a ) QF - FRL of unilamellar liposomes containing 95 mol % of phosphatidylcholine and 5 mol % of a phosphoinositide , labelled with GST - PX . Only liposomes containing PtdIns ( 3 ) P were labelled intensely . GST - PXR58A , a PX - domain mutant that does not bind PtdIns ( 3 ) P , yielded virtually no labelling in PtdIns ( 3 ) P - containing liposomes . Scale bar , 0 . 2 μm .

O O O O O O O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O B-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O O O

a) QF-FRL of unilamellar liposomes containing 95 mol% of phosphatidylcholine and 5 mol% of a phosphoinositide, labelled with GST-PX. Only liposomes containing PtdIns(3)P were labelled intensely. GST-PXR58A, a PX-domain mutant that does not bind PtdIns(3)P, yielded virtually no labelling in PtdIns(3)P-containing liposomes. Scale bar, 0.2 μm.


( b ) QF - FRL of unilamellar liposomes containing 0 - 5 mol % of PtdIns ( 3 ) P . Scale bar , 0 . 2 μm . The labelling density increased roughly in proportion to the PtdIns ( 3 ) P content of the liposomes . The experiment was repeated twice and the number of colloidal gold particles per μm2 was measured in more than 75 randomly selected liposomes . Values for all concentrations are statistically different from those in the adjacent conditions ( for example , 1 versus 2 mol % ; mean ± s . e . m . ; Student ' s t - test ; P0 . 05 ) .

O O O O O O O O B-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O 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) QF-FRL of unilamellar liposomes containing 0-5 mol % of PtdIns(3)P. Scale bar, 0.2 μm. The labelling density increased roughly in proportion to the PtdIns(3)P content of the liposomes. The experiment was repeated twice and the number of colloidal gold particles per μm2 was measured in more than 75 randomly selected liposomes. Values for all concentrations are statistically different from those in the adjacent conditions (for example, 1 versus 2 mol%; mean±s.e.m.; Student's t-test; P0.05).


( b ) QF - FRL in wild - type yeast in log phase . PtdIns ( 3 ) P was labelled with GFP - PX and marked with colloidal gold ( 10 nm ) . The vacuolar membrane PF was labelled intensely , whereas other organellar membranes did not exhibit significant labelling . Scale bar , 0 . 5 μm .

O O O O O O O O O O B-ORGANISM O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O

(b) QF-FRL in wild-type yeast in log phase. PtdIns(3)P was labelled with GFP-PX and marked with colloidal gold (10 nm). The vacuolar membrane PF was labelled intensely, whereas other organellar membranes did not exhibit significant labelling. Scale bar, 0.5 μm.


c ) Quantification of the labelling intensity of PtdIns ( 3 ) P ( mean ± s . e . m . ) . The experiment was repeated three times and the number of colloidal gold particles per μm2 was measured in more than ten randomly selected images .

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c) Quantification of the labelling intensity of PtdIns(3)P (mean±s.e.m.). The experiment was repeated three times and the number of colloidal gold particles per μm2 was measured in more than ten randomly selected images.


( d , e ) Negative controls . Both wild - type yeast incubated with GST - PXR58A ( d ) and vps34Δ yeast incubated with GST - PX ( e ) were virtually unlabelled . Scale bar , 0 . 5 μm .

O O O O O O O O O O O O B-ORGANISM O O B-GENEPROD O O O O O O B-GENEPROD B-ORGANISM O O B-GENEPROD O O O O O O O O O O O O O O O O O O

(d,e) Negative controls. Both wild-type yeast incubated with GST-PXR58A (d) and vps34Δ yeast incubated with GST-PX (e) were virtually unlabelled. Scale bar, 0.5 μm.


( a ) Wild - type yeast cultured in S ( - NC ) for 4 h . PtdIns ( 3 ) P was labelled intensely in the EF of autophagosomal membranes . A lower but still significant amount of labelling was also seen in the PF of autophagosome membranes as well as in the EF of the vacuolar membrane . Scale bar , 0 . 5 μm .

O O O O O O B-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O

(a) Wild-type yeast cultured in S(-NC) for 4 h. PtdIns(3)P was labelled intensely in the EF of autophagosomal membranes. A lower but still significant amount of labelling was also seen in the PF of autophagosome membranes as well as in the EF of the vacuolar membrane. Scale bar, 0.5 μm.


( b ) Autophagosomes in wild - type yeast cultured in S ( - NC ) for 4 h . The autophagosome was identified as a smooth - surfaced double - membrane structure , and seen in two different patterns , as shown in the diagrams in c . Upper panel : convex structures displaying the outer membrane EF and the inner membrane PF . Lower panel : concave structures displaying the outer membrane PF and the inner membrane EF . In both cases , PtdIns ( 3 ) P was labelled more intensely in the EF than in the PF . Scale bars , 0 . 2 μm .

O O O B-SUBCELLULAR O O O O B-ORGANISM O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O

(b) Autophagosomes in wild-type yeast cultured in S(-NC) for 4 h. The autophagosome was identified as a smooth-surfaced double-membrane structure, and seen in two different patterns, as shown in the diagrams in c. Upper panel: convex structures displaying the outer membrane EF and the inner membrane PF. Lower panel: concave structures displaying the outer membrane PF and the inner membrane EF. In both cases, PtdIns(3)P was labelled more intensely in the EF than in the PF. Scale bars, 0.2 μm.


( a ) Wild - type yeast cultured in S ( - NC ) for 4 h . PtdIns ( 3 ) P was labelled intensely in the EF of autophagic body membranes in the vacuolar lumen . A lower amount of labelling was also seen in the PF of autophagic body membranes . Scale bar , 0 . 5 μm . (

O O O O O O B-ORGANISM O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O

(a) Wild-type yeast cultured in S(-NC) for 4 h. PtdIns(3)P was labelled intensely in the EF of autophagic body membranes in the vacuolar lumen. A lower amount of labelling was also seen in the PF of autophagic body membranes. Scale bar, 0.5 μm. (


( c ) Labelling intensity of PtdIns ( 3 ) P in wild - type yeast cultured in S ( - NC ) for 4 h ( average ± s . e . m . ) . PtdIns ( 3 ) P was found in both leaflets of the autophagosome and autophagic body membrane , and the labelling was much stronger in the EF than in the PF . In the vacuolar membrane , significant labelling was found in the EF , albeit at much lower levels than in the PF . The experiment was repeated three times , and the number of colloidal gold particles per μm2 was measured in more than ten randomly selected images .

O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O

(c) Labelling intensity of PtdIns(3)P in wild-type yeast cultured in S(-NC) for 4 h (average±s.e.m.). PtdIns(3)P was found in both leaflets of the autophagosome and autophagic body membrane, and the labelling was much stronger in the EF than in the PF. In the vacuolar membrane, significant labelling was found in the EF, albeit at much lower levels than in the PF. The experiment was repeated three times, and the number of colloidal gold particles per μm2 was measured in more than ten randomly selected images.


( a ) Huh7 cells expressing GFP - LC3 treated with 0 . 25 μM Torin1 and 0 . 4 μM bafilomycin A1 for 1 h . The autophagosome was identified as the IMP - poor double - membrane structure labelled with anti - GFP antibody ( large gold , 10 nm ) . Both PtdIns ( 3 ) P ( small gold , 6 nm ) and GFP - LC3 were seen exclusively in the PF in both convex and concave structures . The small and large gold particles are shown as green and red dots , respectively , in the figures at right . Scale bars , 0 . 1 μm . Figures displaying wider areas , as well as additional examples , are presented in Supplementary Fig . 4c . (

O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) Huh7 cells expressing GFP-LC3 treated with 0.25 μM Torin1 and 0.4 μM bafilomycin A1 for 1 h. The autophagosome was identified as the IMP-poor double-membrane structure labelled with anti-GFP antibody (large gold, 10 nm). Both PtdIns(3)P (small gold, 6 nm) and GFP-LC3 were seen exclusively in the PF in both convex and concave structures. The small and large gold particles are shown as green and red dots, respectively, in the figures at right. Scale bars, 0.1 μm. Figures displaying wider areas, as well as additional examples, are presented in Supplementary Fig. 4c. (


( b ) NIH3T3 cells expressing Atg4C78A were incubated in Earle ' s balanced salt solution for 1 h . The isolation membrane was observed as parallel membranes with few IMPs . PtdIns ( 3 ) P was labelled only in the PF . Scale bar , 0 . 1 μm . Other examples are presented in Supplementary Fig . 5 .

O O O B-CELL_LINE O O B-GENEPROD O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O 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) NIH3T3 cells expressing Atg4C78A were incubated in Earle's balanced salt solution for 1 h. The isolation membrane was observed as parallel membranes with few IMPs. PtdIns(3)P was labelled only in the PF. Scale bar, 0.1 μm. Other examples are presented in Supplementary Fig. 5.


( c ) Labelling density of PtdIns ( 3 ) P in mammalian autophagosomal membrane ( mean ± s . e . m . ) . The experiment was repeated three times and the number of colloidal goldparticles per μm2 was measured in more than ten randomly selected images . Some labelling was observed in the EF but the majority of gold particles in the EF occurred near the EF - PF boundary ( white circles ; see Supplementary Fig . 6c for more examples ) , suggesting that many of them actually represent PtdIns ( 3 ) P in the PF .

