Figure 8 . ( B - D ) ARPE - 19 cells were infected with adenovirus expressing MITF1 - FLAG . 16 h later , cells were incubated with DMSO or 250 nM Torin - 1 for 1 h and analyzed by immunoblotting ( C ) or immunofluorescence ( B and D ) with anti - FLAG antibodies . The region within the dotted box is magnified in the insets . Bars : ( B and D , main images ) 10 µm ; ( D , insets ) 5 µm . ( E ) ARPE - 19 cells were incubated with 250 nM Torin - 1 for 1 h . Cells were then lysed and analyzed by immunoblotting with antibodies against MITF . ( F ) HEK - 293T cells were incubated in medium containing DMSO or Torin - 1 for 1 h . Cells were lysed , and nuclei and membrane plus cytosol fractions were obtained by low speed centrifugation . Proteins from the different fractions were subjected to immunoblotting with antibodies against MITF , Lamp1 , and Histone H3 . ( G ) ARPE - 19 cells expressing active ( RagBGTP / RagDGDP ) or inactive ( RagBGDP / RagDGTP ) Rag heterodimers were immunoprecipitated with the anti - FLAG antibody and immunoblotted with antibodies against FLAG and GST ( used to detect MITF and Rag proteins , respectively ) . ( H ) HEK - 293T cells expressing active ( RagBGTP / RagDGDP ) or inactive ( RagBGDP / RagDGTP ) Rag heterodimers were pulled down using glutathione - Sepharose beads and immunoblotted with antibodies against GST and MITF ( used to detect Rag proteins and endogenous MITF , respectively ) . IP , immunoprecipitation .

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Figure 8.(B-D) ARPE-19 cells were infected with adenovirus expressing MITF1-FLAG. 16 h later, cells were incubated with DMSO or 250 nM Torin-1 for 1 h and analyzed by immunoblotting (C) or immunofluorescence (B and D) with anti-FLAG antibodies. The region within the dotted box is magnified in the insets. Bars: (B and D, main images) 10 µm; (D, insets) 5 µm.(E) ARPE-19 cells were incubated with 250 nM Torin-1 for 1 h. Cells were then lysed and analyzed by immunoblotting with antibodies against MITF.(F) HEK-293T cells were incubated in medium containing DMSO or Torin-1 for 1 h. Cells were lysed, and nuclei and membrane plus cytosol fractions were obtained by low speed centrifugation. Proteins from the different fractions were subjected to immunoblotting with antibodies against MITF, Lamp1, and Histone H3.(G) ARPE-19 cells expressing active (RagBGTP/RagDGDP) or inactive (RagBGDP/RagDGTP) Rag heterodimers were immunoprecipitated with the anti-FLAG antibody and immunoblotted with antibodies against FLAG and GST (used to detect MITF and Rag proteins, respectively).(H) HEK-293T cells expressing active (RagBGTP/RagDGDP) or inactive (RagBGDP/RagDGTP) Rag heterodimers were pulled down using glutathione-Sepharose beads and immunoblotted with antibodies against GST and MITF (used to detect Rag proteins and endogenous MITF, respectively). IP, immunoprecipitation.


Figure 9 . ( A and B ) ARPE - 19 cells were nucleofected with the indicated Rag - and MITF - expressing plasmids . 12 h later , cells were fixed , permeabilized with 0 . 2 % Triton X - 100 , and double stained with antibodies against FLAG and GST ( used to detect MITF and Rag proteins , respectively ) . Bars , 10 µm .

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Figure 9.(A and B) ARPE-19 cells were nucleofected with the indicated Rag- and MITF-expressing plasmids. 12 h later, cells were fixed, permeabilized with 0.2% Triton X-100, and double stained with antibodies against FLAG and GST (used to detect MITF and Rag proteins, respectively). Bars, 10 µm.


Figure 1A Immunoblot of DDX3 expression in lung cancer cell lines . B , C Colony - forming assays in H1299 ( B ) and A549 ( C ) lung cancer cells after knockdown by shRNA lentiviral constructs designed against DDX3 or vector control . Corresponding immunoblots displaying knockdown levels of DDX3 . Mean from 3 replicates with SD . D Proliferation of A549 and H1299 cells after knockdown of DDX3 . Mean from 3 replicates with SD . ( A549 P = 0 . 011 , H1299 P = 0 . 014 ; exponential curve fit , extra sum of squares F - test ) . E β - galactosidase staining in parental A549 cells and A549 DDX3 knockdown cells displaying senescent cells identified by the blue color . F Expression of DDX3 by immunohistochemistry in normal lung tissue . G DDX3 expression in squamous cell carcinoma . H DDX3 expression in adenocarcinoma . I DDX3 expression in small cell carcinoma . J Expression of DDX3 in different histological types of lung cancer . All data sets were compared against each other ( chi - square test , P = 0 . 481 ) . K Survival analysis of lung cancer patients in low and high DDX3 expressing tumors ( Kaplan - Meier curve and log - rank test , P = 0 . 016 ) .

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Figure 1A Immunoblot of DDX3 expression in lung cancer cell lines.B, C Colony-forming assays in H1299 (B) and A549 (C) lung cancer cells after knockdown by shRNA lentiviral constructs designed against DDX3 or vector control. Corresponding immunoblots displaying knockdown levels of DDX3. Mean from 3 replicates with SD.D Proliferation of A549 and H1299 cells after knockdown of DDX3. Mean from 3 replicates with SD. (A549 P = 0.011, H1299 P = 0.014; exponential curve fit, extra sum of squares F-test).E β-galactosidase staining in parental A549 cells and A549 DDX3 knockdown cells displaying senescent cells identified by the blue color.F Expression of DDX3 by immunohistochemistry in normal lung tissue.G DDX3 expression in squamous cell carcinoma.H DDX3 expression in adenocarcinoma.I DDX3 expression in small cell carcinoma.J Expression of DDX3 in different histological types of lung cancer. All data sets were compared against each other (chi-square test, P = 0.481).K Survival analysis of lung cancer patients in low and high DDX3 expressing tumors (Kaplan-Meier curve and log-rank test, P = 0.016).


Figure 2F Immunoblots of pull - down assay of DDX3 with biotin , AK - 298 , and SK - 153 . Lower panels display results using DDX5 and DDX17 antibodies . H Immunoblot displaying increasing concentrations of RK - 33 ( 50 , 100 , 200 nM ) resulting in increased inhibition of unwinding of oligomer products ( lanes 4 - 6 ) . I MTS viability assay of various lung cancer cell lines treated with RK - 33 for 72 h . Mean from 3 replicates with SD . J , K Colony - forming assay of A549 and H3255 cells treated with RK - 33 and with various doses of radiation 4 h later . Curves were fitted with a quadratic polynomial equation . Mean from 2 replicates with SD . P - values were determined by the extra sum of squares F - test .

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Figure 2F Immunoblots of pull-down assay of DDX3 with biotin, AK-298, and SK-153. Lower panels display results using DDX5 and DDX17 antibodies.H Immunoblot displaying increasing concentrations of RK-33 (50, 100, 200 nM) resulting in increased inhibition of unwinding of oligomer products (lanes 4-6).I MTS viability assay of various lung cancer cell lines treated with RK-33 for 72 h. Mean from 3 replicates with SD.J, K Colony-forming assay of A549 and H3255 cells treated with RK-33 and with various doses of radiation 4 h later. Curves were fitted with a quadratic polynomial equation. Mean from 2 replicates with SD. P-values were determined by the extra sum of squares F-test.


Figure 5Following injection of 20 mg / kg of RK - 33 , twice a week for 7 weeks , extensive histopathological examination was carried out following necropsy . Identical patterns were observed both in the control and in the treated mice ( n = 2 ) . Samples were stained with H & amp ; amp ; E . Scale bar is 50 μm . Pharmacokinetics of RK - 33 in SCID mice at various time intervals . Results are mean ± SD from 5 mice . LC - MS / MS method was used to determine concentration of RK - 33 in mouse plasma and tissue . HAPI cells were treated with RK - 33 ( 10 μM ) , HA14 - 1 ( 25 μM ) , or DMSO vehicle , followed by oligomycin ( oligo , 0 . 5 μg / ml ) , FCCP ( 3 μM ) , and antimycin A ( AA , 1 μM ) while oxygen consumption rate ( OCR ) was measured . Pyruvate ( 10 mM ) was added in combination with FCCP to ensure that substrate supply was not rate - limiting for maximal OCR . Data are mean ± SD from 2 to 3 wells and representative of independent experiments performed with two different HAPI passages . OCR is baseline - normalized to the point prior to drug or vehicle addition . HAPI microglial cells were incubated for 1 h in glucose - free XF24 assay medium that was supplemented with 2 - deoxyglucose ( 50 mM ) and pyruvate ( 10 mM ) . RK - 33 ( 10 μM ) , oligomycin ( 0 . 5 μg / ml ) , or vehicle control was additionally present as indicated . Results are mean ± SD from 12 replicates pooled from experiments using two consecutive passages . Significance was assessed by two - sided , unpaired t - test .

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Figure 5Following injection of 20 mg/kg of RK-33, twice a week for 7 weeks, extensive histopathological examination was carried out following necropsy. Identical patterns were observed both in the control and in the treated mice (n = 2). Samples were stained with H&amp;E. Scale bar is 50 μm.Pharmacokinetics of RK-33 in SCID mice at various time intervals. Results are mean ± SD from 5 mice. LC-MS/MS method was used to determine concentration of RK-33 in mouse plasma and tissue.HAPI cells were treated with RK-33 (10 μM), HA14-1 (25 μM), or DMSO vehicle, followed by oligomycin (oligo, 0.5 μg/ml), FCCP (3 μM), and antimycin A (AA, 1 μM) while oxygen consumption rate (OCR) was measured. Pyruvate (10 mM) was added in combination with FCCP to ensure that substrate supply was not rate-limiting for maximal OCR. Data are mean ± SD from 2 to 3 wells and representative of independent experiments performed with two different HAPI passages. OCR is baseline-normalized to the point prior to drug or vehicle addition.HAPI microglial cells were incubated for 1 h in glucose-free XF24 assay medium that was supplemented with 2-deoxyglucose (50 mM) and pyruvate (10 mM). RK-33 (10 μM), oligomycin (0.5 μg/ml), or vehicle control was additionally present as indicated. Results are mean ± SD from 12 replicates pooled from experiments using two consecutive passages. Significance was assessed by two-sided, unpaired t-test.


