F , Effect of chemogenetic inhibition ( F ) of dopaminergicA11 - SDH neurons on DCP - induced scratching . * P < 0 . 05 . Two - way ANOVA followed by Bonferroni ' s test . n = 8 mice / group . Data information : Bars represent mean values . Error bars indicate the SEM .

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F, Effect of chemogenetic inhibition (F) of dopaminergicA11-SDH neurons on DCP-induced scratching. * P < 0.05. Two-way ANOVA followed by Bonferroni's test. n = 8 mice/group. Data information: Bars represent mean values. Error bars indicate the SEM.


G Effect of chemogenetic activation ( G ) of dopaminergicA11 - SDH neurons on DCP - induced scratching . * P < 0 . 05 . Two - way ANOVA followed by Bonferroni ' s test . n = 8 mice / group . Data information : Bars represent mean values . Error bars indicate the SEM .

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G Effect of chemogenetic activation (G) of dopaminergicA11-SDH neurons on DCP-induced scratching. * P < 0.05. Two-way ANOVA followed by Bonferroni's test. n = 8 mice/group. Data information: Bars represent mean values. Error bars indicate the SEM.


A Effect of SCH23390 and sulpiride on scratching behaviors induced by C48 / 80 ( left ) and CQ ( right ) . * * * P < 0 . 001 . One - way ANOVA followed by Bonferroni ' s test . n = 7 - 8 mice / group .

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A Effect of SCH23390 and sulpiride on scratching behaviors induced by C48/80 (left) and CQ (right). *** P < 0.001. One-way ANOVA followed by Bonferroni's test. n = 7-8 mice/group.


C Effect of SCH23390 and sulpiride on DCP - induced scratching behaviors . * P < 0 . 05 . One - way ANOVA followed by Bonferroni ' s test . n = 8 mice / group .

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C Effect of SCH23390 and sulpiride on DCP-induced scratching behaviors. * P < 0.05. One-way ANOVA followed by Bonferroni's test. n = 8 mice/group.


D The Drd1 mRNA level in the spinal cord after DCP or vehicle treatment . * P < 0 . 05 . Student ' s t - test . n = 8 mice / group .

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D The Drd1 mRNA level in the spinal cord after DCP or vehicle treatment. * P < 0.05. Student's t-test. n = 8 mice/group.


G Statistical curve shows the firing frequency evoked by step current injection into DRD1 + neurons that incubated with vehicle or SKF38393 . * * * P < 0 . 001 . Two - way ANOVA . n = 8 neurons / group .

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G Statistical curve shows the firing frequency evoked by step current injection into DRD1+ neurons that incubated with vehicle or SKF38393. *** P < 0.001. Two-way ANOVA. n = 8 neurons/group.


I Statistical shows the resting membrane potential in DRD1 + neurons incubated with vehicle or SKF38393 . * P < 0 . 05 . Paired Student ' s t - test . n = 8 neurons / group .

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I Statistical shows the resting membrane potential in DRD1+ neurons incubated with vehicle or SKF38393. * P < 0.05. Paired Student's t-test. n = 8 neurons/group.


K Representative traces from one spinal DRD1 + neuron show that blue light stimuli did not induce spontaneous firing ( holding potential , - 65 mV , lower panel ) but increased the existed firing of DRD1 + neuron ( holding potential , - 60 mV , upper panel ) . Arrows indicate blue light stimuli ( 2 - 3 mW / cm2 , 5 pulses , 2 ms duration , 20 Hz , repeated every 1 s ) . shows the change of firing frequency from individual DRD1 + neurons after blue light stimuli . * * P < 0 . 01 . Paired Student ' s t - test . n = 8 neurons / group . Data information : Bars represent mean values . Error bars indicate the SEM .

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K Representative traces from one spinal DRD1+ neuron show that blue light stimuli did not induce spontaneous firing (holding potential, -65 mV, lower panel) but increased the existed firing of DRD1+ neuron (holding potential, -60 mV, upper panel). Arrows indicate blue light stimuli (2-3 mW/cm2, 5 pulses, 2 ms duration, 20 Hz, repeated every 1 s). shows the change of firing frequency from individual DRD1+ neurons after blue light stimuli. ** P < 0.01. Paired Student's t-test. n = 8 neurons/group. Data information: Bars represent mean values. Error bars indicate the SEM.


G Effect of chemogenetic activation of spinal DRD1 + neurons on spontaneous itch behaviors . * * * P < 0 . 001 . Student ' s t - test . n = 8 mice / group . Data information : Bars represent mean values . Error bars indicate the SEM .

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G Effect of chemogenetic activation of spinal DRD1+ neurons on spontaneous itch behaviors. *** P < 0.001. Student's t-test. n = 8 mice/group. Data information: Bars represent mean values. Error bars indicate the SEM.


I Effect of chemogenetic inhibition ( left ) or activation ( right ) of spinal DRD1 + neurons on the DCP - induced chronic itch behaviors . * P < 0 . 05 , * * P < 0 . 01 . Two - way ANOVA followed by Bonferroni ' s test . n = 8 mice / group . Data information : Bars represent mean values . Error bars indicate the SEM .

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I Effect of chemogenetic inhibition (left) or activation (right) of spinal DRD1+ neurons on the DCP-induced chronic itch behaviors. * P < 0.05, ** P < 0.01. Two-way ANOVA followed by Bonferroni's test. n = 8 mice/group. Data information: Bars represent mean values. Error bars indicate the SEM.


F - H Representative images show non - overlap of tdTomato with EGFP in the SDH of Drd1 - tdTomato ; Drd2 - EGFP mice . Scale bar , 100 μm .

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F-H Representative images show non-overlap of tdTomato with EGFP in the SDH of Drd1-tdTomato;Drd2-EGFP mice. Scale bar, 100 μm.


I Representative image shows the co - localization of NeuN with tdTomato . Scale bar , 50 μm . Inset scale bar , 25 μm .

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I Representative image shows the co-localization of NeuN with tdTomato. Scale bar, 50 μm. Inset scale bar, 25 μm.


J - M Representative images show the co - localization of NK1R ( J ) , CGRP ( K ) , IB4 ( L ) , and PKCγ ( M ) with tdTomato . Scale bar , 100 μm .

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J-M Representative images show the co-localization of NK1R (J), CGRP (K), IB4 (L), and PKCγ (M) with tdTomato. Scale bar, 100 μm.


N Co - localization of tdTomato fluorescence with vGluT2 - probe . Scale bar , 50 μm . O FISH double - staining of Drd1 - probe with vGluT2 - probe . White arrows and arrowheads show double - labeled neurons and vGluT2 single - labeled neurons , respectively . Scale bar , 10 μm . P Staining of PAX2 in Drd1 - tdTomato mice . Scale bar , 50 μm . Q Fluorescence of tdTomato and EGFP in the SDH of Drd1 - tdTomato ; Gad67 - EGFP mice . Scale bar , 50 μm .

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N Co-localization of tdTomato fluorescence with vGluT2-probe. Scale bar, 50 μm. O FISH double-staining of Drd1-probe with vGluT2-probe. White arrows and arrowheads show double-labeled neurons and vGluT2 single-labeled neurons, respectively. Scale bar, 10 μm. P Staining of PAX2 in Drd1-tdTomato mice. Scale bar, 50 μm. Q Fluorescence of tdTomato and EGFP in the SDH of Drd1-tdTomato;Gad67-EGFP mice. Scale bar, 50 μm.


C Representative images show the reconstitution of pre - and post - mGRASP to form an intact GFP ( green ) , which mapped synaptic connectivity between spinal DRD1 + neurons ( red , as presynaptic constructs ) and GRPR + neurons ( upper panels ) . High - magnification images in the white frames were shown in lower panels . Scale bar , 20 μm in upper panel and 10 μm in lower panel .

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C Representative images show the reconstitution of pre- and post-mGRASP to form an intact GFP (green), which mapped synaptic connectivity between spinal DRD1+ neurons (red, as presynaptic constructs) and GRPR+ neurons (upper panels). High-magnification images in the white frames were shown in lower panels. Scale bar, 20 μm in upper panel and 10 μm in lower panel.


G Representative traces of the whole - cell voltage - clamp show the blue light ( 470 nm , 2 ms ) - evoked EPSCs recorded in a GRPR + neuron before ( black ) and after 20 μM DNQX perfusion ( gray ) . H Representative traces show the effect of TTX alone ( purple ) and the combined presence of 4 - AP ( gray ) superfusion on the blue light - evoked EPSCs ( black ) recorded in a GRPR + neuron .

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G Representative traces of the whole-cell voltage-clamp show the blue light (470 nm, 2 ms)-evoked EPSCs recorded in a GRPR+ neuron before (black) and after 20 μM DNQX perfusion (gray). H Representative traces show the effect of TTX alone (purple) and the combined presence of 4-AP (gray) superfusion on the blue light-evoked EPSCs (black) recorded in a GRPR+ neuron.


I Representative traces show the effect of TTX + 4 - AP ( gray ) on the blue light ( 470 nm , 2 ms ) - evoked EPSCs ( black ) recorded in a GRPR + neuron .

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I Representative traces show the effect of TTX + 4-AP (gray) on the blue light (470 nm, 2 ms)-evoked EPSCs (black) recorded in a GRPR+ neuron.


D Single - cell RT - PCR shows the expression of Drd1 , Grp , NeuN , and Gapdh mRNA in tdTomato + neurons . N , negative control . * indicate Drd1 + Grp + neurons .

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D Single-cell RT-PCR shows the expression of Drd1, Grp, NeuN, and Gapdh mRNA in tdTomato+ neurons. N, negative control. * indicate Drd1+Grp+ neurons.


I Effect of DNQX , RC - 3095 , or vehicle on the spontaneous scratching of mice with spinal DRD1 + neurons expressing mCherry or hM3Dq and activated by CNO . * * P < 0 . 01 , * * * P < 0 . 001 . One - way ANOVA followed by Bonferroni ' s test . n = 8 mice / group . Data information : Bars represent mean values . Error bars indicate the SEM .

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I Effect of DNQX, RC-3095, or vehicle on the spontaneous scratching of mice with spinal DRD1+ neurons expressing mCherry or hM3Dq and activated by CNO. ** P < 0.01, *** P < 0.001. One-way ANOVA followed by Bonferroni's test. n = 8 mice/group. Data information: Bars represent mean values. Error bars indicate the SEM.


