Bone number per mouse at 5 - 6 months of ( n = 23 wa / + , 15 wa / wa )

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Bone tumor number per mouse at 5-6 months of age (n=23 wa/+, 15 wa/wa)


of in tibiae at 5 - 6 months of ( n = 23 wa / + , 15 wa / wa )

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Quantification of tumor size in tibiae at 5-6 months of age (n=23 wa/+, 15 wa/wa)


Alkaline Phosphatase ( ALP ) in serum at endpoint ( = 5 - 6 months ; n = 16 wa / + , 11 wa / wa )

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Alkaline Phosphatase (ALP) levels in the serum at endpoint (age = 5-6 months; n=16 wa/+, 11 wa/wa)


X - ray before ( = 2 months ) after ( = 7 months ) vehicle or Erlotinib

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X-ray analysis before (age = 2 months) and after (age = 7 months) vehicle or Erlotinib treatment


of number during treatment ( n = 6 Vehicle , 5 Erlotinib )

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Quantification of tumor number during treatment (n=6 Vehicle, 5 Erlotinib)


during treatment ( n = 6 Vehicle , 5 Erlotinib )

Tumor size during treatment (n=6 Vehicle, 5 Erlotinib)


Analysis of serum ALP levels during treatment (n=5)


PET summation ( 0 - 90 min ) in horizontal ( upper panel ) coronal view ( lower panel ) depicting [ 11C ] Erlotinib distribution in one H2 - c - fosLTR / Egfrwt mouse ( M125 ) . Anatomical structures are labelled with arrows ( T , ; L , liver ; H , heart ; B , brain ) . Scale bars : 1cm - time curves of [ 11C ] Erlotinib in bone tumors in right scapula of three H2 - c - fosLTR / Egfrwt mice measured with PET . Broken indicates threshold for in vitro effect of Erlotinib ( 1 µM )

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PET summation images (0-90 min) in horizontal (upper panel) and coronal view (lower panel) depicting [11C]Erlotinib distribution in one H2-c-fosLTR/Egfrwt mouse (M125). Anatomical structures are labelled with arrows (T, tumor; L, liver; H, heart; B, brain). Scale bars: 1cm Concentration-time curves of [11C]Erlotinib in bone tumors in right scapula of three H2-c-fosLTR/Egfrwt mice measured with PET. Broken line indicates threshold for in vitro effect of Erlotinib (1 µM)


X - ray of 7 - months - old H2 - c - fosLTR / Egfrwt H2 - c - fosLTR / Egfr ∆ Ob . Scale bars :

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X-ray analysis of 7-months-old H2-c-fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob littermates. Scale bars: 1cm


Bone number per mouse at 6 - 7 months of ( n = 22 wt , 11 ∆ Ob ) of in tibiae ( n = 22 wt , 11 ∆ Ob )

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Bone tumor number per mouse at 6-7 months of age (n=22 wt, 11 ∆Ob) Quantification of tumor size in tibiae (n=22 wt, 11 ∆Ob)


ALP in serum at 7 - month endpoint ( n = 17 wt , 8 ∆ Ob )

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ALP levels in the serum at 7-month endpoint (n=17 wt, 8 ∆Ob)


µPET / CT of 7 - months - old H2 - c - fosLTR / Egfrwt H2 - c - fosLTR / Egfr ∆ Ob Standardized uptake values ( SUV ) of µPET - tracer Na [ 18F ] F in pelvic osteosarcoma of 4 - 7 - months - old H2 - c - fosLTR / Egfrwt H2 - c - fosLTR / Egfr ∆ Ob mice . n = 6 wt , 4 ∆ Ob for 4 month time - point , n = 6 wt , 3 ∆ Ob for 7 month time -

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µPET/CT analysis of 7-months-old H2-c-fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob littermates Standardized uptake values (SUV) of the µPET-tracer Na[18F]F in the pelvic osteosarcoma of 4- and 7-months-old H2-c-fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob mice. n=6 wt, 4 ∆Ob for 4 month time-point, n=6 wt, 3 ∆Ob for 7 month time-point


PCNA Caspase 3 shown as % positive ( for PCNA ) as positive per mm² ( for Caspase 3 ) in OS from H2 - c - fosLTR / Egfrwt ( n = 6 ) H2 - c - fosLTR / Egfr ∆ Ob ( n = 5 ) mice . Scale bars :

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PCNA and cleaved Caspase 3 IHC staining and quantification shown as % positive cells (for PCNA) and as positive cells per mm² (for cleaved Caspase 3) in OS from H2-c-fosLTR/Egfrwt (n=6) and H2-c-fosLTR/Egfr∆Ob (n=5) mice. Scale bars: 100µm


Egfr , Ccnd1 , c - fos transgenic c - fos ( c - fostg ) mRNA expression in tumors of H2 - c - fosLTR / Egfrwt H2 - c - fosLTR / Egfr ∆ Ob mice normalized to Tbp . n = 17 wt , 14 ∆ Ob ( Egfr , Ccnd1 ) , n = 16 wt , 13 ∆ Ob ( c - fos ) , n = 16 wt , 14 ∆ Ob ( c -

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Egfr, Ccnd1, c-fos and transgenic c-fos (c-fostg) mRNA expression levels in tumors of H2-c-fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob mice normalized to Tbp. n=17 wt, 14 ∆Ob (Egfr, Ccnd1), n=16 wt, 13 ∆Ob (c-fos), n=16 wt, 14 ∆Ob (c-fostg


showing pRSK2 ( n = 4 ) , pCREB ( n = 7 wt , 6 ∆ Ob ) c - Fos ( n = 5 ) positive ( % ) in OS from H2 - c - fosLTR / Egfrwt H2 - c - fosLTR / Egfr ∆ Ob . Scale bars :

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IHC staining and quantification showing pRSK2 (n=4), pCREB (n=7 wt, 6∆Ob) and c-Fos (n=5) positive cells (%) in OS from H2-c-fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob littermates. Scale bars: 100µm


Western Blot of OS isolated from H2 ‑ c ‑ fosLTR / Egfrwt H2 - c - fosLTR / Egfr ∆ Ob

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Western Blot analysis of primary OS cells isolated from H2‑c‑fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob mice


c - fos transgenic c - fos ( c fostg ) mRNA expression in H2 - c - fosLTR OS after 4x in vitro passages , cultured under standard conditions ( n = 3 independent lines )

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c-fos and transgenic c-fos (c fostg) mRNA expression levels in primary H2-c-fosLTR OS cells after 4x in vitro passages, cultured under standard conditions (n=3 independent cell lines)


Western Blot of H2 - c - fosLTR / Egfrwt OS treated for 24h with

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Western Blot analysis of H2-c-fosLTR/Egfrwt OS cells treated for 24h with Erlotinib


c - fos c - fostg mRNA expression in H2 - c - fosLTR / Egfrwt OS treated for 24h with Erlotinib ( 10µM ) or DMSO as control ( n = 4 independent lines )

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c-fos and c-fostg mRNA expression levels in H2-c-fosLTR/Egfrwt OS cells treated for 24h with Erlotinib (10µM) or DMSO as control (n=4 independent cell lines)


Western Blot of starved H2 - c - fosLTR / Egfrwt OS , pre - treated with DMSO ( 1 : 1000 ) , Afatinib ( 5µM ) , GSK2233470 ( 10µM ) , Rapamycin ( 10nM ) or U0126 ( 10µM ) for 30 minutes stimulated with EGF ( 50ng / ml ) as

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Western Blot analysis of starved H2-c-fosLTR/Egfrwt OS cells, pre-treated with DMSO (1:1000), Afatinib (5µM), GSK2233470 (10µM), Rapamycin (10nM) or U0126 (10µM) for 30 minutes and stimulated with EGF (50ng/ml) as indicated


Western Blot of OS isolated from a p53f / f Rb1f / f Osx - Cre mouse after 24h Erlotinib

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Western Blot analysis of primary OS cells isolated from a p53f/f Rb1f/f Osx-Cre mouse after 24h Erlotinib treatment


c - fos mRNA expression in p53f / f Rb1f / f Osx - Cre OS treated for 24h with Erlotinib ( 10µM ) ( n = 3 )

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c-fos mRNA expression levels in p53f/f Rb1f/f Osx-Cre OS cells treated for 24h with Erlotinib (10µM) (n=3)


Western Blot of starved p53f / f Rb1f / f Osx - Cre OS , pre - treated with DMSO ( 1 : 1000 ) or Afatinib ( 5µM ) for 30 minutes stimulated with EGF ( 50ng / ml ) as

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Western Blot analysis of starved p53f/f Rb1f/f Osx-Cre OS cells, pre-treated with DMSO (1:1000) or Afatinib (5µM) for 30 minutes and stimulated with EGF (50ng/ml) as indicated


X - ray of 6 - months - old H2 - c - fosLTR H2 - c - fosLTR / ColAREG . Scale bars :

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X-ray analysis of 6-months-old H2-c-fosLTR and H2-c-fosLTR/ColAREG littermates. Scale bars: 1cm


Bone number per mouse at 5 - 6 months of ( n = 22 wt , 13 ColAREG )

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Bone tumor number per mouse at 5-6 months of age (n=22 wt, 13 ColAREG)


of in tibiae at 5 - 6 months of ( n = 22 wt , 13 ColAREG )

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Quantification of tumor size in tibiae at 5-6 months of age (n=22 wt, 13 ColAREG)


Alkaline Phosphatase ( ALP ) in serum at endpoint ( = 5 - 6 months ; n = 29 wt , 19 ColAREG )