O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O

(c) Labelling density of PtdIns(3)P in mammalian autophagosomal membrane (mean±s.e.m.). The experiment was repeated three times and the number of colloidal goldparticles per μm2 was measured in more than ten randomly selected images. Some labelling was observed in the EF but the majority of gold particles in the EF occurred near the EF-PF boundary (white circles; see Supplementary Fig. 6c for more examples), suggesting that many of them actually represent PtdIns(3)P in the PF.


( a ) ymr1Δsjl3Δ cells cultured in S ( - NC ) for 4 h . PtdIns ( 3 ) P was labelled in comparable densities in PF and EF of autophagosomal membranes . Scale bar , 0 . 2 μm .

O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O

(a) ymr1Δsjl3Δ cells cultured in S(-NC) for 4 h. PtdIns(3)P was labelled in comparable densities in PF and EF of autophagosomal membranes. Scale bar, 0.2 μm.


( b ) The PF / EF ratio of the PtdIns ( 3 ) P labelling density in autophagosomal membrane ( mean ± s . e . m . ) . The ratio was small ( that is , PF ) in WT , but close to 1 ( that is , PF ≅ EF ) in ymr1Δsjl3Δ . The experiment was repeated three times and the number of colloidal goldparticles per μm2 was measured in more than ten randomly selected images .

O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O

(b) The PF/EF ratio of the PtdIns(3)P labelling density in autophagosomal membrane (mean±s.e.m.). The ratio was small (that is, PF) in WT, but close to 1 (that is, PF≅EF) in ymr1Δsjl3Δ. The experiment was repeated three times and the number of colloidal goldparticles per μm2 was measured in more than ten randomly selected images.


( A - B ) Live third instar discs incubated in DiBAC . DiBAC fluorescence is observed in the wing imaginal disc in both apical ( A - A ' ) , and basal ( B - B ' ) optical sections . Increased fluorescence is observed in the center of the pouch ( white arrow , B ' ) , and at the dorsoventral ( D - V ) compartment boundary in the anterior compartment ( yellow arrow , B ' , and inset , D ) .

O O O O O O O O B-TISSUE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-TISSUE I-TISSUE I-TISSUE O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-B) Live third instar discs incubated in DiBAC. DiBAC fluorescence is observed in the wing imaginal disc in both apical (A-A'), and basal (B-B') optical sections. Increased fluorescence is observed in the center of the pouch (white arrow, B'), and at the dorsoventral (D-V) compartment boundary in the anterior compartment (yellow arrow, B', and inset, D).


( C - E ) Comparison of DiBAC with the voltage - insensitive dye FM4 - 64 . Incubation of live discs in FM4 - 64 ( E ) shows more uniform fluorescence when compared to DiBAC ( D ) . Quantitative comparison of fluorescence in the two white boxes in each panel is shown in ( C ) . N = 7 discs for each treatment , data compared using an unpaired t test , * * indicates p < 0 . 001 , error bars are standard deviations . The region boxed in yellow in ( D ) is shown at higher magnification to show DiBAC fluorescence at the D - V compartment boundary .

O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-TISSUE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O 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-TISSUE O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O

(C-E) Comparison of DiBAC with the voltage-insensitive dye FM4-64. Incubation of live discs in FM4-64 (E) shows more uniform fluorescence when compared to DiBAC (D). Quantitative comparison of fluorescence in the two white boxes in each panel is shown in (C). N=7 discs for each treatment, data compared using an unpaired t test, ** indicates p<0.001, error bars are standard deviations. The region boxed in yellow in (D) is shown at higher magnification to show DiBAC fluorescence at the D-V compartment boundary.


( F , G ) Incubation in ouabain results in brighter and more uniform DiBAC fluorescence .

O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O

(F, G) Incubation in ouabain results in brighter and more uniform DiBAC fluorescence.


( H - H ' ' ) Live wing discs expressing UAS - RFP anterior to the compartment boundary , under the control of ptc - Gal4 , were incubated in DiBAC , showing that the stripe of increased DiBAC fluorescence coincides with the posterior edge of ptc expression . White arrowhead in ( H ) indicates the stripe in the wing pouch ; yellow arrowheads indicate the stripe in the dorsal and ventral hinge .

O O O O O O O O B-TISSUE I-TISSUE O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE O

(H-H'') Live wing discs expressing UAS-RFP anterior to the compartment boundary, under the control of ptc-Gal4, were incubated in DiBAC, showing that the stripe of increased DiBAC fluorescence coincides with the posterior edge of ptc expression. White arrowhead in (H) indicates the stripe in the wing pouch; yellow arrowheads indicate the stripe in the dorsal and ventral hinge.


( I , I ' ) Early L3 discs incubated in DiBAC . Patterned depolarization is evident throughout the third larval instar , with the stripe of increased fluorescence becoming narrower in more mature discs with developmental time ( Compare I with A , bracket in I indicates width of increased DiBAC fluorescence ) .

O O O O O O O O B-TISSUE O O B-SMALL_MOLECULE O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O

(I, I') Early L3 discs incubated in DiBAC. Patterned depolarization is evident throughout the third larval instar, with the stripe of increased fluorescence becoming narrower in more mature discs with developmental time (Compare I with A, bracket in I indicates width of increased DiBAC fluorescence).


Expression of Rpk is increased anterior to the A - P compartment boundary , and in two rows of cells flanking the D - V compartment boundary in the A compartment .

B-EXP_ASSAY O B-GENEPROD O O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE O O B-TISSUE I-TISSUE O

Expression of Rpk is increased anterior to the A-P compartment boundary, and in two rows of cells flanking the D-V compartment boundary in the A compartment.


( B - B ' ' ) Blockade of DEG / ENaC channels by incubation in amiloride abolishes the stripe of increased DiBAC fluorescence along the A - P compartment boundary ( white arrowhead ) . White boxes indicate regions used to calculate the average DiBAC fluorescence intensity ratio across the compartment boundary = 0 . 98 , standard deviation = 0 . 05 , n = 5 discs . Increased fluorescence at the D - V boundary in the A compartment is still observable ( yellow arrowhead ) .

O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O

(B-B'') Blockade of DEG/ENaC channels by incubation in amiloride abolishes the stripe of increased DiBAC fluorescence along the A-P compartment boundary (white arrowhead). White boxes indicate regions used to calculate the average DiBAC fluorescence intensity ratio across the compartment boundary = 0.98, standard deviation = 0.05, n = 5 discs. Increased fluorescence at the D-V boundary in the A compartment is still observable (yellow arrowhead).


( C , C ' ) Amiloride incubation does not diminish Rpk expression . White arrowhead indicates approximate position of the A - P compartment boundary , yellow arrowhead indicates approximate position of the D - V compartment boundary .

O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE O

(C, C') Amiloride incubation does not diminish Rpk expression. White arrowhead indicates approximate position of the A-P compartment boundary, yellow arrowhead indicates approximate position of the D-V compartment boundary.


( D , E ) Expression of rpk RNAi using dpp - Gal4 results in diminished DiBAC fluorescence along the A - P compartment boundary ( white arrowhead ) , while increased fluorescence at the D - V boundary in the A compartment is still observable ( yellow arrowhead ) .

O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D, E) Expression of rpk RNAi using dpp-Gal4 results in diminished DiBAC fluorescence along the A-P compartment boundary (white arrowhead), while increased fluorescence at the D-V boundary in the A compartment is still observable (yellow arrowhead).


( A - A ' ' ' ) Immunostaining of discs expressing ptc > RFP with antibodies to Rpk and ATPα showing elevated levels of both proteins anterior to the compartment boundary . White arrowheads indicate approximate position of the A - P compartment boundary .

O O O O O O O O B-EXP_ASSAY O B-TISSUE O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE O

(A-A''') Immunostaining of discs expressing ptc>RFP with antibodies to Rpk and ATPα showing elevated levels of both proteins anterior to the compartment boundary. White arrowheads indicate approximate position of the A-P compartment boundary.


( B - C ' ) L3 discs that are temperature - sensitive for hh following shift to the restrictive temperature for 12 h show loss of increased expression of Rpk ( B , B ' ) and ATPα ( C , C ' ) anterior to the compartment boundary . Controls shown in Figure EV2 . White arrowheads indicate approximate position of the A - P compartment boundary .

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(B-C') L3 discs that are temperature-sensitive for hh following shift to the restrictive temperature for 12 h show loss of increased expression of Rpk (B, B') and ATPα (C, C') anterior to the compartment boundary. Controls shown in Figure EV2. White arrowheads indicate approximate position of the A-P compartment boundary.


( D - E ' ' ' ) Clones of cells expressing a constitutively activated form of the transcription factor Ci show elevated Rpk and ATPα expression in the anterior compartment ( white box ) as well as the posterior compartment ( yellow arrowhead ) . ( E - E ' ' ' ) A single clone in the A compartment is shown at higher magnification .

O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O B-TISSUE I-TISSUE O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O

(D-E''') Clones of cells expressing a constitutively activated form of the transcription factor Ci show elevated Rpk and ATPα expression in the anterior compartment (white box) as well as the posterior compartment (yellow arrowhead). (E-E''') A single clone in the A compartment is shown at higher magnification.