Figure 6Confirmation of high expression of Twist1 and DDX3 in the lung tumors of the transgenic Twist1 / KrasG12D mouse . Scale bar is 100 μm . Micro - CT images of transgenic Twist1 / KrasG12D mice treated as in ( C ) , before treatment and 1 week after treatment . Tumors are indicated by arrows and confirmed by H & amp ; amp ; E staining of lung sections ( lower panel ) . Scale bar is 250 μm . Quantification of tumor volume measured by micro - CT in Twist1 / KrasG12D mice , as shown in ( D ) . Significance was assessed by two - sided , unpaired t - test . Error bars represent SD . An orthotopic lung tumor model was generated using A549 human lung cancer cells and treated as in ( C ) . Figure displays H & amp ; amp ; E staining of lung sections from radiation - treated ( upper panel ) and RK - 33 - and radiation - treated mice ( lower panel ) . Scale bar is 2 mm . Quantification of tumor burden ( as tumor surface divided by total lung surface ) in orthotopic A549 lung cancer mouse model , as shown in ( F ) . Significance was assessed by two - sided , unpaired t - test . Error bars represent SD . Micro - CT images of transgenic Twist1 / KrasG12D mice treated as in ( H ) , before treatment and 1 week after treatment . Quantification of tumor volume measured by micro - CT in Twist1 / KrasG12D mice , as shown in ( I ) and expressed as relative tumor size . Significance was assessed by two - sided , unpaired t - test . Error bars represent SEM .

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Figure 6Confirmation of high expression of Twist1 and DDX3 in the lung tumors of the transgenic Twist1/KrasG12D mouse. Scale bar is 100 μm.Micro-CT images of transgenic Twist1/KrasG12D mice treated as in (C), before treatment and 1 week after treatment. Tumors are indicated by arrows and confirmed by H&amp;E staining of lung sections (lower panel). Scale bar is 250 μm.Quantification of tumor volume measured by micro-CT in Twist1/KrasG12D mice, as shown in (D). Significance was assessed by two-sided, unpaired t-test. Error bars represent SD.An orthotopic lung tumor model was generated using A549 human lung cancer cells and treated as in (C). Figure displays H&amp;E staining of lung sections from radiation-treated (upper panel) and RK-33- and radiation-treated mice (lower panel). Scale bar is 2 mm.Quantification of tumor burden (as tumor surface divided by total lung surface) in orthotopic A549 lung cancer mouse model, as shown in (F). Significance was assessed by two-sided, unpaired t-test. Error bars represent SD.Micro-CT images of transgenic Twist1/KrasG12D mice treated as in (H), before treatment and 1 week after treatment.Quantification of tumor volume measured by micro-CT in Twist1/KrasG12D mice, as shown in (I) and expressed as relative tumor size. Significance was assessed by two-sided, unpaired t-test. Error bars represent SEM.


Figure 7A Cell cycle analysis of H1299 cells treated with shDDX3 and processed by flow cytometry . Knockdown of DDX3 led to a decrease of cells in S - phase and an increase of cells in G1 - phase , indicative of a G1 arrest . Significance was assessed by two - sided , unpaired t - test . Error bars represent SD . B , C Cell cycle analysis of A549 and H1299 cells by flow cytometry after treatment with RK - 33 ( 0 , 2 , 4 , and 6 μM ) . RK - 33 induced a G1 cell cycle arrest in both cell lines . Significance was assessed by two - sided , unpaired t - test . Error bars represent SD . D , E Immunoblot of cell cycle - related proteins ( Cyclin D1 and Cyclin E1 ) and cell death - related proteins ( cleaved caspase 7 , cleaved caspase 9 ) in A549 and H1299 cells after treatment with RK - 33 ( 10 μM ) . Initially , a strong decrease of Cyclin D1 was observed . After 8 and 24 h , cleaved caspases 9 and 7 were apparent . F Immunoblot of MAPK pathway - related proteins ERK1 / 2 and phosphorylated ERK1 / 2 in A549 , H1299 , and H3255 ( RK - 33 resistant ) cells 24 h after treatment with RK - 33 ( 7 . 5 μM or 10 μM ) . ERK2 and especially ERK1 become dephosphorylated after treatment with RK - 33 in A549 and H1299 cells but not in H3255 cells . Outlined boxes indicate spliced lanes .

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Figure 7A Cell cycle analysis of H1299 cells treated with shDDX3 and processed by flow cytometry. Knockdown of DDX3 led to a decrease of cells in S-phase and an increase of cells in G1-phase, indicative of a G1 arrest. Significance was assessed by two-sided, unpaired t-test. Error bars represent SD.B, C Cell cycle analysis of A549 and H1299 cells by flow cytometry after treatment with RK-33 (0, 2, 4, and 6 μM). RK-33 induced a G1 cell cycle arrest in both cell lines. Significance was assessed by two-sided, unpaired t-test. Error bars represent SD.D, E Immunoblot of cell cycle-related proteins (Cyclin D1 and Cyclin E1) and cell death-related proteins (cleaved caspase 7, cleaved caspase 9) in A549 and H1299 cells after treatment with RK-33 (10 μM). Initially, a strong decrease of Cyclin D1 was observed. After 8 and 24 h, cleaved caspases 9 and 7 were apparent.F Immunoblot of MAPK pathway-related proteins ERK1/2 and phosphorylated ERK1/2 in A549, H1299, and H3255 (RK-33 resistant) cells 24 h after treatment with RK-33 (7.5 μM or 10 μM). ERK2 and especially ERK1 become dephosphorylated after treatment with RK-33 in A549 and H1299 cells but not in H3255 cells. Outlined boxes indicate spliced lanes.


Figure 8A β - catenin ( red ) and DDX3 ( green ) expression in H1299 cells . After overexpressing β - catenin , both DDX3 and β - catenin accumulate in the nucleus . Scale bar is 10 μm . B Immunoprecipitation with DDX3 or H3K4Me3 ( control ) and immunoblotted with β - catenin in A549 and H1299 cells . Outlined boxes indicate spliced lanes . C Immunoprecipitation with β - catenin or H3K4Me3 ( control ) and immunoblotted with DDX3 in A549 and H1299 cells . Outlined boxes indicate spliced lanes . D , E β - catenin / TCF4 activity was determined by the TOP / FOP reporter assay . Co - transfection with β - catenin is indicated below . F - I H1299 and A549 cells were treated with RK - 33 ( 0 , 1 , 2 , and 3 μM ) and co - transfected with β - catenin in ( F , H ) . Treatment with RK - 33 decreased TCF4 activity in both cell lines . J , K Normalized mRNA expression of TCF4 - regulated proteins ( Axin - 2 , c - Myc , Cyclin D1 ) and DDX3 were measured by qRT - PCR in H1299 cells after knockdown of DDX3 ( J ) and treatment with RK - 33 ( K ) . All experiments were repeated three times .

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Figure 8A β-catenin (red) and DDX3 (green) expression in H1299 cells. After overexpressing β-catenin, both DDX3 and β-catenin accumulate in the nucleus. Scale bar is 10 μm.B Immunoprecipitation with DDX3 or H3K4Me3 (control) and immunoblotted with β-catenin in A549 and H1299 cells. Outlined boxes indicate spliced lanes.C Immunoprecipitation with β-catenin or H3K4Me3 (control) and immunoblotted with DDX3 in A549 and H1299 cells. Outlined boxes indicate spliced lanes.D, E β-catenin/TCF4 activity was determined by the TOP/FOP reporter assay. Co-transfection with β-catenin is indicated below.F-I H1299 and A549 cells were treated with RK-33 (0, 1, 2, and 3 μM) and co-transfected with β-catenin in (F, H). Treatment with RK-33 decreased TCF4 activity in both cell lines.J, K Normalized mRNA expression of TCF4-regulated proteins (Axin-2, c-Myc, Cyclin D1) and DDX3 were measured by qRT-PCR in H1299 cells after knockdown of DDX3 (J) and treatment with RK-33 (K). All experiments were repeated three times.


Figure 9A Immunofluorescence images showing 53BP1 and γH2AX foci in A549 cells after 2 - Gy radiation and A549 cells pre - treated with 6 μM RK - 33 , 12 h before radiation . Overlap of 53BP1 and γH2AX is seen in the merged picture of the co - immunofluorescence staining . Scale bar is 2 μm . B A549 cells were pre - treated with RK - 33 and radiated with 2 Gy , and 53BP1 and γH2AX foci were counted as a measure of DNA damage . Cells with more than 10 foci 53BP1 or γH2AX were counted . More than 400 cells per sample were evaluated . C H1299 cells stably transfected with a homologous recombination ( HR ) reporter construct were treated with RK - 33 . Reporter constructs expressed GFP , which was quantified by flow cytometry . Experiments were repeated three times . D H1299 cells , containing a stable non - homologous end - joining ( NHEJ ) reporter construct , were treated with RK - 33 and knockdown of DDX3 . Reporter construct expressed GFP , which was quantified by flow cytometry . All experiments were repeated three times . E Microarray results from MDA - MB - 231 cells treated with RK - 33 and shDDX3 were validated by qRT - PCR using NHEJ Mechanisms of DSBs Repair PrimePCR plates ( Bio - Rad ) and performed in biological triplicates . F , G DNA repair - related proteins ( ATR and XRCC4 ) , DDX3 , and actin were assessed by immunoblotting in A549 ( F ) and H1299 ( G ) cells . Cells were pretreated for 4 h with vehicle control or 6 μM RK - 33 and then radiated with 0 or 5 Gy . Outlined boxes indicate spliced lanes .

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Figure 9A Immunofluorescence images showing 53BP1 and γH2AX foci in A549 cells after 2-Gy radiation and A549 cells pre-treated with 6 μM RK-33, 12 h before radiation. Overlap of 53BP1 and γH2AX is seen in the merged picture of the co-immunofluorescence staining. Scale bar is 2 μm.B A549 cells were pre-treated with RK-33 and radiated with 2 Gy, and 53BP1 and γH2AX foci were counted as a measure of DNA damage. Cells with more than 10 foci 53BP1 or γH2AX were counted. More than 400 cells per sample were evaluated.C H1299 cells stably transfected with a homologous recombination (HR) reporter construct were treated with RK-33. Reporter constructs expressed GFP, which was quantified by flow cytometry. Experiments were repeated three times.D H1299 cells, containing a stable non-homologous end-joining (NHEJ) reporter construct, were treated with RK-33 and knockdown of DDX3. Reporter construct expressed GFP, which was quantified by flow cytometry. All experiments were repeated three times.E Microarray results from MDA-MB-231 cells treated with RK-33 and shDDX3 were validated by qRT-PCR using NHEJ Mechanisms of DSBs Repair PrimePCR plates (Bio-Rad) and performed in biological triplicates.F, G DNA repair-related proteins (ATR and XRCC4), DDX3, and actin were assessed by immunoblotting in A549 (F) and H1299 (G) cells. Cells were pretreated for 4 h with vehicle control or 6 μM RK-33 and then radiated with 0 or 5 Gy. Outlined boxes indicate spliced lanes.