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Alkaline Phosphatase (ALP) levels in the serum at endpoint (age = 5-6 months; n=29 wt, 19 ColAREG)


c - fos mRNA expression in osteosarcomas of H2 - c - fosLTR ( n = 11 ) H2 - c - fosLTR / ColAREG mice ( n = 14 )

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c-fos mRNA expression levels in osteosarcomas of H2-c-fosLTR (n=11) and H2-c-fosLTR/ColAREG mice (n=14)


Western Blot of bone lysates from H2 - c - fosLTR H2 - c - fosLTR / ColAREG

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Western Blot analysis of bone tumor protein lysates from H2-c-fosLTR and H2-c-fosLTR/ColAREG mice


Representative of OS biopsies stained with antibodies against EGFR c ‑ Fos . Scale bars :

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Representative images of OS biopsies stained with antibodies against EGFR and c‑Fos. Scale bars: 50µm


Kaplan - Meier survival curve comparing survival of with EGFR c - Fos double positive osteosarcomas against without co - expression of both . ( n = 52 )

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Kaplan-Meier survival curve comparing the survival of patients with EGFR and c-Fos double positive osteosarcomas against patients without co-expression of both proteins. (n=52)


Western Blot showing EGFR c - Fos in human OS lines cultured under standard

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Western Blot analysis showing EGFR and c-Fos protein levels in human OS cell lines cultured under standard conditions


viability of 143b or LM7 cultured for 24h in medium ( + 10 % FCS ) with DMSO ( 1 : 2000 ) or Erlotinib ( 10µM ) ( n = 3 , representative result from 3 independent experiments , shown as fold change , normalized to DMSO )

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Cell viability of 143b or LM7 cells cultured for 24h in medium (+10% FCS) with DMSO (1:2000) or Erlotinib (10µM) (n=3, representative result from 3 independent experiments, shown as fold change, normalized to DMSO)


viability of 143b or LM7 cultured for 24h in medium ( + 0 . 5 % FCS ) containing 1xPBS ( contr . , 1 : 200 ) or EGF ( 50ng / ml ) ( n = 3 , representative result from 3 independent experiments , shown as fold change , normalized to control )

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Cell viability of 143b or LM7 cells cultured for 24h in medium (+0.5% FCS) containing 1xPBS (contr., 1:200) or EGF (50ng/ml) (n=3, representative result from 3 independent experiments, shown as fold change, normalized to control)


143b xenograft growth curve during therapeutic regime ( n = 8 vehicle , 6 Erlotinib ; two independent experiments ) 143b at endpoint ( n = 8 Vehicle , 6 Erlotinib ; two independent experiments )

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143b xenograft growth curve during therapeutic regime (n=8 vehicle, 6 Erlotinib; two independent experiments) 143b tumor weight at endpoint (n=8 Vehicle, 6 Erlotinib; two independent experiments)


LM7 xenograft growth curve during therapeutic regime ( n = 7 Vehicle , 5 Erlotinib ; two independent experiments ) LM7 at endpoint . n = 7 vehicle , 5 Erlotinib , two independent

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LM7 xenograft growth curve during therapeutic regime (n=7 Vehicle, 5 Erlotinib; two independent experiments) LM7 tumor weight at endpoint. n=7 vehicle, 5 Erlotinib, two independent experiment


of PCNA Caspase 3 in 143b - derived tumors ( n = 4 ) . Scale bars : 50µm of pCREB c - Fos in 143b - derived tumors . ( n = 5 for pCREB n = 4 for c - Fos ) . Scale bars :

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IHC analysis of PCNA and cleaved Caspase 3 in 143b-derived tumors (n=4). Scale bars: 50µm IHC analysis of pCREB and c-Fos in 143b-derived tumors. (n=5 for pCREB and n=4 for c-Fos analysis). Scale bars: 50µm


Western Blot of pCREB / CREB p - c - Fos / c - Fos expression in lysates directly isolated from 143b xenografts at endpoint , after 14 days of vehicle or Erlotinib

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Western Blot analysis of pCREB/CREB and p-c-Fos/c-Fos protein expression in lysates directly isolated from 143b xenografts at endpoint, after 14 days of vehicle or Erlotinib treatment


( B ) Automatic cluster - mapping of - dependent splicing changes in CHX , on changes in PSI ( spliced in ) . Categories ( right ) were manually assigned .

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(B) Automatic cluster-mapping of the temperature-dependent splicing changes in CHX, based on changes in PSI (percent spliced in). Categories (right) were manually assigned.


( C ) Examples for cold - induced ( blue ) or heat - induced ( red ) AS - decay via exon inclusion or exon skipping . In each example , on top a simplified exon - intron structure is given , PTCs of frameshift inducing exons are highlighted . Predicted splicing changes were validated by radioactive RT - PCR with primers binding to surrounding constitutive exons . Box plots show median PSI of validation PCRs of four different samples from at least two different mice , whiskers show min to max . data points are shown as circles .

O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O

(C) Examples for cold-induced (blue) or heat-induced (red) AS-decay via exon inclusion or exon skipping. In each example, on top a simplified exon-intron structure is given, PTCs and the length of frameshift inducing exons are highlighted. Predicted splicing changes were validated by radioactive RT-PCR with primers binding to the surrounding constitutive exons. Box plots show median PSI of validation PCRs of four different samples from at least two different mice, whiskers show min to max. Individual data points are shown as circles.


( A ) - dependent poison events in Hnrnpdl ( left , see also Figure 1C ) , Hnrnph3 ( middle ) Cirbp ( right ) . For each target , on top a simplified exon - intron structure is given below Sashimi plots show distribution of raw sequencing reads . Exon - Exon junction reads are indicated by numbers connecting exons . Below , conservation across placental species is indicated . Hnrnpdl exhibits a heat - included exon that leads to a PTC , Hnrnph3 a cold - skipped exon that leads to a frameshift , in Cirbp , heat leads to inclusion of an alternative transcript end , which leads to formation of a PTC . ( B ) For each gene from A , triplicate normalized read in DMSO ( left y - axis , green ) of poison isoform in CHX ( right y - axis , orange ) are plotted at two temperatures . represents mean PSI .

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(A) Temperature-dependent poison events in Hnrnpdl (left, see also Figure 1C), Hnrnph3 (middle) and Cirbp (right). For each target, on top a simplified exon-intron structure is given and below Sashimi plots show the distribution of raw sequencing reads. Exon-Exon junction reads are indicated by the numbers connecting the exons. Below, sequence conservation across placental species is indicated. Hnrnpdl exhibits a heat-included exon that leads to a PTC, Hnrnph3 a cold-skipped exon that leads to a frameshift, and in Cirbp, heat leads to inclusion of an alternative transcript end, which leads to formation of a PTC. (B) For each gene from A, the triplicate normalized read counts in DMSO (left y-axis, green) and the percentage of the poison isoform in CHX (right y-axis, orange) are plotted at the two temperatures. Line represents mean PSI.


( A ) - dependent poison events in Hnrnpdl ( left , see also Figure 1C ) , Hnrnph3 ( middle ) Cirbp ( right ) . For each target , on top a simplified exon - intron structure is given below Sashimi plots show distribution of raw sequencing reads . Exon - Exon junction reads are indicated by numbers connecting exons . Below , conservation across placental species is indicated . Hnrnpdl exhibits a heat - included exon that leads to a PTC , Hnrnph3 a cold - skipped exon that leads to a frameshift , in Cirbp , heat leads to inclusion of an alternative transcript end , which leads to formation of a PTC .

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(A) Temperature-dependent poison events in Hnrnpdl (left, see also Figure 1C), Hnrnph3 (middle) and Cirbp (right). For each target, on top a simplified exon-intron structure is given and below Sashimi plots show the distribution of raw sequencing reads. Exon-Exon junction reads are indicated by the numbers connecting the exons. Below, sequence conservation across placental species is indicated. Hnrnpdl exhibits a heat-included exon that leads to a PTC, Hnrnph3 a cold-skipped exon that leads to a frameshift, and in Cirbp, heat leads to inclusion of an alternative transcript end, which leads to formation of a PTC.


( C ) Correlation of poison isoform inclusion GE for RBPs with - dependent AS - decay skipped exon events . Shown are log2 fold change ( FC ) in GE versus ∆ PSI of poison isoform between CHX samples . Top shows GE change between DMSO samples bottom between CHX samples . R² P ( deviation from zero slope ) are indicated . N = 24 .

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(C) Correlation of poison isoform inclusion and GE levels for all RBPs with temperature-dependent AS-decay skipped exon events. Shown are the log2 fold change (FC) in GE versus the ∆PSI of the poison isoform between the CHX samples. Top shows the GE change between the DMSO samples and bottom between the CHX samples. R² and P (deviation from zero slope) are indicated. N=24.


( E ) Splicing reporters , containing only an intron ( GFP ctrl , # 1 ) or additionally - dependent exons with ~ 1kB surrounding introns . Representative radioactive RT - PCRs ( bottom ) confirm cold - induced inclusion of Ythdf3 exon 7 ( # 2 ) human Synerg exon 21 ( # 3 ) after 24 hours at indicated temperatures . Representative are derived from three independent gels . See SourceDataForFigure2E for uncropped versions of these gels .

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(E) Splicing reporters, containing only an intron (GFP ctrl, #1) or additionally temperature-dependent exons with ~1kB surrounding introns. Representative radioactive RT-PCRs (bottom) confirm cold-induced inclusion of Ythdf3 exon 7 (#2) and human Synerg exon 21 (#3) after 24 hours at the indicated temperatures. Representative images are derived from three independent gels. See SourceDataForFigure2E for uncropped versions of these gels.


