B ) Characterization of the Doxycycline - inducible cell lines expressing siRNA resistant wild type ( WT ) or mutant ( TMA or TMD ) FLAG / HA - tagged PALB2 . The cell lines were transfected with UNC ( Negative Control ) or PALB2 siRNA for 24 hours prior to induction with Doxycycline . Immunoblots were performed with indicated antibodies .

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B) Characterization of the Doxycycline-inducible cell lines expressing siRNA resistant wild type (WT) or mutant (TMA or TMD) FLAG/HA-tagged PALB2. The cell lines were transfected with UNC (Negative Control) or PALB2 siRNA for 24 hours prior to induction with Doxycycline. Immunoblots were performed with indicated antibodies.


C ) Cell line characterization of FLAG / HA - tagged PALB2 localization 2 hours following exposure to 15 Gy of IR . Z - stack max intensity image projection and background subtraction was done using ImageJ / Fiji . Scale bar = 5μm .

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C) Cell line characterization of FLAG/HA-tagged PALB2 localization 2 hours following exposure to 15 Gy of IR. Z-stack max intensity image projection and background subtraction was done using ImageJ/Fiji. Scale bar = 5μm.


D ) Kinase assay was performed as described in Figure 1E except that the SDS - PAGE gel was Coomassie stained following electrophoresis . WT was used at two different concentrations ( 1x and 0 . 75x ) .

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D) Kinase assay was performed as described in Figure 1E except that the SDS-PAGE gel was Coomassie stained following electrophoresis. WT was used at two different concentrations (1x and 0.75x).


E ) Doxycycline - inducible stable U2OS cell lines were left untreated or treated with Doxycycline and / or IR ( 15 Gy , 2h recovery ) . Following protein extraction the lysates were immunoprecipitated ( IP ) with IgG or antibody recognizing phosphorylated S / Q sites ( pS / Q ) and analyzed by SDS - PAGE . Immunoblots were performed with indicated antibodies .

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E) Doxycycline-inducible stable U2OS cell lines were left untreated or treated with Doxycycline and/or IR (15 Gy, 2h recovery). Following protein extraction the lysates were immunoprecipitated (IP) with IgG or antibody recognizing phosphorylated S/Q sites (pS/Q) and analyzed by SDS-PAGE. Immunoblots were performed with indicated antibodies.


A ) PALB2 cell lines transfected with PALB2 siRNA followed by inducible expression of siRNA resistant PALB2 versions were exposed to 15 Gy of IR and fixed for IF 2 hours later . Z - stack images were acquired with a 60X oil objective ( Deltavision ) . Z stack max intensity projection , background subtraction , and foci count was done using ImageJ / Fiji . ( * p < 0 . 0001 , unpaired Student ' s t - test ) . Scale bar = 5μm .

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A) PALB2 cell lines transfected with PALB2 siRNA followed by inducible expression of siRNA resistant PALB2 versions were exposed to 15 Gy of IR and fixed for IF 2 hours later. Z-stack images were acquired with a 60X oil objective (Deltavision). Z stack max intensity projection, background subtraction, and foci count was done using ImageJ/Fiji. (* p<0.0001, unpaired Student's t-test). Scale bar = 5μm.


B ) PALB2 cell lines were transfected and the exogenous PALB2 induced as in Figure 3A before they were pulsed with 10 μM EdU for 20 minutes prior to addition of 2 mM HU . 24 hours later the cells were fixed and processed for IF as in Figure 3A . Cells in S - phase ( EdU + ) at the time of HU treatment were Click - IT labeled with an Alexa Fluor 647 azide and RAD51 foci in EdU positive cells were enumerated using ImageJ / Fiji . ( * p < 0 . 0001 , unpaired Student ' s t - test ) . Scale bar = 5μm .

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B) PALB2 cell lines were transfected and the exogenous PALB2 induced as in Figure 3A before they were pulsed with 10 μM EdU for 20 minutes prior to addition of 2 mM HU. 24 hours later the cells were fixed and processed for IF as in Figure 3A. Cells in S-phase (EdU+) at the time of HU treatment were Click-IT labeled with an Alexa Fluor 647 azide and RAD51 foci in EdU positive cells were enumerated using ImageJ/Fiji. (* p<0.0001, unpaired Student's t-test). Scale bar=5μm.


C ) HeLa DR - GFP cell line was used to analyze homologous recombination activity . Cells were transfected with PALB2 siRNA followed by transfection of I - SceI together with empty vector ( EV ) , WT - , TMA - , or TMD - PALB2 the next day . 72 hours later cells were fixed and analyzed by FACS . The percentage of cells with GFP signal from the empty vector sample was set to one ( dotted line ) . The graph shows a representative image from three biological repeats ( error bars = SEM ) .

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C) HeLa DR-GFP cell line was used to analyze homologous recombination activity. Cells were transfected with PALB2 siRNA followed by transfection of I-SceI together with empty vector (EV), WT-, TMA-, or TMD-PALB2 the next day. 72 hours later cells were fixed and analyzed by FACS. The percentage of cells with GFP signal from the empty vector sample was set to one (dotted line). The graph shows a representative image from three biological repeats (error bars=SEM).


D ) Phosphorylation of PALB2 supports RAD51 foci . Cells were transfected with siPALB2 overnight followed by forward transfection of siFBH1 / siBLM for 6 hours then induced for expression of PALB2 ( WT and TMA ) . 24 hours following induction cells were pulsed with EdU and exposed to HU as in Figure 3B . Cells in S - phase ( EdU + ) at the time of HU treatment were Click - IT labeled with an Alexa Fluor 647 azide and RAD51 foci in EdU positive cells were enumerated using ImageJ / Fiji ( * p < 0 . 0001 , unpaired Student ' s t - test ) . Representative images of displaying RAD51 ( green ) and HA - PALB2 ( red ) localization in HU treated EdU positive ( not shown ) cells . Scale bar = 5μm .

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D) Phosphorylation of PALB2 supports RAD51 foci. Cells were transfected with siPALB2 overnight followed by forward transfection of siFBH1/siBLM for 6 hours then induced for expression of PALB2 (WT and TMA). 24 hours following induction cells were pulsed with EdU and exposed to HU as in Figure 3B. Cells in S-phase (EdU+) at the time of HU treatment were Click-IT labeled with an Alexa Fluor 647 azide and RAD51 foci in EdU positive cells were enumerated using ImageJ/Fiji (* p<0.0001, unpaired Student's t-test). Representative images of displaying RAD51 (green) and HA-PALB2 (red) localization in HU treated EdU positive (not shown) cells. Scale bar=5μm.


A ) U2OS and PALB2 cell lines were transfected with UNC ( Negative Control ) or PALB2 siRNA and 24 hours later Doxycycline was added to the PALB2 cell lines . Left panel , cells were treated the following day with IR ( 5 Gy ) and 2 hours later Nocodazole was added for 6 hours , or treated with only Nocodazole for 6 hours . Right panel , cells were treated with IR ( 5 Gy ) and left to recover for 16 hours . Cells were fixed and stained with the pMPM2 antibody to detect the mitotic cells by flow cytometry . The percentages of mitotic cells in the IR + Nocodazole samples were normalized to the Nocodazole samples ( left panel , n = 3 , error bars = SEM ) . For the samples with the 16 hour recovery after IR the percentage of mitotic cells in the WT - PALB2 expressing cells was set to one ( right panel ) . ( n = 3 , * * p < 0 . 005 , * p < 0 . 05 , unpaired Student ' s t - test , error bars = SEM ) .

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A) U2OS and PALB2 cell lines were transfected with UNC (Negative Control) or PALB2 siRNA and 24 hours later Doxycycline was added to the PALB2 cell lines. Left panel, cells were treated the following day with IR (5 Gy) and 2 hours later Nocodazole was added for 6 hours, or treated with only Nocodazole for 6 hours. Right panel, cells were treated with IR (5 Gy) and left to recover for 16 hours. Cells were fixed and stained with the pMPM2 antibody to detect the mitotic cells by flow cytometry. The percentages of mitotic cells in the IR+Nocodazole samples were normalized to the Nocodazole samples (left panel, n=3, error bars=SEM). For the samples with the 16 hour recovery after IR the percentage of mitotic cells in the WT-PALB2 expressing cells was set to one (right panel). (n=3, **p<0.005, *p<0.05, unpaired Student's t-test, error bars=SEM).


B ) The relative intensity of phosphorylated H2AX ( pS139 ) was examined in the total population of WT - and TMA - PALB2 expressing cell lines 18 hours post exposure to IR ( 5 Gy ) . Cell lines were prepared as in above , fixed at the indicated time point , and stained for pH2AX . Cells were imaged with a 20X air objective on a Scan ^ R workstation ( Olympus ) , mean relative pH2AX intensity was calculated from background subtracted images using the Scan ^ R analysis software . ( * p < 0 . 0001 , unpaired Student ' s t - test ) .

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B) The relative intensity of phosphorylated H2AX (pS139) was examined in the total population of WT- and TMA-PALB2 expressing cell lines 18 hours post exposure to IR (5 Gy). Cell lines were prepared as in above, fixed at the indicated time point, and stained for pH2AX. Cells were imaged with a 20X air objective on a Scan^R workstation (Olympus), mean relative pH2AX intensity was calculated from background subtracted images using the Scan^R analysis software. (* p<0.0001, unpaired Student's t-test).


C ) Number of 53BP1 nuclear in TMA - PALB2 expressing G1 daughter cells . Cell lines were prepared as above and left untreated ( NT ) or exposed to IR ( 3 Gy ) , and 16 hours later Cytochalasin B was added for 8 hours to block cytokinesis . The irradiated bi - nucleated cells were scored for 53BP1 nuclear bodies . Scale bar = 10μm .

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C) Number of 53BP1 nuclear in TMA-PALB2 expressing G1 daughter cells. Cell lines were prepared as above and left untreated (NT) or exposed to IR (3 Gy), and 16 hours later Cytochalasin B was added for 8 hours to block cytokinesis. The irradiated bi-nucleated cells were scored for 53BP1 nuclear bodies. Scale bar = 10μm.


B , Mouse embryonic fibroblasts genetically modified using CRISPR to introduce a GFP sequence immediately after the start codon of Syne2 display fluorescence at the nuclear envelope .

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B, Mouse embryonic fibroblasts genetically modified using CRISPR to introduce a GFP sequence immediately after the start codon of Syne2 display fluorescence at the nuclear envelope.


C , Brightfield and fluorescence images of a cell migrating through a 2 µm constriction . Arrows indicate areas of accumulation of nesprin - 2 at the front of the nucleus as it is deformed through the constriction .

