Figure 1 ( B ) Maximum intensity projections ( top ) and representative sections ( bottom ) of typical nuclei of the indicated cell types stained with DAPI . Scale bars , 3 μm . ( C ) Areas of DAPI - dense regions ( top ) , distance of DAPI - dense regions from the nuclear periphery ( middle ) , and variance of DAPI signals ( bottom ) . The point marks the median while the thick and thin lines correspond to 66 % and 95 % intervals , respectively . Number of DAPI dense regions = 950 / 1450 / 839 / 1535 / 736 and number of slices = 90 / 115 / 95 / 135 / 110 for mESC / EpiLC / d2 / d4c7 mPGCLC / GSC . Significances are computed using Wilcoxon rank - sum tests , p - values from top to bottom 4 . 37e - 3 , 1 . 62e - 3 , 2 . 99e - 2 , 2 . 03e - 10 , 4 . 03e - 1 , < 2 . 2e - 16 , 1 . 31e - 3 , 8 . 94e - 3 , 1 . 06e - 4 , 5 . 62e - 2 , 4 . 63e - 5 , 7 . 65e - 13 . P - value symbol brackets : ∗ ∗ ∗ ∗ = [ 0 , 0 . 0001 ) ; ∗ ∗ ∗ = [ 0 . 0001 , 0 . 001 ] ; ∗ ∗ = [ 0 . 001 , 0 . 01 ) ; ∗ = [ 0 . 01 , 0 . 05 ) ; ns = [ 0 . 05 , ( D ) ( left ) Fluorescence in situ hybridization ( FISH ) against chromosome 16 ( red ) with DAPI staining ( grey ) . Z - stacked representative images are paired with magnified views . ( right ) Distributions of surface volumes for chr16 . The point marks the median while the thick and thin lines correspond to 66 % and 95 % intervals , respectively . Number of cells = 51 / 68 / 53 for mESC / EpiLC / GSC . Scale bars , 5 μm . Significances are computed using Wilcoxon rank - sum tests , p - values from left to right : 4 . 16e - 2 , 4 . 33e - 6 , 8 . 68e - 9 .

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 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 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

Figure 1(B) Maximum intensity projections (top) and representative sections (bottom) of typical nuclei of the indicated cell types stained with DAPI. Scale bars, 3 μm.(C) Areas of DAPI-dense regions (top), distance of DAPI-dense regions from the nuclear periphery (middle), and variance of DAPI signals (bottom). The point marks the median while the thick and thin lines correspond to 66% and 95% intervals, respectively. Number of DAPI dense regions = 950/1450/839/1535/736 and number of slices = 90/115/95/135/110 for mESC/EpiLC/d2/d4c7 mPGCLC/GSC. Significances are computed using Wilcoxon rank-sum tests, p-values from top to bottom 4.37e-3, 1.62e-3, 2.99e-2, 2.03e-10, 4.03e-1, <2.2e-16, 1.31e-3, 8.94e-3, 1.06e-4, 5.62e-2, 4.63e-5, 7.65e-13. P-value symbol brackets: ∗∗∗∗ = [0, 0.0001); ∗∗∗ = [0.0001, 0.001]; ∗∗ = [0.001, 0.01); ∗ = [0.01, 0.05); ns = [0.05,(D) (left) Fluorescence in situ hybridization (FISH) against chromosome 16 (red) with DAPI staining (grey). Z-stacked representative images are paired with magnified views. (right) Distributions of surface volumes for chr16. The point marks the median while the thick and thin lines correspond to 66% and 95% intervals, respectively. Number of cells = 51/68/53 for mESC/EpiLC/GSC. Scale bars, 5 μm. Significances are computed using Wilcoxon rank-sum tests, p-values from left to right: 4.16e-2, 4.33e-6, 8.68e-9.


Figure 4 ( H ) Western blot for lamin B1 in different cell types ( bottom ) and quantification normalized by β - actin ( top ) . ( L ) ( top ) Representative images of FISH against major satellite repeats in EpiLCs and GSCs . Scale bars , 10 μm ; ( bottom ) percentage of the pericentromeres detached from the nuclear lamina in EpiLCs and GSCs . The point marks the median while the thick and thin lines correspond to 66 % and 95 % intervals , respectively . Number of cells = 18 / 22 for EpiLC / GSC .

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

Figure 4(H) Western blot for lamin B1 in different cell types (bottom) and quantification normalized by β-actin (top).(L) (top) Representative images of FISH against major satellite repeats in EpiLCs and GSCs. Scale bars, 10 μm; (bottom) percentage of the pericentromeres detached from the nuclear lamina in EpiLCs and GSCs. The point marks the median while the thick and thin lines correspond to 66% and 95% intervals, respectively. Number of cells = 18/22 for EpiLC/GSC.


Figure 5 ( I ) ( left ) Western blot for G9a , GLP , Setdb1 and α - tubulin ; ( right ) quantification normalized by α - Tubulin .

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

Figure 5(I) (left) Western blot for G9a, GLP, Setdb1 and α-tubulin; (right) quantification normalized by α-Tubulin.


Figure 6 ( K ) ( top ) FISH against the Y chromosome ; ( bottom ) sphericity of the Y chromosome FISH signals ; ( right ) distributions Y chromosome surface volumes . The point marks the median while the thick and thin lines correspond to 66 % and 95 % intervals , respectively . Number of cells = 89 / 76 / 69 for mESC / EpiLC / GSC . Scale bar , 10 μm . Significances are computed using Wilcoxon rank - sum tests , p - values

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

Figure 6(K) (top) FISH against the Y chromosome; (bottom) sphericity of the Y chromosome FISH signals; (right) distributions Y chromosome surface volumes. The point marks the median while the thick and thin lines correspond to 66% and 95% intervals, respectively. Number of cells = 89/76/69 for mESC/EpiLC/GSC. Scale bar, 10 μm. Significances are computed using Wilcoxon rank-sum tests, p-values from


Figure 7 ( B ) Maximum intensity projections ( top ) and representative sections ( bottom ) of typical nuclei of GSCs and GSCLCs stained with DAPI . Scale bars , 3 μm . ( C ) Areas of DAPI - dense regions ( left ) , distance of DAPI - dense regions from the nuclear periphery ( middle ) , and variance of DAPI signals ( right ) . The point marks the median while the thick and thin lines correspond to 66 % and 95 % intervals , respectively . Number of DAPI dense regions = 1535 / 736 / 1227 and number of slices = 135 / 110 / 120 for d4c7 mPGCLC / GSC / GSCLC . Significances are computed using Wilcoxon rank - sum tests , p - values from left to right : 2 . 03e - 10 , 1 . 69e - 9 , 0 . 123 , 0 . 00894 , 8 . 02e - 8 , 0 . 0707 , 7 . 65e - 13 , 0 . 417 , 2 . 07e - 12 .

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

Figure 7(B) Maximum intensity projections (top) and representative sections (bottom) of typical nuclei of GSCs and GSCLCs stained with DAPI. Scale bars, 3 μm. (C) Areas of DAPI-dense regions (left), distance of DAPI-dense regions from the nuclear periphery (middle), and variance of DAPI signals (right). The point marks the median while the thick and thin lines correspond to 66% and 95% intervals, respectively. Number of DAPI dense regions = 1535/736/1227 and number of slices = 135/110/120 for d4c7 mPGCLC/GSC/GSCLC. Significances are computed using Wilcoxon rank-sum tests, p-values from left to right: 2.03e-10, 1.69e-9, 0.123, 0.00894, 8.02e-8, 0.0707, 7.65e-13, 0.417, 2.07e-12.


Figure 1F Representative BLI and histology of organotypic cultures with H2030 - BrM cancer cells treated with DMSO or decreasing concentrations of DEBIO - 0932 . Scale bar : 100 µm ; high magnification : 50 µm . G Quantification of the bioluminescence signal emitted by each condition shown in ( F ) at day 3 normalized by the initial value obtained at day 0 and normalized to the organotypic cultures treated with DMSO . Day 0 is considered 12 - 16h after the addition of cancer cells and treatment or DMSO . Values are shown in box - and - whisker plots where each dot is an organotypic culture and the line in the box corresponds to the median . The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value ( n = 8 DMSO , n = 8 BKM120 and n = 7 per concentration of DEBIO - 0932 - treated organotypic cultures , 2 independent experiments ) . P value was calculated using two - tailed t - test .

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

Figure 1F Representative BLI and histology of organotypic cultures with H2030-BrM cancer cells treated with DMSO or decreasing concentrations of DEBIO-0932. Scale bar: 100 µm; high magnification: 50 µm. G Quantification of the bioluminescence signal emitted by each condition shown in (F) at day 3 normalized by the initial value obtained at day 0 and normalized to the organotypic cultures treated with DMSO. Day 0 is considered 12-16h after the addition of cancer cells and treatment or DMSO. Values are shown in box-and-whisker plots where each dot is an organotypic culture and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (n=8 DMSO, n=8 BKM120 and n=7 per concentration of DEBIO-0932-treated organotypic cultures, 2 independent experiments). P value was calculated using two-tailed t-test.


Figure 2B Representative images of organotypic cultures with established metastases with various glial components of the microenvironment labelled . Scale bar : 75 µm . Each individual condition was evaluated in several organotypic cultures ( 3 - 6 slices ) . D Quantification of LDH levels in the conditioned media of organotypic slices cultured during 3 days relative to a lysate of the same preparation . Values are shown as mean + s . e . m . ( n = 3 organotypic cultures per experimental condition , 1 independent experiment ) .

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 O O 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 2B Representative images of organotypic cultures with established metastases with various glial components of the microenvironment labelled. Scale bar: 75 µm. Each individual condition was evaluated in several organotypic cultures (3-6 slices).D Quantification of LDH levels in the conditioned media of organotypic slices cultured during 3 days relative to a lysate of the same preparation. Values are shown as mean + s.e.m. (n=3 organotypic cultures per experimental condition, 1 independent experiment).