( F - G ) GFP expression was quantified using FACS is shown relative to GFP ctrl . In ( F ) transfected were incubated at indicated temperatures for 24 hours ( n = 3 , mean + / - SD ) . In ( G ) were incubated directly after transfection for 12 hours at 39 ° C ( blue ) or 33 ° C ( red ) then shifted for 2 , 4 , 8 12 hours ( n = 3 , mean + / - SD ) .

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(F-G) GFP expression was quantified using FACS and is shown relative to GFP ctrl. In (F) transfected cells were incubated at the indicated temperatures for 24 hours (n=3, mean +/- SD). In (G) cells were incubated directly after transfection for 12 hours at 39°C (blue) or 33°C (red) and then shifted for 2, 4, 8 and 12 hours (n=3, mean +/- SD).


( A ) SR specific - dependent AS - decay . Sashimi plots are shown for heat - induced poison exon inclusion for Srsf2 ( left ) cold - induced poison isoform formation for Srsf10 ( right ) . of GE AS are presented in center ( as in Figure 2B ) , lines represent mean .

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(A) SR protein specific temperature-dependent AS-decay. Sashimi plots are shown for heat-induced poison exon inclusion for Srsf2 (left) and cold-induced poison isoform formation for Srsf10 (right). Quantification of GE and AS are presented in the center (as in Figure 2B), lines represent mean.


( B ) Poison exon inclusion for Srsf2 ( left ) Srsf10 ( right ) in a 24h - rhythm in human . Hek293 were pre - entrained with square - wave cycles ( 12h 34 ° C / 12h 38 ° C ) for 48h . For last 24h , were treated with DMSO or CHX every 4h harvested after 4h analyzed by splicing sensitive RT - PCR ( n = 3 , mean ± SD ) . : 34 ° C ; Red : 38 ° C . In Hek293 , inclusion of Srsf10 exon 3 is coupled to polyadenylation making it a weak decay target .

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(B) Poison exon inclusion for Srsf2 (left) and Srsf10 (right) in a 24h temperature-rhythm in human cells. Hek293 cells were pre-entrained with square-wave temperature cycles (12h 34°C/ 12h 38°C) for 48h. For the last 24h, cells were treated with DMSO or CHX every 4h and harvested after 4h and analyzed by splicing sensitive RT-PCR (n=3, mean ± SD). White area: 34°C; Red area: 38°C. In Hek293 cells, inclusion of Srsf10 exon 3 is coupled to polyadenylation making it a weak decay target.


( C ) AS - decay of human targets at 34 ° C ( blue ) 38 ° C ( red ) was investigated after a square - wave regime ( see B ) . RNAs were harvested after 56 68 hours , respectively ( n = 3 , mean ± SD ) . Statistical significance was determined by 2 - way ANOVA is indicated by asterisks : adjusted p values : not significant ( ns ) , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Box plots show median PSI of validation PCRs , whiskers show min to max . data points are shown as circles .

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(C) AS-decay of human targets at 34°C (blue) and 38°C (red) was investigated after a square-wave temperature regime (see B). RNAs were harvested after 56 and 68 hours, respectively (n=3, mean ± SD). Statistical significance was determined by 2-way ANOVA and is indicated by asterisks: adjusted p values: not significant (ns), **p<0.01, ***p<0.001, ****p<0.0001. Box plots show median PSI of validation PCRs, whiskers show min to max. Individual data points are shown as circles.


( E ) of Srsf2 AS - decay in hamster , rabbit chicken ( n = 2 , mean ± SD ) . Statistical significance was determined by 2 - way ANOVA , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Box plots show median PSI of validation PCRs , whiskers show min to max . data points are shown as circles .

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(E) Quantification of Srsf2 AS-decay in hamster, rabbit and chicken cells (n=2, mean ± SD). Statistical significance was determined by 2-way ANOVA, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Box plots show median PSI of validation PCRs, whiskers show min to max. Individual data points are shown as circles.


Rhythmic Srsf2 productive mRNA ( relative to Gapdh ) were analyzed by RT - qPCR . For each clone , expression is normalized to time point 52h ,

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Rhythmic Srsf2 productive mRNA levels (relative to Gapdh) were analyzed by RT-qPCR. For each clone, expression is normalized to time point 52h,


Rhythmic Srsf10 productive mRNA ( relative to Gapdh ) were analyzed by RT - qPCR . For each clone , expression is normalized to time point 52h For Srsf10 two independently generated clones were investigated ( ∆ E3 A B ) . Statistical significance was determined by unpaired t - is indicated by asterisks : p values : * * p < 0 . 01 , * * * * p < 0 . 0001 .

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Rhythmic Srsf10 productive mRNA levels (relative to Gapdh) were analyzed by RT-qPCR. For each clone, expression is normalized to time point 52h For Srsf10 two independently generated clones were investigated (∆E3 A and B). Statistical significance was determined by unpaired t-test and is indicated by asterisks: p values: **p<0.01, ****p<0.0001.


Comparing - induced expression of clock components Bmal1 in WT Srsf10 ∆ E3 ( relative to Gapdh , normalized to time point 52h ) . Statistical significance was determined by unpaired t - is indicated by asterisks : p values : * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . n = 3 , mean ± .

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Comparing temperature-induced expression of the core clock components Bmal1 in WT and Srsf10 ∆E3 cells (relative to Gapdh, normalized to time point 52h). Statistical significance was determined by unpaired t-test and is indicated by asterisks: p values: *p<0.05, ***p<0.001, ****p<0.0001. n=3, mean ± SEM.


Comparing - induced expression of clock components Per1 in WT Srsf10 ∆ E3 ( relative to Gapdh , normalized to time point 52h ) . Statistical significance was determined by unpaired t - is indicated by asterisks : p values : * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . n = 3 , mean ± .

Comparing temperature-induced expression of the core clock components Per1 in WT and Srsf10 ∆E3 cells (relative to Gapdh, normalized to time point 52h). Statistical significance was determined by unpaired t-test and is indicated by asterisks: p values: *p<0.05, ***p<0.001, ****p<0.0001. n=3, mean ± SEM.


( A ) Normalized GE values of SR in a 48 - hour time course . In first day , were kept at 20 ° C ( red ) shifted to 4 ° C ( blue ) for second day . were under a 12 - hour / regimen ( gray / yellow bars ) . Warm - cold - expressed genes are indicated .

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(A) Normalized GE values of plant SR proteins in a 48-hour time course. In the first day, plants were kept at 20°C (red) and shifted to 4°C (blue) for the second day. Plants were under a 12-hour dark/light regimen (gray/yellow bars). Warm- and cold-expressed genes are indicated.


( C ) Sashimi plots as shown in Figure 2A ( without conservation score ) for decay - inducing AS events upon cold ( At - SCL33 , left ) or warm ( At - SR34 , right ) . Data shown represent , from top to bottom , warm - , warm - , cold - cold - conditions as indicated on right of plots . ( D ) For validation , A . thaliana were incubated for 3 days either at 20 ° C or 4 ° C was investigated for gene expression ( green , left y - axis ) poison isoform formation ( orange , right y - axis ) . Gene expression of At - SCL33 ( left ) or At - SR34 ( right ) is shown relative to Ipp2 . Lines represent mean , data points are shown .

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(C) Sashimi plots as shown in Figure 2A (without conservation score) for decay-inducing AS events upon cold (At-SCL33, left) or warm temperature (At-SR34, right). Data shown represent, from top to bottom, warm-dark, warm-light, cold-dark and cold-light conditions as indicated on the right of the plots. (D) For validation, A. thaliana were incubated for 3 days either at 20°C or 4°C and RNA was investigated for gene expression (green, left y-axis) and poison isoform formation (orange, right y-axis). Gene expression of At-SCL33 (left) or At-SR34 (right) is shown relative to Ipp2. Lines represent mean, individual data points are shown.


( C ) Sashimi plots as shown in Figure 2A ( without conservation score ) for decay - inducing AS events upon cold ( At - SCL33 , left ) or warm ( At - SR34 , right ) . Data shown represent , from top to bottom , warm - , warm - , cold - cold - conditions as indicated on right of plots .

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(C) Sashimi plots as shown in Figure 2A (without conservation score) for decay-inducing AS events upon cold (At-SCL33, left) or warm temperature (At-SR34, right). Data shown represent, from top to bottom, warm-dark, warm-light, cold-dark and cold-light conditions as indicated on the right of the plots.


( E ) Correlation of poison isoform inclusion GE for - dependent poison skipped exon events . Shown are log2 fold change ( FC ) in GE versus ∆ PSI of poison isoform . Decay targets were identified by using data from CHX - treated . R² P ( deviation from zero slope ) are indicated . N = 80

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(E) Correlation of poison isoform inclusion and GE levels for temperature-dependent poison skipped exon events. Shown are the log2 fold change (FC) in GE versus the ∆PSI of the poison isoform. Decay targets were identified by using data from CHX-treated plants. R² and P(deviation from zero slope) are indicated. N=80


( E ) Correlation of poison isoform inclusion GE for - dependent poison skipped exon events . Shown are log2 fold change ( FC ) in GE versus ∆ PSI of poison isoform . Decay targets were identified by using data from CHX - treated . R² P ( deviation from zero slope ) are indicated . N =

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( B ) Correlation of rhythmic Srsf10 expression ( orange , left y - axis ) exon 3 inclusion ( black , right y - axis ) in mouse liver samples from indicated ZTs ( n = 4 , mean + / - ) . Quantifications were obtained by - seq of datasets from ( Atger et al . , 2015 ) .

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(B) Correlation of rhythmic Srsf10 expression (orange, left y-axis) and exon 3 inclusion (black, right y-axis) in mouse liver samples from the indicated ZTs (n=4, mean +/- SEM). Quantifications were obtained by RNA-seq analysis of datasets from (Atger et al., 2015).