C, Brightfield and fluorescence images of a cell migrating through a 2 µm constriction. Arrows indicate areas of accumulation of nesprin-2 at the front of the nucleus as it is deformed through the constriction.


E , Brightfield and fluorescence images of a cell migrating along a 15 µm control channel .

E, Brightfield and fluorescence images of a cell migrating along a 15 µm control channel.


F , Quantification of the intensity of the fluorescent signal around the perimeter of the nucleus in a 2 µm constriction and a 15 µm control channel . The average intensity at the front and the back is normalized to the intensity at the sides , demonstrating an increase in the signal at the front of the nucleus as it is deformed and a reduction after deformation . Error bars correspond to the standard deviation . Constriction : n = 17 cells over 4 biological replicates , 15 µm channels : n = 20 cells over 3 biological replicates . A Wilcoxon signed - rank test is used to compare matched samples , stars ( * P < 0 . 05 , * * P < 0 . 01 ) denote comparisons to the measurement at the side , and pounds ( # P < 0 . 05 , # # P < 0 . 01 ) denote a comparison to the " before " time point .

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F, Quantification of the intensity of the fluorescent signal around the perimeter of the nucleus in a 2 µm constriction and a 15 µm control channel. The average intensity at the front and the back is normalized to the intensity at the sides, demonstrating an increase in the signal at the front of the nucleus as it is deformed and a reduction after deformation. Error bars correspond to the standard deviation. Constriction: n=17 cells over 4 biological replicates, 15 µm channels: n=20 cells over 3 biological replicates. A Wilcoxon signed-rank test is used to compare matched samples, stars (*P < 0.05, **P < 0.01) denote comparisons to the measurement at the side, and pounds (#P < 0.05, ##P < 0.01) denote a comparison to the "before" time point.


G , GFP - nesprin - 2 cells deformed by the pressure gradient in a micropipette aspiration device . Note that the GFP signal does not increase at the tip but increases at sides of the nucleus .

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G, GFP-nesprin-2 cells deformed by the pressure gradient in a micropipette aspiration device. Note that the GFP signal does not increase at the tip but increases at sides of the nucleus.


A , CRISPR - modified cells ( Brightfield , left ) that express GFP - nesprin - 2 ( middle ) and mCh - LAC ( right ) show nesprin ( orange arrow ) but not lamin accumulation at the front of the nucleus as it is deformed through a narrow constriction . B , Quantification of intensity of GFP - nesprin - 2 and mCh - LAC , normalized to the intensity at the sides . Error bars correspond to the standard deviation . Constriction : n = 19 cells over 5 biological replicates , 15 µm : n = 27 over 6 biological replicates . A Wilcoxon signed - rank test is used to compare matched samples , stars ( * P < 0 . 05 , * * P < 0 . 01 ) denote comparisons to the measurement at the side , and pounds ( # P < 0 . 05 , # # P < 0 . 01 ) denote a comparison to the " before " time point .

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A, CRISPR-modified cells (Brightfield, left) that express GFP-nesprin-2 (middle) and mCh-LAC (right) show nesprin (orange arrow) but not lamin accumulation at the front of the nucleus as it is deformed through a narrow constriction. B, Quantification of intensity of GFP-nesprin-2 and mCh-LAC, normalized to the intensity at the sides. Error bars correspond to the standard deviation. Constriction: n=19 cells over 5 biological replicates, 15 µm: n= 27 over 6 biological replicates. A Wilcoxon signed-rank test is used to compare matched samples, stars (*P < 0.05, **P < 0.01) denote comparisons to the measurement at the side, and pounds (#P < 0.05, ##P < 0.01) denote a comparison to the "before" time point.


A , The deformed nucleus of cells migrating through narrow constrictions was bleached at the front and the side to determine mobility . Inset are shown close - up views of the bleached areas at the front of the nucleus . B , Quantification of the recovery . Error bars correspond to the standard error of the mean , n = 24 over 3 biological replicates .

A, The deformed nucleus of cells migrating through narrow constrictions was bleached at the front and the side to determine mobility. Inset are shown close-up views of the bleached areas at the front of the nucleus. B, Quantification of the recovery. Error bars correspond to the standard error of the mean, n=24 over 3 biological replicates.


A , Expression of the artificial mini - nesprin - 2G construct results in significant nesprin accumulation ( orange arrow ) in cells migrating through narrow constrictions .

A, Expression of the artificial mini-nesprin-2G construct results in significant nesprin accumulation (orange arrow) in cells migrating through narrow constrictions.


C , Quantification of the mini - nesprin - 2G accumulation of cells migrating through narrow constrictions . Note that the mutant construct does not accumulate at the front of the nucleus . Error bars correspond to the standard deviation . Mini - nesprin - 2G : n = 10 cells over 4 biological replicates ; Mutant : n = 11 cells over 4 biological replicates . A Wilcoxon signed - rank test is used to compare matched samples , stars ( * P < 0 . 05 , * * P < 0 . 01 ) denote comparisons to the measurement at the side , and pounds ( # P < 0 . 05 , # # P < 0 . 01 ) denote a comparison to the " before " time point .

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C, Quantification of the mini-nesprin-2G accumulation of cells migrating through narrow constrictions. Note that the mutant construct does not accumulate at the front of the nucleus. Error bars correspond to the standard deviation. Mini-nesprin-2G: n=10 cells over 4 biological replicates; Mutant: n=11 cells over 4 biological replicates. A Wilcoxon signed-rank test is used to compare matched samples, stars(*P< 0.05, **P < 0.01) denote comparisons to the measurement at the side, and pounds (#P < 0.05, ##P < 0.01) denote a comparison to the "before" time point.


A , Fixed cells labelled with phalloidin shows filamentous actin accumulation on the edges of the pillars and along the top and bottom surfaces . Cross - sections along the direction of migration ( yellow ) and perpendicular to the direction of migration ( blue , 1 - 4 ) are shown below .

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A, Fixed cells labelled with phalloidin shows filamentous actin accumulation on the edges of the pillars and along the top and bottom surfaces. Cross-sections along the direction of migration (yellow) and perpendicular to the direction of migration (blue, 1-4) are shown below.


B , A confocal slice on the surface of the nucleus indicates that GFP - nesprin - 2 co - localizes with actin cables at the surface of the nucleus . Dashed white lines represent the outlines of the constriction .

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B, A confocal slice on the surface of the nucleus indicates that GFP-nesprin-2 co-localizes with actin cables at the surface of the nucleus. Dashed white lines represent the outlines of the constriction.


C , Spinning disk confocal microscopy on live cells reveals the elongation state of nesprins at the nuclear envelope . Dashed white lines represent the outlines of the constriction . The distance between mCh - LAC and GFP - nesprin - 2 was measured at the back , at the constriction , at the front near the constriction , and at the center of the front . ( N = 17 cells , over 3 biological replicates . The alignment of the two channels was corrected using at least 28 fluorescent beads . )

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C, Spinning disk confocal microscopy on live cells reveals the elongation state of nesprins at the nuclear envelope. Dashed white lines represent the outlines of the constriction. The distance between mCh-LAC and GFP-nesprin-2 was measured at the back, at the constriction, at the front near the constriction, and at the center of the front. (N = 17 cells, over 3 biological replicates. The alignment of the two channels was corrected using at least 28 fluorescent beads.)


D , Two - photon laser ablation reveals that ablation at the front of the nucleus results in a rearward movement of the nucleus , but not ablation at the back . The nuclei of DN - KASH , which show a decrease of endogenous nesprin at the nuclear envelope show a dampened rearward movement upon ablation at the front of nuclei . Bars represent the average value . Error bars represent the standard error on the mean . Back : N = 9 , Front : N = 30 , Cell Front : N = 16 , and DNKASH : N = 31 , 3 biological replicates .

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D, Two-photon laser ablation reveals that ablation at the front of the nucleus results in a rearward movement of the nucleus, but not ablation at the back. The nuclei of DN-KASH, which show a decrease of endogenous nesprin at the nuclear envelope show a dampened rearward movement upon ablation at the front of nuclei. Bars represent the average value. Error bars represent the standard error on the mean. Back: N= 9, Front: N= 30, Cell Front: N = 16, and DNKASH: N=31, 3 biological replicates.


HMRs express RNAs . Heat maps show RNA expression levels in 16 . 5 dpc prospermatogonia . Only uniquely mapped RNA - seq reads were used for the analyses .

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HMRs express RNAs. Heat maps show RNA expression levels in 16.5 dpc prospermatogonia. Only uniquely mapped RNA-seq reads were used for the analyses.


metaplots ( B ) show RNA expression levels in 16 . 5 dpc prospermatogonia . Only uniquely mapped RNA - seq reads were used for the analyses . Positions ( 1 ) - ( 4 ) in ( A and B ) ( green arrows ) indicate the same positions ( ± 2 kbp from the centers ) . The lower chart in ( B ) represents mappability defined as the percentage of unique sequences in all possible 50 - nt sequences ( see Appendix Figure S2B ) . TSS isoforms are included in 55798 TSSs . Low mappability at the centers of HMR _ - L1s is due to high occurrence of repeat ( retrotransposon ) sequences . RPM , reads per million mapped reads .

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metaplots (B) show RNA expression levels in 16.5 dpc prospermatogonia. Only uniquely mapped RNA-seq reads were used for the analyses. Positions (1)-(4) in (A and B) (green arrows) indicate the same positions (±2 kbp from the centers). The lower chart in (B) represents mappability defined as the percentage of unique sequences in all possible 50-nt sequences (see Appendix Figure S2B). TSS isoforms are included in 55798 TSSs. Low mappability at the centers of HMR_-L1s is due to high occurrence of repeat (retrotransposon) sequences. RPM, reads per million mapped reads.


Chromatin marks associated with promoters are observed in hypomethylated regions without young LINE1 retrotransposons ( HMR _ - L1s ) . Metaplots show the average ChIP - seq signals across HMR _ - L1s , transcription start sites ( TSSs ) , and randomly selected sites . The signals were normalized by either H3 or input signal . The results shown in heat maps were not normalized by H3 or input .

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Chromatin marks associated with promoters are observed in hypomethylated regions without young LINE1 retrotransposons (HMR_-L1s). Metaplots show the average ChIP-seq signals across HMR_-L1s, transcription start sites (TSSs), and randomly selected sites. The signals were normalized by either H3 or input signal. The results shown in heat maps were not normalized by H3 or input.


The initiation form of Pol2 with an unphosphorylated carboxy terminal domain ( Pol2 null - P CTD ) is highly enriched in HMR _ - L1s .

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The initiation form of Pol2 with an unphosphorylated carboxy terminal domain (Pol2 null-P CTD) is highly enriched in HMR_-L1s.