J Effect of DNQX , RC - 3095 , or vehicle on the scratching behaviors of mice with spinal DRD1 + neurons expressing mCherry or hM3Dq and activated by CNO in C48 / 80 model ( left ) or CQ model ( right ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . One - way ANOVA followed by Bonferroni ' s test . n = 6 mice / group . Data information : Bars represent mean values . Error bars indicate the SEM .

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J Effect of DNQX, RC-3095, or vehicle on the scratching behaviors of mice with spinal DRD1+ neurons expressing mCherry or hM3Dq and activated by CNO in C48/80 model (left) or CQ model (right). * P < 0.05, ** P < 0.01, *** P < 0.001. One-way ANOVA followed by Bonferroni's test. n = 6 mice/group. Data information: Bars represent mean values. Error bars indicate the SEM.


( C - G ' ' ) Lateral images of the cranial vessels in lyve1b : Kaede transgenic embryos with the CCV photoconverted at 36 hpf ( C ) and followed through to 48 hpf ( D ) and 60 hpf ( E ) , while in a separate larva the PHS has been photoconverted at 36 hpf ( F ) and followed to 48 hpf ( G ) . Unconverted vessels are shown in green ( C - G ) , while photoconverted vessels are shown in red ( C ' - G ' ) . The extent of lymphangioblast contribution from each source is further clarified by false colouring the overlap between red and green fluorescence ( purple ) , with the FLS demarcated ( dotted line ) from the adjacent primary veins ( C ' ' - G ' ' ) .

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(C-G'') Lateral images of the cranial vessels in lyve1b:Kaede transgenic embryos with the CCV photoconverted at 36 hpf (C) and followed through to 48 hpf (D) and 60 hpf (E), while in a separate larva the PHS has been photoconverted at 36 hpf (F) and followed to 48 hpf (G). Unconverted vessels are shown in green (C-G), while photoconverted vessels are shown in red (C'-G'). The extent of lymphangioblast contribution from each source is further clarified by false colouring the overlap between red and green fluorescence (purple), with the FLS demarcated (dotted line) from the adjacent primary veins (C''-G'').


( A - D ' ' ) Ventrolateral ( A , C , D ) and lateral ( B ) images of the cranial vessels in lyve1b : Kaede transgenic embryos with the dorsal ( A ) and ventral ( C ) portions of the VA - L photoconverted at 54 hpf and followed through to 72 hpf ( B ) and 78 hpf ( D ) . Unconverted vessels are shown in green ( A - D ) , while photoconverted vessels are shown in red ( A ' - D ' ) . The extent of lymphatic and arterial contribution from the VA - L to the LFL ( B ' ' ) or HA ( D ' ' ) is further clarified by false colouring the overlap between red and green fluorescence ( purple ) , with the FLS ( A ' ' , C ' ' ) and LFL ( B ' ' , D ' ' ) demarcated ( dotted line ) from the adjacent vessels . Schematic included for anatomical reference . Note , for all images , the overlap of red and green fluorescence in the eye ( labelled E ) is due to a combination of natural pigmentation and auto - fluorescence ; and is not indicative of photoconverted cells ( C ' ' , white arrowheads ) .

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(A-D'') Ventrolateral (A, C, D) and lateral (B) images of the cranial vessels in lyve1b:Kaede transgenic embryos with the dorsal (A) and ventral (C) portions of the VA-L photoconverted at 54 hpf and followed through to 72 hpf (B) and 78 hpf (D). Unconverted vessels are shown in green (A-D), while photoconverted vessels are shown in red (A'-D'). The extent of lymphatic and arterial contribution from the VA-L to the LFL (B'') or HA (D'') is further clarified by false colouring the overlap between red and green fluorescence (purple), with the FLS (A'', C'') and LFL (B'', D'') demarcated (dotted line) from the adjacent vessels. Schematic included for anatomical reference. Note, for all images, the overlap of red and green fluorescence in the eye (labelled E) is due to a combination of natural pigmentation and auto-fluorescence; and is not indicative of photoconverted cells (C'', white arrowheads).


Lateral images of the CCV fixed at 36 hpf ( A ) and 42 hpf ( B ) in lyve1b : EGFP fish that have been fluorescently immunostained with anti - PROX1 ( green ) and anti - GFP ( red ) .

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Lateral images of the CCV fixed at 36 hpf (A) and 42 hpf (B) in lyve1b:EGFP fish that have been fluorescently immunostained with anti-PROX1 (green) and anti-GFP (red).


Lateral images of the PHS at 36 hpf ( C ) , 42 hpf ( D ) and 48 hpf ( E ) in lyve1b : EGFP fish that have been fluorescently immunostained with anti - PROX1 ( green ) and anti - GFP ( red ) . PHS Prox1 staining shown alone for all time points ( C ' - E ' ) with the FLS demarcated ( dotted line ) from the adjacent vessels , and the positions of the PHS - LPP and PHS - LPA indicated .

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Lateral images of the PHS at 36 hpf (C), 42 hpf (D) and 48 hpf (E) in lyve1b:EGFP fish that have been fluorescently immunostained with anti-PROX1 (green) and anti-GFP (red). PHS Prox1 staining shown alone for all time points (C'-E') with the FLS demarcated (dotted line) from the adjacent vessels, and the positions of the PHS-LPP and PHS-LPA indicated.


Lateral images of the VA - L at 36 hpf ( F ) , 42 hpf ( G ) and 48 hpf ( H ) in lyve1b : EGFP fish that have been fluorescently immunostained with anti - PROX1 ( green ) and anti - GFP ( red ) . Note the Prox1 - postive ( green ) , GFP - negative structure near to the VA - L is a prox1 - expressing neuromast ( NM ) .

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Lateral images of the VA-L at 36 hpf (F), 42 hpf (G) and 48 hpf (H) in lyve1b:EGFP fish that have been fluorescently immunostained with anti-PROX1 (green) and anti-GFP (red). Note the Prox1-postive (green), GFP-negative structure near to the VA-L is a prox1-expressing neuromast (NM).


( A - H ) Lateral images of the PHS fixed at 36 hpf ( A - B , E - F ) and 42 hpf ( C - D , G - H ) in lyve1b : EGFP fish that have been fluorescently immunostained with anti - PROX1 ( green ) and anti - GFP ( red ) . These show a decrease in the number of Prox1 - positive PHS ECs in the ccbe1 morpholino - injected ( B , D ) and sFLT4 mRNA - injected embryos ( F , H ) compared with control morpholino - injected ( A , C ) and uninjected embryos ( E , G ) . At 42 hpf , ccbe1 morpholino - injected embryos ( D ) and sFLT4 mRNA - injected embryos ( H ) lack the PHS - LP domains present in the control embryos ( C , G ; large arrow heads ) . ( I - J ) Quantitation of the percentage of PHS ECs that are lymphatic progenitors relative to the total number of PHS ECs in control morpholino - injected embryos at 36 hpf ( n = 27 ) and 42 hpf ( n = 17 ) and ccbe1 morpholino - injected embryos at 36 hpf ( n = 36 ) and 42 hpf ( n = 12 ) ( I ) or in uninjected embryos at 36 hpf ( n = 27 ) and 42 hpf ( n = 21 ) and sFLT4 mRNA - injected embryos at 36 hpf ( n = 26 ) and 42 hpf ( n = 17 ) ( J ) . ( K ) Quantitation of the total number of PHS ECs in control morpholino - injected embryos at 36 hpf ( n = 27 ) and 42 hpf ( n = 17 ) and ccbe1 morpholino - injected embryos at 36 hpf ( n = 36 ) and 42 hpf ( n = 12 ) or in uninjected embryos at 36 hpf ( n = 27 ) and 42 hpf ( n = 21 ) and sFLT4 mRNA - injected embryos at 36 hpf ( n = 26 ) and 42 hpf ( n = 17 )

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(A-H) Lateral images of the PHS fixed at 36 hpf (A-B, E-F) and 42 hpf (C-D, G-H) in lyve1b:EGFP fish that have been fluorescently immunostained with anti-PROX1 (green) and anti-GFP (red). These show a decrease in the number of Prox1-positive PHS ECs in the ccbe1 morpholino-injected (B, D) and sFLT4 mRNA-injected embryos (F, H) compared with control morpholino-injected (A, C) and uninjected embryos (E, G). At 42 hpf, ccbe1 morpholino-injected embryos (D) and sFLT4 mRNA-injected embryos (H) lack the PHS-LP domains present in the control embryos (C, G; large arrow heads). (I-J) Quantitation of the percentage of PHS ECs that are lymphatic progenitors relative to the total number of PHS ECs in control morpholino-injected embryos at 36 hpf (n = 27) and 42 hpf (n = 17) and ccbe1 morpholino-injected embryos at 36 hpf (n = 36) and 42 hpf (n = 12) (I) or in uninjected embryos at 36 hpf (n = 27) and 42 hpf (n = 21) and sFLT4 mRNA-injected embryos at 36 hpf (n = 26) and 42 hpf (n = 17) (J). (K) Quantitation of the total number of PHS ECs in control morpholino-injected embryos at 36 hpf (n = 27) and 42 hpf (n = 17) and ccbe1 morpholino-injected embryos at 36 hpf (n = 36) and 42 hpf (n = 12) or in uninjected embryos at 36 hpf (n = 27) and 42 hpf (n = 21) and sFLT4 mRNA-injected embryos at 36 hpf (n = 26) and 42 hpf (n = 17)


( L - O ) Lateral images of the FLS ( L , N ; dotted line ) or dorsolateral CCV region from where the FLS normally sprouts ( M , O ; dotted line ) fixed at 36 hpf in lyve1b : EGFP embryos that have been fluorescently immunostained with anti - PROX1 ( green ) and anti - GFP ( red ) . These show no change in the number of Prox1 - positive lymphatic progenitors in the ccbe1 morpholino - injected ( M ) and sFLT4 mRNA - injected ( O ) embryos compared with control morpholino - injected ( L ) and uninjected ( N ) embryos . ( P ) Quantitation of the percentage of dorsolateral CCV ECs that are lymphatic progenitors relative to the total number of dorsolateral CCV ECs in control morpholino - injected ( n = 26 ) , ccbe1 morpholino - injected ( n = 27 ) , uninjected ( n = 28 ) and sFLT4 mRNA - injected embryos ( n = 32 ) . ( Q ) Quantitation of the total number of dorsolateral region CCV ECs in 36 hpf embryos that were uninjected or injected with control morpholino ( n = 26 ) , ccbe1 morpholino ( n = 27 ) , or sFLT4 mRNA ( n = 32 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O

(L-O) Lateral images of the FLS (L, N; dotted line) or dorsolateral CCV region from where the FLS normally sprouts (M, O; dotted line) fixed at 36 hpf in lyve1b:EGFP embryos that have been fluorescently immunostained with anti-PROX1 (green) and anti-GFP (red). These show no change in the number of Prox1-positive lymphatic progenitors in the ccbe1 morpholino-injected (M) and sFLT4 mRNA-injected (O) embryos compared with control morpholino-injected (L) and uninjected (N) embryos. (P) Quantitation of the percentage of dorsolateral CCV ECs that are lymphatic progenitors relative to the total number of dorsolateral CCV ECs in control morpholino-injected (n = 26), ccbe1 morpholino-injected (n = 27), uninjected (n = 28) and sFLT4 mRNA-injected embryos (n = 32). (Q) Quantitation of the total number of dorsolateral region CCV ECs in 36 hpf embryos that were uninjected or injected with control morpholino (n = 26), ccbe1 morpholino (n = 27), or sFLT4 mRNA (n = 32).