( C ) Representative Western blot of SRSF10 from mouse liver extracts ( NX ) from different ZTs . different SRSF10 variants are highlighted on right . SRSF10 - fl SRSF10 - 2 are result of differential last exon usage . hnRNP L was used as a loading control . asterisks could represent hyperphosphorylated SRSF10 - fl through higher CLK activity during day . ( D ) of SRSF10 - fl + SRSF10 - 2 relative to ZT0 hnRNP L ( mean of at least 3 mice ± ) . Student ' s unpaired t - derived p values * * * p < 0 . 001 .

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(C) Representative Western blot of SRSF10 protein levels from mouse liver nuclear extracts (NX) from different ZTs. The different SRSF10 variants are highlighted on the right. SRSF10-fl and SRSF10-2 are the result of differential last exon usage. hnRNP L was used as a loading control. The asterisks could represent hyperphosphorylated SRSF10-fl through higher CLK activity during the day. (D) Quantification of SRSF10-fl + SRSF10-2 relative to ZT0 and hnRNP L (mean of at least 3 mice ± SEM). Student's unpaired t test-derived p values ***p<0.001.


( C ) Representative Western blot of SRSF10 from mouse liver extracts ( NX ) from different ZTs . different SRSF10 variants are highlighted on right . SRSF10 - fl SRSF10 - 2 are result of differential last exon usage . hnRNP L was used as a loading control . asterisks could represent hyperphosphorylated SRSF10 - fl through higher CLK activity during day .

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(C) Representative Western blot of SRSF10 protein levels from mouse liver nuclear extracts (NX) from different ZTs. The different SRSF10 variants are highlighted on the right. SRSF10-fl and SRSF10-2 are the result of differential last exon usage. hnRNP L was used as a loading control. The asterisks could represent hyperphosphorylated SRSF10-fl through higher CLK activity during the day.


( A B ) Root ( averages ± SD , n ≥ 15 ) ( A ) of cotyledons root tips ( B ) of Arabidopsis seedlings that were exposed to 100 nM Pep2 for 7 days , 2 days after germination . BAK1 ( D416N ) is a kinase - dead variant .

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(A and B) Root length (averages ± SD, n ≥ 15) (A) and cell death staining of cotyledons and primary root tips (B) of Arabidopsis seedlings that were exposed to 100 nM Pep2 for 7 days, 2 days after germination. BAK1 (D416N) is a kinase-dead variant.


(C) Overview of 3-week-old plants.


( D ) Pep2 induces PEPR1 - FLAG association with SERK1 - , SERK2 - , BKK1 - , BAK1 - HA in N . benthamiana . Experiments were repeated at least three times , with same conclusions . IP IB denote imunoprecipitation immunoblotting with indicated antibodies , respectively . NT indicates non - transformed . ( * , p < 0 . 05 in two - compared to differences ( ± Pep2 ) from corresponding values of WT . )

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(D) Pep2 induces PEPR1-FLAG association with SERK1-, SERK2-, BKK1-, and BAK1-HA in N. benthamiana. Experiments were repeated at least three times, with the same conclusions. IP and IB denote imunoprecipitation and immunoblotting with the indicated antibodies, respectively. NT indicates non-transformed plants. (*, p < 0.05 in two-tailed tests compared to the differences (± Pep2) from the corresponding values of WT plants.)


( A ) Immunoblot for BIK1 - HA in Arabidopsis protoplasts exposed to Pep2 or flg22 for 10 min . Arrowheads indicate phosphorylated BIK1 - HA .

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(A) Immunoblot analysis for BIK1-HA in Arabidopsis protoplasts exposed to Pep2 or flg22 for 10 min. Arrowheads indicate phosphorylated BIK1-HA.


( B ) Immunoblot for Pep2 - triggered MAPK activation . Ponceau S - stained loading controls are shown ( bottom ) .

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(B) Immunoblot analysis for Pep2-triggered MAPK activation. Ponceau S-stained loading controls are shown (bottom).


( C ) Root ( averages ± SD , n ≥ 15 ) of Arabidopsis seedlings that were exposed to 100 nM Pep2 . ( * , p < 0 . 05 in two - compared to differences ( ± Pep2 ) from corresponding values of bak1 - 3 . Two independent experiments were combined for statistical . )

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(C) Root length (averages ± SD, n ≥ 15) of Arabidopsis seedlings that were exposed to 100 nM Pep2. (*, p < 0.05 in two-tailed tests compared to the differences (± Pep2) from the corresponding values of bak1-3 plants. Two independent experiments were combined for statistical analysis.)


( A ) Scatter plots for log2 fold changes of gene expression in WT versus bak1 - 3 treated with Pep2 for 2 10 h . regression of these scatter plots is indicated by red lines .

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(A) Scatter plots for log2 fold changes of gene expression in WT plants versus bak1-3 plants treated with Pep2 for 2 and 10 h. The regression of these scatter plots is indicated by the red lines.


( B ) Hierarchical of genes exhibiting a > 2 - fold change in expression level in bak1 - 3 after Pep2 application for 10 h in a whole - genome microarray . Using Genevestigator v3 , these genes were separately cross - referenced to public databases to show their expression responses to salicylate ( SA ) , methyl - jasmonate ( JA ) , or ethylene ( ET ) .

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(B) Hierarchical analysis of genes exhibiting a >2-fold change in expression level in bak1-3 plants after Pep2 application for 10 h in a whole-genome microarray analysis. Using Genevestigator v3, these genes were separately cross-referenced to public databases to show their expression responses to salicylate (SA), methyl-jasmonate (JA), or ethylene (ET).


( C D ) qRT - PCR of defense - related genes in 10 - day - old seedlings exposed to 0 . 5 µM Pep2 for 10 h . Data are averages ( ± SD ) of three biological replicates . ( * , p < 0 . 05 in two - compared to corresponding bak1 - 4 bak1 - 3 values in C D , respectively . Relative cycle threshold ( Ct ) values of two independent experiments were combined for statistical . )

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(C and D) qRT-PCR analysis of defense-related genes in 10-day-old seedlings exposed to 0.5 µM Pep2 for 10 h. Data are averages (±SD) of three biological replicates. (*, p < 0.05 in two-tailed tests compared to the corresponding bak1-4 and bak1-3 values in C and D, respectively. Relative cycle threshold (Ct) values of two independent experiments were combined for statistical analysis.)


( A ) qRT - PCR of PROPEP genes in 10 - day - old seedlings exposed to 0 . 5 µM Pep2 for 10 h . * , p < 0 . 05 in two - compared to corresponding WT values . Relative Ct values of two independent experiments with three biological replicates each were combined for statistical .

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

(A) qRT-PCR analysis of PROPEP genes in 10-day-old seedlings exposed to 0.5 µM Pep2 for 10 h. *, p < 0.05 in two-tailed tests compared to the corresponding WT values. Relative Ct values of two independent experiments with three biological replicates each were combined for statistical analysis.


( B ) Anti - GFP ( upper ) anti - PROPEP3 ( middle ) immunoblot of PROPEP3 - Venus in seedlings submerged in liquid medium treated with ( + ) or without ( - ) 0 . 5 M Pep2 for indicated times . positions of molecular markers are shown on left . Arrowheads asterisks indicate PROPEP3 - Venus cross - reactive bands , respectively .

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(B) Anti-GFP (upper) and anti-PROPEP3 (middle) immunoblot analysis of PROPEP3-Venus in seedlings submerged in liquid medium and treated with (+) or without (-) 0.5 M Pep2 for the indicated times. The positions of the molecular weight markers are shown on the left. Arrowheads and asterisks indicate PROPEP3-Venus and cross-reactive bands, respectively.


( C , D E ) Immunoblot of PEPR1 - FLAG in 10 - day - old seedlings following Pep1 or flg22 application . ( C D ) were exposed to 1 M Pep1 or flg22 for indicated times . ( E ) were pre - exposed to MG132 ( 50 µM ) , Wortmannin ( Wm ) ( 30 µM ) Tyrphostin A23 ( TyrA23 ) ( 100 µM ) for 1 h before being exposed to Pep1 for 10 h . numbers below immunoblots represent relative intensities of PROPEP - Venus ( B ) PEPR1 - FLAG ( C - E ) bands normalized to backgrounds in Ponceau S - stained loading controls ( bottom ) , with band / background values in WT extracts 24 h ( upper ) 0 h ( middle ) after Pep2 application , respectively , set as 1 . 0 .

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(C, D and E) Immunoblot analysis of PEPR1-FLAG in 10-day-old seedlings following Pep1 or flg22 application. (C and D) Plants were exposed to 1 M Pep1 or flg22 for the indicated times. (E) Plants were pre-exposed to MG132 (50 µM), Wortmannin (Wm) (30 µM) and Tyrphostin A23 (TyrA23) (100 µM) for 1 h before being exposed to Pep1 for 10 h. The numbers below the immunoblots represent relative intensities of the PROPEP-Venus (B) and PEPR1-FLAG (C-E) bands normalized to the backgrounds in the Ponceau S-stained loading controls (bottom), with the band/background values in WT plant extracts 24 h (upper) and 0 h (middle) after Pep2 application, respectively, set as 1.0.


( A ) qRT - PCR of defense - related genes in 10 - day - old seedlings exposed to 0 . 5 µM Pep2 for 10 h . ( * , p < 0 . 05 in two - compared to corresponding WT values . Relative Ct values of two independent experiments with three biological replicates each were combined for statistical . )

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(A) qRT-PCR analysis of defense-related genes in 10-day-old seedlings exposed to 0.5 µM Pep2 for 10 h. (*, p < 0.05 in two-tailed tests compared to the corresponding WT values. Relative Ct values of two independent experiments with three biological replicates each were combined for statistical analysis.)


