qPCR measurements of JARID2 mRNA levels in different cell lines . The RNA levels were measured relative to 18S rRNA . Means and SEs are calculated over at least 2 independent experiments and 3 technical replicates . The data is represented as mean ± SE .

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qPCR measurements of JARID2 mRNA levels in different cell lines. The RNA levels were measured relative to 18S rRNA. Means and SEs are calculated over at least 2 independent experiments and 3 technical replicates. The data is represented as mean ± SE.


Immunoblot showing that the ~ 80kDa band disappears upon treatment of HEK293T and HaCaT cells with siRNAs against exon 3 and exon 15 of JARID2 RNA . Non - silencing control ( NSC ) was used as a transfection control .

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Immunoblot showing that the ~80kDa band disappears upon treatment of HEK293T and HaCaT cells with siRNAs against exon 3 and exon 15 of JARID2 RNA. Non-silencing control (NSC) was used as a transfection control.


qPCR measurements of RNA levels ( n = 3 ) for the three isoforms of JARID2 in HaCaT cells . The RNA levels are plotted relative to 18S rRNA . Level of JARID2 isoform - 1 is significantly higher than that of isoform - 2 ( * * * P < 0 . 001 ) and isoform - 3 ( * * * * P < 0 . 0001 ) .

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qPCR measurements of RNA levels (n=3) for the three isoforms of JARID2 in HaCaT cells. The RNA levels are plotted relative to 18S rRNA. Level of JARID2 isoform-1 is significantly higher than that of isoform-2 (***P<0.001) and isoform-3 (****P<0.0001).


Agarose gel showing RT - PCR products amplified using primers in exon3 and exon15 ( 2 . 9kb ) as well as RT - PCR product corresponding to JARID2 isoform - 1 ( 3 . 7kb ) amplified using the first ( exon 1 ) and the last exon ( exon18 ) . Only one product at the right size is observed indicating that it is unlikely that JARID2 might be a product of an alternatively spliced isoform of JARID2 .

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Agarose gel showing RT-PCR products amplified using primers in exon3 and exon15 (2.9kb) as well as RT-PCR product corresponding to JARID2 isoform-1 (3.7kb) amplified using the first (exon 1) and the last exon (exon18). Only one product at the right size is observed indicating that it is unlikely that JARID2 might be a product of an alternatively spliced isoform of JARID2.


CRISPR - Cas9 knockout of JARID2 using a sgRNA guide designed to target main translation start site as seen in ( E ) . Immunoblot revealed that ~ 80kDa form was removed from JARID2 KO lines .

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CRISPR-Cas9 knockout of JARID2 using a sgRNA guide designed to target main translation start site as seen in (E). Immunoblot revealed that ~80kDa form was removed from JARID2 KO lines.


Immunoblot after a full length JARID2 isoform - 1 was expressed from an exogenous vector in HaCaT cells showed an increase ( 2 - 3 fold ) in ~ 80kDa level .

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Immunoblot after a full length JARID2 isoform-1 was expressed from an exogenous vector in HaCaT cells showed an increase (2-3 fold) in ~80kDa level.


Immunoprecipitation using anti - JARID2 antibody and followed by mass spectrometry identification of the ~ 80kDa band detected JARID2 peptides spanning only C - terminus of JARID2 . The identified peptides are shown as red arrows on schematic of JARID2 sequence .

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Immunoprecipitation using anti-JARID2 antibody and followed by mass spectrometry identification of the ~80kDa band detected JARID2 peptides spanning only C-terminus of JARID2. The identified peptides are shown as red arrows on schematic of JARID2 sequence.


Immunoblot showing increase in expression levels of differentiation markers Involucrin ( IVL ) and Transglutaminase - 1 ( TGase - 1 ) as differentiation progressed indicating that keratinocyte differentiation protocol was successful . Protein levels on day 0 ( D0 ) , day 1 ( D1 ) , day 3 ( D3 ) and day 6 ( D6 ) of differentiation are shown . Immunoblot for JARID2 during D0 , D1 , D3 and D6 of differentiation ( as in B ) are shown .

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Immunoblot showing increase in expression levels of differentiation markers Involucrin (IVL) and Transglutaminase-1 (TGase-1) as differentiation progressed indicating that keratinocyte differentiation protocol was successful. Protein levels on day 0 (D0), day 1 (D1), day 3 (D3) and day 6 (D6) of differentiation are shown. Immunoblot for JARID2 during D0, D1, D3 and D6 of differentiation (as in B) are shown.


Effect of JARID2 removal on levels of differentiation markers Involucrin ( IVL ) , Keratin - 1 ( KRT1 ) and Keratin - 10 ( KRT10 ) mRNAs as measured in qPCR experiment relative to 18S rRNA and the rescue using exogenous expression of ΔN - JARID2 . The effect of exogenous expression of full - length JARID2 ( FL - JARID2 ) in JARID2 knockout is also shown . Data from wildtype HaCaTs , two independent JARID2 knockout ( KO1 and KO2 ) HaCaT lines as well as KO cells exogenously expressing empty vector control , ΔN - JARID2 and FL - JARID2 are shown . Levels of Involucrin ( IVL ) , Keratin - 1 ( KRT1 ) and Keratin - 10 ( KRT10 ) mRNAs as above but measured on day 3 of differentiation .

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Effect of JARID2 removal on levels of differentiation markers Involucrin (IVL), Keratin-1 (KRT1) and Keratin-10 (KRT10) mRNAs as measured in qPCR experiment relative to 18S rRNA and the rescue using exogenous expression of ΔN-JARID2. The effect of exogenous expression of full-length JARID2 (FL-JARID2) in JARID2 knockout is also shown. Data from wildtype HaCaTs, two independent JARID2 knockout (KO1 and KO2) HaCaT lines as well as KO cells exogenously expressing empty vector control, ΔN-JARID2 and FL-JARID2 are shown. Levels of Involucrin (IVL), Keratin-1 (KRT1) and Keratin-10 (KRT10) mRNAs as above but measured on day 3 of differentiation.


A metagene plot showing enrichment of full - length JARID2 in human induced pluripotent cells at down - regulated genes ( blue ) as compared to up - regulated genes ( grey ) in JARID2 KO cells . The plot is centered on Transcription Start Sites ( TSS ) of genes and distance from TSS is indicated on the x - axis .

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A metagene plot showing enrichment of full-length JARID2 in human induced pluripotent cells at down-regulated genes (blue) as compared to up-regulated genes (grey) in JARID2 KO cells. The plot is centered on Transcription Start Sites (TSS) of genes and distance from TSS is indicated on the x-axis.


Metagene plots of average enrichment of EZH2 in ES cells and keratinocytes at down - regulated genes ( blue ) as compared to up - regulated genes ( grey ) in JARID2 KO cells . The plots are centered on Transcription Start Sites ( TSS ) of genes and distance from TSS is indicated on the x - axes .

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Metagene plots of average enrichment of EZH2 in ES cells and keratinocytes at down-regulated genes (blue) as compared to up-regulated genes (grey) in JARID2 KO cells. The plots are centered on Transcription Start Sites (TSS) of genes and distance from TSS is indicated on the x-axes.


Levels of H3K27me3 in WT , JARID2 KO lines and JARID2 KO lines expressing ΔN - JARID2 . Histone H3 and Ponceau staining is used as a loading control .

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Levels of H3K27me3 in WT, JARID2 KO lines and JARID2 KO lines expressing ΔN-JARID2. Histone H3 and Ponceau staining is used as a loading control.


A bar - plot showing percentage of down - and up - regulated genes among genes which lose H3K27me3 vs which do not lose H3K27me3 modification during differentiation .

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A bar-plot showing percentage of down- and up-regulated genes among genes which lose H3K27me3 vs which do not lose H3K27me3 modification during differentiation.


Geometric mean - centered , hierarchical cluster heat map from microarray data . 1077 annotated lncRNAs ( p < 0 . 05 ) were represented in liver CSC ( CD13 + CD133 + ) compared with non - CSC ( CD13 - CD133 - ) cells sorted from three HCC primary cells of three patients . Top ten upregulated lncRNAs in CSCs are shown .

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Geometric mean-centered, hierarchical cluster heat map from microarray data. 1077 annotated lncRNAs (p<0.05) were represented in liver CSC (CD13+CD133+) compared with non-CSC (CD13-CD133-) cells sorted from three HCC primary cells of three patients. Top ten upregulated lncRNAs in CSCs are shown.


In situ hybridization of HAND2 - AS1 in HCC tumor tissues . HAND2 - AS1 staining is shown in tumor tissues ( left panel ) . Quantitation of HAND2 - AS1 + cells in liver tissues was examined in 10 high - power fields ( HPF ) of sections from ten different HCC patients ( right panel ) . Results are shown as means ± SEM . Scale bar , 100 μm .

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In situ hybridization of HAND2-AS1 in HCC tumor tissues. HAND2-AS1 staining is shown in tumor tissues (left panel). Quantitation of HAND2-AS1+ cells in liver tissues was examined in 10 high-power fields (HPF) of sections from ten different HCC patients (right panel). Results are shown as means ± SEM. Scale bar, 100 μm.


Macroscopic appearance of livers in 12 - month - old DEN - treated lncHand2 + / + and lncHand2 knockout ( lncHand2 - / - ) mice ( left panel ) . Black arrows indicate liver tumors . Quantitation of tumor foci numbers and areas in DEN - treated livers ( right panel ) . Results are shown as means ± SD ( n = 12 ) .