Figure 1D . RNA expression heatmap showing the top differentially expressed gene markers for each cluster of the reference single cell atlas dataset . Cells ( column ) are clustered by the expression of the main marker genes ( row ) . E . Average expression level of the genes Fas3 ( left panel ) and zfh1 ( right panel ) used as markers to assign epithelial and myoblast cells , respectively , in the reference dataset . F . Confocal single plane image of third instar larval wing disc and orthogonal views of the disc stained with anti - Zfh1 ( red ) and anti - Fas3 ( green ) . G . Dot plot showing the expression levels of the marker genes identified for the myoblast , epithelial and tracheal cells across the 26 clusters of the reference cell atlas . Color intensity represents the average normalized expression level . Dot diameter represents the fraction of cells expressing each gene in each cluster . Gene expression for each cell was normalized by the total expression , and then the expression of each gene was scaled . H . Confocal single plane image of third instar larval wing disc and orthogonal view of SPARC > GFP ( green ) stained with anti - Ct ( red ) and 4 ´ , 6 - diamidino - 2 - phenylindole ( DAPI , blue ) . Full genotype y - , w - / w - ; UAS - GFP / + ; SPARC - GAL4 / + .

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

Figure 1D. RNA expression heatmap showing the top differentially expressed gene markers for each cluster of the reference single cell atlas dataset. Cells (column) are clustered by the expression of the main marker genes (row).E. Average expression level of the genes Fas3 (left panel) and zfh1 (right panel) used as markers to assign epithelial and myoblast cells, respectively, in the reference dataset.F. Confocal single plane image of third instar larval wing disc and orthogonal views of the disc stained with anti-Zfh1 (red) and anti-Fas3 (green).G. Dot plot showing the expression levels of the marker genes identified for the myoblast, epithelial and tracheal cells across the 26 clusters of the reference cell atlas. Color intensity represents the average normalized expression level. Dot diameter represents the fraction of cells expressing each gene in each cluster. Gene expression for each cell was normalized by the total expression, and then the expression of each gene was scaled.H. Confocal single plane image of third instar larval wing disc and orthogonal view of SPARC>GFP (green) stained with anti-Ct (red) and 4´,6-diamidino-2-phenylindole (DAPI, blue). Full genotype y-, w-/w-; UAS-GFP/+; SPARC-GAL4/+.


Figure 2D . Average expression level of the genes used as known markers to assign each epithelial cluster in the reference atlas dataset . Cells colored by the expression of nub , wg , zfh2 , Sox15 , grn , bnl , eyg , Ance , Idg4 and drm . E . Dot plot showing the expression levels of the top marker genes identified for the epithelial cells across 17 clusters of the reference cell atlas dataset . F . Confocal single plane image of third instar larval wing disc stained with anti - Nub , anti - Wg , anti - Svp , anti - Hairy ( red ) , reporting expression of dpp - lacZ , Dad - lacZ ( anti - β - gal , red ) and counterstained with DAPI ( cyan ) . Scale bars : 100 μm . Full genotypes : P { CaryP } attP2 ( top and bottom panels ) , P { PZ } dpp [ 10638 ] ; + , and y - w - ; + ; P { lacW } Dad [ j1E4 ] .

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Figure 2D. Average expression level of the genes used as known markers to assign each epithelial cluster in the reference atlas dataset. Cells colored by the expression of nub, wg, zfh2, Sox15, grn, bnl, eyg, Ance, Idg4 and drm.E. Dot plot showing the expression levels of the top marker genes identified for the epithelial cells across 17 clusters of the reference cell atlas dataset.F. Confocal single plane image of third instar larval wing disc stained with anti-Nub, anti-Wg, anti-Svp, anti-Hairy (red), reporting expression of dpp-lacZ, Dad-lacZ (anti-β-gal, red) and counterstained with DAPI (cyan). Scale bars: 100 μm. Full genotypes: P{CaryP}attP2 (top and bottom panels), P{PZ}dpp[10638];+, and y- w-;+; P{lacW}Dad[j1E4].


Figure 3B . Dot plots showing the expression levels of the top variable genes between IFM and DFM myoblasts across 7 clusters of the reference cell atlas dataset . Confocal single plane images of third instar larval wing discs stained with : ( C ) anti - Ct ( red ) and anti - Zfh1 ( green ) , ( D ) kirre [ rp298 ] - lacZ ( anti - β - gal , green ) and anti - Zfh1 ( red ) , ( E ) Ten - a [ MI04411 ] : : GFP ( green ) , anti - Ct ( red ) and anti - Zfh1 ( cyan ) , ( F ) anti - Nrt ( red ) and anti - Zfh1 ( cyan ) , ( G ) Argk [ CB3789 ] : : GFP ( green ) , anti - Ct ( red ) and anti - Zfh1 ( cyan ) , ( H ) nkd [ MI00209 ] : : GFP ( green ) , anti - Ct ( red ) and anti - Zfh1 ( cyan ) , ( I ) Ama [ NP1297 ] > GFP ( green ) , anti - Ct ( red ) and anti - Zfh1 ( cyan ) and ( J ) Mef2 > CD8 - GFP ( green ) , and fluorescent in situ hybridization of Ama - RNA probe ( white ) . Confocal single plane images of third instar larval wing discs stained with : K . Ama [ NP1297 ] > gTRACE showing the lineage of Ama - GAL4 ( green ) and the active GAL4 ( red ) , and fluorescent in situ hybridization of Ama - RNA probe ( white ) . L . Confocal single plane images of adult DFM of Ama [ NP1297 ] > gTRACE showing the lineage of Ama - GAL4 ( green ) and the active GAL4 ( red ) stained with DAPI ( white ) dorsal right , anterior up . DFM are numbered in white following ( Lawrence , 1982 ) .

O B-PANEL_START O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3B. Dot plots showing the expression levels of the top variable genes between IFM and DFM myoblasts across 7 clusters of the reference cell atlas dataset.Confocal single plane images of third instar larval wing discs stained with: (C) anti-Ct (red) and anti-Zfh1 (green), (D) kirre[rp298]-lacZ (anti-β-gal, green) and anti-Zfh1 (red), (E) Ten-a[MI04411]::GFP (green), anti-Ct (red) and anti-Zfh1 (cyan), (F) anti-Nrt (red) and anti-Zfh1 (cyan), (G) Argk[CB3789]::GFP (green), anti-Ct (red) and anti-Zfh1 (cyan), (H) nkd[MI00209]::GFP (green), anti-Ct (red) and anti-Zfh1 (cyan), (I) Ama[NP1297]>GFP (green), anti-Ct (red) and anti-Zfh1 (cyan) and (J) Mef2>CD8-GFP (green), and fluorescent in situ hybridization of Ama-RNA probe (white).Confocal single plane images of third instar larval wing discs stained with: K. Ama[NP1297]>gTRACE showing the lineage of Ama-GAL4 (green) and the active GAL4 (red), and fluorescent in situ hybridization of Ama-RNA probe (white). L. Confocal single plane images of adult DFM of Ama[NP1297]>gTRACE showing the lineage of Ama-GAL4 (green) and the active GAL4 (red) stained with DAPI (white) dorsal right, anterior up. DFM are numbered in white following (Lawrence, 1982).


Figure 4A . Dot plot showing the expression levels of the top variable genes between myoblasts across 7 clusters of the reference cell atlas dataset . Confocal single plane images of third instar larval wing discs stained with anti - Ct ( red ) and ( B ) E ( spl ) m6 - BFM - gapGFP ( green ) , ( C ) E ( spl ) m7 - HLH [ VK00037 ] - GFP ( green ) Confocal single plane images of third instar larval wing discs stained with ( D ) E ( spl ) m3 - HLH [ GMR10E12 ] > GFP ( green ) . H - I . Confocal single plane images of third instar larval wing discs stained with anti - Ct ( cyan ) showing the lineage of GAL4 ( green ) and the active GAL4 ( red ) in ( H ) E ( spl ) m3 - HLH [ GMR10E12 ] > gTRACE and ( I ) E ( spl ) m6 - BFM - GAL4 > gTRACE .

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Figure 4A. Dot plot showing the expression levels of the top variable genes between myoblasts across 7 clusters of the reference cell atlas dataset.Confocal single plane images of third instar larval wing discs stained with anti-Ct (red) and (B) E(spl)m6-BFM-gapGFP (green), (C) E(spl)m7-HLH[VK00037]-GFP (green)Confocal single plane images of third instar larval wing discs stained with (D) E(spl)m3-HLH[GMR10E12]>GFP (green).H-I. Confocal single plane images of third instar larval wing discs stained with anti-Ct (cyan) showing the lineage of GAL4 (green) and the active GAL4 (red) in (H) E(spl)m3-HLH[GMR10E12]>gTRACE and (I) E(spl)m6-BFM-GAL4>gTRACE.


Figure 5A - E Confocal single plane images of third instar larval wing discs stained with ( A ) anti - LamC ( green ) and anti - Zfh1 ( red ) , ( B ) edl [ k12907 ] - lacZ ( green ) and anti - Zfh1 ( red ) , ( C ) anti - H ( green ) and anti - Zfh1 ( red ) , ( D ) Cg25C [ k00405 ] - lacZ ( green ) and anti - Zfh1 ( red ) , or ( E ) vgk [ G00454 ] - GFP ( green ) and anti - Ct ( red ) .

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Figure 5A-E Confocal single plane images of third instar larval wing discs stained with (A) anti-LamC (green) and anti-Zfh1 (red), (B) edl[k12907]-lacZ (green) and anti-Zfh1 (red), (C) anti-H (green) and anti-Zfh1 (red), (D) Cg25C[k00405]-lacZ (green) and anti-Zfh1 (red), or (E) vgk[G00454]-GFP (green) and anti-Ct (red).