( B ) Root ( averages ± SD , n ≥ 15 ) of Arabidopsis seedlings exposed to 100 nM Pep2 . ( * , p < 0 . 05 in two - compared to differences ( ± Pep2 ) from corresponding values of WT . Two independent experiments were combined for statistical . )

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(B) Root length (averages ± SD, n ≥ 15) of Arabidopsis seedlings exposed to 100 nM Pep2. (*, p < 0.05 in two-tailed tests compared to the differences (± Pep2) from the corresponding values of WT plants. Two independent experiments were combined for statistical analysis.)


( C ) Fresh ( averages ± SD , n ≥ 25 ) of 2 - week - old grown on soil . ( * , p < 0 . 05 in two - compared to corresponding values of WT . ■ , p < 0 . 05 in two - . Two independent experiments were combined for statistical . )

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(C) Fresh weight (averages ± SD, n ≥ 25) of 2-week-old plants grown on soil. (*, p < 0.05 in two-tailed tests compared to the corresponding values of WT plants. ■, p < 0.05 in two-tailed tests. Two independent experiments were combined for statistical analysis.)


( D ) Immunoblot for BAK1 in 10 - day - old seedlings with or without 0 . 5 M Pep1 for 24 h . numbers below upper panel represent relative intensities of BAK1 band normalized to backgrounds in Ponceau S - stained loading controls , with value in non - elicited WT set as 1 . 0 .

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(D) Immunoblot analysis for BAK1 in 10-day-old seedlings with or without 0.5 M Pep1 for 24 h. The numbers below the upper panel represent relative intensities of the BAK1 band normalized to the backgrounds in the Ponceau S-stained loading controls, with the value in non-elicited WT plants set as 1.0.


( A ) qRT - PCR in 4 - week - old leaves challenged with indicated P . syringae pv . tomato ( Pst ) DC3000 strains for indicated times .

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(A) qRT-PCR analysis in 4-week-old plant leaves challenged with the indicated P. syringae pv. tomato (Pst) DC3000 strains for the indicated times.


( B ) Anti - GFP immunoblot analyses in seedlings challenged with Pst DC3000 ( DC3000 ) Pst DC3000 ∆ hrcC ( ∆ hrcC ) for indicated times .

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(B) Anti-GFP immunoblot analyses in seedlings challenged with Pst DC3000 (DC3000) and Pst DC3000 ∆hrcC (∆hrcC) for the indicated times.


. ( C ) Anti - PROPEP3 immunoblot of PROPEP3 - Venus ( upper ) in tissues ( expressed in ) in extracellular fraction ( released into medium ) , 24 h 48 h after inoculation with ( + ) or without ( - ) Pst DC3000 . Lower panels indicate Ponceau S - stained loading controls . numbers below upper panel represent relative intensities of PROPEP3 - Venus band normalized to backgrounds in Ponceau S - stained loading controls . NT denotes non - transgenic WT .

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

. (C) Anti-PROPEP3 immunoblot analysis of PROPEP3-Venus (upper) in plant tissues (expressed in plants) and in the extracellular fraction (released into medium), 24 h and 48 h after the inoculation with (+) or without (-) Pst DC3000. Lower panels indicate Ponceau S-stained loading controls. The numbers below the upper panel represent relative intensities of the PROPEP3-Venus band normalized to the backgrounds in the Ponceau S-stained loading controls. NT denotes non-transgenic WT plants.


( D ) Trypan blue of Arabidopsis seedlings challenged with Pst DC3000 or Pst DC3000 AvrRpm1 ( AvrRpm1 ) for 48 h .

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(D) Trypan blue cell death staining of Arabidopsis seedlings challenged with Pst DC3000 or Pst DC3000 AvrRpm1 (AvrRpm1) for 48 h.


( E ) Anti - PROPEP3 immunoblot analyses in seedlings exposed to indicated Pst DC3000 strains or 1 M Pep1 for 48 h .

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(E) Anti-PROPEP3 immunoblot analyses in the seedlings exposed to the indicated Pst DC3000 strains or 1 M Pep1 for 48 h.


( F ) Anti - GFP immunoblot for extracellular fraction of seedlings exposed to Pst DC3000 AvrRpm1 for 48 h . ( B , C , E F ) Arrowheads arrows indicate an apparently full - processed form of PROPEP3 - Venus , respectively . Asterisks indicate cross - reactive bands . positions of molecular markers are shown on left of immunoblots .

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

(F) Anti-GFP immunoblot analysis for the extracellular fraction of the seedlings exposed to Pst DC3000 AvrRpm1 for 48 h. (B, C, E and F) Arrowheads and arrows indicate an apparently full-length and processed form of PROPEP3-Venus, respectively. Asterisks indicate cross-reactive bands. The positions of the molecular weight markers are shown on the left of the immunoblots.


( A ) Growth of H . arabidopsidis ( Hpa ) Noco2 in 2 - week - old . Spores were counted at 6 days post inoculation ( dpi ) . Results are averages ± SE of three biological replicates . ( * , p < 0 . 05 in two - compared to corresponding values of Col . ■ , p < 0 . 05 in two - between indicated values . Three independent experiments were combined for statistical . )

O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM 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) Growth of H. arabidopsidis (Hpa) Noco2 in 2-week-old plants. Spores were counted at 6 days post inoculation (dpi). Results are averages ± SE of three biological replicates. (*, p < 0.05 in two-tailed tests compared to the corresponding values of Col plants. ■, p < 0.05 in two-tailed tests between the indicated values. Three independent experiments were combined for statistical analysis.)


( B ) Growth of syringe - infiltrated Pst DC3000 in rosette leaves of 4 - week - old pretreated with water ( Mock ) or 1 M flg22 for 24 h . titers were determined at 3 dpi . Results are averages ± SE of three independent replicates . ( * , p < 0 . 05 , * * , p < 0 . 01 in two - compared to corresponding values of WT . ▲ , p < 0 . 01 in two - compared to differences ( ± flg22 ) from corresponding values of WT . Three independent experiments were combined for statistical . )

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

(B) Growth of syringe-infiltrated Pst DC3000 in rosette leaves of 4-week-old plants pretreated with water (Mock) or 1 M flg22 for 24 h. Bacterial titers were determined at 3 dpi. Results are averages ± SE of three independent replicates. (*, p < 0.05, **, p < 0.01 in two-tailed tests compared to the corresponding values of WT plants. ▲, p < 0.01 in two-tailed tests compared to the differences (± flg22) from the corresponding values of WT plants. Three independent experiments were combined for statistical analysis.)


( C ) Total SA were determined in rosette leaves of 4 - week - old with or without 1 µM flg22 application for 24 h . Data are averages ( ± SD ) of material from three independent . experiments were repeated twice , with similar results . ( ▲ , p < 0 . 01 in two - compared to differences ( ± flg22 ) from corresponding values of WT . Two independent experiments were combined for statistical . )

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(C) Total SA levels were determined in the rosette leaves of 4-week-old plants with or without 1 µM flg22 application for 24 h. Data are averages (±SD) of material from three independent plants. The experiments were repeated twice, with similar results. (▲, p < 0.01 in two-tailed tests compared to the differences (± flg22) from the corresponding values of WT plants. Two independent experiments were combined for statistical analysis.)


( D ) qRT - PCR of PR1 gene in 4 - week - old exposed to 1 µM flg22 . Data are averages ( ± SD ) of three biological replicates . ( * , p < 0 . 05 in two - compared to corresponding WT values . Relative Ct values of four independent experiments were combined for statistical . )

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

(D) qRT-PCR analysis of the PR1 gene in 4-week-old plants exposed to 1 µM flg22. Data are averages (±SD) of three biological replicates. (*, p < 0.05 in two-tailed tests compared to the corresponding WT values. Relative Ct values of four independent experiments were combined for statistical analysis.)


( E ) Growth of syringe - infiltrated Pst DC3000 in rosette leaves of 4 - week - old pretreated with water ( Mock ) or 1 M flg22 for 24 h . titers were determined at 3 dpi . Results are averages ± SE of three independent replicates . ( * * , p < 0 . 01 , * * * , p < 1 . 0e - 10 in two - compared to corresponding values of WT . ▲ , p < 0 . 01 in two - compared to differences ( ± flg22 ) from corresponding values of WT . Three independent experiments were combined for statistical . )

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(E) Growth of syringe-infiltrated Pst DC3000 in rosette leaves of 4-week-old plants pretreated with water (Mock) or 1 M flg22 for 24 h. Bacterial titers were determined at 3 dpi. Results are averages ± SE of three independent replicates. (**, p < 0.01, ***, p < 1.0e-10 in two-tailed tests compared to the corresponding values of WT plants. ▲, p < 0.01 in two-tailed tests compared to the differences (± flg22) from the corresponding values of WT plants. Three independent experiments were combined for statistical analysis.)


( A ) Lesion sizes ( left ) were determined 5 days after C . higginsianum ( Ch ) inoculation . Invasion rates ( right ) of Ch on Arabidopsis leaves were determined 3 days after spore inoculation . An average of 3 replicates is shown ( ± SD ) ( left ) ( ± SE ) ( right ) . ( ■ , p < 0 . 05 in two - between indicated samples . * , p < 0 . 05 in two - compared to corresponding WT values . Two independent experiments were combined for statistical . )

O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM O O O O O O O O O B-ORGANISM O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Lesion sizes (left) were determined 5 days after C. higginsianum (Ch) inoculation. Invasion rates (right) of Ch on Arabidopsis leaves were determined 3 days after spore inoculation. An average of 3 replicates is shown (±SD) (left) and (±SE) (right). (■, p < 0.05 in two-tailed tests between the indicated samples. *, p < 0.05 in two-tailed tests compared to the corresponding WT values. Two independent experiments were combined for statistical analysis.)