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Macroscopic appearance of livers in 12-month-old DEN-treated lncHand2+/+ and lncHand2 knockout (lncHand2-/-) mice (left panel). Black arrows indicate liver tumors. Quantitation of tumor foci numbers and areas in DEN-treated livers (right panel). Results are shown as means ± SD (n=12).


Representative H & E and immunohistological staining with Ki67 antibody of liver sections of 12 - month - old DEN - treated lncHand2 + / + and lncHand2 - / - mice . Scale bar , 100 μm .

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Representative H&E and immunohistological staining with Ki67 antibody of liver sections of 12-month-old DEN-treated lncHand2+/+ and lncHand2-/- mice. Scale bar, 100 μm.


Quantitation of Ki67 - positive cells in non - treated and DEN - treated mice . Bars represent average percentages and SD of cells positively staining for Ki67 cells examined in 10 HPF of sections from five different mice . Data are shown as means ± SD .

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Quantitation of Ki67-positive cells in non-treated and DEN-treated mice. Bars represent average percentages and SD of cells positively staining for Ki67 cells examined in 10 HPF of sections from five different mice. Data are shown as means ± SD.


Representative immunofluorescence staining of livers in 5 . 5 - month - old DEN - treated and untreated lncHand2RFP reporter mice for indicated molecules ( lower panel ) . Scale bar , 100 μm . Upper panel : scheme of targeting strategy for IRES - RFP knockin allele .

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Representative immunofluorescence staining of livers in 5.5-month-old DEN-treated and untreated lncHand2RFP reporter mice for indicated molecules (lower panel). Scale bar, 100 μm. Upper panel: scheme of targeting strategy for IRES-RFP knockin allele.


HAND2 - AS1 knockout causes a declined oncosphere - forming capacity in HCC cells . The right panel represents statistical results as means ± SD ( n = 3 per group ) . Overexpression of HAND2 - AS1 ( oelnc ) rescued the sphere formation reduced by HAND2 - AS1 deletion . Scale bar , 100 μm .

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HAND2-AS1 knockout causes a declined oncosphere-forming capacity in HCC cells. The right panel represents statistical results as means ± SD (n=3 per group). Overexpression of HAND2-AS1 (oelnc) rescued the sphere formation reduced by HAND2-AS1 deletion. Scale bar, 100 μm.


CD13 + CD133 + ( CSC ) subpopulations were detected in HAND2 - AS1 knockout cells by FACS analysis . Results are shown as means ± SD ( n = 4 per group ) .

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CD13+CD133+ (CSC) subpopulations were detected in HAND2-AS1 knockout cells by FACS analysis. Results are shown as means ± SD (n=4 per group).


Limiting diluted HAND2 - AS1 depletion or Ctrl HCC cells were subcutaneously implanted into BALB / c nude mice . n = 12 for each group .

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Limiting diluted HAND2-AS1 depletion or Ctrl HCC cells were subcutaneously implanted into BALB/c nude mice. n=12 for each group.


Estimated frequency of TICs in HAND2 - AS1 deficiency and control cells after the first transplantation using the extreme limiting dilution analysis . Data are shown as the mean and 95 % confidence interval . ( n = 12 grafted tumors per dilution ) .

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Estimated frequency of TICs in HAND2-AS1 deficiency and control cells after the first transplantation using the extreme limiting dilution analysis. Data are shown as the mean and 95% confidence interval. (n=12 grafted tumors per dilution).


Estimated frequency of TICs in HAND2 - AS1 deficiency and control HCC cells during serial transplantations . Grey area indicates the 95 % CI .

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Estimated frequency of TICs in HAND2-AS1 deficiency and control HCC cells during serial transplantations. Grey area indicates the 95% CI.


Estimated frequency of TICs in different populations after the first transplantation ( n = 15 grafted tumors per dilution ) . Data represent the mean and 95 % confidence interval .

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Estimated frequency of TICs in different populations after the first transplantation (n=15 grafted tumors per dilution). Data represent the mean and 95% confidence interval.


Representative whole - body imaging of Huh7 - Luc cells transduced with control or HAND2 - AS1 KO # 1 or KO # 2 vectors . Quantification of tumor numbers of 6 weeks after tumor cells orthotopically implanted to B - NSG mice . Data are shown as means ± SD . One - way ANOVA with Dunnett ' s correction for multiple comparisons .

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Representative whole-body imaging of Huh7-Luc cells transduced with control or HAND2-AS1 KO #1 or KO #2 vectors. Quantification of tumor numbers of 6 weeks after tumor cells orthotopically implanted to B-NSG mice. Data are shown as means ± SD. One-way ANOVA with Dunnett's correction for multiple comparisons.


HAND2 - AS1 expression levels ( normalized to 18S rRNA ) in livers from BALB / c nude mice subcutaneously transplanted xenograft primary HCC cells then intraperitoneally administered 25 mg kg − 1 ASOs on days 15 , 20 , 25 , 30 and 35 ( n = 5 per group ) . Three ASOs ( 1 , 2 , 3 ) against HAND2 - AS1 were used to treat mice and showed similar depletion effects . Results are shown as means ± SD . n = 6 mice per group .

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HAND2-AS1 expression levels (normalized to 18S rRNA) in livers from BALB/c nude mice subcutaneously transplanted xenograft primary HCC cells then intraperitoneally administered 25 mg kg−1 ASOs on days 15, 20, 25, 30 and 35 (n = 5 per group). Three ASOs (1, 2, 3) against HAND2-AS1 were used to treat mice and showed similar depletion effects. Results are shown as means ± SD. n=6 mice per group.


Mice were killed at the 40th day after HCC cell injection , and the tumors were excised and weighed . Representative tumors are shown . Scale bar , 1 cm . Statistical data are shown as means ± SD ( n = 6 mice per group ) ( right panel ) .

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Mice were killed at the 40th day after HCC cell injection, and the tumors were excised and weighed. Representative tumors are shown. Scale bar, 1 cm. Statistical data are shown as means ± SD (n = 6 mice per group) (right panel).


HAND2 - AS1 overexpression enhances the capacity of oncosphere formation . Scale bar , 100 μm . oe : overexpression . Right panel : Data are shown as means ± SD ( n = 3 per group ) .

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HAND2-AS1 overexpression enhances the capacity of oncosphere formation. Scale bar, 100 μm. oe: overexpression. Right panel: Data are shown as means ± SD (n = 3 per group).


Estimated frequency of TICs in HAND2 - AS1 overexpression and control HCC cells during serial transplantations .

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Estimated frequency of TICs in HAND2-AS1 overexpression and control HCC cells during serial transplantations.


Representative whole - body imaging of Huh7 - Luc cells transduced with control or HAND2 - AS1 overexpression vectors . Quantification for tumor numbers one month after tumor cells orthotopically implanted to B - NSG mice . ( n = 5 mice per group ) .

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Representative whole-body imaging of Huh7-Luc cells transduced with control or HAND2-AS1 overexpression vectors. Quantification for tumor numbers one month after tumor cells orthotopically implanted to B-NSG mice. (n = 5 mice per group).


Biotin - RNA pulldowns were performed with nuclear extracts of oncosphere cells using full - length HAND2 - AS1 transcript ( sense ) , antisense , and one HAND2 - AS1 intron control , followed by mass spectrometry . Band 1 : INO80 , band 2 : RUVBL2 .

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Biotin-RNA pulldowns were performed with nuclear extracts of oncosphere cells using full-length HAND2-AS1 transcript (sense), antisense, and one HAND2-AS1 intron control, followed by mass spectrometry. Band 1: INO80, band 2: RUVBL2.


Two core components of the INO80 complex were confirmed by immunoblotting .

Two core components of the INO80 complex were confirmed by immunoblotting.


Immunoprecipitation assays of biotin - labeled CHIRP probes incubated with liver CSC lysates .

Immunoprecipitation assays of biotin-labeled CHIRP probes incubated with liver CSC lysates.


HAND2 - AS1 was visualized by RNA FISH , followed by immunofluorescence staining of INO80 in HCC primary oncosphere cells . Scale bar , 100 μm .

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HAND2-AS1 was visualized by RNA FISH, followed by immunofluorescence staining of INO80 in HCC primary oncosphere cells. Scale bar, 100 μm.


Mapping analysis of INO80 - binding domains of HAND2 - AS1 . Schematic diagram of HAND2 - AS1 full - length and truncated fragments ( top panel ) ; Western blot of INO80 and RUVBL2 in RNA pulldown samples by different HAND2 - AS1 fragments ( middle panel ) ; Different HAND2 - AS1 fragments ( bottom panel ) .

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Mapping analysis of INO80-binding domains of HAND2-AS1. Schematic diagram of HAND2-AS1 full-length and truncated fragments (top panel); Western blot of INO80 and RUVBL2 in RNA pulldown samples by different HAND2-AS1 fragments (middle panel); Different HAND2-AS1 fragments (bottom panel).


EMSA of biotin - labeled HAND2 - AS1 ( nt 83 - 242 ) probes incubated with INO80 protein .

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EMSA of biotin-labeled HAND2-AS1 (nt 83-242) probes incubated with INO80 protein.


Binding affinity of HAND2 - AS1 with INO80 was determined by five independent EMSA assays . Non - linear regression curves were generated by GraphPad Prism . Results are shown as means ± SD .

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Binding affinity of HAND2-AS1 with INO80 was determined by five independent EMSA assays. Non-linear regression curves were generated by GraphPad Prism. Results are shown as means ± SD.


Interaction regions of INO80 with HAND2 - AS1 mutations at nt 83 - 242 of exon 2 were detected using RNA pulldown assays . LR , loop region .