( F - F ' ) A clone of cells in the anterior compartment expressing ci3M following incubation in DiBAC .

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(F-F') A clone of cells in the anterior compartment expressing ci3M following incubation in DiBAC.


( G - G ' ) A clone of cells in the anterior compartment expressing ptc RNAi following incubation in DiBAC . The clones also express RFP .

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(G-G') A clone of cells in the anterior compartment expressing ptc RNAi following incubation in DiBAC. The clones also express RFP.


( A - A ' ' ' ) Immunostaining of Smo protein ( red ) and full - length Ci ( light blue ) in discs expressing en - Venus . Smo is expressed at higher levels in the P compartment and in a stripe 5 - 10 cells wide just anterior to the A - P compartment boundary . White arrowhead indicates cells just anterior to the A - P compartment boundary .

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(A-A''') Immunostaining of Smo protein (red) and full-length Ci (light blue) in discs expressing en-Venus. Smo is expressed at higher levels in the P compartment and in a stripe 5-10 cells wide just anterior to the A-P compartment boundary. White arrowhead indicates cells just anterior to the A-P compartment boundary.


( B - C ) Effect of expressing an RNAi against the ENaC channel rpk in the dorsal compartment of the disc using ap - Gal4 . Knockdown of Rpk in the dorsal compartment results in decreased accumulation of the Hh signal transducer Smo ( B , B ' ) , and the activator form of Ci ( B , B ' ' ) . The expression of ptc , visualized using an anti - Ptc antibody ( C ) , a downstream target gene of Hh signaling , is also diminished . The Rpk RNAi line BL : 39053 is used in all panels shown , but the phenotype was validated with a second RNAi line ( BL : 25847 ) .

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(B-C) Effect of expressing an RNAi against the ENaC channel rpk in the dorsal compartment of the disc using ap-Gal4. Knockdown of Rpk in the dorsal compartment results in decreased accumulation of the Hh signal transducer Smo (B, B'), and the activator form of Ci (B, B''). The expression of ptc, visualized using an anti-Ptc antibody (C), a downstream target gene of Hh signaling, is also diminished. The Rpk RNAi line BL:39053 is used in all panels shown, but the phenotype was validated with a second RNAi line (BL:25847).


( A - C ) Localization of Smo protein ( blue ) following expression of 71B - Gal4 ( A ) , 71B - Gal4 and UAS - NaChBac ( B ) or 71B - Gal4 and UAS - Kir2 . 1 ( C ) . Expression of the sodium channel NaChBac increases membrane localization of Smo while expression of the potassium channel Kir2 . 1 reduces membrane localization , and increases cytosolic Smo ( B , C ) .

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(A-C) Localization of Smo protein (blue) following expression of 71B-Gal4 (A), 71B-Gal4 and UAS-NaChBac (B) or 71B-Gal4 and UAS-Kir2.1 (C). Expression of the sodium channel NaChBac increases membrane localization of Smo while expression of the potassium channel Kir2.1 reduces membrane localization, and increases cytosolic Smo (B, C).


( D ) Quantification of the ratio of membrane Smo fluorescence to cytosol fluorescence . N = 7 salivary glands per genotype . Data were compared using ANOVA followed by Tukey test for significance ( * * indicates p < 0 . 01 , * * * indicates p < 0 . 001 ) , error bars are standard deviations .

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(D) Quantification of the ratio of membrane Smo fluorescence to cytosol fluorescence. N=7 salivary glands per genotype. Data were compared using ANOVA followed by Tukey test for significance (** indicates p<0.01, *** indicates p<0.001), error bars are standard deviations.


( E , F ) Expression of the Hh target gene ptc is visualized using anti - Ptc immunostaining . Compared to 71B - Gal4 ( E ) , Ptc expression is increased in glands expressing 71B - Gal4 and UAS - NaChBac ( F ) .

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(E, F) Expression of the Hh target gene ptc is visualized using anti-Ptc immunostaining. Compared to 71B-Gal4 (E), Ptc expression is increased in glands expressing 71B-Gal4 and UAS-NaChBac (F).


( G - I ) Effect of expression and activation of the depolarizing channelrhodopsin ChR2 on Smo protein membrane abundance . After dissection , glands were kept in either dark conditions ( G ) or exposed to 15 minutes of blue light ( H ) . ( I ) Quantification of membrane fluorescence ; N = 13 glands , data compared using an unpaired t test ( * * indicates p < 0 . 01 ) , error bars are standard deviations .

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(G-I) Effect of expression and activation of the depolarizing channelrhodopsin ChR2 on Smo protein membrane abundance. After dissection, glands were kept in either dark conditions (G) or exposed to 15 minutes of blue light (H). (I) Quantification of membrane fluorescence; N=13 glands, data compared using an unpaired t test (** indicates p<0.01), error bars are standard deviations.


( J - L ) Effect of expression and activation of the Cl - - selective channelrhodopsin ChloC . An overall reduction in Smo immunostaining was observed following channel activation ( K ) , as compared to control glands kept in the dark ( J ) . ( L ) Quantification of membrane fluorescence ; N = 10 glands , data were compared using an unpaired t test , ( * * * indicates p < 0 . 001 ) , error bars are standard deviations .

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(J-L) Effect of expression and activation of the Cl--selective channelrhodopsin ChloC. An overall reduction in Smo immunostaining was observed following channel activation (K), as compared to control glands kept in the dark (J). (L) Quantification of membrane fluorescence; N=10 glands, data were compared using an unpaired t test, (*** indicates p<0.001), error bars are standard deviations.


( M - Q ) Time course of change in Smo localization . Salivary glands expressing the channelrhodopsin ChR2 were subjected to increasing intervals of activating light , then fixed and stained for Smo protein ( M - P ' ' ) . ( Q ) Quantification of membrane - associated fluorescence ; N = 7 glands / timepoint , data were compared using ANOVA followed by Tukey test for significance ( * * indicates p < 0 . 01 ) , error bars are standard deviations .

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(M-Q) Time course of change in Smo localization. Salivary glands expressing the channelrhodopsin ChR2 were subjected to increasing intervals of activating light, then fixed and stained for Smo protein (M-P''). (Q) Quantification of membrane-associated fluorescence; N=7 glands/timepoint, data were compared using ANOVA followed by Tukey test for significance (** indicates p<0.01), error bars are standard deviations.


( A - B ' ' ) Wing discs expressing the channelrhodopsin ChR2 were subjected to 0 minutes or 25 minutes of activating light in culture , then fixed and stained for Smo protein . In ( B ' ' ) , the yellow arrowhead indicates dorsoventral compartment boundary , and the white arrowhead indicates anteroposterior compartment boundary . ( C ) Quantification of the ratio of mean Smo fluorescence intensity in 25µm2 regions in the dorsal anterior compartment compared to the ventral posterior compartment . N = 5 for 0min discs , n = 9 for 25min discs , data were compared using an unpaired t test ( * * * p < 0 . 001 ) , error bars are standard deviations . Identical analyses were carried out comparing dorsal posterior fluorescence to ventral posterior fluorescence ( p = 0 . 132 ) , dorsal posterior fluorescence to dorsal anterior fluorescence ( p = 0 . 12 ) , and ventral posterior fluorescence to ventral anterior fluorescence ( p = 0 . 36 ) .

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(A-B'') Wing discs expressing the channelrhodopsin ChR2 were subjected to 0 minutes or 25 minutes of activating light in culture, then fixed and stained for Smo protein. In (B''), the yellow arrowhead indicates dorsoventral compartment boundary, and the white arrowhead indicates anteroposterior compartment boundary. (C) Quantification of the ratio of mean Smo fluorescence intensity in 25µm2 regions in the dorsal anterior compartment compared to the ventral posterior compartment. N=5 for 0min discs, n=9 for 25min discs, data were compared using an unpaired t test (***p<0.001), error bars are standard deviations. Identical analyses were carried out comparing dorsal posterior fluorescence to ventral posterior fluorescence (p=0.132), dorsal posterior fluorescence to dorsal anterior fluorescence (p=0.12), and ventral posterior fluorescence to ventral anterior fluorescence (p=0.36).


( D - E ' ) Discs expressing the red - light activated channelrhodopsin ReaChR under control of ap - Gal4 were raised on a 12hr light / dark cycle , dissected and stained for Smo protein ( D , D ' ) or Ptc protein ( E , E ' ) .

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(D-E') Discs expressing the red-light activated channelrhodopsin ReaChR under control of ap-Gal4 were raised on a 12hr light/dark cycle, dissected and stained for Smo protein (D, D') or Ptc protein (E, E').


All ORFs for the sense non - AUG - dependent translation of the ( G4C2 ) n ~ 188 , DPR coding for ( GA ) , ( GP ) , ( GR ) can be detected and quantified using a filter - trap binding assay probing for the c - terminal HA tag of the C9 DPR reporter construct . The ( GA ) ORF is the most abundantly detected DPR and GR the lowest in HEK293T cell 24 h post transient transfection of the C9 DPR reporter construct . n = 5 .

