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

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

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

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(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 is observed in wing imaginal disc in both apical ( A - A ' ) , basal ( B - B ' ) optical sections . Increased is observed in center of pouch ( arrow , B ' ) , at dorsoventral ( D - V ) compartment boundary in anterior compartment ( yellow arrow , B ' , inset , D ) .

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

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(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 more uniform DiBAC .

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


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

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(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 third larval instar , with stripe of increased becoming narrower in more mature discs with developmental time ( Compare I with A , bracket in I indicates width of increased DiBAC ) .

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(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 A - P compartment boundary , in two rows of flanking D - V compartment boundary in A compartment .

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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 stripe of increased DiBAC along A - P compartment boundary ( arrowhead ) . boxes indicate regions used to calculate average DiBAC across compartment boundary = 0 . 98 , standard deviation = 0 . 05 , n = 5 discs . Increased at D - V boundary in A compartment is still observable ( yellow arrowhead ) .

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(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 . arrowhead indicates approximate position of A - P compartment boundary , yellow arrowhead indicates approximate position of D - V compartment boundary .

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(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 along A - P compartment boundary ( arrowhead ) , while increased at D - V boundary in A compartment is still observable ( yellow arrowhead ) .

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(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 ATPα showing elevated of both anterior to compartment boundary . arrowheads indicate approximate position of A - P compartment boundary .

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(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 - sensitive for hh following shift to restrictive for 12 h show loss of increased expression of Rpk ( B , B ' ) ATPα ( C , C ' ) anterior to compartment boundary . Controls shown in Figure EV2 . arrowheads indicate approximate position of 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 expressing a constitutively activated form of transcription factor Ci show elevated Rpk ATPα expression in anterior compartment ( box ) as well as posterior compartment ( yellow arrowhead ) . ( E - E ' ' ' ) A single clone in A compartment is shown at higher magnification .

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(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 in anterior compartment expressing ci3M following incubation in DiBAC .

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


( G - G ' ) A clone of in anterior compartment expressing ptc RNAi following incubation in DiBAC . 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 ( red ) full - Ci ( blue ) in discs expressing en - Venus . Smo is expressed at higher in P compartment in a stripe 5 - 10 wide just anterior to A - P compartment boundary . arrowhead indicates just anterior to 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 ENaC channel rpk in dorsal compartment of disc using ap - Gal4 . Knockdown of Rpk in dorsal compartment results in decreased accumulation of Hh signal transducer Smo ( B , B ' ) , activator form of Ci ( B , B ' ' ) . expression of ptc , visualized using an anti - Ptc antibody ( C ) , a downstream target gene of Hh signaling , is also diminished . Rpk RNAi BL : 39053 is used in panels shown , but phenotype was validated with a second RNAi ( 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 ( blue ) following expression of 71B - Gal4 ( A ) , 71B - Gal4 UAS - NaChBac ( B ) or 71B - Gal4 UAS - Kir2 . 1 ( C ) . Expression of sodium channel NaChBac increases localization of Smo while expression of potassium channel Kir2 . 1 reduces localization , increases 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 ) of of Smo to cytosol . N = 7 salivary per genotype . Data were compared using ANOVA followed by Tukey 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 Hh target gene ptc is visualized using anti - Ptc immunostaining . Compared to 71B - Gal4 ( E ) , Ptc expression is increased in expressing 71B - Gal4 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 activation of depolarizing channelrhodopsin ChR2 on Smo abundance . After dissection , were kept in either conditions ( G ) or exposed to 15 minutes of blue ( H ) . ( I ) of ; N = 13 , data compared using an unpaired t ( * * 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 activation of Cl - - selective channelrhodopsin ChloC . An overall reduction in Smo immunostaining was observed following channel activation ( K ) , as compared to control kept in ( J ) . ( L ) of ; N = 10 , data were compared using an unpaired t , ( * * * 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 expressing channelrhodopsin ChR2 were subjected to increasing intervals of activating , then fixed stained for Smo ( M - P ' ' ) . ( Q ) of - associated ; N = 7 / timepoint , data were compared using ANOVA followed by Tukey 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 channelrhodopsin ChR2 were subjected to 0 minutes or 25 minutes of activating in culture , then fixed stained for Smo . In ( B ' ' ) , yellow arrowhead indicates dorsoventral compartment boundary , arrowhead indicates anteroposterior compartment boundary . ( C ) of of mean Smo in 25µm2 regions in dorsal anterior compartment compared to ventral posterior compartment . N = 5 for 0min discs , n = 9 for 25min discs , data were compared using an unpaired t ( * * * p < 0 . 001 ) , error bars are standard deviations . Identical analyses were carried out comparing dorsal posterior to ventral posterior ( p = 0 . 132 ) , dorsal posterior to dorsal anterior ( p = 0 . 12 ) , ventral posterior to ventral anterior ( 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 red - activated channelrhodopsin ReaChR under control of ap - Gal4 were raised on a 12hr / cycle , dissected stained for Smo ( D , D ' ) or Ptc ( 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').