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Interaction regions of INO80 with HAND2-AS1 mutations at nt 83-242 of exon 2 were detected using RNA pulldown assays. LR, loop region.


Representative whole - body imaging of Huh7 - Luc cells transduced with control or INO80 shRNA # 1 or shRNA # 2 vectors . Quantification of tumor numbers of 4 weeks after tumor cells orthotopically implanted to B - NSG mice . Data are shown as means ± SD ( n = 5 mice per group ) . One - way ANOVA with Dunnett ' s correction for multiple comparisons .

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Representative whole-body imaging of Huh7-Luc cells transduced with control or INO80 shRNA #1 or shRNA #2 vectors. Quantification of tumor numbers of 4 weeks after tumor cells orthotopically implanted to B-NSG mice. Data are shown as means ± SD (n = 5 mice per group). One-way ANOVA with Dunnett's correction for multiple comparisons.


Macroscopic appearance of livers in 10 . 5 - month - old DEN - treated Ino80flox / flox ( Ino80 + / + ) and Ino80 knockout ( Ino80 - / - ) ( left panel ) . Black arrows indicate tumors . Quantitation of tumor foci numbers and areas in DEN - treated livers . Results are shown as means ± SD ( n = 10 ) .

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Macroscopic appearance of livers in 10.5-month-old DEN-treated Ino80flox/flox (Ino80+/+) and Ino80 knockout (Ino80-/-) (left panel). Black arrows indicate tumors. Quantitation of tumor foci numbers and areas in DEN-treated livers. Results are shown as means ± SD (n=10).


Heatmap results for HAND2 - AS1 or INO80 deficiency in HCC cells .

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Heatmap results for HAND2-AS1 or INO80 deficiency in HCC cells.


Based on GSEA results , significantly changed genes due to depletion of HAND2 - AS1 and INO80 were attributed to the BMP signaling in HCC primary cells . NES , normalized enrichment score ; FWER , familywise error rate .

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Based on GSEA results, significantly changed genes due to depletion of HAND2-AS1 and INO80 were attributed to the BMP signaling in HCC primary cells. NES, normalized enrichment score; FWER, familywise error rate.


ChIRP - seq analysis of HAND2 - AS1 genomic binding at target sites in liver CSCs , using LacZ probes as negative control . A 10 - kb interval centered on the called HAND2 - AS1 peak is shown .

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ChIRP-seq analysis of HAND2-AS1 genomic binding at target sites in liver CSCs, using LacZ probes as negative control. A 10-kb interval centered on the called HAND2-AS1 peak is shown.


Representative ChIRP - seq of HAND2 - AS1 binding of BMPR1A .

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Representative ChIRP-seq of HAND2-AS1 binding of BMPR1A.


Gel analysis of HAND2 - AS1 RNA binding BMPR1A promoter in liver CSCs . LacZ and PVT1 served as negative controls .

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Gel analysis of HAND2-AS1 RNA binding BMPR1A promoter in liver CSCs. LacZ and PVT1 served as negative controls.


HAND2 - AS1 deletion in HCC cells decreases BMPR1A expression and inactivates BMP signaling .

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HAND2-AS1 deletion in HCC cells decreases BMPR1A expression and inactivates BMP signaling.


ChIRP - immunoblotting analysis of BMPR1A interaction with the INO80 complex in control and HAND2 - AS1 - knockout liver CSCs .

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ChIRP-immunoblotting analysis of BMPR1A interaction with the INO80 complex in control and HAND2-AS1-knockout liver CSCs.


EMSA analysis of the interaction of HAND2 - AS1 , INO80 and RUVBL2 with the BMPR1A promoter region .

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EMSA analysis of the interaction of HAND2-AS1, INO80 and RUVBL2 with the BMPR1A promoter region.


BMPR1A promoter binding region to HAND2 - AS1 / INO80 was deleted ( BMPR1A - p - KO ) in liver CSCs using lenti - Cas9 , followed by HAND2 - AS1 and INO80 overexpression ( oeHAND2 - AS1 , oeINO80 ) for 36 h . BMPR1A mRNA levels were examined by realtime PCR . Data are shown as means ± SD . n = 5 . * * P < 0 . 01 by two - tailed Student ' s t test .

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BMPR1A promoter binding region to HAND2-AS1/INO80 was deleted (BMPR1A-p-KO) in liver CSCs using lenti-Cas9, followed by HAND2-AS1 and INO80 overexpression (oeHAND2-AS1, oeINO80) for 36 h. BMPR1A mRNA levels were examined by realtime PCR. Data are shown as means ± SD. n=5. ** P < 0.01 by two-tailed Student's t test.


BMP signaling was examined in INO80 - silenced liver CSCs by Western blot .

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BMP signaling was examined in INO80-silenced liver CSCs by Western blot.


BMPR1A is highly expressed in HCC tumor tissues provided by Wang ' s cohort ( GSE14520 ) .

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BMPR1A is highly expressed in HCC tumor tissues provided by Wang's cohort (GSE14520).


Kaplan - Meier analyses of liver cancer outcomes in the cancer genome atlas ( TCGA ) . P value for Kaplan - Meier curves was determined using a Mantel - Cox log - rank test .

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Kaplan-Meier analyses of liver cancer outcomes in the cancer genome atlas (TCGA). P value for Kaplan-Meier curves was determined using a Mantel-Cox log-rank test.


Representative immunohistochemistry staining of BMPR1A was detected in HCC samples . Scale bar , 100 μm .

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Representative immunohistochemistry staining of BMPR1A was detected in HCC samples. Scale bar, 100 μm.


BMPR1A overexpression rescues sphere formation ability reduced by HAND2 - AS1 depletion in HCC samples . Representative sphere formation is shown on the left panel . Scale bar , 100 μm . Percentages of sphere - forming cells were calculated as means ± SD ( right panel ) . n = 4 .

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BMPR1A overexpression rescues sphere formation ability reduced by HAND2-AS1 depletion in HCC samples. Representative sphere formation is shown on the left panel. Scale bar, 100 μm. Percentages of sphere-forming cells were calculated as means ± SD (right panel). n=4.


BMPR1A depletion reduces tumor - initiating capacity . n = 12 mice per group .

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BMPR1A depletion reduces tumor-initiating capacity. n=12 mice per group.


Representative whole - body imaging of Huh7 - Luc cells transduced with scramble ( Ctrl ) or BMPR1A shRNAs . Quantification for tumor numbers of 7 weeks after tumor cells orthotopically implanted to B - NSG mice . Calculated data are shown as means ± SD ( n = 5 mice per group ) .

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Representative whole-body imaging of Huh7-Luc cells transduced with scramble (Ctrl) or BMPR1A shRNAs. Quantification for tumor numbers of 7 weeks after tumor cells orthotopically implanted to B-NSG mice. Calculated data are shown as means ± SD (n = 5 mice per group).


Macroscopic appearance of livers from 10 - month - old mice treated by DEN or vehicle . Scale bar , 1 cm . Black arrows indicates tumors .

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Macroscopic appearance of livers from 10-month-old mice treated by DEN or vehicle. Scale bar, 1 cm. Black arrows indicates tumors.


Representative H & E and immunohistological staining with Ki67 antibody in liver sections of 10 - month - old DEN - treated WT and Bmpr1a - / - mice . Scale bar , 100 μm . Right : Quantitation of Ki67 - positive cells in DEN - treated mice . Data are shown as means ± SD . n = 5 .

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Representative H&E and immunohistological staining with Ki67 antibody in liver sections of 10-month-old DEN-treated WT and Bmpr1a-/- mice. Scale bar, 100 μm. Right: Quantitation of Ki67-positive cells in DEN-treated mice. Data are shown as means ± SD. n=5.


BMP signaling inhibitor noggin decreased tumor sphere formation . Scale bar , 100 μm . Right : Data are shown as means ± SD ( n = 4 ) .

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BMP signaling inhibitor noggin decreased tumor sphere formation. Scale bar, 100 μm. Right: Data are shown as means ± SD (n=4).


Representative whole - body imaging of Huh7 - Luc cells in different groups . Mice treated with scramble ASOs and / or siRNAs obtained similar results to vehicle treatment . n ≥ 5 mice in each group .

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Representative whole-body imaging of Huh7-Luc cells in different groups. Mice treated with scramble ASOs and/or siRNAs obtained similar results to vehicle treatment. n≥5 mice in each group.


Quantification of tumor numbers treated with ASOs and / or siRNAs after Huh7 - Luc tumor cells orthotopically implanted to B - NSG mice . Calculated data are shown as means ± SD ( n = 5 ) .

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Quantification of tumor numbers treated with ASOs and/or siRNAs after Huh7-Luc tumor cells orthotopically implanted to B-NSG mice. Calculated data are shown as means ± SD (n = 5).


Tumors were excised from livers of PDX liver cancer models . Scale bar , 0 . 5 cm .

Tumors were excised from livers of PDX liver cancer models. Scale bar, 0.5 cm.


Representative H & E staining in liver sections from tumor - bearing mice treated with ASO1 and siBMPR1A in orthotopic transplants of tumors from HCC # 50 and HCC # 51 . Scale bar , 100 μm .

Representative H&E staining in liver sections from tumor-bearing mice treated with ASO1 and siBMPR1A in orthotopic transplants of tumors from HCC#50 and HCC#51. Scale bar, 100 μm.


Representative immunohistological staining with Ki67 antibody of tumor - bearing mice treated with ASO1 and siBMPR1A . Scale bar , 100 μm . Lower panel : Quantitation of Ki67 - positive cells in different groups . Data are shown as means ± SD ( n = 5 per group ) .