Figure 6The muscle - specific ( A - B ) Mef2 - GAL4 drivers were crossed to UAS - RNAi lines to knockdown the expression of the gene markers for the myoblast clusters . ( A , Viability test quantified by scoring the percentage of viable animals at each developmental stage , including early pupa ( black ) , pharate ( grey ) and adult ( white ) , relative to the total number of animals . Data are expressed as stacked bars showing the median with interquartile range ( n ≥ 3 independent biological replicates ) , unless noted as viable ( NQ ) and lethal ( NQ ) , which stands for animals that were viable and lethal , respectively , but no quantitative analysis were performed . ( B , Flight ability scored by quantifying the percentage of flies landing on each section of the column ( top , middle and bottom ) as indicated on the diagram on the right . Data are expressed as stacked bars . The total number of flies used in each assay was pooled together and displayed next to each bar ( N ≥ 6 ) . n / a stands for non - applicable since animals are not viable . The muscle - specific 151 - GAL4 drivers were crossed to UAS - RNAi lines to knockdown the expression of the gene markers for the myoblast clusters . C ) . Viability test quantified by scoring the percentage of viable animals at each developmental stage , including early pupa ( black ) , pharate ( grey ) and adult ( white ) , relative to the total number of animals . Data are expressed as stacked bars showing the median with interquartile range ( n ≥ 3 independent biological replicates ) , unless noted as viable ( NQ ) and lethal ( NQ ) , which stands for animals that were viable and lethal , respectively , but no quantitative analysis were performed . D ) . Flight ability scored by quantifying the percentage of flies landing on each section of the column ( top , middle and bottom ) as indicated on the diagram on the right . Data are expressed as stacked bars . The total number of flies used in each assay was pooled together and displayed next to each bar ( N ≥ 6 ) . n / a stands for non - applicable since animals are not viable . E . Confocal single plane images of third instar larval wing discs from Mef2 > mCherry - RNAi , ( left panel ) and Mef2 > chinmo [ HM04048 ] - RNAi ( right panel ) stained with anti - Zfh1 ( red ) and DAPI ( cyan ) . Scale bars : 50 μm . F . Quantification of phenotype displayed in ( E ) . Stacked bars showing the percentage of wing discs scored as either normal or as abnormal . Data are expressed as mean ± sem , N = 32 discs per genotype , n = 3 independent experiments . G . Quantification of viability test . Since the UAS - Ama - RNAi [ GD12733 ] transgene is inserted on Chromosome X the genotype of females is Mef2 > Ama - RNAi whereas males are Mef2 > + . Box plot showing the ratio of flies with genotype of interest ( females ) over control flies ( males ) . The whiskers show 5 - 95 percentile , and the middle line the median . * p = 0 . 014 ( Mann - Whitney test , N ≥ 6 independent biological replicates ) . Full genotypes are w , UAS - Dicer2 ; + ; mCherry - RNAi / Mef2 - GAL4 and w , UAS - Dicer2 , UAS - Ama - RNAi [ GD12733 ] ; + ; Mef2 - GAL4 .

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

Figure 6The muscle-specific (A-B) Mef2-GAL4 drivers were crossed to UAS-RNAi lines to knockdown the expression of the gene markers for the myoblast clusters. (A, Viability test quantified by scoring the percentage of viable animals at each developmental stage, including early pupa (black), pharate (grey) and adult (white), relative to the total number of animals. Data are expressed as stacked bars showing the median with interquartile range (n≥ 3 independent biological replicates), unless noted as viable (NQ) and lethal (NQ), which stands for animals that were viable and lethal, respectively, but no quantitative analysis were performed. (B, Flight ability scored by quantifying the percentage of flies landing on each section of the column (top, middle and bottom) as indicated on the diagram on the right. Data are expressed as stacked bars. The total number of flies used in each assay was pooled together and displayed next to each bar (N≥6). n/a stands for non-applicable since animals are not viable.The muscle-specific 151-GAL4 drivers were crossed to UAS-RNAi lines to knockdown the expression of the gene markers for the myoblast clusters. C). Viability test quantified by scoring the percentage of viable animals at each developmental stage, including early pupa (black), pharate (grey) and adult (white), relative to the total number of animals. Data are expressed as stacked bars showing the median with interquartile range (n≥ 3 independent biological replicates), unless noted as viable (NQ) and lethal (NQ), which stands for animals that were viable and lethal, respectively, but no quantitative analysis were performed. D). Flight ability scored by quantifying the percentage of flies landing on each section of the column (top, middle and bottom) as indicated on the diagram on the right. Data are expressed as stacked bars. The total number of flies used in each assay was pooled together and displayed next to each bar (N≥6). n/a stands for non-applicable since animals are not viable.E. Confocal single plane images of third instar larval wing discs from Mef2>mCherry-RNAi, (left panel) and Mef2>chinmo[HM04048]-RNAi (right panel) stained with anti-Zfh1 (red) and DAPI (cyan). Scale bars: 50 μm. F. Quantification of phenotype displayed in (E). Stacked bars showing the percentage of wing discs scored as either normal or as abnormal. Data are expressed as mean ± sem, N = 32 discs per genotype, n = 3 independent experiments. G. Quantification of viability test. Since the UAS-Ama-RNAi[GD12733] transgene is inserted on Chromosome X the genotype of females is Mef2>Ama-RNAi whereas males are Mef2>+. Box plot showing the ratio of flies with genotype of interest (females) over control flies (males). The whiskers show 5-95 percentile, and the middle line the median. * p=0.014 (Mann-Whitney test, N≥6 independent biological replicates). Full genotypes are w, UAS-Dicer2;+; mCherry-RNAi/Mef2-GAL4 and w, UAS-Dicer2, UAS-Ama-RNAi[GD12733];+; Mef2-GAL4.


Figure 7A . Lethality of 1151 > Ama - RNAi [ HMS00297 ] animals at pharate stage was partially rescued by overexpressing the transgene Ama - cDNA . Representative images of adults and pharate pupa are shown . Full genotypes : 1151 - GAL4 ; UAS - myrRFP ; + , 1151 - GAL4 ; UAS - myrRFP ; UAS - Ama [ HMS00297 ] - RNAi , 1151 - GAL4 ; UAS - AmaOE / CyO ; UAS - Ama [ HMS00297 ] - RNAi and 1151 - GAL4 ; UAS - AmaOE ; + . C . Average expression level of the genes Fas3 ( left panel ) and zfh1 ( right panel ) in the 1151 > Ama - RNAi dataset . D . Confocal single plane images of 1151 > mCherry - RNAi and 1151 > Ama - RNAi wing discs stained with anti - Ct ( red ) and anti - Zfh1 ( green ) . E . Dot plots showing the expression levels of the top markers for myoblast clusters identified in the reference cell atlas across the cluster of myoblasts in 1151 > Ama - RNAi and 7 myoblast clusters in control dataset . F . The myoblast cells in 1151 > Ama - RNAi dataset projected into the reference single cell atlas by transferring cell type labels using Seurat . The bar graph represents the total number of cells in each cluster normalized to the total number of epithelial cells per genotype . G - H . Confocal single plane images of 1151 > mCherry - RNAi and 1151 > Ama - RNAi wing discs stained with anti - Zfh1 ( green ) and ( G ) anti - Nrt ( white ) at wandering third instar larval stage ( 110 - 135 h AEL ) or ( H ) DAPI ( red ) at early third instar larval wing discs ( 72 - 110 h AEL ) .

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Figure 7A. Lethality of 1151>Ama-RNAi[HMS00297] animals at pharate stage was partially rescued by overexpressing the transgene Ama-cDNA. Representative images of adults and pharate pupa are shown. Full genotypes: 1151-GAL4;UAS-myrRFP;+ , 1151-GAL4;UAS-myrRFP;UAS-Ama[HMS00297]-RNAi , 1151-GAL4;UAS-AmaOE/CyO;UAS-Ama[HMS00297]-RNAi and 1151-GAL4;UAS-AmaOE;+.C. Average expression level of the genes Fas3 (left panel) and zfh1 (right panel) in the 1151>Ama-RNAi dataset.D. Confocal single plane images of 1151>mCherry-RNAi and 1151>Ama-RNAi wing discs stained with anti-Ct (red) and anti-Zfh1 (green).E. Dot plots showing the expression levels of the top markers for myoblast clusters identified in the reference cell atlas across the cluster of myoblasts in 1151>Ama-RNAi and 7 myoblast clusters in control dataset.F. The myoblast cells in 1151>Ama-RNAi dataset projected into the reference single cell atlas by transferring cell type labels using Seurat. The bar graph represents the total number of cells in each cluster normalized to the total number of epithelial cells per genotype.G-H. Confocal single plane images of 1151>mCherry-RNAi and 1151>Ama-RNAi wing discs stained with anti-Zfh1 (green) and (G) anti-Nrt (white) at wandering third instar larval stage (110 - 135 h AEL) or (H) DAPI (red) at early third instar larval wing discs (72 - 110 h AEL).


Figure 8Confocal single plane images of 1151 > Ama - RNAi animals and 1151 > mCherry - RNAi . A . Forming DFM at 40 h APF stained with anti - Zfh1 ( green ) , anti - Futsch ( 22c10 , red ) and anti - Kettin ( white ) . White arrows point to the wing hinge , wings pointing left , anterior up . B . Forming IFM ( DLM ) at 16 h APF , stained with anti - Futsch ( 22c10 , red ) , anti - Zfh1 ( green ) and DAPI ( cyan ) . Yellow - dashed box indicates magnified area ( bottom panel ) . Anterior up . C . DFM at 96 h APF stained with Phalloidin ( red ) , anti - Kettin ( green ) and DAPI ( blue ) . Anterior up , dorsal right . DFM are numbered in white as in ( Lawrence , 1982 ) . D . IFM at 96 h APF , transverse section ( top ) stained with anti - PS - integrin ( green ) , Phalloidin ( red ) and DAPI ( blue ) , dorsal up ; sagittal section ( bottom ) stained with anti - Kettin ( green ) , Phalloidin ( red ) and DAPI ( blue ) , anterior up , dorsal right .

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

Figure 8Confocal single plane images of 1151>Ama-RNAi animals and 1151>mCherry-RNAi. A. Forming DFM at 40 h APF stained with anti-Zfh1 (green), anti-Futsch (22c10, red) and anti-Kettin (white). White arrows point to the wing hinge, wings pointing left, anterior up. B. Forming IFM (DLM) at 16 h APF, stained with anti-Futsch (22c10, red), anti-Zfh1 (green) and DAPI (cyan). Yellow-dashed box indicates magnified area (bottom panel). Anterior up.C. DFM at 96 h APF stained with Phalloidin (red), anti-Kettin (green) and DAPI (blue). Anterior up, dorsal right. DFM are numbered in white as in (Lawrence, 1982). D. IFM at 96 h APF, transverse section (top) stained with anti-PS-integrin (green), Phalloidin (red) and DAPI (blue), dorsal up; sagittal section (bottom) stained with anti-Kettin (green), Phalloidin (red) and DAPI (blue), anterior up, dorsal right.