( R - U ) Dorsolateral images of the VA - L fixed at 54 hpf in lyve1b : EGFP larva that have been fluorescently immunostained with anti - PROX1 ( green ) and anti - GFP ( red ) . These show that lymphatic progenitor formation still occurs within the VA - L in ccbe1 morpholino - injected ( S ) and sFLT4 mRNA - injected embryos ( U ) , with Prox1 expression resembling that of controls ( R , T ) .

O O O O O 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-MEASURED_VAR O 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-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O

(R-U) Dorsolateral images of the VA-L fixed at 54 hpf in lyve1b:EGFP larva that have been fluorescently immunostained with anti-PROX1 (green) and anti-GFP (red). These show that lymphatic progenitor formation still occurs within the VA-L in ccbe1 morpholino-injected (S) and sFLT4 mRNA-injected embryos (U), with Prox1 expression resembling that of controls (R, T).


( A - B ) Still images from Movie EV3 of early facial lymphatic development in a lyve1b : EGFP ( A ) , kdrl : nlsmCherry ( B ) compound transgenic from 42 - 51 . 5 hpf , with the CCV - L - derived leading tip cell ( green asterisk ) , the PHS - LP domains ( orange and yellow asterisk ) and the distal tip of the VA - L ( purple asterisk ) highlighted ( A ) . Between 42 and 46 hpf , the PHS - LPP ( orange outline ) cells divide ( dark blue parent cells , light blue daughter cells ) before sprouting as lymphangioblasts from the PHS and fusing with the tip ( pink cells ) of the CCV - derived FLS ( green outline ) at 48 hpf ( B ) . Cells ( red ) within the VA - L ( purple outline ) can also be seen migrating towards the now PHS - L - derived FLS tip by 48 hpf . The PHS - LPA is also shown ( yellow outline ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Still images from Movie EV3 of early facial lymphatic development in a lyve1b:EGFP (A), kdrl:nlsmCherry (B) compound transgenic from 42-51.5 hpf, with the CCV-L-derived leading tip cell (green asterisk), the PHS-LP domains (orange and yellow asterisk) and the distal tip of the VA-L (purple asterisk) highlighted (A). Between 42 and 46 hpf, the PHS-LPP (orange outline) cells divide (dark blue parent cells, light blue daughter cells) before sprouting as lymphangioblasts from the PHS and fusing with the tip (pink cells) of the CCV-derived FLS (green outline) at 48 hpf (B). Cells (red) within the VA-L (purple outline) can also be seen migrating towards the now PHS-L-derived FLS tip by 48 hpf. The PHS-LPA is also shown (yellow outline).


( C ) Quantitation of leading lymphangioblast velocity ( µm / min ) before and after fusion with another lymphangioblast ( CCV - L - derived FLS tip ( pink cells ) to PHS - L ( B , blue cells ; n = 5 / 5 ) and PHS - L - derived FLS tip ( blue cells ) to VA - L ( F , red cells ; n = 5 / 5 ) ) or by the VA - L to the PHS - L - derived FLS tip ( B , n = 5 / 5 ) .

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

(C) Quantitation of leading lymphangioblast velocity (µm/min) before and after fusion with another lymphangioblast (CCV-L-derived FLS tip (pink cells) to PHS-L (B, blue cells; n = 5/5) and PHS-L-derived FLS tip (blue cells) to VA-L (F, red cells; n = 5/5)) or by the VA-L to the PHS-L-derived FLS tip (B, n = 5/5).


( D ) Cell migration tracks where each coloured track depicts the distance ( µm ) travelled over 3 hours by the leading tip cell in a separate animal before and after fusion with the CCV - L - derived FLS tip ( pink cells ) and the PHS - Ls ( B , blue cells ; n = 5 / 5 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cell migration tracks where each coloured track depicts the distance (µm) travelled over 3 hours by the leading tip cell in a separate animal before and after fusion with the CCV-L-derived FLS tip (pink cells) and the PHS-Ls (B, blue cells; n = 5/5).


( E - F ) Still images from Movie EV4 of facial lymphatic development in a lyve1b : EGFP kdrl : nlsmCherry compound transgenic from 48 - 62 hpf showing that after the CCV - derived FLS fuses to the PHS - L , these take over as the leading tip cells ( orange asterisks ) , with one PHS - L migrating anteriorly to fuse with PHS - LPA ( yellow asterisk ) , and others migrating ventrally to fuse with the VA - L ( purple asterisk ) After the PHS - derived portion ( orange outline ) of the FLS ( green outline ) fuses to the VA - L ( purple outline ) , migration of the entire facial lymphatic pauses to allow for lymphangioblast proliferation ( dark blue and red parent cells , light blue and pink daughter cells ) before resuming migration anteriorly along the ventral base of the

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

(E-F) Still images from Movie EV4 of facial lymphatic development in a lyve1b:EGFP kdrl:nlsmCherry compound transgenic from 48-62 hpf showing that after the CCV-derived FLS fuses to the PHS-L, these take over as the leading tip cells (orange asterisks), with one PHS-L migrating anteriorly to fuse with PHS-LPA (yellow asterisk), and others migrating ventrally to fuse with the VA-L (purple asterisk) After the PHS-derived portion (orange outline) of the FLS (green outline) fuses to the VA-L (purple outline), migration of the entire facial lymphatic pauses to allow for lymphangioblast proliferation (dark blue and red parent cells, light blue and pink daughter cells) before resuming migration anteriorly along the ventral base of the eye


( G - H ) Cell migration tracks where each coloured track depicts the distance ( µm ) travelled over 3 . 5 hours by the leading tip cell in a separate animal before and after fusion with another lymphangioblast ( PHS - L - derived FLS tip ( blue cells ) to VA - L ( F , red cells ; n = 5 / 5 ) ) or by the VA - L to the PHS - L - derived FLS tip ( F , n = 5 / 5 ) .

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

(G-H) Cell migration tracks where each coloured track depicts the distance (µm) travelled over 3.5 hours by the leading tip cell in a separate animal before and after fusion with another lymphangioblast (PHS-L-derived FLS tip (blue cells) to VA-L (F, red cells; n = 5/5)) or by the VA-L to the PHS-L-derived FLS tip (F, n = 5/5).


( A - B ' ) Lateral ( A ) and ventrolateral ( B , B ' ) images of the cranial vessels in kdrl : nlsKaede ; kdrl : EGFP embryos , with unconverted tissue shown in green ( A - B ) while photoconverted tissue is shown in red ( B ' ) . The early kdrl - positive VA - L was photoconverted in 34 hpf embryos ( A ) , and followed through to 54 hpf ( B , B ' ) . B ' is a higher magnification image of the box in B and is accompanied by a red channel only ( traced photoconverted cells ) image with the VA - L ( dotted line ) demarcated from the adjacent VA . The VA - L photoconverted cells are indicated by arrowheads .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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') Lateral (A) and ventrolateral (B, B') images of the cranial vessels in kdrl:nlsKaede;kdrl:EGFP embryos, with unconverted tissue shown in green (A-B) while photoconverted tissue is shown in red (B'). The early kdrl-positive VA-L was photoconverted in 34 hpf embryos (A), and followed through to 54 hpf (B, B'). B' is a higher magnification image of the box in B and is accompanied by a red channel only (traced photoconverted cells) image with the VA-L (dotted line) demarcated from the adjacent VA. The VA-L photoconverted cells are indicated by arrowheads.


( C ) Still images from Movie EV5 of VA - L development in a kdrl : EGFP ; kdrl : nlsmCherry compound transgenic from 31 - 35 . 5 hpf . This movie shows that the VA - L begins expressing kdrl just after 32 hpf , in a manner that is isolated and independent of any neighbouring kdrl - positive tissue or vessel . Putative kdrl - positive primitive myeloid cells ( light blue asterisks ) can also be seen migrating along the surface of the yolk in the immediate vicinity of the VA and VA - L . ( D ) Still images from Movie EV6 depicting a 3D reconstruction of the 35 . 5 hpf timepoint from Movie EV5 . A dorsal view that scrolls through the Y - stack of the VA - L is shown , which illustrates that the VA - L is closely associated with , but not connected to the VA .

O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Still images from Movie EV5 of VA-L development in a kdrl:EGFP;kdrl:nlsmCherry compound transgenic from 31-35.5 hpf. This movie shows that the VA-L begins expressing kdrl just after 32 hpf, in a manner that is isolated and independent of any neighbouring kdrl-positive tissue or vessel. Putative kdrl-positive primitive myeloid cells (light blue asterisks) can also be seen migrating along the surface of the yolk in the immediate vicinity of the VA and VA-L. (D) Still images from Movie EV6 depicting a 3D reconstruction of the 35.5 hpf timepoint from Movie EV5. A dorsal view that scrolls through the Y-stack of the VA-L is shown, which illustrates that the VA-L is closely associated with, but not connected to the VA.