RNA - immunoprecipitation ( RIP ) analyses of EGFP - MIWI2 and EGFP . EGFP - MIWI2 and EGFP were immunoprecipitated from 16 . 5 dpc prospermatogonia , and long RNAs bound to these proteins and input RNAs were sequenced . RNAs expressed from HMR _ - L1s and piRNA clusters , but not Ensembl and prospermatogonium - expressed mRNAs , are overall enriched by immunoprecipitation ( IP ) of EGFP - MIWI2 ( left ) . This enrichment is not observed when EGFP is immunoprecipitated ( right ) . The numbers of mRNAs , piRNA clusters , and HMR _ - L1s analyzed were shown in parentheses . Prospermatogonium - expressed mRNAs are those that are highly and specifically expressed in prospermatogonia . The bottom and top of boxes represent 25 % percentile and 75 % percentile , respectively . The line in the box represents the median . Circles represent outliers , and the bottom ( top ) of whiskers represents the minimum ( maximum ) values except for outliers .

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RNA-immunoprecipitation (RIP) analyses of EGFP-MIWI2 and EGFP. EGFP-MIWI2 and EGFP were immunoprecipitated from 16.5 dpc prospermatogonia, and long RNAs bound to these proteins and input RNAs were sequenced. RNAs expressed from HMR_-L1s and piRNA clusters, but not Ensembl and prospermatogonium-expressed mRNAs, are overall enriched by immunoprecipitation (IP) of EGFP-MIWI2 (left). This enrichment is not observed when EGFP is immunoprecipitated (right). The numbers of mRNAs, piRNA clusters, and HMR_-L1s analyzed were shown in parentheses. Prospermatogonium-expressed mRNAs are those that are highly and specifically expressed in prospermatogonia. The bottom and top of boxes represent 25% percentile and 75% percentile, respectively. The line in the box represents the median. Circles represent outliers, and the bottom (top) of whiskers represents the minimum (maximum) values except for outliers.


RIP analyses of EGFP - MIWI2 in newborn testes from WT and Mili KO mice . RNA bound to EGFP - MIWI2 and input RNA were sequenced . All RNAs derived from each class was counted for this analysis .

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RIP analyses of EGFP-MIWI2 in newborn testes from WT and Mili KO mice. RNA bound to EGFP-MIWI2 and input RNA were sequenced. All RNAs derived from each class was counted for this analysis.


HMR _ - L1s preferentially generate piRNAs . X - and Y - axes represent expression levels of long RNAs ( 16 . 5 dpc prospermatogonia ) as determined by RNA - seq and MIWI2 - bound piRNAs ( 18 . 5 dpc testes ) as determined by small RNA - seq , respectively . Circles in the charts represent individual mRNAs , piRNA clusters and HMR _ - L1s . More than half of the HMR _ - L1s ( right ) are located in the area of y > x ( upper part of the diagonal ) , whereas most mRNAs ( left ) are located in the area of y < x ( lower part of the diagonal ) . RPKM , reads per kilobase per million mapped reads .

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HMR_-L1s preferentially generate piRNAs. X- and Y-axes represent expression levels of long RNAs (16.5 dpc prospermatogonia) as determined by RNA-seq and MIWI2-bound piRNAs (18.5 dpc testes) as determined by small RNA-seq, respectively. Circles in the charts represent individual mRNAs, piRNA clusters and HMR_-L1s. More than half of the HMR_-L1s (right) are located in the area of y > x (upper part of the diagonal), whereas most mRNAs (left) are located in the area of y < x (lower part of the diagonal). RPKM, reads per kilobase per million mapped reads.


Frequent 10 - nt overlap between sense and antisense piRNAs derived from HMR _ - L1s . Top 100 HMR _ - L1s that generate the largest number of unique piRNAs were analyzed . Frequencies of 1 − 18 - nt overlaps were analyzed for each HMR _ - L1 .

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Frequent 10-nt overlap between sense and antisense piRNAs derived from HMR_-L1s. Top 100 HMR_-L1s that generate the largest number of unique piRNAs were analyzed. Frequencies of 1−18-nt overlaps were analyzed for each HMR_-L1.


piRNAs derived from HMR _ - L1s are enriched in adenine at their 10th nucleotide . HMR _ - L1s generating more than 40 unique piRNAs were analyzed ( 678 HMR _ - L1s ) . The proportions of piRNAs with adenine ( left ) and uridine ( right ) at the indicated positions were analyzed for each HMR _ - L1 . See A for the explanation of box plots .

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piRNAs derived from HMR_-L1s are enriched in adenine at their 10th nucleotide. HMR_-L1s generating more than 40 unique piRNAs were analyzed (678 HMR_-L1s). The proportions of piRNAs with adenine (left) and uridine (right) at the indicated positions were analyzed for each HMR_-L1. See A for the explanation of box plots.


DNA methylation status of a portion of the Rasgrf1 differentially methylated region ( DMR ) in 0 − 2 - dpp Mili + / − and Mili − / − prospermatogonia . Regions corresponding to B , C , D , E , and F are demethylated in Mili − / − prospermatogonia . Vertical bars represent DNA methylation levels at CpG sites determined by whole genome bisulfite sequencing ( see below ) . The top dark gray bar represents a portion of the Rasgrf1 gametic DMR ( Tomizawa et al , 2011 ) . The CpG cluster is shown . The deleted RMER4B sequence is indicated .

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DNA methylation status of a portion of the Rasgrf1 differentially methylated region (DMR) in 0−2-dpp Mili+/− and Mili−/− prospermatogonia. Regions corresponding to B, C, D, E, and F are demethylated in Mili−/− prospermatogonia. Vertical bars represent DNA methylation levels at CpG sites determined by whole genome bisulfite sequencing (see below). The top dark gray bar represents a portion of the Rasgrf1 gametic DMR (Tomizawa et al, 2011). The CpG cluster is shown. The deleted RMER4B sequence is indicated.


DNA methylation status of the Rasgrf1 DMR in Rasgrf1ΔRMER4B / ΔRMER4B and control spermatogonia are shown . The regions examined are shown in ( A ) . Black ( white ) circles represent methylated ( unmethylated ) CpGs .

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DNA methylation status of the Rasgrf1 DMR in Rasgrf1ΔRMER4B/ΔRMER4B and control spermatogonia are shown. The regions examined are shown in (A). Black (white) circles represent methylated (unmethylated) CpGs.


Rasgrf1 RMER4B functions in cis . Wild - type and mutant alleles of the Rasgrf1 DMR in Rasgrf1 + / ΔRMER4B spermatogonia were analyzed separately .

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Rasgrf1 RMER4B functions in cis. Wild-type and mutant alleles of the Rasgrf1 DMR in Rasgrf1+/ΔRMER4B spermatogonia were analyzed separately.


Structure of the chr7 piRNA cluster . Top gray bars represent a putative piRNA precursor predicted by H3K4me3 ChIP - seq , RNA - seq , and small RNA - seq . Only uniquely mapped reads were used for displaying the sequencing result . For RNA - seq and small RNA - seq , only the results of minus strand are shown , because plus strand does not express appreciable level of RNA . A promoter region deleted is shown above the putative piRNA precursor . RPM , Read Per Million .

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Structure of the chr7 piRNA cluster. Top gray bars represent a putative piRNA precursor predicted by H3K4me3 ChIP-seq, RNA-seq, and small RNA-seq. Only uniquely mapped reads were used for displaying the sequencing result. For RNA-seq and small RNA-seq, only the results of minus strand are shown, because plus strand does not express appreciable level of RNA. A promoter region deleted is shown above the putative piRNA precursor. RPM, Read Per Million.


Low expression level of piRNAs from chr7 cluster in chr7 piRNA clusterΔpromoter / Δpromoter testes . Unchanged expression of piRNAs from chr10 cluster ( bottom ) shows chr7 cluster is specifically affected . Only uniquely mapped piRNAs are shown .

Low expression level of piRNAs from chr7 cluster in chr7 piRNA clusterΔpromoter/Δpromoter testes. Unchanged expression of piRNAs from chr10 cluster (bottom) shows chr7 cluster is specifically affected. Only uniquely mapped piRNAs are shown.


DNA methylation status of the Rasgrf1 differentially methylated region ( DMR ) in spermatogonia from chr7 piRNA clusterΔpromoter / Δpromoter . See Figures 4A and B for information about the region examined .

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DNA methylation status of the Rasgrf1 differentially methylated region (DMR) in spermatogonia from chr7 piRNA clusterΔpromoter/Δpromoter. See Figures 4A and B for information about the region examined.


Depletion of piRNAs from chr7 piRNA cluster RMER4B sequence in chr7 piRNA clusterΔRMER4B / ΔRMER4B mice . Top panel shows overall view of chr7 piRNA cluster , and lower panel shows magnified view of RMER4B region in chr7 piRNA cluster . Only uniquely mapped piRNAs are shown .

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Depletion of piRNAs from chr7 piRNA cluster RMER4B sequence in chr7 piRNA clusterΔRMER4B/ΔRMER4B mice. Top panel shows overall view of chr7 piRNA cluster, and lower panel shows magnified view of RMER4B region in chr7 piRNA cluster. Only uniquely mapped piRNAs are shown.


DNA methylation status of the Rasgrf1 differentially methylated region ( DMR ) in spermatogonia from chr7 piRNA clusterΔRMER4B / ΔRMER4B mice .

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DNA methylation status of the Rasgrf1 differentially methylated region (DMR) in spermatogonia from chr7 piRNA clusterΔRMER4B/ΔRMER4B mice.


DNA methylation status of the Rasgrf1 differentially methylated region ( DMR ) in spermatocytes , spermatids , and sperms ( D ) from chr7 piRNA clusterΔRMER4B / ΔRMER4B mice .

DNA methylation status of the Rasgrf1 differentially methylated region (DMR) in spermatocytes, spermatids, and sperms (D) from chr7 piRNA clusterΔRMER4B/ΔRMER4B mice.


DNA methylation pattern in the progeny of male Rasgrf1ΔRMER4B / ΔRMER4B mice . DNA methylation status of the Rasgrf1 DMR in E12 . 5 whole embryos is shown . E12 . 5 whole embryos were obtained by the crosses shown in the panel . Two independent bisulfite - treated DNA sample were examined for Homo13 embryo .

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DNA methylation pattern in the progeny of male Rasgrf1ΔRMER4B/ΔRMER4B mice. DNA methylation status of the Rasgrf1 DMR in E12.5 whole embryos is shown. E12.5 whole embryos were obtained by the crosses shown in the panel. Two independent bisulfite-treated DNA sample were examined for Homo13 embryo.


Conditioned medium was collected from pericytes 48 h after adenoviral transduction . Western blotting verified the secretion of Sema3CΔ13 ( 81 kDa ) and Sema3Cp60 ( 60 kDa ) isoforms .