Figure 1B ) Larval brains at 6 h ALH from grh - Gal4 ; UAS - CD8 - GFP were labeled with Sas - 4 , Dpn , and GFP . C ) Larval brains at 0 h ALH from grh - Gal4 ; UAS - CD8 - GFP were labeled with γ - tubulin ( γ - tub ) , Asl , and GFP and imaged under super - resolution microscopy . D ) Larval brains at 0 h ALH from grh - Gal4 ; UAS - CD8 - GFP were labeled with Centrosomin ( CNN ) , Asl and GFP and imaged under spinning disc super - resolution microscopy . E ) Larval brains at 0 h ALH from Jupiter - GFP ( G147 ) were labeled with GFP , Msps , and Dpn . F ) Larval brains at 24 h ALH from Jupiter - GFP were raised on normal food ( fed ) and food depleted of amino acids ( nutritional restriction / NR ) and labelled with GFP and Dpn .

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

Figure 1B) Larval brains at 6 h ALH from grh-Gal4; UAS-CD8-GFP were labeled with Sas-4, Dpn, and GFP.C) Larval brains at 0 h ALH from grh-Gal4; UAS-CD8-GFP were labeled with γ-tubulin (γ-tub), Asl, and GFP and imaged under super-resolution microscopy. D) Larval brains at 0 h ALH from grh-Gal4; UAS-CD8-GFP were labeled with Centrosomin (CNN), Asl and GFP and imaged under spinning disc super-resolution microscopy. E) Larval brains at 0 h ALH from Jupiter-GFP (G147) were labeled with GFP, Msps, and Dpn. F) Larval brains at 24 h ALH from Jupiter-GFP were raised on normal food (fed) and food depleted of amino acids (nutritional restriction/NR) and labelled with GFP and Dpn.


Figure 2 A ) Larval brains at 16 h ALH , in which kin - lacZ was expressed under the control of insc - Gal4 , tub - Gal80ts , were labelled with β - Gal , Msps , and Dpn . The primary protrusion of qNSCs was marked by Msps . Quiescent NSCs at the ventral nerve cord ( VNC ) are shown . Arrows indicate the localization of Kin - lacZ in qNSCs . B ) Larval brains at 6 h ALH from insc - Gal4 ; UAS - nod - lacZ were labeled with β - Gal , Msps , and Dpn . Quiescent NSCs at both CB and VNC are shown . Arrows indicate the localization of Nod - lacZ in qNSCs

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 A) Larval brains at 16 h ALH, in which kin-lacZ was expressed under the control of insc-Gal4, tub-Gal80ts, were labelled with β-Gal, Msps, and Dpn. The primary protrusion of qNSCs was marked by Msps. Quiescent NSCs at the ventral nerve cord (VNC) are shown. Arrows indicate the localization of Kin-lacZ in qNSCs. B) Larval brains at 6 h ALH from insc-Gal4; UAS-nod-lacZ were labeled with β-Gal, Msps, and Dpn. Quiescent NSCs at both CB and VNC are shown. Arrows indicate the localization of Nod-lacZ in qNSCs


Figure 3 A ) Larval brains at 24 h ALH from control ( insc - Gal4 ; UAS - dicer2 / UAS - β - Gal RNAi ) and msps RNAi ( VDRC # 21982 ) controlled under insc - Gal4 were analyzed for EdU incorporation . NSCs were marked by Dpn and Mira . B ) Quantification graph of EdU - negative NSCs per brain lobe for genotypes in ( A ) . n = 13 BL for both control and msps RNAi . * * * * p < 0 . 0001 C ) Larval brains at 24 h ALH from wild - type and various msps loss - of - function alleles and msps810 with a genomic rescue construct ( g - msps ) were analyzed for EdU incorporation . NSCs were marked by Dpn and Mira . D ) Quantification graph of EdU - negative NSCs per brain lobe for genotypes in ( C ) . n = 13 BL for control ; n = 13 BL for msps810 ; n = 17 BL for msps924 ; n = 14 BL for mspsP18 ; n = 6 BL for mspsP ; n = 12 BL for g - msps ; msps924 . * * * * p < 0 . 0001 ; p = 0 . 7502 ( ns ) . E ) Quantification graph of the diameter of the cell body in NSCs at 24 h ALH from various genotypes . n = 238 NSCs for control ; n = 219 NSC for msps924 ; n = 170 NSCs for mspsP . * * * * p < 0 . 0001 . F ) Quantification graph of the percentage of qNSCs with a primary protrusion in wild - type , msps924 , and mspsP18 larval brains . The protrusion was labelled by grh > CD8 - GFP . n = 5 BL for control ; n = 6 BL for msps924 ; n = 4 BL for mspsP . * * * * p < 0 . 0001 G ) Larval brains at 24 h ALH from wild - type and tacc loss - of - function tacc74 were examined for EdU incorporation . NSCs were marked by Dpn and Mira . H ) Quantification graph of EdU - negative NSCs per brain lobe for genotypes in ( G ) . n = 17 BL for control ; n = 15 BL for tacc74 . * * * * p < 0 . 0001 . I ) Quantification graph of qNSCs retaining primary protrusion per brain lobe for genotypes in ( G ) . n = 15 BL for both control and tacc74 . * * * * p < 0 . 0001 . J ) Quantification graph of NSCs are positive for mitotic marker PH3 per brain lobe for genotypes in ( G ) . n = 15 BL for both control and tacc74 . * * * * p < 0 . 0001

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 A) Larval brains at 24 h ALH from control (insc-Gal4; UAS-dicer2/UAS-β-Gal RNAi) and msps RNAi (VDRC#21982) controlled under insc-Gal4 were analyzed for EdU incorporation. NSCs were marked by Dpn and Mira. B) Quantification graph of EdU-negative NSCs per brain lobe for genotypes in (A). n=13 BL for both control and msps RNAi. ****p< 0.0001  C) Larval brains at 24 h ALH from wild-type and various msps loss-of-function alleles and msps810 with a genomic rescue construct (g-msps) were analyzed for EdU incorporation. NSCs were marked by Dpn and Mira. D) Quantification graph of EdU-negative NSCs per brain lobe for genotypes in (C). n=13 BL for control; n=13 BL for msps810; n=17 BL for msps924; n=14 BL for mspsP18; n=6 BL for mspsP; n=12 BL for g-msps; msps924. ****p< 0.0001; p=0.7502 (ns). E) Quantification graph of the diameter of the cell body in NSCs at 24 h ALH from various genotypes. n=238 NSCs for control; n=219 NSC for msps924; n=170 NSCs for mspsP. ****p< 0.0001. F) Quantification graph of the percentage of qNSCs with a primary protrusion in wild-type, msps924, and mspsP18 larval brains. The protrusion was labelled by grh>CD8-GFP. n=5 BL for control; n=6 BL for msps924; n=4 BL for mspsP. ****p< 0.0001  G) Larval brains at 24 h ALH from wild-type and tacc loss-of-function tacc74 were examined for EdU incorporation. NSCs were marked by Dpn and Mira. H) Quantification graph of EdU-negative NSCs per brain lobe for genotypes in (G). n=17 BL for control; n=15 BL for tacc74. ****p< 0.0001. I) Quantification graph of qNSCs retaining primary protrusion per brain lobe for genotypes in (G). n=15 BL for both control and tacc74. ****p< 0.0001. J) Quantification graph of NSCs are positive for mitotic marker PH3 per brain lobe for genotypes in (G). n=15 BL for both control and tacc74. ****p< 0.0001


Figure 4 B ) Quantification graph of fold changes in the number of EB1 - GFP comets in the primary protrusion of qNSCs 6 h ALH from various genotypes compared with the control in ( A ) . n = 26 NSCs for control ; n = 18 NSCs for mspsP18 ; n = 21 NSCs for mspsP18 / P . * * * * p < 0 . 0001 . C ) Quantification graph of the percentage of retrograde EB1 - GFP comets in the primary protrusion of qNSCs from the control , mspsP18 with EB1 - GFP driven by grh - Gal4 at 6 h ALH . As EB1 - GFP comets were almost lost in mspsP18 , we were unable to capture adequate EB1 - GFP comets for each individual brain to calculate the standard deviation . 38 and 41 qNSCs were analyzed in control and mspsP18 , respectively . In control qNSCs , among total 383 EB1 - GFP comets , 353 were anterograde and 30 were retrograde movements . In mspsP18 qNSCs , we only found 12 EB1 - GFP comets with 6 anterograde and 6 retrograde movements D ) Larval brains at 24 h ALH from control ( grh - Gal4 UAS - CD8 - GFP + UAS - nod - lacZ ) and msps810 , UAS - nod - lacZ under the control of grh - Gal4 UAS - CD8 - GFP were labeled with β - Gal , Dpn , and GFP . Quiescent NSCs in the central brain were shown . Arrows point at the localization of Nod - lacZ in qNSCs . E ) Quantification graph of Nod - β - Gal localization in qNSCs from genotypes in ( D ) . n = 55 NSCs for control ; n = 45 NSCs for msps810 . * * * * p < 0 . 0001 F ) Larval brains at 16 h ALH from control ( grh - Gal4 UAS - CD8 - GFP ; UAS - dicer2 + UAS - nod - lacZ ) and msps RNAi , UAS - nod - lacZ ( VDRC # 21982 ) with grh - Gal4 UAS - CD8 - GFP ; UAS - dicer2 were labeled with β - Gal , Dpn , and GFP . Quiescent NSCs in the central brain are shown . Arrows indicate the localization of Nod - lacZ in qNSCs . G ) Quantification graph of Nod - β - Gal localization in qNSCs from genotypes in ( F ) . n = 60 NSCs for control ; n = 68 NSCs for msps RNAi . * * * * p < 0 . 0001 H ) Larval brains at 24 h ALH from wild - type control ( grh - Gal4 > UAS - CD8 - GFP ) , msps924 , and mspsP18 expressing grh > CD8 - GFP were labeled with Dpn and GFP . The arrow indicates the second protrusion in msps924 . I ) Quantification graph of the thickness of the primary protrusion of qNSCs from wild - type , msps924 , and mspsP18 expressing grh > CD8 - GFP . The thickness was measured at the middle point of the primary protrusion . n = 18 NSCs for control ; n = 52 NSCs for msps924 ; n = 32 NSCs for mspsP18 . * * * * p < 0 . 0001 L ) Larval brains at 24 h ALH from control ( grh - Gal4 UAS - CD8 - GFP + UAS - nod - lacZ ) and tacc74 , UAS - nod - lacZ under the control of grh - Gal4 UAS - CD8 - GFP ; tacc59 were labeled with β - Gal , Dpn , and GFP . Quiescent NSCs in the central brain are shown . M ) Quantification graph of Nod - β - Gal localization in qNSCs from genotypes in ( L ) . n = 51 NSCs for control ; n = 65 NSCs for tacc59 / 74 . * * p = 0 . 0051