B ) Immunoblot ( left ) qRT - PCR ( right ) analyses following Ch challenge for indicated times ( dpi ) . numbers below immunoblots represent relative intensities of indicated bands normalized to backgrounds , with values at 0 dpi set as 1 . 0 . Experiments were performed three times with same conclusions .

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B) Immunoblot (left) and qRT-PCR (right) analyses following Ch challenge for the indicated times (dpi). The numbers below the immunoblots represent relative intensities of the indicated bands normalized to the backgrounds, with the values at 0 dpi set as 1.0. Experiments were performed three times with the same conclusions.


E . Microscale thermophoresis binding experiment for PlexA1 - 4 - mVenus Sema1becto ( red ) or Sema1becto - mutA ( blue ) or Sema1becto - mutB ( green , ) or Sema1becto - mutA + B ( orange ) . Sema1becto wild - type binding results are as reported previously ( Rozbesky et al . , 2019 ) . data were collected at same time , error bars represent s . d . of three technical replicates .

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E. Microscale thermophoresis binding experiment for PlexA1-4-mVenus and Sema1becto (red) or Sema1becto-mutA (blue) or Sema1becto-mutB (green,) or Sema1becto-mutA+B (orange). The Sema1becto wild-type binding results are as reported previously (Rozbesky et al., 2019). All data were collected at the same time, error bars represent s.d. of three technical replicates.


A . FRET - FLIM in live COS - 7 shows surface PlexA - Sema1b cis interaction . Sema1b mutants reveal that both head - to - head side - on orientations of PlexA - Sema1b are involved in cis interaction . cis interaction between Sema1a PlexA is not observed . boxcharts represent average lifetime . Box limits indicate 25th 75th percentiles , centred lines show median , squares represent sample means , whiskers extend 1 . 5 - fold interquartile range from 25th 75th percentiles , p - value was calculated by one - way of variance ( ANOVA ) . * P < 0 . 05 , * * * P < 0 .

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A. FRET-FLIM in live COS-7 cells shows cell surface PlexA-Sema1b cis interaction. Sema1b mutants reveal that both head-to-head and side-on orientations of the PlexA-Sema1b complex are involved in the cis interaction. The cis interaction between Sema1a and PlexA is not observed. The boxcharts represent the average lifetime. Box limits indicate the 25th and 75th percentiles, centred lines show the median, squares represent sample means, whiskers extend 1.5-fold the interquartile range from the 25th and 75th percentiles, p-value was calculated by one-way analysis of variance (ANOVA). *P<0.05, ***P<0.001


B . Representative negative stain class averages of PlexAecto correlate well with ring - like conformation of mouse PlxnA1 . ring - like conformation of mouse PlxnA1 is shown in surface representation rainbow colour ramped from blue ( N terminus ) to red ( C terminus ) . Scale bar , 10 nm .

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B. Representative negative stain class averages of PlexAecto correlate well with the ring-like conformation of mouse PlxnA1. The ring-like conformation of mouse PlxnA1 is shown in surface representation rainbow colour ramped from blue (N terminus) to red (C terminus). Scale bar, 10 nm.


E . In vitro FRET - FLIM measurement of PlexA - FRET Biosensor shows that PlexA ectodomain undergoes a conformational change upon treatment with Sema1b resulting in more open conformation . boxcharts represent average lifetime . Box limits indicate 25th 75th percentiles , centred lines show median , squares represent sample means , whiskers extend 1 . 5 - fold interquartile range from 25th 75th percentiles , p - value was calculated by one - way of variance ( ANOVA ) . * * * P < 0 . 001 .

O O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. In vitro FRET-FLIM measurement of the PlexA-FRET Biosensor shows that the PlexA ectodomain undergoes a conformational change upon treatment with Sema1b resulting in more open conformation. The boxcharts represent the average lifetime. Box limits indicate the 25th and 75th percentiles, centred lines show the median, squares represent sample means, whiskers extend 1.5-fold the interquartile range from the 25th and 75th percentiles, p-value was calculated by one-way analysis of variance (ANOVA). ***P<0.001.


F . Relative change of COS - 7 surface upon treatment with Sema1a - Fc . expressing PlexAFL - mClover or PlexAFL - mCover Sema1b - mRuby2 wild - type or mutants were treated with purified Sema1a - Fc at a final of 5 . 8 µM . were acquired every minute for 30 minutes . surface was calculated using ImageJ before after stimulation . Data are presented as means ± . * P < 0 . 05 , * * * P < 0 . 001 , p - value calculated by one - way of variance ( ANOVA ) .

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

F. Relative change of COS-7 cells surface area upon treatment with Sema1a-Fc. Cells expressing PlexAFL-mClover or PlexAFL-mCover and Sema1b-mRuby2 wild-type or mutants were treated with purified Sema1a-Fc at a final concentration of 5.8 µM. Images were acquired every minute for 30 minutes. Cell surface area was calculated using ImageJ before and after stimulation. Data are presented as means ± sem. *P<0.05, ***P<0.001, p-value calculated by one-way analysis of variance (ANOVA).


A . Cultured dorsal root ganglion ( DRG ) neurons from E12 . 5 Sema6A knockout ( KO ) were transfected with EGFP , EGFP - tagged wild - type ( WT ) Sema6A ( Sema6A - WT ) or different EGFP - tagged Sema6A mutants ( Sema6A - mutA , Sema6A - mutB , Sema6A - mutA + B ) at 1 day in vitro ( DIV1 ) treated with purified Sema6A - Fc at a of 75 nM for 1 hour at 37 ° C at DIV4 . Transfection of EGFP was used as a control ( full collapse ) . DRG neurons from Sema6A KO acquired sensitivity to Sema6A growth cone collapse was observed in three independent experiments . Transfection of Sema6A - WT , Sema6A - mutA Sema6A - mutB but not of EGFP or Sema6A - mutantA + B prevented Sema6A - mediated growth cone collapse ( Fisher ' s exact p < 0 . 0001 ) . of growth cone collapse was performed using a growth cone matrix ( lower part panel A ) . Data is presented as of morphologically distinct growth cones , divided ( represented by dotted ) in two categories , from uncollapsed ( 1 - 4 ) to fully collapsed ( 5 - 8 ) . Totally , 20 - 40 growth cones were analyzed for each condition per experiment ( n = 3 experiments ) . Scale bar 20 µm .

O O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O B-CELL_TYPE O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-TISSUE B-CELL_TYPE O B-GENEPROD O O O O B-GENEPROD B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O

A. Cultured dorsal root ganglion (DRG) neurons from E12.5 Sema6A knockout (KO) embryos were transfected with EGFP, EGFP-tagged wild-type (WT) Sema6A (Sema6A-WT) or different EGFP-tagged Sema6A mutants (Sema6A-mutA, Sema6A-mutB, and Sema6A-mutA+B) at 1 day in vitro (DIV1) and treated with purified Sema6A-Fc at a concentration of 75 nM for 1 hour at 37°C at DIV4. Transfection of EGFP was used as a control (full collapse). DRG neurons from Sema6A KO embryos acquired sensitivity to Sema6A and growth cone collapse was observed in three independent experiments. Transfection of Sema6A-WT, Sema6A-mutA and Sema6A-mutB but not of EGFP or Sema6A-mutantA+B prevented Sema6A-mediated growth cone collapse (Fisher's exact test p<0.0001). Quantification of growth cone collapse was performed using a growth cone morphology matrix (lower part panel A). Data is presented as percentage of morphologically distinct growth cones, divided (represented by the dotted line) in two categories, from uncollapsed (1-4) to fully collapsed (5-8). Totally, 20-40 growth cones were analyzed for each condition per experiment (n=3 experiments). Scale bar 20 µm.


B . Representative of growth cones of mouse DRG neurons transfected with WT or mutant constructs treated with 75 nM Sema6A - Fc visualized with immunofluorescent to detect GFP ( green ) phalloidin ( red ) . Average for different constructs are shown in grey color . Scale bar 20 µm .

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B. Representative images of growth cones of mouse DRG neurons transfected with WT or mutant constructs treated with 75 nM Sema6A-Fc and visualized with immunofluorescent staining to detect GFP (green) and phalloidin (red). Average intensity images for all different constructs are shown in grey color. Scale bar 20 µm.


( A ) Hep3B hepatocytes were treated with nontargeting control siRNA ( siNT ) or siRNA targeting Dyn2 ( siDyn2 ) , followed by re - expression of either vector alone , or GFP - tagged versions of Dyn2 , loaded with 150 µM oleate overnight starved for 48 h in medium containing 0 . 1 % FBS . LDs were visualized by immunofluorescence using Oil Red O . boundaries are outlined those re - expressing GFP , GFP - wtDyn2 , or GFP - - K44A are denoted with asterisks . Bars , 20 µM . ( B ) Representative blot showing efficiency of Dyn2 knockdown in these . ( C D ) Quantitation of average LD number ( in pixels2 ) per from three independent experiments . data are represented as mean ± SE . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 . NS , not significant .