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


C9orf72 NRE non - AUG - dependent reporter constructs recapitulate localization patterns pathological features identified in C9orf72 NRE models . Dendra2 ( green ) c - terminal fusion was used to monitor localization for each DPR translated from G4C2 strand in HEK293T transiently cotransfected with an NES - mIFP - mIFP - NES ( NES - mIFP ) fusion 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 - type variability in number of that are DPR positive overall DPR . Transient cotransfections of HEK293T robustly undergo G4C2 - Dendra2 non - AUG - dependent translation compared to NSC34 or rat cortical neurons . DPR positive DPR were analyzed calculated 24 h post - transfection only in expressing co - transfected NES - mIFP construct . DPR was normalized to NES - mIFP to adjust for specific AUG - dependent translation ; represented as fold - change relative to HEK293T . HEK293T : n = 5 with m > 500 analyzed per n . NSC34 : n = 5 with m > 500 per n . Rat cortical neuron : n = 5 with m > 40 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 microscopy imaging shows a dose - dependent increase in number of C9 DPR reporter positive with increasing concentrations of an apoptotic stimulator , staurosporine ( SSP ) . C9 DPR reporter in 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 DPR are increased for sense - coding C9 DPR reporter when NSC34 are challenged with stress - inducing compounds . These stress - inducing compounds broadly cover distinct or overlapping pathways ( see Table EV2 ) Antibodies specific for C9orf72 NRE DPRs , poly - GA , - GP , or - GR , were used to quantify DPR normalized to GAPDH 24 h post transient transfection of 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 microscopy imaging shows that NSC34 , rat cortical neurons have increased relative DPR following compound induced stress . In NES - mIFP cotransfection positive NSC34 rat cortical neurons there is a significant increase in C9 DPR reporter 24 post treatment with most stress - inducing compounds . was not normalized to NES - mIFP . NSC34 : n = 10 with m > 500 analyzed per n . Cortical neuron : n = 6 with m > 40 per n . Endogenously expression of DPRs in iPS spinal motor neurons ( iPS sMN ) derived from C9orf72 NRE increases following induction of stress . DPR in iPS sMN was measured using DPR - specific antibodies described above . IPS sMN : n = 4 with m > 20 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.


microscopy shows Glutamate - induced ( Glut ) excitotoxic stress increases DPR in rat cortical neurons expressing C9 DPR reporter in iPS sMN derived from C9orf72 NRE . Glutamate - induced DPR are reduced by pretreating with AMPA NMDA specific antagonists NBQX MK801 , respectively . Relative demonstrates there is a significant increase in DPR when cortical neurons sMNs are challenged with excitotoxic stressors : glutamate , AMPA , NMDA , HC . Blocking either AMPA or NMDA receptors reduces DPR in presence of excitotoxic stressors . NES - mIFP or MAP2 were used to mark transfection positive rat cortical neurons or iPS sMNs , respectively , for . DPR in iPS sMN was measured using DPR - specific antibodies . Changes in DPR are relative to non - stressed ( CTRL ) Cortical neuron : n = 6 with m > 40 per n . IPSC sMN : n = 4 with m > 25 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 show increased DPR from medium high frequency stimulation of ChR2 in cortical neurons transfected with 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 shows a significant increase in DPR with increases in neuronal activity through medium high stimulation in cortical neurons . No stimulation low stimulation frequencies did not increase of DPR . C9 DPR reporter , monitoring GP DPR ( ORF2 ) , was cotransfected with ChR2 . Cortical neuron : n = 4 with m > 10 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 are enhanced by stressors . Significant increases following stress treatments are seen in of phospho - eif2α to total eif2α as well as phospho - PERK ATF4 compared to untreated controls . Changes in ISR are normalized to GAPDH shown relative to non - stressed or DMSO - only treated ( CTRL ) depending on 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.


measurements show of two ISR - related , PERK relative phospho - eif2α ( phospho - eif2α / total eif2α ) , are increased following different excitotoxic stress treatments in cortical neurons . Representative indicate differences for phospho - eif2α phospho - PERK 24 h post treatment with different excitotoxic stressor paradigms . Blocking of AMPA NMDA receptors using known antagonists significantly reduces ISR - related , which parallels reduced DPR ( Figure 3B ) . Changes in ISR are relative to non - stressed ( 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 shows a significant decrease in C9 DPR reporter when stressor challenged NSC34 were treated with compounds inhibiting ISR - related or translation - targeting pathways . Dibenzoylmethane ( 1 , 3DBM ; 20 µM ) , which inhibits eif2α - P activity , had most significant decreases in DPR production . Trazodone ( Traz ; 20 µM ) showed similar significant reduction in DPR as 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 shows a significant decrease in C9 DPR reporter when glutamate stressed cortical neurons were treated with compounds Trazodone ( Traz ; 20 µM ) or Dibenzoylmethane ( 1 , 3DBM ; 20 µM ) . Similar to treatments in NSC34 , 1 , 3DBM shows 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 INSL5 in immortalized NPEC NPC lines after inhibition of secretion by BFA , 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 showing secreted INSL5 in supernatants of NPECs NPC 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 , of plasma INSL5 in healthy controls ( normal EBV ( - ) ) , non - individuals with VCA - IgA positive ( normal EBV ( + ) ) NPC , 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 , of plasma INSL5 in healthy controls ( normal EBV ( - ) ) , non - individuals with VCA - IgA positive ( normal EBV ( + ) ) NPC , in validation cohort 1 ( GZ ) . F , of plasma INSL5 in healthy controls ( normal EBV ( - ) ) , non - individuals with VCA - IgA positive ( normal EBV ( + ) ) NPC , 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 overall survival ( OS ) curves of NPC with low or high INSL5 ( A ) , low ( ≤ 4000 copy / ml ) or high ( > 4000 copy / ml ) EBV copy number ( B ) , combination of INSL5 with EBV 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 disease - free survival ( DFS ) curves of NPC with low or high INSL5 ( D ) , low or high EBV copy number ( E ) , combination of INSL5 with EBV 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 . Representative immunoblotting ( upper panel ) immunofluorescent ( lower panel ) showed stable exogenous expression of INSL5 in both CNE1 , CNE2 HK1 NPC 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 of vector control or INSL5 overexpressing CNE1 , CNE2 HK1 NPC lines ( upper panel ) either transfected with control siRNA ( NC ) or GPCR142 siRNA ( # 1 # 2 ) ( lower panel ) . n = 4 biological replicates for CNE1 CNE2 , 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 D ) of vector control or INSL5 overexpressing CNE1 , CNE2 HK1 NPC lines either transfected with control siRNA ( NC ) or GPCR142 siRNA ( # 1 # 2 ) . Representative are shown in ( C ) for colony formation Number of colonies per field of view were plotted in D 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 F ) of vector control or INSL5 overexpressing CNE1 , CNE2 HK1 NPC lines either transfected with control siRNA ( NC ) or GPCR142 siRNA ( # 1 # 2 ) . Representative are shown in ( E ) Brdu incorporation of Brdu positive were plotted in 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 H ) of vector control or INSL5 overexpressing CNE1 , CNE2 HK1 NPC lines either transfected with control siRNA ( NC ) or GPCR142 siRNA ( # 1 # 2 ) . Representative are shown in ( G ) for migration assays migrated per field of view were plotted in H , respectively . 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 growth of INSL5 over expression NPC HK1 stable lines in nude mice . . ( 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 growth of INSL5 over expression NPC HK1 stable lines in nude mice . . 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 overexpressed with INSL5 or vector control . genes are shaded with green , black or red in 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 , signal pathway of subset of genes involved in metabolism was assessed by Gene Set Enrichment .