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All ORFs for the sense non-AUG-dependent translation of the (G4C2)n~188, DPR coding for (GA), (GP), (GR) can be detected and quantified using a filter-trap binding assay probing for the c-terminal HA tag of the C9 DPR reporter construct. The (GA) ORF is the most abundantly detected DPR and GR the lowest in HEK293T cell 24 h post transient transfection of the C9 DPR reporter construct. n = 5.


The C9orf72 NRE non - AUG - dependent reporter constructs recapitulate localization patterns and pathological features identified in C9orf72 NRE models . The Dendra2 ( green ) c - terminal fusion was used to monitor cellular localization and fluorescence intensity for each DPR translated from the G4C2 strand in HEK293T cells transiently cotransfected with an NES - mIFP - mIFP - NES ( NES - mIFP ) fusion protein cytoplasmic marker .

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The C9orf72 NRE non-AUG-dependent reporter constructs recapitulate localization patterns and pathological features identified in C9orf72 NRE models. The Dendra2 (green) c-terminal fusion was used to monitor cellular localization and fluorescence intensity for each DPR translated from the G4C2 strand in HEK293T cells transiently cotransfected with an NES-mIFP-mIFP-NES (NES-mIFP) fusion protein cytoplasmic marker.


C9 DPR reporter constructs show cell - type variability in the number of cells that are DPR positive and the overall DPR levels . Transient cotransfections of HEK293T cells robustly undergo G4C2 - Dendra2 non - AUG - dependent translation compared to NSC34 or rat primary cortical neurons . DPR positive cells and DPR fluorescent intensity were analyzed and calculated 24 h post - transfection only in cells expressing the co - transfected NES - mIFP construct . DPR fluorescent intensity was normalized to NES - mIFP fluorescent intensity to adjust for cell specific AUG - dependent translation levels ; represented as fold - change relative to HEK293T . HEK293T : n = 5 with m > 500 cells analyzed per n . NSC34 : n = 5 with m > 500 cells per n . Rat primary cortical neuron : n = 5 with m > 40 cells per n . ( * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 ) See also Figure EV1 - 2 .

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C9 DPR reporter constructs show cell-type variability in the number of cells that are DPR positive and the overall DPR levels. Transient cotransfections of HEK293T cells robustly undergo G4C2 -Dendra2 non-AUG-dependent translation compared to NSC34 or rat primary cortical neurons. DPR positive cells and DPR fluorescent intensity were analyzed and calculated 24 h post-transfection only in cells expressing the co-transfected NES-mIFP construct. DPR fluorescent intensity was normalized to NES-mIFP fluorescent intensity to adjust for cell specific AUG-dependent translation levels; represented as fold-change relative to HEK293T. HEK293T: n = 5 with m > 500 cells analyzed per n. NSC34: n = 5 with m > 500 cells per n. Rat primary cortical neuron: n = 5 with m > 40 cells per n. (***P < 0.001, **P < 0.01, *P <0.05) See also Figure EV1-2.


Quantitative fluorescent microscopy imaging shows a dose - dependent increase in the number of C9 DPR reporter positive cells with increasing concentrations of an apoptotic stimulator , staurosporine ( SSP ) . The C9 DPR reporter in the GP frame was utilized for these experiments . n = 4 .

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Quantitative fluorescent microscopy imaging shows a dose-dependent increase in the number of C9 DPR reporter positive cells with increasing concentrations of an apoptotic stimulator, staurosporine (SSP). The C9 DPR reporter in the GP frame was utilized for these experiments. n = 4.


Filter trap binding assays using DPR - specific antibodies show all DPR levels are increased for the sense - coding C9 DPR reporter when NSC34 cells are challenged with stress - inducing compounds . These stress - inducing compounds broadly cover distinct or overlapping cellular pathways ( see Table EV2 ) Antibodies specific for the C9orf72 NRE DPRs , poly - GA , - GP , or - GR , were used to quantify DPR levels normalized to GAPDH 24 h post transient transfection of the C9 DPR reporter constructs . n = 4 .

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Filter trap binding assays using DPR-specific antibodies show all DPR levels are increased for the sense-coding C9 DPR reporter when NSC34 cells are challenged with stress-inducing compounds. These stress-inducing compounds broadly cover distinct or overlapping cellular pathways (see Table EV2) Antibodies specific for the C9orf72 NRE DPRs, poly-GA, -GP, or -GR, were used to quantify DPR levels normalized to GAPDH 24 h post transient transfection of the C9 DPR reporter constructs. n = 4.


Quantitative fluorescent microscopy imaging shows that NSC34 , rat primary cortical neurons have increased relative DPR fluorescent intensity levels following compound induced cellular stress . In NES - mIFP cotransfection positive NSC34 and rat primary cortical neurons there is a significant increase in C9 DPR reporter fluorescent intensity levels 24 post treatment with most stress - inducing compounds . Fluorescent intensity was not normalized to NES - mIFP fluorescent levels . NSC34 : n = 10 with m > 500 cells analyzed per n . Cortical neuron : n = 6 with m > 40 cells per n . Endogenously expression of DPRs in iPS spinal motor neurons ( iPS sMN ) derived from C9orf72 NRE patients increases following induction of cellular stress . DPR fluorescent intensity in iPS sMN was measured using the DPR - specific antibodies described above . IPS sMN : n = 4 with m > 20 cells per n .

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Quantitative fluorescent microscopy imaging shows that NSC34, rat primary cortical neurons have increased relative DPR fluorescent intensity levels following compound induced cellular stress. In NES-mIFP cotransfection positive NSC34 and rat primary cortical neurons there is a significant increase in C9 DPR reporter fluorescent intensity levels 24 post treatment with most stress-inducing compounds. Fluorescent intensity was not normalized to NES-mIFP fluorescent levels. NSC34: n = 10 with m > 500 cells analyzed per n. Cortical neuron: n = 6 with m > 40 cells per n. Endogenously expression of DPRs in iPS spinal motor neurons (iPS sMN) derived from C9orf72 NRE patients increases following induction of cellular stress. DPR fluorescent intensity in iPS sMN was measured using the DPR-specific antibodies described above. IPS sMN: n = 4 with m > 20 cells per n.


Fluorescent microscopy images shows Glutamate - induced ( Glut ) excitotoxic stress increases DPR levels in rat cortical neurons expressing the C9 DPR reporter and in iPS sMN derived from C9orf72 NRE patients . Glutamate - induced DPR levels are reduced by pretreating cells with the AMPA and NMDA specific antagonists NBQX and MK801 , respectively . Relative fluorescent intensity quantification demonstrates there is a significant increase in DPR levels when primary cortical neurons and sMNs are challenged with the excitotoxic stressors : glutamate , AMPA , NMDA , and HC . Blocking either the AMPA or NMDA receptors reduces DPR levels in the presence of excitotoxic stressors . NES - mIFP or MAP2 were used to mark transfection positive rat cortical neurons or iPS sMNs , respectively , for quantification . DPR fluorescent intensity in iPS sMN was measured using DPR - specific antibodies . Changes in DPR levels are relative to non - stressed cells ( CTRL ) Cortical neuron : n = 6 with m > 40 cells per n . IPSC sMN : n = 4 with m > 25 cells per n .

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Fluorescent microscopy images shows Glutamate-induced (Glut) excitotoxic stress increases DPR levels in rat cortical neurons expressing the C9 DPR reporter and in iPS sMN derived from C9orf72 NRE patients. Glutamate-induced DPR levels are reduced by pretreating cells with the AMPA and NMDA specific antagonists NBQX and MK801, respectively. Relative fluorescent intensity quantification demonstrates there is a significant increase in DPR levels when primary cortical neurons and sMNs are challenged with the excitotoxic stressors: glutamate, AMPA, NMDA, and HC. Blocking either the AMPA or NMDA receptors reduces DPR levels in the presence of excitotoxic stressors. NES-mIFP or MAP2 were used to mark transfection positive rat cortical neurons or iPS sMNs, respectively, for quantification. DPR fluorescent intensity in iPS sMN was measured using DPR-specific antibodies. Changes in DPR levels are relative to non-stressed cells (CTRL) Cortical neuron: n = 6 with m > 40 cells per n. IPSC sMN: n = 4 with m > 25 cells per n.


Microscopy images show increased DPR fluorescent intensity levels from medium and high frequency stimulation of ChR2 in primary cortical neurons transfected with the C9 DPR reporter .

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Microscopy images show increased DPR fluorescent intensity levels from medium and high frequency stimulation of ChR2 in primary cortical neurons transfected with the C9 DPR reporter.


Relative fluorescent intensity quantification shows a significant increase in DPR levels with increases in neuronal activity through medium and high stimulation in primary cortical neurons . No stimulation and low stimulation frequencies did not increase levels of DPR . The C9 DPR reporter , monitoring the GP DPR ( ORF2 ) , was cotransfected with ChR2 . Cortical neuron : n = 4 with m > 10 cells per n . ( * * * * P < 0 . 0001 ) .

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Relative fluorescent intensity quantification shows a significant increase in DPR levels with increases in neuronal activity through medium and high stimulation in primary cortical neurons. No stimulation and low stimulation frequencies did not increase levels of DPR. The C9 DPR reporter, monitoring the GP DPR (ORF2), was cotransfected with ChR2. Cortical neuron: n = 4 with m > 10 cells per n. (****P < 0.0001).