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(A) Hep3B hepatocytes were treated with nontargeting control siRNA (siNT) or siRNA targeting Dyn2 (siDyn2), followed by re-expression of either vector alone, or GFP-tagged versions of Dyn2, loaded with 150 µM oleate overnight and starved for 48 h in medium containing 0.1% FBS. LDs were visualized by immunofluorescence using Oil Red O staining. Cell boundaries are outlined and those cells re-expressing GFP, GFP-wtDyn2, or GFP--K44A are denoted with asterisks. Bars, 20 µM. (B) Representative blot showing the efficiency of the Dyn2 knockdown in these cells. (C and D) Quantitation of the average LD number and area (in pixels2) per cell from three independent experiments. The data are represented as mean ± SE. *, P ≤ 0.05; **, P ≤ 0.01. NS, not significant.


HuH - 7 ( A ) Hep3B ( B ) hepatocytes were loaded with 150 µM oleate overnight starved for 48 h in medium containing 0 . 1 % FBS in presence of Dyn2 inhibitors or DMSO as indicated . Representative of inhibitor - treated control ( stained with Oil Red O ) are shown in A B together with quantitation of average LD per from three independent experiments . Pharmacological inhibitors used were : Dynasore ( inhibits Dyn2 GTPase activity ) , MiTMAB ( targets PH domain interferes with binding ) , Dynole 34 - 2 ( allosteric GTPase inhibitor ) , Dynole 31 - 2 ( negative control for Dynole 34 - 2 ) . Bars , 20 µM .

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HuH-7 (A) and Hep3B (B) hepatocytes were loaded with 150 µM oleate overnight and starved for 48 h in medium containing 0.1% FBS in the presence of Dyn2 inhibitors or DMSO as indicated. Representative images of inhibitor-treated and control cells (stained with Oil Red O) are shown in A and B together with the quantitation of the average LD area per cell from three independent experiments. Pharmacological inhibitors used were: Dynasore (inhibits Dyn2 GTPase activity), MiTMAB (targets PH domain and interferes with membrane binding), Dynole 34-2 (allosteric GTPase inhibitor), and Dynole 31-2 (negative control for Dynole 34-2). Bars, 20 µM.


( C ) Representative from control Dyn2 knockout MEFs after an overnight lipid loading with 400 µM Dyn2 for 17 h . Knockout of Dyn2 was induced by treatment with 2 µM 4 - hydroxy - tamoxifen for 7 d was confirmed by immunostaining of endogenous Dyn2 ( top row ) by immunoblot ( D ) . Bars , 20 µM . ( E F ) Average LD number ( E ) ( F ) per from five independent experiments . data are represented as mean ± SE . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 .

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(C) Representative images from control and Dyn2 knockout MEFs after an overnight lipid loading with 400 µM Dyn2 for 17 h. Knockout of Dyn2 was induced by treatment with 2 µM 4-hydroxy-tamoxifen for 7 d and was confirmed by immunostaining of endogenous Dyn2 (top row) and by immunoblot (D). Bars, 20 µM. (E and F) Average LD number (E) and area (F) per cell from five independent experiments. All data are represented as mean ± SE. *, P ≤ 0.05; **, P ≤ 0.01.


( G ) Whole - lysates LD fractions isolated from HuH - 7 hepatocytes under resting or starved ( 2 h HBSS starvation ) conditions .

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(G) Whole-cell lysates and LD fractions isolated from HuH-7 hepatocytes under resting or starved (2 h HBSS starvation) conditions.


( H ) hepatocyte expressing Dyn2 - GFP , showing an absence of colocalization with LD surface ( stained with Oil Red O ) . Bar , 20 µM . Inset shows magnification of boxed region ( bar , 2 µM ) .

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(H) Primary hepatocyte expressing Dyn2-GFP, showing an absence of colocalization with the LD surface (stained with Oil Red O). Bar, 20 µM. Inset shows magnification of boxed region (bar, 2 µM).


Hep3B treated with either a nontargeting control siRNA ( A B , siNT ) or an siRNA targeting human Dyn2 ( C D , siDyn2 ) were fixed co - stained with antibodies specific for LAMP1 ( red ) LC3 ( green ) . After Dyn2 knockdown , a juxtanuclear aggregation enlargement of LAMP1 - positive compartment is observed ( C D , arrows ) . Increased labeling of LC3 is also detectable after knockdown of Dyn2 .

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Hep3B cells treated with either a nontargeting control siRNA (A and B, siNT) or an siRNA targeting human Dyn2 (C and D, siDyn2) were fixed and co-stained with antibodies specific for LAMP1 (red) and LC3 (green). After Dyn2 knockdown, a juxtanuclear aggregation and enlargement of the LAMP1-positive compartment is observed (C and D, arrows). Increased labeling of LC3 is also detectable after knockdown of Dyn2.


( E ) Western blotting of Hep3B lysates after a 3 - d treatment with either control or Dyn2 - targeted siRNA further treatment with or without 50 µM leupeptin . Densitometry - of six independent experiments is shown at bottom of figure .

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(E) Western blotting of Hep3B lysates after a 3-d treatment with either the control or Dyn2-targeted siRNA and further treatment with or without 50 µM leupeptin. Densitometry-based analysis of six independent experiments is shown at the bottom of the figure.


( F ) Western blotting of Hep3B lysates after treatment for 2 h with DMSO or 80 µM Dynasore , in presence or absence of 50 µM leupeptin . Quantitation of LC3 - II relative to control are shown below blots . data are represented as mean ± SE ; * , P ≤ 0 . 05 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Western blotting of Hep3B lysates after treatment for 2 h with DMSO or 80 µM Dynasore, in the presence or absence of 50 µM leupeptin. Quantitation of LC3-II levels relative to control are shown below the blots. The data are represented as mean ± SE; *, P ≤ 0.05.


Dyn2 knockdown results in formation of enlarged autophagic structures . ( A - D ) Transmission electron ( TEMs ) of oleate - loaded Hep3B hepatocytes treated with nontargeting control ( siNT ) or Dyn2 ( siDyn2 ) siRNA for 72 h . Bars : ( A B ) 2 µm ; ( A ′ B ′ ) 1 µm ; ( C D ) 0 . 5 µm . Insets in A B show of LAMP1 - stained ( bars , 10 µM ) . Control ( A ) contain an abundance of small ( < 1 µm ) electron - dense lysosomes autolysosomes ( arrowheads ) . Under conditions in which Dyn2 expression is suppressed ( B - D ) , far fewer small lysosomes are observed . Instead , are populated by larger autolysosomes ( ≥ 1 µm ) with aberrant morphologies containing putative LDs ( * ) . ( E ) Quantitative measure of autolysosomal from in which Dyn2 was knocked down as compared with control . Values represent fold - increase in number of autophagic structures of a given diameter ( measured in microns ) observed over siNT - treated control . Data were obtained from a single experiment with n = 6 examined by electron microscopy from each condition .

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

Dyn2 knockdown results in the formation of enlarged autophagic structures. (A-D) Transmission electron micrographs (TEMs) of oleate-loaded Hep3B hepatocytes treated with nontargeting control (siNT) or Dyn2 (siDyn2) siRNA for 72 h. Bars: (A and B) 2 µm; (A′ and B′) 1 µm; (C and D) 0.5 µm. Insets in A and B show fluorescent micrographs of LAMP1-stained cells (bars, 10 µM). Control cells (A) contain an abundance of small (<1 µm) electron-dense lysosomes and autolysosomes (arrowheads). Under conditions in which Dyn2 expression is suppressed (B-D), far fewer small lysosomes are observed. Instead, the cells are populated by larger autolysosomes (≥1 µm) with aberrant morphologies and containing putative LDs (*). (E) Quantitative measure of autolysosomal size from cells in which Dyn2 was knocked down as compared with control cells. Values represent the fold-increase in the number of autophagic structures of a given diameter (measured in microns) observed over siNT-treated control cells. Data were obtained from a single experiment with n = 6 cells examined by electron microscopy from each condition.


Hep3B treated with either a nontargeting control siRNA ( A B , siNT ) or an siRNA targeting human Dyn2 ( C D , siDyn2 ) were fixed stained with an antibody specific for LAMP1 after a 24 - h starvation period in media containing 0 . 1 % FBS . Control displayed normal lysosomal compartments , with LAMP1 - stained structures dispersed throughout cytosol , some containing short reformation tubules ( B ′ ) . After Dyn2 knockdown , many contained enlarged autolysosomes with a noticeable increase in persistent lengthy reformation tubules emanating from LAMP1 - positive compartments ( C ′ D ′ ) . Bars : ( A - D ) 20 µM ; ( A ′ B ′ ) 5 µM ; ( C ′ D ′ ) 10 µM . ( E F ) Measurements of ( E ) distribution ( F ) of LAMP1 - stained ALR tubules in control ( siNT ) siDyn2 - treated . Data represent mean derived from measurements of tubules in a minimum of 40 for each experimental group over 6 independent experiments . Error bars represent SE ; * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 .

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

Hep3B cells treated with either a nontargeting control siRNA (A and B, siNT) or an siRNA targeting human Dyn2 (C and D, siDyn2) were fixed and stained with an antibody specific for LAMP1 after a 24-h starvation period in media containing 0.1% FBS. Control cells displayed normal lysosomal compartments, with LAMP1-stained structures dispersed throughout the cytosol, some containing short reformation tubules (B′). After Dyn2 knockdown, many cells contained enlarged autolysosomes with a noticeable increase in persistent and lengthy reformation tubules emanating from LAMP1-positive compartments (C′ and D′). Bars: (A-D) 20 µM; (A′ and B′) 5 µM; (C′ and D′) 10 µM. (E and F) Measurements of the length (E) and distribution (F) of LAMP1-stained ALR tubules in control (siNT) and siDyn2-treated cells. Data represent the mean derived from measurements of tubules in a minimum of 40 cells for each experimental group over 6 independent experiments. Error bars represent SE; *, P ≤ 0.05; **, P ≤ 0.01.