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


D , of glycolytic gene expression by immunoblot in INSL5 overexpressed or knock - down 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 HK1 ) or seven ( CNE2 ) independent samples of two vector INSL5 overexpressed lines analyzed by liquid chromatography - mass spectrometry ( LC - MS ) . Red : glycolytic intermediate metabolites . Green : tricarboxylic acid cycle ( TCA cycle ) intermediate metabolites . PPP : pentose phosphate pathway . color scale bar represents Z - score of 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 , extracellular acidification rate ( ECAR ) was measured in 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 of effects of INSL5 overexpression on glycolytic activity .

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


H , CNE1 - vector CNE1 - INSL5 were grown in presence of glucose analog 2 - NBDG ( Invitrogen ) for 15min , glucose uptake was then quantified using flow cytometry ( FACS ) ( left panel ) . Statistical of glucose uptake was showed in 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 lactate production ( I ) in CNE1 - vector CNE1 - INSL5 .

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


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

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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 CNE1 - INSL5 .

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


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

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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 were starved in FBS - free medium for 24h then treated with INSL5 peptide ( 50ng / mL ) in 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 of STAT5 was performed using CNE1 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 , from vector or INSL5overexpressing were separated subjected to immunoblot as indicated . marker Lamin B1 marker GAPDH were used to demonstrate 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 STAT5 phosphorylation , not STAT3 in HK1 . INSL5 overexpressing HK1 were treated with MK2206 ( 50uM ) , U0126 ( 20uM ) , MK2206 + U0126 , or Ruxolitinib ( 5uM ) for 24h . Western blotting was performed to evaluate effects of those inhibitors on STAT5 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 occupancy of potential E - boxes by STAT5 in genes of HK2 , Glut3 PFK1 in INSL5 - overexpressing CNE1 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 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 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 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 of vector control or INSL5 overexpressing HK1 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 for CNE1 stimulated with 100ng / ml INSL5 peptide in 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 invasion for CNE1 stimulated with 100ng / ml INSL5 peptide in 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 lines stably expressing INSL5 or vector 48 h post - treatment with indicated dose of glycolysis inhibitor , 2 - DG . CNE1 ( C ) , CNE2 ( D ) HK1 ( E ) . n = 4 biological replicates for CNE1 CNE2 , 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 lines stably expressing INSL5 or vector 48 h post - treatment with indicated dose of cis - platinum ( DDP ) 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.


( G ) of nude mice burdened with NPC PDX 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.


( H ) of nude mice burdened with NPC PDX 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 showing unsupervised consensus clustering of TCGA melanomas ( n = 445 : 356 metastases 89 melanomas ) . identified clusters are indicated below : cluster 1 ( n = 55 , red ) , cluster 2 ( n = 304 , blue ) cluster 3 ( n = 86 , green ) . Type of melanoma samples ( or metastatic ) of metastatic site ( subcutaneous , lymph node or distant ) within each cluster are indicated below above , respectively . B showing unsupervised consensus clustering of TCGA melanomas ( n = 89 ) using 1 , 000 most variable expressed genes in same dataset . identified signature clearly divides TCGA dataset in two groups : - A ( n = 34 , red ) - 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 distributions of normalized rank - sums of up - regulated ( left panel ) down - regulated ( right panel ) genes in identified signature . Down - regulated genes in - B melanomas better discriminate two groups of melanoma samples ( p = 5 . 9e - 15 , Wilcoxon rank - sum ) . 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 whiskers ( from 5th to 95th percentile ) plots depicting TINCR in TCGA SKCM - seq dataset of melanoma samples . TINCR expression is higher in A melanomas , its reduction in - B melanomas groups of metastatic samples is statistically significant ( Mann - Whitney , * * 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 expression of TINCR normalized rank - sums s of down - regulated ( upper panel ) up - regulated ( lower panel ) genes in melanoma signature . TINCR expression strongly correlates ( Spearman ' s correlation coefficient of 0 . 79 , Spearman rank p - value : * * p < 0 . 01 ) with expression of 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 of TINCR expression in ( 8 ) metastatic ( 8 ) melanoma samples from surgical specimens or - derived xenografts ( 4 8 metastatic PDXs ) . TINCR relative to L32P housekeeping gene are shown . Paired metastasis PDXs are marked with an asterisk . difference between metastatic samples is significant ( Mann - Whitney , * * p < 0 . 01 ) . Median , minimum , maximum , 25th , 75th whiskers of expression values are shown .