Filter trap binding assays show that ISR proteins are enhanced by all stressors . Significant increases following cellular stress treatments are seen in the ratio of phospho - eif2α to total eif2α as well as phospho - PERK and ATF4 levels compared to untreated controls . Changes in ISR proteins are normalized to GAPDH and shown relative to non - stressed or DMSO - only treated cells ( CTRL ) depending on the vehicle compounds were dissolved in . n = 4 .

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Filter trap binding assays show that ISR proteins are enhanced by all stressors. Significant increases following cellular stress treatments are seen in the ratio of phospho-eif2α to total eif2α as well as phospho-PERK and ATF4 levels compared to untreated controls. Changes in ISR proteins are normalized to GAPDH and shown relative to non-stressed or DMSO-only treated cells (CTRL) depending on the vehicle compounds were dissolved in. n = 4.


Fluorescent intensity measurements show the levels of two ISR - related proteins , PERK and relative phospho - eif2α ( phospho - eif2α / total eif2α ) , are increased following different excitotoxic stress treatments in cortical neurons . Representative images indicate fluorescent intensity differences for phospho - eif2α and phospho - PERK levels 24 h post treatment with different excitotoxic stressor paradigms . Blocking of AMPA and NMDA receptors using known antagonists significantly reduces ISR - related protein levels , which parallels reduced DPR levels ( Figure 3B ) . Changes in ISR proteins are relative to non - stressed cells ( CTRL ) .

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Fluorescent intensity measurements show the levels of two ISR-related proteins, PERK and relative phospho-eif2α (phospho-eif2α / total eif2α), are increased following different excitotoxic stress treatments in cortical neurons. Representative images indicate fluorescent intensity differences for phospho-eif2α and phospho-PERK levels 24 h post treatment with different excitotoxic stressor paradigms. Blocking of AMPA and NMDA receptors using known antagonists significantly reduces ISR-related protein levels, which parallels reduced DPR levels (Figure 3B). Changes in ISR proteins are relative to non-stressed cells (CTRL).


Relative fluorescent intensity quantification shows a significant decrease in C9 DPR reporter levels when stressor challenged NSC34 cells were treated with compounds inhibiting ISR - related or translation - targeting pathways . Dibenzoylmethane ( 1 , 3DBM ; 20 µM ) , which inhibits eif2α - P activity , had the most significant decreases in DPR production . Trazodone ( Traz ; 20 µM ) showed similar significant reduction in DPR levels as the PERK inhibitor , GSK2606414 ( 500 nM ) .

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Relative fluorescent intensity quantification shows a significant decrease in C9 DPR reporter levels when stressor challenged NSC34 cells were treated with compounds inhibiting ISR-related or translation-targeting pathways. Dibenzoylmethane (1,3DBM; 20 µM), which inhibits eif2α-P activity, had the most significant decreases in DPR production. Trazodone (Traz; 20 µM) showed similar significant reduction in DPR levels as the PERK inhibitor, GSK2606414 (500 nM).


Relative fluorescent intensity quantification shows a significant decrease in C9 DPR reporter levels when glutamate stressed primary cortical neurons were treated with the compounds Trazodone ( Traz ; 20 µM ) or Dibenzoylmethane ( 1 , 3DBM ; 20 µM ) . Similar to the treatments in NSC34 , 1 , 3DBM shows the most significant decrease in DPR production .

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Relative fluorescent intensity quantification shows a significant decrease in C9 DPR reporter levels when glutamate stressed primary cortical neurons were treated with the compounds Trazodone (Traz; 20 µM) or Dibenzoylmethane (1,3DBM; 20 µM). Similar to the treatments in NSC34, 1,3DBM shows the most significant decrease in DPR production.


A , Western blotting showing cellular INSL5 in the immortalized NPEC and NPC cell lines after inhibition of protein secretion by BFA , and β - actin was used as a loading control .

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A, Western blotting showing cellular INSL5 in the immortalized NPEC and NPC cell lines after inhibition of protein secretion by BFA, and β-actin was used as a loading control.


B , ELISA assay showing secreted INSL5 in the supernatants of NPECs and NPC cell lines . For NPECs , n = 3 ; for NPC , n = 7 .

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B, ELISA assay showing secreted INSL5 in the supernatants of NPECs and NPC cell lines. For NPECs, n=3; for NPC, n=7.


C , The concentration of plasma INSL5 in healthy controls ( normal EBV ( - ) ) , non - tumor individuals with VCA - IgA positive ( normal EBV ( + ) ) and NPC patients , in training cohort .

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C, The concentration of plasma INSL5 in healthy controls (normal EBV (-)), non-tumor individuals with VCA-IgA positive (normal EBV (+)) and NPC patients, in training cohort.


E , The concentration of plasma INSL5 in healthy controls ( normal EBV ( - ) ) , non - tumor individuals with VCA - IgA positive ( normal EBV ( + ) ) and NPC patients , in validation cohort 1 ( GZ ) . F , The concentration of plasma INSL5 in healthy controls ( normal EBV ( - ) ) , non - tumor individuals with VCA - IgA positive ( normal EBV ( + ) ) and NPC patients , in validation cohort 2 ( ZS ) .

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E, The concentration of plasma INSL5 in healthy controls (normal EBV (-)), non-tumor individuals with VCA-IgA positive (normal EBV (+)) and NPC patients, in validation cohort 1(GZ). F, The concentration of plasma INSL5 in healthy controls (normal EBV (-)), non-tumor individuals with VCA-IgA positive (normal EBV (+)) and NPC patients, in validation cohort 2(ZS).


A - C , Kaplan - Meier curves showing the overall survival ( OS ) curves of NPC patients with low or high INSL5 concentration ( A ) , low ( ≤ 4000 copy / ml ) or high ( > 4000 copy / ml ) EBV DNA copy number ( B ) , and the combination of INSL5 concentration with EBV DNA copy number ( C ) .

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A-C, Kaplan-Meier curves showing the overall survival (OS) curves of NPC patients with low or high INSL5 concentration (A), low (≤4000 copy/ml) or high (>4000 copy/ml) EBV DNA copy number (B), and the combination of INSL5 concentration with EBV DNA copy number (C).


D - F , Kaplan - Meier curves showing the disease - free survival ( DFS ) curves of NPC patients with low or high INSL5 concentration ( D ) , low or high EBV DNA copy number ( E ) , and the combination of INSL5 concentration with EBV DNA copy number ( F ) .

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D-F, Kaplan-Meier curves showing the disease-free survival (DFS) curves of NPC patients with low or high INSL5 concentration (D), low or high EBV DNA copy number (E), and the combination of INSL5 concentration with EBV DNA copy number (F).


A . Exogenous expression of INSL5 in NPC cells . Representative immunoblotting ( upper panel ) and immunofluorescent staining ( lower panel ) showed stable exogenous expression of INSL5 in both CNE1 , CNE2 and HK1 NPC cell lines . Scale bars represent 20μm .

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A. Exogenous expression of INSL5 in NPC cells. Representative immunoblotting (upper panel) and immunofluorescent staining (lower panel) showed stable exogenous expression of INSL5 in both CNE1, CNE2 and HK1 NPC cell lines. Scale bars represent 20μm.B


MTT assay of vector control or INSL5 overexpressing CNE1 , CNE2 and HK1 NPC cell lines ( upper panel ) either transfected with control siRNA ( NC ) or GPCR142 siRNA ( # 1 and # 2 ) ( lower panel ) . n = 4 biological replicates for CNE1 and CNE2 cell line , n = 6 biological replicates for HK1 .

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MTT assay of vector control or INSL5 overexpressing CNE1, CNE2 and HK1 NPC cell lines (upper panel) either transfected with control siRNA (NC) or GPCR142 siRNA (#1 and #2) (lower panel). n=4 biological replicates for CNE1 and CNE2 cell line, n=6 biological replicates for HK1.


colony formation ( C and D ) of vector control or INSL5 overexpressing CNE1 , CNE2 and HK1 NPC cell lines either transfected with control siRNA ( NC ) or GPCR142 siRNA ( # 1 and # 2 ) . Representative images are shown in ( C ) for colony formation Number of colonies per field of view were plotted in D The results are from three different experiments .

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colony formation (C and D) of vector control or INSL5 overexpressing CNE1, CNE2 and HK1 NPC cell lines either transfected with control siRNA (NC) or GPCR142 siRNA (#1 and #2). Representative images are shown in (C) for colony formation Number of colonies per field of view were plotted in D The results are from three different experiments.