Figure 3A - C Immunofluorescence against HSP90 in mouse brains with established metastases . ( A ) HSP90 positive structures in areas not affected by the metastasis includes the medial habenula , where neurons co - localize with the chaperone . Scale bars : 100 µm ( low magnification ) , 50 µm ( medial habenula nucleus ) , 12 µm ( high magnification neurons ) . ( B ) Established metastases from different primary origins and oncogenomic profiles stained with HSP90 . Dotted lines delineate the metastasis ( cc : cancer cells ) . Scale bars : 75 µm . ( C ) Iba1 colocalizes with HSP90 within areas affected by metastases . BB : bisbenzamide . Scale bar : 75 µm ( low magnification ) , 12 µm ( high magnification ) . E Representative human brain metastases showing different intensities or scores for HSP90 . Scale bar : 50 µm . F Quantification of HSP90 in human brain metastases . 59 out of 60 ( 98 % ) showed positive staining of HSP90 in the tumor , 15 ( 25 % ) scored with 3 ( strong ) , 36 ( 60 % ) with 2 ( moderate ) and 8 ( 13 % ) with 1 ( weak ) according to the signal intensity of HSP90 in the cytoplasm of cancer cells . Representative human brain metastases showing different percentages of nuclear HSP90 . Scale bars : ( J ) 50 µm Black arrows point to cancer cells positive for HSP90 in the nucleus . Representative human brain metastases showing different percentages of nuclear HSP90 . Scale bars : ( K ) low magnification : 100 µm ; high magnification : 10 µm . L Quantification of nuclear HSP90 in human brain metastases . 54 out of 60 samples ( 90 % ) showed positive nuclear HSP90 in the tumor . 27 ( 45 % ) showed 1 - 5 % ( moderate ) and 27 ( 45 % ) showed > 5 % ( high ) of nuclear HSP90 . M Quantification of nuclear HSP90 in human primary tumors . 19 out of 30 ( 63 % ) showed positive nuclear HSP90 in the tumor . 9 ( 30 % ) showed 1 - 5 % ( moderate ) and 10 ( 33 % ) showed > 5 % ( high ) of nuclear HSP90 .

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

Figure 3A-C Immunofluorescence against HSP90 in mouse brains with established metastases. (A) HSP90 positive structures in areas not affected by the metastasis includes the medial habenula, where neurons co-localize with the chaperone. Scale bars: 100 µm (low magnification), 50 µm (medial habenula nucleus), 12 µm (high magnification neurons). (B) Established metastases from different primary origins and oncogenomic profiles stained with HSP90. Dotted lines delineate the metastasis (cc: cancer cells). Scale bars: 75 µm. (C) Iba1 colocalizes with HSP90 within areas affected by metastases. BB: bisbenzamide. Scale bar: 75 µm (low magnification), 12 µm (high magnification).E Representative human brain metastases showing different intensities or scores for HSP90. Scale bar: 50 µm. F Quantification of HSP90 in human brain metastases. 59 out of 60 (98%) showed positive staining of HSP90 in the tumor, 15 (25%) scored with 3 (strong), 36 (60%) with 2 (moderate) and 8 (13%) with 1 (weak) according to the signal intensity of HSP90 in the cytoplasm of cancer cells.Representative human brain metastases showing different percentages of nuclear HSP90. Scale bars: (J) 50 µm Black arrows point to cancer cells positive for HSP90 in the nucleus.Representative human brain metastases showing different percentages of nuclear HSP90. Scale bars: (K) low magnification: 100 µm; high magnification: 10 µm.L Quantification of nuclear HSP90 in human brain metastases. 54 out of 60 samples (90%) showed positive nuclear HSP90 in the tumor. 27 (45%) showed 1-5% (moderate) and 27 (45%) showed >5% (high) of nuclear HSP90. M Quantification of nuclear HSP90 in human primary tumors. 19 out of 30 (63%) showed positive nuclear HSP90 in the tumor. 9 (30%) showed 1-5% (moderate) and 10 (33%) showed >5% (high) of nuclear HSP90.


Figure 4B Representative in vivo and ex vivo images of vehicle and DEBIO - 0932 treated mice 5 weeks ( experimental endpoint ) after intracardiac inoculation of H2030 - BrM cells . E Representative sections of brains from vehicle and DEBIO - 0932 treated mice in ( B - D ) . The dotted lines surround the metastases ( GFP + ) . Representative field of view of metastasis stained with GFP and cleaved caspase 3 . Scale bars : slices , 1mm ; cleaved caspase 3 , 50 µm . F Quantification of established metastases found in vehicle and DEBIO - 0932 treated brains from panel ( E ) . Values are shown in box - and - whisker plots where every dot represents a different brain and the line in the box corresponds to the median . The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value ( vehicle : n = 10 brains ; DEBIO - 0932 : n = 14 brains ) . P value was calculated using two - tailed t - test . G Quantification of number of cleaved caspase 3 ( CC3 + ) in cancer cells found in vehicle and DEBIO - 0932 treated brains from panel ( E ) . Values are shown in box - and - whisker plots where every dot is a metastatic lesion and the line in the box corresponds to the median . The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value ( n = 8 metastatic lesions from 4 brains per condition ) . P value was calculated using two - tailed t - test . I Representative BrM - PDOC stained with proliferation markers ( BrdU ) and markers of the microenvironment ( GFAP for astrocytes , Iba1 for microglia / macrophages ) . Scale bar : 50 µm .

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

Figure 4B Representative in vivo and ex vivo images of vehicle and DEBIO-0932 treated mice 5 weeks (experimental endpoint) after intracardiac inoculation of H2030-BrM cells.E Representative sections of brains from vehicle and DEBIO-0932 treated mice in (B-D). The dotted lines surround the metastases (GFP+). Representative field of view of metastasis stained with GFP and cleaved caspase 3. Scale bars: slices,1mm; cleaved caspase 3, 50 µm. F Quantification of established metastases found in vehicle and DEBIO-0932 treated brains from panel (E). Values are shown in box-and-whisker plots where every dot represents a different brain and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (vehicle: n=10 brains; DEBIO-0932: n=14 brains). P value was calculated using two-tailed t-test. G Quantification of number of cleaved caspase 3 (CC3+) in cancer cells found in vehicle and DEBIO-0932 treated brains from panel (E). Values are shown in box-and-whisker plots where every dot is a metastatic lesion and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (n=8 metastatic lesions from 4 brains per condition). P value was calculated using two-tailed t-test.I Representative BrM-PDOC stained with proliferation markers (BrdU) and markers of the microenvironment (GFAP for astrocytes, Iba1 for microglia/ macrophages). Scale bar: 50 µm.


Figure 5F qRT - PCR of H2030 - BrM brain metastases obtained from animals during neurosurgery compared to relapsed metastases from the corresponding animals . A panel of five genes related to HSP90 pathway is evaluated . Values are shown in box - and - whisker plots where every dot represents a different animal and the line in the box corresponds to the median . The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value ( n = 5 mice per experimental condition ) . P value was calculated using two - tailed t - test . H Representative images of vehicle and DEBIO - 0932 treated mice after neurosurgery until experimental endpoint at 6 and 8 weeks for vehicle and DEBIO - 0932 treated mice , respectively .

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

Figure 5F qRT-PCR of H2030-BrM brain metastases obtained from animals during neurosurgery compared to relapsed metastases from the corresponding animals. A panel of five genes related to HSP90 pathway is evaluated. Values are shown in box-and-whisker plots where every dot represents a different animal and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (n=5 mice per experimental condition). P value was calculated using two-tailed t-test.H Representative images of vehicle and DEBIO-0932 treated mice after neurosurgery until experimental endpoint at 6 and 8 weeks for vehicle and DEBIO-0932 treated mice, respectively.


Figure 6C Volcano plot with deregulated proteins ( red : upregulated ; green : downregulated ) found in brain metastases treated with DEBIO - 0932 compared to DMSO ( n = 3 biological replicates ( mice ) per condition , n ≥ 12 brain metastases per mouse were pooled together ) . Proteins with a P value < 0 . 05 and a log2 ratio > 1 or < - 1 were defined as deregulated . Gray dotted lines indicate P value and log2 ratio cut offs . The names of the top deregulated proteins are shown . G Kaplan - Meier curves showing significant correlation between worse survival post - brain metastasis and high expression levels of the HSP90 signature ( AHR , DDA1 , UBE4B , GPATCH8 ) in a cohort of 45 breast cancer brain metastasis patients . H Representative images ( selected cases obtained from Fig EV6M ) and histological score of AHR , DDA1 and UBE4B in human brain metastases ( n = 16 ) according to the signal intensity of the corresponding protein in cancer cells . K Quantification of ex vivo BLI of brains and thoracic regions at the endpoint of the experiment . Values are shown in box - and - whisker plots where every dot represents a different animal and the line in the box corresponds to the median . The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value ( n = 9 shControl mice and n = 10 shAHR # 1 mice ) . P value was calculated using two - tailed t - test . N Representative images of primary tumors with high ( red dot ) or low ( grey dot ) UBE4B levels . A few cases of matched primary - metastases allowed to evaluate the HSP90 - dependent protein . Scale bar : : 100 µm . P Kaplan - Meier curve comparing relapse - free survival of primary tumors with high and low values of UBE4B . P value was calculated using log - rank ( Mantel - Cox ) test .

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

Figure 6C Volcano plot with deregulated proteins (red: upregulated; green: downregulated) found in brain metastases treated with DEBIO-0932 compared to DMSO (n=3 biological replicates (mice) per condition, n≥12 brain metastases per mouse were pooled together). Proteins with a P value <0.05 and a log2 ratio >1 or <-1 were defined as deregulated. Gray dotted lines indicate P value and log2 ratio cut offs. The names of the top deregulated proteins are shown.G Kaplan-Meier curves showing significant correlation between worse survival post-brain metastasis and high expression levels of the HSP90 signature (AHR, DDA1, UBE4B, GPATCH8) in a cohort of 45 breast cancer brain metastasis patients.H Representative images (selected cases obtained from Fig EV6M) and histological score of AHR, DDA1 and UBE4B in human brain metastases (n=16) according to the signal intensity of the corresponding protein in cancer cells.K Quantification of ex vivo BLI of brains and thoracic regions at the endpoint of the experiment. Values are shown in box-and-whisker plots where every dot represents a different animal and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (n=9 shControl mice and n=10 shAHR#1 mice). P value was calculated using two-tailed t-test.N Representative images of primary tumors with high (red dot) or low (grey dot) UBE4B levels. A few cases of matched primary-metastases allowed to evaluate the HSP90-dependent protein. Scale bar: : 100 µm.P Kaplan-Meier curve comparing relapse-free survival of primary tumors with high and low values of UBE4B. P value was calculated using log-rank (Mantel-Cox) test.