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Representative immunohistological staining with Ki67 antibody of tumor-bearing mice treated with ASO1 and siBMPR1A. Scale bar, 100 μm. Lower panel: Quantitation of Ki67-positive cells in different groups. Data are shown as means ± SD (n=5 per group).


Northern blotting of HAND2 - AS1 and immunobloting of BMPR1A in tumors from PDX HCC # 50 after treatment .

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Northern blotting of HAND2-AS1 and immunobloting of BMPR1A in tumors from PDX HCC#50 after treatment.


Kaplan - Meier survival analysis of B - NSG mice orthotopically transplanted with PDXs HCC # 50 ( I ) and HCC # 51 ( J ) . Treatment with ASO1 combined siBMPR1A causes synergistic therapeutic effect than each treatment alone ( n = 10 per group ) . P value for Kaplan - Meier curves was determined using a Mantel - Cox log - rank test .

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Kaplan-Meier survival analysis of B-NSG mice orthotopically transplanted with PDXs HCC#50 (I) and HCC#51 (J). Treatment with ASO1 combined siBMPR1A causes synergistic therapeutic effect than each treatment alone (n = 10 per group). P value for Kaplan-Meier curves was determined using a Mantel-Cox log-rank test.


Time lapse images of T6SS activity in tagged parental strain ( VipA - mCherry2 ) and ∆ tssA mutant background . The parental strain forms multiple dynamic structures per cell . ∆ tssA mutant predominantly forms dynamic sheath spots ( white arrow ) and few WT - like sheath structures ( blue arrow ) . Scale bars : 2 µm .

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Time lapse images of T6SS activity in tagged parental strain (VipA-mCherry2) and ∆tssA mutant background. The parental strain forms multiple dynamic structures per cell. ∆tssA mutant predominantly forms dynamic sheath spots (white arrow) and few WT-like sheath structures (blue arrow). Scale bars: 2 µm.


H2 - T6SS dynamics in tagged parental strain ( ∆ retS TssB2 - mCherry2 , referred to as WT H2 ) and ΔtssA2PA strain . Blue arrow indicates extending and contracting T6SS sheath structures , white arrow points to dynamic sheath spots . The WT H2 strain harbors multiple dynamic sheath structures per cell , while the ΔtssA2PA strain displays very few dynamic spots or extended and contracting structures . Scale bars : 2 µm .

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H2-T6SS dynamics in tagged parental strain (∆retS TssB2-mCherry2, referred to as WT H2) and ΔtssA2PA strain. Blue arrow indicates extending and contracting T6SS sheath structures, white arrow points to dynamic sheath spots. The WT H2 strain harbors multiple dynamic sheath structures per cell, while the ΔtssA2PA strain displays very few dynamic spots or extended and contracting structures. Scale bars: 2 µm.


Time lapse images of T6SS activity in tagged strain ( VipA - mCherry2 TssAVC - mNeonGreen ) . White arrow indicates path of TssAVC localization during one cycle of T6SS dynamics . Scale bar : 1 µm .

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Time lapse images of T6SS activity in tagged strain (VipA-mCherry2 TssAVC-mNeonGreen). White arrow indicates path of TssAVC localization during one cycle of T6SS dynamics. Scale bar: 1 µm.


Kymographs of dynamics observed for TssAVC . Frequency of specific dynamics is indicated in the upper right corner of the merge image . ( B ) Sheath and TssAVC dynamics from A . TssAVC localizes to T6SS initiation site and to distal end of growing sheath ( white arrow ) . ( C ) Once sheath reaches cell periphery , TssAVC dissociates from distal end ( white arrow ) and sheath stays extended a prolonged period of time . ( D ) TssAVC dissociates from growing sheath structure and re - associates at a later time point ( white arrows ) to continue catalysis of polymerization . ( E ) TssAVC stays attached to sheath distal end after contraction ( white arrow ) .

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Kymographs of dynamics observed for TssAVC. Frequency of specific dynamics is indicated in the upper right corner of the merge image. (B) Sheath and TssAVC dynamics from A. TssAVC localizes to T6SS initiation site and to distal end of growing sheath (white arrow). (C) Once sheath reaches cell periphery, TssAVC dissociates from distal end (white arrow) and sheath stays extended a prolonged period of time. (D) TssAVC dissociates from growing sheath structure and re-associates at a later time point (white arrows) to continue catalysis of polymerization. (E) TssAVC stays attached to sheath distal end after contraction (white arrow).


Fluorescence microscopy of T6SS dynamics in a double tagged strain ( ∆ retS TssB2 - mCherry2 TssA2PA - mNeonGreen ) . White arrow indicates full cycle of extending and contracting T6SS sheath . Scale bar : 1 µm .

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Fluorescence microscopy of T6SS dynamics in a double tagged strain (∆retS TssB2-mCherry2 TssA2PA-mNeonGreen). White arrow indicates full cycle of extending and contracting T6SS sheath. Scale bar: 1 µm.


Kymographs of TssB2 and TssA2PA dynamics . TssA2PA localizes at sheath initiation site and on distal end during polymerization ( G ) , dissociates before contraction ( H ) , dissociates and re - associated during sheath extension ( I ) or stays attached to distal end after contraction ( J ) .

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Kymographs of TssB2 and TssA2PA dynamics. TssA2PA localizes at sheath initiation site and on distal end during polymerization (G), dissociates before contraction (H), dissociates and re-associated during sheath extension (I) or stays attached to distal end after contraction (J).


Top view ( XY - plane ) and side view ( XZ - plane ) of the TssAVC Nt2 domain cryo - EM reconstruction ( pink , non transparent ) shown with fitted Nt2 - dimer model ( left ) . Partial Nt2 - CTD and Nt1 - Nt2 linker densities are highlighted with black arrows . Top and side views of ribbon diagram of Nt2 - dimer ( middle ) . Representative 2D class averages of TssAVC particles with visible connections between Nt2 - dimer and CTD ( top right ) , representative 2D class averages of Nt2 - dimer particles , Nt2 - CTD linker and Nt1 - Nt2 linker densities are highlighted with blue and yellow arrows ( bottom right ) .

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Top view (XY-plane) and side view (XZ-plane) of the TssAVC Nt2 domain cryo-EM reconstruction (pink, non transparent) shown with fitted Nt2-dimer model (left). Partial Nt2-CTD and Nt1-Nt2 linker densities are highlighted with black arrows. Top and side views of ribbon diagram of Nt2-dimer (middle). Representative 2D class averages of TssAVC particles with visible connections between Nt2-dimer and CTD (top right), representative 2D class averages of Nt2-dimer particles, Nt2-CTD linker and Nt1-Nt2 linker densities are highlighted with blue and yellow arrows (bottom right).


The ∆ tagA mutant displays long and bent but still dynamic structures . Scale bar : 2 µm . Frames from time lapse movies showing sheath dynamics in the ∆ tagA mutant . Sheath polymerization does not stop when the distal end reaches opposing membrane ( white arrow ) . Continued polymerization causes the sheath to tilt and distal end to move towards the bacterial cell pole , ultimately effecting bending of the whole structure . C . Sheath detaches from its membrane anchor ( orange arrow ) , keeps extending to maximum cell length and then contracts . Scale bars : 1 µm .

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The ∆tagA mutant displays long and bent but still dynamic structures. Scale bar: 2 µm. Frames from time lapse movies showing sheath dynamics in the ∆tagA mutant. Sheath polymerization does not stop when the distal end reaches opposing membrane (white arrow). Continued polymerization causes the sheath to tilt and distal end to move towards the bacterial cell pole, ultimately effecting bending of the whole structure. C. Sheath detaches from its membrane anchor (orange arrow), keeps extending to maximum cell length and then contracts. Scale bars: 1 µm.


Time lapse series of T6SS activity in tagged strain ( VipA - mCherry2 TagAVC - mNeonGreen ) . Orange arrow indicates sheath distal end , white arrow indicates initiation site of another T6SS sheath . Scale bar : 1 µm .

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Time lapse series of T6SS activity in tagged strain (VipA-mCherry2 TagAVC-mNeonGreen). Orange arrow indicates sheath distal end, white arrow indicates initiation site of another T6SS sheath. Scale bar: 1 µm.


Kymographs of dynamics observed for TagAVC . E . TagAVC localizes at the distal end when sheath reaches the cell periphery . Signal of TagAVC - mNeonGreen disappeared and reappeared on this site repeatedly . F . TagAVC localizes at the distal end when sheath reaches the cell periphery and stays associated to this site until contraction . G . TagAVC briefly localizes at T6SS initiation site . H . TagAVC briefly localizes at T6SS initiation site and at distal end , where it stays attached after contraction .

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Kymographs of dynamics observed for TagAVC. E. TagAVC localizes at the distal end when sheath reaches the cell periphery. Signal of TagAVC-mNeonGreen disappeared and reappeared on this site repeatedly. F. TagAVC localizes at the distal end when sheath reaches the cell periphery and stays associated to this site until contraction. G. TagAVC briefly localizes at T6SS initiation site. H. TagAVC briefly localizes at T6SS initiation site and at distal end, where it stays attached after contraction.


H1 - T6SS dynamics in tagged parental strain ( ∆ retS TssB1 - mCherry2 , referred to as WT H1 ) and ΔtssA1PA strain . Blue arrow indicates extending and contracting T6SS sheath structures , white arrow points to dynamic sheath spots . Scale bars : 2 µm .

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H1-T6SS dynamics in tagged parental strain (∆retS TssB1-mCherry2, referred to as WT H1) and ΔtssA1PA strain. Blue arrow indicates extending and contracting T6SS sheath structures, white arrow points to dynamic sheath spots. Scale bars: 2 µm.