Brdu incorporation ( E and F ) of vector control or INSL5 overexpressing CNE1 , CNE2 and HK1 NPC cell lines either transfected with control siRNA ( NC ) or GPCR142 siRNA ( # 1 and # 2 ) . Representative images are shown in ( E ) Brdu incorporation the percentage of Brdu positive cells were plotted in The results are from three different experiments . Scale bars represent 20μm in ( E )

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Brdu incorporation (E and F) of vector control or INSL5 overexpressing CNE1, CNE2 and HK1 NPC cell lines either transfected with control siRNA (NC) or GPCR142 siRNA (#1 and #2). Representative images are shown in (E) Brdu incorporation the percentage of Brdu positive cells were plotted in The results are from three different experiments. Scale bars represent 20μm in (E)


migration assays ( G and H ) of vector control or INSL5 overexpressing CNE1 , CNE2 and HK1 NPC cell lines either transfected with control siRNA ( NC ) or GPCR142 siRNA ( # 1 and # 2 ) . Representative images are shown in ( G ) for migration assays migrated cells per field of view were plotted in H , respectively . The results are from three different experiments . Scale bars represent 100μm in ( G )

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migration assays (G and H) of vector control or INSL5 overexpressing CNE1, CNE2 and HK1 NPC cell lines either transfected with control siRNA (NC) or GPCR142 siRNA (#1 and #2). Representative images are shown in (G) for migration assays migrated cells per field of view were plotted in H, respectively. The results are from three different experiments. Scale bars represent 100μm in (G)


I , J , Xenograft tumor growth of INSL5 over expression NPC HK1 stable cell lines in nude mice . . Tumor size ( I ) n = 11 mice per group .

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I, J, Xenograft tumor growth of INSL5 over expression NPC HK1 stable cell lines in nude mice.. Tumor size (I) n=11 mice per group.


Xenograft tumor growth of INSL5 over expression NPC HK1 stable cell lines in nude mice . . Tumor weight of two groups . n = 11 mice per group .

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Xenograft tumor growth of INSL5 over expression NPC HK1 stable cell lines in nude mice.. Tumor weight of two groups. n=11 mice per group.


A , Gene expression profiles from CNE2 cells overexpressed with INSL5 or the vector control . The genes are shaded with green , black or red in the heat map to indicate low , intermediate or high expression , respectively .

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A, Gene expression profiles from CNE2 cells overexpressed with INSL5 or the vector control. The genes are shaded with green, black or red in the heat map to indicate low, intermediate or high expression, respectively.


C , The signal pathway of the subset of genes involved in metabolism was assessed by Gene Set Enrichment Analysis .

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C, The signal pathway of the subset of genes involved in metabolism was assessed by Gene Set Enrichment Analysis.


D , Analysis of glycolytic gene expression by immunoblot in INSL5 overexpressed or knock - down cell lines .

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D, Analysis of glycolytic gene expression by immunoblot in INSL5 overexpressed or knock-down cell lines.


E , Metabolites were purified from five ( CNE1 cell line and HK1 cell line ) or seven ( CNE2 cell line ) independent samples of two vector and INSL5 overexpressed cell lines and analyzed by liquid chromatography - mass spectrometry ( LC - MS ) . Red : glycolytic intermediate metabolites . Green : tricarboxylic acid cycle ( TCA cycle ) intermediate metabolites . PPP : pentose phosphate pathway . The color scale bar represents Z - score of the metabolites ' level .

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E, Metabolites were purified from five (CNE1 cell line and HK1 cell line) or seven (CNE2 cell line) independent samples of two vector and INSL5 overexpressed cell lines and analyzed by liquid chromatography-mass spectrometry (LC-MS). Red: glycolytic intermediate metabolites. Green: tricarboxylic acid cycle (TCA cycle) intermediate metabolites. PPP: pentose phosphate pathway. The color scale bar represents Z-score of the metabolites' level.


F , The extracellular acidification rate ( ECAR ) was measured in cells with or without INSL5 overexpression using a Seahorse XF96 Extracellular Flux analyzer . Glu , glucose ; Oligo , oligomycin .

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F, The extracellular acidification rate (ECAR) was measured in cells with or without INSL5 overexpression using a Seahorse XF96 Extracellular Flux analyzer. Glu, glucose; Oligo, oligomycin.


G , Statistical analysis of the effects of INSL5 overexpression on glycolytic activity .

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G, Statistical analysis of the effects of INSL5 overexpression on glycolytic activity.


H , CNE1 - vector and CNE1 - INSL5 cells were grown in the presence of the fluorescent glucose analog 2 - NBDG ( Invitrogen ) for 15min , and glucose uptake was then quantified using flow cytometry ( FACS ) ( left panel ) . Statistical analysis of the glucose uptake was showed in the right panel .

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H, CNE1-vector and CNE1-INSL5 cells were grown in the presence of the fluorescent glucose analog 2-NBDG (Invitrogen) for 15min, and glucose uptake was then quantified using flow cytometry (FACS) (left panel). Statistical analysis of the glucose uptake was showed in the right panel.


glucose consumption and lactate production ( I ) in CNE1 - vector and CNE1 - INSL5 cells .

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glucose consumption and lactate production (I) in CNE1-vector and CNE1-INSL5 cells.


basic ATP level or the ATP level of cell which was treated by 1uM oligomycin or 50uM 2 - DG for 24h ( J ) in CNE1 - vector and CNE1 - INSL5 cells .

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basic ATP level or the ATP level of cell which was treated by 1uM oligomycin or 50uM 2-DG for 24h (J) in CNE1-vector and CNE1-INSL5 cells.


HK2 enzyme activity ( K ) in CNE1 - vector and CNE1 - INSL5 cells .

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HK2 enzyme activity (K) in CNE1-vector and CNE1-INSL5 cells.


L , ATP concentration , HK2 enzyme activity and lactate production in INSL5 wide - type or knock down HNE1 - EBV cells .

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L, ATP concentration, HK2 enzyme activity and lactate production in INSL5 wide-type or knock down HNE1-EBV cells.


B , phosphorylation of STAT5 under INSL5 stimulation . CNE1 cells were starved in FBS - free medium for 24h and then treated with INSL5 peptide ( 50ng / mL ) in the presence of an anti - INSL5 antibody at 100ug / ml , or an isotype - matched IgG control ( IgG ) for 30min .

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B, phosphorylation of STAT5 under INSL5 stimulation. CNE1 cells were starved in FBS-free medium for 24h and then treated with INSL5 peptide (50ng/mL) in the presence of an anti-INSL5 antibody at 100ug/ml, or an isotype-matched IgG control (IgG) for 30min.


C , Localization of STAT5 . A confocal analysis of STAT5 staining was performed using CNE1 cells transfected vector or INSL5 . Scale bars represent

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C, Localization of STAT5. A confocal analysis of STAT5 staining was performed using CNE1 cells transfected vector or INSL5. Scale bars represent 20μm


D , Cytoplasmic and nuclear proteins from vector or INSL5overexpressing cells were separated and subjected to immunoblot as indicated . The nuclear marker Lamin B1 and the cytoplasmic marker GAPDH were used to demonstrate the purity of fractions .

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D, Cytoplasmic and nuclear proteins from vector or INSL5overexpressing cells were separated and subjected to immunoblot as indicated. The nuclear marker Lamin B1 and the cytoplasmic marker GAPDH were used to demonstrate the purity of fractions.


E , INSL5 overexpression activated AKT , ERK , JAK1 and STAT5 phosphorylation , not STAT3 in HK1 cells . INSL5 overexpressing HK1 cells were treated with MK2206 ( 50uM ) , U0126 ( 20uM ) , MK2206 + U0126 , or Ruxolitinib ( 5uM ) for 24h . Western blotting was performed to evaluate the effects of those inhibitors on STAT5 and STAT3 phosphorylation level .

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E, INSL5 overexpression activated AKT, ERK, JAK1 and STAT5 phosphorylation, not STAT3 in HK1 cells. INSL5 overexpressing HK1 cells were treated with MK2206 (50uM), U0126(20uM), MK2206+U0126, or Ruxolitinib (5uM) for 24h. Western blotting was performed to evaluate the effects of those inhibitors on STAT5 and STAT3 phosphorylation level.


F , ChIP - qPCR analyzed the occupancy of potential E - boxes by STAT5 in the genes of HK2 , Glut3 and PFK1 in INSL5 - overexpressing CNE1 cells using IgG or anti - STAT5 antibodies .

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F, ChIP-qPCR analyzed the occupancy of potential E-boxes by STAT5 in the genes of HK2, Glut3 and PFK1 in INSL5-overexpressing CNE1 cells using IgG or anti-STAT5 antibodies.


G , Luciferase activity of different glycolytic gene promoter reporters in 293T cells transfected with STAT5 or empty vector .

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G, Luciferase activity of different glycolytic gene promoter reporters in 293T cells transfected with STAT5 or empty vector.


H , Glucose uptake assessed by 2 - NBDG incubation in INSL5 overexpressed CNE1 cells with STAT5 knock down .

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H, Glucose uptake assessed by 2-NBDG incubation in INSL5 overexpressed CNE1 cells with STAT5 knock down.


I , Glucose uptake was assessed by 2 - BNDG incubation in INSL5 overexpressed HK1 cells treated with JAK kinase inhibitor ( Ruxolitinib ) .

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I, Glucose uptake was assessed by 2-BNDG incubation in INSL5 overexpressed HK1 cells treated with JAK kinase inhibitor (Ruxolitinib).


J , MTT assay of vector control or INSL5 overexpressing HK1 cells either treated with STAT5 siRNAs or JAK kinase inhibitor .

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J, MTT assay of vector control or INSL5 overexpressing HK1 cells either treated with STAT5 siRNAs or JAK kinase inhibitor.


A , Brdu assay for CNE1 cells stimulated with 100ng / ml INSL5 peptide in the presence of an anti - INSL5 antibody at 50 or 100ug / ml , or an isotype - matched IgG control ( IgG ) for 24h . Scale bars represent 20μm .