Figure 7D Representative organotypic cultures from the experiment in panel ( C ) . RFP is an internal control probe labelling cancer cells independent of autophagy flux and GFP indicate GFP - LC3 + puncta . The dotted line in the upper panel delimits a high magnification area shown in the lower panel respect to the GFP signal derived from GFP - LC3 accumulation . Dotted lines in lower panel surround individual cancer cells . Asterisk labels the area in the cell magnified in the high magnification panel showing the GFP - LC3 + puncta . Scale bar : low magnificantion , 25 µm ; high magnification ( cells ) , 10 µm ; high magnification ( puncta ) , 2 . 5 µm . E Quantification of GFP - LC3 + vesicles per cell of the experiment in panel ( C ) . Values are shown in box - and - whisker plots where every dot represents a field of view of an organotypic culture and the line in the box corresponds to the median . The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value ( DMSO : n = 15 fields of view , 2232 cancer cells from 3 organotypic cultures ; DEBIO - 0932 : n = 20 fields of view , 3260 cancer cells from 4 organotypic cultures ) . P value was calculated using two - tailed t - test .

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Figure 7D Representative organotypic cultures from the experiment in panel (C). RFP is an internal control probe labelling cancer cells independent of autophagy flux and GFP indicate GFP-LC3+ puncta. The dotted line in the upper panel delimits a high magnification area shown in the lower panel respect to the GFP signal derived from GFP-LC3 accumulation. Dotted lines in lower panel surround individual cancer cells. Asterisk labels the area in the cell magnified in the high magnification panel showing the GFP-LC3+ puncta. Scale bar: low magnificantion, 25 µm; high magnification (cells), 10 µm; high magnification (puncta), 2.5 µm. E Quantification of GFP-LC3+ vesicles per cell of the experiment in panel (C). Values are shown in box-and-whisker plots where every dot represents a field of view of an organotypic culture and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (DMSO: n=15 fields of view, 2232 cancer cells from 3 organotypic cultures; DEBIO-0932: n=20 fields of view, 3260 cancer cells from 4 organotypic cultures). P value was calculated using two-tailed t-test.


Figure 1A Representative Gli1ZF / DNA structure extrapolated from MD trajectories . Gli1ZF is shown as blue cartoon , residues involved in binding to DNA ( based on single point mutation study ) are shown as magenta sticks , and Zn ions as gray spheres . B Effect of Gli1ZF mutants on the binding affinity to DNA as predicted by in silico alanine scanning . The ∆ ∆ G was calculated along MD trajectories as the difference between the ∆ G of each Gli1ZF alanine mutant and Gli1ZF ‐ WT . Results are shown as ∆ ∆ G values in kcal / mol calculated by means of the MM ‐ PBSA methods ± SEM . C Effect of Gli1ZF mutants on Gli1 ‐ dependent transcriptional activation . Luciferase assay was performed in HEK293T cells transfected with 12XGliBS ‐ Luc ( GliBS , Gli binding site ) , pRL ‐ TK Renilla ( normalization control ) , Flag ‐ Gli1 WT or the indicated Flag ‐ Gli1 mutants . Data show the mean ± SD of three independent experiments . * P 0 . 01 ; * * P 0 . 05 versus Gli1 WT . Western blot analysis of Flag ‐ Gli1 WT or the indicated Flag ‐ Gli1 mutant expression levels ( bottom panel ) . E Gli1 / DNA binding . Double ‐ stranded oligonucleotide containing the canonical GliBS sequence ( 5 ′ - TTGCCTACCTGGGTGGTCTCTCCACTT - 3 ′ ) or mutated GliBS sequence used as control ( 5 ′ - TTGCCTACCTCCCACTTCTCTCCACTT - 3 ′ ) was used as probe ( P ) in EMSA experiments . The assay was performed using recombinant GST ‐ Gli1ZF ‐ WT ( Gli1 zinc finger fragment : aa 242 - 424 ) , GST ‐ Gli1ZF ‐ K350A and GST ‐ Gli1ZF ‐ K340A . The graph on the right indicates ratio ( mean arbitrary units ± SD from three independent experiments ) of GST ‐ Gli1ZF ‐ WT or GST ‐ Gli1ZF mutants bound to the labeled GliBS probe / GliBS ‐ free probe normalized to the amount of GST ‐ Gli1ZF ‐ WT / DNA binding ( as described in ) . * P 0 . 05 versus Gli1 WT .

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 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 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 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

Figure 1A Representative Gli1ZF/DNA structure extrapolated from MD trajectories. Gli1ZF is shown as blue cartoon, residues involved in binding to DNA (based on single point mutation study) are shown as magenta sticks, and Zn ions as gray spheres.B Effect of Gli1ZF mutants on the binding affinity to DNA as predicted by in silico alanine scanning. The ∆∆G was calculated along MD trajectories as the difference between the ∆G of each Gli1ZF alanine mutant and Gli1ZF‐WT. Results are shown as ∆∆G values in kcal/mol calculated by means of the MM‐PBSA methods ± SEM.C Effect of Gli1ZF mutants on Gli1‐dependent transcriptional activation. Luciferase assay was performed in HEK293T cells transfected with 12XGliBS‐Luc (GliBS, Gli binding site), pRL‐TK Renilla (normalization control), Flag‐Gli1 WT or the indicated Flag‐Gli1 mutants. Data show the mean ± SD of three independent experiments. *P 0.01; **P 0.05 versus Gli1 WT. Western blot analysis of Flag‐Gli1 WT or the indicated Flag‐Gli1 mutant expression levels (bottom panel).E Gli1/DNA binding. Double‐stranded oligonucleotide containing the canonical GliBS sequence (5′-TTGCCTACCTGGGTGGTCTCTCCACTT-3′) or mutated GliBS sequence used as control (5′-TTGCCTACCTCCCACTTCTCTCCACTT-3′) was used as probe (P) in EMSA experiments. The assay was performed using recombinant GST‐Gli1ZF‐WT (Gli1 zinc finger fragment: aa 242-424), GST‐Gli1ZF‐K350A and GST‐Gli1ZF‐K340A. The graph on the right indicates ratio (mean arbitrary units ± SD from three independent experiments) of GST‐Gli1ZF‐WT or GST‐Gli1ZF mutants bound to the labeled GliBS probe/GliBS‐free probe normalized to the amount of GST‐Gli1ZF‐WT/DNA binding (as described in ). *P 0.05 versus Gli1 WT.


Figure 2A Inhibition of Gli1 ‐ induced transcription in transfected HEK293T cells . HEK293T cells were transfected with 12XGliBS ‐ Luc and pRL ‐ TK Renilla ( normalization control ) plus control ( empty ) or Gli1 vector and treated with increasing concentrations of GlaB or GANT61 . Treatment time was 24 h , and control cells were treated with DMSO only . B GlaB chemical structure with numbering scheme for NMR analysis . C Inhibition of Gli1 ‐ induced transcription in transfected Smo − / − MEF cells . Smo − / − MEF cells were transfected with 12XGliBS ‐ Luc and pRL ‐ TK Renilla ( normalization control ) plus control ( empty ) or Gli1 vector and treated for 24 h with increasing concentrations of GlaB or DMSO only as control . D The graphs show the Hh target gene expression levels in Ptch1 − / − MEFs treated for 48 h with GlaB and DMSO as a control . mRNA levels were determined by quantitative real ‐ time PCR ( qRT - PCR ) normalized to endogenous control ( β2 ‐ microglobulin and HPRT ) . Pfkfb3 gene was used as a negative control . F SuFu − / − MEFs were treated for 48 h with GlaB and DMSO as a control . Gli1 and Ptch1mRNA levels were determined by qRT - PCR normalized to β2 ‐ microglobulin and HPRT expression . Pfkfb3 gene was used as a negative control . Promoter occupancy of Gli1 is prevented by GlaB treatment . MEF WT cells were transfected with Flag ‐ tagged Gli1 or empty vectors , and chromatin immunoprecipitation ( ChIP ) was carried out . qRT - PCR was performed using primers encompassing the Gli ‐ BS of mouse Ptch1 promoter ( right , schematic representation ) . Results are indicated as fold difference , relative to empty ( pcDNA3 ) control . H Ptch1 mRNA expression levels ( left panel ) were determined by qRT - PCR in Daoy cells transfected with siRNA specific for Gli1 and Gli2 ( siGli1 / 2 ) or a non ‐ specific control siRNA ( siCtr ) and treated for 24 h with GlaB or DMSO as a control . ( right panel ) The graph shows Gli1 and Gli2mRNA expression levels determined by qRT - PCR in Daoy cells transfected with siGli1 / 2 or siCtr . Results are expressed as fold repression relative to control , and data were normalized to GAPDH and HPRT expression .

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

Figure 2A Inhibition of Gli1‐induced transcription in transfected HEK293T cells. HEK293T cells were transfected with 12XGliBS‐Luc and pRL‐TK Renilla (normalization control) plus control (empty) or Gli1 vector and treated with increasing concentrations of GlaB or GANT61. Treatment time was 24 h, and control cells were treated with DMSO only.B GlaB chemical structure with numbering scheme for NMR analysis.C Inhibition of Gli1‐induced transcription in transfected Smo−/−MEF cells. Smo−/−MEF cells were transfected with 12XGliBS‐Luc and pRL‐TK Renilla (normalization control) plus control (empty) or Gli1 vector and treated for 24 h with increasing concentrations of GlaB or DMSO only as control.D The graphs show the Hh target gene expression levels in Ptch1−/−MEFs treated for 48 h with GlaB and DMSO as a control. mRNA levels were determined by quantitative real‐time PCR (qRT-PCR) normalized to endogenous control (β2‐microglobulin and HPRT). Pfkfb3 gene was used as a negative control.F SuFu−/−MEFs were treated for 48 h with GlaB and DMSO as a control. Gli1 and Ptch1mRNA levels were determined by qRT-PCR normalized to β2‐microglobulin and HPRT expression. Pfkfb3 gene was used as a negative control.Promoter occupancy of Gli1 is prevented by GlaB treatment. MEF WT cells were transfected with Flag‐tagged Gli1 or empty vectors, and chromatin immunoprecipitation (ChIP) was carried out. qRT-PCR was performed using primers encompassing the Gli‐BS of mouse Ptch1 promoter (right, schematic representation). Results are indicated as fold difference, relative to empty (pcDNA3) control.H Ptch1 mRNA expression levels (left panel) were determined by qRT-PCR in Daoy cells transfected with siRNA specific for Gli1 and Gli2 (siGli1/2) or a non‐specific control siRNA (siCtr) and treated for 24 h with GlaB or DMSO as a control. (right panel) The graph shows Gli1 and Gli2mRNA expression levels determined by qRT-PCR in Daoy cells transfected with siGli1/2 or siCtr. Results are expressed as fold repression relative to control, and data were normalized to GAPDH and HPRT expression.