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Conditioned medium was collected from pericytes 48 h after adenoviral transduction. Western blotting verified the secretion of Sema3CΔ13 (81 kDa) and Sema3Cp60 (60 kDa) isoforms.


HUVECs were suspended in conditioned medium with VEGF ( 25 ng / ml ) and plated on Matrigel . Branch points of the capillary - like network were counted after 18 h . n = 4 independent experiments ; scale bar , 200 μm . Control vs . Sema3C , * P = 0 . 0375 and control vs . Sema3CΔ13 , * P = 0 . 0162 .

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HUVECs were suspended in conditioned medium with VEGF (25 ng/ml) and plated on Matrigel. Branch points of the capillary-like network were counted after 18 h. n = 4 independent experiments; scale bar, 200 μm. Control vs. Sema3C, *P = 0.0375 and control vs. Sema3CΔ13, *P = 0.0162.


HUVEC spheroids in collagen were treated with conditioned medium with or without VEGF ( 25 ng / ml ) . After 24 h , the cumulative sprout length per spheroid was quantified . Data are representative of three independent experiments with ten spheroids per group . Scale bar , 100 μm . Control vs . Sema3C ( − VEGF ) , * P = 0 . 0276 ; control vs . Sema3CΔ13 ( − VEGF ) , * P = 0 . 0443 ; control vs . Sema3C ( + VEGF ) , * P = 0 . 028 and control vs . Sema3CΔ13 ( + VEGF ) , * P = 0 . 032 .

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HUVEC spheroids in collagen were treated with conditioned medium with or without VEGF (25 ng/ml). After 24 h, the cumulative sprout length per spheroid was quantified. Data are representative of three independent experiments with ten spheroids per group. Scale bar, 100 μm. Control vs. Sema3C (− VEGF), *P = 0.0276; control vs. Sema3CΔ13 (− VEGF), *P = 0.0443; control vs. Sema3C (+ VEGF), *P = 0.028 and control vs. Sema3CΔ13 (+ VEGF), *P = 0.032.


Microvessels within the plugs stained against human - specific CD34 . Cell nuclei were stained with DAPI . Scale bar , 100 μm . The area of CD34 - positive microvessels was quantified and normalized to the total area of plug . Results represent the means ± s . e . m of 5 plugs ( control ) and 7 plugs ( Sema3C ) , * P = 0 . 018 .

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Microvessels within the plugs stained against human-specific CD34. Cell nuclei were stained with DAPI. Scale bar, 100 μm.The area of CD34-positive microvessels was quantified and normalized to the total area of plug. Results represent the means ± s.e.m of 5 plugs (control) and 7 plugs (Sema3C), *P = 0.018.


Vessel coverage was assessed by αSMA staining . Scale bar , 100 μm . Vessel coverage was quantified as the ratio of endothelial cell - associated mural cells to total amount of vessels ( n = 5 ( control ) and n = 7 ( Sema3C ) ) . Vessel perfusion was evaluated by i . v . injection of FITC - lectin ( 0 . 15 mg ) 30 min before mice were sacrificed . The data show the percentage of perfused vessels in the plug and normalized to total vessel area ( n = 5 ( control ) and n = 7 ( Sema3C ) ) .

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Vessel coverage was assessed by αSMA staining. Scale bar, 100 μm.Vessel coverage was quantified as the ratio of endothelial cell-associated mural cells to total amount of vessels (n = 5 (control) and n = 7 (Sema3C)).Vessel perfusion was evaluated by i.v. injection of FITC-lectin (0.15 mg) 30 min before mice were sacrificed. The data show the percentage of perfused vessels in the plug and normalized to total vessel area (n = 5 (control) and n = 7 (Sema3C)).


A C57BL / 6N mice pups at P3 received 1 μg of mouse Sema3C - Fc ( right eye ) or control IgG - Fc ( left eye ) . The retinal vasculature was examined at P5 by whole - mount CD31immunofluorescence staining . Representative images show the total retinal vasculature and sprouting tips at the vascular front ( iii and iv , arrows ) and the enlarged intercapillary space ( ii and iv , asterisks ) . Scale bar , 100 μm and 20 μm ( iii and iv ) . B , C ( B ) Retinal vessel outgrowth measured as the total area from the central optic disk to the vascular front normalized to control IgG - Fc treatment , * * * P = 0 . 0009 . ( C ) Vessel density was quantified by CD31 - stained vessels and normalized to the total vessel area , * * * P = 0 . 0002 . ( Mean ± s . e . m ; n = 23 retinae in each group from four independent experiments ; unpaired Student ' s t - test . ) D , E ( D ) Number of sprouting tips per retina , * P < 0 . 0001 . ( E ) Branch points in the capillary plexus represented as the ratio to the control IgG - treated retinae , * * * P < 0 . 0001 . ( Mean ± s . e . m ; n = 18 retinae in each group from three independent experiments ; unpaired Student ' s t - test . )

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A C57BL/6N mice pups at P3 received 1 μg of mouse Sema3C-Fc (right eye) or control IgG-Fc (left eye). The retinal vasculature was examined at P5 by whole-mount CD31immunofluorescence staining. Representative images show the total retinal vasculature and sprouting tips at the vascular front (iii and iv, arrows) and the enlarged intercapillary space (ii and iv, asterisks). Scale bar, 100 μm and 20 μm (iii and iv).B,C (B) Retinal vessel outgrowth measured as the total area from the central optic disk to the vascular front normalized to control IgG-Fc treatment, ***P = 0.0009. (C) Vessel density was quantified by CD31-stained vessels and normalized to the total vessel area, ***P = 0.0002. (Mean ± s.e.m; n = 23 retinae in each group from four independent experiments; unpaired Student's t-test.)D,E (D) Number of sprouting tips per retina, *P < 0.0001. (E) Branch points in the capillary plexus represented as the ratio to the control IgG-treated retinae, ***P < 0.0001. (Mean ± s.e.m; n = 18 retinae in each group from three independent experiments; unpaired Student's t-test.)


F Pups received same treatment as described in ( A ) . The astrocyte layer was examined at P5 by GFAP staining . Scale bar , 100 μm ( upper panel ) and 20 μm ( lower panel ) . Representative images of six retinae per group .

F Pups received same treatment as described in (A). The astrocyte layer was examined at P5 by GFAP staining. Scale bar, 100 μm (upper panel) and 20 μm (lower panel). Representative images of six retinae per group.


G Six - week - old C57BL / 6N male mice were intravitreally injected with Sema3C - Fc ( right eye ) or control IgG - Fc ( left eye ) , and retinae were collected 2 days later . The retinal vessel network was examined with CD31immunostaining . Scale bar , 100 μm ( upper panel ) and 20 μm ( lower panel ) . Representative images of four retinae per group .

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G Six-week-old C57BL/6N male mice were intravitreally injected with Sema3C-Fc (right eye) or control IgG-Fc (left eye), and retinae were collected 2 days later. The retinal vessel network was examined with CD31immunostaining. Scale bar, 100 μm (upper panel) and 20 μm (lower panel). Representative images of four retinae per group.


HUVECs were treated with Sema3C - or GFP - conditioned medium for 24 h . Cell proliferation was quantified by BrdU incorporation ( mean ± s . e . m ; n = 3 ; n . s . , non - significant ) .

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HUVECs were treated with Sema3C- or GFP-conditioned medium for 24 h. Cell proliferation was quantified by BrdU incorporation (mean ± s.e.m; n = 3; n.s., non-significant).


Apoptosis of HUVEC was measured by caspase - 3 and caspase - 7 activities after 3 h of incubation with Sema3C - or control - conditioned medium ( mean ± s . e . m ; n = 3 , * P = 0 . 018 using unpaired Student ' s t - test ) .

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Apoptosis of HUVEC was measured by caspase-3 and caspase-7 activities after 3 h of incubation with Sema3C- or control-conditioned medium (mean ± s.e.m; n = 3, *P = 0.018 using unpaired Student's t-test).


Serum - starved HUVECs were incubated with conditioned medium or conditioned medium + VEGF ( 50 ng / ml ) for 15 min . Cell lysates were subjected to SDS - PAGE and immunoblotted with anti - phospho - AKT antibody . Quantification of phospho - AKT normalized to total AKT levels ( mean ± s . e . m ; n = 4 ; control vs . Sema3C ( − VEGF ) , * P = 0 . 034 and control vs . Sema3C ( + VEGF ) , * P = 0 . 024 using unpaired Student ' s t - test ) .

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Serum-starved HUVECs were incubated with conditioned medium or conditioned medium + VEGF (50 ng/ml) for 15 min. Cell lysates were subjected to SDS-PAGE and immunoblotted with anti-phospho-AKT antibody.Quantification of phospho-AKT normalized to total AKT levels (mean ± s.e.m; n = 4; control vs. Sema3C (− VEGF), *P = 0.034 and control vs. Sema3C (+ VEGF), *P = 0.024 using unpaired Student's t-test).


HUVECs were adenovirally transduced with VE - cadherin - GFP and plated on ibidi slides ( n = 4 ; control vs . Sema3C ( − VEGF ) at 20 × magnification ( Supplementary Movies S1 and S2 ) ) . Representative pictures are shown at time points 0 and 1 h . Arrowheads indicate gaps between endothelial cells . Scale bar , 100 μm .

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HUVECs were adenovirally transduced with VE-cadherin-GFP and plated on ibidi slides (n = 4; control vs. Sema3C (− VEGF) at 20× magnification (Supplementary Movies S1 and S2)). Representative pictures are shown at time points 0 and 1 h. Arrowheads indicate gaps between endothelial cells. Scale bar, 100 μm.


C57BL / 6Nmicepups at P5 received 1 μg of recombinant Sema3C or IgG - Fc proteins . Endothelial cell junctions were examined by whole - mount VE - cadherinimmunofluorescence staining 6 h later . Representative images show VE - cadherin at cell junctions ( arrows ) . The linear structure ( i ) was disrupted in Sema3C - treated retinae ( ii ) . Scale bar , 100 μm ( upper panel ) and 20 μm ( lower panel ) .

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C57BL/6Nmicepups at P5 received 1 μg of recombinant Sema3C or IgG-Fc proteins. Endothelial cell junctions were examined by whole-mount VE-cadherinimmunofluorescence staining 6 h later. Representative images show VE-cadherin at cell junctions (arrows). The linear structure (i) was disrupted in Sema3C-treated retinae (ii). Scale bar, 100 μm (upper panel) and 20 μm (lower panel).