( E - E ' ) Still images from Movie EV7 of VA - L development in an etv2 : EGFP ( E ) , kdrl : nlsmCherry ( E ' ) compound transgenic from 28 - 35 . 2 hpf . This movie shows that the VA - L begins expressing etv2 ( E ) at 29 hpf and is independent of any neighbouring vessel . kdrl expression ( red arrowheads ) occurs 4 hours later in the VA - L ( demarcated by the green dotted line ) ( E ' ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E-E') Still images from Movie EV7 of VA-L development in an etv2:EGFP (E), kdrl:nlsmCherry (E') compound transgenic from 28-35.2 hpf. This movie shows that the VA-L begins expressing etv2 (E) at 29 hpf and is independent of any neighbouring vessel. kdrl expression (red arrowheads) occurs 4 hours later in the VA-L (demarcated by the green dotted line) (E').


( F - G ' ) Lateral images of the cranial vessels in etv2 : Kaede embryos , with unconverted tissue shown in green ( F - G ) while photoconverted tissue is shown in red ( G ' ) . The earliest visible form of the VA - L was photoconverted at 29 hpf ( F ) , then followed through to 54 ( G ) . G ' is a higher magnification image of the box in G and is accompanied by a red channel only ( traced photoconverted cells ) image with the VA - L ( dotted line ) demarcated from the adjacent VA , and VA - L photoconverted cells indicated ( arrowheads ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G') Lateral images of the cranial vessels in etv2:Kaede embryos, with unconverted tissue shown in green (F-G) while photoconverted tissue is shown in red (G'). The earliest visible form of the VA-L was photoconverted at 29 hpf (F), then followed through to 54 (G). G' is a higher magnification image of the box in G and is accompanied by a red channel only (traced photoconverted cells) image with the VA-L (dotted line) demarcated from the adjacent VA, and VA-L photoconverted cells indicated (arrowheads).


( A - G ) Lateral ( A - B ) , ventrolateral ( C - F ) and ventral ( G ) images of the cranial vessels in an etv2 : EGFP ; kdrl : nlsmCherry compound transgenic embryo , which has had the etv2 - expressing angioblast population ( early VA - L ) on the left lateral side of the yolk sac ablated . Images were taken at 30 hpf before ( A ) and after ( B ) ablation of the early VA - L , with the site of ablation indicated ( B ; blue dotted circle ) . Note that the LDA and VA adjacent to the VA - L become photobleached during the ablation process ( B ) , however these were not ablated as they can be seen developing normally at 54 hpf ( D ) . Follow - up images were taken of both the contralateral ( C , E ) and ablated ( D , F ) sides of the embryo ' s head at 54 hpf ( C - D ) and 78 hpf ( E - G ) . Although the VA - L ( light blue dotted line ) is seen migrating towards the FLS ( white dotted lined ) on the contralateral side at 54 hpf ( C ) , it is missing from the ablated side ( D ) . Subsequently , the LFL ( white dotted line ) of the ablated side is short ( F ) compared to that of the contralateral side ( E ) . In addition , the HA ( pink dotted line ) is underdeveloped on the ablated side compared to the contralateral side , as it no longer extends towards nor connects to the left AA1 ( G ) . ( H ) Quantitation of the length of the LFL ( µm ) on the contralateral ( control ) side and on the ablated side at 78 hpf ( n = 16 ) . ( I ) Quantitation of the total number of cells within the LFL on the contralateral ( control ) side and from the ablated side at 78 hpf ( n = 16 ) .

O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G) Lateral (A-B), ventrolateral (C-F) and ventral (G) images of the cranial vessels in an etv2:EGFP;kdrl:nlsmCherry compound transgenic embryo, which has had the etv2-expressing angioblast population (early VA-L) on the left lateral side of the yolk sac ablated. Images were taken at 30 hpf before (A) and after (B) ablation of the early VA-L, with the site of ablation indicated (B; blue dotted circle). Note that the LDA and VA adjacent to the VA-L become photobleached during the ablation process (B), however these were not ablated as they can be seen developing normally at 54 hpf (D). Follow-up images were taken of both the contralateral (C, E) and ablated (D, F) sides of the embryo's head at 54 hpf (C-D) and 78 hpf (E-G). Although the VA-L (light blue dotted line) is seen migrating towards the FLS (white dotted lined) on the contralateral side at 54 hpf (C), it is missing from the ablated side (D). Subsequently, the LFL (white dotted line) of the ablated side is short (F) compared to that of the contralateral side (E). In addition, the HA (pink dotted line) is underdeveloped on the ablated side compared to the contralateral side, as it no longer extends towards nor connects to the left AA1 (G). (H) Quantitation of the length of the LFL (µm) on the contralateral (control) side and on the ablated side at 78 hpf (n = 16). (I) Quantitation of the total number of cells within the LFL on the contralateral (control) side and from the ablated side at 78 hpf (n = 16).


( A ) Top panel displays structure of each CDK4 / 6 inhibitor tested . Bottom panel shows dose response curves with these inhibitors displaying percentage of G1 - arrested RPE1 - FUCCI cells . To obtain dose response curves , the number of mKO - Cdt1 positive ( G1 - arrested ) cells were calculated following 24hr drug addition ( dark blue solid lines ) or 24h after subsequent drug washout ( light blue dotted lines ) . Cmax values observed in patients ( taken from ( He et al . , 2017 ; Klein et al . , 2018 ) ) are represented on each graph with red dotted lines . Graphs display mean data - / + SEM from 3 experiments , with at least 500 cells counted per condition per experiment . ( B ) Percentage of G1 - arrested RPE1 - FUCCI cells , calculated as in panel A , but using a fixed concentration of CDK4 / 6 inhibitor for different durations of time , as indicated . Note , this is a fixed assay that quantifies the percentage of G1 - arrested cells prior to , or 24 hrs following , CDK4 / 6 inhibitor washout . Each bar displays mean data + SEM from 3 experiments , with at least 500 cells counted per condition per experiment . (

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O 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) Top panel displays structure of each CDK4/6 inhibitor tested. Bottom panel shows dose response curves with these inhibitors displaying percentage of G1-arrested RPE1-FUCCI cells. To obtain dose response curves, the number of mKO-Cdt1 positive (G1-arrested) cells were calculated following 24hr drug addition (dark blue solid lines) or 24h after subsequent drug washout (light blue dotted lines). Cmax values observed in patients (taken from (He et al., 2017; Klein et al., 2018)) are represented on each graph with red dotted lines. Graphs display mean data -/+ SEM from 3 experiments, with at least 500 cells counted per condition per experiment. (B) Percentage of G1-arrested RPE1-FUCCI cells, calculated as in panel A, but using a fixed concentration of CDK4/6 inhibitor for different durations of time, as indicated. Note, this is a fixed assay that quantifies the percentage of G1-arrested cells prior to, or 24 hrs following, CDK4/6 inhibitor washout. Each bar displays mean data + SEM from 3 experiments, with at least 500 cells counted per condition per experiment. (


( A ) Dose response curves displaying the percentage of G1 - arrested p53 - WT ( blue ) or KO ( green ) RPE1 - FUCCI cells following 24hr incubation with palbociclib ( dark solid lines ) or 24h after subsequent washout ( light dotted lines ) . Graphs display mean data - / + SEM from 3 experiments , with at least 500 cells counted per condition per experiment .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O 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) Dose response curves displaying the percentage of G1-arrested p53-WT (blue) or KO (green) RPE1-FUCCI cells following 24hr incubation with palbociclib (dark solid lines) or 24h after subsequent washout (light dotted lines). Graphs display mean data -/+ SEM from 3 experiments, with at least 500 cells counted per condition per experiment.


( C ) Representative images and quantifications of colony forming assays in p53 - WT or KO RPE1 cells treated with palbociclib ( 1 . 25μM ) for 1 , 4 or 7 days and then grown at low density without inhibitor for 10 days . Each bar displays mean data + SEM from 3 experiments .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Representative images and quantifications of colony forming assays in p53-WT or KO RPE1 cells treated with palbociclib (1.25μM) for 1, 4 or 7 days and then grown at low density without inhibitor for 10 days. Each bar displays mean data + SEM from 3 experiments.


( E ) Quantification of cell cycle profiles from cells treated as in D . Graph shows the mean percentage of cells + SD that divide at each round of division , with 150 cells analysed in total from 3 experimental repeats .

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

(E) Quantification of cell cycle profiles from cells treated as in D. Graph shows the mean percentage of cells + SD that divide at each round of division, with 150 cells analysed in total from 3 experimental repeats.


( A ) Representative immunofluorescence images of p21 levels in p53 - WT or KO RPE1 cells , 48 hours after release from 1 , 4 or 7 days palbociclib ( 1 . 25μM ) treatment . Zoom inserts are 3x magnification of the indicated regions . Scale bars = 250 μM . ( B ) Quantification of p21 intensities in cells treated as in panel A . At least 100 cells were analysed per experiment and graph shows data from 3 experimental repeats . Violin plots display the variation in intensities between individual cells . Horizontal lines display the median , and error bars show 95 % confidence intervals . (

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(A) Representative immunofluorescence images of p21 levels in p53-WT or KO RPE1 cells, 48 hours after release from 1, 4 or 7 days palbociclib (1.25μM) treatment. Zoom inserts are 3x magnification of the indicated regions. Scale bars = 250 μM. (B) Quantification of p21 intensities in cells treated as in panel A. At least 100 cells were analysed per experiment and graph shows data from 3 experimental repeats. Violin plots display the variation in intensities between individual cells. Horizontal lines display the median, and error bars show 95% confidence intervals. (


( D - E ) Quantification of nuclear morphologies ( D ) and γH2AX - positive DNA damage foci ( E ) following palbociclib ( 1 . 25μM ) treatment in p53 WT and KO RPE1 cells . Cells were treated for 1 , 4 or 7 days and then analysed before or after drug washout for 48 hours . 100 cells ( nuclear morphology ) or 50 cells ( γH2AX foci ) were scored per condition per experiment , and bar graphs represent mean data + SEM from 6 experiments .

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(D-E) Quantification of nuclear morphologies (D) and γH2AX-positive DNA damage foci (E) following palbociclib (1.25μM) treatment in p53 WT and KO RPE1 cells. Cells were treated for 1, 4 or 7 days and then analysed before or after drug washout for 48 hours. 100 cells (nuclear morphology) or 50 cells (γH2AX foci) were scored per condition per experiment, and bar graphs represent mean data +SEM from 6 experiments.