Fluorescence microscopy of T6SS dynamics in ∆ retS TssB1 - mCherry2 strain harboring pPSV35 plasmid with TssA1PA - mNeonGreen fusion . Scale bar : 2 µm .

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Fluorescence microscopy of T6SS dynamics in ∆retS TssB1-mCherry2 strain harboring pPSV35 plasmid with TssA1PA-mNeonGreen fusion. Scale bar: 2 µm.


Kymographs of TssB1 and TssA1PA dynamics . TssA1PA localizes exclusively at sheath initiation site ( B - E ) , disappears after contraction ( C , D ) or remains in place for longer timeframe ( C , E ) . TssA1PA signal appears in neighboring cell shortly after T6SS attack ( B , C ) .

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Kymographs of TssB1 and TssA1PA dynamics. TssA1PA localizes exclusively at sheath initiation site (B-E), disappears after contraction (C,D) or remains in place for longer timeframe (C,E). TssA1PA signal appears in neighboring cell shortly after T6SS attack (B,C).


( D - E ) Western blot analysis of RIPA soluble - fraction and RIPA - insoluble , urea - soluble fraction of proteins prepared from HEK293T cells expressing empty plasmid ( control ) , FLAG - KIF5A WT , FLAG - ΔExon27 . Insoluble / soluble KIF5A fractions were detected with anti - FLAG and actin was used as a loading control , three biological replicates . Statistical analysis was performed using student t - test ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) . Data represent the mean ± s . d .

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(D-E) Western blot analysis of RIPA soluble-fraction and RIPA-insoluble, urea-soluble fraction of proteins prepared from HEK293T cells expressing empty plasmid (control), FLAG-KIF5A WT, FLAG-ΔExon27. Insoluble/soluble KIF5A fractions were detected with anti-FLAG and actin was used as a loading control, three biological replicates. Statistical analysis was performed using student t-test (*p < 0.05, **p<0.01, ***p<0.001). Data represent the mean ± s.d.


( B ) Pull down analysis showing the association of ΔExon27 with WT KIF5A , two biological replicates .

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(B) Pull down analysis showing the association of ΔExon27 with WT KIF5A, two biological replicates.


( A ) Sanger sequencing chromatograms of cDNA from control and patient - derived iMNs ( P1 - P3 ) showing the effect of mutations on KIF5A transcripts . While the c . 3020 + 2T > C mutation causes exon 27 skipping , the c . 2993 - 1G > A mutation results in an aberrant transcript with a splice acceptor shifted only by a single nucleotide , leading to a frameshift but containing exon 27 . The respective protein product predicted for both mutations comprise the same C - terminal aberrant 39 amino acid end .

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(A) Sanger sequencing chromatograms of cDNA from control and patient-derived iMNs (P1-P3) showing the effect of mutations on KIF5A transcripts. While the c.3020+2T>C mutation causes exon 27 skipping, the c.2993-1G>A mutation results in an aberrant transcript with a splice acceptor shifted only by a single nucleotide, leading to a frameshift but containing exon 27. The respective protein product predicted for both mutations comprise the same C-terminal aberrant 39 amino acid end.


( B ) Western blot analysis for validation of the custom mutant - specific antibody using transfected HEK293T cells . While an antibody against the total KIF5A ( lower panel ) recognized both wild type and mutant protein , the custom antibody targeting the aberrant " neopeptide " detected ΔExon27 specifically ( upper panel ) . NT : No transfection .

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(B) Western blot analysis for validation of the custom mutant-specific antibody using transfected HEK293T cells. While an antibody against the total KIF5A (lower panel) recognized both wild type and mutant protein, the custom antibody targeting the aberrant "neopeptide" detected ΔExon27 specifically (upper panel). NT: No transfection.


( C ) Mutant KIF5A proteins are endogenously expressed in iMNs , detected by using the custom mutant specific antibody directed against the C - terminal aberrant 39 amino acid sequence . CTL : Control , P1 : Patient 1 , P2 : Patient 2 , P3 : Patient 3 , two independent experiments .

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(C) Mutant KIF5A proteins are endogenously expressed in iMNs, detected by using the custom mutant specific antibody directed against the C-terminal aberrant 39 amino acid sequence. CTL: Control, P1: Patient 1, P2: Patient 2, P3: Patient 3, two independent experiments.


( D ) Representative images of KIF5A distribution ( arrowheads ) in control and patient iMNs . Scale bar : 20 µm . ( E ) Abundance of KIF5A inclusions after normalizing to total neuron numbers . Graph represents data from n = 146 control 1 ( CTL1 ) , n = 134 control 2 ( CTL2 ) , n = 154 patient 1 ( P1 ) , n = 162 patient 2 ( P2 ) and n = 133 patient 3 ( P3 ) iMNs from 3 individual experiments . Bars indicate mean ± s . d . Statistical analysis was performed using Mann Whitney Wilcoxon Test ( * p < 0 . 05 ) .

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(D) Representative images of KIF5A distribution (arrowheads) in control and patient iMNs. Scale bar: 20 µm. (E) Abundance of KIF5A inclusions after normalizing to total neuron numbers. Graph represents data from n=146 control 1 (CTL1), n = 134 control 2 (CTL2), n = 154 patient 1 (P1), n = 162 patient 2 (P2) and n = 133 patient 3 (P3) iMNs from 3 individual experiments. Bars indicate mean ± s.d. Statistical analysis was performed using Mann Whitney Wilcoxon Test (*p<0.05).


B , ITC experiment shows the CBS - pair domain of CNNM3 binds PRL2 with a nanomolar Kd and one - to - one stoichiometry .

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B, ITC experiment shows the CBS-pair domain of CNNM3 binds PRL2 with a nanomolar Kd and one-to-one stoichiometry.


D , Structure of CBS - pair domain of CNNM2 ( PDB 4IY0 ) with the residues DPFYE indicated .

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D, Structure of CBS-pair domain of CNNM2 (PDB 4IY0) with the residues DPFYE indicated.


F , ITC data of CBS - pair domain mutants binding to PRL2 identifies aspartic acid 426 as essential for binding .

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F, ITC data of CBS-pair domain mutants binding to PRL2 identifies aspartic acid 426 as essential for binding.


G , Kinetics of hydrolysis of a synthetic substrate by PRL2 shows burst kinetics . Addition of the wild - type CBS - pair domain but not the D426A mutant inhibits phosphatase activity .

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G, Kinetics of hydrolysis of a synthetic substrate by PRL2 shows burst kinetics. Addition of the wild-type CBS-pair domain but not the D426A mutant inhibits phosphatase activity.


A , Treatment of PRL2 with a synthetic substrate leads to complete phosphorylation of the catalytic residue , cysteine 101 . Recombinant PRL2 was treated with 3 - O - methylfluorescein phosphate ( OMFP ) and analyzed by SDS - PAGE with the Phos - tag reagent . Phosphocysteine is hydrolyzed by boiling .

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A, Treatment of PRL2 with a synthetic substrate leads to complete phosphorylation of the catalytic residue, cysteine 101. Recombinant PRL2 was treated with 3-O-methylfluorescein phosphate (OMFP) and analyzed by SDS-PAGE with the Phos-tag reagent. Phosphocysteine is hydrolyzed by boiling.


B , Detection of phosphorylated by electrospray mass spectrometry . OMFP - treatment increased the mass of the intact protein by 79 . 99 amu but had no effect on the C101A mutant .

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B, Detection of phosphorylated by electrospray mass spectrometry. OMFP-treatment increased the mass of the intact protein by 79.99 amu but had no effect on the C101A mutant.


C , 31P NMR spectrum of the thioPO3 signal from phosphocysteine in PRL2 . The broad peak is TCEP in the buffer .

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C, 31P NMR spectrum of the thioPO3 signal from phosphocysteine in PRL2. The broad peak is TCEP in the buffer.


D , Cysteine phosphorylated PRL2 does not bind to CNNM3 . Recombinant PRL2 was phosphorylated by treatment with OMFP and subjected to pull - down by the CBS - pair domain of CNNM3 as a GST fusion . Only unphosphorylated PRL2 was observed in the pull - down lanes while phosphorylated PRL2 was in the flow - through lanes .

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D, Cysteine phosphorylated PRL2 does not bind to CNNM3. Recombinant PRL2 was phosphorylated by treatment with OMFP and subjected to pull-down by the CBS-pair domain of CNNM3 as a GST fusion. Only unphosphorylated PRL2 was observed in the pull-down lanes while phosphorylated PRL2 was in the flow-through lanes.


E , ITC measurement of the affinity of PRL2 for the CNNM3 GST - CBS - pair domain . Phosphorylation by OMFP completely blocked binding while oxidization of PRL2 by diamide decreased the affinity 200 - fold .

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E, ITC measurement of the affinity of PRL2 for the CNNM3 GST-CBS-pair domain. Phosphorylation by OMFP completely blocked binding while oxidization of PRL2 by diamide decreased the affinity 200-fold.


F , The cysteine residues in the PRL2 active site are not required for binding the CNNM3 CBS - pair domain .

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F, The cysteine residues in the PRL2 active site are not required for binding the CNNM3 CBS-pair domain.


A , Mutations in the CBS - pair loop of CNNM4 prevent binding to PRL3 . Lysates of COS7 cells transfected with the indicated constructs were subjected to immunoprecipitation ( IP ) and immunoblotting ( IB ) with the indicated antibodies .