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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 of TINCR expression in MMC70 WM902B lines , independently infected with two shRNA targeting TINCR expression ( shTINCR - 1 - 2 ) . transduced with shScamble ( shSCR ) were used as control . TINCR relative to L32P housekeeping gene control ( mean ± sd ) measured in three independent biological experiments are shown . In both lines TINCR knockdown efficiency is more than 70 % with two shRNAs . Statistical analyses were performed using Student t - ( * * : 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 of MMC70 WM902B 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 - ( * * * : 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 proliferation of shSCR or shTINCR infected MMC70 WM902B assayed 72h after infection at indicated time points using CellTiter - Glo luminescent kit ( Promega ) . growth from three biological replicates is indicated as proliferation rate relative to control ( mean ± sd ) . difference between control TINCR - silenced samples at time points is not significant ( Student t - ) .

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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 in NSG mice . 600 , 000 WM902B per mouse were inoculated . volumes ( mean ± sd ) were measured 8 after transplantation . Results from two independent experiments ( four per group ) are shown . Statistical between control TINCR - silenced groups was performed using Student t - ( * * = p < 0 . 01 ) . Lower panel : qRT - PCR of TINCR expression in tumors derived from in vivo transplant of shSCR shTINCR WM902B ( 8 after injection ) . TINCR relative to L32P housekeeping gene control ( mean ± sd ) from four biological replicates ( ) 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 after treatment with BRAF ( Vemurafenib ; from 10 to 10 , 000 nM ) MEK ( Trametinib ; from 4 to 2 , 000 nM ) inhibitors . shSCR - ( control ) shTINCR - WM902B were treated with increasing concentrations of drugs their viability assayed after 72 hours using CellTiter - Glo luminescent kit ( Promega ) . Error bars represent s . e . m . of three biological replicates . 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 of a panel of that are specifically associated to proliferative or invasive phenotype . Total lysates were extracted from shSCR - shTINCR - WM902B ( lane 1 2 ) or shSCR - shTINCR - MMC70 ( lane 3 4 ) 10 days after infection analyzed by immunoblot using 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 of TINCR expression ( left ) of MM13 MM2 PDX infected with lentiviral vectors expressing full - TINCR cDNA . PDX infected with empty vector ( Empty ) were used as control . TINCR relative to L32P housekeeping gene control ( mean ± sd ) from three biological replicates are shown . Statistical analyses were performed using Student t - ( * * * : 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 MM2 TINCR overexpressing ( TINCR ) compared to control ( Empty ) . Statistical analyses were performed using Student t - ( * * * : 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 proliferation was assayed 72 hours post infection in Empty TINCR MM13 MM2 PDX at indicated time points ( 1 - 5 days ) using CellTiter - Glo viability . 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 ) to Trametinib BRAF inhibitor Vemurafenib of TINCR MM2 ( BRAF - mutant PDX ) . Empty ( control ) TINCR expressing MM13 were treated with increasing concentrations of drug ( ranging from 5 to 5000 nM ) viability was assayed after 72 hours by CellTiter - Glo viability . Error bars represent s . e . m . of three biological replicates . 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 of expression of invasive proliferative phenotype markers EPHA2 , EGFR , AXL , PDGFRβ , SOX10 , MITF in TINCR - overexpressing MM13 MM2 . Total lysates were extracted from PDX 7 days after infection analyzed by immunoblot using indicated antibodies . Vinculin was used as loading control . This experiment is representative of three independent experiments showing comparable 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 TINCR cDNA expressing MM13 following intradermal injection into NSG mice . Four ( 200 , 000 / mouse ) per group were used in each experiment . growth ( mean ± sd ) was monitored weekly volume ( mean ± sd ) measured at 4 for tumors ( originated in site of injection , left panel ) at 8 for spontaneous metastases which colonized lymph nodes ( right panel ) . results of two independent experiments are shown . Statistical analyses of control TINCR - overexpressing groups from both experiments were performed using Student t - ( * * : p < 0 . 01 ) .

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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).


G Representative pictures of lymph node metastases derived from Empty TINCR expressing MM13 PDX are shown . Hematoxylin - eosin of sections of liver lung metastases derived from Empty TINCR MM13 PDX . Metastasis formation was evaluated 8 after transplantation into NSG mice . Pictures of representative lungs livers infiltrated with metastases are shown in inset . Scale bars : 200μm .

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G Representative pictures of lymph node metastases derived from Empty and TINCR expressing MM13 PDX cells are shown. Hematoxylin-eosin staining of sections of liver and lung metastases derived from Empty and TINCR MM13 PDX cells. Metastasis formation was evaluated 8 weeks after transplantation into NSG mice. Pictures of representative lungs and livers infiltrated with metastases are shown in the inset. Scale bars: 200μm.