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A, Brdu assay for CNE1 cells stimulated with 100ng/ml INSL5 peptide in the presence of an anti-INSL5 antibody at 50 or 100ug/ml, or an isotype-matched IgG control (IgG) for 24h. Scale bars represent 20μm.


B , similar to ( A ) , migration and invasion assay for CNE1 cells stimulated with 100ng / ml INSL5 peptide in the presence of an anti - INSL5 antibody at 50 or 100ug / ml , or an isotype - matched IgG control ( IgG ) for 24h . Scale bars represent 100μm .

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B, similar to (A), migration and invasion assay for CNE1 cells stimulated with 100ng/ml INSL5 peptide in the presence of an anti-INSL5 antibody at 50 or 100ug/ml, or an isotype-matched IgG control (IgG) for 24h. Scale bars represent 100μm.


C - E Growth curves of cell lines stably expressing INSL5 or vector 48 h post - treatment with the indicated dose of glycolysis inhibitor , 2 - DG . CNE1 cells ( C ) , CNE2 cells ( D ) and HK1 cells ( E ) . n = 4 biological replicates for CNE1 and CNE2 cell line , n = 6 biological replicates for

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C-E Growth curves of cell lines stably expressing INSL5 or vector 48 h post-treatment with the indicated dose of glycolysis inhibitor, 2-DG. CNE1 cells (C), CNE2 cells (D) and HK1 cells (E). n=4 biological replicates for CNE1 and CNE2 cell line, n=6 biological replicates for HK1


F , Growth curves of cell lines stably expressing INSL5 or vector 48 h post - treatment with the indicated dose of cis - platinum ( DDP ) and 2 - DG . n = 4 biological replicates per group .

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F, Growth curves of cell lines stably expressing INSL5 or vector 48 h post-treatment with the indicated dose of cis-platinum (DDP) and 2-DG. n=4 biological replicates per group.


Tumor size ( G ) of nude mice burdened with NPC PDX tumor and intraperitoneally injected with anti - INSL5 , anti - GPCR142 or isotype - matched IgG control ( IgG ) every two days . n = 6 mice per group .

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Tumor size (G) of nude mice burdened with NPC PDX tumor and intraperitoneally injected with anti-INSL5, anti-GPCR142 or isotype-matched IgG control (IgG) every two days. n=6 mice per group.


tumor weight ( H ) of nude mice burdened with NPC PDX tumor and intraperitoneally injected with anti - INSL5 , anti - GPCR142 or isotype - matched IgG control ( IgG ) every two days . n = 6 mice per group .

tumor weight (H) of nude mice burdened with NPC PDX tumor and intraperitoneally injected with anti-INSL5, anti-GPCR142 or isotype-matched IgG control (IgG) every two days. n=6 mice per group.


A Heatmap showing the unsupervised consensus clustering of TCGA melanomas ( n = 445 : 356 metastases and 89 primary melanomas ) . The identified clusters are indicated below the heatmap : cluster 1 ( n = 55 , red ) , cluster 2 ( n = 304 , blue ) and cluster 3 ( n = 86 , green ) . Type of melanoma samples ( primary or metastatic ) and of metastatic site ( subcutaneous , lymph node or distant ) within each cluster are indicated below and above the heatmap , respectively . B Heatmap showing the unsupervised consensus clustering of TCGA primary melanomas ( n = 89 ) using the 1 , 000 most variable expressed genes in the same dataset . The identified signature clearly divides the TCGA dataset in two groups : Primary - A ( n = 34 , red ) and Primary - B ( n = 55 , pink ) , as indicated .

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A Heatmap showing the unsupervised consensus clustering of TCGA melanomas (n=445: 356 metastases and 89 primary melanomas). The identified clusters are indicated below the heatmap: cluster 1 (n=55, red), cluster 2 (n=304, blue) and cluster 3 (n=86, green). Type of melanoma samples (primary or metastatic) and of metastatic site (subcutaneous, lymph node or distant) within each cluster are indicated below and above the heatmap, respectively. B Heatmap showing the unsupervised consensus clustering of TCGA primary melanomas (n=89) using the 1,000 most variable expressed genes in the same dataset. The identified signature clearly divides the TCGA dataset in two groups: Primary-A (n=34, red) and Primary-B (n=55, pink), as indicated.


C Boxplots showing the distributions of the normalized rank - sums of the up - regulated ( left panel ) and down - regulated ( right panel ) genes in the identified signature . Down - regulated genes in Primary - B melanomas better discriminate the two groups of primary melanoma samples ( p = 5 . 9e - 15 , Wilcoxon rank - sum test ) . Median , minimum , maximum , 25th , 75th whiskers of ranksum distribution are shown .

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C Boxplots showing the distributions of the normalized rank-sums of the up-regulated (left panel) and down-regulated (right panel) genes in the identified signature. Down-regulated genes in Primary-B melanomas better discriminate the two groups of primary melanoma samples (p=5.9e-15, Wilcoxon rank-sum test). Median, minimum, maximum, 25th, 75th whiskers of ranksum distribution are shown.


A Box and whiskers ( from 5th to 95th percentile ) plots depicting TINCR RNA levels in the TCGA SKCM RNA - seq dataset of melanoma samples . TINCR expression is higher in Primary A melanomas , and its reduction in Primary - B melanomas and all groups of metastatic samples is statistically significant ( Mann - Whitney test , * * p < 0 . 01 ) . Median , minimum , maximum , 25th , 75th whiskers of expression values are shown .

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A Box and whiskers (from 5th to 95th percentile) plots depicting TINCR RNA levels in the TCGA SKCM RNA-seq dataset of melanoma samples. TINCR expression is higher in Primary A melanomas, and its reduction in Primary-B melanomas and all groups of metastatic samples is statistically significant (Mann-Whitney test, **p< 0.01). Median, minimum, maximum, 25th, 75th whiskers of expression values are shown.


B Scatter - plot showing correlation between the expression of TINCR RNA and normalized rank - sums s of the down - regulated ( upper panel ) and up - regulated ( lower panel ) genes in the primary melanoma signature . TINCR expression strongly correlates ( Spearman ' s correlation coefficient of 0 . 79 , Spearman rank p - value : * * p < 0 . 01 ) with the expression of the down - regulated genes .

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B Scatter-plot showing correlation between the expression of TINCR RNA and normalized rank-sums s of the down-regulated (upper panel) and up-regulated (lower panel) genes in the primary melanoma signature. TINCR expression strongly correlates (Spearman's correlation coefficient of 0.79, Spearman rank p-value: **p<0.01) with the expression of the down-regulated genes.


C Dot plot showing qRT - PCR analysis of TINCR expression in primary ( 8 ) and metastatic ( 8 ) melanoma samples from surgical specimens or patient - derived xenografts ( 4 primary and 8 metastatic PDXs ) . TINCR RNA levels relative to the L32P housekeeping gene are shown . Paired primary and metastasis PDXs are marked with an asterisk . The difference between primary and metastatic samples is significant ( Mann - Whitney test , * * p < 0 . 01 ) . Median , minimum , maximum , 25th , 75th whiskers of expression values are shown .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O B-DISEASE O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O

C Dot plot showing qRT-PCR analysis of TINCR expression in primary (8) and metastatic (8) melanoma samples from surgical specimens or patient-derived xenografts (4 primary and 8 metastatic PDXs). TINCR RNA levels relative to the L32P housekeeping gene are shown. Paired primary and metastasis PDXs are marked with an asterisk. The difference between primary and metastatic samples is significant (Mann-Whitney test, **p< 0.01). Median, minimum, maximum, 25th, 75th whiskers of expression values are shown.


A qRT - PCR quantification of TINCR RNA expression in MMC70 and WM902B cell lines , independently infected with two shRNA targeting TINCR expression ( shTINCR - 1 and - 2 ) . Cells transduced with shScamble ( shSCR ) were used as control . TINCR RNA levels relative to L32P housekeeping gene and control ( mean ± sd ) measured in three independent biological experiments are shown . In both cell lines TINCR knockdown efficiency is more than 70 % with the two shRNAs . Statistical analyses were performed using Student t - test ( * * : p < 0 . 01 ) .

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A qRT-PCR quantification of TINCR RNA expression in MMC70 and WM902B cell lines, independently infected with two shRNA targeting TINCR expression (shTINCR-1 and -2). Cells transduced with shScamble (shSCR) were used as control. TINCR RNA levels relative to L32P housekeeping gene and control (mean±sd) measured in three independent biological experiments are shown. In both cell lines TINCR knockdown efficiency is more than 70% with the two shRNAs. Statistical analyses were performed using Student t -test (**: p<0.01).


B In vitro migration assay of MMC70 and WM902B cells transduced with shTINCR - 1 , shTINCR - 2 or shSCR . Relative migration ( mean ± sd ) from three independent experiments is indicated . Statistical analyses were performed using Student t - test ( * * * : p < 0 . 001 ) .

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B In vitro migration assay of MMC70 and WM902B cells transduced with shTINCR-1, shTINCR-2 or shSCR. Relative migration (mean±sd) from three independent experiments is indicated. Statistical analyses were performed using Student t -test (***: p<0.001).