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B) Quantification graph of fold changes in the number of EB1-GFP comets in the primary protrusion of qNSCs 6 h ALH from various genotypes compared with the control in (A). n=26 NSCs for control; n=18 NSCs for mspsP18; n=21 NSCs for mspsP18/P. ****p<0.0001. C) Quantification graph of the percentage of retrograde EB1-GFP comets in the primary protrusion of qNSCs from the control, mspsP18 with EB1-GFP driven by grh-Gal4 at 6 h ALH. As EB1-GFP comets were almost lost in mspsP18, we were unable to capture adequate EB1-GFP comets for each individual brain to calculate the standard deviation. 38 and 41 qNSCs were analyzed in control and mspsP18, respectively. In control qNSCs, among total 383 EB1-GFP comets, 353 were anterograde and 30 were retrograde movements. In mspsP18 qNSCs, we only found 12 EB1-GFP comets with 6 anterograde and 6 retrograde movements  D) Larval brains at 24 h ALH from control (grh-Gal4 UAS-CD8-GFP + UAS-nod-lacZ) and msps810,UAS-nod-lacZ under the control of grh-Gal4 UAS-CD8-GFP were labeled with β-Gal, Dpn, and GFP. Quiescent NSCs in the central brain were shown. Arrows point at the localization of Nod-lacZ in qNSCs. E) Quantification graph of Nod-β-Gal localization in qNSCs from genotypes in (D). n=55 NSCs for control; n=45 NSCs for msps810. ****p<0.0001  F) Larval brains at 16 h ALH from control (grh-Gal4 UAS-CD8-GFP; UAS-dicer2 + UAS -nod-lacZ) and msps RNAi, UAS-nod-lacZ (VDRC#21982) with grh-Gal4 UAS-CD8-GFP; UAS-dicer2 were labeled with β-Gal, Dpn, and GFP. Quiescent NSCs in the central brain are shown. Arrows indicate the localization of Nod-lacZ in qNSCs. G) Quantification graph of Nod-β-Gal localization in qNSCs from genotypes in (F). n=60 NSCs for control; n=68 NSCs for msps RNAi. ****p<0.0001  H) Larval brains at 24 h ALH from wild-type control (grh-Gal4>UAS-CD8-GFP), msps924, and mspsP18 expressing grh>CD8-GFP were labeled with Dpn and GFP. The arrow indicates the second protrusion in msps924. I) Quantification graph of the thickness of the primary protrusion of qNSCs from wild-type, msps924, and mspsP18 expressing grh>CD8-GFP. The thickness was measured at the middle point of the primary protrusion. n=18 NSCs for control; n=52 NSCs for msps924; n=32 NSCs for mspsP18. **** p<0.0001  L) Larval brains at 24 h ALH from control (grh-Gal4 UAS-CD8 -GFP + UAS-nod-lacZ) and tacc74,UAS-nod-lacZ under the control of grh-Gal4 UAS-CD8-GFP; tacc59 were labeled with β-Gal, Dpn, and GFP. Quiescent NSCs in the central brain are shown. M) Quantification graph of Nod-β-Gal localization in qNSCs from genotypes in (L). n=51 NSCs for control; n=65 NSCs for tacc59/74. **p=0.0051


Figure 5A ) Larval VNCs at 24 h ALH from the control ( grh - Gal4 UAS - CD8 - GFP ) and msps810 with grh - Gal4 UAS - CD8 - GFP were labeled with E - cadherin , Dpn , and GFP . NSC - neuropil contact were marked by dashed lines , and NSC - neuropil contact points were circled . B ) Quantification of E - cadherin basal localization at NSC - neuropil contact sites in qNSCs from genotypes in ( A ) . " No E - cad " means absent or strongly reduced E - cad observed at the basal region of qNSCs . n = 32 NSCs for control ; n = 61 NSCs for msps810 . * * p = 0 . 0028 . C ) Quantification graph of the basal E - cad intensity at NSC - neuropil contact sites by normalizing against Dpn intensity in qNSCs from genotypes in ( A ) . n = 34 NSCs for control ; n = 40 NSCs for msps810 . * * * * p < 0 . 0001 . D ) Larval VNCs at 24 h ALH from control ( grh - Gal4 UAS - CD8 - GFP ) and msps924 with grh - Gal4 UAS - CD8 - GFP were labeled with E - cadherin , Dpn , and GFP . NSC - neuropil contact were marked by dashed lines , and NSC - neuropil contact sites were circled . E ) Quantification of E - cadherin basal localization at NSC - neuropil contact sites in qNSCs from genotypes in ( D ) . " No E - cad " means absent or strongly reduced E - cad observed at the basal region of qNSCs . n = 77 NSCs for control ; n = 30 NSCs for msps924 . * * p = 0 . 0080 . F ) Quantification graph of the basal E - cad intensity at NSC - neuropil contact sites by normalizing against Dpn intensity in qNSCs from genotypes in ( D ) . n = 60 NSCs for control ; n = 37 NSCs for msps924 . * * * * p < 0 . 0001 . H ) Larval brains at 16 h ALH from control t - GRASP under the control of single driver grh - Gal4 or nSyb - QF2 ( Q - system ) and t - GRASP under the control of driver pairs grh - Gal4 and nSyb - QF2 ( Q - system ) were analyzed for E - Cad , GFP ( reconstituted GFP ) , and Dpn . Arrows pointing at the cell body of qNSCs on the two different focal planes . NSC - neuropil contact sites were circled by dashed lines .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Larval VNCs at 24 h ALH from the control (grh-Gal4 UAS-CD8-GFP) and msps810 with grh-Gal4 UAS-CD8-GFP were labeled with E-cadherin, Dpn, and GFP. NSC-neuropil contact were marked by dashed lines, and NSC-neuropil contact points were circled. B) Quantification of E-cadherin basal localization at NSC-neuropil contact sites in qNSCs from genotypes in (A). "No E-cad" means absent or strongly reduced E-cad observed at the basal region of qNSCs. n=32 NSCs for control; n=61 NSCs for msps810. **p=0.0028. C) Quantification graph of the basal E-cad intensity at NSC-neuropil contact sites by normalizing against Dpn intensity in qNSCs from genotypes in (A). n=34 NSCs for control; n=40 NSCs for msps810. ****p<0.0001. D) Larval VNCs at 24 h ALH from control (grh-Gal4 UAS-CD8-GFP) and msps924 with grh-Gal4 UAS-CD8-GFP were labeled with E-cadherin, Dpn, and GFP. NSC-neuropil contact were marked by dashed lines, and NSC-neuropil contact sites were circled. E) Quantification of E-cadherin basal localization at NSC-neuropil contact sites in qNSCs from genotypes in (D). "No E-cad" means absent or strongly reduced E-cad observed at the basal region of qNSCs. n=77 NSCs for control; n=30 NSCs for msps924. **p=0.0080. F) Quantification graph of the basal E-cad intensity at NSC-neuropil contact sites by normalizing against Dpn intensity in qNSCs from genotypes in (D). n=60 NSCs for control; n=37 NSCs for msps924. ****p<0.0001. H) Larval brains at 16 h ALH from control t-GRASP under the control of single driver grh-Gal4 or nSyb-QF2 (Q-system) and t-GRASP under the control of driver pairs grh-Gal4 and nSyb-QF2 (Q-system) were analyzed for E-Cad, GFP (reconstituted GFP), and Dpn. Arrows pointing at the cell body of qNSCs on the two different focal planes. NSC-neuropil contact sites were circled by dashed lines.


Figure 6 A ) Larval brains at 16 h ALH from wild - type ( yw ) and E - cadR69 were analyzed for EdU incorporation , and larval brains were labeled with EdU , Dpn and Mira E ) Larval brains at 24 h ALH from the wild - type control ( yw ) and klp64Dk5h / Df , kap3V6 and kap3 V6 ; g - kap3 # 11 were stained with EdU , Dpn , and Mira I ) Larval brains at 24h ALH from wild - type control ( yw ) , klp64Dk5 / Df , and kap3V5 were analyzed E - cad at the NSC - neuropil contact sites , and NSCs were labeled with Dpn and Msps

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 A) Larval brains at 16 h ALH from wild-type (yw) and E-cadR69 were analyzed for EdU incorporation, and larval brains were labeled with EdU, Dpn and Mira  E) Larval brains at 24 h ALH from the wild-type control (yw) and klp64Dk5h/Df, kap3V6 and kap3 V6; g-kap3#11 were stained with EdU, Dpn, and Mira I) Larval brains at 24h ALH from wild-type control (yw), klp64Dk5/Df, and kap3V5 were analyzed E-cad at the NSC-neuropil contact sites, and NSCs were labeled with Dpn and Msps


Figure 7A ) Larval brains at 24 h ALH from wild - type control ( UAS - β - Gal RNAi ; UAS - β - Gal RNAi ) , msps RNAi control ( UAS - GFP + UAS - msps RNAi ( 21982 ) ) , and rescued animals ( UAS - klp64D + msps RNAi ( 21982 ) ) under the control of insc - Gal4 were analyzed for EdU incorporation , and larval brains were labeled with EdU and Dpn . B ) Quantification graph of EdU - negative NSCs per brain lobe for genotypes in ( A ) . n = 13 BL for contorl ; n = 13 BL for msps RNAi ; n = 20 BL for UAS - klp64D + msps RNAi . * * * * p < 0 . 0001 . D ) Larval brains at 24 h ALH from wild - type control ( UAS - β - Gal RNAi ) , RNAi control ( UAS - msps RNAi ( 21982 ) ) , and rescued animals ( g - kap3 + msps RNAi ( 21982 ) ) driven by insc - Gal4 were examined for EdU incorporation , and larval brains were stained with EdU and Dpn . E ) Quantification graph of NSCs negative for EdU incorporation per brain lobe for genotypes in ( D ) . n = 13 BL for control ; n = 15 BL for msps RNAi ; n = 19 BL for g - kap3 + msps RNAi . * * * * p < 0 . 0001 . G ) 24 h ALH Larval brains from wild - type control ( UAS - β - Gal RNAi ; UAS - β - Gal RNAi ) , RNAi control ( UAS - GFP + UAS - msps RNAi ( 21982 ) ) , and rescued animals ( UAS - E - cad7 + msps RNAi ( 21982 ) ) were assessed for EdU incorporation , and larval brains were stained with EdU and Dpn . H ) Quantification graph of NSCs negative for EdU incorporation per brain lobe for genotypes in ( G ) . n = 21 BL for control ; n = 21 BL for msps RNAi ; n = 22 BL for UAS - E - cad7 + msps RNAi . * * * * p < 0 . 0001 . J ) 24 h ALH Larval brains from wild - type control ( UAS - β - Gal RNAi ) , mutant control ( UAS - β - Gal RNAi + klp64Dk5h ) , and rescued animals ( UAS - E - cad7 + klp64Dk5h ) were processed for EdU incorporation , and larval brains were probed by with EdU , Dpn , and Mira . K ) Quantification graph of NSCs negative for EdU incorporation per brain lobe for genotypes in ( J ) . n = 10 BL for control ; n = 12 BL for klp64Dk5h ; n = 10 BL for UAS - E - cad7 + klp64Dk5h . * * * * p < 0 . 0001 ; p = 0 . 3006 ( ns ) .