Whole - mount immunostaining of the vascular bed by CD31 and apoptotic cells by cleaved caspase - 3 in the retinae at P5 8 h after intravitreal injection of Sema3C or IgG - Fc . Representative images show active caspase - 3 signals that co - localize with endothelial cells ( arrows ) . Scale bar , 100 μm . Quantification of cleaved caspase - 3 normalized to area of CD31 - stained vessels ( mean ± s . e . m ; n = 5 ; * * P = 0 . 007 using paired Student ' s t - test ) .

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Whole-mount immunostaining of the vascular bed by CD31 and apoptotic cells by cleaved caspase-3 in the retinae at P5 8 h after intravitreal injection of Sema3C or IgG-Fc. Representative images show active caspase-3 signals that co-localize with endothelial cells (arrows). Scale bar, 100 μm.Quantification of cleaved caspase-3 normalized to area of CD31-stained vessels (mean ± s.e.m; n = 5; **P = 0.007 using paired Student's t-test).


A HUVECs were transduced with adenoviral vector expressing RFP - conjugated F - actin binding peptide ( LifeAct ) . Time - lapse imaging of F - actin in HUVEC spheroids was obtained at 20 × magnification ( Supplementary Movies S3 and S4 ) . Representative images are shown at 0 and 2 h . Arrowheads indicate detached cells after Sema3C treatment . Scale bar , 100 μm .

A HUVECs were transduced with adenoviral vector expressing RFP-conjugated F-actin binding peptide (LifeAct). Time-lapse imaging of F-actin in HUVEC spheroids was obtained at 20× magnification (Supplementary Movies S3 and S4). Representative images are shown at 0 and 2 h. Arrowheads indicate detached cells after Sema3C treatment. Scale bar, 100 μm.


B Endothelial cell migration was determined using a scratch wound assay with a co - culture model . A wound was generated in a confluent HUVEC monolayer , and cells were cultured in basal medium with or without VEGF ( 25 ng / ml ) in a 24 - well plate . At the same time , the transwells containing Sema3C or GFP - expressing pericytes . Representative pictures show remaining gaps within the HUVEC monolayer . Scale bar , 200 μm . C Migration distance was measured after 24 h and normalized to control treatment ( mean ± s . e . m ; n = 3 independent experiments , control vs . Sema3C ( − VEGF ) , * P = 0 . 021 and control vs . Sema3C ( + VEGF ) , * P = 0 . 039 using unpaired Student ' s t - test ) .

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B Endothelial cell migration was determined using a scratch wound assay with a co-culture model. A wound was generated in a confluent HUVEC monolayer, and cells were cultured in basal medium with or without VEGF (25 ng/ml) in a 24-well plate. At the same time, the transwells containing Sema3C or GFP-expressing pericytes. Representative pictures show remaining gaps within the HUVEC monolayer. Scale bar, 200 μm.C Migration distance was measured after 24 h and normalized to control treatment (mean ± s.e.m; n = 3 independent experiments, control vs. Sema3C (− VEGF), *P = 0.021 and control vs. Sema3C (+ VEGF), *P = 0.039 using unpaired Student's t-test).


D , E ( D ) Serum - starved HUVECs were treated with Sema3C - or control - conditioned medium with or without VEGF ( 50 ng / ml ) for 10 min . Cell lysates were subjected to SDS - PAGE and probed with anti - phosphoT180 / Y182 p38MAPK antibody . Quantification of p38MAPK activation was normalized to total p38MAPK and presented as the ratio to the cells with control medium without VEGF treatment . Control vs . Sema3C ( − VEGF ) , * P = 0 . 046 and control vs . Sema3C ( + VEGF ) , * P = 0 . 043 . ( E ) HUVECs were treated as described before and the activation of FAK detected by anti - phosphoY397 FAK antibody and normalized to the total amount of FAK . Control vs . Sema3C ( − VEGF ) , * P = 0 . 033 and control vs . Sema3C ( + VEGF ) , * P = 0 . 047 . ( Mean ± s . e . m ; n = 3 independent experiments ; unpaired Student ' s t - test . )

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D,E (D) Serum-starved HUVECs were treated with Sema3C- or control-conditioned medium with or without VEGF (50 ng/ml) for 10 min. Cell lysates were subjected to SDS-PAGE and probed with anti-phosphoT180/Y182 p38MAPK antibody. Quantification of p38MAPK activation was normalized to total p38MAPK and presented as the ratio to the cells with control medium without VEGF treatment. Control vs. Sema3C (− VEGF), *P = 0.046 and control vs. Sema3C (+ VEGF), *P = 0.043. (E) HUVECs were treated as described before and the activation of FAK detected by anti-phosphoY397 FAK antibody and normalized to the total amount of FAK. Control vs. Sema3C (− VEGF), *P = 0.033 and control vs. Sema3C (+ VEGF), *P = 0.047. (Mean ± s.e.m; n = 3 independent experiments; unpaired Student's t-test.)


F Serum - starved HUVECs were treated with conditioned medium for 15 min . Focal adhesion proteins were examined with anti - phosphoY397FAK and anti - vinculin antibodies . F - actin was stained with Alexa Fluor 546 - conjugated phalloidin . Merged images show the coupling of stress fibers with focal adhesion complexes in the cells treated with control medium . Inset , phospho - Y397FAK and vinculin formed focal adhesions that were stained in an elongated dot - like structure ( i ) . Lower panel ( ii ) , Sema3C treatment induced F - actin disassembly and membrane ruffling . Phospho - Y397FAK and vinculin were randomly distributed at the peripheral membrane . Scale bars , 10 μm .

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F Serum-starved HUVECs were treated with conditioned medium for 15 min. Focal adhesion proteins were examined with anti-phosphoY397FAK and anti-vinculin antibodies. F-actin was stained with Alexa Fluor 546-conjugated phalloidin. Merged images show the coupling of stress fibers with focal adhesion complexes in the cells treated with control medium. Inset, phospho-Y397FAK and vinculin formed focal adhesions that were stained in an elongated dot-like structure (i). Lower panel (ii), Sema3C treatment induced F-actin disassembly and membrane ruffling. Phospho-Y397FAK and vinculin were randomly distributed at the peripheral membrane. Scale bars, 10 μm.


HUVECs were transfected with siRNA against PlexinD1 or control NS ( non - silence ) siRNA . qPCR analyses and Western blotting were performed after 48 h ( n = 3 ; paired Student ' s t - test , NS siRNA vs . PlexinD1 siRNA , * * * P = 0 . 0004 ) . Cell lysates were subjected to SDS - PAGE and probed with anti - PlexinD1 .

HUVECs were transfected with siRNA against PlexinD1 or control NS (non-silence) siRNA. qPCR analyses and Western blotting were performed after 48 h (n = 3; paired Student's t-test, NS siRNA vs. PlexinD1 siRNA, ***P = 0.0004). Cell lysates were subjected to SDS-PAGE and probed with anti-PlexinD1.


HUVECs were transfected with siRNA against PlexinD1 and plated on gelatin - coated coverslips . Confluent monolayers were treated with Sema3C - , Sema3Cp60 - or control - conditioned medium for 30 min . Cells were fixed and stained with Alexa - Fluor 488 - conjugated phalloidin to visualize F - actin . Arrows indicate Sema3C - induced cell membrane ruffling . Scale bar , 20 μm . Cell repellence was quantified as the number of cells with membrane ruffling normalized to the number of nuclei ( n = 5 ; mean ± s . e . m . , paired Student ' s t - test , * * P = 0 . 001 ) .

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HUVECs were transfected with siRNA against PlexinD1 and plated on gelatin-coated coverslips. Confluent monolayers were treated with Sema3C-, Sema3Cp60- or control-conditioned medium for 30 min. Cells were fixed and stained with Alexa-Fluor 488-conjugated phalloidin to visualize F-actin. Arrows indicate Sema3C-induced cell membrane ruffling. Scale bar, 20 μm. Cell repellence was quantified as the number of cells with membrane ruffling normalized to the number of nuclei (n = 5; mean ± s.e.m., paired Student's t-test, **P = 0.001).


Silencing of Nrp - 1 by lentiviral particles expressing shRNA . After culturing 72 h in selection medium , RNA was harvested for qPCR analysis . NS shRNA vs . Nrp - 1 shRNA : # 1 , * P = 0 . 017 and # 2 , * P = 0 . 02 ; n = 3 ; mean ± s . e . m . Cell lysates were subjected to SDS - PAGE and probed with anti - Nrp - 1 .

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Silencing of Nrp-1 by lentiviral particles expressing shRNA. After culturing 72 h in selection medium, RNA was harvested for qPCR analysis. NS shRNA vs. Nrp-1 shRNA: #1, *P = 0.017 and #2, *P = 0.02; n = 3; mean ± s.e.m. Cell lysates were subjected to SDS-PAGE and probed with anti-Nrp-1.


Depletion of Nrp - 1 abolished the response to Sema3C . Sema3Cp60 had no effect on HUVEC adhesion . Arrows indicate membrane ruffling . Scale bar , 20 μm . Cell repellence was quantified as the number of cells with membrane ruffling normalized to the number of nuclei ( n = 5 ; mean ± s . e . m . ; paired Student ' s t - test , * * * P = 0 . 0003 ) .

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Depletion of Nrp-1 abolished the response to Sema3C. Sema3Cp60 had no effect on HUVEC adhesion. Arrows indicate membrane ruffling. Scale bar, 20 μm. Cell repellence was quantified as the number of cells with membrane ruffling normalized to the number of nuclei (n = 5; mean ± s.e.m.; paired Student's t-test, ***P = 0.0003).


HUVECs were treated with Sema3C - conditioned medium for 10 min and lysed , and Nrp - 1 was immunoprecipitated . Bound Sema3C was detected by Western blotting .

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HUVECs were treated with Sema3C-conditioned medium for 10 min and lysed, and Nrp-1 was immunoprecipitated. Bound Sema3C was detected by Western blotting.


RNA scope analyses showed Sema3C and PlexinD1 expression in pre - retinal tufts ( OIR model , P19 ) . The retinae were subsequently stained against collagen IV . Sema3CmRNA was detected in peri - endothelial cells . Scale bar : 20 μm .

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RNA scope analyses showed Sema3C and PlexinD1 expression in pre-retinal tufts (OIR model, P19). The retinae were subsequently stained against collagen IV. Sema3CmRNA was detected in peri-endothelial cells. Scale bar: 20 μm.


Nrp - 1 expression in pre - retinal tufts ( OIR model , P17 ) . Abundant expression of Nrp1 in endothelial cells . Pericytes at vascular tufts were immunostained with anti - NG2 . Scale bar , 20 μm .

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Nrp-1 expression in pre-retinal tufts (OIR model, P17). Abundant expression of Nrp1 in endothelial cells. Pericytes at vascular tufts were immunostained with anti-NG2. Scale bar, 20 μm.