Figure 3A The predicted binding mode of GlaB ( green sticks ) to Gli1ZF ( blue cartoons ) . Residues highlighted by the single ‐ point mutation study to impact on Gli1 binding to DNA are shown as blue sticks ; K340 and K350 of the GlaB binding site are colored magenta . GlaB protons highlighted by the NMR study are shown as orange spheres . B Detail of GlaB binding to Gli1ZF ‐ WT , Gli1ZF ‐ K340A , GLI1ZF ‐ K350A , GLI1ZF ‐ K340A / K350A mutants and respective theoretical affinity values . Gli1 is shown as blue transparent surface ; K340 and K350 are colored magenta . GlaB is shown as green sticks . C Inhibition of Gli1 / DNA binding by GlaB . EMSA using recombinant GST ‐ Gli1ZF ‐ WT or GST ‐ Gli1ZF ‐ K340A in the presence of different concentrations of GlaB or with DMSO only . The shifted complex is competed with a 50 × excess of cold probe . The graph on the right indicates ratio ( mean arbitrary units ± SD from three independent experiments ) of GST ‐ Gli1ZF ‐ WT or GST ‐ Gli1ZF ‐ K340A bound to the labeled GliBS probe / GliBS ‐ free probe normalized to the amount of GST ‐ Gli1ZF ‐ WT / DNA binding in absence of GlaB . * P 0 . 05 versus DMSO ; * * P 0 . 05 K340A + GlaB versus Gli1 WT + GlaB . D HEK293T cells were transfected with 12XGliBS ‐ Luc and pRL ‐ TK Renilla ( normalization control ) plus control ( empty vector ) or Gli1 or Gli1K340A mutant and treated with increasing concentrations of GlaB . Treatment time was 24 h , and control cells were treated with DMSO only . Data show the mean ± SD of three independent experiments . * P 0 . 05 versus DMSO ; * * P 0 . 05 K340A versus Gli1 WT .

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Figure 3A The predicted binding mode of GlaB (green sticks) to Gli1ZF (blue cartoons). Residues highlighted by the single‐point mutation study to impact on Gli1 binding to DNA are shown as blue sticks; K340 and K350 of the GlaB binding site are colored magenta. GlaB protons highlighted by the NMR study are shown as orange spheres.B Detail of GlaB binding to Gli1ZF‐WT, Gli1ZF‐K340A, GLI1ZF‐K350A, GLI1ZF‐K340A/K350A mutants and respective theoretical affinity values. Gli1 is shown as blue transparent surface; K340 and K350 are colored magenta. GlaB is shown as green sticks.C Inhibition of Gli1/DNA binding by GlaB. EMSA using recombinant GST‐Gli1ZF‐WT or GST‐Gli1ZF‐K340A in the presence of different concentrations of GlaB or with DMSO only. The shifted complex is competed with a 50× excess of cold probe. The graph on the right indicates ratio (mean arbitrary units ± SD from three independent experiments) of GST‐Gli1ZF‐WT or GST‐Gli1ZF‐K340A bound to the labeled GliBS probe/GliBS‐free probe normalized to the amount of GST‐Gli1ZF‐WT/DNA binding in absence of GlaB. *P 0.05 versus DMSO; **P 0.05 K340A + GlaB versus Gli1 WT + GlaB.D HEK293T cells were transfected with 12XGliBS‐Luc and pRL‐TK Renilla (normalization control) plus control (empty vector) or Gli1 or Gli1K340A mutant and treated with increasing concentrations of GlaB. Treatment time was 24 h, and control cells were treated with DMSO only. Data show the mean ± SD of three independent experiments. *P 0.05 versus DMSO; **P 0.05 K340A versus Gli1 WT.


Figure 4A qRT - PCR shows Hh target mRNA expression levels determined in 6 ‐ day ‐ old mouse cerebellar progenitors after s . c . injections of GlaB . B Representative EGLHE staining of 6 ‐ day ‐ old mousecerebellum after s . c . injections of GlaB ( upper panel ) ; immunohistochemistry shows Gli1 ( middle panel ) and Ki67 stainings ( lower panel ) . In all images , the scale bar represents 50 μm . C The graph shows the percentage of EGL thickness reduction in GlaB ‐ treated mouse cerebella in comparison with vehicle mouse cerebella . Data show the mean ± SD of cerebella ( n = 10 ) for each treatment . * P 0 . 05 versus Ctr . D BrdU assay in GCPs . Cerebellargranule cell progenitors ( GCPs ) isolated from 4 ‐ day ‐ old mice were treated with Shh ( recombinant mouseSonic Hedgehog , amino ‐ terminal peptide , ShhN , 3 μg / ml ) and with different concentrations of GlaB , as indicated , for 48 h . Inhibition of cell proliferation was measured as percentage of BrdU incorporation in comparison with DMSO ‐ treated sample . Shown is the mean of three independent experiments . Error bars indicate SD . * P 0 . 05 Shh versus DMSO ; * * P 0 . 05 Shh + GlaB versus Shh . E qRT - PCR and show Hh and proliferation target mRNA and protein expression levels determined in ex vivo GCP culture derived from 6 ‐ day ‐ old mousecerebella after s . c . injections of GlaB . In all qRT - PCR experiments , the results were normalized to endogenous control ( β2 ‐ microglobulin and HPRT ) . * P 0 . 05 versus Ctr . F Western blot analysis show Hh and proliferation target mRNA and protein expression levels determined in ex vivo GCP culture derived from 6 ‐ day ‐ old mousecerebella after s . c . injections of GlaB .

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

Figure 4A qRT-PCR shows Hh target mRNA expression levels determined in 6‐day‐old mouse cerebellar progenitors after s.c. injections of GlaB.B Representative EGLHE staining of 6‐day‐old mousecerebellum after s.c. injections of GlaB (upper panel); immunohistochemistry shows Gli1 (middle panel) and Ki67 stainings (lower panel). In all images, the scale bar represents 50 μm.C The graph shows the percentage of EGL thickness reduction in GlaB‐treated mouse cerebella in comparison with vehicle mouse cerebella. Data show the mean ± SD of cerebella (n = 10) for each treatment. *P 0.05 versus Ctr.D BrdU assay in GCPs. Cerebellargranule cell progenitors (GCPs) isolated from 4‐day‐old mice were treated with Shh (recombinant mouseSonic Hedgehog, amino‐terminal peptide, ShhN, 3 μg/ml) and with different concentrations of GlaB, as indicated, for 48 h. Inhibition of cell proliferation was measured as percentage of BrdU incorporation in comparison with DMSO‐treated sample. Shown is the mean of three independent experiments. Error bars indicate SD. *P 0.05 Shh versus DMSO; **P 0.05 Shh + GlaB versus Shh.E qRT-PCR and show Hh and proliferation target mRNA and protein expression levels determined in ex vivo GCP culture derived from 6‐day‐old mousecerebella after s.c. injections of GlaB. In all qRT-PCR experiments, the results were normalized to endogenous control (β2‐microglobulin and HPRT). *P 0.05 versus Ctr.F Western blot analysis show Hh and proliferation target mRNA and protein expression levels determined in ex vivo GCP culture derived from 6‐day‐old mousecerebella after s.c. injections of GlaB.


Figure 5A , B Ex vivo cell cultures from Ptch1 + / − mice MBs were treated with GlaB ( 5 μM ) , GANT61 ( 10 μM ) or DMSO only . After the indicated times , a trypan blue count was performed ( A ) to determine the growth rate of viable cells . A , B Ex vivo cell cultures from Ptch1 + / − miceMBs were treated with GlaB ( 5 μM ) , GANT61 ( 10 μM ) or DMSO only . After the indicated times , Gli1 mRNA expression levels were determined by qRT - PCR ( B ) normalized to endogenous control ( β2 ‐ microglobulin and HPRT ) . C - F GlaB inhibits MB ‐ SCs ' self ‐ renewal and proliferation . ( C ) Suspension of single MB ‐ SCs isolated from Ptch1 + / − mice . MBs were cultured in stem cell medium to allow the formation of primary neurospheres . Primary neurospheres were dissociated and treated with increasing concentrations of GlaB or DMSO only . After 7 days of treatment , the number of secondary neurospheres derived from a known number of single cells was counted . The self ‐ renewal MB ‐ SCs ' capability is expressed as percentage of neurosphere ‐ forming cells ( left ) . Representative bright field images of tumor neurospheres after GlaB treatment are also shown ( right ) . C - F GlaB inhibits MB ‐ SCs ' self ‐ renewal and proliferation . ( D , E ) MB ‐ SCs isolated from Ptch1 + / − miceMBs were treated for 48 h with GlaB ( 5 μM ) or DMSO only . qRT - PCR analysis show Hh , proliferation and stemness target mRNA expression levels . For qRT - PCR , results were normalized to endogenous control ( β2 ‐ microglobulin and HPRT ) . C - F GlaB inhibits MB ‐ SCs ' self ‐ renewal and proliferation . ( D , E ) MB ‐ SCs isolated from Ptch1 + / − miceMBs were treated for 48 h with GlaB ( 5 μM ) or DMSO only . Western blot analysis show Hh , proliferation and stemness target protein expression levels . C - F GlaB inhibits MB ‐ SCs ' self ‐ renewal and proliferation . ( F ) BrdU assay in MB ‐ SCs treated with GlaB ( 5 μM ) for 24 or 48 h and plated on polylysinated chamber slides . Inhibition of cell proliferation was measured as percentage of BrdU incorporation in comparison with DMSO ‐ treated sample . G TUNEL assay in MB ‐ SCs . MB ‐ SCs isolated from Ptch1 + / − miceMBs were treated with GlaB ( 5 μM ) and compared to DMSO ‐ treated sample . Bottom panel shows a Western blot of caspase ‐ 3 in GlaB ‐ treated versus control cells .