A Western blot of integrated stress response activation in WM902B MMC70 . were harvested 96 h after shRNA infection western blot performed using indicated antibodies .

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A Western blot analysis of integrated stress response activation in WM902B and MMC70 cells. Cells were harvested 96 h after shRNA infection and western blot performed using the indicated antibodies.


B Representative comparison of translational efficiency ( TE ) in control ( shSCR ) shTINCR WM902B 72h post infection . genes with statistically significant TE up - downregulation ( FDR < 0 . 1 ) are highlighted in red blue respectively . ATF4 is circled among upregulated genes .

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B Representative comparison of translational efficiency (TE) in control (shSCR) and shTINCR WM902B cells 72h post infection. The genes with statistically significant TE up- and downregulation (FDR< 0.1) are highlighted in red and blue respectively. ATF4 is circled among the upregulated genes.


C representing top translationally upregulated down regulated genes in TINCR WM902B . plotted values are log2 fold TE change values in each biological replicate ( rep1 rep2 ) relative to control ( shSCR ) .

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C Heatmap representing top translationally upregulated and down regulated genes in TINCR WM902B cells. The plotted values are the log2 fold TE change values in each biological replicate (rep1 and rep2) relative to control (shSCR).


D Bar - graph representing ribosome protected fragments ( RPF ) normalized coverage ( mean ± sd ) mapping to ATF4 CDS in control ( shSCR ) TINCR ( shTINCR ) depleted WM902B from two biological replicates is shown .

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D Bar-graph representing ribosome protected fragments (RPF) normalized coverage (mean±sd) mapping to ATF4 CDS in control (shSCR) and TINCR (shTINCR) depleted WM902B cells from two biological replicates is shown.


E UCSC genome browser tracks of ribosome footprint data illustrating RPF density distribution across ATF4 mRNA in shSCR shTINCR WM902B .

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E UCSC genome browser tracks of ribosome footprint data illustrating RPF density distribution across ATF4 mRNA in shSCR and shTINCR WM902B cells.


B Q - PCR validation of TINCR ATF4 recovery in TINCR antisense pulldown . Enrichment relative to input ( mean ± sd ) from three independent pulldown experiments is shown . Statistical analyses were performed using Student t - ( * * : p < 0 . 01 , * * * : p < 0 . 001 ) .

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B Q-PCR validation of TINCR and ATF4 RNA recovery in TINCR antisense pulldown. Enrichment relative to input (mean±sd) from three independent pulldown experiments is shown. Statistical analyses were performed using Student t -test (**: p<0.01, ***: p<0.001).


C UCSC genome browser tracks of TINCR - RIA - seq sequencing reads illustrating coverage enrichment across ATF4 transcript in WM902B MMC70 .

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C UCSC genome browser tracks of TINCR-RIA-seq sequencing reads illustrating coverage and enrichment across ATF4 transcript in WM902B and MMC70 cells.


( A ) Remdesivir inhibition of SARS - CoV - 2 infections of Vero E6 . drugs , , were used at indicated concentrations . Viral level was determined by qRT - PCR 15 hours after inoculation of SARS - CoV - 2 ( Swedish isolate , 106 PFU ) .

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(A) Remdesivir inhibition of SARS-CoV-2 infections of Vero E6 cells. drugs, , were used at the indicated concentrations. Viral RNA level was determined by qRT-PCR 15 hours after inoculation of SARS-CoV-2 (Swedish isolate, 106 PFU).


( B ) hrsACE2 inhibition of SARS - CoV - 2 infections of Vero E6 . murine recombinant soluble ACE2 ( mrsACE2 , control treatment ) , were used at indicated concentrations . Viral level was determined by qRT - PCR 15 hours after inoculation of SARS - CoV - 2 ( Swedish isolate , 106 PFU ) .

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(B) hrsACE2 inhibition of SARS-CoV-2 infections of Vero E6 cells. murine recombinant soluble ACE2 (mrsACE2, control treatment), were used at the indicated concentrations. Viral RNA level was determined by qRT-PCR 15 hours after inoculation of SARS-CoV-2 (Swedish isolate, 106 PFU).


( B ) viability of isolated clones WT AN3 - 12 following 72h remdesivir treatment with indicated doses . Mean ± of 2 - 4 biological replicates is displayed .

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(B) Cell viability of isolated cell clones and WT AN3-12 cells following 72h remdesivir treatment with the indicated doses. Mean ±SEM of 2-4 biological replicates is displayed.


( A ) Treatment of SARS - CoV - 2 ( 106 PFU ) infected Vero - E6 with Human recombinant soluble ACE 2 ( hrsACE2 ) ( 200µg / ml ) remdesivir ( Remd . 4µM ) . Viral level was determined at 15 hours after inoculation .

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(A) Treatment of SARS-CoV-2 (106 PFU) infected Vero-E6 cells with Human recombinant soluble ACE 2 (hrsACE2) (200µg/ml) and remdesivir (Remd. 4µM). Viral RNA level was determined at 15 hours after virus inoculation.


( B ) Treatment of SARS - CoV - 2 ( 106 PFU ) infected human kidney organoids with hrsACE2 ( 200µg / ml ) / or remdesivir ( Remd . 4µM ) ) . Viral was determined by qRT - PCR 72 hours after inoculation of 106 PFU of SARS - CoV - 2 .