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A, Mutations in the CBS-pair loop of CNNM4 prevent binding to PRL3. Lysates of COS7 cells transfected with the indicated constructs were subjected to immunoprecipitation (IP) and immunoblotting (IB) with the indicated antibodies.


B C , Wild - type CNNM4 - dependent magnesium efflux is inhibited by co - transfection by PRL3 but the efflux by the CNNM4 D485A mutant , which is unable to bind PRL3 , is resistant to inhibition . HEK293 cells transfected with the indicated constructs were loaded with Magnesium Green and Mg2 + removed from the medium at the indicated time point ( arrowhead ) . The means of relative fluorescence intensities of 10 cells are graphed as a function of time in panel B and the mean and s . e . m . at 5 min plotted in panel C . P - values were calculated using one way ANOVA followed by Bonferroni ' s multiple comparison test . n . s . , not significant .

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B C, Wild-type CNNM4-dependent magnesium efflux is inhibited by co-transfection by PRL3 but the efflux by the CNNM4 D485A mutant, which is unable to bind PRL3, is resistant to inhibition. HEK293 cells transfected with the indicated constructs were loaded with Magnesium Green and Mg2+ removed from the medium at the indicated time point (arrowhead). The means of relative fluorescence intensities of 10 cells are graphed as a function of time in panel B and the mean and s.e.m. at 5 min plotted in panel C. P-values were calculated using one way ANOVA followed by Bonferroni's multiple comparison test. n.s., not significant.


A , The CBS - pair domain ( yellow ) forms a dimer and binds two molecules of PRL2 via an extended loop . The positions of the PRL2 C - terminal CAAX prenylation motif [ 42 ] and CNNM3 N - terminus suggest that the membrane would sit above the complex .

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A, The CBS-pair domain (yellow) forms a dimer and binds two molecules of PRL2 via an extended loop. The positions of the PRL2 C-terminal CAAX prenylation motif [42] and CNNM3 N-terminus suggest that the membrane would sit above the complex.


B , Contacts between the CNNM3 CBS2 loop and the PRL2 active site .

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B, Contacts between the CNNM3 CBS2 loop and the PRL2 active site.


C , Stereoview of the 2FO - FC omit map of the PRL2 binding loop of CNNM3 contoured at 1 .

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C, Stereoview of the 2FO-FC omit map of the PRL2 binding loop of CNNM3 contoured at 1 .


D , Detail of complex with reduced PRL2 , showing how CNNM3 Asp426 inserts into the pocket adjacent to the catalytic cysteine Cys101 .

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D, Detail of complex with reduced PRL2, showing how CNNM3 Asp426 inserts into the pocket adjacent to the catalytic cysteine Cys101.


E , Model of phosphocysteine preventing CNNM binding . Crystal structure of PRL1 with a bound sulfate shows phosphocysteine would prevent Asp426 binding . Residues are numbered according to the PRL1 structure ( PDB 1XM2 ) .

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E, Model of phosphocysteine preventing CNNM binding. Crystal structure of PRL1 with a bound sulfate shows phosphocysteine would prevent Asp426 binding. Residues are numbered according to the PRL1 structure (PDB 1XM2).


A , PRLs expressed in HEK293 cells are intrinsically phosphorylated on cysteine . Phos - tag analysis of cell lysates shows close to 50 % of transiently expressed FLAG - PRL1 , - 2 , and - 3 are phosphorylated . The phosphorylated PRL bands are marked by an asterisk .

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A, PRLs expressed in HEK293 cells are intrinsically phosphorylated on cysteine. Phos-tag analysis of cell lysates shows close to 50% of transiently expressed FLAG-PRL1, -2, and -3 are phosphorylated. The phosphorylated PRL bands are marked by an asterisk.


B , Immunoblots of PRL1 expressed in HEK293 cells show phosphorylation requires the catalytic cysteine ( C104 ) but not the adjacent cysteine ( C49 ) in the active site .

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B, Immunoblots of PRL1 expressed in HEK293 cells show phosphorylation requires the catalytic cysteine (C104) but not the adjacent cysteine (C49) in the active site.


C , Co - immunoprecipitation of Myc - PRL by FLAG - CNNMs shows only the unphosphorylated forms of PRL1 binds CNNM4 .

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C, Co-immunoprecipitation of Myc-PRL by FLAG-CNNMs shows only the unphosphorylated forms of PRL1 binds CNNM4.


D , Endogenous PRLs in HeLa cells are cysteine phosphorylated .

D, Endogenous PRLs in HeLa cells are cysteine phosphorylated.


E , Levels of unphosphorylated PRL increase in cultured cells in response to removal of magnesium from the cultured medium .

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E, Levels of unphosphorylated PRL increase in cultured cells in response to removal of magnesium from the cultured medium.


A ) In our chemoattraction - assay setup , nematode chemoattraction behavior is quantified using a measurable chemoattraction index ( C . I . ) ; the higher the C . I . measured in the assay , the stronger the attraction of the males to the sex pheromone . Data information : We assayed 400 males from each strain by using the sex pheromone chemoattraction assay . Two biological replicates were combined into a single value .

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A) In our chemoattraction-assay setup, nematode chemoattraction behavior is quantified using a measurable chemoattraction index (C.I.); the higher the C.I. measured in the assay, the stronger the attraction of the males to the sex pheromone. Data information: We assayed 400 males from each strain by using the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value.


B ) After vaporization ( VAC ) at 4 ° C for 24 h , the C . remanei female extract failed to attract wild - type C . elegans males . Conversely , a drop of the sex pheromone extract placed on the lid of a 60 - mm Petri dish evoked chemoattractive behavior in wild - type C . elegans males ( volatility test ) . Thus , the functional components in C . remanei female sex pheromone are volatile substances . Data information : We assayed 400 males from each strain by using the sex pheromone chemoattraction assay . Two biological replicates were combined into a single value . Significance was determined using one - way ANOVA with Bonferroni correction : * * * P < 0 . 001 . Means ± SEM ( error bars ) are shown .

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B) After vaporization (VAC) at 4°C for 24 h, the C. remanei female extract failed to attract wild-type C. elegans males. Conversely, a drop of the sex pheromone extract placed on the lid of a 60-mm Petri dish evoked chemoattractive behavior in wild-type C. elegans males (volatility test). Thus, the functional components in C. remanei female sex pheromone are volatile substances. Data information: We assayed 400 males from each strain by using the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.


C ) Mutant males with established defects in amphid neurons responded to C . remanei sex pheromone to a lesser degree relative to wild - type , demonstrating that some or all of the amphid neurons are required for sex pheromone perception . Data information : We assayed 400 males from each strain by using the sex pheromone chemoattraction assay . Two biological replicates were combined into a single value . Significance was determined using one - way ANOVA with Bonferroni correction : * * * P < 0 . 001 . Means ± SEM ( error bars ) are shown .

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C) Mutant males with established defects in amphid neurons responded to C. remanei sex pheromone to a lesser degree relative to wild-type, demonstrating that some or all of the amphid neurons are required for sex pheromone perception. Data information: We assayed 400 males from each strain by using the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.


A ) Inspection of specific amphid - neuron - defective mutants revealed that AWA neurons are most likely responsible for pheromone perception . Here , an edge connects neurons to a genetic mutant if the neurons are documented to be affected in the mutant . The color code of edges represents the average C . I . of multiple genetic mutants with impaired neuronal functions or specified abnormal neuron identities . The region colored green shows the range of wild - type sex pheromone response in the male . We assayed 400 males from each strain in the sex pheromone chemoattraction assay . Two biological replicates were combined into a single value . Significance was determined using one - way ANOVA with Bonferroni correction : * * * P < 0 . 001 . Means ± SEM ( error bars ) are shown .

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A) Inspection of specific amphid-neuron-defective mutants revealed that AWA neurons are most likely responsible for pheromone perception. Here, an edge connects neurons to a genetic mutant if the neurons are documented to be affected in the mutant. The color code of edges represents the average C.I. of multiple genetic mutants with impaired neuronal functions or specified abnormal neuron identities. The region colored green shows the range of wild-type sex pheromone response in the male. We assayed 400 males from each strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.


B ) AWA - laser - ablated males were not attracted by the sex pheromone in single - worm chemoattraction assays . Both AWA - defective mutant males ( lin - 11 and odr - 7 ) and AWA - laser - ablated males failed to reach the sex pheromone within 30 min . Wild - type C . elegans males responded normally to the sex pheromone and wild - type C . elegans hermaphrodites were not attracted by same - sex pheromone . The single - worm chemoattraction assay was performed as described in Materials and Methods . Sample size of males in AWA - laser - ablation experiment : 12 ; all other experiments : 100 worms .

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B) AWA-laser-ablated males were not attracted by the sex pheromone in single-worm chemoattraction assays. Both AWA-defective mutant males (lin-11 and odr-7) and AWA-laser-ablated males failed to reach the sex pheromone within 30 min. Wild-type C. elegans males responded normally to the sex pheromone and wild-type C. elegans hermaphrodites were not attracted by same-sex pheromone. The single-worm chemoattraction assay was performed as described in Materials and Methods. Sample size of males in AWA-laser-ablation experiment: 12; all other experiments: 100 worms.


B ) Mutant males of 4 GPCR signaling cascade components failed to respond to C . remanei sex pheromone , suggesting that the sex pheromone receptor is a GPCR and that both aforementioned signaling pathways are involved in pheromone perception . We assayed 400 males from each strain in the sex pheromone chemoattraction assay . Two biological replicates were combined into a single value . Significance was determined using one - way ANOVA with Bonferroni correction : * * * P < 0 . 001 . Means ± SEM ( error bars ) are shown .