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

Figure 5A, B Ex vivo cell cultures from Ptch1+/−mice MBs were treated with GlaB (5 μM), GANT61 (10 μM) or DMSO only. After the indicated times, a trypan blue count was performed (A) to determine the growth rate of viable cells.A, B Ex vivo cell cultures from Ptch1+/−miceMBs were treated with GlaB (5 μM), GANT61 (10 μM) or DMSO only. After the indicated times, Gli1 mRNA expression levels were determined by qRT-PCR (B) normalized to endogenous control (β2‐microglobulin and HPRT).C-F GlaB inhibits MB‐SCs' self‐renewal and proliferation. (C) Suspension of single MB‐SCs isolated from Ptch1+/−mice. MBs were cultured in stem cell medium to allow the formation of primary neurospheres. Primary neurospheres were dissociated and treated with increasing concentrations of GlaB or DMSO only. After 7 days of treatment, the number of secondary neurospheres derived from a known number of single cells was counted. The self‐renewal MB‐SCs' capability is expressed as percentage of neurosphere‐forming cells (left). Representative bright field images of tumor neurospheres after GlaB treatment are also shown (right).C-F GlaB inhibits MB‐SCs' self‐renewal and proliferation. (D, E) MB‐SCs isolated from Ptch1+/−miceMBs were treated for 48 h with GlaB (5 μM) or DMSO only. qRT-PCR analysis show Hh, proliferation and stemness target mRNA expression levels. For qRT-PCR, results were normalized to endogenous control (β2‐microglobulin and HPRT).C-F GlaB inhibits MB‐SCs' self‐renewal and proliferation. (D, E) MB‐SCs isolated from Ptch1+/−miceMBs were treated for 48 h with GlaB (5 μM) or DMSO only. Western blot analysis show Hh, proliferation and stemness target protein expression levels.C-F GlaB inhibits MB‐SCs' self‐renewal and proliferation. (F) BrdU assay in MB‐SCs treated with GlaB (5 μM) for 24 or 48 h and plated on polylysinated chamber slides. Inhibition of cell proliferation was measured as percentage of BrdU incorporation in comparison with DMSO‐treated sample.G TUNEL assay in MB‐SCs. MB‐SCs isolated from Ptch1+/−miceMBs were treated with GlaB (5 μM) and compared to DMSO‐treated sample. Bottom panel shows a Western blot of caspase‐3 in GlaB‐treated versus control cells.


Figure 6A Change of tumor volume during GlaB or vehicle treatment period ( 18 days ) . B Representative flank allografts average volumes . Error bars indicate SD . * P 0 . 05 . C Representative Masson ' s trichromestaining of tumors ; immunohistochemistry of Gli1 , Ki67 and TUNEL stainings . Scale bars represent 35 μm for Masson ' s trichromestaining , 25 μm for Gli1 and TUNEL , and 10 μm for Ki67 stainings . D Quantification of Ki67 and TUNEL stainings from immunohistochemistry shown in ( C ) . Error bars indicate SD . * P 0 . 05 . E qRT - PCR analysis show Hh and proliferation target mRNA expression levels . For qRT - PCR , results were normalized to endogenous control ( β2 ‐ microglobulin and HPRT ) . Data show the mean ± SD of tumor ( n = 6 ) for each treatment . * P 0 . 05 versus Ctr . F Western blot analysis show Hh and proliferation target protein expression levels . Data show the mean ± SD of tumor ( n = 6 ) for each treatment . * P 0 . 05 versus Ctr .

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

Figure 6A Change of tumor volume during GlaB or vehicle treatment period (18 days).B Representative flank allografts average volumes. Error bars indicate SD. *P 0.05.C Representative Masson's trichromestaining of tumors; immunohistochemistry of Gli1, Ki67 and TUNEL stainings. Scale bars represent 35 μm for Masson's trichromestaining, 25 μm for Gli1 and TUNEL, and 10 μm for Ki67 stainings.D Quantification of Ki67 and TUNEL stainings from immunohistochemistry shown in (C). Error bars indicate SD. *P 0.05.E qRT-PCR analysis show Hh and proliferation target mRNA expression levels. For qRT-PCR, results were normalized to endogenous control (β2‐microglobulin and HPRT). Data show the mean ± SD of tumor (n = 6) for each treatment. *P 0.05 versus Ctr.F Western blot analysis show Hh and proliferation target protein expression levels. Data show the mean ± SD of tumor (n = 6) for each treatment. *P 0.05 versus Ctr.


Figure 7A Representative images ( low and high magnification ) of HE ( upper panel ) and Ki67immunohistochemistry staining ( lower panel ) of a human Daoy MB cell ‐ derived orthotopic tumor in NOD / SCIDmicecerebella after i . p . injections of GlaB . Scale bars represent 500 or 200 μm for HE staining , and 150 μm or 50 μm for Ki67 staining . B Representative tumor average volumes at the end of treatment . Error bars indicate SD . * P 0 . 05 . C Quantification of Ki67 staining from immunohistochemistry shown in ( A ) . Error bars indicate SD . * P 0 . 05 .

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

Figure 7A Representative images (low and high magnification) of HE (upper panel) and Ki67immunohistochemistry staining (lower panel) of a human Daoy MB cell‐derived orthotopic tumor in NOD/SCIDmicecerebella after i.p. injections of GlaB. Scale bars represent 500 or 200 μm for HE staining, and 150 μm or 50 μm for Ki67 staining.B Representative tumor average volumes at the end of treatment. Error bars indicate SD. *P 0.05.C Quantification of Ki67 staining from immunohistochemistry shown in (A). Error bars indicate SD. *P 0.05.


Figure 8A , B ASZ001 BCC cells were treated with GlaB ( 5 μM ) or DMSO only . After the indicated times , a trypan blue count was performed to determine ( A ) the growth rate and ( B ) the percentage of cell death . Data show the mean ± SD of three independent experiments . * P 0 . 05 versus DMSO . C Gli1 mRNA expression levels were determined by qRT - PCR after treatment of ASZ001 BCC cells with GlaB or DMSO only for the indicated times . Results were normalized to endogenous control ( β2 ‐ microglobulin and HPRT ) . Data show the mean ± SD of three independent experiments . * P 0 . 05 versus DMSO . D - H GlaB inhibits Gli1 ‐ dependent BCC tumor growth in ASZ001 BCC allografts in vivo . ( D ) Change of tumor volume during GlaB or vehicle treatment period ( 18 days ) . D - H GlaB inhibits Gli1 ‐ dependent BCC tumor growth in ASZ001 BCC allografts in vivo . ( E ) Representative flank allografts average volumes ( upper panel ) . D - H GlaB inhibits Gli1 ‐ dependent BCC tumor growth in ASZ001 BCC allografts in vivo . ( F ) HE and immunohistochemistry staining of Gli1 , Ki67 and TUNEL of allograft tumor samples . Scale bars represent 50 μm for HE , 25 μm for Gli1 , 25 μm for Ki67 and 30 μm for TUNEL stainings . ( G ) Quantification of Ki67 and TUNEL stainings from immunohistochemistry shown in ( F ) D - H GlaB inhibits Gli1 ‐ dependent BCC tumor growth in ASZ001 BCC allografts in vivo . ( H ) qRT - PCR of Hh and proliferation target mRNA expression levels . Results were normalized to endogenous control ( β2 ‐ microglobulin and HPRT ) . Shown is the mean ± SD of tumor ( n = 6 ) for each treatment . * P 0 . 05 versus Ctr .

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

Figure 8A, B ASZ001 BCC cells were treated with GlaB (5 μM) or DMSO only. After the indicated times, a trypan blue count was performed to determine (A) the growth rate and (B) the percentage of cell death. Data show the mean ± SD of three independent experiments. *P 0.05 versus DMSO.C Gli1 mRNA expression levels were determined by qRT-PCR after treatment of ASZ001 BCC cells with GlaB or DMSO only for the indicated times. Results were normalized to endogenous control (β2‐microglobulin and HPRT). Data show the mean ± SD of three independent experiments. *P 0.05 versus DMSO.D-H GlaB inhibits Gli1‐dependent BCC tumor growth in ASZ001 BCC allografts in vivo. (D) Change of tumor volume during GlaB or vehicle treatment period (18 days).D-H GlaB inhibits Gli1‐dependent BCC tumor growth in ASZ001 BCC allografts in vivo. (E) Representative flank allografts average volumes (upper panel).D-H GlaB inhibits Gli1‐dependent BCC tumor growth in ASZ001 BCC allografts in vivo. (F) HE and immunohistochemistry staining of Gli1, Ki67 and TUNEL of allograft tumor samples. Scale bars represent 50 μm for HE, 25 μm for Gli1, 25 μm for Ki67 and 30 μm for TUNEL stainings. (G) Quantification of Ki67 and TUNEL stainings from immunohistochemistry shown in (F)D-H GlaB inhibits Gli1‐dependent BCC tumor growth in ASZ001 BCC allografts in vivo. (H) qRT-PCR of Hh and proliferation target mRNA expression levels. Results were normalized to endogenous control (β2‐microglobulin and HPRT). Shown is the mean ± SD of tumor (n = 6) for each treatment. *P 0.05 versus Ctr.


Figure 1 . Autophagy is activated in dying nurse cells during late oogenesis in D . melanogaster . ( A - C ) Confocal micrographs of egg chambers of flies expressing the UASp - GFP - mCherry - DrAtg8a transgene exclusively in the germline ( genotype : UASp - GFP - mCherry - DrAtg8a / + ; nanos - VP - 16 Gal4 / + ) . Expression of UASp - GFP - mCherry - DrAtg8a exhibits punctate staining pattern ( arrows ) . Note that during developmental stages early 12 ( A ) and late 12 ( B ) , the punctate dots are yellow ( merge ) . In late stage 13 ( C ) , large red dots are evident , indicating the presence of acidic compartments ( autolysosomes ; arrows ) . The arrowheads in A point to the physical connection between the oocyte and the nurse cells during early stage 12 ( not observed during late stage 12 ) . ( D and E ) Transmission electron micrographs of nurse cells in stage 12 ( D ) and in stage 13 ( E ) egg chambers . Autophagosomes ( inset in D ) and autolysosomes ( arrow in E ) are evident in the remaining nurse cell cytoplasm ( arrow in D indicates actin bundles ) . Note that the autolysosome shown in E contains condensed material resembling the material of the condensed and fragmented nurse cell nucleus ( NCN ) , providing evidence for nuclear autophagy ( F ) Nurse cell of a late stage 13 egg chamber expressing UASp - mCherry - DrAtg8a exclusively in the germline ( genotype : UASp - mCherry - DrAtg8a / + ; nanos - VP - 16 Gal4 / + ) . mCherry - DrAtg8a puncta are attached to or located adjacent to the fragmented nucleus ( arrows and insets ) . Hoechst staining ( blue ) was performed to visualize the nuclei . Nurse cells are outlined with a white line . FC , follicle cell ; FCN , follicle cell nucleus ; OC , oocyte . Bars : ( A - C ) 10 µm ; ( D - F ) 1 µm .