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(B) Treatment of SARS-CoV-2 (106 PFU) infected human kidney organoids with hrsACE2 (200µg/ml) and/or remdesivir (Remd.4µM)). Viral RNA was determined by qRT-PCR 72 hours after the inoculation of 106 PFU of SARS-CoV-2.


( C ) Treatment of SARS - CoV - 2 ( 106 PFU ) infected Vero - E6 with clinical doses of hrsACE2 ( 5 10µg / ml ) remdesivir ( Remd . 4µM ) ) .

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(C) Treatment of SARS-CoV-2 (106 PFU) infected Vero-E6 cells with clinical doses of hrsACE2 (5 and 10µg/ml) and remdesivir (Remd. 4µM)).


( D ) Treatment of SARS - CoV - 2 ( 106 PFU ) infected kidney organoids with hrsACE2 ( 10µg / ml ) remdesivir ( 4µM ) .

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(D) Treatment of SARS-CoV-2 (106 PFU) infected kidney organoids with hrsACE2 (10µg/ml) and remdesivir (4µM).


( E , F ) Progeny released from untreated Vero - E6 or Vero - E6 treated with clinical doses of hrsACE2 ( 5 10µg / ml ) remdesivir ( Remd . 4µM ) . Progeny was determined ( E ) 15 hours ( F ) 48 hours post - infection ( hpi ) .

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(E,F) Progeny virus released from untreated Vero-E6 cells or Vero-E6 cells treated with clinical doses of hrsACE2 (5 and 10µg/ml) and remdesivir (Remd. 4µM). Progeny was determined (E) 15 hours and (F) 48 hours post-infection (hpi).


( A , B ) A brightfield image showing zebrafish thyroid along with surrounding tissue . thyroid follicles reside in soft tissue surrounding ventral aorta , which extends from outflow tract of heart into gills towards basibranchial cartilage in lower jaw . thyroid follicular , or thyrocytes , are labeled in green in Tg ( tg : nls - mVenus - NTR ) transgenic ( B ' ) . Scale bars : 500

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(A , B) A brightfield image showing the zebrafish thyroid gland along with surrounding tissue. The thyroid follicles reside in the soft tissue surrounding the ventral aorta, which extends from the outflow tract of the heart into the gills towards the basibranchial cartilage in the lower jaw. The thyroid follicular cells, or thyrocytes, are labeled in green in the Tg(tg:nls-mVenus-NTR) transgenic line (B'). Scale bars: 500 µm


( C ) Maximum projection of 3D confocal stack obtained from dissected thyroid of Tg ( tg : nls - EGFP ) labeled with DAPI . ( D ) Confocal scan of a transverse section across dissected thyroid from Tg ( tg : nls - EGFP ) at 3 mpf . Sections were stained with DAPI to visualize surrounding thyroid follicles . ( E ) Boxplot depicting proportion of thyrocytes present in transverse sections obtained from three Tg ( tg : nls - EGFP ) at 3 mpf . Each dot represents a transverse section . Tukey boxplot marks 25th percentile , median 75th percentile with whiskers extending from smallest to largest values .

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(C) Maximum intensity projection of 3D confocal stack obtained from the dissected thyroid gland of Tg(tg:nls-EGFP) animal and labeled with DAPI. (D) Confocal scan of a transverse section across the dissected thyroid gland from Tg(tg:nls-EGFP) animal at 3 mpf. Sections were stained with DAPI to visualize cells surrounding thyroid follicles. (E) Boxplot depicting the proportion of thyrocytes present in transverse sections obtained from three Tg(tg:nls-EGFP) animals at 3 mpf. Each dot represents a transverse section. The Tukey boxplot marks the 25th percentile, median and 75th percentile with whiskers extending from smallest to largest values.


( F , G ) Representative FACS plot of single from Tg ( tg : nls - mVenus - T2A - NTR ) at 2 mpf ( F ) 8 mpf ( G ) . Calcein ( Pacific Blue ) labels live , while green ( FITC ) labels thyrocytes . values represent proportion of calcein + thyrocytes within total calcein + .

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(F , G) Representative FACS plot of single cells from Tg(tg:nls-mVenus-T2A-NTR) animals at 2 mpf (F) and 8 mpf (G). Calcein (Pacific Blue) labels live cells, while green fluorescence (FITC) labels thyrocytes. Percentage values represent proportion of calcein+ thyrocytes within total calcein+ cells.


( A ) A t - SNE plot displaying 6249 single - profiled in zebrafish thyroid atlas . colors represent clusters denoting a specific - type . ( B - D ) Cluster # 1 represents thyrocytes that express tg , slc5a5 ( NIS ) tpo . color scale represents normalized expression for each gene ranging from lowest ( grey ) to highest ( red ) .

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(A) A t-SNE plot displaying the 6249 single-cells profiled in the zebrafish thyroid gland atlas. The colors represent cell clusters denoting a specific cell-type. (B-D) Cluster #1 represents the thyrocytes that express tg, slc5a5 (NIS) and tpo. The color scale represents the normalized expression counts for each gene ranging from lowest (grey) to highest (red).


( A ) depicting five genes specifically expressed in each one of seven clusters of zebrafish thyroid atlas . color scale represents expression as a z - score ranging from - 2 ( purple ) to 2 ( yellow ) . NFE : Non - Follicular Epithelium .

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(A) Heatmap depicting five genes specifically expressed in each one of the seven clusters of the zebrafish thyroid gland atlas. The color scale represents expression as a z-score ranging from -2 (purple) to 2 (yellow). NFE: Non-Follicular Epithelium.