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B) Mutant males of 4 GPCR signaling cascade components failed to respond to C. remanei sex pheromone, suggesting that the sex pheromone receptor is a GPCR and that both aforementioned signaling pathways are involved in pheromone perception. We assayed 400 males from each strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.


C ) In males from both AWA - specific - rescue odr - 3 mutant strain and odr - 3 rescued mutant strain , the sex pheromone response was significantly restored in single male worm arrival assays . However , males from the CEM - specific - rescue odr - 3 mutant strain failed to reach the sex pheromone within 30 min . In odr - 3 and him - 5 strain experiments , the sample sizes were 12 and 20 worms , respectively ; in all other experiments , 40 worms were used .

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C) In males from both AWA-specific-rescue odr-3 mutant strain and odr-3 rescued mutant strain, the sex pheromone response was significantly restored in single male worm arrival assays. However, males from the CEM-specific-rescue odr-3 mutant strain failed to reach the sex pheromone within 30 min. In odr-3 and him-5 strain experiments, the sample sizes were 12 and 20 worms, respectively; in all other experiments, 40 worms were used.


A ) srd - 1 mutant males were not attracted to the sex pheromone but were attracted to the positive - control chemoattractants , 1 : 1000 diacetyl and 10 mg / mL pyrazine . This demonstrated that SRD - 1 is required and specific for sex pheromone perception . Data information : For each transgenic strain , 3 independent lines were examined . We assayed 400 males from each strain in the sex pheromone chemoattraction assay . Two biological replicates were combined into a single value . Significance was determined using one - way ANOVA with Bonferroni correction : * * * P < 0 . 001 . Means ± SEM ( error bars ) are shown .

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A) srd-1 mutant males were not attracted to the sex pheromone but were attracted to the positive-control chemoattractants, 1:1000 diacetyl and 10 mg/mL pyrazine. This demonstrated that SRD-1 is required and specific for sex pheromone perception. Data information: For each transgenic strain, 3 independent lines were examined. We assayed 400 males from each strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.


B ) Summary of srd - 1 expression pattern . The transcriptional reporter Psrd - 1 : : gfp was detected in both male and hermaphrodite ASI neurons in the head region , and only in male AWA and ADF neurons .

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B) Summary of srd-1 expression pattern. The transcriptional reporter Psrd-1::gfp was detected in both male and hermaphrodite ASI neurons in the head region, and only in male AWA and ADF neurons.


C ) SRD - 1 : : GFP fusion construct revealed predominant SRD - 1 localization at the cilia of AWAs in C . elegans males . Scale bar = 5 μm .

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C) SRD-1::GFP fusion construct revealed predominant SRD-1 localization at the cilia of AWAs in C. elegans males. Scale bar = 5 μm.


D ) Examination of srd - 1 transcriptional reporter revealed that srd - 1 is expressed in AWA neurons in males but not hermaphrodites . A transcriptional reporter for odr - 7 indicated the locations of AWAs ( arrowheads ) . Scale bar = 50 μm .

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D) Examination of srd-1 transcriptional reporter revealed that srd-1 is expressed in AWA neurons in males but not hermaphrodites. A transcriptional reporter for odr-7 indicated the locations of AWAs (arrowheads). Scale bar = 50 μm.


F ) AWA - specific rescue of srd - 1 restored sex pheromone perception ability in both males and hermaphrodites . Data information : For each transgenic strain , 3 independent lines were examined . We assayed 400 males from each strain in the sex pheromone chemoattraction assay . Two biological replicates were combined into a single value . Significance was determined using one - way ANOVA with Bonferroni correction : * * * P < 0 . 001 . Means ± SEM ( error bars ) are shown .

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F) AWA-specific rescue of srd-1 restored sex pheromone perception ability in both males and hermaphrodites. Data information: For each transgenic strain, 3 independent lines were examined. We assayed 400 males from each strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.


G ) Ectopic expression of srd - 1 in AWB neurons elicited a distinct repulsive behavior toward the sex pheromone in both males and hermaphrodites . Data information : For each transgenic strain , 3 independent lines were examined . We assayed 400 males from each strain in the sex pheromone chemoattraction assay . Two biological replicates were combined into a single value . Significance was determined using one - way ANOVA with Bonferroni correction : * * * P < 0 . 001 . Means ± SEM ( error bars ) are shown .

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G) Ectopic expression of srd-1 in AWB neurons elicited a distinct repulsive behavior toward the sex pheromone in both males and hermaphrodites. Data information: For each transgenic strain, 3 independent lines were examined. We assayed 400 males from each strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.


A ) The GCaMP5 reporter was used to visualize activation of the SRD - 1 receptor by the sex pheromone in AWA neurons . To facilitate real - time imaging of the process , the heads of the transgenic worms were immobilized on soft - agar pads permeable to sex pheromone .

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A) The GCaMP5 reporter was used to visualize activation of the SRD-1 receptor by the sex pheromone in AWA neurons. To facilitate real-time imaging of the process, the heads of the transgenic worms were immobilized on soft-agar pads permeable to sex pheromone.


B ) Excitation of the GCaMP5 reporter in AWA neurons could be observed through both lateral and top views but the top view was clearer and more suitable for quantification . Arrowheads : AWA neurons . Scale bar = 50 μm .

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B) Excitation of the GCaMP5 reporter in AWA neurons could be observed through both lateral and top views but the top view was clearer and more suitable for quantification. Arrowheads: AWA neurons. Scale bar = 50 μm.


C ) Relative fluorescence - intensity change was quantified through calcium imaging . Wild - type males showed receptor excitation by sex pheromones from C . elegans hermaphrodites or C . remanei females . This response was lost in srd - 1 - mutant - strain males but was restored following AWA - specific rescue of srd - 1 . Red triangles : time points of sex pheromone addition . Three males were assayed from each strain .

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

C) Relative fluorescence-intensity change was quantified through calcium imaging. Wild-type males showed receptor excitation by sex pheromones from C. elegans hermaphrodites or C. remanei females. This response was lost in srd-1-mutant-strain males but was restored following AWA-specific rescue of srd-1. Red triangles: time points of sex pheromone addition. Three males were assayed from each strain.


B ) Wild - type C . elegans males are less attracted to sex pheromone than wild - type C . remanei males . Data information : We assayed 400 males from each wild - type strain in the sex pheromone chemoattraction assay . Two biological replicates were combined into a single value . Significance was determined using one - way ANOVA with Bonferroni correction : * * * P < 0 . 001 . Mean ± SEM ( error bars ) are shown .

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B) Wild-type C. elegans males are less attracted to sex pheromone than wild-type C. remanei males. Data information: We assayed 400 males from each wild-type strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Mean ± SEM (error bars) are shown.


C ) Expression level of srd - 1 cDNA in srd - 1 transgenic lines . srd - 1 mutant males were rescued using , separately , wild - type C . elegans and wild - type C . remanei srd - 1 cDNAs . Transgenic lines expressing the molecule at a similar level were selected for use in chemoattraction assays . Four independent transgenic lines were selected , and the expression - level data were combined into a single bar . The expression levels were measured using qPCR . Data information : For transgenic - line studies , each bar represents a total of 1200 males from 4 independent transgenic lines ; 20 assays were performed for 2 independent transgenic lines and 10 assays were performed for the other 2 independent transgenic lines , and the tests were conducted over 2 - 4 separate days . Significance was determined using one - way ANOVA with Bonferroni correction : * * * P < 0 . 001 . Mean ± SEM ( error bars ) are shown .

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

C) Expression level of srd-1 cDNA in srd-1 transgenic lines. srd-1 mutant males were rescued using, separately, wild-type C. elegans and wild-type C. remanei srd-1 cDNAs. Transgenic lines expressing the molecule at a similar level were selected for use in chemoattraction assays. Four independent transgenic lines were selected, and the expression-level data were combined into a single bar. The expression levels were measured using qPCR. Data information: For transgenic-line studies, each bar represents a total of 1200 males from 4 independent transgenic lines; 20 assays were performed for 2 independent transgenic lines and 10 assays were performed for the other 2 independent transgenic lines, and the tests were conducted over 2-4 separate days. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Mean ± SEM (error bars) are shown.


D ) srd - 1 mutant males rescued with Ce - srd - 1 cDNA showed lower responsiveness than those rescued with Cre - srd - 1 cDNA , which indicates that sex pheromone perception ability depends primarily on the protein coding region and not the regulatory sequences in the promoter . The sex pheromone perception defect of srd - 1 mutants was not rescued in males expressing CT - truncated SRD - 1 proteins . The findings show that the CT region is essential for sex pheromone perception . Data information : We assayed 400 males from each wild - type strain in the sex pheromone chemoattraction assay . Two biological replicates were combined into a single value . Significance was determined using one - way ANOVA with Bonferroni correction : * * * P < 0 . 001 . Mean ± SEM ( error bars ) are shown .

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

D) srd-1 mutant males rescued with Ce-srd-1 cDNA showed lower responsiveness than those rescued with Cre-srd-1 cDNA, which indicates that sex pheromone perception ability depends primarily on the protein coding region and not the regulatory sequences in the promoter. The sex pheromone perception defect of srd-1 mutants was not rescued in males expressing CT-truncated SRD-1 proteins. The findings show that the CT region is essential for sex pheromone perception. Data information: We assayed 400 males from each wild-type strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Mean ± SEM (error bars) are shown.