( J - K ) Representative immunofluorescence images ( J ) and quantifications ( K ) of mitotic DNA replication assays ( MiDAS ) in p53 - KO RPE1 cells released from 7 days of palbociclib ( 1 . 25μM ) treatment or following 0 . 4uM aphidicolin treatment for 40Hrs . EdU foci were quantified in nocodazole - arrested cells . Scale bar = 5 μM , zoom inserts = 3x magnification of highlighted areas . 10 cells were analysed per experiment and the bar chart shows the mean + SEM from 3 experimental repeats .

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(J-K) Representative immunofluorescence images (J) and quantifications (K) of mitotic DNA replication assays (MiDAS) in p53-KO RPE1 cells released from 7 days of palbociclib (1.25μM) treatment or following 0.4uM aphidicolin treatment for 40Hrs. EdU foci were quantified in nocodazole-arrested cells. Scale bar= 5 μM, zoom inserts = 3x magnification of highlighted areas. 10 cells were analysed per experiment and the bar chart shows the mean + SEM from 3 experimental repeats.


( A ) Volcano plot of proteins up or downregulated following prolonged palbociclib ( 1 . 25μM ) treatment in RPE1 cells . The top 10 significantly upregulated and downregulated proteins are shown in blue and red , respectively .

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(A) Volcano plot of proteins up or downregulated following prolonged palbociclib (1.25μM) treatment in RPE1 cells. The top 10 significantly upregulated and downregulated proteins are shown in blue and red, respectively.


( D ) Representative western blots of whole cell lysates from RPE1 - WT cells treated with palbociclib ( 1 . 25μM ) for 1 , 4 or 7 days , or treated identically , and then washed out for the indicated times to reflect when the majority of cells are in S - phase ( see Figure 1C ) .

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(D) Representative western blots of whole cell lysates from RPE1-WT cells treated with palbociclib (1.25μM) for 1, 4 or 7 days, or treated identically, and then washed out for the indicated times to reflect when the majority of cells are in S-phase (see Figure 1C).


( F - G ) Representative images ( F ) and quantifications ( G ) of colony forming assays with RPE1 cells treated with palbociclib ( 1 . 25μM ) for indicated times , and then grown at low density without palbociclib for 10 days . DMSO ( control ) or indicated genotoxic drugs were applied for the first 24 h after palbociclib washout . Each bar displays mean data + SD from 4 experiments .

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(F-G) Representative images (F) and quantifications (G) of colony forming assays with RPE1 cells treated with palbociclib (1.25μM) for indicated times, and then grown at low density without palbociclib for 10 days. DMSO (control) or indicated genotoxic drugs were applied for the first 24 h after palbociclib washout. Each bar displays mean data + SD from 4 experiments.


( A ) Representative western blots of whole cell lysates from MCF7 cells treated with palbociclib ( 1 μM ) for 1 , 4 or 7 days , or treated identically , and then washed out for the indicated times ( see Figure 1C ) . ( B ) Analysis of adjusted relative density from 3 independent western blot experiments . Bars display mean values - / + SD . Significance determined by unpaired Student ' s T test comparing treated target protein to asynchronous target control . ( * < 0 . 01 , * * < 0 . 001 , * * * < 0 . 0001 ) .

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(A) Representative western blots of whole cell lysates from MCF7 cells treated with palbociclib (1 μM) for 1, 4 or 7 days, or treated identically, and then washed out for the indicated times (see Figure 1C). (B) Analysis of adjusted relative density from 3 independent western blot experiments. Bars display mean values -/+ SD. Significance determined by unpaired Student's T test comparing treated target protein to asynchronous target control. (* < 0.01, ** < 0.001, *** < 0.0001).


( C , D ) Quantification of the nuclear morphologies ( C ) or γH2AX - positive DNA damage foci ( D ) from MCF7 , MCF7 p53 KO or T47D cells that were treated with palbociclib ( 1μM ) for 0 , 1 , 4 , or 7 days and then analysed 48hr after drug washout . Either 100 cells ( nuclear morphology ) or 50 cells ( γH2AX ) were scored per condition per experiment and bar graphs represent mean data + SEM from 3 experiments .

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(C,D) Quantification of the nuclear morphologies (C) or γH2AX-positive DNA damage foci (D) from MCF7, MCF7 p53 KO or T47D cells that were treated with palbociclib (1μM) for 0, 1, 4, or 7 days and then analysed 48hr after drug washout. Either 100 cells (nuclear morphology) or 50 cells (γH2AX) were scored per condition per experiment and bar graphs represent mean data + SEM from 3 experiments.


( F ) Quantification of colony forming assays of MCF7 , MCF7 p53 KO and T47D cells treated with CDK4 / 6 inhibitor for 0 , 1 , 4 or 7 days and then grown at a low density without palbociclib for 14 - 21 days . Bar graphs displays mean data + SEM from 4 - 5 experiments .

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(F) Quantification of colony forming assays of MCF7, MCF7 p53 KO and T47D cells treated with CDK4/6 inhibitor for 0, 1, 4 or 7 days and then grown at a low density without palbociclib for 14-21 days. Bar graphs displays mean data + SEM from 4-5 experiments.


( B , C ) Quantification of the nuclear morphologies ( B ) or γH2AX - positive DNA damage foci ( C ) from different cancer lines , as indicated , treated continuously with palbociclib ( 1μM ) for 0 , 1 , 2 , or 3 weeks . Either 100 cells ( nuclear morphology ) or 50 cells ( γH2AX ) were scored per condition per experiment and bar graphs represent mean data + SEM from 3 experiments .

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(B,C) Quantification of the nuclear morphologies (B) or γH2AX-positive DNA damage foci (C) from different cancer lines, as indicated, treated continuously with palbociclib (1μM) for 0, 1, 2, or 3 weeks. Either 100 cells (nuclear morphology) or 50 cells (γH2AX) were scored per condition per experiment and bar graphs represent mean data + SEM from 3 experiments.


( D ) Quantification of weekly proliferation rate in cells treated as in B . A total of 60 , 000 cells of each cell line were cultured in 10cm dishes with 1uM palbociclib for a total of 3 weeks . Every 7 days , cells were trypsinised and total cell counts were determined . The 0 - week timepoint shows the 7 - day fold increase of untreated cells . The data points show fold increase in cell count over each 7 - day period , and bars represent the mean from 2 experiments .

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(D) Quantification of weekly proliferation rate in cells treated as in B. A total of 60,000 cells of each cell line were cultured in 10cm dishes with 1uM palbociclib for a total of 3 weeks. Every 7 days, cells were trypsinised and total cell counts were determined. The 0-week timepoint shows the 7-day fold increase of untreated cells. The data points show fold increase in cell count over each 7-day period, and bars represent the mean from 2 experiments.


Mean ± SEM CB2 mRNA levels in cerebellar extracts of WT and ASM - KO mice ( n = 5 mice per group ) .

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Mean ± SEM CB2 mRNA levels in cerebellar extracts of WT and ASM-KO mice (n = 5 mice per group).


Western blot against CB2 and PSD - 95 ( used as loading control ) and graph showing mean ± SEM CB2 protein levels in cerebellar extracts of WT and ASM - KO mice ( n = 6 mice per group ) .

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Western blot against CB2 and PSD-95 (used as loading control) and graph showing mean ± SEM CB2 protein levels in cerebellar extracts of WT and ASM-KO mice (n = 6 mice per group).


Mean ± SEM CB1 mRNA levels in cerebellar extracts of WT and ASM - KO mice ( * * P = 0 . 0013 , n = 6 mice per group , Student ' s t - test ) .

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Mean ± SEM CB1 mRNA levels in cerebellar extracts of WT and ASM-KO mice (**P = 0.0013, n = 6 mice per group, Student's t-test).


Western blot against CB1 and GAPDH ( used as loading control ) and graph showing mean ± SEM CB1 protein levels in cerebellar extracts of WT and ASM - KO mice ( n = 3 mice per group ) .

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Western blot against CB1 and GAPDH (used as loading control) and graph showing mean ± SEM CB1 protein levels in cerebellar extracts of WT and ASM-KO mice (n = 3 mice per group).


Immunofluorescence images against CB1 and the following cellular markers : Calbindin for Purkinje cells ( E ) , GFAP for astrocytes ( F ) and F4 / 80 for microglia ( G ) in the cerebellum of WT and ASM - KO mice . DAPI stains cell nuclei . Graphs to the left show mean ± SEM intensity associated to CB1 in the Purkinje cells ( shown by white arrows , E ) , astrocytes ( F ) and microglia ( G ) expressed as percentage of the values obtained in WT mice . Graphs to the right show the Mander ' s coefficient that indicates degree of co - localization between CB1 and the cellular markers for Purkinje cells ( E ) , astrocytes ( F ) and microglia ( G ) ( E : * * PFluorecence intensity = 0 . 0082 , * PMander ' s Coefficient = 0 . 0158 ; F : * PMander ' s Coefficient = 0 . 0115 ) ; n = 5 mice per group , Student ' s t - test ) . Scale bar , 100 μm .

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Immunofluorescence images against CB1 and the following cellular markers: Calbindin for Purkinje cells (E), GFAP for astrocytes (F) and F4/80 for microglia (G) in the cerebellum of WT and ASM-KO mice. DAPI stains cell nuclei. Graphs to the left show mean ± SEM intensity associated to CB1 in the Purkinje cells (shown by white arrows, E), astrocytes (F) and microglia (G) expressed as percentage of the values obtained in WT mice. Graphs to the right show the Mander's coefficient that indicates degree of co-localization between CB1 and the cellular markers for Purkinje cells (E), astrocytes (F) and microglia (G) (E: **PFluorecence intensity = 0.0082, *PMander's Coefficient = 0.0158; F: *PMander's Coefficient = 0.0115); n = 5 mice per group, Student's t-test). Scale bar, 100 μm.


H . Immunofluorescence images against CB1 and the Purkinje cell marker calbindin in the cerebellum of age - matched control and ASMD - affected children . Graph shows mean ± SEM intensity associated to CB1 in the Purkinje cells expressed as percentage of the values obtained in the control child ( 16 and 15 replicates in control and ASMD , respectively ) . Scale bar , 10 μm .

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H. Immunofluorescence images against CB1 and the Purkinje cell marker calbindin in the cerebellum of age-matched control and ASMD-affected children. Graph shows mean ± SEM intensity associated to CB1 in the Purkinje cells expressed as percentage of the values obtained in the control child (16 and 15 replicates in control and ASMD, respectively). Scale bar, 10 μm.


Mean ± SEM CB1 mRNA levels in cultured hippocampal neurons from WT and ASM - KO mice ( * * * * P < 0 . 0001 , n = 5 independent cultures , Student ' s t - test ) .

