic Red Cathepsin B + and LAMP1 + / Magic Red Cathepsin B − lysosomes ( terminal storage lysosomes ) per cell ( n = 3 experiments , > 50 cells / experiment ) ( B ) . Yellow arrows indicate LAMP1 + / MR Cathepsin B + lysosomes , and white arrows indicate terminal storage lysosomes . Data information : Data is presented as mean ± SD . The insets at the lower right of each image are higher power regions of the boxed areas . Scale bar is 10 µm in A , p values determined by two - way ANOVA with Holm - Sidak post - hoc test in BC Timelapse snapshots of LAMP1 - mCherry signals from MCF - 7 cells expressing GFP - INPP4B or GFP - vector captured by spinning disk microscopy . Maximum intensity projections of 3 z - planes taken 0 . 27 µm apart are shown . Arrows indicate lysosome reformation . Data information : The insets at the lower right of each image are higher power regions of the boxed areas . Scale bar is 10 µm in C . E Data represent the number of LAMP1 + reformation events per cell per minute ( n = 12 GFP - vector cells , n = 15 GFP - INPP4B cells ) . Data information : Data is presented as mean ± SD . p values determined by two - tailed unpaired t test in E .

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 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

Figure 4A, B Snapshots of LAMP1-GFP, Magic Red cathepsin B substrate and Hoechst 33342 captured in live HeLa cells transfected with INPP4B or NT siRNA (A). Data represent the number of LAMP1+/Magic Red Cathepsin B+ and LAMP1+/Magic Red Cathepsin B− lysosomes (terminal storage lysosomes) per cell (n=3 experiments, >50 cells/experiment) (B).Yellow arrows indicate LAMP1+/MR Cathepsin B+ lysosomes, and white arrows indicate terminal storage lysosomes. Data information: Data is presented as mean ± SD. The insets at the lower right of each image are higher power regions of the boxed areas. Scale bar is 10 µm in A, p values determined by two-way ANOVA with Holm-Sidak post-hoc test in BC Timelapse snapshots of LAMP1-mCherry signals from MCF-7 cells expressing GFP-INPP4B or GFP-vector captured by spinning disk microscopy. Maximum intensity projections of 3 z-planes taken 0.27 µm apart are shown. Arrows indicate lysosome reformation. Data information: The insets at the lower right of each image are higher power regions of the boxed areas. Scale bar is 10 µm in C.E Data represent the number of LAMP1+ reformation events per cell per minute (n=12 GFP-vector cells, n=15 GFP-INPP4B cells). Data information: Data is presented as mean ± SD. p values determined by two-tailed unpaired t test in E.


Figure 5A , B MCF - 7 cells expressing GFP - INPP4B or GFP - vector were treated with 5 µM YM201636 for 2 hours . Cells were fixed and immunostained with recombinant GST - 2xFYVE and LAMP2 antibodies , and co - stained with DAPI ( A ) . Data represent the proportion of 2xFYVE + lysosomes ( n = 3 experiments , > 30 cells / experiment ) ( B ) . Arrows show co - localization between 2xFYVE and LAMP2 . Data information : Data is presented as mean ± SD . The insets at the lower right or bottom of each image are higher power regions of the boxed areas . Scale bar is 10 µm in A p values determined by one - way ANOVA in BD - F MCF - 7 cells expressing GFP - INPP4B or GFP - vector were treated with 5 µM YM201636 or DMSO as a vehicle control for 2 hours , then YM201636 was washed out for 1 , 2 or 4 hours . Cells were fixed and immunostained with LAMP1 antibodies , and co - stained with DAPI and phalloidin ( D ) . Data represent the number of LAMP1 + puncta relative to cell area ( µm2 ) ( E ) , and the lysosome fold change relative to the YM201636 - treated condition for each cell line ( F ) ( n = 3 experiments , > 30 cells per experiment ) . Data information : Data is presented as mean ± SD . The insets at the lower right or bottom of each image are higher power regions of the boxed areas . Scale bar is 10 µm in D . p values determined by one - way ANOVA in E , or by two - tailed unpaired t test of the area under the curve in F . G , H MCF - 7 cells expressing GFP - INPP4B or GFP - vector were treated with 1 , 2 or 5 µM YM201636 or DMSO as a vehicle control for 4 hours . Cells were lysed and immunoblotted with LC3B antibodies , and GAPDH antibodies as a loading control ( G ) . Data represent the relative LC3B - II levels normalized to GAPDH , and expressed relative to DMSO - treated GFP - vector cells which were assigned an arbitrary value of 1 ( n = 3 experiments ) ( H ) . Data information : Data is presented as mean ± SD . p values determined by one - way ANOVA in H ,

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 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 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

Figure 5A, B MCF-7 cells expressing GFP-INPP4B or GFP-vector were treated with 5 µM YM201636 for 2 hours. Cells were fixed and immunostained with recombinant GST-2xFYVE and LAMP2 antibodies, and co-stained with DAPI (A). Data represent the proportion of 2xFYVE+ lysosomes (n=3 experiments, >30 cells/experiment) (B). Arrows show co-localization between 2xFYVE and LAMP2. Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in A p values determined by one-way ANOVA in BD-F MCF-7 cells expressing GFP-INPP4B or GFP-vector were treated with 5 µM YM201636 or DMSO as a vehicle control for 2 hours, then YM201636 was washed out for 1, 2 or 4 hours. Cells were fixed and immunostained with LAMP1 antibodies, and co-stained with DAPI and phalloidin (D). Data represent the number of LAMP1+ puncta relative to cell area (µm2) (E), and the lysosome fold change relative to the YM201636-treated condition for each cell line (F) (n=3 experiments, >30 cells per experiment). Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in D. p values determined by one-way ANOVA in E, or by two-tailed unpaired t test of the area under the curve in F.G, H MCF-7 cells expressing GFP-INPP4B or GFP-vector were treated with 1, 2 or 5 µM YM201636 or DMSO as a vehicle control for 4 hours. Cells were lysed and immunoblotted with LC3B antibodies, and GAPDH antibodies as a loading control (G). Data represent the relative LC3B-II levels normalized to GAPDH, and expressed relative to DMSO-treated GFP-vector cells which were assigned an arbitrary value of 1 (n=3 experiments) (H). Data information: Data is presented as mean ± SD. p values determined by one-way ANOVA in H,