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Figure 7A) Larval brains at 24 h ALH from wild-type control (UAS-β-Gal RNAi; UAS-β-Gal RNAi), msps RNAi control (UAS-GFP + UAS-msps RNAi (21982)), and rescued animals (UAS-klp64D+ msps RNAi (21982)) under the control of insc-Gal4 were analyzed for EdU incorporation, and larval brains were labeled with EdU and Dpn. B) Quantification graph of EdU-negative NSCs per brain lobe for genotypes in (A). n=13 BL for contorl; n=13 BL for msps RNAi; n=20 BL for UAS-klp64D + msps RNAi. ****p<0.0001.D) Larval brains at 24 h ALH from wild-type control (UAS-β-Gal RNAi), RNAi control (UAS-msps RNAi (21982)), and rescued animals (g-kap3 + msps RNAi (21982)) driven by insc-Gal4 were examined for EdU incorporation, and larval brains were stained with EdU and Dpn. E) Quantification graph of NSCs negative for EdU incorporation per brain lobe for genotypes in (D). n=13 BL for control; n=15 BL for msps RNAi; n=19 BL for g-kap3 + msps RNAi. ****p<0.0001. G) 24 h ALH Larval brains from wild-type control (UAS-β-Gal RNAi; UAS-β-Gal RNAi), RNAi control (UAS-GFP + UAS-msps RNAi (21982)), and rescued animals (UAS-E-cad7 + msps RNAi (21982)) were assessed for EdU incorporation, and larval brains were stained with EdU and Dpn. H) Quantification graph of NSCs negative for EdU incorporation per brain lobe for genotypes in (G). n=21 BL for control; n=21 BL for msps RNAi; n=22 BL for UAS-E-cad7 + msps RNAi. ****p<0.0001. J) 24 h ALH Larval brains from wild-type control (UAS-β-Gal RNAi), mutant control (UAS-β-Gal RNAi + klp64Dk5h), and rescued animals (UAS-E-cad7 + klp64Dk5h) were processed for EdU incorporation, and larval brains were probed by with EdU, Dpn, and Mira. K) Quantification graph of NSCs negative for EdU incorporation per brain lobe for genotypes in (J). n=10 BL for control; n=12 BL for klp64Dk5h; n=10 BL for UAS-E-cad7 + klp64Dk5h. ****p<0.0001; p=0.3006 (ns).


Figure 1B . Western blot of PTEN variants in a panel of human cancer cell lines using a rabbit monoclonal antibody against the C - terminal region of PTEN The PTEN null prostate cancer cell line PC3 was used as a negative control .

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Figure 1B. Western blot of PTEN variants in a panel of human cancer cell lines using a rabbit monoclonal antibody against the C-terminal region of PTEN The PTEN null prostate cancer cell line PC3 was used as a negative control.


Figure 3D . Verification of expression of FLAG - tagged PTENα , PTENβ , and PTENε in PtenFLAG knock - in mice . Various tissues from PtenFLAG knock - in mice or control wild - type mice were lysed for immunoprecipitation with anti - FLAG M2 magnetic beads before being immunoblotted with the anti - PTENα

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Figure 3D. Verification of expression of FLAG-tagged PTENα, PTENβ, and PTENε in PtenFLAG knock-in mice. Various tissues from PtenFLAG knock-in mice or control wild-type mice were lysed for immunoprecipitation with anti-FLAG M2 magnetic beads before being immunoblotted with the anti-PTENα antibody


Figure 4B . Reduction of PTENε in response to eIF2A knockdown . Hela cells were infected with lentivirus expressing eIF2A shRNA , eIF5 shRNA , or scramble shRNA separately . The knockdown of eIF2A or eIF5 was validated by western blot ( left panel ) . Lysates of Hela cells were collected and subjected to western blot analysis with antibodies against PTEN , and GAPDH ( right panel ) . C . Overexpression of eIF2A induces the relative expression of PTENε . FLAG - tagged eIF2A or eIF5 were overexpressed in DU145 cells before western blot analysis of PTENε expression with an antibody against PTEN . GAPDH was used as a loading control .

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Figure 4B. Reduction of PTENε in response to eIF2A knockdown. Hela cells were infected with lentivirus expressing eIF2A shRNA, eIF5 shRNA, or scramble shRNA separately. The knockdown of eIF2A or eIF5 was validated by western blot (left panel). Lysates of Hela cells were collected and subjected to western blot analysis with antibodies against PTEN, and GAPDH (right panel).C. Overexpression of eIF2A induces the relative expression of PTENε. FLAG-tagged eIF2A or eIF5 were overexpressed in DU145 cells before western blot analysis of PTENε expression with an antibody against PTEN. GAPDH was used as a loading control.


Figure 5C . The transfected Hela PTEN - / - cells were stained with an anti - β - catenin antibody and DAPI before being imaged with confocal microscopy . To determine the sequences that are critical for the membrane localization of PTENε . Hela were transfected with constructs Thirty - six hours after transfection , transfected Hela cells were stained with anti - β - catenin antibody and DAPI , followed by imaging with confocal microscopy . The scale bars represent 5 μm ( F ) . The transfected HEK293 cells were conducted with cell fractionation before western blotting with GFP , E - cadherin , and β - tubulin antibodies ( G ) . C : cytoplasm . M : cell plasma membrane .

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Figure 5C. The transfected Hela PTEN-/- cells were stained with an anti-β-catenin antibody and DAPI before being imaged with confocal microscopy.To determine the sequences that are critical for the membrane localization of PTENε. Hela were transfected with constructs Thirty-six hours after transfection, transfected Hela cells were stained with anti-β-catenin antibody and DAPI, followed by imaging with confocal microscopy. The scale bars represent 5 μm (F).The transfected HEK293 cells were conducted with cell fractionation before western blotting with GFP, E-cadherin, and β-tubulin antibodies (G). C: cytoplasm. M: cell plasma membrane.


Figure 6D . Images of immunofluorescence staining for PTENε and its interacting proteins ( VASP and FSCN1 ) . Hela PTEN - / - cells were co - transfected by a plasmid expressing C - terminal GFP - tagged PTENε with FLAG - tagged targets ( FSCN1 , VASP ) separately , followed by staining with an anti - FLAG antibody and DAPI , and were imaged by confocal microscopy . E . The cell plasma membrane localization of PTENε is required for its interaction with downstream targets . FLAG - tagged CDC42 , VASP , ACTR2 , or FSCN1 plasmid was co - transfected with a plasmid expressing PTENε with or without 69 - 81aa deletion carrying C - terminal S - tag and HA tag in HEK293 cells . Cell lysates were incubated with S protein agarose before western blotting with antibodies against the FLAG tag and HA tag . ( H , I ) Purified ACTR2 or VASP was phosphorylated in vitro by NIK or PKA and subsequently used in a phosphatase assay with His - PTENε or without purified PTENε protein as a control , followed by immunoblotting with antibodies against p - ACTR2 ( Thr237 and Thr238 ) and p - VASP ( Ser157 ) . Purified proteins ( PTENε , PKA , NIK , ACTR , and VASP ) were detected by anti - His antibody ( ZSGB - BIO , TA - 02 ) .

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Figure 6D. Images of immunofluorescence staining for PTENε and its interacting proteins (VASP and FSCN1). Hela PTEN-/- cells were co-transfected by a plasmid expressing C-terminal GFP-tagged PTENε with FLAG-tagged targets (FSCN1, VASP) separately, followed by staining with an anti-FLAG antibody and DAPI, and were imaged by confocal microscopy.E. The cell plasma membrane localization of PTENε is required for its interaction with downstream targets. FLAG-tagged CDC42, VASP, ACTR2, or FSCN1 plasmid was co-transfected with a plasmid expressing PTENε with or without 69-81aa deletion carrying C-terminal S-tag and HA tag in HEK293 cells. Cell lysates were incubated with S protein agarose before western blotting with antibodies against the FLAG tag and HA tag.(H, I) Purified ACTR2 or VASP was phosphorylated in vitro by NIK or PKA and subsequently used in a phosphatase assay with His-PTENε or without purified PTENε protein as a control, followed by immunoblotting with antibodies against p-ACTR2 (Thr237 and Thr238) and p-VASP (Ser157). Purified proteins (PTENε, PKA, NIK, ACTR, and VASP) were detected by anti-His antibody (ZSGB-BIO, TA-02).


Figure 7A . Immunofluorescence staining of F - actin in PTENε or PTENε Y210L overexpressed Hela PTEN - / - cells and control cells . PTENε - GFP , PTENε Y210L - GFP , or mock construct was introduced into HeLa PTEN - / - cells respectively . The transfected cells were stained with Phalloidin and DAPI before being imaged with confocal microscopy . The scale bars represent 5 μm ( left panel ) . White arrows shown in the magnified immunofluorescence images indicate the filopodia in the cell membrane . B . Quantification of the number of filopodia per cell by FiloQuant software and the data are presented as mean ± SD of three independent experiments and were analyzed with the unpaired t - test . * * * * , p < 0 . 001 . G . Left panel : immunoblotting analyses were used to verify the level of overexpressed PTENε or PTENε Y210L in B16 cells . Middle panel : representative images showing the lung metastasis of PTENε or PTENε Y210L overexpressed B16 cells or control cells in the mouse pulmonary metastasis model ( n = 4 ) . H & E , hematoxylin - eosin staining . The scale bars represent 600 mm ( upper panel ) and 500μm ( lower panel ) separately . Right panel : the representative bar graphs showing the quantification of tumor metastatic ability was displayed by measuring the number of tumor nodules ( data are presented as mean ± SD based on three independent replicates ) . The overexpression of PTENε attenuated tumor metastatic ability compared with the vector control group . * * * * , p < 0 . 0001 in the unpaired t - test .