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Mean ± SEM CB1 mRNA levels in cultured hippocampal neurons from WT and ASM-KO mice (****P < 0.0001, n = 5 independent cultures, Student's t-test).


Western blot against CB1 and GAPDH ( used as loading control ) . Images belong to the same blot but to non - consecutive lanes as indicated by the boxes . Graph showing mean ± SEM CB1 protein levels in cultured neurons from WT and ASM - KO mice ( * * * P = 0 . 0009 , n = 5 independent cultures , Student ' s t - test ) .

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Western blot against CB1 and GAPDH (used as loading control). Images belong to the same blot but to non-consecutive lanes as indicated by the boxes. Graph showing mean ± SEM CB1 protein levels in cultured neurons from WT and ASM-KO mice (***P= 0.0009, n = 5 independent cultures, Student's t-test).


Immunofluorescence images against CB1 and the lysosomal marker Lamp1 in cultured neurons from WT and ASM - KO mice . TOPRO stains cell nuclei . Graphs show mean ± SEM intensity associated to CB1 in the neuronal processes expressed in arbitrary units ( left ) or the Mander ´ s coefficient for the colocalization of CB1 with Lamp1 ( right ) ( * * * * P < 0 . 0001 , n = 3 independent cultures , > 30 neurons per culture , Student ' s t - test ) . Scale bar , 10 μm .

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Immunofluorescence images against CB1 and the lysosomal marker Lamp1 in cultured neurons from WT and ASM-KO mice. TOPRO stains cell nuclei. Graphs show mean ± SEM intensity associated to CB1 in the neuronal processes expressed in arbitrary units (left) or the Mander´s coefficient for the colocalization of CB1 with Lamp1 (right) (****P < 0.0001, n = 3 independent cultures, > 30 neurons per culture, Student's t-test). Scale bar, 10 μm.


Immunofluorescence images against CB1 and the lysosomal marker Lamp1 in Purkinje cells of the cerebellum from WT and ASM - KO mice . Graph shows mean ± SEM Mander ´ s coefficient indicative of the degree of colocalization of CB1 and Lamp1 ( * P = 0 . 0213 , n = 4 mice per group , Student ' s t - test ) . Scale bar , 5 μm .

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Immunofluorescence images against CB1 and the lysosomal marker Lamp1 in Purkinje cells of the cerebellum from WT and ASM-KO mice. Graph shows mean ± SEM Mander´s coefficient indicative of the degree of colocalization of CB1 and Lamp1 (*P= 0.0213, n = 4 mice per group, Student's t-test). Scale bar, 5 μm.


Graphs show mean ± SEM SM levels , expressed as percentage of WT values , in extracts containing the same amount of protein of cultured neurons from WT and ASM - KO mice ( E )

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Graphs show mean ± SEM SM levels, expressed as percentage of WT values, in extracts containing the same amount of protein of cultured neurons from WT and ASM-KO mice (E)


Graphs show mean ± SEM SM levels , expressed as percentage of WT values , in extracts containing the same amount of protein of cultured neurons from WT mice incubated or not with 40μΜ SM ( F ) .

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Graphs show mean ± SEM SM levels, expressed as percentage of WT values, in extracts containing the same amount of protein of cultured neurons from WT mice incubated or not with 40μΜ SM (F).


Mean ± SEM CB1 mRNA levels in cultured neurons from WT mice incubated or not with 40μM SM ( * * P = 0 . 0021 , n = 5 independent cultures , Student ' s t - test ) .

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Mean ± SEM CB1 mRNA levels in cultured neurons from WT mice incubated or not with 40μM SM (**P = 0.0021, n = 5 independent cultures, Student's t-test).


Western blot against CB1 and GAPDH ( used as loading control ) and graph showing mean ± SEM CB1 protein levels in cultured neurons from WT mice incubated or not with 40μΜ SM ( n = 2 independent cultures , Student ' s t - test )

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Western blot against CB1 and GAPDH (used as loading control) and graph showing mean ± SEM CB1 protein levels in cultured neurons from WT mice incubated or not with 40μΜ SM (n = 2 independent cultures, Student's t-test)


Immunofluorescence images against CB1 and Lamp1 in cultured neurons from WT mice incubated or not with 40 μΜ SM . Graphs show mean ± SEM intensity associated to CB1 in the neuronal processes expressed in arbitrary units ( left ) or the Mander ´ s coefficient for the colocalization of CB1 with Lamp1 ( right ) ( * * * P < 0 . 0001 , n = 3 independent cultures , > 30 neurons per culture , Student ' s t - test ) . Scale bar , 10 μm .

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Immunofluorescence images against CB1 and Lamp1 in cultured neurons from WT mice incubated or not with 40 μΜ SM. Graphs show mean ± SEM intensity associated to CB1 in the neuronal processes expressed in arbitrary units (left) or the Mander´s coefficient for the colocalization of CB1 with Lamp1 (right) (***P <0.0001, n = 3 independent cultures, > 30 neurons per culture, Student's t-test). Scale bar, 10 μm.


Western blot against CB1 and GAPDH ( used as loading control ) and graph showing mean ± SEM CB1 protein levels in cultured neurons from WT mice treated with vehicle ( veh ) , with 40μM SM ( SM ) , with 0 , 1μM Bafilomycin ( BafA1 ) or with 40μM SM and 0 , 1μM Bafilomyecin ( SM + BafA1 ) . Graph shows mean ± SEM CB1 levels normalized to GAPDH in arbitrary units ( n = 2 independent cultures , one - way ANOVA ) .

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Western blot against CB1 and GAPDH (used as loading control) and graph showing mean ± SEM CB1 protein levels in cultured neurons from WT mice treated with vehicle (veh), with 40μM SM (SM), with 0,1μM Bafilomycin (BafA1) or with 40μM SM and 0,1μM Bafilomyecin (SM+BafA1). Graph shows mean ± SEM CB1 levels normalized to GAPDH in arbitrary units (n = 2 independent cultures, one - way ANOVA).


Mean ± SEM SM levels in cultured neurons from ASM - KO mice incubated or not with exogenous SMase ( * P = 0 . 0157 , n = 3 independent cultures , Student ' s t - test ) .

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Mean ± SEM SM levels in cultured neurons from ASM-KO mice incubated or not with exogenous SMase (*P = 0.0157, n = 3 independent cultures, Student's t-test).


Mean ± SEM CB1 mRNA levels in cultured neurons from ASM - KO mice incubated or not with exogenous SMase ( * * P = 0 . 0015 , n = 5 independent cultures , Student ' s t - test ) .

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Mean ± SEM CB1 mRNA levels in cultured neurons from ASM-KO mice incubated or not with exogenous SMase (**P= 0.0015, n = 5 independent cultures, Student's t-test).


Western blot against CB1 and GAPDH ( used as loading control ) . Images belong to the same blot but to non - consecutive lanes as indicated by the boxes . Graph showing mean ± SEM CB1 protein levels normalized to GAPDH in extracts from cultured neurons from ASM - KO mice incubated or not with exogenous SMase ( n = 5 independent cultures , Student ' s t - test ) .

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Western blot against CB1 and GAPDH (used as loading control). Images belong to the same blot but to non-consecutive lanes as indicated by the boxes. Graph showing mean ± SEM CB1 protein levels normalized to GAPDH in extracts from cultured neurons from ASM-KO mice incubated or not with exogenous SMase (n = 5 independent cultures, Student's t-test).


Immunofluorescence images against CB1 and the lysosomal marker Lamp1 in cultured neurons from ASM - KO mice incubated or not with exogenous SMase . DAPI stains cell nuclei . Graph shows mean ± SEM Mander ´ s coefficient for the colocalization of CB1 with Lamp1 ( * P = 0 . 0189 , n = 3 independent cultures , > 30 neurons per culture , Student ' s t - test ) . Scale bar , 10 μm .

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Immunofluorescence images against CB1 and the lysosomal marker Lamp1 in cultured neurons from ASM-KO mice incubated or not with exogenous SMase. DAPI stains cell nuclei. Graph shows mean ± SEM Mander´s coefficient for the colocalization of CB1 with Lamp1 (*P = 0.0189, n = 3 independent cultures, > 30 neurons per culture, Student's t-test). Scale bar, 10 μm.


Mean ± SEM SM levels , expressed as percentage of vehicle values , in cultured neurons from ASM - KO mice treated with vehicle or with the indicated concentrations of AEA ( * * * P < 0 . 0001 , n = 3 independent cultures , one - way ANOVA , Bonferroni post hoc ) .

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Mean ± SEM SM levels, expressed as percentage of vehicle values, in cultured neurons from ASM-KO mice treated with vehicle or with the indicated concentrations of AEA (***P<0.0001, n = 3 independent cultures, one - way ANOVA, Bonferroni post hoc).


Mean ± SEM SM levels , expressed as percentage of vehicle values , in cultured neurons from ASM - KO mice treated with vehicle , the inhibitor of NSM GW , AEA or the combination of GW and AEA ( * * * P < 0 . 0001 , n = 3 independent cultures , one - way ANOVA , Bonferroni post hoc ) .

O O O B-MEASURED_VAR 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-CONTROLLED_VAR B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

Mean ± SEM SM levels, expressed as percentage of vehicle values, in cultured neurons from ASM-KO mice treated with vehicle, the inhibitor of NSM GW, AEA or the combination of GW and AEA (***P <0.0001, n = 3 independent cultures, one - way ANOVA, Bonferroni post hoc).


Western blot against NSM and GAPDH ( used as loading control ) and graph showing mean ± SEM NSM protein levels in extracts from cultured neurons treated with vehicle or with 50μM AEA ( * P = 0 . 0230 , n = 6 independent cultures , Student ' s t - test ) .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

Western blot against NSM and GAPDH (used as loading control) and graph showing mean ± SEM NSM protein levels in extracts from cultured neurons treated with vehicle or with 50μM AEA (*P = 0.0230, n = 6 independent cultures, Student's t-test).