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

Figure 1.Autophagy is activated in dying nurse cells during late oogenesis in D. melanogaster. (A-C) Confocal micrographs of egg chambers of flies expressing the UASp-GFP-mCherry-DrAtg8a transgene exclusively in the germline (genotype: UASp-GFP-mCherry-DrAtg8a/+; nanos-VP-16 Gal4/+). Expression of UASp-GFP-mCherry-DrAtg8a exhibits punctate staining pattern (arrows). Note that during developmental stages early 12 (A) and late 12 (B), the punctate dots are yellow (merge). In late stage 13 (C), large red dots are evident, indicating the presence of acidic compartments (autolysosomes; arrows). The arrowheads in A point to the physical connection between the oocyte and the nurse cells during early stage 12 (not observed during late stage 12).(D and E) Transmission electron micrographs of nurse cells in stage 12 (D) and in stage 13 (E) egg chambers. Autophagosomes (inset in D) and autolysosomes (arrow in E) are evident in the remaining nurse cell cytoplasm (arrow in D indicates actin bundles). Note that the autolysosome shown in E contains condensed material resembling the material of the condensed and fragmented nurse cell nucleus (NCN), providing evidence for nuclear autophagy(F) Nurse cell of a late stage 13 egg chamber expressing UASp-mCherry-DrAtg8a exclusively in the germline (genotype: UASp-mCherry-DrAtg8a/+; nanos-VP-16 Gal4/+). mCherry-DrAtg8a puncta are attached to or located adjacent to the fragmented nucleus (arrows and insets). Hoechst staining (blue) was performed to visualize the nuclei. Nurse cells are outlined with a white line. FC, follicle cell; FCN, follicle cell nucleus; OC, oocyte. Bars: (A-C) 10 µm; (D-F) 1 µm.


Figure 2 . Genetic inhibition of autophagy in the germline prevents DNA fragmentation . Confocal micrographs of stage 13 / 14 egg chambers after TUNEL staining . ( A ) Wild - type ( wt ) stage 13 egg chamber contains nurse cell nuclei exhibiting fragmented DNA ( positive TUNEL staining ; arrows ) . ( B ) Wild - type stage 14 egg chambers do not contain nurse cells . Arrows point to the respiratory appendages ( RA ) . ( C - E ) Stage 14 atg1 − / − ( C ) , atg13 − / − ( D ) , and vps34 − / − ( E ) germline mutant chambers have persisting nurse cell nuclei that do not contain fragmented DNA ( negative TUNEL staining ; arrows ) . Nurse cells are outlined with a white line . Draq5 / Hoechst staining ( blue ) was performed to visualize the nuclei . PhC , phase contrast . Bars , 50 µm .

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

Figure 2.Genetic inhibition of autophagy in the germline prevents DNA fragmentation. Confocal micrographs of stage 13/14 egg chambers after TUNEL staining. (A) Wild-type (wt) stage 13 egg chamber contains nurse cell nuclei exhibiting fragmented DNA (positive TUNEL staining; arrows). (B) Wild-type stage 14 egg chambers do not contain nurse cells. Arrows point to the respiratory appendages (RA). (C-E) Stage 14 atg1−/− (C), atg13−/− (D), and vps34−/− (E) germline mutant chambers have persisting nurse cell nuclei that do not contain fragmented DNA (negative TUNEL staining; arrows). Nurse cells are outlined with a white line. Draq5/Hoechst staining (blue) was performed to visualize the nuclei. PhC, phase contrast. Bars, 50 µm.


Figure 3 . Germline autophagy mutant egg chambers exhibit reduced expression of cleaved caspase - 3 . Confocal micrographs of stage 13 / 14 egg chambers stained for cleaved caspase - 3 ( casp - 3 ) . ( A ) Wild - type ( wt ) late stage 13 egg chamber showing cleaved caspase - 3 staining in the degenerating nurse cell cluster . ( B ) In wild - type stage 14 egg chamber , nurse cells are completely degraded ( arrows ) . ( C - E ) atg1 − / − ( C ) , atg13 − / − ( D ) , and vps34 − / − ( E ) germline mutant stage 14 egg chambers exhibit significantly reduced staining for cleaved caspase - 3 and contain persisting nurse cell nuclei . Nurse cells are outlined with a white line . Draq5 / Hoechst staining ( blue ) was performed to visualize the nuclei . PhC , phase contrast . ( F ) Quantification of mean intensity of cleaved caspase - 3 staining observed in the nurse cells of stage 12 - 14 egg chambers in wild - type and germline autophagy mutant egg chambers . Wild type ( WT ) : three independent experiments , n = 30 egg chambers ; atg1 − / − GLCs : four independent experiments , n = 30 egg chambers ; atg13 − / − GLCs : four independent experiments , n = 30 egg chambers ; vps34 − / − GLCs : three independent experiments , n = 30 egg chambers . Data are presented as mean ± SD . Difference was significant with P < 0 . 001 for all values versus wild type . Bars , 20 µm .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3.Germline autophagy mutant egg chambers exhibit reduced expression of cleaved caspase-3. Confocal micrographs of stage 13/14 egg chambers stained for cleaved caspase-3(casp-3). (A) Wild-type (wt) late stage 13 egg chamber showing cleaved caspase-3 staining in the degenerating nurse cell cluster. (B) In wild-type stage 14 egg chamber, nurse cells are completely degraded (arrows). (C-E) atg1−/− (C), atg13−/− (D), and vps34−/− (E) germline mutant stage 14 egg chambers exhibit significantly reduced staining for cleaved caspase-3 and contain persisting nurse cell nuclei. Nurse cells are outlined with a white line. Draq5/Hoechst staining (blue) was performed to visualize the nuclei. PhC, phase contrast. (F) Quantification of mean intensity of cleaved caspase-3 staining observed in the nurse cells of stage 12-14 egg chambers in wild-type and germline autophagy mutant egg chambers. Wild type (WT): three independent experiments, n = 30 egg chambers; atg1−/− GLCs: four independent experiments, n = 30 egg chambers; atg13−/− GLCs: four independent experiments, n = 30 egg chambers; vps34−/− GLCs: three independent experiments, n = 30 egg chambers. Data are presented as mean ± SD. Difference was significant with P < 0.001 for all values versus wild type. Bars, 20 µm.


Figure 4 . dBruce colocalizes with the autophagic marker Atg8a - GFP in the nurse cells during late oogenesis and is accumulated in autophagy germline mutants . Confocal micrographs of late stage egg chambers expressing Atg8a - GFP and stained for dBruce . ( A - C ) Stage 10 ( A ) , early stage 12 ( B ) , and late stage 12 ( C ) . dBruce colocalizes with Atg8a - GFP in punctuate structures during stages 12 and 13 ( insets in B and insets and arrows in C ) and not during stage 10 ( insets in A ) . ( D - G ) Confocal micrographs of stage 14 wild - type ( D ) and autophagy mutant egg chambers ( E - G ) stained for dBruce . dBruce is accumulated in atg1 , atg13 , and vps34 mutant nurse cells ( arrows ) . Draq5 / Hoechst staining ( blue ) was performed to visualize the nuclei . Nurse cells are outlined with a white line . OC , oocyte ; PhC , phase contrast . Bars , 10 µm .

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 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

Figure 4.dBruce colocalizes with the autophagic marker Atg8a-GFP in the nurse cells during late oogenesis and is accumulated in autophagy germline mutants. Confocal micrographs of late stage egg chambers expressing Atg8a-GFP and stained for dBruce. (A-C) Stage 10 (A), early stage 12 (B), and late stage 12 (C). dBruce colocalizes with Atg8a-GFP in punctuate structures during stages 12 and 13 (insets in B and insets and arrows in C) and not during stage 10 (insets in A).(D-G) Confocal micrographs of stage 14 wild-type (D) and autophagy mutant egg chambers (E-G) stained for dBruce. dBruce is accumulated in atg1, atg13, and vps34 mutant nurse cells (arrows). Draq5/Hoechst staining (blue) was performed to visualize the nuclei. Nurse cells are outlined with a white line. OC, oocyte; PhC, phase contrast. Bars, 10 µm.


Figure 5 . dBruce is required for nurse cell survival by controlling DNA fragmentation during oogenesis . ( A ) Confocal micrographs of dBrucee00984 mutant ovarioles stained for TUNEL and DNA . Arrows point to degenerating stage 8 / 9 egg chambers that are TUNEL positive . ( B and C ) Confocal micrographs of vps34 / dBruceE81 and atg , dBruceE81 double mutant egg chambers stained for TUNEL ( green and red , respectively ) and DNA . ( B ) vps34 − / − GLCs ; dBruceE81 / dBruceE81 stage 14 mutant egg chamber contain persisting TUNEL - positive nurse cell nuclei ( outlined ; arrow ) . ( C ) atg1 − / − GLCs , dBruceE81 / dBruceE81 stage 14 mutant egg chamber contain persisting TUNEL - positive nurse cell nuclei . atg1 mutant nurse cells are identified by the lack of GFP staining ( outlined ; arrow ) . Draq5 / Hoechst staining ( blue ) was performed to visualize the nuclei . PhC , phase contrast . Bars , 50 µm .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.dBruce is required for nurse cell survival by controlling DNA fragmentation during oogenesis. (A) Confocal micrographs of dBrucee00984 mutant ovarioles stained for TUNEL and DNA. Arrows point to degenerating stage 8/9 egg chambers that are TUNEL positive.(B and C) Confocal micrographs of vps34/dBruceE81 and atg, dBruceE81 double mutant egg chambers stained for TUNEL (green and red, respectively) and DNA. (B) vps34−/−GLCs; dBruceE81/dBruceE81 stage 14 mutant egg chamber contain persisting TUNEL-positive nurse cell nuclei (outlined; arrow). (C) atg1−/−GLCs, dBruceE81/dBruceE81 stage 14 mutant egg chamber contain persisting TUNEL-positive nurse cell nuclei. atg1 mutant nurse cells are identified by the lack of GFP staining (outlined; arrow). Draq5/Hoechst staining (blue) was performed to visualize the nuclei. PhC, phase contrast. Bars, 50 µm.