C In vitro cell proliferation of shSCR or shTINCR infected MMC70 and WM902B cells assayed 72h after infection at the indicated time points using CellTiter - Glo luminescent kit ( Promega ) . Cell growth from three biological replicates is indicated as proliferation rate relative to control cells ( mean ± sd ) . The difference between control and TINCR - silenced samples at all time points is not significant ( Student t - test ) .

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C In vitro cell proliferation of shSCR or shTINCR infected MMC70 and WM902B cells assayed 72h after infection at the indicated time points using CellTiter-Glo luminescent kit (Promega). Cell growth from three biological replicates is indicated as proliferation rate relative to control cells (mean±sd). The difference between control and TINCR-silenced samples at all time points is not significant (Student t-test).


D Upper panel : in vivo growth of tumors derived from intradermal injection of shTINCR - 2 or shSCR WM902B cells in NSG mice . 600 , 000 WM902B cells per mouse were inoculated . Tumor volumes ( mean ± sd ) were measured 8 weeks after transplantation . Results from two independent experiments ( four animals per group ) are shown . Statistical analysis between control and TINCR - silenced groups was performed using the Student t - test ( * * = p < 0 . 01 ) . Lower panel : qRT - PCR analysis of TINCR RNA expression in tumors derived from in vivo transplant of shSCR and shTINCR WM902B cells ( 8 weeks after injection ) . TINCR RNA levels relative to L32P housekeeping gene and control ( mean ± sd ) from four biological replicates ( animals ) from both experiments are shown .

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D Upper panel: in vivo growth of tumors derived from intradermal injection of shTINCR-2 or shSCR WM902B cells in NSG mice. 600,000 WM902B cells per mouse were inoculated. Tumor volumes (mean±sd) were measured 8 weeks after transplantation. Results from two independent experiments (four animals per group) are shown. Statistical analysis between control and TINCR-silenced groups was performed using the Student t -test (**=p<0.01). Lower panel: qRT-PCR analysis of TINCR RNA expression in tumors derived from in vivo transplant of shSCR and shTINCR WM902B cells (8 weeks after injection). TINCR RNA levels relative to L32P housekeeping gene and control (mean±sd) from four biological replicates (animals) from both experiments are shown.


E Drug - response curves showing increased resistance of shTINCR - WM902B cells after treatment with BRAF ( Vemurafenib ; from 10 to 10 , 000 nM ) and MEK ( Trametinib ; from 4 to 2 , 000 nM ) inhibitors . The shSCR - ( control ) and shTINCR - WM902B cells were treated with increasing concentrations of drugs and their viability assayed after 72 hours using CellTiter - Glo luminescent kit ( Promega ) . Error bars represent s . e . m . of three biological replicates . The dose response curve was fit with nonlinear regression ( GraphPad Prism ) .

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E Drug-response curves showing increased resistance of shTINCR-WM902B cells after treatment with BRAF (Vemurafenib; from 10 to 10,000 nM) and MEK (Trametinib; from 4 to 2,000 nM) inhibitors. The shSCR- (control) and shTINCR-WM902B cells were treated with increasing concentrations of drugs and their viability assayed after 72 hours using CellTiter-Glo luminescent kit (Promega). Error bars represent s.e.m. of three biological replicates. The dose response curve was fit with nonlinear regression (GraphPad Prism).


G Western blot analysis of a panel of proteins that are specifically associated to the proliferative or invasive phenotype . Total protein lysates were extracted from shSCR - and shTINCR - WM902B cells ( lane 1 and 2 ) or shSCR - and shTINCR - MMC70 cells ( lane 3 and 4 ) 10 days after infection and analyzed by immunoblot assay using the indicated antibodies . Vinculin was used as loading control .

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G Western blot analysis of a panel of proteins that are specifically associated to the proliferative or invasive phenotype. Total protein lysates were extracted from shSCR- and shTINCR-WM902B cells (lane 1 and 2) or shSCR- and shTINCR-MMC70 cells (lane 3 and 4) 10 days after infection and analyzed by immunoblot assay using the indicated antibodies. Vinculin was used as loading control.


A qRT - PCR analysis of TINCR expression ( left ) of MM13 and MM2 PDX cells infected with lentiviral vectors expressing full - length TINCR cDNA . PDX cells infected with empty vector ( Empty ) were used as control . TINCR RNA levels relative to L32P housekeeping gene and control ( mean ± sd ) from three biological replicates are shown . Statistical analyses were performed using Student t - test ( * * * : p < 0 . 001 ) .

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A qRT-PCR analysis of TINCR expression (left) of MM13 and MM2 PDX cells infected with lentiviral vectors expressing full-length TINCR cDNA. PDX cells infected with empty vector (Empty) were used as control. TINCR RNA levels relative to L32P housekeeping gene and control (mean±sd) from three biological replicates are shown. Statistical analyses were performed using Student t -test (***: p<0.001).


B In vitro relative migration ( mean ± sd ) from three independent experiments ( biological replicates ) is shown in MM13 and MM2 TINCR overexpressing ( TINCR ) compared to control ( Empty ) cells . Statistical analyses were performed using Student t - test ( * * * : p < 0 . 001 ) .

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B In vitro relative migration (mean±sd) from three independent experiments (biological replicates) is shown in MM13 and MM2 TINCR overexpressing (TINCR) compared to control (Empty) cells. Statistical analyses were performed using Student t -test (***: p<0.001).


C In vitro cell proliferation was assayed 72 hours post infection in Empty and TINCR MM13 and MM2 PDX cells at the indicated time points ( 1 - 5 days ) using CellTiter - Glo viability assay . Relative proliferation rate ( mean ± sd ) of three independent experiments are shown .

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C In vitro cell proliferation was assayed 72 hours post infection in Empty and TINCR MM13 and MM2 PDX cells at the indicated time points (1-5 days) using CellTiter-Glo viability assay. Relative proliferation rate (mean±sd) of three independent experiments are shown.


D Drug - response curves showing sensitization to MEK inhibitor Trametinib of MM13 ( NRAS - mutant PDX ) and to Trametinib and BRAF inhibitor Vemurafenib of TINCR MM2 cells ( BRAF - mutant PDX ) . The Empty ( control ) and TINCR expressing MM13 cells were treated with increasing concentrations of drug ( ranging from 5 to 5000 nM ) and cell viability was assayed after 72 hours by CellTiter - Glo viability assay . Error bars represent s . e . m . of three biological replicates . The dose response curve was fit with nonlinear regression ( GraphPad Prism ) .

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D Drug-response curves showing sensitization to MEK inhibitor Trametinib of MM13 (NRAS-mutant PDX) and to Trametinib and BRAF inhibitor Vemurafenib of TINCR MM2 cells (BRAF-mutant PDX). The Empty (control) and TINCR expressing MM13 cells were treated with increasing concentrations of drug (ranging from 5 to 5000 nM) and cell viability was assayed after 72 hours by CellTiter-Glo viability assay. Error bars represent s.e.m. of three biological replicates. The dose response curve was fit with nonlinear regression (GraphPad Prism).


E Western blot analysis of the expression of the invasive and proliferative phenotype markers EPHA2 , EGFR , AXL , PDGFRβ , SOX10 , and MITF in TINCR - overexpressing MM13 and MM2 cells . Total protein lysates were extracted from PDX cells 7 days after infection and analyzed by immunoblot assay using the indicated antibodies . Vinculin was used as loading control . This experiment is representative of three independent experiments showing comparable levels of TINCR overexpression ( 8 - 10 folds ) .

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E Western blot analysis of the expression of the invasive and proliferative phenotype markers EPHA2, EGFR, AXL, PDGFRβ, SOX10, and MITF in TINCR-overexpressing MM13 and MM2 cells. Total protein lysates were extracted from PDX cells 7 days after infection and analyzed by immunoblot assay using the indicated antibodies. Vinculin was used as loading control. This experiment is representative of three independent experiments showing comparable levels of TINCR overexpression (8-10 folds).


F In vivo growth of Empty vector and TINCR cDNA expressing MM13 cells following intradermal injection into NSG mice . Four animals ( 200 , 000 cells / mouse ) per group were used in each experiment . Tumor growth ( mean ± sd ) was monitored weekly and tumor volume ( mean ± sd ) measured at 4 weeks for primary tumors ( originated in the site of injection , left panel ) and at 8 weeks for spontaneous metastases which colonized lymph nodes ( right panel ) . The results of two independent experiments are shown . Statistical analyses of control and TINCR - overexpressing groups from both experiments were performed using Student t - test ( * * : p < 0 . 01 ) .

O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O B-ORGANISM O O O O O O O O O B-ORGANISM O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O 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-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O

F In vivo growth of Empty vector and TINCR cDNA expressing MM13 cells following intradermal injection into NSG mice. Four animals (200,000 cells/mouse) per group were used in each experiment. Tumor growth (mean±sd) was monitored weekly and tumor volume (mean±sd) measured at 4 weeks for primary tumors (originated in the site of injection, left panel) and at 8 weeks for spontaneous metastases which colonized lymph nodes (right panel). The results of two independent experiments are shown. Statistical analyses of control and TINCR-overexpressing groups from both experiments were performed using Student t -test (**: p<0.01).