E ) srd - 1 mutant males rescued by using Ce - srd - 1 cDNA together with Cre - srd - 1 CT cDNA were more responsive to sex pheromone than those rescued using Cre - srd - 1 cDNA with Ce - srd - 1 CT cDNA . The results of the CT - swapping experiment demonstrated that sex pheromone perception ability is determined by the CT region and not the rest of the SRD - 1 protein . Data information : We assayed 400 males from each wild - type strain in the sex pheromone chemoattraction assay . Two biological replicates were combined into a single value . Significance was determined using one - way ANOVA with Bonferroni correction : * * * P < 0 . 001 . Mean ± SEM ( error bars ) are shown .

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

E) srd-1 mutant males rescued by using Ce-srd-1 cDNA together with Cre-srd-1 CT cDNA were more responsive to sex pheromone than those rescued using Cre-srd-1 cDNA with Ce-srd-1 CT cDNA. The results of the CT-swapping experiment demonstrated that sex pheromone perception ability is determined by the CT region and not the rest of the SRD-1 protein. Data information: We assayed 400 males from each wild-type strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Mean ± SEM (error bars) are shown.


PS decarboxylase activity in wild - type ( WT , W303 ) and the indicated mutant strains expressed as ( B ) the molecular species composition of 13C315N - labeled PS ( * PS ) and 13C215N - labeled PE ( * PE ) , after 20 min incubation with 13C315N - serine .

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

PS decarboxylase activity in wild-type (WT, W303) and the indicated mutant strains expressed as (B) the molecular species composition of 13C315N-labeled PS (*PS) and 13C215N-labeled PE (*PE), after 20 min incubation with 13C315N-serine.


( C ) The corresponding molecular signatures of PSD activity represented by the label distribution per molecular species between * PS and * PE as percentages of the total amount of label incorporated in the molecular species indicated .

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(C) The corresponding molecular signatures of PSD activity represented by the label distribution per molecular species between *PS and *PE as percentages of the total amount of label incorporated in the molecular species indicated.


PS decarboxylase activity in a psd1Δpsd2Δdpl1Δ mutant expressing Psd1 , Psd1 ( ER ) or Psd1 ( Mt ) expressed as ( B ) the molecular species composition of * PS and * PE after 20 min incubation with 13C315N -

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PS decarboxylase activity in a psd1Δpsd2Δdpl1Δ mutant expressing Psd1, Psd1(ER) or Psd1(Mt) expressed as (B) the molecular species composition of *PS and *PE after 20 min incubation with 13C315N-serine


PS decarboxylase activity in a psd1Δpsd2Δdpl1Δ mutant expressing Psd1 , Psd1 ( ER ) or Psd1 ( Mt ) expressed as ; ( C ) the corresponding molecular signatures of PSD activity .

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PS decarboxylase activity in a psd1Δpsd2Δdpl1Δ mutant expressing Psd1, Psd1(ER) or Psd1(Mt) expressed as ; (C) the corresponding molecular signatures of PSD activity.


( B ) Acyl chain composition of WT and psd1 ∆ cells overexpressing SCT1 ( pSCT1 ) vs . empty vector control ( pEmpty ) . Data is presented as mean ± SD ( n > 10 ) . * * * * p < 0 . 0001 , multiple t - test .

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(B) Acyl chain composition of WT and psd1∆ cells overexpressing SCT1 (pSCT1) vs. empty vector control (pEmpty). Data is presented as mean ± SD (n>10). **** p < 0.0001, multiple t-test.


( C ) Ratio of the proportions of saturated over unsaturated acyl chains ( SFA / UFA ) of WT , psd1 ∆ , crd1 ∆ and psd2 ∆ overexpressing SCT1 vs . empty vector control . Data is presented as mean ± SD ( n ≥ 3 ) . * * p < 0 . 01 , * * * * p < 0 . 0001 , unpaired two - tailed t - test .

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(C) Ratio of the proportions of saturated over unsaturated acyl chains (SFA/UFA) of WT, psd1∆, crd1∆ and psd2∆ overexpressing SCT1 vs. empty vector control. Data is presented as mean ± SD (n≥3). ** p<0.01, **** p < 0.0001, unpaired two-tailed t-test.


( D ) OLE1 transcript levels in WT and psd1 ∆ transformed with pSCT1 vs . pEmpty as determined by RT - qPCR . Data were normalized to endogenous ACT1 mRNA , and to the average of WT pEmpty ( n = 3 ) .

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(D) OLE1 transcript levels in WT and psd1∆ transformed with pSCT1 vs. pEmpty as determined by RT-qPCR. Data were normalized to endogenous ACT1 mRNA, and to the average of WT pEmpty (n=3).


( A ) SFA / UFA ratios of WT and indicated mutant strains overexpressing SCT1 ( pSCT1 ) vs . control ( pEmpty ) . Data is presented as mean ± SD ( n ≥ 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , n . s . not significant , unpaired two - tailed t - test of the indicated bar compared to WT pSCT1 .

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(A) SFA/UFA ratios of WT and indicated mutant strains overexpressing SCT1 (pSCT1) vs. control (pEmpty). Data is presented as mean ± SD (n≥3). * p < 0.05, ** p < 0.01, **** p < 0.0001, n.s. not significant, unpaired two-tailed t-test of the indicated bar compared to WT pSCT1.


( B ) Growth of WT and indicated mutant strains overexpressing SCT1 ( pSCT1 ) vs . pEmpty . Serial dilutions ( 10 - 1 - 10 - 4 ) were spotted on SGal + 0 . 05 % glucose and incubated for 3 days at 30 ° C .

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(B) Growth of WT and indicated mutant strains overexpressing SCT1 (pSCT1) vs. pEmpty. Serial dilutions (10-1 - 10-4) were spotted on SGal + 0.05% glucose and incubated for 3 days at 30°C.


PS decarboxylase activity in wild - type ( WT , BY4741 ) and the indicated mutant strains transformed with pEmpty , expressed , and ( D ) molecular species composition of 13C315N - labeled PS ( * PS ) and 13C215N - labeled PE ( * PE ) after 20 min incubation with 13C315N - serine . The differences in molecular species profiles of * PS and * PE between the BY4741 WT shown here and those obtained by 2H3 - serine labeling ( Figure 4A ) are attributed to the different culture media , lipid extraction procedures and mass spectrometry techniques used .

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PS decarboxylase activity in wild-type (WT, BY4741) and the indicated mutant strains transformed with pEmpty, expressed , and (D) molecular species composition of 13C315N-labeled PS (*PS) and 13C215N-labeled PE (*PE) after 20 min incubation with 13C315N-serine. The differences in molecular species profiles of *PS and *PE between the BY4741 WT shown here and those obtained by 2H3-serine labeling (Figure 4A) are attributed to the different culture media, lipid extraction procedures and mass spectrometry techniques used.


( E ) The corresponding molecular signatures of PSD activity represented by the label distribution per molecular species between * PS and * PE as percentages of the total amount of label incorporated in the molecular species indicated .

(E) The corresponding molecular signatures of PSD activity represented by the label distribution per molecular species between *PS and *PE as percentages of the total amount of label incorporated in the molecular species indicated.


SFA / UFA - ratio of WT and indicated mutant strains overexpressing SCT1 cultured without ( data taken from Fig 5A ) and with 1 mM ethanolamine ( etn ) or 1 mM choline ( cho ) as indicated . Data is presented as average ± SD ( n ≥ 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , unpaired t - test of the indicated bar compared to the corresponding condition of WT . Underlying data for this figure can be found in Appendix Table S1 .

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SFA/UFA-ratio of WT and indicated mutant strains overexpressing SCT1 cultured without (data taken from Fig 5A) and with 1 mM ethanolamine (etn) or 1 mM choline (cho) as indicated. Data is presented as average ± SD (n≥3). * p < 0.05, ** p < 0.01, *** p < 0.001, unpaired t-test of the indicated bar compared to the corresponding condition of WT. Underlying data for this figure can be found in Appendix Table S1.


( b ) The CD spectra of the Beclin 1 CC domain at different temperatures .

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(b) The CD spectra of the Beclin 1 CC domain at different temperatures.


( c ) . Dynamic exchange assay of wild - type Beclin 1 . His - tagged and untagged Beclin 1 CC domain were co - incubated at the indicated temperatures and coprecipitated at preset temperatures by Ni2 + - nitrilotriacetic acid ( NTA ) agarose beads . The presence of heterodimeric Beclin 1 CC domain , consisting of one monomer with his - tag and the other monomer without tag , was checked by SDS gel and indicated by the arrow .

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(c). Dynamic exchange assay of wild-type Beclin 1. His-tagged and untagged Beclin 1 CC domain were co-incubated at the indicated temperatures and coprecipitated at preset temperatures by Ni2+- nitrilotriacetic acid (NTA) agarose beads. The presence of heterodimeric Beclin 1 CC domain, consisting of one monomer with his-tag and the other monomer without tag, was checked by SDS gel and indicated by the arrow.


( d ) Self - association pattern of Beclin 1 in vivo . Both GFP - and AsRed - tagged full - length Beclin 1 are co - transfected into HEK 293T cells and their self - association is probed by co - IP using GFP antibody for immunoprecipitation , followed by western blot with anti - AsRed antibody .

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(d) Self-association pattern of Beclin 1 in vivo. Both GFP- and AsRed-tagged full-length Beclin 1 are co-transfected into HEK 293T cells and their self-association is probed by co-IP using GFP antibody for immunoprecipitation, followed by western blot with anti-AsRed antibody.


( a , b ) ITC titration profile of Beclin 1 CC domain with Atg14L ( a ) or UVRAG ( b ) .

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(a, b) ITC titration profile of Beclin 1 CC domain with Atg14L (a) or UVRAG (b).