Figure 1B - E ) Representaitve pictures showing that the withdrawal of dietary leu but not his significantly reduced stem cells and follicle cell proliferation ( pH3 , red ) in the adult ovary after 10 days , quantified in ( E ) ( n = 13 , 14 , 13 ) , scale bar = 100 μm . withdrawal of dietary leu but not his significantly reduced stem cells and follicle cell proliferation ( pH3 , red ) in the adult ovary after 10 days , quantified in ( E ) ( n = 13 , 14 , 13 ) , scale bar = 100 μm . wildtype larval NB clonal growth was significantly reduced after 6 days of dietary leucine depletion ( 25 % leu ) , and significantly increased by dietary histidine depletion ( 25 % his ) , quantified in ( F ) ( n = 28 , 22 , 7 ) , scale bar = 50 μmF - H ) Representaitve pictures showing that wildtype larval NB clonal growth was significantly reduced after 6 days of dietary leucine depletion ( 25 % leu ) , and significantly increased by dietary histidine depletion ( 25 % his ) , I - K ) Representaitve pictures showing that adult nerfin - 1 clonal growth was significantly reduced on - his diet compared to CDD ( measured at day 0 , and day 9 ) quantified in ( K ) ( n = 27 , 110 , 136 ) . Scale bar = 75μm . * * * p < 0 . 001 L - N ) Representaitve pictures showing that larval nerfin - 1 clonal growth was significantly reduced on 25 % his diet compared to CDD ( after 6 days ) , quantified in ( N ) ( n = 53 , 36 ) . Scale bar = 50 μm . O - Q ) Representaitve pictures showing that adult pros clonal growth was not significantly altered on - his diet compared to CDD ( after 7 days ) , quantified in ( Q ) ( n = 12 , 23 , 16 ) . Scale bar = 75μm . R - T ) Representaitve pictures showing that larval pros clonal growth was not significantly altered by dietary histidine reduction ( 25 % his ) compared to CDD ( after 6 days ) , quantified in ( T ) ( n = 19 , 13 ) . Scale bar = 50 μm . U - W ) Representative pictures showing that the growth of larval type II lineages overexpressing NACT was significantly reduced dietary histidine reduction ( 25 % his ) compared to CDD , quantified in ( W ) ( n = 27 , 19 ) . Scale bar = 100 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 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 1B-E) Representaitve pictures showing that the withdrawal of dietary leu but not his significantly reduced stem cells and follicle cell proliferation (pH3, red) in the adult ovary after 10 days, quantified in (E) (n=13, 14,13), scale bar = 100 μm.withdrawal of dietary leu but not his significantly reduced stem cells and follicle cell proliferation (pH3, red) in the adult ovary after 10 days, quantified in (E) (n=13, 14,13), scale bar = 100 μm.wildtype larval NB clonal growth was significantly reduced after 6 days of dietary leucine depletion (25% leu), and significantly increased by dietary histidine depletion (25% his), quantified in (F) (n=28,22,7), scale bar = 50 μmF-H) Representaitve pictures showing that wildtype larval NB clonal growth was significantly reduced after 6 days of dietary leucine depletion (25% leu), and significantly increased by dietary histidine depletion (25% his),I-K) Representaitve pictures showing that adult nerfin-1 clonal growth was significantly reduced on -his diet compared to CDD (measured at day 0, and day 9) quantified in (K) (n= 27, 110, 136). Scale bar = 75μm. ***p<0.001 L-N) Representaitve pictures showing that larval nerfin-1 clonal growth was significantly reduced on 25% his diet compared to CDD (after 6 days), quantified in (N) (n=53,36). Scale bar = 50 μm. O-Q) Representaitve pictures showing that adult pros clonal growth was not significantly altered on -his diet compared to CDD (after 7 days), quantified in (Q) (n = 12, 23,16). Scale bar = 75μm. R-T) Representaitve pictures showing that larval pros clonal growth was not significantly altered by dietary histidine reduction (25% his) compared to CDD (after 6 days), quantified in (T) (n=19,13). Scale bar = 50 μm. U-W) Representative pictures showing that the growth of larval type II lineages overexpressing NACT was significantly reduced dietary histidine reduction (25% his) compared to CDD, quantified in (W) (n=27,19). Scale bar = 100 μm.


Figure 2B ) Concentration of histidine and 3 - methyl - L - histidine from whole Drosophila adults extracts measured by 1H NMR after 3 days of feeding on either CDD or - his diet ( n = 3 , 3 ) . Both compounds are reduced to below detection levels ( < 0 . 3mM ) upon dietary his depletion . C ) Dietary supplementation of 3 - methyl - L - his for 4 days significantly increased adult nerfin - 1 clonal growth , and rescued nerfin - 1 growth inhibition due to his dietary withdrawal ( n = 18 , 14 , 7 , 9 ) . D ) Dietary supplementation of histamine does not significantly increase adult nerfin - 1 clone size , but can rescue nerfin - 1 clonal growth inhibition due to his dietary withdrawal ( n = 42 , 14 , 23 , 25 ) . E ) Larval nerfin - 1 clonal growth is significantly decreased upon overexpression of HDC RNAi ( n = 23 , 36 ) . G ) Hdc inhibition significantly reduced the percentage of Dpn + NBs ( n = 16 , 17 ) , and significantly increased the percentage of Elav + neurons per clone ( n = 16 , 17 ) . H ) HdcRi overexpression reduced cellular growth of nerfin - 1 NBs ( n = 17 , 19 ) and neurons ( n = 36 , 19 ) measured as the ratio of nucleolus / nuclear volume .

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

Figure 2B) Concentration of histidine and 3-methyl-L-histidine from whole Drosophila adults extracts measured by 1H NMR after 3 days of feeding on either CDD or -his diet (n=3,3). Both compounds are reduced to below detection levels (<0.3mM) upon dietary his depletion.C) Dietary supplementation of 3-methyl-L-his for 4 days significantly increased adult nerfin-1 clonal growth, and rescued nerfin-1 growth inhibition due to his dietary withdrawal (n =18,14, 7,9).D) Dietary supplementation of histamine does not significantly increase adult nerfin-1 clone size, but can rescue nerfin-1 clonal growth inhibition due to his dietary withdrawal (n =42,14,23,25).E) Larval nerfin-1 clonal growth is significantly decreased upon overexpression of HDC RNAi (n=23,36).G) Hdc inhibition significantly reduced the percentage of Dpn+ NBs (n=16,17), and significantly increased the percentage of Elav+ neurons per clone (n=16,17).H) HdcRi overexpression reduced cellular growth of nerfin-1 NBs (n=17,19) and neurons (n=36,19) measured as the ratio of nucleolus/nuclear volume.


Figure 3A - E ) Representative images of nerfin - 1 clones in the adult VNC after 6 days of feeding on CDD or - his diet , scale bar = 100μm . Histidine dietary withdrawal resulted in clones significantly altering the amount of cell death per clone quantified in ( C ) ( n = 6 , 7 ) . Histidine dietary withdrawal resulted in clones containing significantly more differentiated Elav + neurons ( red ) quantified in ( D ) ( n = 16 , 17 ) and significantly fewer Mira + NBs ( blue ) , quantified in ( E ) ( n = 12 , 22 ) , F - G ) Larval nerfin - 1 clones consisted of significantly greater proportion of differentiated neurons ( n = 10 , 7 ) and significantly reduced proportion of NBs per clone upon his dietary depletion ( 25 % his , n = 9 , 11 ) . Representative images of adult nerfin - 1 ( I - J ' , green ) and pros clones ( K - L ' , green ) , scale bar = 5μm . In nerfin - 1 clones , the size of the NBs ( Ase + , blue ) was significantly reduced upon 6 days of histidine dietary withdrawal , quantified in ( H ) ( n = 33 , 20 ) . In pros clones , the size of the NBs ( Ase + blue ) , and cellular growth measured as the ratio of nucleolus ( Fib , white ) / nuclear ( Ase , blue ) volume was not significantly altered upon 6 days of histidine dietary withdrawal , quantified in ( H ) ( n = 9 , 8 ) Cellular growth measured as the ratio of nucleolus ( Fib , white ) / nuclear ( Ase , blue ) volume was also significantly reduced , quantified in ( M ) ( n = 33 , 20 ) . Cellular growth measured as the ratio of nucleolus / nuclear volume was significantly reduced in larval nerfin - 1 NBs after 6 days his dietary reduction ( 25 % his compared to CDD , n = 14 , 6 ) quantified in ( N ) . In pros clones , the size of the NBs ( Ase + blue ) , and cellular growth measured as the ratio of nucleolus ( Fib , white ) / nuclear ( Ase , blue ) volume was not significantly altered upon 6 days of histidine dietary withdrawal , quantified in ( O ) ( n = 9 , 7 ) . Panel ( P ) shows that the larval nerfin - 1 neuron to NB reversion requires 15 - fold increase in cellular volume ( n = 10 ) and pros GMC to NB reversion requires approximately 3 . 5 - fold increase in cellular volume ( n = 31 ) .