Mean ± SEM SM levels , expressed as percentage of vehicle values , in cultured neurons from ASM - KO mice treated with vehicle , or with JNJ , PF or URB in the presence or absence of AEA ( * * PJNJ = 0 . 0013 , * PPF = 0 . 0143 , * * * PURB = 0 . 0002 , * * PJNJ + AEA = 0 . 0069 , * * PPF + AEA = 0 . 0077 , * * * PURB + AEA < 0 . 0001 , n = 3 independent cultures , one - way ANOVA , Bonferroni post hoc ) .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O 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-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

Mean ± SEM SM levels, expressed as percentage of vehicle values, in cultured neurons from ASM-KO mice treated with vehicle, or with JNJ, PF or URB in the presence or absence of AEA (**PJNJ = 0.0013, *PPF = 0.0143, ***PURB = 0.0002, **PJNJ+AEA = 0.0069, **PPF+AEA = 0.0077, ***PURB+AEA <0.0001, n = 3 independent cultures, one - way ANOVA, Bonferroni post hoc).


Mean ± SEM SM levels , expressed as percentage of vehicle values , in cultured neurons from ASM - KO mice treated with vehicle , with PF or with SR141716 + PF ( * PPF = 0 . 0109 , * PSR + PF = 0 . 0343 , n = 3 independent cultures , one - way ANOVA , Bonferroni post hoc ) .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

Mean ± SEM SM levels, expressed as percentage of vehicle values, in cultured neurons from ASM-KO mice treated with vehicle, with PF or with SR141716 + PF (*PPF = 0.0109, *PSR+PF = 0.0343, n = 3 independent cultures, one - way ANOVA, Bonferroni post hoc).


Immunofluorescences against the dendritic marker MAP2 of cultured neurons from ASM - KO mice treated with vehicle , AEA , JNJ , PF or URB . Scale bar , 50 μm .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O

Immunofluorescences against the dendritic marker MAP2 of cultured neurons from ASM-KO mice treated with vehicle, AEA, JNJ, PF or URB. Scale bar, 50 μm.


Mean ± SEM number of apoptotic cells measured by TUNEL assays in cultured neurons from ASM - KO mice treated with H2O2 ( as positive control ) , vehicle , AEA , JNJ , PF or URB ( n = 3 independent cultures ) .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O

Mean ± SEM number of apoptotic cells measured by TUNEL assays in cultured neurons from ASM-KO mice treated with H2O2 (as positive control), vehicle, AEA, JNJ, PF or URB (n = 3 independent cultures).


Weekly mean ± SEM body weight of WT and ASM - KO mice treated or not with PF ( * * * P < 0 . 0001 , n = 7 mice per group , statistics reflect the significant difference between the slope of the data in the PF - treated ASM - KO with respect to the other three groups , Linear Regression test ) .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O

Weekly mean ± SEM body weight of WT and ASM-KO mice treated or not with PF (***P <0.0001, n = 7 mice per group, statistics reflect the significant difference between the slope of the data in the PF-treated ASM-KO with respect to the other three groups, Linear Regression test).


Mean ± SEM time spent on the rod in the 4 trials of the rotarod test by WT and ASM - KO mice after two months of vehicle or PF treatment ( * PT3 = 0 . 0488 , * PT4 = 0 . 361 , n = 4 mice per group , two - way ANOVA , Bonferroni post hoc ) .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mean ± SEM time spent on the rod in the 4 trials of the rotarod test by WT and ASM-KO mice after two months of vehicle or PF treatment (*PT3 = 0.0488, *PT4 = 0.361, n = 4 mice per group, two - way ANOVA, Bonferroni post hoc).


Mean ± SEM time spent in the new arm of the Y maze test by WT and ASM - KO mice after two months of vehicle or PF treatment ( * * PWTVeh Vs KO Veh = 0 . 0024 , * * PWT PF Vs KO Veh = 0 . 0016 , * PKO PF Vs KO Veh = 0 . 0391 , n = 9 mice per group , two - way ANOVA , Bonferroni post hoc ) .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

Mean ± SEM time spent in the new arm of the Y maze test by WT and ASM-KO mice after two months of vehicle or PF treatment (**PWTVeh Vs KO Veh = 0.0024, **PWT PF Vs KO Veh = 0.0016, *PKO PF Vs KO Veh = 0.0391, n = 9 mice per group, two - way ANOVA, Bonferroni post hoc).


Mean ± SEM time of immobility in the Tail suspension test of WT and ASM - KO mice after two months of vehicle or PF treatment ( * PWTVeh Vs KO Veh = 0 . 0198 , * * PWT PF Vs KO Veh = 0 . 0067 , n = 6 mice per group , two - way ANOVA , Bonferroni post hoc ) .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

Mean ± SEM time of immobility in the Tail suspension test of WT and ASM-KO mice after two months of vehicle or PF treatment (*PWTVeh Vs KO Veh = 0.0198, **PWT PF Vs KO Veh = 0.0067, n = 6 mice per group, two - way ANOVA, Bonferroni post hoc).


Mean ± SEM time spent in the open arms of the Elevated Plus Maze by WT and ASM - KO mice after two months of vehicle or PF treatment ( * PWTVeh Vs KO Veh = 0 . 0342 , n = 7 mice per group , two - way ANOVA , Bonferroni post hoc ) .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mean ± SEM time spent in the open arms of the Elevated Plus Maze by WT and ASM-KO mice after two months of vehicle or PF treatment (*PWTVeh Vs KO Veh = 0.0342, n = 7 mice per group, two - way ANOVA, Bonferroni post hoc).


Percentage of survival with respect to weeks of age in WT and ASM - KO mice treated with vehicle or PF ( * * P = 0 . 0031 , n = 5 mice in WT vehicle , WT PF treated and KO vehicle , 6 mice in KO PF treated , Mantel - Cox )

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O

Percentage of survival with respect to weeks of age in WT and ASM-KO mice treated with vehicle or PF (**P = 0.0031, n = 5 mice in WT vehicle, WT PF treated and KO vehicle, 6 mice in KO PF treated, Mantel-Cox)


Mean ± SEM AEA levels in cerebellar extracts from WT and ASM - KO mice after two months of vehicle or PF treatment ( * * PWT Veh vs KO Veh = 0 . 0063 ; * * * * PWT Veh vs KO PF < 0 . 0001 ; * * * * PWT PF vs KO PF < 0 . 0001 ; * * * * PKO Veh vs KO PF < 0 . 0001 , n = 4 mice per group , one - way ANOVA , Bonferroni post hoc ) .

O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

Mean ± SEM AEA levels in cerebellar extracts from WT and ASM-KO mice after two months of vehicle or PF treatment (**PWT Veh vs KO Veh = 0.0063; ****PWT Veh vs KO PF < 0.0001; ****PWT PF vs KO PF < 0.0001; ****PKO Veh vs KO PF < 0.0001, n = 4 mice per group, one - way ANOVA, Bonferroni post hoc).


Mean ± SEM SM levels , expressed as percentage of vehicle values , in cerebellar extracts from WT and ASM - KO mice after two months of vehicle or PF treatment ( * * * PWTVeh Vs KO Veh < 0 . 0001 , * PKO Veh Vs KO PF = 0 . 0465 ) , n = 4 mice per group , one - way ANOVA , Bonferroni post hoc ) .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

Mean ± SEM SM levels, expressed as percentage of vehicle values, in cerebellar extracts from WT and ASM-KO mice after two months of vehicle or PF treatment (***PWTVeh Vs KO Veh <0.0001, *PKO Veh Vs KO PF = 0.0465), n = 4 mice per group, one - way ANOVA, Bonferroni post hoc).


Western blot against NSM and GAPDH ( used as loading control ) and graph showing mean ± SEM NSM protein levels in cerebellar extracts from WT and ASM - KO mice after two months of vehicle or PF treatment ( n = 3 mice per group , one - way ANOVA , Bonferroni post hoc ) .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

Western blot against NSM and GAPDH (used as loading control) and graph showing mean ± SEM NSM protein levels in cerebellar extracts from WT and ASM-KO mice after two months of vehicle or PF treatment (n = 3 mice per group, one - way ANOVA, Bonferroni post hoc).


Mean ± SEM CB1 mRNA levels in cerebellar extracts from WT and ASM - KO mice after two months of vehicle or PF treatment ( * * PWTVeh Vs KO Veh = 0 . 0026 , * PKO Veh Vs KO PF = 0 . 0345 ) , n = 4 mice per group , one - way ANOVA , Bonferroni post hoc ) .

O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

Mean ± SEM CB1 mRNA levels in cerebellar extracts from WT and ASM-KO mice after two months of vehicle or PF treatment (**PWTVeh Vs KO Veh = 0.0026, *PKO Veh Vs KO PF = 0.0345), n = 4 mice per group, one - way ANOVA, Bonferroni post hoc).


Immunofluorescence against CB1 in Purkinje cells of the cerebellum of WT and ASM - KO mice after two months of vehicle or PF treatment . Graph shows mean ± SEM intensity associated to CB1 , expressed as percentage of values in vehicle - treated mice ( n = 4 mice per group , > 20 Purkinje cells per

O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

Immunofluorescence against CB1 in Purkinje cells of the cerebellum of WT and ASM-KO mice after two months of vehicle or PF treatment. Graph shows mean ± SEM intensity associated to CB1, expressed as percentage of values in vehicle-treated mice (n = 4 mice per group, > 20 Purkinje cells per mouse


Immunofluorescence against CB1 and Lamp1 in the Purkinje cell layer of the cerebellum of WT and ASM - KO mice after two months of vehicle or PF treatment . Graphs show Mander ´ s coefficient for the colocalization of CB1 with Lamp1 ( left ) and mean ± SEM lysosomal size ( right ) ( * * * PWT Veh vs . KO Veh = 0 . 0002 ; * * PKO Veh vs . KO PF = 0 . 0052 , n = 4 mice per group , > 20 lysosomes per mouse , one - way ANOVA , Bonferroni post hoc ) . Scale bar , 5 μm .

O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunofluorescence against CB1 and Lamp1 in the Purkinje cell layer of the cerebellum of WT and ASM-KO mice after two months of vehicle or PF treatment. Graphs show Mander´s coefficient for the colocalization of CB1 with Lamp1 (left) and mean ± SEM lysosomal size (right) (***PWT Veh vs. KO Veh = 0.0002; **PKO Veh vs. KO PF = 0.0052, n = 4 mice per group, >20 lysosomes per mouse, one - way ANOVA, Bonferroni post hoc). Scale bar, 5 μm.