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Figure 7A. Immunofluorescence staining of F-actin in PTENε or PTENε Y210L overexpressed Hela PTEN-/- cells and control cells. PTENε-GFP, PTENε Y210L-GFP, or mock construct was introduced into HeLa PTEN-/- cells respectively. The transfected cells were stained with Phalloidin and DAPI before being imaged with confocal microscopy. The scale bars represent 5 μm (left panel). White arrows shown in the magnified immunofluorescence images indicate the filopodia in the cell membrane. B. Quantification of the number of filopodia per cell by FiloQuant software and the data are presented as mean ± SD of three independent experiments and were analyzed with the unpaired t-test. ****, p<0.001.G. Left panel: immunoblotting analyses were used to verify the level of overexpressed PTENε or PTENε Y210L in B16 cells. Middle panel: representative images showing the lung metastasis of PTENε or PTENε Y210L overexpressed B16 cells or control cells in the mouse pulmonary metastasis model (n=4). H & E, hematoxylin-eosin staining. The scale bars represent 600 mm (upper panel) and 500μm (lower panel) separately. Right panel: the representative bar graphs showing the quantification of tumor metastatic ability was displayed by measuring the number of tumor nodules (data are presented as mean ± SD based on three independent replicates). The overexpression of PTENε attenuated tumor metastatic ability compared with the vector control group. ****, p<0.0001 in the unpaired t-test.


Figure 1A Scheme of the lentiviral vector with inducible FlagOmomyc ( top ) and immunoblotting ( bottom ) of FlagOmomyc and actin loading control upon Dox treatment of BT168FOcells for 0 - 48 hours . B Proliferation curves of BT168FO and BT168GFPcells upon Dox treatment for 0 - 96 h ( n = 3 ; mean ± SD ) . Viable cells were counted using a haemocytometer . C - D Self - renewal assay upon Dox treatment . C : histograms showing the percentage of cells capable of re - forming a neurosphere seven days after dissociation ( n = 3 ; mean ± SD ) . D : representative micrographs of BT168FO cell neurospheres . E qRT - PCR of proliferation , stem cell and differentiation markers ( CCND1 , PTEN , SOX2 , NOTCH1 , NESTIN , MYC ) in BT168FO cells after 48 h Dox treatment , compared to uninduced cells ( n = 3 ; mean ± SD ) . Expression levels in non - induced cells were set as 1 . F . Transwell migration assay of BT168FO cells after 3 days with or without Dox ( n = 3 ; mean ± SD ) . 10 fields were counted per assay . G . Immunofluorescence analyses of GSC differentiation . To stimulate differentiation BT168FO were grown as monolayer in the presence of serum and treated with doxycycline for up to 7 days . The upper panel displays immunofluorescence images of NESTIN , GFAP , ß - tubulin III , SOX2 , and FlagOmomyc expression . FlagOmomyc blunted SOX2 expression and decreased GFAP and NESTIN protein levels , while inducing the onset of ß - tubulin III . The lower panel shows the percentage of positive cells for each cell marker evaluated ( n = 4 ; mean ± SD ) . 16 fields for each assay were examined ; scale bar = 100 μm .

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Figure 1A Scheme of the lentiviral vector with inducible FlagOmomyc (top) and immunoblotting (bottom) of FlagOmomyc and actin loading control upon Dox treatment of BT168FOcells for 0-48 hours.B Proliferation curves of BT168FO and BT168GFPcells upon Dox treatment for 0-96 h (n = 3; mean ± SD). Viable cells were counted using a haemocytometer.C-D Self-renewal assay upon Dox treatment. C: histograms showing the percentage of cells capable of re-forming a neurosphere seven days after dissociation (n = 3; mean ± SD). D: representative micrographs of BT168FO cell neurospheres.E qRT-PCR of proliferation, stem cell and differentiation markers (CCND1, PTEN, SOX2, NOTCH1, NESTIN, MYC) in BT168FO cells after 48 h Dox treatment, compared to uninduced cells (n = 3; mean ± SD). Expression levels in non-induced cells were set as 1.F. Transwell migration assay of BT168FO cells after 3 days with or without Dox (n = 3; mean ± SD). 10 fields were counted per assay.G. Immunofluorescence analyses of GSC differentiation. To stimulate differentiation BT168FO were grown as monolayer in the presence of serum and treated with doxycycline for up to 7 days. The upper panel displays immunofluorescence images of NESTIN, GFAP, ß-tubulin III, SOX2, and FlagOmomyc expression. FlagOmomyc blunted SOX2 expression and decreased GFAP and NESTIN protein levels, while inducing the onset of ß-tubulin III. The lower panel shows the percentage of positive cells for each cell marker evaluated (n = 4; mean ± SD). 16 fields for each assay were examined; scale bar = 100 μm.


Figure 2A - H Immunostaining of representative xenografts derived from naïve ( control ) and Omomyc expressing BT168FO cells . A - D ) Flag , Ki67 , CD31 , and DCX ( doublecortin ) immunostaining of adjacent sections in control and Omomycxenografts . E - G ) Differentiation phenotype investigated by GFAP , ß - tubulin III , and OLIG2 staining . H ) Immunostaining for anti - human nuclei . Representative images are shown . Scale bar = 100μm .

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Figure 2A-H Immunostaining of representative xenografts derived from naïve (control) and Omomyc expressing BT168FO cells. A-D) Flag, Ki67, CD31, and DCX (doublecortin) immunostaining of adjacent sections in control and Omomycxenografts. E-G) Differentiation phenotype investigated by GFAP, ß-tubulin III, and OLIG2 staining. H) Immunostaining for anti-human nuclei. Representative images are shown. Scale bar = 100μm.


Figure 3A - B Seqminer heatmaps of MYC and Omomyc levels around TSSs of all MYC promoter - target genes in BT168FO ( A ) cells , treated or not with Dox for 24 h . TSS regions are ranked by decreasing MYC occupancy in untreated cells . Each row shows the ± 5 kb region centred on TSSs . Colour scaled intensities are in units of tags per 50bp . The plots adjacent to each heatmap depict MYC and Omomyc binding patterns at genes in the two gene clusters denoted by arrows , in cells treated or not with Dox . A - B Seqminer heatmaps of MYC and Omomyc levels around TSSs of all MYC promoter - target genes in U87FO ( B ) cells , treated or not with Dox for 24 h . TSS regions are ranked by decreasing MYC occupancy in untreated cells . Each row shows the ± 5 kb region centred on TSSs . Colour scaled intensities are in units of tags per 50bp . The plots adjacent to each heatmap depict MYC and Omomyc binding patterns at genes in the two gene clusters denoted by arrows , in cells treated or not with Dox .

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Figure 3A-B Seqminer heatmaps of MYC and Omomyc levels around TSSs of all MYC promoter-target genes in BT168FO (A) cells, treated or not with Dox for 24 h. TSS regions are ranked by decreasing MYC occupancy in untreated cells. Each row shows the ± 5 kb region centred on TSSs. Colour scaled intensities are in units of tags per 50bp. The plots adjacent to each heatmap depict MYC and Omomyc binding patterns at genes in the two gene clusters denoted by arrows, in cells treated or not with Dox.A-B Seqminer heatmaps of MYC and Omomyc levels around TSSs of all MYC promoter-target genes in U87FO (B) cells, treated or not with Dox for 24 h. TSS regions are ranked by decreasing MYC occupancy in untreated cells. Each row shows the ± 5 kb region centred on TSSs. Colour scaled intensities are in units of tags per 50bp. The plots adjacent to each heatmap depict MYC and Omomyc binding patterns at genes in the two gene clusters denoted by arrows, in cells treated or not with Dox.


Figure 4A Heatmap of RNA Pol II levels around TSSs of MYC promoter - target genes in BT168FO cells , treated or not with Dox for 24 h . TSS regions are ranked by decreasing MYC occupancy in untreated cells . Each row shows the ± 5 kb region centred on TSSs . Colour scaled intensities are in tags / 50bp . B Tracks of Pol II binding signals ( ChIP - seq ) at NCL , miR - 17 - 92 , OLIG2 , HDAC1 , and DUSP10 genes in BT168FO cells treated or not with Dox for 24 h . The y - axis shows Pol II binding signals as tags / 500bp / million reads . The x - axis displays genomic positions ; introns as lines , exons as boxes . Arrowheads denote direction of transcription . Blue and green bars denote the presence of MYC or Omomyc peaks . Grey boxes are TSS regions . C Transcript levels ( FPKM from RNA - seq data ) in BT168FO cells of the genes shown in B . Data are means of 3 independent biological replicates . D Ratio between fold enrichments ( specific antibody / IgG signal ) derived from qChIPs of Pol II - Ser2p and Pol II - Ser5p ( n = 3 ; mean ± SD ) . E RNA Polymerase II traveling ratio distribution from the Pol II ChIP - seqs , in the presence or absence of Dox .

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Figure 4A Heatmap of RNA Pol II levels around TSSs of MYC promoter-target genes in BT168FO cells, treated or not with Dox for 24 h. TSS regions are ranked by decreasing MYC occupancy in untreated cells. Each row shows the ± 5 kb region centred on TSSs. Colour scaled intensities are in tags/50bp.B Tracks of Pol II binding signals (ChIP-seq) at NCL, miR-17-92, OLIG2, HDAC1, and DUSP10 genes in BT168FO cells treated or not with Dox for 24 h. The y-axis shows Pol II binding signals as tags/500bp/million reads. The x-axis displays genomic positions; introns as lines, exons as boxes. Arrowheads denote direction of transcription. Blue and green bars denote the presence of MYC or Omomyc peaks. Grey boxes are TSS regions.C Transcript levels (FPKM from RNA-seq data) in BT168FO cells of the genes shown in B. Data are means of 3 independent biological replicates.D Ratio between fold enrichments (specific antibody / IgG signal) derived from qChIPs of Pol II-Ser2p and Pol II-Ser5p (n = 3; mean ± SD).E RNA Polymerase II traveling ratio distribution from the Pol II ChIP-seqs, in the presence or absence of Dox.