( B - G ) t - SNE plots overlaid with expression of a gene specific to each of cluster . endothelium cluster ( cluster # 2 ) is a mix of blood vessels lymphatic vessels while immune cluster ( cluster # 4 ) is a mix of macrophages , neutrophils lymphocytes color scale represents normalized expression for each gene ranging from lowest ( grey ) to highest ( blue ) .

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(B - G) t-SNE plots overlaid with the expression of a gene specific to each of the cluster. The endothelium cluster (cluster #2) is a mix of blood vessels and lymphatic vessels while the immune cell cluster (cluster #4) is a mix of macrophages, neutrophils and lymphocytes The color scale represents the normalized expression counts for each gene ranging from lowest (grey) to highest (blue).


( A - D ) show immunofluorescence labeling of thyroid from adult zebrafish . Transverse sections were utilized for imaging . organ was isolated from tissue - specific transgenic lines to allow marking of a particular - type adjacent to thyroid follicle . Blood vessels were marked using Tg ( kdrl : EGFP ) ( A ) , macrophages using Tg ( mpeg1 . 1 : mCherry ) ( B ) stroma using Tg ( col1a2 : mCherry ) ( C ) . Thyrocytes were labeled with pax2amKO2 expression in ( A ) Tg ( tg : nls - EGFP ) expression in ( B - C ) . ( D ) NFE was labeled using antibody against TP63 in sections of thyroid isolated from Tg ( tg : nls - EGFP ) . Gills are marked on their morphological appearance . DAPI labels nuclei . Scale bars : 10 µm ( A - B ) , 50 µm ( C - D ) .

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(A-D) Images show immunofluorescence labeling of thyroid gland from adult zebrafish. Transverse sections were utilized for imaging. The organ was isolated from tissue-specific transgenic lines to allow marking of a particular cell-type adjacent to the thyroid follicle. Blood vessels were marked using Tg(kdrl:EGFP) (A), macrophages using Tg(mpeg1.1:mCherry) (B) and stroma using Tg(col1a2:mCherry) (C). Thyrocytes were labeled with pax2amKO2 expression in (A) and Tg(tg:nls-EGFP) expression in (B - C). (D) NFE was labeled using antibody against TP63 in sections of the thyroid gland isolated from Tg(tg:nls-EGFP) animals. Gills are marked based on their morphological appearance. DAPI labels nuclei. Scale bars: 10 µm (A - B), 50 µm (C - D).


( C ) A dotplot depicting examples of paracrine autocrine signaling in thyroid . of dots represent of expressing gene in a particular cluster , while color scale represents average expression of gene in cluster after scaling .

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(C) A dotplot depicting examples of paracrine and autocrine signaling in the thyroid gland. The size of the dots represent percentage of cells expressing the gene in a particular cluster, while the color scale represents average expression of the gene in the cluster after scaling.


( A ) Dot plot depicting expression entropy on Y - axis against average gene expression on X - axis for thyrocyte population . Each dot depicts a gene , with red dots depicting genes that show statistically significant ( p - value < 0 . 05 ) difference in entropy from expected value . Expected value is represented by black regression . pax2a ctsba are marked on graph .

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(A) Dot plot depicting expression entropy on Y-axis against average gene expression on X-axis for the thyrocyte population. Each dot depicts a gene, with red dots depicting genes that show statistically significant (p-value < 0.05) difference in entropy from expected value. Expected value is represented by black regression line. pax2a and ctsba are marked on the graph.


( B ) t - SNE plot of thyrocyte cluster with expression of tg , pax2a ctsba . color scale represents normalized expression for each gene ranging from lowest ( yellow ) to highest ( red ) . ( B ' ) Histogram overlaid with density plots depicting distribution of normalized gene expression for tg , pax2a ctsba .

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(B) t-SNE plot of the thyrocyte cluster with expression of tg, pax2a and ctsba. The color scale represents the normalized expression counts for each gene ranging from lowest (yellow) to highest (red). (B') Histogram overlaid with density plots depicting the distribution of normalized gene expression for tg, pax2a and ctsba.


( B ) Whole mount immunofluorescence of 9 . 5hpf pax2amKO2 stained with anti - mKO2 antibody ( red ) anti - PAX2A antibody ( green ) . Anterior is to left , dorsal side is to top . Scale bar : 0 . 1 mm .

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(B) Whole mount immunofluorescence of 9.5hpf pax2amKO2 embryos stained with anti-mKO2 antibody (red) and anti-PAX2A antibody (green). Anterior is to the left, and dorsal side is to the top. Scale bar: 0.1 mm.


( C ) Whole mount immunofluorescence of 55 hpf pax2amKO2 ; Tg ( tg : nls - EGFP ) stained with PAX2A antibody ( PAX2A - Ab ) displays an overlap of mKO2 PAX2A - Ab signal . otic vesicle ( OV ) , mid - hindbrain barrier ( MHB ) , interneurons ( IN ) thyroid ( THY ) is labelled . dashed represents outline of zebrafish larvae . Scale bar : 100 µm . Anterior to right .

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(C) Whole mount immunofluorescence of 55 hpf pax2amKO2; Tg(tg:nls-EGFP) stained with PAX2A antibody (PAX2A-Ab) displays an overlap of mKO2 and PAX2A-Ab signal. The otic vesicle (OV), mid-hindbrain barrier (MHB), interneurons (IN) and thyroid gland (THY) is labelled. White dashed line represents the outline of the zebrafish larvae. Scale bar: 100 µm. Anterior to the right.