Immunofluorescence against the Purkinje cell marker Calbindin in the cerebellum of WT and ASM - KO mice after two months of vehicle or PF treatment . Graph shows mean ± SEM number of calbindin positive cells per area ( * * * PWTVeh Vs KO Veh < 0 . 0001 , * * PKO PF Vs KO Veh = 0 . 00642 ) , n = 4 mice per group , one - way ANOVA , Bonferroni post hoc ) . Scale bar , 100 μm . Immunofluorescence against the astrocytic marker GFAP in the cerebellum of WT and ASM - KO mice after two months of vehicle or PF treatment . Graph shows mean ± SEM intensity associated to GFAP in arbitrary units ( * * * PWTVeh Vs KO Veh < 0 . 0001 , * * PKO PF Vs KO Veh = 0 . 0083 ) , n = 4 mice per group , one - way ANOVA , Bonferroni post hoc ) . Scale bar , 10 μm . Immunofluorescence against the microglia marker Iba1 in the cerebellum of WT and ASM - KO mice after two months of vehicle or PF treatment . Graph shows mean ± SEM number ( left ) and area ( right ) of Iba1 - positive cells ( Left * * * PWTVeh Vs KO Veh < 0 . 0001 , * * PKO PF Vs KO Veh = 0 . 0034 ; Right * * * PWTVeh Vs KO Veh < 0 . 0001 , PKO PF Vs KO Veh = 0 . 00123 ) , n = 4 mice per group , > 20 cells analyzed per mouse , one - way ANOVA , Bonferroni post hoc ) . Scale bar , 10 μm .

O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunofluorescence against the Purkinje cell marker Calbindin in the cerebellum of WT and ASM-KO mice after two months of vehicle or PF treatment. Graph shows mean ± SEM number of calbindin positive cells per area (***PWTVeh Vs KO Veh <0.0001, **PKO PF Vs KO Veh = 0.00642), n = 4 mice per group, one - way ANOVA, Bonferroni post hoc). Scale bar, 100 μm. Immunofluorescence against the astrocytic marker GFAP in the cerebellum of WT and ASM-KO mice after two months of vehicle or PF treatment. Graph shows mean ± SEM intensity associated to GFAP in arbitrary units (***PWTVeh Vs KO Veh < 0.0001, **PKO PF Vs KO Veh = 0.0083), n = 4 mice per group, one - way ANOVA, Bonferroni post hoc). Scale bar, 10 μm. Immunofluorescence against the microglia marker Iba1 in the cerebellum of WT and ASM-KO mice after two months of vehicle or PF treatment. Graph shows mean ± SEM number (left) and area (right) of Iba1-positive cells (Left ***PWTVeh Vs KO Veh < 0.0001, **PKO PF Vs KO Veh = 0.0034; Right ***PWTVeh Vs KO Veh < 0.0001, PKO PF Vs KO Veh = 0.00123), n = 4 mice per group, > 20 cells analyzed per mouse, one - way ANOVA, Bonferroni post hoc). Scale bar, 10 μm.


H & E staining of cerebellum , hippocampus , cortex and liver from WT and ASM - KO mice 48 hours after one single administration of vehicle or the indicated doses of PF . Scale bar , 100 μm .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O

H&E staining of cerebellum, hippocampus, cortex and liver from WT and ASM-KO mice 48 hours after one single administration of vehicle or the indicated doses of PF. Scale bar, 100 μm.


Mean ± SEM SM levels , expressed as percentage of vehicle values , in cerebellar , hippocampal , cortical and liver extracts from WT and ASM - KO mice 48 hours after one single administration of vehicle or the indicated doses of PF . ( * PKOVeh Vs KO 5 = 0 . 0304 , n = 3 mice per group , one - way ANOVA , Bonferroni post hoc )

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mean ± SEM SM levels, expressed as percentage of vehicle values, in cerebellar, hippocampal, cortical and liver extracts from WT and ASM-KO mice 48 hours after one single administration of vehicle or the indicated doses of PF. (*PKOVeh Vs KO 5 = 0.0304, n = 3 mice per group, one - way ANOVA, Bonferroni post hoc)


Immunofluorescences against the microglia marker Iba1 in the cerebellum , hippocampus , cortex or against the macrophage marker F4 / 80 in the liver from WT and ASM - KO mice 48 hours after one single administration of vehicle or the indicated doses of PF . Graphs show mean ± SEM number of microglia per area unit in cerebellum , hippocampus and cortex or mean ± SEM area of macrophages in the liver ( Hippocampus * * PKOVeh Vs KO 5 = 0 . 0015 , Cortex * PKOVeh Vs KO 5 = 0 . 0364 , Liver * PKOVeh Vs KO 5 = 0 . 0152 , n = 6 mice per group , > 20 cells analyzed per mouse , one - way ANOVA , Bonferroni post hoc ) . Scale bar , 10 μm .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunofluorescences against the microglia marker Iba1 in the cerebellum, hippocampus, cortex or against the macrophage marker F4/80 in the liver from WT and ASM-KO mice 48 hours after one single administration of vehicle or the indicated doses of PF. Graphs show mean ± SEM number of microglia per area unit in cerebellum, hippocampus and cortex or mean ± SEM area of macrophages in the liver (Hippocampus **PKOVeh Vs KO 5 = 0.0015, Cortex *PKOVeh Vs KO 5 = 0.0364, Liver *PKOVeh Vs KO 5 = 0.0152, n = 6 mice per group, > 20 cells analyzed per mouse, one - way ANOVA, Bonferroni post hoc). Scale bar, 10 μm.


Western blot against CB1 and GAPDH ( used as loading control ) and graph showing mean ± SEM CB1 protein levels in cerebellar extracts from WT and Npc1nmf164 mice ( n = 6 ) .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O

Western blot against CB1 and GAPDH (used as loading control) and graph showing mean ± SEM CB1 protein levels in cerebellar extracts from WT and Npc1nmf164 mice (n = 6).


Immunofluorescence images against CB1 and Calbindin in the cerebellum of WT and Npc1nmf164 mice . DAPI stains cell nuclei . Graphs show mean ± SEM intensity associated to CB1 in the Purkinje cells ( indicated by arrows ) expressed in arbitrary units ( * * P = 0 . 0085 , n = 5 mice per group , Student ' s t - test ) . Scale bar , 10 μm .

O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunofluorescence images against CB1 and Calbindin in the cerebellum of WT and Npc1nmf164 mice. DAPI stains cell nuclei. Graphs show mean ± SEM intensity associated to CB1 in the Purkinje cells (indicated by arrows) expressed in arbitrary units (**P= 0.0085, n = 5 mice per group, Student's t-test). Scale bar, 10 μm.


Immunofluorescence images against CB1 in Purkinje cells of the cerebelllum of age - matched control and NPC affected children . Graph shows mean ± SEM intensity associated to CB1 in the Purkinje cells expressed as percentage of the control values ( 16 and 57 cells in control and NPC , respectively ) . Scale bar , 10 μm .

O O O B-MEASURED_VAR 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunofluorescence images against CB1 in Purkinje cells of the cerebelllum of age-matched control and NPC affected children. Graph shows mean ± SEM intensity associated to CB1 in the Purkinje cells expressed as percentage of the control values (16 and 57 cells in control and NPC, respectively). Scale bar, 10 μm.


Graph shows mean ± SEM SM levels , expressed as percentage of vehicle values , in cultured fibroblasts from a NPC patient treated with vehicle or with PF at 50μM or 100μM ( n = 2 different cultures )

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O

Graph shows mean ± SEM SM levels, expressed as percentage of vehicle values, in cultured fibroblasts from a NPC patient treated with vehicle or with PF at 50μM or 100μM (n= 2 different cultures)


Graph shows mean ± SEM cholesterol levels , expressed as percentage of vehicle values , in cultured fibroblasts from a NPC patient treated with vehicle or with PF at 50μM or 100μM ( n = 2 different cultures )

Graph shows mean ± SEM cholesterol levels, expressed as percentage of vehicle values, in cultured fibroblasts from a NPC patient treated with vehicle or with PF at 50μM or 100μM (n=2 different cultures)


Immunofluorescence images of CB1 and Lamp1 in cultured fibroblasts from control subject and NPC patient treated with vehicle or with PF at 100μM . Graph shows mean ± SEM Mander ´ s coefficient for the colocalization of CB1 with Lamp1 ( n = 2 different cultures ) . Scale bar , 5 μm .

O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O

Immunofluorescence images of CB1 and Lamp1 in cultured fibroblasts from control subject and NPC patient treated with vehicle or with PF at 100μM. Graph shows mean ± SEM Mander´s coefficient for the colocalization of CB1 with Lamp1 (n= 2 different cultures). Scale bar, 5 μm.


Graphs show mean ± SEM SM and cholesterol levels expressed as percentage of the values obtained in the vehicle treated mice in the cerebellum of Npc1nmf164 after 48 hours of a single dose of 5mg / kg PF ( n = 3 mice )

O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O 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-CONTROLLED_VAR O O O O O O

Graphs show mean ± SEM SM and cholesterol levels expressed as percentage of the values obtained in the vehicle treated mice in the cerebellum of Npc1nmf164 after 48 hours of a single dose of 5mg/kg PF (n=3 mice)


Immunofluorescence images against the microglia marker F4 / 80 in the cerebellum of WT and Npc1nmf164 after 48 hours of a single dose of 5mg / kg PF . Graphs show mean ± SEM number ( upper ) and area ( lower ) of F4 / 80 positive cells ( upper graph * * * * P < 0 . 0001 ; lower graph * * * PWT Veh vs NPC Veh = 0 . 0008 ; * PWT Veh vs . NPC PF = 0 . 0286 ; * * * * PWT PF vs . NPC Veh < 0 . 0001 ; * * PWT PF vs . NPC PF = 0 . 0022 , n = 3 mice per group , > 20 cells analyzed per mouse , one - way ANOVA , Bonferroni post hoc ) . Scale bar , 10 μm .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunofluorescence images against the microglia marker F4/80 in the cerebellum of WT and Npc1nmf164 after 48 hours of a single dose of 5mg/kg PF. Graphs show mean ± SEM number (upper) and area (lower) of F4/80 positive cells (upper graph ****P< 0.0001; lower graph ***PWT Veh vs NPC Veh = 0.0008; *PWT Veh vs. NPC PF = 0.0286; ****PWT PF vs. NPC Veh < 0.0001; **PWT PF vs. NPC PF = 0.0022, n = 3 mice per group, > 20 cells analyzed per mouse, one - way ANOVA, Bonferroni post hoc). Scale bar, 10 μm.