( G ) of siDyn2 - treated Hep3B hepatocyte exhibiting enlarged autolysosomal structures with extensive tubulation ( arrows ) . Bar , 2 µm . ( G ′ ) Inset showing enlargement of autolysosomal tubule ( arrows ) . Bar , 1 µm . ( H ) Tubulating autolysosome with engulfed lipid droplet . Bar , 0 . 5 µm .

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(G) TEM of siDyn2-treated Hep3B hepatocyte exhibiting enlarged autolysosomal structures with extensive tubulation (arrows). Bar, 2 µm. (G′) Inset showing enlargement of autolysosomal tubule (arrows). Bar, 1 µm. (H) Tubulating autolysosome with engulfed lipid droplet. Bar, 0.5 µm.


Acute inhibition of Dyn2 reversibly disrupts autophagic lysosomal reformation ( ALR ) lysosomal tubule scission . ( A - D ) Still frames from time - lapse movies of Hep3B expressing LAMP1 - mCherry . were starved for 2 h in HBSS subsequently treated for 30 min with either DMSO ( A B ) or 40 µM Dynasore ( C D ) , which induced extensive tubulation of LAMP1 - positive compartments . Bars ( A - D ) : 20 µM ; ( A ′ - B ′ ) 2 µM ; ( C ′ - D ′ ) 10 µM . ( E - G ) To demonstrate reversibility of this tubulation , Dynasore - treated were washed extensively with drug - free media containing 10 % FBS monitored by time - lapse microscopy for 45 min . Frequently , after drug washout , LAMP1 - positive tubules exhibited noticeable varicosities ( E F , arrows ; bars , 10 µM ) along their . These sites are suggestive of areas of scission resumed budding of nascent protolysosomes from reformation tubules ( G ; bars , 10 µM ) . ( H ) Tubules from undergoing drug washout were quantified by tracing their lengths at beginning end of these movies . Still frames from a representative movie show tubule content at t = 10 45 min after drug washout . Bars , 20 µM . ( I ) of five independent movies showed an average decrease in total tubulation of ∼ 50 % after drug washout . Data represent average relative change in total tubule between first last frames of time - lapse movies . Error bars represent SE ; * , P < 0 . 05 .

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

Acute inhibition of Dyn2 reversibly disrupts autophagic lysosomal reformation (ALR) and lysosomal tubule scission. (A-D) Still frames from time-lapse movies of Hep3B cells expressing LAMP1-mCherry. Cells were starved for 2 h in HBSS and subsequently treated for 30 min with either DMSO (A and B) or 40 µM Dynasore (C and D), which induced extensive tubulation of LAMP1-positive compartments. Bars (A-D): 20 µM; (A′-B′) 2 µM; (C′-D′) 10 µM. (E-G) To demonstrate the reversibility of this tubulation, Dynasore-treated cells were washed extensively with drug-free media containing 10% FBS and monitored by time-lapse microscopy for 45 min. Frequently, after drug washout, LAMP1-positive tubules exhibited noticeable varicosities (E and F, arrows; bars, 10 µM) along their length. These sites are suggestive of areas of scission and resumed budding of nascent protolysosomes from the reformation tubules (G; bars, 10 µM). (H) Tubules from cells undergoing drug washout were quantified by tracing their lengths at the beginning and end of these movies. Still frames from a representative movie show tubule content at t = 10 and 45 min after drug washout. Bars, 20 µM. (I) Analysis of five independent movies showed an average decrease in total tubulation of ∼50% after drug washout. Data represent the average relative change in total tubule length between the first and last frames of the time-lapse movies. Error bars represent SE; *, P < 0.05.


( A B ) Subcellular density gradient fractionation of Hep3B hepatocytes starved for 2 h in HBSS treated with 40 µM Dynasore , to induce tubule formation , as in Fig . 5 . were lysed ( WCL ) , post - supernatant ( PNS ) was pelleted by centrifugation to produce a crude lysosomal fraction ( CLF ) high - speed supernatant ( HSS ) . CLF was subsequently loaded onto an 8 - 27 % discontinuous iodixanol ( OptiPrep ) gradient for separation by ultracentrifugation . Nine fractions were collected from top of gradient blotted for Dyn2 LAMP1 resident , LAMP1 . Lysosomal acid phosphatase activity roughly correlates with LAMP1 in each fraction . of Dyn2 are highest in fraction 2 , corresponding with both peak of LAMP1 lysosomal activity . ( A ) data shown are from a single representative experiment out of three repeats . ( B ) Blotting for subcellular components shows that Dyn2 is specific for these same fractions , unlike other organelle markers such as EEA1 ( early endosomes ) COXIV ( mitochondria ) .

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(A and B) Subcellular density gradient fractionation of Hep3B hepatocytes starved for 2 h in HBSS and treated with 40 µM Dynasore, to induce tubule formation, as in Fig. 5. Cells were lysed (WCL), and the post-nuclear supernatant (PNS) was pelleted by centrifugation to produce a crude lysosomal fraction (CLF) and high-speed supernatant (HSS). The CLF was subsequently loaded onto an 8-27% discontinuous iodixanol (OptiPrep) gradient for separation by ultracentrifugation. Nine fractions were collected from the top of the gradient and blotted for Dyn2 and the LAMP1 resident protein, LAMP1. Lysosomal acid phosphatase activity roughly correlates with LAMP1 protein levels in each fraction. Levels of Dyn2 are highest in fraction 2, corresponding with both the peak levels of LAMP1 and lysosomal activity. (A) The data shown are from a single representative experiment out of three repeats. (B) Blotting for subcellular components shows that Dyn2 is specific for these same fractions, unlike other organelle markers such as EEA1 (early endosomes) and COXIV (mitochondria).


( C D ) Dyn2 localizes to surface of LAMP1 - positive compartments . imaging of Hep3B hepatocytes transfected with Dyn2 - GFP LAMP1 - mCherry under resting ( C C ′ ) or starvation ( D ) conditions . ( C ′ ′ ) Dyn2 - GFP localizing to LAMP1 - labeled lysosome structures under starvation conditions . Arrows indicate regions of colocalization . Dyn2 ( arrows ) is present at site of scission of LAMP1 - positive tubules ( arrowhead ) from large autolysosomal structures . Bars : ( C ) 5 µM ; ( C ′ , C ′ ′ , D ) 3 µM .

O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-EXP_ASSAY O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O

(C and D) Dyn2 localizes to the surface of LAMP1-positive compartments. Fluorescence imaging of Hep3B hepatocytes transfected with Dyn2-GFP and LAMP1-mCherry under resting (C and C′) or starvation (D) conditions. (C′′) Dyn2-GFP localizing to LAMP1-labeled lysosome structures under starvation conditions. Arrows indicate regions of protein colocalization. Dyn2 (arrows) is present at the site of scission of LAMP1-positive tubules (arrowhead) from large autolysosomal structures. Bars: (C) 5 µM; (C′, C′′, and D) 3 µM.


A ) Box blot of vitrealANG - 1 from newly diagnosed diagnosed with wAMD , DR , proliferative DR , RVO compared to controls ( macular whole ) . B ) Box blot of vitrealANG - 2 from as in A ) . interquartile range of data is indicated by box . A non - parametric Kruskal - Wallis ( * * , P = 0 . 029 ) followed by Dunn ' s method for multiple comparisons was used to show significant differences of groups to control which are indicated by an asterisks . ANG - 1 did not differ significantly , but ANG - 2 were significantly different : control vs AMD ( * , P = 0 . 0451 ) , vs DR ( * , P < 0 . 0001 ) , pDR ( * , P < 0 . 0001 ) RVO ( P < 0 . 0001 ) .

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

A) Box blot of vitrealANG-1 levels from newly diagnosed patients diagnosed with wAMD, DR, proliferative DR, and RVO compared to controls (macular whole).B) Box blot of vitrealANG-2 levels from patients as in A). The interquartile range of the data is indicated by the box. A non-parametric Kruskal-Wallis analysis (**, P = 0.029) followed by Dunn's method for multiple comparisons was used to show significant differences of the groups to control which are indicated by an asterisks. ANG-1 levels did not differ significantly, but ANG-2 levels were significantly different:control vs AMD (*, P = 0.0451), vs DR (*, P < 0.0001), pDR (*,P < 0.0001) and RVO (P < 0.0001).


D ) 24 - h of ANG - 2 in supernatants of basal site of culture stimulated with 5 ng / ml of VEGF - A , 200 ng / ml of ANG - 1 or combination . Error bars show with one sided ANOVA ( * * * * , P < 0 . 0001 ) Tukey ' s multiple T - for five independent experiments indicating significance for control vs VEGF - A ( * * , P = 0 . 0028 ) ; VEGF - A vs ANG - 1 ( * * * * , P < 0 . 0001 ) ; ANG - 1 vs VEGF - A ANG1 ( * * , P = 0 . 0061 ) ; VEGF - A vs VEGF - A ANG - 1 ( ns , P = 0 . 0625 ) .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O

D) 24-h concentration of ANG-2 in supernatants of the basal site of the culture stimulated with 5 ng/ml of VEGF-A, 200 ng/ml of ANG-1 or the combination. Error bars show SEM with one sided ANOVA (****, P < 0.0001) and Tukey's multiple T-test for five independent experiments indicating significance for control vs VEGF-A (**, P = 0.0028); VEGF-A vs ANG-1 (****, P <0.0001); ANG-1 vs VEGF-A ANG1 (**, P = 0.0061); VEGF-A vs VEGF-A ANG-1 (ns, P = 0.0625).