H & amp ; amp ; E staining shows neovascular tufts ( arrows ) . Representative of five retinae per group in two independent experiments . Scale bar , 50 μm .

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H&amp;E staining shows neovascular tufts (arrows). Representative of five retinae per group in two independent experiments. Scale bar, 50 μm.


Whole - mount CD31 immunostaining . The white line defines the whole retinal area , and red line outlines the vaso - obliterative zone . Inset image shows large numbers of peri - retinal tufts ( i , arrow heads ) in control IgG - Fc - treated retinae compared to Sema3C - treated retinae ( ii ) . Scale bar , 500 μm ( upper panel ) and 100 μm ( lower panel ) . The avascular area is presented as percentage of the whole retinal area ( n = 10 retinae in each group ; n . s . , non - significant using paired Student ' s t - test ) . The formation of pre - retinal tufts was quantified in the superficial layer of the retinal vasculature and normalized to whole retinal area ( n = 6 retinae in each group ; * P = 0 . 033 using paired Student ' s t - test ) .

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Whole-mount CD31 immunostaining. The white line defines the whole retinal area, and red line outlines the vaso-obliterative zone. Inset image shows large numbers of peri-retinal tufts (i, arrow heads) in control IgG-Fc-treated retinae compared to Sema3C-treated retinae (ii). Scale bar, 500 μm (upper panel) and 100 μm (lower panel).The avascular area is presented as percentage of the whole retinal area (n = 10 retinae in each group; n.s., non-significant using paired Student's t-test).The formation of pre-retinal tufts was quantified in the superficial layer of the retinal vasculature and normalized to whole retinal area (n = 6 retinae in each group; *P = 0.033 using paired Student's t-test).


Confocal images of mPS - PI stained 6 - day - old seedlings of Col - 0 ( A ) , bravo - 2 ( B ) , wox5 - 1 ( C ) and bravo - 2 wox5 - 1 ( D ) mutants . Left black arrows indicate QC cells and right white arrows indicate CSC . Scale bar : 50 µm .

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Confocal images of mPS-PI stained 6-day-old seedlings of Col-0 (A), bravo-2 (B), wox5-1 (C) and bravo-2 wox5-1 (D) mutants. Left black arrows indicate QC cells and right white arrows indicate CSC. Scale bar: 50 µm.


Quantification of the QC divisions in 6 - day - old roots expressed in percentage ( n > 50 , 3 replicates ) . D : QC divided ; ND : QC non divided .

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Quantification of the QC divisions in 6-day-old roots expressed in percentage (n>50, 3 replicates). D: QC divided; ND: QC non divided.


Quantification of CSC layers in 6 - day - old roots expressed in percentage ( n > 50 , 3 replicates ) .

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Quantification of CSC layers in 6-day-old roots expressed in percentage (n>50, 3 replicates).


Lateral root density ( number of lateral roots per mm of root length ) of 7 - day - olf WT , bravo - 2 , wox5 - 1 and bravo - 2 wox5 - 1 mutants ( n > 40 , 2 replicates ) . Different letters indicate statistically significant differences ( p - value < 0 . 05 Student ´ s t - test ) . In the boxplot , box width represents the interquartile range ( IQR = Q3 - Q1 ) , with the horizontal line denoting the median , while whiskers extend from Q1 - 1 . 5IQR to Q3 + 1 . 5IQR . White dots are the outliers .

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Lateral root density (number of lateral roots per mm of root length) of 7-day-olf WT, bravo-2, wox5-1 and bravo-2 wox5-1 mutants (n>40, 2 replicates). Different letters indicate statistically significant differences (p- value < 0.05 Student´s t-test). In the boxplot, box width represents the interquartile range (IQR=Q3-Q1), with the horizontal line denoting the median, while whiskers extend from Q1-1.5IQR to Q3+1.5IQR. White dots are the outliers.


Confocal images of PI - stained 6 - day - old roots . GFP - tagged expression is shown in yellow . A - C ) pBRAVO : GFP in WT ( A ) , bravo - 2 ( B ) and wox5 - 1 ( C ) knockout backgrounds . D - G ) pWOX5 : GFP in the WT ( D ) , bravo - 2 ( E ) , wox5 - 1 ( F ) and bravo - 2 wox5 - 1 ( G ) knockout backgrounds . Scale bar : 50 µm . Quantification of the GFP fluorescent signal of the roots in A - C ( H ) and D - G ( I ) . Boxplot indicating the average pixel intensity of the GFP in the stem cell niche ( n > 25 , 3 biological replicates , * p - value < 0 . 05 Student ´ s t - test for each genotype versus the WT in the same condition ) . Quantification was done by integrating the GFP signal in each root across defined areas that included the whole SCN ( Appendix Figure S7 ) . In the boxplot , box width represents the interquartile range ( IQR = Q3 - Q1 ) , with the horizontal line denoting the median , while whiskers extend from Q1 - 1 . 5IQR to Q3 + 1 . 5IQR . White dots are the outliers and black dots are the experimental observations .

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Confocal images of PI-stained 6-day-old roots. GFP-tagged expression is shown in yellow. A-C) pBRAVO:GFP in WT (A), bravo-2 (B) and wox5-1 (C) knockout backgrounds. D-G) pWOX5:GFP in the WT (D), bravo-2 (E), wox5-1 (F) and bravo-2 wox5-1 (G) knockout backgrounds. Scale bar: 50 µm. Quantification of the GFP fluorescent signal of the roots in A-C (H) and D-G (I). Boxplot indicating the average pixel intensity of the GFP in the stem cell niche (n>25, 3 biological replicates, *p-value < 0.05 Student´s t- test for each genotype versus the WT in the same condition). Quantification was done by integrating the GFP signal in each root across defined areas that included the whole SCN (Appendix Figure S7). In the boxplot, box width represents the interquartile range (IQR=Q3-Q1), with the horizontal line denoting the median, while whiskers extend from Q1-1.5IQR to Q3+1.5IQR. White dots are the outliers and black dots are the experimental observations.


Parameter space exploration of ( C ) the alleviation and the ( D ) activation models . Boxplots showing the distribution of stationary promoter pBRAVO ( pB ) and pWOX5 ( pW ) fold - changes in each mutant and overexpression lines over the WT obtained for different parameter values . Superindexes denote the genotype , standing bravo for bravo mutant , wox5 for wox5 mutant , dm for the bravo wox5 double mutant , Woe for WOX5 overexpression and Boe for BRAVO overexpression . Box width represents the interquartile range ( IQR = Q3 - Q1 ) , with the horizontal line denoting the median , while whiskers extend from Q1 - 1 . 5IQR to Q3 + 1 . 5IQR . The results are obtained by solving N = 1000 different runs of each model at the stationary state ( red and blue stripplots ) for the WT , mutants and overexpressor scenarios . These N runs differ in the parameter values , which are chosen at random from a uniform distribution

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Parameter space exploration of (C) the alleviation and the (D) activation models. Boxplots showing the distribution of stationary promoter pBRAVO (pB) and pWOX5 (pW) fold-changes in each mutant and overexpression lines over the WT obtained for different parameter values. Superindexes denote the genotype, standing bravo for bravo mutant, wox5 for wox5 mutant, dm for the bravo wox5 double mutant, Woe for WOX5 overexpression and Boe for BRAVO overexpression. Box width represents the interquartile range (IQR=Q3-Q1), with the horizontal line denoting the median, while whiskers extend from Q1-1.5IQR to Q3+1.5IQR. The results are obtained by solving N=1000 different runs of each model at the stationary state (red and blue stripplots) for the WT, mutants and overexpressor scenarios. These N runs differ in the parameter values, which are chosen at random from a uniform distribution between


Interaction of BRAVO with WOX5 ( B ) , BES1 ( C ) , BES1D ( D ) and TPL ( E ) ; and interaction of WOX5 with BRAVO ( G ) , BES1 ( H ) , BES1D ( I ) and TPL ( J ) measured by FRET - FLIM . GFP fluorescence lifetime τ [ ns ] was measured in transiently expressing Nicotiana benthamiana leaf epidermal cells . GFP fluorescence lifetime fitted pixel - wise with a mono - exponential model of BRAVO and WOX5 interactions . mV , mVenus ; mCh , mCherry . Scale bar : 5 µm .

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Interaction of BRAVO with WOX5 (B), BES1 (C), BES1D (D) and TPL (E); and interaction of WOX5 with BRAVO (G), BES1 (H), BES1D (I) and TPL (J) measured by FRET-FLIM. GFP fluorescence lifetime τ [ns] was measured in transiently expressing Nicotiana benthamiana leaf epidermal cells. GFP fluorescence lifetime fitted pixel-wise with a mono-exponential model of BRAVO and WOX5 interactions. mV, mVenus; mCh, mCherry. Scale bar: 5 µm.


Fluorescence - weighted average lifetimes of BRAVO and WOX5 interactions fitted with a double - exponential model of the indicated samples are summarized in box plots . Statistical significance was tested by one - way ANOVA with a Sidakholm post - hoc test . Different letters indicate statistically significant differences ( p - value < 0 . 01 ) . For each combination two to three independent experiments were carried out and in total more than 20 biological replicates were measured . In the boxplot , box width represents the interquartile range ( IQR = Q3 - Q1 ) , with the horizontal line denoting the median , while whiskers extend from Q1 - 1 . 5IQR to Q3 + 1 . 5IQR . White dots are the outliers .

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Fluorescence-weighted average lifetimes of BRAVO and WOX5 interactions fitted with a double-exponential model of the indicated samples are summarized in box plots. Statistical significance was tested by one-way ANOVA with a Sidakholm post-hoc test. Different letters indicate statistically significant differences (p-value < 0.01). For each combination two to three independent experiments were carried out and in total more than 20 biological replicates were measured. In the boxplot, box width represents the interquartile range (IQR=Q3-Q1), with the horizontal line denoting the median, while whiskers extend from Q1-1.5IQR to Q3+1.5IQR. White dots are the outliers.


Yeast two - hybrid assay showing BRAVO interacting with WOX5 , BES1 - D and TPL ; and BES1 - D interacting with TPL . In the left column yeast cells were grown on control media , and in the right column yeast cells were grown on control media lacking Leu , Trp and His , indicating an interaction between the proteins .

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Yeast two-hybrid assay showing BRAVO interacting with WOX5, BES1-D and TPL; and BES1-D interacting with TPL. In the left column yeast cells were grown on control media, and in the right column yeast cells were grown on control media lacking Leu, Trp and His, indicating an interaction between the proteins.