( D - F ) Confocal microscopy imaging of a sagittal section of a 55 hpf pax2amKO2 ( red ) ; Tg ( tg : nls - EGFP ) ( green ) immunostained with PAX2A antibody ( cyan ) showing co - localization of mKO2 pax2a in pronephros ( D ) , thyroid ( E ) mid - hindbrain barrier ( F ) . In thyroid , mKO2 ( red ) , PAX2A - Ab ( cyan ) thyrocyte - specific GFP ( green ) show co - localization . Scale bar : 50 µm .

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(D - F) Confocal microscopy imaging of a sagittal section of a 55 hpf pax2amKO2 (red); Tg(tg:nls-EGFP) (green) embryos immunostained with PAX2A antibody (cyan) showing co-localization of mKO2 and pax2a in the pronephros (D), thyroid gland (E) and mid-hindbrain barrier (F). In the thyroid gland, mKO2 (red), PAX2A-Ab (cyan) and thyrocyte-specific GFP (green) show co-localization. Scale bar: 50 µm.


( G , H ) Snapshots from live imaging of 55 hpf pax2amKO2 ; Tg ( tg : nls - EGFP ) injected with sgRNA targeting pax2a coding . anterior part of a representative control ( G ) is shown alongside a representative crispant ( H ) . Crispants display a strong reduction of mKO2 , as well as an absence of GFP signal suggesting absence of thyroid ( THY ) tissue . dashed represents outline of zebrafish larvae . Scale bar : 100 µm . Anterior to right .

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(G , H) Snapshots from live imaging of 55 hpf pax2amKO2; Tg(tg:nls-EGFP) embryos injected with sgRNA targeting pax2a coding sequence. The anterior part of a representative control embryo (G) is shown alongside a representative crispant (H). Crispants display a strong reduction of mKO2 fluorescence, as well as an absence of GFP signal suggesting absence of thyroid (THY) tissue. White dashed line represents the outline of the zebrafish larvae. Scale bar: 100 µm. Anterior to the right.


( A - D ) of 3 mpf thyroid from pax2amKO2 zebrafish shows heterogeneity in pax2a reporter expression . ( A ) Whole mount confocal imaging of a 3 mpf pax2amKO2 thyroid labelled with mKO2 ( red ) , E - cadherin ( cyan , not shown in ' A ' for clarity reasons ) DAPI ( blue ) for localisation . ( B - D ) Optical sections of three follicles , with mKO2 - Low labelled with arrows . E - cadherin ( B ' - D ' ) DAPI ( B ' ' - D ' ' ) shows that absence of mKO2 signal does not correspond to an absence of . Anterior to bottom of pictures .

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(A-D) Analysis of 3 mpf thyroid gland from pax2amKO2 zebrafish shows heterogeneity in pax2a reporter expression. (A) Whole mount confocal imaging of a 3 mpf pax2amKO2 thyroid labelled with mKO2 (red), E-cadherin (cyan, not shown in 'A' for clarity reasons) and DAPI (dark blue) for nuclear localisation. (B - D) Optical sections of three follicles, with mKO2-Low cells labelled with arrows. E-cadherin (B' - D') and DAPI (B'' - D'') staining shows that absence of mKO2 signal does not correspond to an absence of cells. Anterior to the bottom of the pictures.


( E , F ) ( E ) Confocal image of thyroid section from Tg ( tg : nls - EGFP ) at 4 mpf stained with PAX2A antibody DAPI . dotted region is displayed at high magnification in ( F ) . Arrows marks thyrocytes displaying low PAX2A . Notably , PAX2A - Low thyrocytes display tg - driven EGFP expression , demonstrating their differentiated status .

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(E,F) (E) Confocal image of thyroid gland section from Tg(tg:nls-EGFP) at 4 mpf stained with PAX2A antibody and DAPI. The dotted region is displayed at high magnification in (F). Arrows marks thyrocytes displaying low PAX2A staining. Notably, PAX2A-Low thyrocytes display tg-driven EGFP expression, demonstrating their differentiated status.


( A - C ) from thyroid of 5 mpf Tg ( tg : nls - GFP ) ; pax2amKO2 were labelled with calcein ( live marker ) analysed using FACS . ( A ) A FACS plot showing calcein on Y - axis GFP on X - axis . box encompassing GFP + represents thyrocyte population , which was gated for further . ( B ) Histogram showing distribution of GFP in thyrocytes . ( C ) Histogram showing distribution of mKO2 in thyrocytes . Thyrocytes were selected by gating for GFP + population . Horizontal lines indicate mKO2 - Low mKO2 - High expression level , with values representing proportion of thyrocytes with mKO2 - Low mKO2 - High expression .

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(A - C) Cells from the thyroid gland of 5 mpf Tg(tg:nls-GFP); pax2amKO2 animals were labelled with calcein (live cell marker) and analysed using FACS. (A) A FACS plot showing calcein on Y-axis and GFP on X-axis. The box encompassing the GFP+ cells represents the thyrocyte population, which was gated for further analysis. (B) Histogram showing the distribution of GFP intensity in thyrocytes. (C) Histogram showing the distribution of mKO2 intensity in thyrocytes. Thyrocytes were selected by gating for GFP+ population. Horizontal lines indicate the mKO2-Low and mKO2-High expression level, with percentage values representing proportion of thyrocytes with mKO2-Low and mKO2-High expression.