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

Figure 3A-E) Representative images of nerfin-1 clones in the adult VNC after 6 days of feeding on CDD or -his diet, scale bar = 100μm. Histidine dietary withdrawal resulted in clones significantly altering the amount of cell death per clone quantified in (C) (n = 6, 7).Histidine dietary withdrawal resulted in clones containing significantly more differentiated Elav+ neurons (red) quantified in (D) (n = 16,17) and significantly fewer Mira+ NBs (blue), quantified in (E) (n = 12,22),F-G) Larval nerfin-1 clones consisted of significantly greater proportion of differentiated neurons (n=10,7) and significantly reduced proportion of NBs per clone upon his dietary depletion (25% his, n=9,11).Representative images of adult nerfin-1 (I-J', green ) and pros clones (K-L', green), scale bar = 5μm. In nerfin-1 clones, the size of the NBs (Ase+, blue) was significantly reduced upon 6 days of histidine dietary withdrawal, quantified in (H) (n=33,20). In pros clones, the size of the NBs (Ase+ blue), and cellular growth measured as the ratio of nucleolus (Fib, white)/nuclear (Ase, blue) volume was not significantly altered upon 6 days of histidine dietary withdrawal, quantified in (H) (n=9,8)Cellular growth measured as the ratio of nucleolus (Fib, white)/nuclear (Ase, blue) volume was also significantly reduced, quantified in (M) (n=33,20).Cellular growth measured as the ratio of nucleolus /nuclear volume was significantly reduced in larval nerfin-1 NBs after 6 days his dietary reduction (25% his compared to CDD, n=14,6) quantified in (N).In pros clones, the size of the NBs (Ase+ blue), and cellular growth measured as the ratio of nucleolus (Fib, white)/nuclear (Ase, blue) volume was not significantly altered upon 6 days of histidine dietary withdrawal, quantified in (O) (n=9,7).Panel (P) shows that the larval nerfin-1 neuron to NB reversion requires 15-fold increase in cellular volume (n=10) and pros GMC to NB reversion requires approximately 3.5-fold increase in cellular volume (n=31).


Figure 4A - D ) Representative pictures showing that pros NBs and GMCs are smaller than wildtype ( NBs and GMCs are distinguished at telophase , as a doublet of unequal size ) , quantified in C ( n = 25 , 31 , 21 , 16 ) . pros NBs exhibit similar nucleolus / nuclear ratio ( white arrows , nucleolus marked by Fib ) compared to wild type and pros ; TorDN NBs , quantified in D ( n = 10 , 18 , 15 ) . E - F ) Representative pictures showing pros clones ( green ) generated in myc hypomorphic background did not significantly alter pros nucleolus / nuclear ratio ( E - F ) pros clones ( green ) generated in myc hypomorphic background did not significantly alter pros nucleolus / nuclear ratio ( E - F ) , nucleolus labelled with Fib ( red ) [ quantified in G ( n = 44 , 43 ) ] , clone size [ quantified in H ( n = 41 , 34 ) ] , or the proportion of Dpn + NBs and Elav + neurons per clone [ quantified in I ( n = 15 , 15 , 15 , 15 ) ] . J - K ) Representative pictures showing that nerfin - 1 clones ( green ) generated in myc hypomorphic background ( J - K ) nerfin - 1 clones ( green ) generated in myc hypomorphic background ( J - K ) exhibited significantly reduced nucleolus / nuclear ratio , [ nucleolus labelled with Fib ( red ) ] and reduced clone size , quantified in L ( n = 34 , 27 ) and M ( n = 106 , 75 ) , and displayed an increased proportion of differentiated Elav + neurons , and reduced proportion of Dpn + NBs per clone , quantified in N ( n = 20 , 20 , 20 , 20 ) . scale bar = 10μmO ) Reducing Myc dosage with the hypomorphic Myc allele , mycP0 , abolished the increase in nerfin - 1 clone size mediated by 2x histidine dietary intake , 2x his ( n = 41 , 18 , 10 , 57 ) . P ) Overexpression of myc restored the growth of nerfin - 1 clones inhibited by histidine dietary reduction , 25 % his ( n = 51 , 36 , 39 , 29 ) . Q , R ) pros ( Q ) and nerfin - 1 ( R ) clones are reduced upon Tor inhibition ( n = 22 , 63 ) compared to control ( n = 51 , 65 ) , but their growth differentially depend on amino acid transporter Slif ( n = 12 , 32 ) and downstream growth regulators eIF4E ( n = 21 , 47 ) and S6K ( n = 39 , 37 ) .

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 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 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 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 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 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

Figure 4A-D) Representative pictures showing that pros NBs and GMCs are smaller than wildtype (NBs and GMCs are distinguished at telophase, as a doublet of unequal size), quantified in C (n=25,31,21,16). pros NBs exhibit similar nucleolus /nuclear ratio (white arrows, nucleolus marked by Fib) compared to wild type and pros;TorDN NBs, quantified in D (n=10,18,15).E-F) Representative pictures showing pros clones (green) generated in myc hypomorphic background did not significantly alter pros nucleolus/nuclear ratio (E-F)pros clones (green) generated in myc hypomorphic background did not significantly alter pros nucleolus/nuclear ratio (E-F), nucleolus labelled with Fib (red) [quantified in G (n=44,43)], clone size [quantified in H (n=41,34)], or the proportion of Dpn+ NBs and Elav+ neurons per clone [quantified in I (n=15,15,15,15)].J-K) Representative pictures showing that nerfin-1 clones (green) generated in myc hypomorphic background (J-K)nerfin-1 clones (green) generated in myc hypomorphic background (J-K) exhibited significantly reduced nucleolus/nuclear ratio, [nucleolus labelled with Fib (red)] and reduced clone size, quantified in L (n=34,27) and M (n= 106,75), and displayed an increased proportion of differentiated Elav+ neurons, and reduced proportion of Dpn+ NBs per clone, quantified in N (n=20,20,20,20). scale bar =10μmO) Reducing Myc dosage with the hypomorphic Myc allele, mycP0, abolished the increase in nerfin-1 clone size mediated by 2x histidine dietary intake, 2x his (n=41,18,10,57).P) Overexpression of myc restored the growth of nerfin-1 clones inhibited by histidine dietary reduction, 25% his (n=51,36,39,29).Q, R) pros (Q) and nerfin-1 (R) clones are reduced upon Tor inhibition (n=22,63) compared to control (n=51,65), but their growth differentially depend on amino acid transporter Slif ( n=12,32) and downstream growth regulators eIF4E (n= 21,47) and S6K (n=39,37).


Figure 1The graphs depict the recovery of CAR1 - mcherry ( left ) and tPH - CynA - KikGR ( right ) . Mean ± SEM is shown for n = 2 cells . Time - lapse confocal images of tPH - CynA - KikGR expressing AX3 cells showing docking events at the back of a migrating Dictyostelium cell . Front and back of the cell are shown .

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

Figure 1The graphs depict the recovery of CAR1-mcherry (left) and tPH-CynA-KikGR (right). Mean ± SEM is shown for n = 2 cells.Time-lapse confocal images of tPH-CynA-KikGR expressing AX3 cells showing docking events at the back of a migrating Dictyostelium cell. Front and back of the cell are shown.


Figure 2Time - lapse confocal images of AX3 cell co - expressing PH - Crac - GFP ( green ) and another established PI ( 3 , 4 ) P2 biosensor tPH - TAPP1 - RFP ( red ) . Box shows the vesicle . Time - lapse confocal images of tPH - CynA - KikGR ( green ) expressing wild - type AX3 cells . FM4 - 64 ( red ) was added at time 0 during live cell imaging . Front and back of the cell are shown . Box shows the vesicle .

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

Figure 2Time-lapse confocal images of AX3 cell co-expressing PH-Crac-GFP (green) and another established PI(3,4)P2 biosensor tPH-TAPP1-RFP (red). Box shows the vesicle.Time-lapse confocal images of tPH-CynA-KikGR (green) expressing wild-type AX3 cells. FM4-64 (red) was added at time 0 during live cell imaging. Front and back of the cell are shown. Box shows the vesicle.


Figure 3Time - lapse confocal images of wild - type AX3 cells expressing tPH - CynA - KikGR ( green ) and PH - crac ( red ) before and after PI3K inhibition by 5 µM Wortmannin . Confocal images of tPH - CynA - KikGR ( green ) and PH - Crac ( red ) expressing wild - type Dictyostelium AX3 cells ( left ) and PI3K1 - 2 - Dictyostelium cells ( right ) treated with 5 µM Latrunculin A .

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Figure 3Time-lapse confocal images of wild-type AX3 cells expressing tPH-CynA-KikGR (green) and PH-crac (red) before and after PI3K inhibition by 5 µM Wortmannin.Confocal images of tPH-CynA-KikGR (green) and PH-Crac (red) expressing wild-type Dictyostelium AX3 cells (left) and PI3K1-2- Dictyostelium cells (right) treated with 5 µM Latrunculin A.


Figure 4Time - lapse confocal images showing dynamic localization of PI ( 3 , 4 ) P2 . Nocodazole was added at time 0 during the live cell imaging . Quantification of vesicle number , vesicle number / min , and vesicle lifetime ( s ) from experiment in E . T - test was carried out by GraphPad Prism , * * P ≤ 0 . 01 versus After Nocodazole group ; mean ± SEM ( n = 10 ) . ( L ) Representative confocal images of tPH - CynA - KikGR in wild - type Dictyostelium AX2 or RacH − cells treated with 5 μM latrunculin A Scale bar : 5 μm . ( M ) Ratio of membrane to cytosol intensity of tPH - CynA - KikGR in wild - type Dictyostelium AX2 and RacH − cells . T - test was carried out by GraphPad Prism , * * * * P ≤ 0 . 0001 versus AX2 group ; mean ± SEM ( n = 33 ) .

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

Figure 4Time-lapse confocal images showing dynamic localization of PI(3,4)P2. Nocodazole was added at time 0 during the live cell imaging.   Quantification of vesicle number, vesicle number/min, and vesicle lifetime (s) from experiment in E. T-test was carried out by GraphPad Prism, **P ≤ 0.01 versus After Nocodazole group; mean±SEM (n = 10).   (L) Representative confocal images of tPH-CynA-KikGR in wild-type Dictyostelium AX2 or RacH− cells treated with 5 μM latrunculin A Scale bar: 5 μm. (M) Ratio of membrane to cytosol intensity of tPH-CynA-KikGR in wild-type Dictyostelium AX2 and RacH− cells. T-test was carried out by GraphPad Prism, ****P ≤ 0.0001 versus AX2 group; mean ± SEM (n = 33).


Figure 5Time - lapse confocal images of tPH - CynA - KikGR expressing Dictyostelium AX3 cells . The macropinosome at the front of the cell was photoconverted at time 0 . White arrow points to PI ( 3 , 4 ) P2 localization at the back of the membrane . Cell front and back are shown , box showing the photoconverted region .

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

Figure 5Time-lapse confocal images of tPH-CynA-KikGR expressing Dictyostelium AX3 cells. The macropinosome at the front of the cell was photoconverted at time 0. White arrow points to PI(3,4)P2 localization at the back of the membrane. Cell front and back are shown, box showing the photoconverted region.