Expression of the 380 common BRAVO and WOX5 regulated genes from ( B ) . Expression of the 53 genes from ( C ) showing similar expression in bravo and wox5 mutants . Expression of the 41 genes from ( C ) showing opposite expression levels in bravo and wox5 mutants .

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Expression of the 380 common BRAVO and WOX5 regulated genes from (B). Expression of the 53 genes from (C) showing similar expression in bravo and wox5 mutants. Expression of the 41 genes from (C) showing opposite expression levels in bravo and wox5 mutants.


Immune response and physiology of monkeys immunized with recombinant adenovirus vaccine . Time course of antibody response . Serum samples were diluted 40 - fold prior to ELISA analysis . Animals in the high - dose and low - dose group were immunized with 1 × 109 and 1 × 108 pfu virus each time , respectively . Arrows indicate the time point for immunization .

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Immune response and physiology of monkeys immunized with recombinant adenovirus vaccine. Time course of antibody response. Serum samples were diluted 40-fold prior to ELISA analysis. Animals in the high-dose and low-dose group were immunized with 1×109 and 1×108 pfu virus each time, respectively. Arrows indicate the time point for immunization.


Immune response and physiology of monkeys immunized with recombinant adenovirus vaccine . Determination of virus neutralization antibody titer using 96 - well microneutralization assay . An arbitrate titer of 2 was assigned to animals in the placebo and control group since there was no detectable neutralization activity in these samples . Statistical significance between different data sets was calculated using t - test .

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Immune response and physiology of monkeys immunized with recombinant adenovirus vaccine. Determination of virus neutralization antibody titer using 96-well microneutralization assay. An arbitrate titer of 2 was assigned to animals in the placebo and control group since there was no detectable neutralization activity in these samples. Statistical significance between different data sets was calculated using t-test.


Immune response and physiology of monkeys immunized with recombinant adenovirus vaccine . ELISPOT analysis of IFN - γ response in PBMC samples . Pool A and B contain peptides corresponding to S1 - orf8 fusion and N , respectively . Data represent two animals from each group .

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Immune response and physiology of monkeys immunized with recombinant adenovirus vaccine. ELISPOT analysis of IFN-γ response in PBMC samples. Pool A and B contain peptides corresponding to S1-orf8 fusion and N, respectively. Data represent two animals from each group.


Immune response and physiology of monkeys immunized with recombinant adenovirus vaccine . Average food consumption after live virus challenge .

Immune response and physiology of monkeys immunized with recombinant adenovirus vaccine. Average food consumption after live virus challenge.


Histology and IHC study on postmortem lung sections from rhesus macaques infected with live SARS - CoV . All animals were euthanized 7 days after viral challenge , and anti - SARS - CoV monoclonal antibody was used to identify SARS antigens in the tissue . Representative data from lung sections were shown . Histology samples include : a . Control group . b . High - dose group . c . Placebo group . The arrowheads point to alveoli space filled with proteinaceous fluid . IHC samples include : d . Control group . e . High - dose group . f . Placebo group . Arrows point to cells that were positive for SARS antigen expression .

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Histology and IHC study on postmortem lung sections from rhesus macaques infected with live SARS-CoV. All animals were euthanized 7 days after viral challenge, and anti-SARS-CoV monoclonal antibody was used to identify SARS antigens in the tissue. Representative data from lung sections were shown. Histology samples include: a. Control group. b. High-dose group. c. Placebo group. The arrowheads point to alveoli space filled with proteinaceous fluid. IHC samples include: d. Control group. e. High-dose group. f. Placebo group. Arrows point to cells that were positive for SARS antigen expression.


mRNA levels of TMX1 ( A ) and NFAT1 ( B ) in primary melanocytes , keratinocytes and melanoma cell lines . data are are normalized to the expression of TBP and are presented as mean ± SEM ( n ≥ 3 ) .

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mRNA levels of TMX1 (A) and NFAT1 (B) in primary melanocytes, keratinocytes and melanoma cell lines. data are are normalized to the expression of TBP and are presented as mean ± SEM (n≥3).


Representative immunoblot ( from 3 ) depicting protein abundance of TMX1 and NFAT1 in melanocytes and melanoma cell lines ; actin was used as a loading control . Indicated band intensities were normalized to actin and background signal was subtracted .

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Representative immunoblot (from 3) depicting protein abundance of TMX1 and NFAT1 in melanocytes and melanoma cell lines; actin was used as a loading control. Indicated band intensities were normalized to actin and background signal was subtracted.


Melan - A ( brown ) , TMX1 ( red - brown ) and NFAT1 ( deep red ) staining ( IHC ) in a primary stage 4 high risk nodular melanoma patient I ( out of four ) , the enlarged regions show healthy tissue ( panels 2 , 5 and 8 ) and tumor tissue ( panels 3 , 6 and 9 ) . Arrows indicate Melan - A positive melanocytes .

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Melan-A (brown), TMX1 (red-brown) and NFAT1 (deep red) staining (IHC) in a primary stage 4 high risk nodular melanoma patient I (out of four), the enlarged regions show healthy tissue (panels 2, 5 and 8) and tumor tissue (panels 3, 6 and 9). Arrows indicate Melan-A positive melanocytes.


TMX1 ( red - brown ) and NFAT1 ( deep red ) staining of healthy human skin and increasing melanoma stages ; P1 - P13 refer to the donor patient number .

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TMX1 (red-brown) and NFAT1 (deep red) staining of healthy human skin and increasing melanoma stages; P1-P13 refer to the donor patient number.


Insulin ( 50 µg / mL ) induced the translocation of NFAT1 - GFP to the nucleus of WM3734 cells . ( A ) Ratio images ( F / F0 ) , ( B ) corresponding endpoint quantification ( n = 5 ) .

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Insulin (50 µg/mL) induced the translocation of NFAT1-GFP to the nucleus of WM3734 cells. (A) Ratio images (F/F0), (B) corresponding endpoint quantification (n=5).


Reduced nuclear translocation of NFAT1 - GFP in TMX1 or TMX3 silenced ( siRNA ) melanoma cells . ( C ) Images show NFAT1 - GFP fluorescence intensity before and after stimulation with thapsigargin ( Tg ; 1 µM ) in WM3734 cells . ( D ) Corresponding time - dependent nuclear import of NFAT1 as a change of F / F0 . ( E ) Normalized endpoint quantification . Same analysis was performed with Mel Juso cells with ( F ) images , ( G ) time - dependent nuclear import and ( H ) normalized endpoint quantification . data are presented as mean ± SEM ( n values : WM3734 , control = 142 , TMX1 kd = 116 , TMX3 kd = 148 ; Mel Juso , control = 75 , TMX1 kd = 47 , TMX3 kd = 67 ) .

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Reduced nuclear translocation of NFAT1-GFP in TMX1 or TMX3 silenced (siRNA) melanoma cells. (C) Images show NFAT1-GFP fluorescence intensity before and after stimulation with thapsigargin (Tg; 1 µM) in WM3734 cells. (D) Corresponding time-dependent nuclear import of NFAT1 as a change of F/F0. (E) Normalized endpoint quantification. Same analysis was performed with Mel Juso cells with (F) images, (G) time-dependent nuclear import and (H) normalized endpoint quantification. data are presented as mean ± SEM (n values: WM3734, control=142, TMX1 kd=116, TMX3 kd=148; Mel Juso, control=75, TMX1 kd=47, TMX3 kd=67).


Thapsigargin ( Tg ) induced Fura - 2 based cytosolic Ca2 + imaging in Ringer ' s buffer containing 0 . 25 mM Ca2 + . ( B ) Quantification of basal cytosolic calcium levels and ( C ) SOCE quantification ( plateau - basal ) for WM3734 after stable silencing of TMX1 ( two clones ) . : In ( B - C ) , data are presented as mean ± SEM ( n values : WM3734 , control = 939 , TMX1 kds 1 = 988 , TMX1 kds 2 = 508 ) .

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Thapsigargin (Tg) induced Fura-2 based cytosolic Ca2+ imaging in Ringer's buffer containing 0.25 mM Ca2+. (B) Quantification of basal cytosolic calcium levels and (C) SOCE quantification (plateau-basal) for WM3734 after stable silencing of TMX1 (two clones). : In (B-C), data are presented as mean ± SEM (n values: WM3734, control=939, TMX1 kds 1=988, TMX1 kds 2=508).


Cytosolic Ca2 + imaging ( Fura - 2 ) and ( E ) SOCE quantification ( plateau - basal ) for WM3734 after transient silencing of TMX1 or TMX3 . In ( E ) , data are presented as mean ± SEM ( n values : WM3734 , control = 30 , TMX1 kd = 49 , TMX3 kd = 52 ) .

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Cytosolic Ca2+ imaging (Fura-2) and (E) SOCE quantification (plateau-basal) for WM3734 after transient silencing of TMX1 or TMX3. In (E), data are presented as mean ± SEM (n values: WM3734, control=30, TMX1 kd=49, TMX3 kd=52).


Cellular H2O2 ( HyPer ) was evaluated in two melanoma cell lines upon TMX1 kd and TMX3 kd . ( F ) Exemplary ratiometric images ( F505 nm / F420 nm ) are shown for WM3734 and Mel Juso . Quantification of basal cellular H2O2 in WM3734 ( G ) and Mel Juso ( H ) cells . In ( G - H ) , data are presented as mean ± SEM ( n values : WM3734 : control = 168 , TMX1 kd = 209 , TMX3 kd = 192 ; Mel Juso : control = 297 , TMX1 kd = 343 , TMX3 kd = 440 ) . In ( F ) scale bars : 10 µm ; color code : WM3734 : color code : blue = 0 , red = 3 ; Mel Juso : color code : blue = 0 , red = 1 . 5 .

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Cellular H2O2 (HyPer) was evaluated in two melanoma cell lines upon TMX1 kd and TMX3 kd. (F) Exemplary ratiometric images (F505 nm / F420 nm) are shown for WM3734 and Mel Juso. Quantification of basal cellular H2O2 in WM3734 (G) and Mel Juso (H) cells. In (G-H), data are presented as mean ± SEM (n values: WM3734: control=168, TMX1 kd=209, TMX3 kd=192; Mel Juso: control=297, TMX1 kd=343, TMX3 kd=440). In (F) scale bars: 10 µm; color code: WM3734: color code: blue=0, red=3; Mel Juso: color code: blue=0, red=1.5.


WM3734 cells were pre - incubated for 5 min with extracellular H2O2 ( 0 - 500 µM ) . Tg - stimulated NFAT1 - GFP nuclear translocation was measured as F / F0 .

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WM3734 cells were pre-incubated for 5 min with extracellular H2O2 (0 - 500 µM). Tg-stimulated NFAT1-GFP nuclear translocation was measured as F/F0.


The IC50 of H2O2 inhibition of NFAT1 nuclear import was determined to be 13 . 2 µM ( red mark ) .

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The IC50 of H2O2 inhibition of NFAT1 nuclear import was determined to be 13.2 µM (red mark).


Tg - induced NFAT1 - GFP nuclear import in transient TMX1 silenced WM3734 cells and after pre - incubation with 100 µM N - acetylcysteine ( NAC ) for 48 h .

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Tg-induced NFAT1-GFP nuclear import in transient TMX1 silenced WM3734 cells and after pre-incubation with 100 µM N-acetylcysteine (NAC) for 48 h.


Endpoint quantification of the data from ( C ) and upon treatment with NAC , PEG - catalase ( 50 U / mL ) , or DTT ( 1 mM ) . data are presented as mean ± SEM ( n values : control = 73 , control + NAC = 19 , TMX1 kd = 57 , TMX1 kd + NAC = 63 , TMX1 kd + catalase = 58 , TMX1 kd + DTT = 22 ) .

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Endpoint quantification of the data from (C) and upon treatment with NAC, PEG-catalase (50 U/mL), or DTT (1 mM). data are presented as mean ± SEM (n values: control=73, control+NAC= 19, TMX1 kd=57, TMX1 kd+NAC=63, TMX1 kd+catalase=58, TMX1 kd+DTT=22).


Cytosolic calcineurin activity ( CaNAR2 ) in WM3734 cells with transient knockdown of TMX1 or TMX3 measured upon Tg stimulation .

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Cytosolic calcineurin activity (CaNAR2) in WM3734 cells with transient knockdown of TMX1 or TMX3 measured upon Tg stimulation.


Quantification of the basal calcineurin activity and the maximal calcineurin activity ( plateau - basal ) . data are presented as mean ± SEM ( n values : control = 49 , TMX1 kd = 48 , TMX3 kd = 63 ) .

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Quantification of the basal calcineurin activity and the maximal calcineurin activity (plateau-basal). data are presented as mean ± SEM (n values: control=49, TMX1 kd=48, TMX3 kd=63).


Normalized Tg - induced cytosolic calcineurin activity in Mel Juso cells after 5 min pre - incubation with extracellular H2O2 ( 0 - 200 µM ) . Quantification of the maximum cytosolic calcineurin activity ( plateau - basal ) .

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Normalized Tg-induced cytosolic calcineurin activity in Mel Juso cells after 5 min pre-incubation with extracellular H2O2 (0 - 200 µM). Quantification of the maximum cytosolic calcineurin activity (plateau-basal).


Quantification of the basal calcineurin activity and the maximal calcineurin activity ( plateau - basal ) 8 h after treatment with the antioxidant NAC ( 100 µM ) in WM3734 with transient knockdown of TMX1 . data are presented as mean ± SEM ( n values : control = 15 , TMX1 kd = 24 , TMX1 kd + NAC = 19 ) .

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Quantification of the basal calcineurin activity and the maximal calcineurin activity (plateau-basal) 8 h after treatment with the antioxidant NAC (100 µM) in WM3734 with transient knockdown of TMX1. data are presented as mean ± SEM (n values: control=15, TMX1 kd=24, TMX1 kd+NAC=19).


Mitochondrial H2O2 ( mito - HyPer ) was measured in two cell lines upon transient TMX knockdown . Exemplary ratiometric images ( F505 nm / F420 nm ) are shown ( A ) ; corresponding quantification for ( B ) WM3734 and ( C ) Mel Juso . , data are presented as mean ± SEM ( n values : WM3734 : control = 546 , TMX1 kd = 510 , TMX3 kd = 621 ; Mel Juso : control = 416 , TMX1 kd = 418 , TMX3 kd = 442 ) .

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Mitochondrial H2O2 (mito-HyPer) was measured in two cell lines upon transient TMX knockdown. Exemplary ratiometric images (F505 nm / F420 nm) are shown (A); corresponding quantification for (B) WM3734 and (C) Mel Juso. , data are presented as mean ± SEM (n values: WM3734: control=546, TMX1 kd=510, TMX3 kd=621; Mel Juso: control=416, TMX1 kd=418, TMX3 kd=442).


Mitochondrial Ca2 + uptake in WM3734 with stable TMX1 knockdown was measured using a mitochondria - targeted calcium sensor ( 4mt - D3cpV ) ( D ) . Cells were exposed to 0 . 25 mM Ca2 + containing Ringer ' s buffer and Tg . ( E ) Quantification of basal mitochondrial Ca2 + levels and ( F ) mitochondrial Ca2 + uptake ( basal - plateau ) . , data are presented as mean ± SEM ( n values : control = 62 , TMX1 kds = 45 ) .

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Mitochondrial Ca2+ uptake in WM3734 with stable TMX1 knockdown was measured using a mitochondria-targeted calcium sensor (4mt-D3cpV) (D). Cells were exposed to 0.25 mM Ca2+ containing Ringer's buffer and Tg. (E) Quantification of basal mitochondrial Ca2+ levels and (F) mitochondrial Ca2+ uptake (basal-plateau). , data are presented as mean ± SEM (n values: control=62, TMX1 kds=45).


Mitochondrial volume was determined using MitoTracker DeepRed in WM3734 cells with stable knockdown of TMX1 . The mean mitochondrial volume was modeled based on microscopy , the quantification of mean volume ( µm3 ) and mean surface ( µm2 ) is depicted in ( G ) and ( H ) respectively . data are presented as boxplots ( n values : control = 101 , TMX1 kds = 90 ; center line : median ; box : 25 % and 75 % percentile ; whiskers : 1 . 5 times interquartile range ; outliers are shown as dots ) .

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Mitochondrial volume was determined using MitoTracker DeepRed in WM3734 cells with stable knockdown of TMX1. The mean mitochondrial volume was modeled based on microscopy, the quantification of mean volume (µm3) and mean surface (µm2) is depicted in (G) and (H) respectively. data are presented as boxplots (n values: control=101, TMX1 kds=90; center line: median; box: 25 % and 75 % percentile; whiskers: 1.5 times interquartile range; outliers are shown as dots).


Peripheral mitochondria were quantified in WM3734 cells with stable knockdown of TMX1 . A peripheral mask was applied based on the membrane staining ( CellMask Green ) and mitochondria covered area ( MitoTracker DeepRed ) was evaluated ( see Fig . 9 ) . Representative images ( I ) and quantification ( J ) . In ( J ) , data are presented as mean ± SEM ( n values : control = 163 , TMX1 kds = 116 ) .

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Peripheral mitochondria were quantified in WM3734 cells with stable knockdown of TMX1. A peripheral mask was applied based on the membrane staining (CellMask Green) and mitochondria covered area (MitoTracker DeepRed) was evaluated (see Fig. 9). Representative images (I) and quantification (J). In (J), data are presented as mean ± SEM (n values: control=163, TMX1 kds=116).


Representative electron micrographs of control and 1205Lu cells with stable TMX1 knockdown ( K ) . Corresponding quantification of the distance between ( L ) mitochondria and plasma membrane , ( M ) mitochondria and ER , and ( N ) the length of the MAM ( mitochondria - ER contact site ) . In ( L ) , data are presented as mean ± SEM ( n values : control = 100 , TMX1 kds = 100 ) . In ( M - N ) , data are presented as mean ± SEM ( n values : control = 60 , TMX1 kds = 60 ) .

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Representative electron micrographs of control and 1205Lu cells with stable TMX1 knockdown (K). Corresponding quantification of the distance between (L) mitochondria and plasma membrane, (M) mitochondria and ER, and (N) the length of the MAM (mitochondria-ER contact site). In (L), data are presented as mean ± SEM (n values: control=100, TMX1 kds= 100). In (M-N), data are presented as mean ± SEM (n values: control=60, TMX1 kds= 60).


Cellular H2O2 concentration ( HyPer ) 48 h after transient silencing of TMX1 and / or NOX4 in WM3734 cells or after inhibiting NOX4 with GKT137831 ( 140 nM ) ( O ) Representative ratiometric images ( F505 nm / F420 nm ) and ( P ) quantification . In ( P ) , data are presented as mean ± SEM ( n values : control = 837 , TMX1 kd = 888 , TMX1 kd + NOX4 kd = 793 , TMX1 kd + GKT = 844 ) .

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Cellular H2O2 concentration (HyPer) 48 h after transient silencing of TMX1 and / or NOX4 in WM3734 cells or after inhibiting NOX4 with GKT137831 (140 nM) (O) Representative ratiometric images (F505 nm / F420 nm) and (P) quantification. In (P), data are presented as mean ± SEM (n values: control=837, TMX1 kd=888, TMX1 kd + NOX4 kd=793, TMX1 kd + GKT=844).


Proliferation ( 48 h ) of melanoma cell lines measured after transient knockdown of TMX1 or TMX3 .

Proliferation (48 h) of melanoma cell lines measured after transient knockdown of TMX1 or TMX3.


Proliferation ( 48 h ) of melanoma cell lines with stable TMX1 knockdown ( two clones ) .

Proliferation (48 h) of melanoma cell lines with stable TMX1 knockdown (two clones).


Proliferation ( 24 h ) of melanoma cell lines after transient knockdown of NFAT1 .

Proliferation (24 h) of melanoma cell lines after transient knockdown of NFAT1.


Proliferation of WM3734 48 h after transient knockdown of TMX1 and treatment with NAC ( 100 µM ) or mTEMPO ( 100 nM ) .

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Proliferation of WM3734 48 h after transient knockdown of TMX1 and treatment with NAC (100 µM) or mTEMPO (100 nM).


Transwell migration ( 48 h ) of WM3734 after transient knockdown of TMX1 or TMX3 .

Transwell migration (48 h) of WM3734 after transient knockdown of TMX1 or TMX3.


Transwell migration ( 48 h ) of WM3734 after transient knockdown of TMX1 and treatment with NAC ( 100 µM ) or mTEMPO ( 1 µM ) .

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Transwell migration (48 h) of WM3734 after transient knockdown of TMX1 and treatment with NAC (100 µM) or mTEMPO (1 µM).


Transwell migration ( 48 h ) of melanoma cell lines after preventing NFAT - calcineurin interaction with dipyridamole ( 40 µM ) or inhibition of calcineurin with cyclosporine A ( CsA , 2 µM ) .

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Transwell migration (48 h) of melanoma cell lines after preventing NFAT-calcineurin interaction with dipyridamole (40 µM) or inhibition of calcineurin with cyclosporine A (CsA, 2 µM).


Invasion of WM3734 with stable TMX1 knockdown ( two clones ) through Matrigel for 96 h .

Invasion of WM3734 with stable TMX1 knockdown (two clones) through Matrigel for 96 h.